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PULMONARY DELIVERY FOR BIOCONJUGATION
FIELD OF THE INVENTION
This invention relates to the field of therapeutic and diagnostic agents
in medicine. In particular, this invention relates to the field of delivery,
in
particular, pulmonary delivery, of therapeutic and diagnostic agents wherein
the agents are capable of covalently bonding to a site of interest in vivo, to
provide increased bioavailability and pharmacodynamic duration of
therapeutic and diagnostic benefit for the given agent.
BACKGROUND. OF THE INVENTION
Peptide and protein drugs are being used increasingly in major
research and development programs in the pharmaceutical industry and are
also an important class of therapeutic agents due to advances in genetic
engineering and biotechnology. Systemic delivery of these macromolecular
drugs and other therapeutic and diagnostic agents,however, has been limited
to the parenteral route largely because of their extensive presystemic
elimination when taken orally. Faced with this dilemma concerning the
systemic delivery of these macromolecules with their unique conformational
complexity for therapeutic activity, pharmaceutical scientists are continually
evaluating the potential of various non-oral routes of administration as
alternatives.
Despite the tremendous efforts that have been devoted to this
problem, only limited success has been achieved--mostly with small peptides.
An alternative, non-invasive means for systemic delivery of therapeutic and
diagnostic agents is via the pulmonary system. Delivery via the pulmonary
system is advantageous because the lungs provide a large but extremely thin
absorptive mucosal membrane for increased absorption and delivery to the
blood stream. However, pulmonary delivery of therapeutic and diagnostic
agents is complicated by the complexity of the anatomic structure of the
human respiratory system; the effect of respiration on drug deposition and an
instability of the drugs resulting from degradation in either the lungs or
plasma.
There is thus a need to improve and enhance delivery of therapeutic
and diagnostic agents, especially pulmonary delivery of of therapeutic and
diagnostic agents through increasing the stability and blood absorption of the
agents.
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SUMMARY OF THE INVENTION
In order to meet these needs, the present invention is directed to
therapeutic and diagnostic agents capable of forming covalent bonds to blood
and pulmonary fluid proteins or other components ex vivo or in vivo. The
therapeutic agents of this invention have a long duration of action for the
management of disease. The invention relates to ex vivo and in vivo.
bioconjugation of therapeutic agents to protein (e.g. albumin), as well as an
intrapulmonary in vivo bioconjugation of therapeutic agents to endogenous
pulmonary fluid proteins or other components to dramatically increase the half
life of the therapeutic agents and avoid the need for parenteral
administration.
The present invention reflects the ability to bioconjugate selected
therapeutic agents to blood and pulmonary pulmonary fluid proteins, including
albumin, for processing as a particulate for intrapulmonary drug delivery. The
pulmonary fluid protein conjugate is targeted to provide a stable drug
substance that retains biological activity for prolonged periods of time. This
invention provides prolonged local retention of therapeutic agent activity in
the
airways for use with selected therapeutic agents in managing pulmonary
disease.
The invention is further directed to methods of facilitating systemic
drug delivery of protein-therapeutic agent bioconjugates and to agents
capable of forming bioconjugates to protein in vivo via pulmonary delivery
with subsequent transcytosis across the alveolar and pulmonary mucosa. The
invention is further directed to methods of facilitating systemic drug
delivery of
protein-therapeutic agent bioconjugates via pulmonary delivery of agents
capable of forming bioconjugates in vivo, the agents crossing the epithelium
of the alveolar or pulmonary mucosa, either through diffusion or receptor-
mediated transport, to conjugate with blood proteins. The methods of this
invention result in long acting, systemic therapeutics that are stabilized by
ex
vivo or in vivo conjugation to pulmonary fluid proteins and/or blood proteins.
This invention is further directed to site-specific and protein-specific
bioconjugation of a therapeutic agent to albumin. Albumin possesses a
unique nucleophilic moiety, specifically, the thiol functionality on cysteine
34
that is capable of undergoing a nucleophilic attack on electrophile present on
a therapeutic agent modified according to the invention. This selective
covalent bonding enables bioconjugation to extracellular as well as
intracellular albumin for prolonged retention and bioavailabilty of the
therapeutic agent.
This invention is further directed to the use of reactive chemistries
including: N-hydroxy sulfosuccinimide, maleimide-benzoyl-succinimide,
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gamma-maleimido-butyryloxy succinimide ester, maleimidopropionic acid,
isocyanate, thiolester, thionocarboxylic acid ester, imino ester, and
carbodiimide anhydride. Maleimidopropionic acid is the preferred reactive
chemistry, but the invention also contemplates the selection of the above and
like reactive chemistries as an electrophilic moeity for bioconjugations with
albumin or other proteins.
This invention is further directed to the use of a composition for the
manufacture of a medicament where the composition comprises a derivative
of an antihistamine and analogs thereof wherein the derivative includes a
reactive functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds, said
reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anhistamine effect.
The modified antihistamine may be cetirizine, loratidine and analogs
thereof.
This invention is further directed to the use of a composition for for the
manufacture of a medicament where the composition comprises a derivative
of an anti-angina agent and analogs thereof wherein the derivative includes a
reactive functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds, said
reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anti-angina effect.
The modifed anti-angina agent may be tirofiban or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament where the composition comprises a derivative
of an anti-hypertensive agent and analogs thereof wherein the derivative
includes a reactive functional group which reacts with amino groups, hydroxyl
groups, or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anti-hypertensive effect.
The anti-hypetensive agent may be enalapril or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament where the composition comprising a derivative
of an anti-arrhythmic agent and analogs thereof wherein the derivative
includes a reactive functional group which reacts with amino groups, hydroxyl
groups, or thiol groups on blood components to form stable covalent bonds,
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said reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anti-arrhythmic effect.
The anti-arrhythmic agent may be capobenic acid or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament where the composition comprising a derivative
of an anti-depressant agent and analogs thereof wherein the derivative
includes a reactive functional group which reacts with amino groups, hydroxyl
groups, or thiol groups on blood components to form stable covalent bonds,
said reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anti-depressan effect.
The anti-depressant agent may be fluoxetine or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament said composition comprising a derivative of a
bronchodilator and analogs thereof wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups, or thiol
groups on blood components to form stable covalent bonds, said reactive
functional group being selected from N-hydroxysuccinimide, N-hydroxy-
sulfosuccinimide and a maleimide group for use in the treatment of the human
body to provide a bronchodilation effect.
The bronchodilator may be theobromineacetamine or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament said composition comprising a derivative of an
opioid and analogs thereof, wherein the derivative includes a reactive
functional group which reacts with amino groups, hydroxyl groups, or thiol
groups on blood components to form stable covalent bonds, said reactive
functional group being selected from N-hydroxysuccinimide, N-hydroxy-
sulfosuccinimide and a maleimide group for use in the treatment of the human
body to provide an analgesic effect.
The opioid may be fentanyl or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament said composition comprising a derivative of an
anti-inflammatory agent and analogs thereof, wherein the derivative includes
a reactive functional group which reacts with amino groups, hydroxyl groups,
or thiol groups on blood components to form stable covalent bonds, said
reactive functional group being selected from N-hydroxysuccinimide, N-
hydroxy-sulfosuccinimide and a maleimide group for use in the treatment of
the human body to provide an anti-inflammatory effect.
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The anti-inflammatory agent may be loxoprofen or analogs thereof.
This invention is further directed to the use of a composition for the
manufacture of a medicament where the composition comprising a derivative
of an anti-thyroid deficiency agent and analogs thereof, wherein the
derivative includes a reactive functional group which reacts with amino
groups, hydroxyl groups, or thiol groups on blood components to form stable
covalent bonds, said reactive functional group being selected from N-
hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide group for
use in the treatment of the human body to provide an anti-thyroid deficiency
effect.
the anti-thyroid deficiency agent may be thyroxin or analogs thereof.
This invention is further directed to composition comprising one or more
compounds selected from the group consisting of
2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-
maleimidopropionylacetamide; 11-(N-maleimidopropionyl-4-piperidylidene)-8-
chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine;
N-(1 (S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-
prolinylmaleimidopropionilamide;
Maleimidopropynamyl-E-(3,4,5-trimethoxybenz-amido)-caproicamide;
Maleimidopropionamyl-1-theobromineacetamide;
Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide
N-maleimidopropionyl-N-methyl-3-(p-trifluoromethylphenoxy)-3-
phenylpropylamine; 4-anilino-1-(2-phenethyl)piperdine and
Maleimidopropionamyl-3,5-3',5' tetraiodothyroninamide.
This invention is further directed to the an aerosol composition for
delivery of a therapeutic agent to the pulmonary system of a host comprising
an aerosolized aqueous solution containing a modified therapeutic agent
conjugated to a blood protein.
This invention is further directed to the use of a particulate formulation
for delivery of a therapeutic agent to the pulmonary system of a host
comprising:
a dispersable dry powder containing a modified therapeutic agent, the
modified therapeutic agent comprising a therapeutic agent and a reactive
group which reacts with amino groups, hydroxyl groups or thiol groups on
pulmonary components to form a stable covalent bond wherein said
therapeutic agent is covalently bonded to a blood protein.
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DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
To ensure a complete understanding of the invention the following
definitions are provided:
Therapeutic Agents: Therapeutic agents are agents that have a
therapeutic effect and inlcude peptides and non-peptide organic molecules.
Therapeutic agents include but are not limited to wound healing agents,
antibiotics, anti-infectives, anti-oxidants, chemotherapeutic agents, anti-
cancer agents, anti-inflammatory agents, and antiproliferative drugs.
Therapeutic agents also include abortifacients, ace-inhibitor, a-adrenergic
agonists, ~i-adrenergic agonists, a-adrenergic blockers, ~3-adrenergic
blockers, adrenocortical steroids, adrenocortical supressants,
adrenocorticotrophic hormones, alcohol deterrents, aldose reductase
inhibitors, aldosterone antagonists, 5-alpha reductase inhibitors, anabolics,
analgesics, analgesics, analgesics, androgens, anesthetics, anesthetics,
angiotensin coverting enzyme inhibitors, anorexics, antacids, anthelmintics,
antiacne agents, antiallergic agents, antialopecia agents, antiamebic agents,
antiandrogen agents, antianginal agents, antiarrhythmic agents,
antiarteriosclerotic agents, antiarthritic/antirheumatic agents, antiasthmatic
agents, antibacterial agents, aminoglycosides, amphenicols, ansamycins, ~3-
lactams, lincosamides, macrolides, polypeptides, tetracyclines, antibacterial
agents, 2,4-diaminopyrimidines, nitrofurans, quinolones and analogs,
sulfonamides, sulfones, antibiotics, anticholelithogenic agents,
anticholesteremic agents, anticholinergic agents, anticoagulant agents,
anticonvulsant agents, antidepressant agents, hydrazides/hydrazines,
pyrrolidones, tetracyclics, antidiabetic agents, biguanides, hormones,
sulfonylurea derivatives, antidiarrheal agents, antiduretic agents, antidotes,
antidote, antidote, antidote, antidote, antidyskinetic, antieczematic,
antiemetic
agents, antiepileptic agents, antiestrogen agents, antifibrotic agents,
antiflatulent agents, antifungal agents, polyenes, allylamines, imidazoles,
triazoles and antiglaucoma agents.
Other therapetic agents include anti-viral agents, anti-fusogenic
agents, blood brain barrier peptides (BBB peptides), RGD peptides,
glucagon-like peptides, antigonadotropin, antigout, antihemorrhagic and
antihistaminic agents; alkylmaine derivatives, aminoalkyl ethers,
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ethylenediamine derivatives, piperazines and tricyclics,
antihypercholesterolemic, antihyperlipidemic, anthyperlipidemic and
antihyperlipoproteinemic agents, aryloxyalkanoic acid derivatives, bile acid
sequesterants, hmg coa reductase inhibitors, nicotine acid derivatives,
thyroid
hormones/analogs, antihyperphosphatemic, antihypertensive agents,
arlethanolamine derivatives, arloxypropanolamine derivatives,
benzothiadiazine derivatives, n-carboxyalkyl derivatives, dihydropyridine
derivatives, guanidine derivatives, hydrazines/phthalazines, imidazole
derivatives, quaternary ammonium compounds, quinazolinyl piperazine
derivatives, reserpine derivatives, sulfonamide derivatives, antihyperthyroid
agents, antihypotensive agents, antihypothyroid agents, anti-infective agents,
anti-inflammatory agents, anti-inflammatory agents, aminoarylcarboxylic acid
derivatives, arylacetic acid derivatives, arylbutyric acid derivatives and
arylcarboxylic acids.
Therapeutic agents also include arylpropionic acid derivatives,
pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
antileprotic, antileukemic, antilipemic, antilipidemic, antimalarial,
antimanic,
antimethemoglobinemic, antimigraine, antimycotic, antinauseant,
antineoplastic and alkylating agents, antimetabolites, enzymes, androgens,
antiadrenals, antiandrogens, antiestrogens, Ih-rh analogs, progestogens,
adjunct, folic acid replenisher, uroprotective and antiosteporotic agents
Therapeutic agents also include antipagetic, antiparkinsonian,
antiperistaltic, antipheochromocytoma, antipneumocystis, antiprostatic
hypertrophy, antiprotozoal, antiprozoal" antipruritic, antipsoriatic and
antipsychotic agents, butyrophenes, phenothiazines, thioxanthenes,
antipyretic, antirheumatic, antirickettsial, antiseborreheic and
antiseptic/disinfectant agetns, alcohols, aldehydes, dyes, guanidines,
halogens/halogen compounds, mercurial compounds, nitrofurans,
peroxides/permanganates, phenols, quinolines, silver compounds, others,
antispasmodic,antisyphilitic, antithrombotic, antitubercular, antitumor,
antitussive, antiulcerative, antiurolithic, antivenin, antivertigo and
antiviral
agents, purines/pyrimidinomes, anxiolytic, arylpiperazines, benzodiazepine
derivatives, carbamates, astringent, benzodiazepine antagonist, beta-blocker,
bronchodilator, ephedrine derivatives, calcium channel blockers,
arylalkylamines, dihydropyridine derivatives, piperazine derivatives, calcium
regulators, calcium supplements, cancer chemotherapy agents, capillary
protectants, carbonic anhydrase inhibitors, cardiac depressants, cardiotonic,
cathartic, cation-exchange resin, cck antagonists, central stimulants,cerebral
vasodilators, chelating agents, cholecystokinn antagonists, choleitholytic
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agents, choleretic agents, cholinergic agents, cholinesterase inhibitors,
cholinesterase reactivators, cns stimulants, cognition activators,
contraceptives, agents to control intraocular pressure, converting-enzyme
inhibitors, coronary vasodilators, cytoprotectants, debriging agents,
decongestants, depigmentora, dermatitis herpretiformis suppresanta,
diagnostic aids, digestive aids, diuretics, benthothiadiazine derivatives,
organomercurials, pteridines, purines, steroids, sulfanamide derivatives,
uracils, others, dopamine and receptor agonists.
Therapeutic agents also include dopamine receptor antagonists,
ectoparasiticides, electrolyte replenishers, emetics, enzymes, digestive
agents, mucolytic agents, penicillin inactivating agents, proteolytic agents,
enzyme inducers, estrogen antagonists, expectorant gastric and pancreatic
secreation stimulantd, gastric proton pump inhibitor, gastric secretion
inhibitord, glucocorticoidd, a-glucosidase inhibitord, gonad-stimulating
principled, gonadotrophic hormoned, gout suppressant, growth hormone
inhibitor, growth hormone releasing factor, growth stimulant, hematinic,
hemolytic, demostatic, heparin antagonist, hepatoprotectant, histamine h,-
receptor antagonists, histamine h2 receptor antagonists, hmg coa reductase
inhibitor, hypnotic, hypocholesteremic and hypolipidemic agents.
Therapeutic agents also include hypotensive, immunomodulators,
immunosuppressants, inotrophic agents, keratolytic agents, lactation
stimulating hormone, laxative/cathargic, Ih-rh agonists, lipotrophic agents,
local anesthetics, lupus erythematosus suppressants, major tranquilizers,
mineralocorticoids, minor tranquilizers, miotic agents, monoamine oxidase
ihibitors, mucolytic agents, muscle relaxants, mydriatic agents, narcotic
agents; analgesics, narcotic antagonists, nasal decongestants, neuroleptic
agents, neuromuscular blocking agents, neuroprotective agents, nmda
antagonists, nootropic agents, nsaid agents, opioid analgesics, oral
contraceptives and ovarian hormones.
Therapeutic agents also include oxytocic agents, blood brain barrier
protiens, GP-41 peptides, insulinotropic peptides parasympathomimetic
agents, pediculicides, pepsin inhibitors, peripheral vasodilators, peristaltic
stimulants, pigmentation agents, plasma volume expanders, potassium
channel activators./openers, pressor agents, progestogen, prolactin
inhibitors,
prostaglandin/prostaglandin analogs, protease inhibitors, proton pump
inhibitors, 5a-reductase inhibitors, replenishers/supplements, respiratory
stimulants, reverse transcriptase inhibitors, scabicides, sclerosing agents,
sedative/hypnotic agents, acyclic ureides, alcohols, amides, barbituric acid
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derivatives, benzodiazepine derivatives, bromides, carbamates, chloral
derivatives, quinazolone derivatives and piperidinediones.
Therapeutic agents also include serotonin receptor agonists, serotonin
receptor antagonists, serotonin uptake inhibitors, skeletal muscle relaxants,
somatostatin analogs, spasmolytic agents, stool softeners, succinylcholine
synergists, sympathomimetics, thrombolytics, thyroid hormone, thyroid
inhibitors, thyrotrophic hormone, tocolytic, topical protectants, uricosurics,
vasodilators, vasopressors, vasoprotectants, vitamin/vitamin sources,
antichitic, antiscorbutic and antixerophthalmic agents, enzyme co-factors,
hematopoietic, prombogenic agents and xanthene oxidase inhibitors.
Diagnostic Imaging Agents: Diagnostic imaging agents are agents
useful in imaging the mammalian vascular system and include such agents as
position emission tomography (PET) agents, computerized tomography (CT)
agents, magnetic resonance imaging (MRI) agents, nuclear magnetic imaging
agents (NMI), fluroscopy agents and ultrasound contrast agents. Diagnostic
agents of interest include radioisotopes of such elements as iodine (I),
including 'z31, 'z51, '3' I, etc., barium (Ba), gadolinium (Gd); technetium
(Tc),
including 99Tc, phosphorus (P), including 3'P, iron (Fe), manganese (Mn),
thallium (TI), chromium (Cr), including 5'Cr, carbon (C.), including'4C, or
the
like, fluorescently labeled compounds, etc.
Wound Healing Agents: Wound healing agents are agents that
promote wound healing. Wound healing agents include integrins, cell
adhesion molecules such as ICAM, ECAM, ELAM and the like, antibiotics,
growth factors such as EGF, PDGF, IGF, bFGF, aFGF and KGF, fibrin,
thrombin, RGD peptides and the like.
Antiproliferatives: Antiproliferatives include antimetabolites,
topoisomerase inhibitors, folic acid antagonists like methotrexate, purine
antagonists like mercaptopurine, azathioprine, and pyrimidine antagonists like
fluorouracil, cytarabine and the like.
Antioxidants: Antioxidants are agents that prevents oxidative damage
to tissue and include aspartate, orotate, tacophenol derivative (vitamin E),
and free radical scavengers such as SOD, glutathione and the like.
Mammalian cells are continuously exposed to activated oxygen
species such as superoxide, hydrogen peroxide, hydroxyl radical, and singlet
oxygen. These reactive oxygen intermediates are generated in vivo by cells
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in response to aerobic metabolism, catabolism of drugs and other
xenobiotics, ultraviolet and x-ray radiation, and the respiratory burst of
phagocytic cells (such as white blood cells) to kill invading bacteria such as
those introduced through wounds. Hydrogen peroxide, for example, is
produced during respiration of most living organisms especially by stressed
and injured cells.
Active oxygen species can injure cells. An important example of such
damage is lipid peroxidation which involves the oxidative degradation of
unsaturated lipids. Lipid peroxidation is highly detrimental to membrane
structure and function and can cause numerous cytopathological effects.
Cells defend against lipid peroxidation by producing radical scavengers such
as superoxide dismutase, catalase, and peroxidase. Injured cells have a
decreased ability to produce radical scavengers. Excess hydrogen peroxide
can react with DNA to cause backbone breakage, produce mutations, and
alter and liberate bases. Hydrogen peroxide can also react with pyrimidines
to open the 5,6-double bond, which reaction inhibits the ability of
pyrimidines
to hydrogen bond to complementary bases, Hallaender et al. (1971). Such
oxidative biochemical injury can result in the loss of cellular membrane
integrity, reduced enzyme activity, changes in transport kinetics, changes in
membrane lipid content, and leakage of potassium ions, amino acids, and
other cellular material.
Antioxidants have been shown to inhibit damage associated with active
oxygen species. For example, pyruvate and other alpha-ketoacids have been
reported to react rapidly and stoichiometrically with hydrogen peroxide to
protect cells from cytolytic effects, O'Donnell-Tormey et al., J. Exp. Med.,
165,
pp. 500-514 (1987).
Anti-Infective Agents: Anti-infective agents are agents that inhibit
infection and include anti-viral agents, anti-fungal agents and antibiotics.
Anti-Viral Agents: Anti-viral agents are agents that inhibit virus and
include vidarabine, acyclovir and trifluorothymidine.
Anti-Fungal Agents: Anti-fungal agents are agents that inhibit fungal
growth. Anti-fungal agents include anphoterecin B, myconazole, terconazole,
econazole, isoconazole, thioconazole, biphonazole, clotrimazole,
ketoconazole, butaconazole, itraconazole, oxiconazole, phenticonazole,
nystatin, naphthyphene, zinoconazole, cyclopyroxolamine and fluconazole.
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Antibiotics: Antibiotics are natural chemical substances of relatively
low molecular weight produced by various species of microorganisms, such
as bacteria (including Bacillus species), actinomycetes (including
Streptomyces) and fungi, that inhibit growth of or destroy other
microorganisms. Substances of similar structure and mode of action may be
synthesized chemically, or natural compounds may be modified to produce
semi-synthetic antibiotics. These biosynthetic and semi-synthetic derivatives
are also effective as antibiotics. The major classes of antibiotics are (1 )
the
beta-lactams, including the penicillins, cephalosporins and monobactams; (2)
the aminoglycosides, e.g. gentamicin, tobramycin, netilmycin, and amikacin;
(3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the
fluoroquinolones, e.g. ciprofloxacin, norfloxacin, and ofloxacin; (6)
vancomycin; (7) the macrolides, which include for example, erythromycin,
azithromycin, and clarithromycin; and (8) other antibiotics, e.g., the
polymyxins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several
mechanisms of action which can be generally grouped as follows: (1) agents
acting on the bacterial cell wall such as bacitracin, the cephalosporins,
cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2)
agents
affecting the cell membrane or exerting a detergent effect, such as colistin,
novobiocin and polymyxins; (3) agents affecting cellular mechanisms of
replication, information transfer, and protein synthesis by their effects on
ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol,
clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other
streptomycins, and the macrolide antibiotics such as erythromycin and
oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the
fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin,
rifamycins; and (5) drugs affecting intermediary metabolism, such as the
sulfonamides, trimethoprim, and the tuberculostatic agents isoniazid and
para-aminosalicylic acid. Some agents may have more than one primary
mechanism of action, especially at high concentrations. In addition,
secondary changes in the structure or metabolism of the bacterial cell often
occur after the primary effect of the antimicrobial drug.
Anti-Cancer Agents: Anti-cancer agents (chemotherapeutic agents)
are natural or synthetic molecules which are effective against one or more
forms of cancer. This definition includes molecules which by their mechanism
of action are cytotoxic (anti-cancer chemotherapeutic agents), those which
stimulate the immune system (immune stimulators) and modulators of
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angiogenesis. The outcome in either case is the slowing of the growth of
cancer cells.
Anti-cancer therapy include radioactive isotopes such as 32P used in
the treatment of polycythemia vera and in chronic leukemia. Radioactive
phosphorus has a biological half-life of about 8 days in humans. It emits beta
rays that exert a destructive effect on the rapidly multiplying cells. 32P is
usually administered in doses of about 1 me daily for 5 days. Either the oral
or intravenous route may be used and the doses are not greatly different.
Radioactive iodine'3'I, radioactive gold'98Au, and other isotopes are not as
useful as 32P. Nevertheless, '3'I has some limited applications in metastatic
thyroid carcinoma. Other radioactive isotopes can be used with our
technology either as complexes of radioactive metal such as 5'Cr, SzMn ,
s2Mg, s' Ni, 55Co and SsP, ssFe , 'o3pd,'921r, 6°Cu and 6'Cu or as
chelates of
these metals using bifunctional chelating agents like (BFCs), 6-[p-
(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-
tetraacetic acid (BAT), 6-[p-(isothiocyanato)benzyl]-1,4,8,11-
tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (SCN-TETA),
4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid (CPTA), and 1-
[(1,4,7,10,13-pentaazacyclopentadec-1-yl)methyl]benzoic acid (PCBA).
Numerous drugs fall into the category of chemotherapeutic agents
useful in the treatment of neoplastic disease that are amenable to the
embodiment of this application. Such agents derivitized with this technology
can include anti-metabolites such as metotrexate (folic acid derivatives),
fluoroaucil, cytarabine, mercaptopurine, thioguanine, petostatin (pyrimidine
and purine analogs or inhibitors), a variety of natural products such as
vincristine and vinblastine (vinca alkaloid), etoposide and teniposide,
various
antibiotics such as miotomycin, plicamycin, bleomycin, doxorubicin,
danorubicin, dactomycin; a variety of biological response modifiers including
interferon-alpha; a variety of miscellaneous agents and hormonal modulators
including cisplatin, hydroxyurea, mitoxantorne, procarbozine,
aminogultethimide, prednisone, progestins, estrogens, antiestorgens such as
tamoxifen, androgenic steroids, antiadrogenic agents such as flutamide,
gonadotropin releasing hormones analogs such as leuprolide, the matrix
metalloprotease inhibitors (MMPIs) as well as anti-cancer agents including
Taxol (paclitaxel) and related molecules collectively termed taxoids, taxines
or
taxanes.
Included within the definition of "taxoids" are various modifications and
attachments to the basic ring structure (taxoid nucleus) as may be shown to
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be efficacious for reducing cancer cell growth and which can be constructed
by organic chemical techniques known to those skilled in the art.
Chemotherapeutics include podophyllotoxins and their derivatives and
analogues. Another important class of chemotherapeutics useful in this
invention are camptothecins.
Another preferred class of chemotherapeutics useful in this invention
are the anthracyclines (adriamycin and daunorubicin).
Another important class of chemotherapeutics are compounds which
are drawn from the following list: Taxotere, Amonafide, Illudin S, 6-
hydroxymethylacylfulvene Bryostatin 1, 26-succinylbryostatin 1, Palmitoyl
Rhizoxin, DUP 941, Mitomycin B, Mitomycin C, Penclomedine, angiogenesis
inhibitor compounds, Cisplatin hydrophobic complexes such as 2-hydrazino-
4,5-dihydro-1 H-imidazole with platinum chloride and 5-hydrazino-3,4-dihydro-
2H-pyrrole with platinum chloride, vitamin A, vitamin E and its derivatives,
particularly tocopherol succinate.
Other compounds useful in the invention include: 1,3-bis(2-
chloroethyl)-1-nitrosurea ("carmustine" or "BCNU"), 5-fluorouracil,
doxorubicin
("adriamycin"), epirubicin, aclarubicin, Bisantrene (bis(2-imidazolen-2-
ylhydrazone)-9,10-anthracenedicarboxaldehyde, mitoxantrone, methotrexate,
edatrexate, muramyl tripeptide, muramyl dipeptide, lipopolysaccharides,
vidarabine and its 2-fluoro derivative, resveratrol, retinoic acid and
retinol,
carotenoids, and tamoxifen.
Other chemotherapeutic agents useful in the application of this
invention include: Decarbazine, Lonidamine, Piroxantrone, Anthrapyrazoles,
Etoposide, Camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin,
camptothecin-11 ("Irinotecan'), Topotecan, Bleomycin, the Vinca alkaloids
and their analogs [Vincristine, Vinorelbine, Vindesine, Vintripol, Vinxaltine,
Ancitabine], 6-aminochrysene, and Navelbine.
Other compounds useful in the application of the invention are
mimetics of taxol, eleutherobins, sarcodictyins, discodermolides and
epothiolones.
Antineoplastic Agents-- Antineoplastic agents are anti-cancer agents
such as fluoropyrimidines, pyrimidine nucleosides, purines, platinum analogs,
anthracyclines/anthracenediones, podophyllotoxins, camptothecins,
hormones and hormonal analogs, enzymes, proteins and antibodies, vinca
alkaloids, taxanes, atihormonal agents, antifolates, antimicrotubule agents,
alkylating agents (classical and non-classical), antimetabolites, antibiotics,
topoisomerase inhibitors, antivirals, and miscellaneous cytotoxic agents, for
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example hydroxyurea, mitotane, fusion toxins, PZA, bryostatin, retinoids,
butyric acid and derivatives, pentosan, fumagillin, and others. The objective
of
all antineoplastic drugs is to eliminate (cure) or to retard the growth and
spread (remission) of cancer cells. The majority of the above listed
antineoplastic agents pursue this objective by possessing primary cytotoxic
activity, effecting a direct kill on the cancer cells. Other antineoplastic
drugs
stimulate the body's natural immunity to effect cancer cell death.
Matrix metalloprotease inhibitors (MMPIs) - Also known as matrix
~ metalloproteinase inhibitors, MMPIs are inhibitors of the matrix
metalloproteases. The metalloproteases are a family of enzymes containing
zinc at the active site, which facilitate the catalytic hydrolysis of various
protein substrates. A subfamily of the metalloprotease family is known as the
matrix metalloproteases (MMPs) because these enzymes are capable of
degrading the major components of articular cartilage and basement
membranes. The matrix metalloproteases include stromelysin, collagenase,
matrylisin and gelatinase, among other. The action of matrix
metalloptoreases is inhibited by MMPIs used in the preparation of the
derivatized MMPIs of the present invention. Some characterized MMPs and
their preferred substrates are illustrated in the following table.
The nomenclature used to describe the interaction of proteases and
their substrates is widely used in the protease literature. In this system,
the
binding site for a polypeptide substrate on a protease is envisioned as a
series of subsites; each subsite interacts with one amino acid reside of the
substrate. By convention, the substrate amino acid residues are called P (for
peptide); the subsites on the protease that interact with the substrate are
called S (for subsite). The subsites are in the catalytic or active site of
the
protease. The amino acid residues on the amino-terminal side of the scissile
bond (bond that is cleaved on the substrate) are numbered P,, P2, P3, etc.,
and the residues on the carboxy-terminal side of the scissile bond are
numbered P,', P2', P3', etc. The residues can be numbered up to P6 on each
side of the scissile bond. The subsites on the protease are termed S3, S2, S,,
S,', S2', S3', etc. to complement the substrate residues that interact with
the
enzyme.
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Characterized MMPs and their preferred substrates.
MATRIX MMP PREFERRED
METALLOPROTEINASE NUMB SUBSTRATE
ER
CLASS I
n ers i is co agenase i n ar co agens, pe , ,
eu rop i i n ar co agens, ype , ,
_ _ _ ,_ ____ _, _
collagenase
o agenase- i n ar co agens, pe , ,
o agenase-
a atinase a o agen ypes , , ge a in
a a inase a o agen ypes , , ge a m
eta oe as ase as in
trome ysin- aminin, i ronec in,
proteoglycans
trome ysin- aminin, i ronec in,
proteoglycans
atry isin pump aminin, i ronec in,
proteoglycans
rome ysin- -an i rypsin
em rane- ype ro-ge a inase
In this application, the term MMPI should be understood to include
matrix metalloprotease inhibitors as well as analogs thereof. In addition, the
term MMPI includes optical isomers and diastereomers; as well as the
racemic and resolved, enantiomerically pure R and S stereoisomers; as well
as other mixtures of the R and S stereoisomers and pharmaceutically
acceptable salts thereof.
Oxytocin - Oxytocin is a hormone involved in the enhancement of
lactation, contraction of the uterus, and relaxation of the pelvis prior to
childbirth. Oxytocin secretion in nursing women is stimulated by direct neural
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feedback obtained by stimulation of the nipple during suckling. Its
physiological effects include the contraction of mammary gland myoepithelial
cells, which induces the ejection of milk from mammary glands, and the
stimulation of uterine smooth muscle contraction leading to childbirth.
Oxytocin causes myoepithelial cells surrounding secretory acini of mammary
glands to contract, pushing milk through ducts. In addition, it stimulates the
release of prolactin, and prolactin is trophic on the breast and stimulates
acinar formation of milk.
Cholecystokinin (CCK)- CCK is a polypeptide of 33 amino acids
originally isolated from pig small intestine that stimulates gallbladder
contraction and bile flow and increases secretion of digestive enzymes from
pancreas. It exists in multiple forms, including CCK-4 and CCK-8, with the
octapeptide representing the dominant molecular species showing the
greatest activity. It belongs to the CCK/gastrin peptide family and is
distributed centrally in the nervous system and peripherally in the
gastrointestinal system. It has many biological roles, including stimulation
of
pancreatic secretion, gall bladder contraction and intestinal mobility in the
GI
tract as well as the possible mediation of satiety and painful stimuli.
Antihypertensive Agents - Antihypertensive agents are various
agents that can be used to treat hypertension, including but not limited to
enalapril, acebutolol, and doxazosin. Enarlapril is a pro-drug that is
activated
to the angiotensin-converting enzyme (ACE) inhibitor, enalaprilat. This pro-
drug inhibits the conversion of angiotensin I to angiotensin II and exerts an
antihypertensive effect by suppressing the renin-angiotensin-aldosterone
system. Acebutolol is in a class of drugs called beta-blockers, which affect
the heart and circulatory system. Acebutolol is used to lower blood pressure,
lower heart rate, and reduce angina (chest pain). Doxazosin is a member of
the alpha blocker family of drugs used to lower blood pressure in people with
hypertension. Doxazosin is also used to treat symptoms of benign prostatic
hyperplasia (BPH). Doxazosin works by relaxing blood vessels so that blood
passes through them more easily, which helps to lower blood pressure.
Methylprednisolone - Methylprednisolone is a synthetic steroid that
suppresses acute and chronic inflammation. In addition, it stimulates
gluconeogenesis, increases catabolism of proteins and mobilization of free
fatty acids. In addition, it potentiates vascular smooth muscle relaxation by
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beta adrenergic agonists, and may alter airway hyperactivity. It is also a
potent inhibitor of the inflammatory response.
GP-41 Peptides - GP-41 is an HIV transmembrane protein which has
been shown to be essential for the virus to fuse with and infect healthy
cells.
Anti-viral and antifusogenic peptides: Anti-viral peptides refers to
peptides that inhibit viral infection of cells, by, for example, inhibiting
cell-cell
fusion or free virus infection. The route of infection may involve membrane
fusion, as occurs in the case of enveloped viruses, or some other fusion event
involving viral and cellular structures. Peptides that inhibit viral infection
by a
particular virus may be referenced with respect to that particular virus,
e.g.,
anti-HIV peptide, anti-RSV peptide, etc. Antifusogenic peptides are peptides
demonstrating an ability to inhibit or reduce the level of membrane fusion
events between two or more entities, e.g., virus-cell or cell-cell, relative
to the
level of membrane fusion that occurs in the absence of the peptide.
In particular, anti-viral and antifusogenic peptides include the DP107
and DP178 peptides and analogs thereof, as well as peptides comprised of
amino acid sequences from other (non-HIV) viruses that correspond to the
gp41 region of HIV from which DP107 and DP178 are derived, and that
exhibit anti-viral or anti-fusogenic activity. Thhese peptides can exhibit
anti-
viral activity against not only HIV, but other viruses including human
respiratory syncytial virus (RSV), human parainfluenza virus (HPV), measles
virus (MeV) and simian immunodeficiency virus (SIV).
In particular, anti-HIV peptides refer to peptides that exhibit anti-viral
activity against HIV, including inhibiting CD-4+ cell infection by free virus
and/or inhibiting HIV-induced syncytia formation between infected and
uninfected CD-4+ cells. Anti-SIV peptides are peptides that exhibit anti-viral
activity against SIV, including inhibiting of infection of cells by the SIV
virus
and inhibiting syncytia formation between infected and uninfected cells. Anti-
RSV peptides are peptides that exhibit anti-viral activity against RSV,
including inhibiting mucous membrane cell infection by free RSV virus and
syncytia formation between infection and uninfected cells. Anti-HPV peptides
are peptides that exhibit anti-viral activity against HPV, including
inhibiting
infection by free HPV virus and syncytia formation between infected and
uninfected cells. Anti-MeV peptides are peptides that exhibit anti-viral
activity
against MeV, including inhibiting infection by free MeV virus and syncytia
formation between infected and uninfected cells.
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Blood Brain Barrier (BBB) Peptides - The "blood-brain barrier" is a
layer of cells that controls which substances may penetrate from the general
circulation into the brain. BBB proteins can traverse this barrier through
protein transduction. Small sections of these proteins (10-16 residues long),
i.e. BBB peptides, are responsible for this transduction.
RGD Peptides: The RGD peptide for conjugation to tissues or fixed
endogenous proteins in accordance with the present invention includes a
sequence of amino acids, preferably naturally occurring L-amino acids and
glycine, having the following formula:
R,-Arg-Gly-Asp-R2
In this formula, R, and RZ represent an amino acid or a sequence of more
than one amino acid or a derivatized or chemically modified amino acid or
more than one derivatized or chemically modified amino acids.
Insulinotropic Peptides: Insulinotropic peptides (ITPs) are peptides
with insulinotropic activity. Insulinotropic peptides stimulate, or cause the
stimulation of, the synthesis or expression of the hormone insulin. Such
peptides include precursors, analogues, fragments of peptides such as
Glucagon-like peptide, exendin 3 and exendin 4 and other peptides with
insulinotropic activity.
Glucagon-Like Peptide: Glucagon-Like Peptide (GLP) and GLP
derivatives are intestinal hormones which generally simulate insulin secretion
during hyperglycemia, suppresses glucagon secretion, stimulates (pro) insulin
biosynthesis and decelerates gastric emptying and acid secretion. Some
GLPs and GLP derivatives promote glucose uptake by cells but do not
simulate insulin expression as disclosed in U.S. Patent No. 5,574,008 which
is hereby incorporated by reference.
Pulmonary Condition: A pulmonary condition is a disease which
affects lung function. Such conditions may result from a defect in a gene or
genes associated with lung function (e.g., cystic fibrosis), asthma,
allergies,
an immune or autoimmune disorder, a microbial infection (e.g. bacterial,
viral,
fungal or parasitic infection), or a mechanical injury to the lungs.
Exemplary pulmonary conditions contemplated by the subject invention
include cystic fibrosis, asthmatic bronchitis, tuberculosis, bronchitis,
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bronchiectasis, laryngotracheobronchitis, bronchiolitis, emphysema, bronchial
pneumonia, allergic bronchopneumonia, viral pneumonia, pertussis,
diphtheria, spasmodic croup, pulmonary phthisis, encephalitis with retained
secretions, and pulmonary edema. Other pulmonary conditions, such as
those which develop as a result of injury or surgery (e.g., after
tracheotomy),
as well as those associated with insufficient surfactant secretion in the
lungs
of premature infants, are also contemplated by the subject invention.
Pulmonary conditions amenable to treatment by the subject method may also
develop as a result of activity associated with inhalation of particulate
matter
e.g. smoking, exposure to construction areas or other high dust areas,
occupational hazards associated with inhalation of particulates, exposure to
environmental particulates (e.g. smog, pollen, asbestos, siliconis), pulmonary
delivery of pharmaceutical agents (e.g. bronchodilators) or inhalation of
cocaine.
Other pulmonary conditions include diffuse parenchyma) lung disease
from infectious cases, such as cytomegaloviral pneumonia or miliary
tuberculosis, drug-induced lung disease (after administration of penicillin,
nitrofurantoin), neoplastic lung disease having lymphangitic spread pattern or
bronchoalveolar cell carcinoma, granulomatous disease (infectious or
noninfectious), hypersensitivity pneumonitis, histoplasmosis, tuberculosis,
idiophatic pulmonary fibrosis (aka cryptogenic fibrosing alveolitis),
hereditary
pulmonary disorders, such as alveolar microlithiasis and bronchiectasis,
eosinophilic granuloma, lympphangioleimyomatosis, and plumonary alveolar
proteinosis disorder.
Symptoms of a Pulmonary Condition: Symptoms of a pulmonary
condition are symptoms associated with any of the pulmonary conditions
described above. The classic symptoms associated with such pulmonary
conditions may include coughing, exertional dyspnea, wheezing, chest pain
and purulent sputum production. Other components of the syndrome which
may accompany a pulmonary condition include hypoxia, C02 narcosis,
hyperventilation, decreased expiration volume, and decreased lung capacity.
Pulmonary Fluid: Pulmonary fluid is the fluid which bathes the apical
surface of the lung epithelium, particularly the alveolar epithelium and
contains fixed and mobile pulmonary fluid components.
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Pulmonary Delivery Agent: Pulmonary delivery agents are agents
that may be delivered to the lungs. Such agents include therapeutic agents.
Fixed Pulmonary Components: Fixed pulmonary components are
non-mobile pulmonary components and include tissues, membrane receptors,
interstitial proteins, fibrin proteins, collagens, platelets, endothelial
cells,
epithelial cells and their associated membrane and membraneous receptors,
somatic body cells, skeletal and smooth muscle cells, neuronal components,
osteocytes and osteoclasts.
Mobile Pulmonary Components: Mobile pulmonary components are
pulmonary components that do not have a fixed situs for any extended period
of time, generally not exceeding 5, more usually one minute. Mobile
pulmonary components are components of the pulmonary or lung fluid and
include such soluble proteins such as immunoglobulins, serum albumin,
ferritin, transferrin and the like.
Blood Components: Blood components may be either fixed or
mobile. Fixed blood components are non-mobile blood components and
include tissues, membrane receptors, interstitial proteins, fibrin proteins,
collagens, platelets, endothelial cells, epithelial cells and their associated
membrane and membraneous receptors, somatic body cells, skeletal and
smooth muscle cells, neuronal components, osteocytes and osteoclasts and
all body tissues especially those associated with the circulatory and
lymphatic
systems. Mobile blood components are blood components that do not have a
fixed situs for any extended period of time, generally not exceeding 5, more
usually one minute. These blood components are not membrane-associated
and are present in the blood for extended periods of time and are present in a
minimum concentration of at least 0.1 pg/ml. Mobile blood components
include serum albumin, transferrin, ferritin and immunoglobulins such as IgM
and IgG. The half-life of mobile blood components is at least about 12 hours.
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Inhaler Device: An inhaler device is any device useful in the
administration of the inhalable medicament of the invention. Examples of
inhaler devices include nebulizers, metered dose inhalers, dry powder
inhalers, intermittent positive pressure breathing apparatuses, humidifiers,
bubble environments, oxygen chambers, oxygen masks and artificial
respirators.
Reactive Groups: Reactive groups are chemical groups capable of
forming a covalent bond. Such reactive groups are coupled or bonded to a
therapeutic or diagnositic agent. Reactive groups will generally be stable in
an aqueous environment and will usually be carboxy, phosphoryl, or
convenient acyl group, either as an ester or a mixed anhydride, an imidate or
maleimide, thereby capable of forming a covalent bond with functionalities
such as an amino group, a hydroxy or a thiol at the target site on pulmonary
components. For the most part, the esters will involve phenolic compounds,
or be thiol esters, alkyl esters, phosphate esters, or the like. Prefereably,
the
reactive group will be a maleimide group.
Functionalities: Functionalities are groups on pulmonary components
to which reactive groups on modified therapeutic agents react to form
covalent bonds: Functionalities include hydroxyl groups for bonding to ester
reactive entities; thiol groups for bonding to maleimides, imidates and
thioester groups; amino groups for bonding to carboxy, phosphoryl or acyl
groups and carboxyl groups for bonding to amino groups.
IC5°: Concentration of an enzyme inhibitor at which 50% of the
enzymatic activity is inhibited.
Protective Groups: Protective groups are chemical moieties utilized to
protect reactive entities from reacting with other functionalities. Various
protective groups are disclosed in U.S. 5,493,007 which is hereby
incorporated by reference. Such protective groups include acetyl,
fluorenylmethyloxycarbonyl (FMOC), t-butyloxy carbonyl (BOC),
benzyloxycarbonyl (CBZ), and the like. For small organic molecules all
protecting groups like tetrahydropyranyl (THP), all silyl derivatives,
acetals,
thioacetals and the like.
Linking Groups: Linking groups are chemical moieties that link or connect
reactive groups to therapeutic agents. Linking groups may comprise one or more
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alkyl moeities, alkoxy moeity, alkenyl moeity, alkynyl moeity or amino moeity
substituted by alkyl moeities, cycloalkyl moeity, polycyclic moeity, aryl
moeity,
polyaryl moeities, substituted aryl moeities, heterocyclic moeities, and
substituted
heterocyclic moeities. Linking groups may also comprise poly ethoxy amino
acids,
such as AEA ((2-amino) ethoxy acetic acid) or a preferred linking group AEEA
([2-(2-
amino) ethoxy)] ethoxy acetic acid.
Sensitive functional groups -A sensitive functional group is a group
of atoms that represents a potential reaction site on a therapeutic agent. If
present, a sensitive functional group may be chosen as the attachment point
for the linking group-reactive group modification. Sensitive functional groups
include but are not limited to carboxyl, amino, thiol, and hydroxyl groups.
Modified Therapeutic and Diagnostic Agents - Modified therapeutic
and diagnostic agents are agents that have been modified by attaching a
reactive group. The reactive group may be attached to the therapeutic agent
either via a linking group, or optionally without using a linking group.
Modified
therapeutic and diagnostic agents may be administered in Vivo such that
conjugation with blood or pulmonary components occurs in vivo, or they may
be first conjugated to blood or pulmonary components in vitro and the
resulting conjugated therapeutic agent (as defined below) administered in
VIVO.
Conjugated Therapeutic and Diagnostic Agents - Conjugated
therapeutic and diagnostic agents are modified therapeutic and diagnostic
agents that have been conjugated to a blood or pulmonary component via a
covalent bond formed between the reactive group of the modified therapeutic
agent and the functionalities of the pulmonary component, with or without a
linking group. As used throughout this application, the term "conjugated
therapeutic agent" can be made more specific to refer to particular conjugated
therapeutic agents, for example "conjugated antihistamine."
Taking into account these definitions, the present invention is directed
to modified therapeutic and diagnostic agents capable of reacting with
available functionalities on pulmonary or blood components via covalent
linkages. The invention is also directed to methods of making the modified
agents and their use. The modified therapeutic agents of the present
invention are capable of reacting in vivo to form conjugates with pulmonary
and/or blood components, such as pulmonary or blood proteins, thereby
extending the half-life and improving bioavailability of the therapeutic agent
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without deterioiusly altering the agent's therapeutic effect. In preferred
embodiments of this invention, the functionality on the protein will be a
thiol
group and the reactive group on the modified therapeutic agent will be a
maleimido-containing group such as gamma-maleimide-butyralamide
(GMBA), maleimidopropionic acid (MPA) or maleimide-benzoyl-succinimide
(MBS).
The invention in one aspect contemplates delivery of the modified
agents to the blood of a host for conjugation to blood components, including
blood proteins. While pulmonary administration is further described as such a
route of administration, it will be understood that the invention is not
limited to
such routes of adminstration, and also contemplated administration of the
modified agents to a patient's blood stream using other methods, including
parenterally, such as intravenously (IV), intraarterially (IA),
intramuscularly
(IM), subcutaneously (SC) and the like.
For pulmonary delivery, a wide variety of devices and carrier molecules
have been utilized to enhance pulmonary drug delivery and can be used with
the modified agents of the present invention. These devices and methods
include metered dosing, carriers such as liposomes (Meisner et al, 1989)
actide/glycolide copolymer (PLGA) nanospheres (Niwa et al., 1995), albumin
microspheres (Feinstein et al., 1990), and other physical art forms to created
aerosols or nanoparticulates.
A new type of inhalation aerosol, characterized by particles of small
mass density and large size, has permitted the highly efficient delivery of
inhaled therapeutics (e.g. insulin , testosterone) into the systemic
circulation.
Particles with mass densities less than 0.4 gram per cubic centimeter and
mean diameters exceeding 5 micrometers have been reported to avoid the
lungs' natural clearance mechanisms providing higher bioavailability than that
of conventional inhaled therapeutic particles. (Edwards et al., 1997). For
most
of these therapies, aerosols are designed to comprise small spherical
droplets or particles of mass density near 1 g/cm3 and mean geometric
diameter between approximately 1 and 3 micron, suitable for particle
penetration into the airways or lung periphery.
Studies performed primarily with liquid aerosols have shown that these
characteristics of inhaled aerosols lead to optimal therapeutic effect, both
for
local and systemic therapeutic delivery. Inefficient drug delivery can still
arise,
owing to excessive particle aggregation in an inhaler, deposition in the mouth
and throat, and overly rapid particle removal from the lungs by mucocilliary
or
phagocytic clearance mechanisms.
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To address these problems, particle surface chemistry and surface
roughness are traditionally manipulated. Recent data indicate that major
improvements in aerosol particle performance may also be achieved by
lowering particle mass density and increasing particle size, since large,
porous particles display less tendency to agglomerate than (conventional)
small and nonporous particles. Also, large, porous particles inhaled into the
lungs can potentially release therapeutic substances for long periods of time
by escaping phagocytic clearance from the lung periphery, thus enabling
therapeutic action for periods ranging from hours to many days. (Edwards et
al., 1998)
It has been previously reported that specific transport receptors for
albumin (GP60, albondin) exist in the endothelium that function as unique
albumin carriers (U.S. PAT. 5,254,342). These transcytosis proteins facilitate
the movement of albumin and albumin carriers across the lining of the airway
and result in extensive plasma levels of these proteins or protein carriers.
As
further described, modified agents according to the present invention can be
prepared that react with albumin, and upon pulmonary delivery, the resulting
conjugates can pass to the bloodstream via such carriers.
Pulmonary drug delivery is also advantageous for local treatment of
the lung in that it promotes an increase in drug retention-time in the lung
and
more importantly, a reduction in extrapulmonary side-effects, invariably
resulting in enhanced therapeutic efficacies. (Shek, 1994). A key advantage
of pulmonary delivery includes reduced systemic toxicity and increased drug
concentration at the site of action (e.g. infection or inflammation site.
(Stout
and Derendorf, 1987).
The use of in vivo or ex vivo bioconjugation associated with pulmonary
drug delivery includes the following non-limited benefits. Retention of the
drug at the site of placement is enhanced due to covalently attachment of the
drug to the airway site. Additionally, prolonged duration of action of the
drug
is made possible, both in the lung by in situ attachment to soluble proteins
for
localized intrapulmonary activity, as well as systemic absorption and
conjugation to blood proteins.
Drug stability is improved, both locally and systemically, as conjugation
affords protection against enzymatic degradation that occurs in the pulmonary
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mucosal fluid or in the plasma. Also, in the deep lungs, alveolar
macrophages can rapidly deposited particles; the reactivity of the modified
agents of the invention with epithelial cells will allow for localized
retention of
the agent.
Localized delivery to the pulmonary tissues also reduces toxicity and
reduces systemic exposure as there is no first-pass liver effect. Systemic
delivery also exhibits reduced extravascular side effects through conjugation
to, for example, albumin, due to, for example, the limitation of hepatic,
central
nervous system (CNS) or renal toxicity due to the limited clearance of
albumin into these organs.
Pulmonary delivery of the modified agents of the invention also
provides advantages of improved patient compliance due to prolonged
duration of action of the modified agents. In turn, cost benefits can be
achieved through, e.g., reduced costs of goods per course of therapy due to
prolonged duration of action, and outpatient use of medications that would
otherwise have limited use or complicated dosing titration schedules.
Pulmonary delivery can also reduce difficulties associated with oral dosing,
including low solubility, interactions with food, and low bioavailability.
A further advantage of pulmonary delivery of modified agents of the
present invention is the ability to deliver systemically large macromolecule
drugs, such as insulin, growth hormones, beta-interferon, calcitonin, and
others that, due to their large size and instability, are typically delivered
by
injection. The present invention provides an alternative and more convenient
route of adminstering these drugs. In addition, many drugs and therapeutic
peptides are more stable in solid, dry form, rather than solubilized. Dry
pulmonary formulations of such drugs modified according to the present
invention provide for a more stable form of the drug, as well as the
convenience of pulmonary administration.
1. Therapeutic Agents
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A wide variety of therapeutic agents are contemplated for use in the
present invention, including peptide therapeutics and small organic
molecules, provided they can be modified as described.
In addition to therapeutic agents discussed above, the following
therapeutic agents are within the scope of this invention.
Sympathomimetic compounds mimic the action of endogenous
catecholamines (adrenalinelike neurotransmitters) at peripheral sympatnenc
neurons in addition to CNS effects. Adrenaline systems control important
body functions like blood pressure regulation and wakefulness. A vast
panoply of compounds has been developed that allows one to selectively
tweak these systems. These include agonists, used as decongestants and
antiasthmatics and antagonists, used as antihypertensives).
Nonselective adrenergic agents
0
HO OH OH OH
i w
HO-~N' N; N
epinephrine phenylpropanolamine ephedrine
Alpha agonists
OH OH -0 OH
i v / H 0 -~ f . M
N~ N
~0
phenylephrine synephrine methoxamine
0 G j.
i \ ~' / w N N ~ N
N~ CI N , N
0 ~/
ibopamine clonidine naphazoline
Beta agonists
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HO OH HO OH HO OH
HO ! w I~ HO ! ~ /~ r w /
N. N. N
HO
albuterol (Proventil) isoproterenol metaproterenol (hAetaprel)
HO ~H H0 0H HO OH
HO ~_~ l~ ~ w j' ~ ' j.
~N~ HO N, HO N
! ~ OH
isoetharine (Isuprel) terbutaline (Brethine) fenoterol
HO
Ho ! ~ l
N.
OH
0
r OH ~ ' 0 N
0
dobutamine
bitolterol
Selective agents include alpha agonists such as phenylephrine (Neo-
Synephrine) and more lipophilic agents such as naphazoline. Clonidine, an
antihypertensive, is sometimes used in ethanol withdrawal, and has been tried
in cigarette smoking cessation. It probably has a net antagonist effect
through
autoreceptors, i.e. presynaptic receptors detect an excess of adrenergic
agonist
and decrease norepinephrine release. Ibopamine, a cardiotonic, has diuretic
and dopamine agonist activity.
The beta agonists are popular antiasthma medications. Isoproterenol and
dobutamine are fairly selective for the beta1 receptor; the latter is used as
a
cardiotonic.
Nonselective adrenergic antagonists
.~N 0 OH
Ho ! w j.
N.
i w
labetolol
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Alpha antagonists
-o
HO ~~ ~ N
~N N~ r w N r ~ N
N
S N
0 p OH
N~ I
'j~~~0
prazosin ~ ~ phentolamine yohimbine
Beta antagonists
r w 0 OH r0 ! ~ 0 OH
N N N 0 U
I ~. N.
propranolol metoprolol atenolol w
0 pH ~0
N / w 0 OH 0 N r ~ 0 OH
/ \ Nr ..y f.
N H H 1. ~N~
I
p~actolol ., nadolol acebutolol
Alpha blockers such as phentolamine and prazosin are also used as ,
antihypertensives. Yohimbine, a selective alpha2 blocker, is a popular
aphrodisiac for males, purported to prolong or intensify erection. Alpha2
receptors are inhibitory, so that inhibiting them produces a stimulating
effect.
Alpha2 and alpha3 receptors are alsopresent in fat storage sites; antagonizing
them may provide a way to promote fat catabolism without resorting to central
stimulants.
Beta antagonists include propranolol, which has been used for some time to
calm the nerves in event-specific anxiety (stage fright) as well as its more
traditional role in hypertension. Acebutolol is a cardioselective beta blocker
used in angina.
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Antidepressants
Antidepressants work by altering the concentration of catecholamines and/or
serotonin in CNS neurons emanating from the limbic system into the frontal
lobe. Raising levels of catecholamines - excitatory, adrenalin-type
neurotransmitters - causes stimulation. Elevating serotonin, an inhibitory
neurotransmitter, produces a calming action, and results in subsequent
upregulation of catecholamine systems as a mechanism of habituation.
Selective serotonin reuptake inhibitors
F
F xr
Br N F~ F F
o a v
,N' - :/4\ CI o~0 . N /0-~0 N
,rN
zimeldine fluoxetine (Prozac) sertraline (Loloft) paroxetine (Paxil)
femoxetine (Malexil)
F
F
F 0
OH. \\ /' N ~ F
0
p-~ I~ ~ HO
N,.
venlataxine (Effexor) fluvoxamine (Luvox) citalopram (Celexa)
N N .
1
\. F ~ 1. i N 0
~'~YJ , N
F F N ~.~N~.
prolintane oxaflazone indalpine indeloxazine dr~datine
N~ ~' i w N~
~ N
i w 0~ N~N\ CI ~ N~ CI ~~- CI ~~~ N
,~ ~ ' /N O ~ ' /N~
.. 0
nefazodone (Serzone) trazodone(Desyrel) m-chlorcpiperazine etoperidone
The selective serotonin reuptake inhibitors (SSRIs) inhibit reuptake of
serotonin
without significantly affecting adrenergic systems. The adverse effect
profiles are
much less than the tricyclics, since muscarinic, histaminic and adrenergic
binding
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is much reduced.
The effect of these serotonin agents on mood has led to more complex
theories of how antidepressants work. According to one hypothesis, the
noradrenergic systems (dopamine, noradrenalin) which underlie the serotonin
systems respond to an increase in the inhibitory serotonin function by
upregulating, or increasing the number of receptors on the individual post-
synaptic neural surface. This increase in adrenaline-type neural function
might
then account for the antidepressant activity which is delayed from the onset
of
serotonin reuptake inhibition by several weeks. Another possibility is that
the
serotonin receptors take a while to register the excess serotonin and resopond
with similar mechanisms. In addition, recent evidence suggests interaction
with
DNA through transcriptases, increasing production of neurotransmitter by
producing more synthase enzymes, for example.
Antihistamines, antiasthmatics & histamine agonists
Antihistamines are compounds that block histamine from activating histamine
receptors. Since histamine functions to mediate allergic response, blocking
histamine at H1 receptors stops the body's characteristic responses, i.e.
inflammation and vasoconstriction. H2 receptors in the stomach regulate the
release of gastric acid; hence the new class of H2-blockers such as Zantac and
Tagamet stop the secretion of acid by selectively blocking these receptors
without
affecting the H1 receptors responsible for allergic response.
Histamine is concentrated in mast cells, cells whose function is essentially
to
release histamine and immunoglobins when tissue damage occurs. Receptors on
the cell surface trigger lysing (breaking open) of the cell, releasing these
allergic
mediators. Mast cells are especially numerous in parts of the body that are
injured
often, such as the fingers and toes, or which enjoy frequent contact with the
environment, such as the mucosa of the lips, nose, etc.
Histamine is also a neurotransmitter in the CNS and a typical problem with
some antihistamines is drowsiness. The effort has been to produce compounds
that do not enter the brain very well.
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Antihistamines
S r v r ~ I ~ CI r_ r w
r ~ N ~N . r y ~ v r w
N
N / 0 N O~N~.~ O~N~
.- I.~ ' /~ I
fenethazine loratadine cyproheptadine diphenhydramine dimenhydrinate
(Claritin) (Periactin) (Benadryl, Nytol, Sominex) (Dramamine)
r
CI Br r ~
r_w / ~ r_~
r ~ r ~ r_~
N 0 N N
~I~ ~~~ N
.. .. . . r w
doxylamine chlorpheniramine brompheniramine cinnanz~ne
(Unisom) (Chlor-Trimeton) (Dimetapp) (Stugeron)
HO r ~ CI HO r ~
r_w
r ~ r ~
N cetirizine terfenadine
[Lyrtec) (Seldane)
N N N
0 0
fexofenadine
(Wlegra) HO OH HO
0
HO
CI
CI
r ~ _ 0
- N Na + 0 0
0 N ~ ~ r ' 0 0
(j~OH ~ .+
0 ~ ~r--i~p Na
0
0
hydroxyzine ~ azelastine cromolyn
(Ptarax, lAstaril) OH (Astelin) (Nasalcrom)
Fenethazine represents a tricyclic antihistamine very similar to Thorazine, a
strong antipsychotic dopamine blocker. Cyproheptadine (Periactin), which also
acts at serotonin receptors, resembles the now-popular Claritin. Periactin has
been prescribed psychiatrically for anxiety. Benadryl is probably the most
familiar
of the class; it has strong sedating qualities. Hydroxyzine also has been
prescribed as a sedative. Dimenhydrinate has been marketed as an anti-nausea
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medication as Dramamine. Cetirizine (Zyrtec) is a metabolic product of
hydroxyzine; since hydroxyzine is available as a generic, it is cheaper than
the
newer drug and just as effective.
Azelastine (Astelin) has a novel structure but also acts on both H1 and H2
receptors. Cromolyn works by a distinct mechanism; it prevents release of
histamine following immunoglobin binding on mast cells (prevents mast
degranulation).
Histamine blockers (H2 blockers)
1I yI 1I.
'~S~N~ ~N~N~
-N S N02
cimetidine I ~1, I ~ nizatidine
(fagamet) ~ (Aocid)
'r
N
SI. 5I~
~.N~N~~S~ y 0 S~N~N'..
~N~ S -N J~N02
I famotidine I ~ ranitidine
(Pepcid) (Zantac)
H2-selective antihistamines have become popular as treatments for excess
stomach acid. These histamine blockers are very similar structurally and
mechanistically. All four have about the same bioavailability, half lives, and
antagonist activity.
Proton pump inhibitors
0 0
ii ii
7=' N S 1-/- N S / \
-0 0~ O~F
~'~ F
F
omeprazole (Prilosec) lansoprazole (Prevacid)
Omeprazole (Prilosec) and lansoprazole (Prevacid) belong to a class of
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antisecretory compounds, the substituted benzimidazoles, that do not exhibit
anticholinergic or histamine H2-receptor antagonist properties, but that
suppress gastric acid secretion by specific inhibition of the (H+,K+)-ATPase
enzyme system at the secretory surface of the gastric parietal cell. These
proton pump inhibitors have emerged as a therapeutic alternative to histamine
antagonists for the treatment of gastric disorders, especially acid reflux
disease
("heartburn"). .
Histamine agonists
N N , sI
N
N' N! ~ N N
. ~ '1
2-methylhistamine 4methylhistamine impromidine
For any receptor system it is possible to find drugs that act as mimetics or
agonists. With histamine receptors these drugs do not find much clinical
usefulness, but it is gratifying - to some! - to enumerate them nevertheless.
2-
methylhistamine acts as a selective agonist at H1 receptors; 4-methylhistamine
is relatively selective at H2. Impromidine antagonizes H2 receptors but
functions as an agonist at H3.
Antiasthmatics
Asthma is essentially an accentuated allergic response to the environment,
i.e.
an autoimmune disorder. The process of allergic response is complex, which
gives one many points at which to attack the problem. First, immunoglobin and
antigens bind to the surface of mast cells. Mast cells then release histamine,
leukotrienes, peptides, which bind to tissue receptor sites and modify
intracellular
chemical processes governing various functions such as blood pressure
regulation, smooth muscle tone, fluid disposition, etc. Compounds that inhibit
any
of these steps can be used to treat asthma and allergy, beginning with the
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antihistamines listed above.
methylxanthine
txonchodilators
~OH
0~_'~'0 0~-7 - ~-7 - ~-7 0 / '1
N OH
N N N N N ~ ;7
N N N N~~
~TN ~lN STN ~- -
~ N N
0 0 0 \ 0 \ p ,~ N
~ 0
urio xanthine theophyllinecaffeine
acid
(1 ,3,7-trimethylxaMhine)enprotyllinedyphylline
leukotriene
antagonist
N 0 0 0
0~ N - i ~ - I
0 p
zafirlukast
beta agxiists
HO OH Hp OH Hp OH Hp OH Hp OH
HO ! \ /~ HO ! ~ /~ / \ I~ HO ! \
N N. N- N- N
HO ~ HO
albuterol (ProveMil) isoproterenol metaproterenol (AAMaprel) isoetharine
(Iwprel) terbutaline (Brethine)
Perhaps the oldest method for reducing asthma symptoms is
bronchodilation by methylxanthine compounds like caffeine and
theophylline. These are currently outmoded by other compounds that
perform the same function more selectively like the beta2-adrenergic
agonists (beta agonists). Methylxanthines act by inhibiting the enzyme
which effects cAMP degradation (phosphodiesterase) and by antagonizing
adenosine, which causes bronchoconstriction. The beta agonists, like
metaproterenol, isoetharine, isoproterenol, terbutaline, and albuterol, mimic
adrenaline at a subset of adrenaline receptor which controls the tone of
smooth muscle like that of the bronchi. Another older class of drugs,
antimuscarinics, exemplified by atropine, has enjoyed some historical use.
Zafirlukast (Accolate) represents a line of attack on the leukotriene
compounds released along with histamine from mast cells. Leukotrienes are
mediators like histamine which bind to receptors on tissue cells to signal an
allergic reaction. Blocking them blocks the signal to the (bronchial) cells
and
thus the undesirable response.
The final mechanism one can attack is the slow inflammatory response to
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binding by leukotrienes and/or histamine. The body regulates inflammation
with glucocorticoid steroids, and synthetic compounds such as fluticasone
(Flonase) and beclomethasone (Beclonase) are effective mimetics.
Antihyperlipidemics
Antihyperlipidemics are relaively new drugs which lower blood cholesterol
levels and help to prevent atherosclerosis by inhibiting the formation of
plaque on arterial and vascular linings. The formation of this plaque is
dependent on the proportion of various types of blood-fats, particularly on
the ratio of high-density lipoproteins (HDLs) to low-density lipoproteins
(LDLs). This proportion is in turn influenced by genetics and by the amount
of certain substances in the diet, particularly cholesterol and saturated fat.
Cholesterol is essential in the formation of VLDLs, large lipoproteins
produced by the liver; on catabolysis by lipoprotein lipase, VLDLs produce
the smaller LDLs, which are the so-called "bad cholesterol," HDLs being
termed "good cholesterol" in the common parlance. Because the production
of these lipoproteins is complex, several points can be targeted for action by
various drugs.
HMG-CoA reductase inhibitors ("Statin drugs")
0
OH
OH OH HO
0\' ~ 0', ~ 0' 0 HO
0 ~ 0 ~ 0 ~ 0
/ ~~[1 \ v /~[~ \ \
\ \ \ \
HO
simvastatin mevastatin lovastatin pravastatin
(Zocor) (Compadin) (Mevaeor) (Pravaehol)
The most commonly used antihyperlipidemics are simvastatin analogs,
especially simvastatin (Zocor) itself. These drugs, known as HMG-CoA
reductase inhibitors, decrease production of cholesterol by inhibiting the
first
step in sterol synthesis which involves production of mevalonate ion from
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CoA-S-mevalonate. Decreased cholesterol formation results in a reduction
in VLDLs and hence LDLs. Pravastatin is the active metabolite of
mevastatin; lovastatin and simvastatin are inactive prodrugs that work
through their hydroxyl derivatives, obtained by similar ring-opening.
These drugs also stimulate receptor-mediated clearance of LDLs. LDL
receptors undergo upregulation (increase in receptor density) and VLDL
catabolysis is increased.
Recent studies suggest a reduction in osteoporosis as a result of
treatment with statin drugs.
Clofibrate analogs
0 0
0~ ~~0 ~o
1 0 H~ 0 0
-OH
0 0
dofiDrate pemfibrozil 0 bezafibrate ~ H fenofibrate
(Atromid) (LoDi~ (Bezatol) (FenoDrate) 0
Other drugs like clofibrate and gemfibrozil increase the activity of
lipoprotein
lipase, reducing the level of VLDLs. This enzyme is an extracellular species
present in the blood and gut. Effects on other lipase enzymes may account
for the nausea common to this drug. Cholesterol production in the liver is
reduced, probably as a secondary effect due to lower VLDL levels, while fecal
cholesterol excretion is enhanced. Clofibrate is fairly toxic and may be
carcinogenic.
Antihypertensives
The body controls blood pressure by a complex feedback mechanism
between baroreceptors and effector nerves, primarily adrenergic in nature.
This
system is modulated by a peptide systems (angiotensin/renin).
Pharmacologic control of blood pressure acts through four basic
mechanisms. Sympathoplegics reduce peripheral vascular resistance, inhibit
cardiac function and increase venous pooling by a number of mechanisms
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involving adrenergic nerves.
Direct-acting vasodilators decrease blood pressure by increasing blood
volume. Veins and arteries are forms of smooth muscle, and relaxing the
muscle results in larger volume and lower pressure. Angiotensin antagonists
work on peptide systems with effects on smooth muscle. Finally, diuretics
decrease sodium content, decreasing blood volume and thus blood pressure.
Because the body responds to exogenous agents by homeostatic regulation,
concomitant use of several agents working on different mechanisms is
frequently used, rather than simply increasing the dose of a single med.
Adrenoceptor drugs
w 0 ~0 / ~ 0 OH
0
N N 0
I L~ N.
' I
propranolol metoprolol atenolol
0 OH 0
N r w 0~ 0 N _ 0
i ~ N
N~ ~H H I ~ N
prac2olol I, nadolol . acebutolol
'\ 0 CI HO
:N ' ' OH ;. r ~ N HO i_~
HO
N ~ CI ~ N
N HO
0
i
labdold ~ clonidine methyldopa
CI \/, t CI \/~ t v/~ t ~ v/~ t
Na !N'. ~N~N_. ~N~N~N', ~o~N~N'.
CI N - ~N 0 ~N ~ N~ ~N
/S ~S /\ ~1
guanabenz guanfacine guanethidine guanadrel
-0 \0 -0 \0 _
~r ~ r ' ~ N
~NNN .iN N 0 - N 0-
0 ~ '0 r ~ 0
0 ~~ 0 ~ 0~ 0 0 '
p~azosin N~ terazosin N reserpine
r v
r 0
Sympathoplegics or sympatholytics antagonize the function of adrenalin
compounds. Beta blockers block beta-1 receptors in hart muscle, decreasing
cardiac output. The include propranolol (Inderal), metoprolol (Lopressor),
labetolol (Normodyne), nadolol (Corgard), atenolol (Tenormin), and
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acebutolol (Sectrol). Some adrenoceptor-activating compounds have a
hypotensive effect by acting centrally at alpha receptors. These include
clonidine (Catapres), guanfacine (Tenex), guanabenz (Wytensin), and
methyldopa. These probably work by activating presynaptic autoreceptors,
decreasing norepinephrine (agonist) release.
Compounds that block alpha-1 receptors peripherally, in blood vessels,
include prazosin (Minipress) and terazosin (Hymn).
Reserpine (Serpasil) blocks amine uptake, while guanethidine (Ismelin)
and guanadrel (Hylorel) block sympathetic nerve terminals.
Propranolol, which was formerly the most prescribed drug of the class,
leads to accumulation of bradykinin, which contributes to the
antihypertensive effect.
Cholinergic drugs
o - +/s~
O~~s ~ ~N~ ~ r
0 0
0
Il
:~ N'
mecamylamine i:rimethamapan camsylate
Some anticholinergic drugs (ganglion blockers) are effective as hypotensives,
but they are less popular because of side effects. These include trimethapan
(Arfonad) and mecamylamine (Inversine).
Direct vasodilators
0
II
N +
N ' ..\
0 ~ _ iN N . N _Ni.
N -~ N ~. Fey
CI ! ~S N~ -N~N~N~ N N _
diazoxide hydralazine . .'minoxidil n'rtroprusside IN
(Hyperstat) (Apresoline) (Lon'rten, Rogaine) (Nipride) I
Diazoxide acts by opening potassium channels and relaxing smooth venous
and arterial muscle. Despite structural similarities, it has no diuretic
properties.
It has a relatively long half-life of about 24 hours, and is often used
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parenterally (by injection) in emergencies. The drug also inhibits insulin
release and is used in diabetes. Minoxidil is mainly used orally, also opening
potassium channels.
Hydralazine and minoxidil dilate arterioles but not veins. Nitroprusside (as
the sodium salt) dilates both, by a mechanism involving activation of guanylyl
cyclase, resulting in formation of cGMP and relaxation of smooth muscle.
Calcium channel blockers
o-
i
0
o~;
i0 0 i ~ ~l. 0. . .N - 0 i ~ S
N ~ w 0 ~~ 0 0 N - ~ ~ N 0
N 0
iN'
verdpamil nifedipine dihiazem I
(Calan) (Procardia) (Cardizem)
The calcium channel blockers are a popular means to control hypertension.
Smooth muscle contraction depends on calcium influx to control muscle tone.
The reaction path is complex, involving the peptides calmodulin and myosin
light chain kinase (MLCK). When the latter enzyme is activated it
phosphorylates myosin which acts with actin to contract muscle. Blocking the
channel statistically prevents calcium ion influx and decreases tension in
blood
vessel smooth muscle. Skeletal muscles rely on intracellular calcium ion, and
are not affected by these drugs. Cardiac muscle is highly dependent on
calcium channel action.
Calcium channels also exist at presynaptic nerve terminals in adrenergic
neurons. These are voltage-dependent ion channels embedded in nerve
membranes at the ends of adrenergic neurons. When a voltage pulse arrives
at the end of a neuron (propagated by sequential firing of sodium channels),
the calcium channels detect the change in voltage and allow influx of Ca++
ions. This triggers binding of vesicles and release of vesicular adrenalin,
noradrenalin or dopamine, along with cotransmitters such as peptides, ATP,
etc. One of the acid tests as to whether a substance is a neurotransmitter is
whether its release is calcium-dependent.
Verapamil is the oldest and prototypical calcium channel blocker. It is highly
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bound to blood plasma proteins and suffers about an 80% hepatic first-pass
elimination on oral administration. This means that most of the drug absorbed
through the intestine is removed by the kidney before reaching the target
tissues (heart and major blood vessels). Nifedipine is significantly less
active
at cardiac sites than diltiazem or verapamil, and is also highly plasma-bound.
Diltiazem is much less plasma bound. Despite plasma binding, all three drugs
have fairly short half lives (3-6 hours).
Ion channel blockers generally act by lodging in an open channel and
blocking it. Similarities in ion channels mean that some sodium channel
blockade occurs with calcium channel blockers. This is more of a problem with
verapamil than the other drugs. In addition to treatment of hypertension,
these
drugs are used to treat angina, migraine, and atherosclerosis.
Diuretics
0
_~ 1 ° ° ~~ rr
s
N IIS / w
S
0 Oi
~1
benzthiazide
(Diuril)
Diuretics reduce blood volume by causing excretion of water through the
kidney. This reduces blood pressure very effectively. The drugs used for this
purpose are typically thiazides; a main problem is potassium depletion.
Potassium-sparing diuretics have also been developed.
Angiotensin antagonists 8~ ACE inhibitors
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sar-arg-val-iyr-val-his-pro-ala
saralasin
0 0
H0~1
N SH HO ~N ~ N i
01 HO
' 0
captopril enalapril
(Capoten)
(Vasotec)
p 0 0
III
~N N ~i P
HO 01. HO 0 HO
lisinopril fosinopril
(Zestril)
N (Monopril) 0
~0
Recent studies have shown the ACE inhibitors to be extremely safe drugs.
ACE is an enzyme which converts angiotensin I to angiotensin II, the active
form.
Angiotensin receptors modulate the tension of smooth muscle, including venous
and arterial tissue. Inhibiting the enzyme decreases the amount of active
peptide
extant in body tissues.
Antipsychotics, lithium, mood stabilizers & dopamine agonists
Antipsychotics are used to calm mania or racing thoughts, to control
aggression, or to block spurious thoughts in schizophrenia, including auditory
hallucination (hearing voices). They exert their tranquilizing effects by
blocking
the excitatory neurotransmitter dopamine at post-synaptic terminals. Dopamine
neurons are abundant in the limbic system and its projections into the
cerebrum, especially the originating in the substantia nigra. Blocking of
adrenergic, histaminic and serotonergic receptors also contribute to the CNS
effects of these drugs.
Although they are potent psychotropics, these drugs are not commonly
abused, since they inhibit the brain's pleasure pathways which emanate from
the limbic system into the frontal lobe.
Thorazine analogs
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r w ~ r w g r w g r w
i ~ N i ~ N i ~ N r_~
0
CI ~ ',. CI ~ ',. S'\ N~~.
~\ N ./N ~\ r 0
-N
chlorpromazine prochlorperazine ~~ cymemazine ~ \ thiothixene N
(Ihordzine) (Compazine) N- ~ (Navane) ~ ~ °H
I ..
Tricyclic compounds like Thorazine, known as phenothiazines, are the oldest
compounds, and are the least selective, blocking several subtypes of dopamine
receptors. Thorazine was discovered by accident while seeking better
antihistaminic agents. It has shown efficacy in blocking the effects of LSD,
confirming the dopamine agonist activity of that drug.
Haloperidol analogs
F
OH F OH
CI r ~ r
N N N
./
r ~ F ../ , \ F ../ i ~ F
° 0 0
melperone haloperidol trifluperidol
(Haldol)
N~0 N~0 N~0
~~ N CI -~-- N ~-~ N
r~ .r~ r~
N N ~ ~~ N
,~ i w F ..~ N './ r ~ F
0 O~N r ~
benperidol domperidone pimozide
(Orap) F
0 iN ~N
F .\ N 0 - N~ /:
N
vN~N~ rN r w F .~N r w F
r ' v i
risperidone ttuspirilene amperozide
(Risperdal) F (Hogpax) F
Newer compounds of the haloperidol class (bytyrophenones), first
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synthesized in the late 1950s, are more selective for D2 subreceptors.
Sulpiride analogs
0
~N~ o o .~ o o .~~ o o ,
CI ~p ~ - N ~~ N ~~ N ~~ N
- N ~ . /l I N // I N ~b ~ N
N 0 ./~ 0 -N ,/~ 0 ./
cleDopride s~lpiride amisulpiride sultoprida
Clebopride and sulpiride analogs represent another structural class of
antipsychotics with similar actions. The binding profile of all these groups
(indeed, of each compound) will be slightly different.
Clozapine analogs & novel neuroleptics
..\N- ..\N- ..\N_ ..\N-
N N N N
~CI ~~ CI ~CI
clozapine loxapine clothiapine olanzapine
(Lyprexa)
-0H i ~ -0
0 ~0 _\
N N)
quetiapine 0 /
N (Seroquel)
N HO
~~ CI
S butaclamol tetrabenazine
The clozapine analogs represent another structure type with a different
neurological profile. They are not as selective for D2 as the haldol class,
but
may be more effective at controlling some types of psychosis. These drugs
show significant 5-HT2 receptor blockade and may be more selective for
limbic dopamine systems (as opposed to those involved in motor control),
reducing the extrapyramidal syndrome (EPS) and dyskinesias common to
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antipsychotic meds. The newer agents, such as Zyprexa and Seroquel, seem
not to impart the agranulocytosis common with clozapine.
Clozapine is extensively metabolized and some of its metabolites show
anti-AIDS activity.
Other novel structural classes which function as neuroleptics are
represented by butaclamol (a pentacyclic) and tetrabenazine. The latter is a
dopamine depleter, a drug which can chemically induce depression.
Mood stabilizers
c1
'j
._ N s~
°. y ° S~ 'N .° ~° °~ _, s
HO H° ° 0 OH
baclofen gabapentine vigabatrin valproic acid tiagabine
(Neurontin) (Depakote) (Gabitril)
CI CI 0
~ N N v. N~° -' ~ I_ 0 ~ 0~ II
_ S _
°'~-N~' o II
.. ; 0 0
lamotrigine phenytoin carbamazepine ~ topiramate
(Lamictal) (Dilantin) (Tegretol) (Topamax)
° 1I. 0-
CI f ~ ~ ' ° i \
N ~-- ° O H i N / w 0
v
CI ~ N , N
N
clonidine propanolol verapamil
The use of anticonvulsant medications for psychotropic purposes has
recently grown, primarily to prophylact against manic and/or panic syndromes.
Phenytoin and carbamazepine probably work by affecting ion-gating systems
(particularly sodium channels) in excitable membranes. Phenytoin structurally
resembles the barbiturates while carbamazepine has a tricyclic structure like
the tricyclic antidepressants.
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Gabapentine and valproic acid probably work on GAGA systems. The latter
inhibits the enzyme responsible for degrading GAGA at high concentrations,
but probably works by other mechanisms at lower, therapeutic levels.
Lamotrigine is another antiepileptic used as a mood stabilizer. It reduces
release of glutamate, an excitatory amino acid. Topamax, a fructose
derivative, enhances GAGA systems while blocking glutamate.
Propanolol is a beta-adrenergic blocker prescribed for performance phobia
or stage fright. Clonidine is an alpha agonist sometimes used to calm
peripheral tremor as in alcohol withdrawal. Verapamil, a calcium-channel
blocker, is also used for this purpose.
Dopamine agonists
HO OH / HO
r w r ~ N r ~ g r w 0~ 0
N~. 0 0
N
I o
.. N'
naxagolide
apomorphinepergolide (Dopazinol) ibopamine
Pergolide and naxagolide are dopamine agonists employed against
Parkinsonism, which results from decreasing dopamine function in the CNS
with aging. Apomorphine is a selective D2 agonist, while ibopamine serves
agonist function at both dopamine and adrenergic receptors.
Cholinergic drugs
Acetylcholine neurons convey sensory information to the brain and control
muscular tension, including peristalsis and motor control. Cholinergic neurons
are dominant in inhibitory activity inherent to so-called parasympathetic
neurons whic comlpement dopamine/norepinephrine based neurons in parallel
sympathtic structures. Two cholinergic receptor subtypes have been identified
by selective agonists: nicotinic and muscarinic. At least two subtypes of
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muscarinic receptors (M1 and M2) have been identified.
In addition to direct agonists, selective antagonists, enzyme inhibitors, and
antidotes to enzyme inhibitors have been developed. Cholinoceptors also
serve as heteroreceptors, presynaptically governing the release of
norepinephrine and other neurotransmitters.
Cholinoceptor agonists
~ CI _N~O~ ~ = CI _N~O~ ~ ' CI
/ ~ f
acetylcholine chloride carbachol bethanechol
N ~ 0
HO ~ + N ~ N ~N
- 0 ~ ~\ ~L~OH
muscarine nicotine N-(hydroxymethyl)nicotinamide
SI
N ~ N 0
~ W O~N~ CI
%N"N\ I\
i
guanidine . pilocarpine ~ ~ lachesine
Nicotine is a selective agonist at nicotinic receptors: it defines this subset
of
cholinergic receptors. Muscarine defines the other subset, with further
distinctions of M1 and M2 (at least) existing. Muscarine is produced in trace
amounts in the fly agaric mushroom. Other species of fungus produce
greater amounts. Fly agaric also contains muscarinic antagonists (atropine)
and GABA agonists (muscimol). Atropine used to be applied as an antidote
to poisoning by muscarine in this fungus, before the role of muscimol was
elucidated.
The N-hydroxymethyl amide of nicotinic acid is also active as an agonist at
nicotinic cholinoceptors. Carbachol is used opthalmically as a miotic, i.e. to
dilate the pupils. It is also used in large animals, mainly in atonic
conditions of
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the gut, since its formal positive charge prevents it from entering the brain
and limits its absorption in the gut. In addition to receptor action, it
probably
promotes acetylcholine release. Lachesine is a selective muscarinic agonist.
Guanidine exists as the guanidium ion at physiologic pH; it is used as a
pro-cholinergic, antiviral, antifungal, antipyretic and muscle stimulant.
Bethanechol activates M1 and M2 subreceptors, releases IP3 (inositol
triphosphate), and activates guanylyl cyclase. Again, as a quaternary,
positively charged species, it is used mainly to mimic acetylcholine in the
gut.
It is sometimes given to relieve the antimuscarinic constipation caused by
tricyclic antidepressants or other meds. Pilocarpine is a cholinomimetic which
also increases gastric acid secretion.
GABA drugs
GABA (gamma-aminobutyric acid) is the most important inhibitory
neurotransmitter in the CNS. By gating negative chloride (CI-) ions into the
interior of nerve cells, GABA inhibits the presynaptic release of
neurotransmitter due to a positive voltage polarization pulse. Such inhibition
is extremely common: GABA receptors can be found at 60 - 80% of CNS
neurons.
Subtypes of GABA receptors can be activated by the mushroom toxin
muscimol (at the A subtype) as well as the antispasmodic amino acid
baclofen (B subtype). These drugs directly mimic the action of GABA at the
receptor.
Allosteric facilitation of GABA receptors occurs at several distinct sites;
the
compounds which bind there are used as sedatives and anxiolytics. These
compounds bend the receptor open to indirectly facilitate GAGA binding.
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GABA agonists / facilitators
0 F
r_~ F
N~0 CI i ~ OH F~0
S N ~:
~N' /N F i w N v
I
muscimol progabide p N~ riluzole
CI
~v
i N S,
0~ S._ 0 N~ y N :..0 0 ~ S.
S o
HO HO 7 0 . OH
baclofen gabapentine vigabatrin valproic acid tiagabine
(Neurontin) (Depakote) (Gabitril)
CI CI 0 ~ 0
N N / w N~0 '\ ~I~ 0 0~S\
O~Ni.. o II
.. ~ 0 0
lamotrigine phenytoin carbamazepine ~ topiramate
(Lamictal) (Dilantin) (Tegretol) (Topamax)
Progabide is a pro-drug which decomposes to GABA in the CNS. It crosses
the blood-brain barrier, which GABA itself, being a zwitterion (doubly-ionized
amino acid), does not. Vigabatrin (gamma-vinyl-GAGA) inhibits GABA-
aminotransferase (GABA-T), the enzyme responsible for degrading GAGA in
the synapse. It thus prolongs the sojourn of GAGA molecules and promotes
binding in this way.
Depakote (valproic acid) seems to act on nerve membranes to reduce
susceptibility to seizure. At high doses it acts like vigabatrin to inhibit
GABA-
T. Gabapentine is another recently marketed antiepileptic (Neurontin) that is
also finding psychiatric application as a mood stabilizer. The neurological
rationale for this application is that panic attacks (or mania in bipolar
disorder)
resemble epilepsy in that they are characterized by a pre-panic "kindling"
phenomenon, characterized by repetitive neural firings, leading to a critical
stage. Gabapentine may encourage production of or discourage degradation
of GABA. Lamotrigine probably works by reducing release of glutamate, an
excitatory neurotransmitter usually governed by the inhibitory GAGA.
Novel GABA drugs represent one of the most active areas of psychotropic
research. Riluzole, for instance, is a GABA uptake inhibitor with
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anticonvulsant and hypnotic properties; it also blocks sodium channels and
inhibits glutamate release.
Opiate narcotics
Opiates, derived from the poppy plant, contain alkaloids which activate the
brain's endogenous endorphin receptors to produce analgesia, euphoria, and
respiratory suppression. Poppy opiates possess a polycyclic phenanthrene
nucleus with various substituents that determine the fit into the receptor.
Although morphinelike compounds have been found in mammalian brain
tissue, it is generally agreed that the enkephalins and endorphins represent
the endogenous compounds which poppy constituents mimic.
Opiate receptors of several varieties are responsible for the major
pharmacologic effects. These subtypes are given Greek names like mu
(analgesia, euphoria), sigma (dysphoria, cardiac stimulation), kappa
(sedation, spinal cord analgesia, miosis), delta, etc. Antitussive properties,
emesis (vomiting), and anticholinergic (constipation) effects also occur,
indicating a wide variety of receptor types and actions. The sigma receptor is
now surmised to be related to glutamate function.
Opiate receptors exert effects on synaptic transmission by presynaptically
modulating the release of neurotransmitters, including acetylcholine,
norepinephrine, dopamine, serotonin, and substance P. The latter compound
is a peptide neurotransmitter involved in nociceptive (pain-related) neurons.
Opiate receptors act on G-peptides, transmembranal macromolecules linked
to post-synaptic intracellular enzymes (such as adenylyl cyclase) or ion
channels (such as K+, Ca++). In high doses the opiates cause generalized
CNS depression sufficient for surgical anesthesia.
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Phenathrenes
HO N 0 0 HO _ N~ HO _ N
0 0 0 0 OH
i
OH 0 0 0
morphine heroin hydromorphone oxymorphone
(0.0-diacetylmorphine) (Dilaudid) (Numorphan)
N~ ~ N-' ~0 N,-- HO N
0
p 0 0 OH 0 .~ OH
OH
HO 0 0 - 0
HO
codeine hydrocodone oxycodone etorphine
(Tylenol 3.4) (lAcodin, Lorocet) (Peroocet, Tylox) (Immobilon)
Phenylheptylamines
r
r . r r
N ' N \ N ' N '
~ / ~ / ~ / ~ / .'
0~ ~'' ~ HO ~r 0
ll 0
methadone methadyl acetate dimeheptanol isomethadone
(methadol)
~ N~
~/
N.~ N \
\ 0
0
_ ~
0
0
0
dipipanone dimenoxadol propoxyphene
(Darvon)
Phenylpiperidines
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N' N' N.' N' N
r w r ~ r ' r w ~ r w
0 0 0 0 ! ~ N
0 0 0 0 0
0 0
meperidine properidine alphaprodine beta-promedol alfentanyl
(Demerol) (Plfenta)
I~ _ I~ _ I~ I
N 0 N D N 0 N
D r ~ D i ~
N ~ ~ N ~ ~ N ~ ~ N
I ~ ! ~ I ~ I ~
o ' o ' o ' o
fentanyl carfentanyl lofentanil suferrtanil
(Sublimaze) (Sufenta)
Codeine is a mild analgesic which retains agonist activity at other receptor
subtypes including those controlling respiration, peristalsis and euphoria.
Morphine is among the most potent of the phenanthrene class. The less
amphoteric heroin crosses the blood-brain barrier more readily but
decomposes into morphine once there. Oxycodone, the main active
constituent in Percodan and Percocet, is somewhat less potent.
Meperidine (Demerol) is a synthetic drug that has approximately the same
analgesic activity as morphine. Methadone, invented by the Nazis and
originally named dolophine, is famous for its use in assuaging the heroin
withdrawal syndrome. Its half-life is substantially greater than that of
heroin,
and while it is bound to receptors it blocks newly administered heroin. Its
analgesic activity is also approximately equal to morphine's, but it imparts
less euphoria.
Fentanyl constitutes one of the most potent synthetics, propoxyphene
(Darvon) one of the least. Methoxy compounds such as codeine and
oxycodone are less susceptible to first-pass reactions (typically conjugation
to
a glucuronide) and therefore have a higher oral-to-parenteral ratio. Less-
amphoteric compounds (compounds with more definite acid or base
properties) pass the blood-brain barrier more easily.
Partial agonist-antagonists
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0
0
N~ HO HO
0 ~ ~ ' 'N
v o ON OH
0
~0
noscapine pentazocine butorphanol nalbuphine
(falwin) (Stadol) (Nubain)
Alteration of the phenanthrene skeleton produces drugs with mixed
agonist/antagonist properties at opiopeptin subreceptors. These drugs are
being used variously as pain killers, aids in withdrawal from heroin and even
alcohol addiction, and (illegally) to increase athletic stamina. Stadol has
been
used nasally to relieve migraines. Although mixed agonists retain analgesic
properties, they often impart dysphoric effects.
Narcotic antagonists
HO H ~ NO N HO H HO H
0 OH 0 OH 0 OH 0 OH
i
0 OH 0
naloxone nalorphine naltrexone nalmefene
(Halline) (ReVia)
. l~
aar rao ~~t~o'~ p
l/
o ~ ox
0
nadide
(Enzopride)
Narcotic antagonists are especially useful in cases of overdose, where they
can reverse the CNS depression caused by opiate agonists. Naloxone is the
most often used, most effective, and prototypal narcotic antagonist.
Naloxone, nalmefene, and nadide are among several other compounds used
to antagonize morphine receptors.
Naltrexone has recetnly been used to reduce the craving for alcohol among
recovering alcoholics and heroin addicts (as ReVia).
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Nootropics & smart drugs
Nootropics, also known as smart drugs or cognition activators, are drugs
that enhance mental function. Several mechanisms that affect nerve function
may be attacked. Compounds that are used by the body to manufacture
neurotransmitters constitute one group (precursors). Reuptake and
degradation inhibitors form another. Mimetics of excitatory neurotransmitters
and antagonists of inhibitory ones can both stimulate neural function.
Antianoxics enhance the ability of neurons to burn glucose. Phospholipid
compounds affect the fatty excitable membranes of nerve cells, which are
responsible for transporting a depolarization pulse down dendrites and axons.
Steroid compounds also affect membrane chemistry. Vasodilators which act
in the CNS increase blood supply to brain cells. Still other drugs increase
the
flexibility of red blood cells so they can gain access to more neurons more
often. All these effects be theoretically be used to enhance neurological
function in the CNS.
Precursors ~ mimetics
,+ - r+ , ,
p'--~N~ O-'~N' =N~N'- =N~N
. I .
glycine dimethylglycine glycinamide milacemide
(DMG)
0 0 0 0 0
r+ - ~+ a~ o
O_-= N O-:i N =N ~ ~ o
0 + r ~O o ~ '' A _
- ,~N~
0 0 ---
- 0 calcium-N-carbamoylaspartate
aspartate glutamate aceglutamide
0
0 0 OH O 0
O~N' O~N' O~N'
GAHA cam'rtine acetyl-L-camrtine
(ALC)
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Glycine systems perform inhibitory functions in the CNS. Enhancement of
these pathways imparts antianxiety effects and so stabilizes mood. Glycine
itself is a zwitterion and so does not pass the blood-brain barrier very well.
Dimethylglycine is stabilized by the methyl groups; its greater lipophilicity
results in better transport to the CNS, where it is converted to glycine.
Milacemide is a pro compound which decomposes (via MAO-B) to
glycinamide and then glycine in the CNS.
Glutamate and aspartate are another group of excitatory neurotransmitter
prominent in the CNS. Since they are acidic amino acids they have difficulty
crossing the blood-brain barrier, but standard tricks can be used to deliver
them to the CNS. Making an amide out of a carboxy acid is one of these (as
in glutamine and aceglutamide); a somewhat more radical method is to make
a covalent salt with calcium, as in calcium-N-carbamoylaspartate.
Carnitine is a catabolic (tearing-down) amino acid which serves as a
neuroprotectant at NMDA receptors (a subset of glutamate/aspartate
receptors). Acetylation of the hydroxy group gives ALC, which again has the
effect of promoting transport into the CNS.
Steroids
0
OH
0
HO OH HO
0
testosterone estradiol pregnenolone dehydriepiandrosterone
(DHF~jI
o OH o OH
0
OH HO OH
HO
0 0
cortisone cortisol cholesterol
(hydrocortisone)
Several steroids have been used to bolster mental function and libido. Both
testosterone and estrogens have been administered historically to increase
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vitality and sexual drive as people grow older (replacement therapy).
Precursors to estrogen and androgen steroids such as DHEA and
pregnenolone have recently been marketed as nutrients. These steroids do
not have significant estrogenic or androgenic properties until converted by
the
body to active forms. As with all precursors, one trusts the body's
homeostatic mechanisms to regulate formation of active molecules by rate-
limiting steps, competitive mechanisms, and tachyphylaxis (tolerance).
Mood stabilizers
c1
'j
s~
0 S_ 0 1 N~ y N : . 0 0 0 i S
HO H0 ~ + f 0 - 0 OH
baclofen gabapentine vigabatrin valproic acid tiagabine
(Neurontin) (Depakote) (Gabitril)
o~
CI CI 0 0
i ~ N N t . N"~0
I ' 0~'" ~ o I I
.. \ 0 0 .
lamotrigine phenytoin carbamazepine ~ topiramate
(Lamictal) (Dilantin) (Tegretol) (Topamax)
0 ~ /. 0-
N ~0 OH i0 ~ ~ 1 N / ~ Ov
CI ~ N ~ N
N
clonidine propanolol verapamil
The use of anticonvulsant medications for psychotropic purposes has
recently grown, primarily to prophylact against manic and/or panic
syndromes. Phenytoin and carbamazepine probably work by affecting ion-
gating systems in excitable membranes. Phenytoin structurally resembles the
barbiturates while carbamazepine has a tricyclic structure like the tricyclic
antidepressants. Propanolol, a beta-blocker, has been used to calm
peripheral reactions to stress, such as stage fright.
Propanolol is a beta-adrenergic blocker prescribed for performance phobia
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or stage fright. Clonidine is an alpha agonist sometimes used to calm
peripheral tremor as in alcohol withdrawal. Verapamil, a calcium-channel
blocker, is also used for this purpose.
Antianoxics
° o . 0 0
~ ~.N ~ ~ N ~°H
HOOH -OH i ~N i ~N
J. 0 ~. 0~' '0I S 0
glutamic acid pyrogl~tamate piracetam oxiracetam
.I
0 NON
1 ~~ ~1 1
-0
aniracetam prdmirdcetam
The piracetam group of antianoxic compounds work by several
mechanisms to invigorate neural function. By supplying glutamic acid analogs
to the Krebs cycle they enhance glucose utilization in aerobic respiration,
the
major means by which animal cells extract chemical energy from sugars via
ATP formation. This in turn raises phospholipid cAMP levels, enhancing the
function of dopamine and acetylcholine neurons. Additionally they function as
antioxidants (compare the structure to that of vitamin C) and retard
lipofuscin
formation. Experimentally, piracetam has been shown to increase athletic
performance, to reverse alcohol-induces brain degeneration, and has been
tried as a treatment for dyslexia.
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Cerebral vasodilators & anticoagulants
0 0
0
~N ~ o ~ o N o
o~N~N-" N N o~N~N
N ~~ o~~~_ N r o~~~ N ' v N
caffeine N~ ~0
(1,3,7-trimethylxanthine) perrtoxyfylline properrtofylline uric acid
0 /~ N ~ N
0
CI O~N 0 N N
02N / ~ N'~ ~ ~ ~ \
~1N 0 N HO
.iN~ I 0 0
' ~ 0 0
nizofenone nimod~pine vinpocetine vincamine
N
0 H0
~o i ~ S\
OH g
0 OH N
Idebenone py~i~l HO HO
Methylxanthines are used as bronchodilators in the treatment of asthma
(typically theophylline) and in conjunction with analgesics to treat headache.
Pentoxyfylline and propentofylline have central and peripheral vasodilatory
properties. Increased blood supply to brain tissue probably accounts for
whatever nootropic properties they have. Pentoxfylline also increases the
elasticity of red blood cells, enabling them to better squeeze through
constricted capillaries. Such drugs are called anti-ischemics, ischemia
referring to a lack of blood supply to a tissue.
Pyritinol is another vasodilator which has been used against dementia
senilis (senility). Idebenone resembles ubiquinone, a compound which
catalyzes mitochondria) metabolic processes. It promotes secretion of nerve
growth factor (NGF) and may also protect cell membranes against lipid
peroxidation. Ergocryptine is an ergot alkaloid which has been used to
combat age-related memory loss and Alzheimer's. It dilates blood vessels by
blocking alpha-adrenoceptors. It has been used in accident victims to
increase blood flow to the brain following trauma to prevent tissue damage by
anoxia. Vinpocetine and vincamine are two alkaloids from the vinca plant
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which also have anticoagulant and vasodilation effects.
Non-steroidal anti-inflammatory drugs (NSAIDs)
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the drugs of choice for
mild to moderate pain and to reduce fever (antipyretics). They interfere with
the formation of prostaglandins by inhibiting the enzyme cyclooxygenase,
which closes a bond on arachidonic acid, an essential oil. The cyclical
prostaglandin compounds are potent, short-lived mediators of the
inflammation response. They are not stored in cells but are synthesized as
needed in response to injury or irritation. Interfering with their production
peripherally turns off the inflammation response in the body.
Aspirin and the other NSAIDs may also act at a site in the CNS. Some of
the NSAIDs (e.g. ketoprofen) inhibit other enzymes such as lipoxygenase,
further retarding the allergic/inflammation response.
C~-~ o ~. . 0 0 0 0
~0- ~0- r ~ OH ~ ~ OH
0
aspirin methyl salicylate ibuprofen ketoprofen
(acetylsalicylic acid) (Advil, Motrin) (Aaron)
0 0
~0 ~ ~ w o OH ~D v , ~ ~ ~ ~~~H 0''
N
HS
naproxen nabumetone oxaprozin
(Al eve, Naprosyn) (Relafen) (Daypro) penicillamine
Aspirin (acetylsalicylic acid) has been used for centuries along with its
relative, methylsalicylate. The latter, known as oil of wintergreen and often
used topically, is even more toxic than aspirin in overdose. Aspirin
interferes
with platelet aggregation and retards coagulation of blood. This property
probably accounts for its use in the long-term prevention of heart attacks.
In recent years drugs like ibuprofen and ketoprofen have become available
over-the-counter. The more lipophilic of these drugs, such as naproxen
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(Aleve, Naprosyn), ketoprofen (Actron) and nabumetone (Relafen), have
longer half-lives, requiring less frequent dosing, but are probably no more
effective analgesics than ibuprofen. Liver, kidney and GI problems, of varying
seriousness commensurate with dosage history, are common. Penicillamine
has been used as a long-acting NSAID, but it is fairly toxic, causing
reduction
of healing and a host of autoimmmune and histological disorders.
Cyclooxygenase inhibitors
F
1 1
~ rN~ F , ~ 0
N 1 II
' 1/ ~ N F
0 i 0 i 0
celecoxib rofecoxib
(Celebrex) (Vioxx)
A new class of NSAIDs inhibit cyclooxygenase-2, an enzyme responsible
for interconversion of prostaglandins. These COX-2 inhibitors are intended to
preserve the formation of cytoprotective prostaglandins while targeting
inhibition of the compounds responsible for pain and inflammation, reducing
stomach irritation, Celebrex (celecoxib) is one such drug. Vioxx is another
new drug in this class.
Non-narcotic analgesics
v/
N
0 0 0 00
~ 0
0 ! ~ N~ 0 r ~ N~ HO ~ ~ N~ O~N
i \= i \: i \: i \:
/ 1
phenacetin butacetin acetaminophen acetamidoquinone nefopam
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Glucosamine and chondroitin
HO HO 0 HO OS03
OH
HO ~ HO ~ 0 ~ ~ 0
HO HO OH HO 0 0
HO N HO ,iN~
OH 1. '
0
glucose glucosamine chondroitin
Recent studies have suggested that a pair of aminated sugar compounds
can assist in repairing damage to cartilage in osteoarthritis. Glucosamine, a
monomer, and chondroitin, a polymer, are being marketed as nutrients for
this purpose. In cartilage, sugar polymers form a flexible connecting matrix
around the tough protein strands in cartilage (a composite material).
Sedatives & alcohol
Benzodiazepines
,O, OH . _ N ~OH . _.N ,O, OH . _ N ~OH
l \ I N / \ ~I'~/N CI l \ J~I~/N CI / \ ~I~[/N I \ J~I"C/N
CI ~ \ CI ~ \ 0 N ~ \ CI ~ \ CI
2
diazepam (Valium) lorazepam (L~tivan) clonazepam (bonopin) oxazepam (Serax)
temazepam (Restoril)
N~.N N~N ~N~ 0
~N fN _N ' 0H '_N
/ 1 ~ N I \ ~ N CI / \ ~~''~~/N CI I \ f N F
CI / \ CI / \ CI / \ OZN / \
alprazolam (><anax) triazolam (Halcion) chlondiazepoxide (librium)
flun'rtrazepam (Rohypnol)
The benzodiazepine sedatives include Valium, Librium, Halcion, Xanax,
Ativan, Serax, and Klonopin, to name just a few. In addition to potential
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effects on lipophosphate nerve membranes, these drugs work by
allosterically enhancing the effect of the inhibitory neurotransmitter GAGA at
post-synaptic receptors. That is, they "bend" the receptor slightly so that
GABA molecules attach to and activate their receptors more effectively and
more often. Their chief advantage over the barbiturates, such as seconal,
nembutal and Phenobarbital, is that they do not act directly to open chloride
ion channels.
Serotonin drugs
Serotonin is an inhibitory neurotransmitter which complements excitatory
sympathetic systems like adrenaline and dopamine in the CNS. Like the "fight
or flight" adrenaline compounds, serotonin is released not only at specific
synaptic sites, but also in a broadcast manner into brain tissue from sets of
"diffuse" neurons emanating from the emotional centers in the limbic system
into the frontal lobe. This diffuse release sets the biochemical tone of large
areas of neural functioning, controlling mood and motivation. Serotonin's
inhibitory action is however more complex and selective than that of GABA
sedatives like Valium or Xanax, which act more globally. Because of their
effects on mood, serotonin-active drugs are used as antidepressants and
anxiolytics (anti-anxiety) drugs.
Serotonin antagonists
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-o
/~ ~p p N
N
N ~ mescaline p
r \ . . N ' t pY
J~ i \ i N _~~ N ~ F
N \ N ~p
N i-
0
p ~ ~N 0
ondansetron granisetron
(Kytril) ketanserin oxetorone
CI ~N-
i ,,~'//w
r w / 1 l ~ 1 l S
N
N N N N
N ,/~ /~ ~ 1 l= ~ 1 J.
homoohlorcyclizine cyproheptadiene pizotyline mirtazapine mianserin perlapine
(Periadin) (Remeron)
Ondansetron is a selective 5-HT3 antagonist. This receptor subtype is
found on cholinergic neurons; when it is activated it inhibits release of
acetylcholine. Along with its chemical relatives such as granisetron and
zatosetron, it may thus be useful in reviving memory function in the aged.
Granisetron is also used as an antiemetic (Kytril) in chemotherapy.
Ketanserin, a selective 5-HT2 antagonist, also acts on alpha-1
adrenoceptors to lower blood pressure. Mescaline, a hallucinogen,
antagonizes 5-HT2 terminals and has been tried as an alternative to
dopamine blocking antipsychotics (without much success; it facilitates
dopamine function). Oxetorone is a relatively new antagonist used against
migraine, as is pizotyline. Cyproheptadiene is an older serotonin antagonist
and antihistaminic. Mirtazapine (Remeron) causes serotonin release, but
blocks the 5-HT2 and 5-HT3 subreceptors, effectively augmenting serotonin
action at 5-HT1 receptors. Mianserin and homochlorcyclizine also antagonize
serotonin receptors.
Serotonin agonists
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0
~Nr 0 ~Nr ~--N ~Nr ~Nr
U -~~ / ~ / ~ N /_
HO N\ W N.~S N\ N\ N _N
r ~~
0
2-methyl-serotonin sumahiptan zolmitriptan rizatriptan
(Imitrex) (Zomig) (Maxalt)
,d''~ ~N~N ~ ~N~N ~ ~N~N
H0~ ' N N N 0
I N 011
N_ r\. /N~ ~. ,N~ ~. ~N~ ~S ~ i
\\ N ~N N
[I 0 0 0
8-hydroxy-DPAT ' gepirone 0 buspirone D ~ ipsapirone
Sumatriptan activates 5-HT1d terminals, and is used against migraine
under the trade name Imitrex. Zolmitriptan (Zomig) and rizatriptan (Maxal) are
similar, recently approved, antimigraine serotonin drugs.
Buspirone, ipsapirone and gepirone enjoy 5-HT1 agonist properties with
only weak D2 blocking effects. Buspirone is used against anxiety as an
alternative to GABA-mimetic sedatives. 8-hydroxy-DPAT acts selectively at 5-
HT1 a receptors, while 2-methylserotonin activates 5-HT3 terminals.
Steroids ~ reproductive drugs
Steroids are fat-soluble (lipophilic) hormones with a tetracyclic base
structure. The steroid structure is synthesized from smaller structures called
terpenes to precursor molecules, which then undergo extensive and subtle
alterations for a rich variety of uses: to control systems, often in fatty
tissues,
as diverse as meiosis, carbohydrate metabolism, fat storage, muscle growth,
immune function, and nerve cell membrane chemistry. Because of their high
lipophilicity, they can pass through cell membranes, which are fatty bilayers,
and influence DNA transcription and thereby alter protein synthesis. By
binding to specific sites on the DNA, they release a kind of molecular "boot"
(hsp90) that, in the absence of steroid, locks up the DNA and prevents a
short sequence from being expressed into a protein. The action of the
enzymes or active peptides generated by the activation of the DNA can
persist for long periods of time, explaining the long duration of action of
many
steroids.
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Steroids may be separated into the broad groups of gonadal compounds
and glucocorticoids, depending on the site of synthesis, which is in the ovary
or testis for the gonadal variety and in the adrenal cortex for
glucocorticoids.
They may also be divided according to function, with the usual designations
being androgens, estrogens, and progestogens (typically for the gonadal
hormones) and anabolics and catabolics (typically for the glucocorticoids).
It is important to realize, however, that these terms are not totally
exclusive.
That is, even the gonadal hormone testosterone is synthesized in small
quantities by the adrenal gland, and imparts anabolic properties separate
from its effects on gonad function. Moreover, all hormones act in coordination
with other compounds to produce a net result.
Gonadal steroids
OH
aH a~
' m
~a v ~ Ho t ~
0
testosterone estradiol ethinylestradiol norethindrone
Testosterone is the prototype of the androgen group of gonadal steroids.
Androgens impart features typified by males of mammalian species. These
include morphological features such as a protruding browridge, robust bone
structure, and large canines. It is also responsible for aggression and
libido. It
also acts as an anabolic, aiding muscle formation in response to exercise.
Estradiol, meanwhile, is the prototypical estrogen, imparting female
characteristics such as breast growth and storage of subcutaneous fat.
Estrogens also prevent heart disease (women get it statistically less than
men).
Oral contraceptives reformulate the body's steroid chemistry to mimic that
of pregnancy to prevent ovulation. This is usually done in accordance with the
natural 28 day menstrual cycle, although it is possible to trick the body into
delaying ovulation for longer periods. This is accomplished by a mixture of an
estrogen and a progestogen. The most popular preparation is norethindrone
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(a progestogen) and ethinylestradiol (an estrogen). This combination, taken in
a large dose just after unprotected sex, can also prevent pregnancy by the
same mechanism. Replacement therapy for gonadal steroids in the form of
testosterone for men and estrogen/progesterones (depot ProVera) and
recently also testosterone for women has been tried to combat the symptoms
of aging, including diminished sex drive. In men, testosterone helps libido
and
may improve cardiovascular fitness and general vigor. In women, the drop in
estrogen after menopause imparts some changes, but the drop is relatively
modest (20% or so) compared to the drop in progesterone, which causes
osteoblasts to make new bone tissue and inhibits cancer cell formation. Since
estrogens, being anabolic or tissue-building compounds, can promote cancer
cell growth, modern replacement formulations should compensate more for
progesterones than estrogens. Non-steroidal soybean estrogens are now
marketed as treatments for menopausal hot flashes, and testosterone creams
and tablets to increase sex drive.
Glucocorticoids
o OH o OH
0 0
OH H° OH °~ off
I I / F /J
0 0 0 oU
cortisone cortisol dexamethasone prednisone
(hydrocortisone)
0 OH 0 ~F
S
HO HO
HO ~ 0
HO ~ ~I Ci ~~ F ~F 0
0 0
cholesterol beclomethasone fluticasone
Cholesterol is heavily involved in membrane in metabolic chemistries. One
of its main uses in the body is to decrease the permeability of phospholipid
cell walls to ionic species such as Na+, K+, and Ca'+. In recent years it has
been implicated in aiding the accumulation of plaque on the interior surfaces
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of veins and arteries. Animal meat is typically rich in cholesterol. Through
natural selection, carnivores such as cats and dogs have developed different
chemistries adjusted for processing higher amounts of cholesterol,
cholestanol (the saturated from), and other steroids, which make diets higher
in meat safer for them than for us. Cholesterol is excreted through gall, a
fatty
digestive substance secreted from the liver. It is present in high quantities
in
gallstones. Bile is also used to excrete fat-soluble substances such as
bilirubin (from the heme group in decomposed red blood cells). Cholesterol is
also used endogenously to synthesize vitamin D.
Cortisone, another prototypical glucocorticoid, controls healing processes
associated with the immune system, as well as regulating membrane and
other functions. Hydrocortisone, also known as cortisol, works similarly to
inhibit histamine-mediated allergic reaction and regulates the body's
response to stress by modulating the chemistry of neuronal excitable
membranes. Prednisone is a synthetic compound used regularly in place of
cortisone. Dexamethasone has been used to diagnose depression, i.e. in the
dexamethasone suppression test, where the body is "stressed" by
introduction of the steroid and its response is measured.
Nonsteroidal estrogens
HO ~ v ~ r w OH HO ~ r ~ OH
diethylstilbestrol hexestrol
Hexestrol and diethylstilbestrol are two examples of polycyclic, non-steroid
compounds which activate estrogen receptors. The local structure in the
bound configuration resembles that of steroids. The other main category of
non-steroidal estrogens is the isoflavones.
Environmental estrogens have become a health concern since cattle are
commonlty fed estrogens due to their anabolic (weight-gain) properties.
Ingestion of meat therefore equates to absorbing some estrogens. Some
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pesticides are non-steroidal estrogens, as is THC, the main psychoactive
constituent of marijuana. Compounds such as diethylstilbestrol have been
shown to be carcinogens, though this is not due to action on DNA.
Tamoxifen & raloxifene
HO
r w r w 0
r ~ 0 _
~ 0 S ~ ~ H ~ CI
/ ~ H~ r
I
tamoxifen raloxifene
Recently, two nonsteroidal estrogen agents have shown great medical
promise in several women's health issues. These selective estrogen receptor
modulators (SERMs) mimic the effects of estrogens in some tissues but not
others.
Tamoxifen has been used for years following detection of breast cancer. By
blocking estrogen receptors, it discourages tumor growth. New studies show
it may also prevent breast cancer, probably by the same mechanism.
However, the benefits of this prevention must be weighed against the
increased risk of uterine cancer and other potential risks.
Raloxifene retains the ability to prormote bone maintenance and prevent
osteoporosis; it cuts the risk of breast cancer by as much as 60%, and
decreases levels of LDLs ("bad" cholesterol).
Finasteride (Propecia)
O SI.
N
N
0 finasteride
(Propecia)
Finasteride (Proscar, Propecia) is presently being marketed as a systemic
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(oral) anti-baldness medication with a mechanism distinct from that of
minoxidil, which is a topical vasodilator which stimulates follicular activity
by
improving blood flow. Finasteride works by inhibiting the formation of 5-alpha-
dihydrotestosterone, a potent androgen, from the less potent parent
compound, testosterone. It has also been used to treat benign prostatic
hypertrophy (enlargement of the prostate).
The side effects of reducing androgens in the body can be essentially
termed feminization: atrophy of the male gonads, breast augmentation,
decrease in aggressive behavior, increased risk of osteoporosis, etc.
Plant steroids
0 0
,o
o~ , o
OH
o OH \ OH
0 0 _ 0
~o
HO
oleandrin OH o dig'rtalin
0
OH
OH
Steroids are not confined to the animal kingdom but are synthesized by
plants as well. The well-known cardiotonic digitalis is derived from the
foxglove plant which synthesizes several glucoside steroids (i.e. steroids
bonded to sugar moieties).
The oleander shrub generates several steroids with similar effects on
cardiac conduction, including oleandrin and oleandrigenin.
Viagra & gossypol
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0 \N_N
-N \ ~ 0 ~ 0 0
N - ~~ HO OH HO OH
Nil N N HO ~ ~' ' i ~ OH
0
_ S
.' ~ N 11
0 sildenafil caffeine gossypol
(Viagra)
-N
Sildenafil (Viagra) is a erection facilitator. Erection depends on an
interaction of adrenergic and cholinergic neurons in which muscles must relax
to let blood into erectile tissue. The presence of nitrous oxide (NO) species
is
involved, and Viagra may affect the enzymes responsible for generating NO.
Viagra is also a selective inhibitor of cGMP phophodiesterase, which acts in
some GI vascular smooth-muscles. Caffeine, wose central ring structure
resembles Viagra's, works similarly on the more widespread cAMP
phosphodiesterase, a more widespread second messenger system.
Yohimbine, a selective alpha2 adrenergic blocker, has also been touted as
being able to prolong or intensify erection.
Gossypol, isolated from the cotton plant, has the ability to inhibit
production
of viable sperm in men. It damages the epithelial lining of seminferous
vesicles, inhibiting sperm formation. It also poisons the oxygen-carrying
capacity of blood.
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Among peptide therapeutic candidates are peptides which
demonstrate anti-neoplastic activity, such as the RGD peptides including
peptide SEQ ID NOS. 1-3.
Also included are peptides that are active against HIV, including the
GP-41 peptides including peptide SEQ ID NOS. 4-6.
Anti-viral peptides which demonstrate the ability to disrupt fusogenic
events common in viral infections. These include the RSV peptides which
demonstrate the ability to treat or prevent infection by respiratory syncytial
virus (RSV) as well as acquired immune deficiency syndrome (AIDS) caused
by infection of the human immunodeficiency virus (HIV). Such peptides
include peptide SEQ NOS. 7-9.
Also included are GLP-1 peptides including those peptides depicted in
SEQ ID NOS. 10-11.
Also included are Kringle or K5 peptides including those peptides
depicted in SEQ ID NOS. 12-13; BBB peptides (TAT) including those
peptides depicted in SEQ ID NOS. 14-15 and analgesic peptides, such as
dynorphins, are also useful, including peptide SEQ ID NO. 16.
3. Modified Therapeutic Agents
The modified therapeutic agents of the present invention comprise
therapeutic agents that have been modified by attaching a reactive group.
The reactive group may be attached to the therapeutic agent via a linking
group, or optionally without using a linking group. The modified therapeutic
agents can react with the available functionalitieson blood or pulmonary
components or blood components via covalent linkages. The invention also
relates to such modifications, such combinations with pulmonary components
or blood components, and methods for their use. These methods include
extending the effective therapeutic life of the conjugated therapeutic agents
as compared to administration of unconjugated therapeutic agents.
To form covalent bonds with functionalities on a protein, one may use
as a reactive group a wide variety of active carboxyl groups, particularly
esters, where the hydroxyl moiety is physiologically acceptable at the levels
required to modify the therapeutic agent. While a number of different
hydroxyl groups may be employed, the most convenient would be N-
hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS),
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maleimide, maleimide acids including but not limited to maleimidopropionic
acid (MPA), and maleimide esters. In the preferred embodiments of this
invention, the functionality on the blood component will be a thiol group and
the reactive group will a maleimide.
Primary amines are the principal targets for NHS esters. Accessible
a-amine groups present on the N-termini of proteins react with NHS esters.
However, a-amino groups on a protein may not be desirable or available for
the NHS coupling. While five amino acids have nitrogen in their side chains,
only the s-amine of lysine reacts significantly with NHS esters. An amide
bond is formed when the NHS ester conjugation reaction reacts with primary
amines releasing N-hydroxysuccinimide as demonstrated in the schematic
below.
o °e
O O H
Rv~~ ~~--.': + R'~HZ H~ R ~~N~-R~ + HO-N
O~ O
NHS-Ester Reaction Scheme
In the preferred embodiments of this invention, the functional group on
this protein will be a thiol group and the chemically reactive group will be a
maleimido-containing group such as MPA or GMBA (gamma-maleimide-
butyralamide). The maleimido group is most selective for sulfhydryl groups
on peptides when the pH of the reaction mixture is kept between 6.5 and 7.4.
At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls is 1000-
fold faster than with amines. A stable thioether linkage between the
maleimido group and the sulfhydryl is formed which cannot be cleaved under
physiological conditions, as demonstrated in the following schematic:
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0 0
H
N-R
H ~ N-R ~ p
O.
O
O .
N-R ~ HO-~C=H .CAN--R
O
Maleimide Reaction Scheme
A. Specific Labeling.
Preferably, the modified therapeutic agents of this invention are
designed to specifically react with thiol groups on pulmonary proteins or
mobile blood proteins. Such reaction is preferably established by covalent
bonding of the therapeutic agent modified with a maleimide link (e.g. prepared
from GMBS, MPA or other maleimides) to a thiol group on a pulmonary
protein, such as intra- or extra-cellular albumin, or a mobile blood protein
such as serum albumin or IgG.
Under certain circumstances, specific labeling with maleimides offers
several advantages over non-specific labeling of proteins with groups such as
NHS and sulfo-NHS. Thiol groups are less abundant in vivo than amino
groups. Therefore, the maleimide-modified therapeutic agents of this
invention, i.e., maleimide therapeutic agents, will covalently bond to fewer
proteins. For example, in albumin (the most abundant blood protein) there is
only a single thiol group. Thus, therapeutic agent-maleimide-albumin
conjugates will tend to comprise approximately a 1:1 molar ratio of
therapeutic agent to albumin. In addition to albumin, IgG molecules (class II)
also have free thiols. In the case of systemic delivery, since IgG molecules
and serum albumin make up the majority of the soluble protein in blood they
also make up the majority of the free thiol groups in blood that are available
to
covalently bond to maleimide-modified therapeutic agents.
Further, even among free thiol-containing blood proteins, including
. IgGs, specific labeling with maleimides leads to the preferential formation
of
therapeutic agent-maleimide-albumin conjugates, due to the unique
characteristics of albumin itself. The single free thiol group of albumin,
highly
conserved among species, is located at amino acid residue 34 (Cyst). It has
been demonstrated recently that the Cys34 of albumin has increased reactivity
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relative to free thiols on other free thiol-containing proteins. This is due
in
part to the very low pK value of 5.5 for the Cys~ of albumin. This is much
lower than typical pK values for cysteine residues in general, which are
typically about 8. Due to this low pK, under normal physiological conditions
Cys34 of albumin is predominantly in the ionized form, which dramatically
increases its reactivity. In addition to the low pK value of Cys34, another
factor
which enhances the reactivity of Cys34 is its location, which is in a crevice
close to the surface of one loop of region V of albumin. This location makes
Cys34 very available to ligands of all kinds, and is an important factor in
Cys34's biological role as free radical trap and free thiol scavenger. These
properties make Cys34 highly reactive with maleimide-therapeutic agents, and
the reaction rate acceleration can be as much as 1000-fold relative to rates
of
reaction of maleimide-therapeutic agents with other free-thiol containing
proteins.
Another advantage of therapeutic agent-maleimide-albumin conjugates
is the reproducibility associated with the 1:1 loading of therapeutic agent to
albumin specifically at Cys34. Other techniques, such as glutaraldehyde,.
DCC, EDC and other chemical activations of, e.g, free amines, lack this
selectivity. For example, albumin contains 52 lysine residues, 25-30 of which
are located on the surface of albumin and therefore accessible for
conjugation. Activating these lysine residues, or alternatively modifying
therapeutic agents to couple through these lysine residues, results in a
heterogenous population of conjugates. Even if 1:1 molar ratios of
therapeutic agent to albumin are employed, the yield will consist of multiple
conjugation products, some containing 0, 1, 2 or more therapeutic agents per
albumin, and each having therapeutic agents randomly coupled at any one or
more of the 25-30 available lysine sites. Given the numerous possible
combinations, characterization of the exact composition and nature of each
conjugate batch becomes difficult, and batch-to-batch reproducibility is all
but
impossible, making such conjugates less desirable as a therapeutic.
Additionally, while it would seem that conjugation through lysine residues of
albumin would at least have the advantage of delivering more therapeutic
agent per albumin molecule, studies have shown that a 1:1 ratio of
therapeutic agent to albumin is preferred. In an article by Stehle, et al.,
"The
Loading Rate Determines Tumor Targeting properties of Methotrexate-
Albumin Conjugates in Rats," Anti-Cancer Drugs, Vol. 8, pp. 677-685 (1988),
incorporated herein in its entirety, the authors report that a 1:1 ratio of
the
anti-cancer methotrexate to albumin conjugated via glutaraldehyde gave the
most promising results. These conjugates were preferentially taken up by
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tumor cells, whereas conjugates bearing 5:1 to 20:1 methotrexate molecules
had altered HPLC profiles and were quickly taken up by the liver in vivo. It
is
postulated that at these higher ratios, conformational changes to albumin
diminish its effectiveness as a therapeutic carrier.
Through controlled administration of maleimide-therapeutic agents in
vivo, one can control the specific labeling of albumin and IgG in vivo. For
systemic delivery via pulmonary administration, in typical administrations, 80-
90% of the administered maleimide-therapeutic agents that reach the
bloodstream will label albumin and less than 5% will label IgG. Trace labeling
of free thiols such as glutathione will also occur. Such specific labeling is
preferred for in vivo use as it permits an accurate calculation of the
estimated
half life of the administered agent.
In addition to providing controlled specific in vivo labeling, maleimide-
therapeutic agents can provide specific labeling of albumin or other proteins
ex vivo. Such ex vivo labeling involves the addition of maleimide-therapeutic
agents to a saline solution containing albumin or other protein. Once
conjugation has occurred ex vivo with the maleimide-therapeutic agents, the
saline solution can be administered via pulmonary delivery for in vivo
treatment.
In contrast to NHS-therapeutic agents, maleimide-therapeutic agents
are generally quite stable in the presence of aqueous solutions and in the
presence of free amines. Since maleimide-therapeutic agents will only react
with free thiols, protective groups are generally not necessary to prevent the
maleimide-therapeutic agents from reacting with itself. In addition, the
increased stability of the modified therapeutic agent permits the use of
further
purification steps such as HPLC to prepare highly purified products suitable
for in vivo use. Lastly, the increased chemical stability provides a product
with a longer shelf life.
B. Non-Specific Labeling.
The therapeutic agents of the invention may also be modified for non-
specific labeling of pulmonary or blood components. Bonds to amino groups
will also be employed, particularly with the formation of amide bonds for non-
specific labeling. To form such bonds, one may use as a chemically reactive
group a wide variety of active carboxyl groups, particularly esters, where the
hydroxyl moiety is physiologically acceptable at the levels required. While a
number of different hydroxyl groups may be employed in these linking agents,
the most convenient would be N-hydroxysuccinimide (NHS) and N-hydroxy-
sulfosuccinimide (sulfo-NHS).
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Other linking agents which may be utilized are described in U.S. Patent
5,612,034, which is hereby incorporated herein.
The various sites with which the chemically reactive group of the
modified therapeutic agents may react in vivo include cells, particularly the
alveolar cells and capillary endothelial cells that make up the alveoli in the
lungs as well as red blood cells (erythrocytes) and platelets in the blood
itself.
The agents may also react with pulmonary proteins, including membrane
bound receptors, intra- and extra-cellular albumin, immunoglobulins, ferritin,
and transferrin, and serum proteins of the blood, such as immunoglobulins,
including IgG and IgM, serum albumin, ferritin, steroid binding proteins,
transferrin, thyroxin binding protein, a- 2-macroglobulin, and the like. Those
receptors with which the modified therapeutic agents react, which are not
long-lived, will generally be eliminated from the human host within about
three
days. The proteins indicated above (including the proteins of the cells) will
remain at least three days, and may remain five days or more (usually not
exceeding 60 days, more usually not exceeding 30 days) particularly as to the
half life, based on the concentration in the blood.
For the most part, for systemic delivery of the therapeutic agent,
reaction will be with mobile components in the blood, particularly blood
proteins and cells, more particularly blood proteins and erythrocytes. By
"mobile" is intended that the component does not have a fixed situs for any
extended period of time, generally not exceeding 5 minutes, more usually one
minute, although some of the blood component may be relatively stationary
for extended periods of time. Initially, there will be a relatively
heterogeneous
population of functionalized proteins and cells. However, for the most part,
the population within a few days will vary substantially from the initial
population, depending upon the half-life of the functionalized proteins in the
.
blood stream. Therefore, usually within about three days or more, IgG will
become the predominant functionalized protein in the blood stream.
Usually, by day 5 post-administration, IgG, serum albumin and
erythrocytes will be at least about 60 mole %, usually at least about 75 mole
%, of the conjugated components in blood, with IgG, IgM (to a substantially
lesser extent) and serum albumin being at least about 50 mole %, usually at
least about 75 mole %, more usually at least about 80 mole %, of the non-
cellular conjugated components.
The desired conjugates of non-specific modified therapeutic agents to
blood components may be prepared in vivo by pulmonary administration of
the modified therapeutic agents to the patient, which may be a human or
other mammal. If desired, the subject conjugates may also be prepared ex
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vivo by combining a carrier protein or protein solution with modified
therapeutic agents of the present invention, allowing covalent bonding of the
modified therapeutic agents to functionalitieson the protein and then
administering the conjugated mixture to the host via pulmonary delivery.
3. Diagnostic Agents and Modified Diagnostic Agents
Diagnostic agents are agents useful in imaging the mammalian
vascular system and include such agents as position emission tomography
(PET) agents, computerized tomography (CT) agents, magnetic resonance
imaging (MRI) agents, nuclear magnetic imaging agents (NMI), fluroscopy
agents and ultrasound contrast agents.
The modified diagnostic agent of the present invention will, for the
most part, have the following formula: X-Y-Z.
In the formula, X is a diagnostic agent selected from PET agent, CT
agents, MRI agents, NMI agents, fluroscopy agents and ultrasound contrast
agents. Diagnostic agents of interest include radioisotopes of such elements
as iodine (I), including '231, ,251, '3'I, etc., barium (Ba), gadolinium (Gd),
technetium (Tc), including 99Tc, phosphorus (P), including 3'P, iron (Fe),
manganese (Mn), thallium (TI), chromium (Cr), including 5'Cr, carbon (C),
including '4C, or the like, fluorescently labeled compounds, etc.
In the formula, Y is a linking group of from 0-30, more usually of from
2-12, preferably of from 4-12 atoms, particularly carbon, oxygen,
phosphorous and nitrogen, more particularly carbon and oxygen, where the
oxygen is preferably present as oxy ether, where Y may be alkylene,
oxyalkylene, or polyoxyalkylene, where the oxyalkylene group has from 2-3
carbon atoms, and the like. A linking group of 0 atoms is preferred when it is
desired to place X as close to Z as possible.
In the formula, Z is a reactive entity, such as carboxy, carboxy ester,
where the ester group is of 1-8, more usually 1-6 carbon atoms, particularly a
physiologically acceptable leaving group which activates the carboxy carbonyl
for reaction with amino groups in an aqueous system, e.g. N
hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide, (sulfo-NHS),
maleimide, maleimide esters, maleimide acids, maleimide-benzoyl-
succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS)
and maleimidopropionic acid (MPA), N-hydroxysuccinimide isocyanate,
isothiocyanate, thiolester, thionocarboxylic acid ester, imino ester, mixed
anhydride, e.g. carbodiimide anhydride, carbonate ester, etc. and the like.
The reactive entity Z will covalently bond to functionalities in vivo or ex
vivo.
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4. Synthesis of Peptide Therapeutic A ents
A. Peptide Synthesis
Therapeutic agents according to the present invention that are
peptides may be synthesized by standard methods of solid phase peptide
chemistry known to those of ordinary skill in the art. For example, peptides
may be synthesized by solid phase chemistry techniques following the
procedures described by Steward and Young (Steward, J. M. and Young, J.
D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company,
Rockford, III., (1984) using an Applied Biosystem synthesizer. Similarly,
multiple peptide fragments may be synthesized then linked together to form
larger peptides. These synthetic peptides can also be made with amino acid
substitutions at specific locations.
For solid phase peptide synthesis, a summary of the many techniques
may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide
Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer,
Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York),
1973. For classical solution synthesis see G. Schroder and K. Lupke, The
Peptides, Vol. 1, Acacemic Press (New York). In general, these methods
~0 comprise the sequential addition of one or more amino acids or suitably
protected amino acids to a growing peptide chain. Normally, either the amino
or carboxyl group of the first amino acid is protected by a suitable
protecting
group. The protected or derivatized amino acid is then either attached to an
inert solid support or utilized in solution by adding the next amino acid in
the
sequence having the complimentary (amino or carboxyl) group suitably
protected and under conditions suitable for forming the amide linkage. The
protecting group is then removed from this newly added amino acid residue
and the next amino acid (suitably protected) is added, and the process is
repeated.
After all the desired amino acids have been linked in the proper
sequence, any remaining protecting groups (and any solid support) are
removed sequentially or concurrently to afford the final polypeptide. By
simple modification of this general procedure, it is possible to add more than
one amino acid at a time to a growing chain, for example, by coupling (under
conditions which do not racemize chiral centers) a protected tripeptide with a
properly protected dipeptide to form, after deprotection, a pentapeptide.
A particularly preferred method of preparing compounds of the present
invention involves solid phase peptide synthesis wherein the amino acid a-N-
terminal is protected by an acid or base sensitive group. Such protecting
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groups should have the properties of being stable to the conditions of peptide
linkage formation while being readily removable without destruction of the
growing peptide chain or racemization of any of the chiral centers contained
therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),
t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),
biphenylisopropyloxycarbonyl , t-amyloxycarbonyl, isobornyloxycarbonyl, a,
a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-
butyloxycarbonyl, and the like. The 9-fluorenyl-methyloxycarbonyl (Fmoc)
protecting group is particularly preferred for the synthesis of the peptides
of
the present invention. Other preferred side chain protecting groups are, for
side chain amino groups like lysine and arginine, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-
methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine,
benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-
Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and
tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and
2,4-
dinitrophenyl; for tryptophan, formyl; for asparticacid and glutamic acid,
benzyl and t-butyl and for cysteine, triphenylmethyl (trityl).
In the solid phase peptide synthesis method, the a-C-terminal amino
acid is attached to a suitable solid support or resin. Suitable solid supports
useful for the above synthesis are those materials which are inert to the
reagents and reaction conditions of the stepwise condensation-deprotection
reactions, as well as being insoluble in the media used. The preferred solid
support for synthesis of a-C-terminal carboxy peptides is 4-
hydroxymethylphenoxymethyl-copoly(styrene-1 % divinylbenzene). The
preferred solid support for a-C-terminal amide peptides is the 4-(2',4'-
dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available
from Applied Biosystems (Foster City, Calif.). The a-C-terminal amino acid is
coupled to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-
diisoprop~~lcarbodiimide (DIC) or O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluronium-hexafluorophosphate (HBTU), with or without 4-
dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT),
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate
(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCI), mediated
coupling for from about 1 to about 24 hours at a temperature of between
10°
and 50°C in a solvent such as dichloromethane or DMF.
When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a
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secondary amine, preferably piperidine, prior to coupling with the a-C-
terminal
amino acid as described above. The preferred method for coupling to the
deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-
acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluoro-phosphate (HBTU, 1 equiv.) and 1-
hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive
protected amino acids can be carried out in an automatic polypeptide
synthesizer as is well known in the art. In a preferred embodiment, the a-N-
terminal amino acids of the growing peptide chain are protected with Fmoc.
The removal of the Fmoc protecting group from the a-N-terminal side of the
growing peptide is accomplished by treatment with a secondary amine,
preferably piperidine. Each protected amino acid is then introduced in about
3-fold molar excess, and the coupling is preferably carried out in DMF. The
coupling agent is normally O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-
hydroxybenzotriazole (HOBT, 1 equiv.).
At the end of the solid phase synthesis, the polypeptide is removed
from the resin and deprotected, either in successively or in a single
operation.
Removal of the polypeptide and deprotection can be accomplished in a single
operation by treating the resin-bound polypeptide with a cleavage reagent
comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. In
cases
wherein the a-C-terminal of the polypeptide is an alkylamide, the resin is
cleaved by aminolysis with an alkylamine. Alternatively, the peptide may be
removed by transesterification, e.g. with methanol, followed by aminolysis or
by direct transamidation. The protected peptide may be purified at this point
or taken to the next step directly. The removal of the side chain protecting
groups is accomplished using the cleavage cocktail described above. The
fully deprotected peptide is purified by a sequence of chromatographic steps
employing any or all of the following types: ion exchange on a weakly basic
resin (acetate form); hydrophobic adsorption chromatography on underivitized
polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel
adsorption chromatography; ion exchange chromatography on
carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25,
LH-20 or countercurrent distribution; high performance liquid chromatography
(HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica
bonded phase column packing.
Molecular weights of these ITPs are determined using Fast Atom
Bombardment (FAB) Mass Spectroscopy.
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(1 ) N-Terminal Protective Groups
As discussed above, the term "N-protecting group" refers to those
groups intended to protect the a-N-terminal of an amino acid or peptide or to
otherwise protect the amino group of an amino acid or peptide against
undesirable reactions during synthetic procedures. Commonly used N-
protecting groups are disclosed in Greene, "Protective Groups In Organic
Synthesis," (John Wiley & Sons, New York (1981)), which is hereby
incorporated by reference. Additionally, protecting groups can be used as
pro-drugs which are readily cleaved in vivo, for example, by enzymatic
hydrolysis, to release the biologically active parent. a-N-protecting groups
comprise loweralkanoyl groups such as formyl, acetyl ("Ac"), propionyl,
pivaloyl, t-butylacetyl and the like; other acyl groups include 2-
chloroacetyl, 2-
bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
-
chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and
the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the
like; carbamate forming groups such as benzyloxycarbonyl, p-
chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-
nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-
bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-
dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-
ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-
trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, a,a-
dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-
butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl,
ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-
trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-
9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl,
cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groups such
as benzyl, triphenylmethyl, benzyloxymethyl, 9-fluorenylmethyloxycarbonyl
(Fmoc) and the like and silyl groups such as trimethylsilyl and the like.
(2) Carboxy Protective Groups
As discussed above, the term "carboxy protecting group" refers to a
carboxylic acid protecting ester or amide group employed to block or protect
the carboxylic acid functionality while the reactions involving other
functional
sites of the compound are performed. Carboxy protecting groups are
disclosed in Greene, "Protective Groups in Organic Synthesis" pp. 152-186
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(1981), which is hereby incorporated by reference. Additionally, a carboxy
protecting group can be used as a pro-drug whereby the carboxy protecting
group can be readily cleaved in vivo, for example by enzymatic hydrolysis, to
release the biologically active parent. Such carboxy protecting groups are
well known to those skilled in the art, having been extensively used in the
protection of carboxyl groups in the penicillin and cephalosporin fields as
described in U.S. Pat. Nos. 3,840,556 and 3,719,667, the disclosures of
which are hereby incorporated herein by reference. Representative carboxy
protecting groups are C, -C8 loweralkyl (e.g., methyl, ethyl or t-butyl and
the
like); arylalkyl such as phenethyl or benzyl and substituted derivatives
thereof
such as alkoxybenzyl or nitrobenzyl groups and the like; arylalkenyl such as
phenylethenyl and the like; aryl and substituted derivatives thereofsuch as 5-
indanyl and the like; dialkylaminoalkyl such as dimethylaminoethyl and the
like); alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl,
valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-
1-ethyl, 1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,
pivaloyloxymethyl, propionyloxymethyl and the like; cycloalkanoyloxyalkyl
groups such as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,
cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like;
aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl and the like;
arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl, 2-
benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl or
cycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;
alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such as
methoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, 1-
ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and the like;
aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl, 2-(5-
indanyloxycarbonyloxy)ethyl and the like; alkoxyalkylcarbonyloxyalkyl such as
2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;
arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl and the
like; arylalkenyloxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-
yloxycarbonyloxy)ethyl and the like; alkoxycarbonylaminoalkyl such as t-
butyloxycarbonylaminomethyl and the like; alkylaminocarbonylaminoalkyl
such as methylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl
such as acetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as
4-methylpiperazinylcarbonyloxymethyl and the like; dialkylaminocarbonylalkyl
such as dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl and the
like; (5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-t-butyl-2-oxo-
1,3-
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dioxolen-4-yl)methyl and the like; and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl
such as (5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.
Representative amide carboxy protecting groups are aminocarbonyl
and loweralkylaminocarbonyl groups.
Preferred carboxy-protected compounds of the invention are
compounds wherein the protected carboxy group is a loweralkyl, cycloalkyl or
arylalkyl ester, for example, methyl ester, ethyl ester, propyl ester,
isopropyl
ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl
ester,
octyl ester, cyclohexyl ester, phenylethyl ester and the like or an
alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or an
arylalkylcarbonyloxyalkyl ester. Preferred amide carboxy protecting groups
are loweralkylaminocarbonyl groups. For example, aspartic acid may be
protected at the a-C-terminal by an acid labile group (e.g. t-butyl) and
protected at the ~-C-terminal by a hydrogenation labile group (e.g. benzyl)
then deprotected selectively during synthesis.
B. Peptide Modification
The manner of producing the modified peptides of the present
invention will vary widely, depending upon the nature of the various elements
comprising the peptide. The synthetic procedures will be selected so as to be
simple, provide for high yields, and allow for a highly purified stable
product.
Normally, the chemically reactive group will be created at the last stage of
the
synthesis, for example, with a carboxyl group, esterification to form an
active
ester. Specific methods for the production of modified peptides of the present
invention are described below.
Specifically, the selected peptide is modified with the linking group only
at either the N-terminus, C-terminus or interior of the peptide. The
therapeutic activity of this modified peptide-linking group is then assayed.
If
the therapeutic activity is not reduced dramatically (i.e., reduced less than
10
fold), then the stability of the modified peptide-linking group is measured by
its
in vivo lifetime. If the stability is not improved to a desired level, then
the
peptide is modified at an alternative site, and the procedure is repeated
until a
desired level of therapeutic and stability is achieved.
More specifically, each peptide selected to undergo modification with a
linking group and a reactive group will be modified according to the following
criteria: if a terminal carboxylic group is available on the peptide and is
not
critical for the retention of therapeutic activity, and no other sensitive
functional group is present on the peptide, then the carboxylic acid will be
chosen as attachment point for the linking group-reactive group modification.
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If the terminal carboxylic group is involved in therapeutic activity, or if no
carboxylic acids are available, then any other sensitive functional group not
critical for the retention of therapeutic activity will be selected as the
attachment point for the linking group-reactive entity modification. If
several
sensetive functional groups are available on a a peptide, a combination of
protecting groups will be used in such a way that after addition of the
linking
group/reactive entity and deprotection of all the protected sensetive
functional
groups, retention of therapeutic activity is still obtained. If no sensetive
functional groups are available on the peptide, or if a simpler modification
route is desired, synthetic efforts will allow for a modification of the
original
peptide in such a way that retention of therapeutic is maintained. In this
case
the modification will occur at the opposite end of the peptide
An NHS derivative may be synthesized from a carboxylic acid in
absence of other sensetive functional groups in the peptide. Specifically,
such a peptide is reacted with N-hydroxysuccinimide in anhydrous CH2C12
and EDC, and the product is purified by chromatography or recrystallized
from the appropriate solvent system to give the NHS derivative.
Alternatively, an NHS derivative may be synthesized from a peptide
that contains an amino and/or thiol group and a carboxylic acid. When a free
amino or thiol group is present in the molecule, it is preferable to protect
these
sensetive functional groups prior to perform the addition of the NHS
derivative. For instance, if the molecule contains a free amino group, a
transformation of the amine into a Fmoc or preferably into a tBoc protected
amine is necessary prior to perform the chemistry described above. The
amine functionality will not be deprotected after preparation of the NHS
derivative. Therefore this method applies only to a compound whose amine
group is not required to be freed to induce the desired therapeutic effect. If
the amino group needs to be freed to retain the original properties of the
molecule, then another type of chemistry described below has to be
performed.
In addition, an NHS derivative may be synthesized from a peptide
containing an amino or a thiol group and no carboxylic acid. When the
selected molecule contains no carboxylic acid, an array of bifunctional
linking
groups can be used to convert the molecule into a reactive NHS derivative.
For instance, ethylene glycol-bis(succinimydylsuccinate) (EGS) and
triethylamine dissolved in DMF and added to the free amino containing
molecule (with a ratio of 10:1 in favor of EGS) will produce the mono NHS
derivative. To produce an NHS derivative from a thiol derivatized molecule,
one can use N-[ -maleimidobutyryloxy]succinimide ester (GMBS) and
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triethylamine in DMF. The maleimido group will react with the free thiol and
the NHS derivative will be purified from the reaction mixture by
chromatography on silica or by HPLC.
An NHS derivative may also be synthesized from a peptide containing
multiple sensetive functional groups. Each case will have to be analyzed and
solved in a different manner. However, thanks to the large array of protecting
groups and bifunctional linking groups that are commercially available, this
invention is applicable to any peptide with preferably one chemical step only
to modify the peptide (as described above) or two steps (as described above
involving prior protection of a sensitive group) or three steps (protection,
activation and deprotection). Under exceptional circumstances only, would
multiple steps (beyond three steps) synthesis be required to transform a
peptide into an active NHS or maleimide derivative.
A maleimide derivative may also be synthesized from a peptide
containing a free amino group and a free carboxylic acid. To produce a
maleimide derivative from a amino derivatized molecule, one can use N-[y-
maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine in DMF.
The succinimide ester group will react with the free amino and the maleimide
derivative will be purified from the reaction mixture by crystallization or by
chromatography on silica or by HPLC.
Finally, a maleimide derivative may be synthesized from a peptide
containing multiple other sensetive functional groups and no free carboxylic
acids. When the selected molecule contains no carboxylic acid, an array of
bifunctional crosslinking reagents can be used to convert the molecule into a
reactive NHS derivative. For instance maleimidopropionic acid (MPA) can be
coupled to the free amine to produce a maleimide derivative through reaction
of the free amine with the carboxylic group of MPA using HBTU/HOBt/DIEA
activation in DMF.
Many other commercially available heterobifunctional crosslinking
reagents can alternatively be used when needed. A large number of
bifunctional compounds are available for linking to entities. Illustrative
reagents include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-
[2'-pyridyldithio)propionamide), bis-sulfosuccinimidyl suberate, dimethyl
adipimidate, disuccinimidyl tartrate, N-y-maleimidobutyryloxysuccinimide
ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-
azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-
iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl 4-[N-
maleimidomethyl]cyclohexane-1-carboxylate.
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5. Synthesis of Modified Organic Therapeutic Agents
Similar to procedures for modified peptide therapeutics, the synthetic
procedures used to prepare modified organic therapeutics will also be
selected so as to be simple, provide for high yields, and allow for a highly
purified product. Normally, the chemically reactive group will be created as
the last stage, for example, with a carboxyl group, esterification to form an
active ester will be the last step of the synthesis. Methods for the
production
and/or modification of organic therapeutic agents of the present invention are
described in the Examples below.
Each organic therapeutic agent selected to undergo the derivatization
with a linking group and a reactive group will be modified according to the .
following criteria: Generally, the therapeutic agents are commercially
available. If not, they can be synthesized by procedures well known in the
art. As a first step, the therapeuticlly active region of the therapeutic
agent is
identified. Next, the therapeutic agent is modified at a site sufficiently far
away from the active portion to prevent a potential interference between the
modified drug and the target site of the drug, such that the modified agent
substantially retains its therapeutic activity, (i.e. the therapeutic activity
is
reduced by no more than 10 fold). Finally, keeping constant the site of
chemical modification, optimize the biological activity of the modified agent
by
modifying the length and nature of the linking group.
If a carboxylic group, not critical for the retention of pharmacological
activity is available on the original molecule and no other reactive
functionality
is present on the molecule, then the carboxylic acid will be chosen as
attachment point for the linking group-reactive entity modification. If no
carboxylic acids are available, then any other functionalities not critical
for the
retention of pharmacological activity will be selected as attachment point for
the linking group-reactive entity modification. If several functionalities are
available on a therapeutic agent, a combination of protecting groups will be
used in such a way that after addition of the linking group/reactive entity
and
deprotection of all the protected functionalities, retention of
pharmacological
activity is still obtained. If no functionalitiesare available on the
therapeutic
agent, synthetic efforts will allow for a modification of the original parent
drug
in such a way that retention of biological activity and retention of receptor
or
target specificity is obtained.
Where the derivatized therapeutic agent of the present invention
represents a derivatized enzyme inhibitor will generally have substantially
lower ICSO's generally in the range of about 0.5-0.01 of the ICso of the
parent
molecule. Desirably, the ICSO should be not less than 0.05, preferably not
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than about 0.1. In view of the varying ICS°'s, the amount of the
derivatized
therapeutic agent administered will also vary.
The determination of the nature and length of the linking group will be
performed through an empirical optimization phase and will be measured by
the retention or the loss of biological activity. For instance, with a given
inhibitor enzyme interactions, an iteration of the modification of the nature
and
the length of the linking group and a measure of the biological enzymatic
activity may be necessary to determine the most favored linking group length
and nature. Preferably a short hydrophilic 4-12 atom linking group easily
synthesized will be favored to start the iteration process.
In the case of radiolabeled therapeutic agents, a minimum distance
from the target has to be respected based on the nature of the isotope and its
penetration. The length and nature of the linking groups are not as important
as they are for an enzyme inhibitor combination. For instance an isotope that
emits a beta rays like 3ZP should be positioned within 5 mm from the target to
have maximum efficiency (99%) with limited or no effect coming from a small
change on the nature and length of the linking group.
6. Synthesis of Modified Diagnostic Agents
The manner of producing the modified diagnostic agents of the present
invention will vary widely, depending upon the nature of the various elements
comprising the molecule. The synthetic procedures will be selected so as to
be simple, provide for high yields, and allow for a highly purified product.
Normally, the chemically reactive group will be created as the last stage, for
example, with a carboxyl group, esterification to form an active ester will be
the last step of the synthesis. Methods for the production of the diagnostic
agents of the present invention are described in the Examples.
7. Pulmonary Formulations and Delivery Methods
A further aspect of the invention is directed to aerosol compositions for
treatment of the symptoms of pulmonary conditions. With therapeutic agents
capable of forming a covalent bond with a pulmonary fluid protein.
Alternatively, the therapeutic agent may be first conjugated with the
pulmonary solution protein prior to administration.
The aerosol compositions may be composed of an aqueous solution
suitable for inhalation consisting of at least 2.5% by weight (more preferably
between about 3% and 10% by weight, and most preferably at least about 5%
by weight) of a derivatized therapeutic agent. The droplets of the aerosol
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should be 10 ~, or less in diameter to maximize deposition in the lung alveoli
rather than the throat or upper respiratory tract.
The invention also features inhaler devices for administration of the
inhalable compositions (or medicaments) of the subject invention. In one
aspect of the invention, the inhaler device comprises a housing defining a
chamber which contains a dry powder. The dry powder is composed of
therapeutic agent compound present in an amount that, upon administration
will bind to pulmonary proteins. Alternatively, the agent may be covalently
bonded to pulmonary proteins ex vivo prior to administration. At least 50%
(preferably at least 70%, and more preferably at least 90%) of the powder
consists of primary particles which have a diameter of 10~m or less, and
which may be agglomerated into larger particles or agglomerates which
readily break down into primary particles upon inhalation from the device. The
chamber has an opening through which the medicament can be drawn by.
inhalation by a patient.
In another aspect of the invention, the inhaler device comprises a
vessel containing an inhalable medicament in the form of an aqueous solution
suspended in a compressed or liquified propellant gas. At least 2.5% by
weight (preferably at least 3%, more preferably at least 4%, even more
preferably at least 5% and most preferably between 6 and 10%) of the
aqueous solution is a pH-raising buffer compound.
The inhaler device may also have a housing defining a port onto which
the vessel is mounted, a lumen in communication with the port, and a
mechanism for controllably releasing the propellant from the vessel into the
lumen, thereby releasing the suspended medicament from the vessel into the
lumen. The lumen is configured to route the medicament suspended in the
propellant into the respiratory system of the patient.
8. Therapeutic Uses of Modified Therapeutic Agents
The modified therapeutic agents of the invention find numerous uses,
as enumerated below.
A. Therapeutic Uses of Modified Antineoplastic Agent
The antineoplastic agents of the invention, including but not limited to
those specified in the examples, possess anti-angiogenic activity. As
modified antineoplastic agents having anti-angiogenic activity, the compounds
of the present invention are useful in the treatment of a variety of diseases,
for example primary and metastatic solid tumors and carcinomas of the
breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach.
Pancreas; liver; gallbladder; bile ducts; small intestine; urinary tract
including
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kidney, bladder and urothelium; female genital tract including cervix, uterus,
endometrium, ovaries, choriocarcinoma and festational trophoblastic disease;
male genital tract including prostate, seminal vesicles, testes and germ cells
tumors; endocrine glands including thyroid, adrenal and pituitary; skin
including hmangiomas, melanomas, sarcomas arising from bone of soft
tissues and Karposi's sarcoma, Wilm's tumor, rhabdomyosarcoma; tumor of
the head and neck, brain, nerves, eyes, and meninges including
astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas; tumors of the bone marrow and hematopoeitic tumors, solid
tumors arising from hematopoietic malignancies such as leukemias and
including chloromas, plasmocytomas, plagues and tumors of mycosis
fungoides and cutaneous T-cell lymphoma/leukemia; acute lymphotic, actute
granulocytic and chronic granulocytic leukemia; lymphomas including both
Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmune
diseases including rheumatoid, immune and degenerative arthritis; ocular
diseases including diabethic retinopathy; retinopathy of prematurity; corneal
graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,
retinal
neovascularization due to macular degeneration and hypoxia; abnormal
neovascularization conditions of the eye; skin diseases including psoriasis;
blood vessel diseases including hemagiomas and capillary proliferatrion
withinatherosclerotic plaques; myocardial angiogenesis; plaque
neovascularization; hemophiliac joints; angiofibroma; wound granulation;
dieases charadterized by excessive or abnormal stimulation of endothelial
cells including intestinal adhesion, Crohn's disease, atheroscelrosis,
scleroderma and hypertrophic scars and diseases which have angiogenesis
as a pathological consequence including ulcers (Helicobacter pilori);
rheumatoid arthritis, osteogenic sarcoma, osteoarthritis, osteopenias such as
osteoporosis, periodontitis, gingivitis, corneal epidermal or gastric
ulceration,
and tumor metastasis, invasion and growth, retinopathies, wound healing
(ocular inflammation, soft and osseous tissue disease, gingivitis/periodontal
disease), vascular disease (restenosis) annuerysm inflammation, autoimmune
diseases, and rare cancers such as choriocarcinoma.
The compounds of the present invention may also be useful for the
prevention of metastases from the tumors described above either when used
alone or in combination with radiotherapy and /or other chemotherapeutic
treatments conventionally administered to patients for treating angiogenic
diseases. For example, when used in the treatment of solid tumors,
compounds of the present invention may be administered with
chemotherapeutic agents such as alpha-inferon, COMP (cyclophosphamnide,
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vincristine, methotraxate and prednisone), etoposide, mBACOD
(methotraxate, bleomycin, doxorubicin, cyclophosphamide, vincristine and
dexamethasone), PROMACE/MOPP (prednisone, methotrexate, doxirubicin,
cyclophaophamide, taxol, etoposide/mechloetamine, vincristine, prednisone
and procarbazine), vincristine, vinblastine, angioinhibins, TNP-470, pentosan
polysulfate, platelet factor-4, angiostatin, LM-609, SU-101, CM-101,
techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents
include alkylating agents such as nitrogen mustards including
mechloethamine, melphanchloambucil, cyclophaosphamide, and ifosfamide;
nirrosdoureas including carmustine, lomustine, semustine and streptozocin;
alkyl sulfonates icluding busulfan; triazines including dacarbazine;
ethyenimines including thiotepa na dhexamethylmelanine; folic acid analogs
including methotraxate; pyrimidine analogs including 5-FU, cytosine
arabinoside; purine analogs including 6-mercaptopurine and 6-thioguanine;
antitumor antibiotics including actinomycin D; the anthraqcyclines including
doxorubicin, bleomycin, mitomycin C and methramycin; hormones and
hormones antagonists including tamoxifen and corticosteroids and
mioscellaneous agnets including cisplatin and brequinar; fragments of
plasminogen (kringle-5) as well as fragments from other integrin-binding
substrates. For insnance, a tumor may be treated conventionally with
surgery, radiation or chemiotherapy and administration of modified
antineoplastic agents to extend the dormancy of micrometastasis and to
inhibit the growth of any residual primary tumor.
B. Therapeutic Uses of Modified Matrix Metalloprotease Inhibitors
The MMPIs of the invention, including but not limited to those specified
in the examples, possess anti-angiogenic activity. As modified matrix
metalloprotease inhibitors having anti-angiogenic activity, the compounds of
the present invention are useful in the treatment of a variety of diseases,
for
example primary and metastatic solid tumors and carcinomas of the breast;
colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach.
Pancreas; liver; gallbladder; bile ducts; small intestine; urinary tract
including
kidney, bladder and urothelium; female genital tract including cervix, uterus,
ovaries, choriocarcinoma and festational trophoblastic disease; male genital
tract including prostate, seminal vesicles, testes and germ cells tumors;
endocrine glands including thyroid, adrenal and pituitary; skin including
hmangiomas, melanomas, sarcomas arising from bone of soft tissues and
Karposi's sarcoma; tumor of the nrain, nerves, eyes, and meninges including
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astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas; tumors of the bone marrow and hematopoeitic tumors, solid
tumors arising from hematopoietic malignancies such as leukemias and
including chloromas, plasmocytomas, plagues and tumors of mycosis
fungoides and cutaneous T-cell lymphoma/leukemia; lymphomas including
both Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmune
diseases including rheumatoid, immune and degenerative arthritis; ocular
diseases including diabethic retinopathy; retinopathy of prematurity; corneal
graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,
retinal
neovascularization due to macular degeneration and hypoxia; abnormal
neovascularization conditions of the eye; skin diseases including psoriasis;
blood vessel diseases including hemagiomas and capillary proliferatrion
withinatherosclerotic plaques; myocardial angiogenesis; plaque
neovascularization; hemophiliac joints; angiofibroma; wound granulation;
dieases charadterized by excessive or abnormal stimulation of endothelial
cells including intestinal adhesion, Crohn's disease, atheroscelrosis,
scleroderma and hypertrophic scars and diseases which have angiogenesis
as a pathological consequence including ulcers (Helicobacter pilori);
rheumatoid arthritis, osteoarthritis, osteopenias such as osteoporosis,
periodontitis, gingivitis, corneal epidermal or gastric ulceration, and tumor
metastasis, invasion and growth, retinopathies, wound healing (ocular
inflammation, soft and osseous tissue disease, gingivitis/periodontal
disease),
vascular disease (restenosis) annuerysm inflammation and and autoimmune
diseases. Another use is as birth control agent which inhibits ovulation and
establishment of the placenta.
The compounds of the present invention may also be useful for the
prevention of metastases from the tumors described above either when used
alone or in combination with radiotherapy and /or other chemotherapeutic
treatments conventionally administered to patients for treating angiogenic
diseases. For example, when used in the treatment of solid tumors,
compounds of the present invention may be administered with
chemotherapeutic agents such as alpha-inferon, COMP (cyclophosphamnide,
vincristine, methotraxate and prednisone), etoposide, mBACOD
(methotraxate, bleomycin, doxorubicin, cyclophosphamide, vincristine and
dexamethasone), PROMACE/MOPP (prednisone, methotrexate, doxirubicin,
cyclophaophamide, taxol, etoposide/mechloetamine, vincristine, prednisone
and procarbazine), vincristine, vinblastine, angioinhibins, TNP-470, pentosan
polysulfate, platelet factor-4, angiostatin, LM-609, SU-101, CM-101,
techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents
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include alkylating agents such as nitrogen mustards including
mechloethamine, melphanchloambucil, cyclophaosphamide, and ifosfamide;
nirrosdoureas including carmustine, lomustine, semustine and streptozocin;
alkyl sulfonates icluding busulfan; triazines including dacarbazine;
ethyenimines including thiotepa na dhexamethylmelanine; folic acid analogs
including methotraxate; pyrimidine analogs including 5-FU, cytosine
arabinoside; purine analogs including 6-mercaptopurine and 6-thioguanine;
antitumor antibiotics including actinomycin D; the anthraqcyclines including
doxorubicin, bleomycin, mitomycin C and methramycin; hormones and
hormones antagonists including tamoxifen and corticosteroids and
mioscellaneous agnets including cisplatin and brequinar; fragments of
plasminogen (kringle-5) as well as fragments from other integrin-binding
substrates. For instance, a tumor may be treated conventionally with surgery,
radiation or chemiotherapy and the modified MMPI molecules of the invention
to extend the dormancy of micrometastasis and to inhibit the growth of any
residual primary tumor.
It has also been found that hydroxamic acid MMPIs can inhibit the
production of the cytokine tumor necrosis factor (TNF) (Mohler et al., Nature,
1994, 370, 218-220; Gearing AJH et al., Nature 1994, 370, 555-557;
McGeehan GM et al., Nature 1994, 370, 558-561 ). Compounds which inhibit
the production or action of TNF are thought to be potentially useful for the
treatment or prophylaxis of many inflammatory, infectious, immunological or
malignant diseases. These include, but are not restricted to, septic shock,
haemodynamic shock and sepsis syndrome, post ischaemic reperfusion
injury, malaria, Crohn's disease, mycobacterial infection, meningitis,
psoriasis,
congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer,
autoimmune disease, rheumatic arthritis, multiple scleroris, radation damage,
toxicity following administration of immunosuppressive monoclonal antibodies
such as OKT3 or CAMPATH-1 _and hyperoxic alveolar injury. Since excessive
TNF production has been noted in several diseases or conditions also
characterized by MMP-mediated tissue degradation, compounds which inhibit
both MMPs and TNF production may have particular advantages in the
treatment or prophylaxis of diseases or conditions in which both mechanisms
are involved.
The compounds of the present invention inhibit various enzymes from
the matrix metalloproteinase family such as collagenase, which initiates
collagen breakdown, stromelysin (protoglycanase), and gelatinase, and hence
are useful for the treatment of matrix metallo endoproteinase diseases. There
is evidence implicating collagenase as one of the key enzymes in the
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breakdown of articular cartilage and bone in rheumatoid arthritis (Arthritis
and
Rheumatism, 20, 1231-1239, 1977). Potent inhibitors of collagenase and
other metalloproteases involved in tissue degradation are useful in the
treatment of rheumatoid arthritis and related diseases in which collagenolytic
activity is important. Inhibitors of metalloproteases of this type can
therefore
be used in treating or preventing conditions which involve tissue breakdown;
they are therefore useful in the treatment of arthropathy, dermatological
conditions, bone resorption, inflammatory diseases and tumour invasion and
in the promotion of wound healing. Specifically, compounds of the present
invention may be useful in the treatment of osteopenias such as osteoporosis,
rheumatoid arthritis, osteoarthritis, periodontitis, gingivitis, corneal
ulceration
and tumour invasion.
C. Therapeutic Uses of Oxytocin
A conjugated oxytocin may be used to aid lactation and help relax the
pelvis prior to birth. It could also be used to prevent post partum uterine
hemorrage.
D. Therapeutic Uses of Cholecystokinin i(CCK~
A conjugated CCK could be used in diagnostic studies of the gall
bladder or in chronic cholecystisis.
E. Therapeutic Uses of Antihyrpertensive Agents
Antihypertensive agents are used to treat hypertension.
F. Therapeutic Uses of Methylprednisolone
Methylprednisolone is used to treat a wide range of disorders such as
asthma and arthritis. In gastroenterology, it is effective in the treatment of
several inflammatory conditions such as ulcerative and microscopic colitis,
Crohn's disease and autoimmune hepatitis. A newer usage is for reduction of
post-traumatic spinal cord edema.
G. Therapeutic Uses of GP-41 Pelotides
GP-41, an HIV transmembrane protein, can be used to create
therapeutic and diagnostic agents against HIV. For example, antibodies can
be constructed to recognize epitopes of gp41. The structure of this antibody
will provide important information regarding antibody/antigen interaction,
guide chemists in the selection of superior antigenic peptides for HIV
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detection, and will provide important information for future recombinant
experiments with genetically engineered antibodies.
H. Therapeutic Uses of Blood Brain Barrier (BBBI Peutides
As BBB peptides can traverse the blood brain barrier through protein
transduction, these peptides can be covalently linked to compounds,
peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame
fusions with full-length proteins, to allows these compounds to enter any cell
type in a receptor- and transporter-independent fashion. This effectively
delivers these compounds past the blood brain barrier.
Therapeutic Uses of Modified Cell Adhesion i(RGD~~eptides
The RGD peptides of the invention and their derivatives and analogs
find multiple uses including use as a treatment for neoplastic diseases and
inflammatory diseases such as rheumatoid arthritis, lupus.
1. Anti-neoalastic Treatments
The modified cell adhesion peptides of the invention or their derivatives
or analogs generally will target directly to cancer cells via peptide-specific
receptors. It has been shown that receptors for these peptides are expressed
at elevated levels on the surface of tumor cells. Thus, the modified peptides
or their derivatives or analogs can be used to preferentially target drugs to
metastatic tumor cells. Therefore, the modified cell adhesion peptides or
their
derivatives or analogs are useful as agents for the treatment of different
types
of cancers such as breast carcinoma, melanoma, and fibrosarcoma.
The use of an effective amount of modified cell adhesion peptides or
their derivatives or analogs as a treatment for cancer has the advantage of
being more potent than non modified cell adhesion peptides. Since the
modified cell adhesion peptides or their derivatives or analogs are more
stable in vivo, smaller amounts of the molecule can be administered for
effective treatment.
The derivatives and conjugates of the modified cell adhesion peptides
and their analogs may be used in several different ways and to achieve
several different ends. As mentioned above, these materials may be used in
place of typical cell adhesion peptide drugs as an anti-adhesive agent. As
compared with cell adhesion peptide drugs currently available, the materials
of this invention can reduce clot formation with less side effects and are
available for reducing clot formation for a substantially longer time than
conventionally administered cell adhesion peptide drugs. In addition, the
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derivatized cell adhesion peptides of this invention may be utilized (in
accordance with U.S. Patent Numbers 5,443.827; 5,439,88 and 5,433,940
and PCT application number WO/97/01093 which are hereby incorporated by
reference) in conjunction with various other anti-adhesive or anti-cancer
therapies. Such anti-cancer therapies include the use of radiation or
treatment with antineoplastic agents such as, for example, vinca alkaloids,
alkylating agents, doxorubicin, etoposide, methotrexate, tamoxifen,
vinblastine, asparaginase, biclutamide, bleomycin, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubisin, docetaxel, floxuridine, fludarabine,
fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, interferon
alpha, irnotecan, leuprolide, mechlorethamine, megestrol, melphalan,
mercaptopurine, mitomycin, mitoxantrone, paclitaxel, plicamycin, por lrmer,
procarbazine, streptozocin, teniposide, thioguanine, thiotepa, topotecan,
trastuzumab, vincristine, vinorelbine, and the like.
The present invention also provides for a method for treating cancer in
an individual, wherein said method comprises providing an amount of
modified cell adhesion peptide sufficient to treat cancer; where the
composition contains a modified cell adhesion peptide.
2. Treatment Of Inflammatoryi Disease
The modified cell adhesion peptides of the invention and their
derivatives and analogs also find use as anti-inflammatories. In one aspect of
the invention, there is provided a method of treating a mammalian subject
with an abnormality resulting in increased inflammation of the joints or
tissues
using the modified cell adhesion peptides of the invention or their
derivatives
or analogs. The method comprises administering a modified cell adhesion
peptide or its derivative or analog to the subject in an amount sufficient to
produce an anti-inflammatory effect on the subject. The modified cell
adhesion peptide may be administered intracerebroventriculary, orally,
subcutaneously, intramuscularly, or intravenously.
The peptides of the present invention, their derivatives, analogs, and
conjugates can be used to treat acute or chronic inflammatory disorders
involving ischemia, infection, tissue swelling, and/or bone and cartilage
degradation. Inflammatory disease refers to a condition in which activation of
leukocytes leads to an impairment of normal physiologic function. Examples
of such conditions include acute and chronic inflammation such as
osteoarthritis, sepsis, ARDS, immune and autoimmune disorders, rheumatoid
arthritis, IBD (inflammatory bowel disease), lupus, MS, graft rejection,
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cirrhosis, sarcoidosis, granulomatous lesions, periodontitis/gingivitis, graft-
vs.-
host disease, contact dermatitis, and the like. Included among autoimmune
disorders which may be treated using the present method are chronic active
hepatitis, Graves' disease, insulin-dependent diabetes mellitus (type I), and
Hasshimoto's thyroiditis. Included among inflammatory disorders which may
be treated using the present method are inflammatory brain disease,
inflammatory demyelinating disease, inflammatory vasculitis, inflammatory
myopathies, osteomyelitis, Crohn's disease and interstitial cystitis.
Additional
examples of inflammatory diseases include myocardial diseases, infectious
diseases, pulmonary diseases and graft rejection.
J. Therapeutic Uses of Modified Insulinotropic Peptides
such as GLP-1
The modified insulinotropic peptides (ITPs) such as GLP-1 of the
invention find multiple uses including use as a treatment for diabetes, a
sedative, a treatment of nervous system disorders, use to induce an anxiolytic
effect on the CNS, use to activate the CNS, use for post surgery treatment
and as a treatment for insulin resistance.
1. Diabetes Treatments
The modified ITPs of the invention generally will normalize
hyperglycemia through glucose-dependent, insulin-dependent and insulin-
independent mechanisms. As such, the modified ITPs are useful as primary
agents for the treatment of type I I diabetes mellitus and as adjunctive
agents
for the treatment of type I diabetes mellitus.
The use of an effective amount of modified ITPs as a treatment for
diabetes mellitus has the advantage of being more potent than non modified
ITPs. Since the modified ITPs are move stable in vivo, smaller amounts of the
molecule can be administered for effective tratment. The present invention is
especially suited for the treatment of patients with diabetes, both type I and
type II, in that the action of the peptide is dependent on the glucose
concentration of the blood, and thus the risk of hypoglycemic side effects are
greatly reduced over the risks in using current methods of treatment.
The present invention also provides for a method for treating diabetes
mellitus in an individual, wherein said method comprises providing an amount
of modified ITP sufficient to treat diabetes; where the composition contains a
modified ITP.
2. Treatment Of Nervous Slrstem Disorders
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The modified ITPs of the invention also find use as a sedative. In one
aspect of the invention, there is provided a method of sedating a mammalian
subject with an abnormality resulting in increased activation of the central
or
peripheral nervous system using the modified ITPs of the invention. The
method comprises administering a modified ITP to the subject in an amount
sufficient to produce a sedative or anxiolytic effect on the subject. The
. modified ITP may be administered intracerebroventriculary, orally,
subcutaneously, intramuscularly, or intravenously. Such methods are useful
to treat or ameliorate nervous system conditions such as anxiety, movement
disorder, aggression, psychosis, seizures, panic attacks, hysteria and sleep
disorders.
In a related aspect, the invention encompasses a method of increasing
the activity of a mammalian subject, comprising administering a modified ITP
to the subject in an amount sufficient to produce an activating effect on the
subject. Preferably, the subject has a condition resulting in decreased
activation of the central or peripheral nervous system. The modified ITPs find
particular use in the treatment or amelioration of depression, schizoaffective
disorders, sleep apnea, attention deficit syndromes with poor concentration,
memory loss, forgetfulness, and narcolepsy, to name just a few conditions in
which arousal of the central nervous system may be advantageous.
The modified ITPs of the invention may be used to induce arousal for
the treatment or amelioration of depression, schizoaffective disorders, sleep
apnea, attention deficit syndromes with poor concentration, memory loss,
forgetfulness, and narcolepsy. The therapeutic efficacy of the modified ITP
treatment may be monitored by patient interview to assess their condition, by
psychological/neurological testing, or by amelioration of the symptoms
associated with these conditions. For example, treatment of narcolepsy may
be assessed by monitoring the occurrence of narcoleptic attacks. As another
example, effects of modified ITPs on the ability of a subject to concentrate,
or
on memory capacity, may be tested using any of a number of diagnostic tests
well known to those of skill in art.
3. Post Surgery Treatment
The modified ITPs of the invention may be utilized for post surgery
treatments. A patient is in need of the modified ITPs of the present invention
for about 1-16 hours before surgery is performed on the patient, during
surgery on the patient, and after the patient's surgery for a period of not
more
than about 5 days.
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The modified ITPs of the present invention are administered from
about sixteen hours to about one hour before surgery begins. The length of
time before surgery when the compounds used in the present invention
should be administered in order to reduce catabolic effects and insulin
resistance is dependent on a number of factors. These factors are generally
known to the physician of ordinary skill, and include, most importantly,
whether the patient is fasted or supplied with a glucose infusion or beverage,
or some other form of sustenance during the preparatory period before
surgery. Other important factors include the patient's sex, weight and age,
the severity of any inability to regulate blood glucose, the underlying causes
of any inability to regulate blood glucose, the expected severity of the
trauma
caused by the surgery, the route of administration and bioavailability, the
persistence in the body, the formulation, and the potency of the compound
administered. A preferred time interval within which to begin administration
of
the modified ITPs used in the present invention is from about one hour to
about ten hours before surgery begins. The most preferred interval to begin
administration is between two hours and eight hours before surgery begins.
Insulin resistance following a particular type of surgery, elective
abdominal surgery, is most profound on the first post-operative day, lasts at
least five days, and may take up to three weeks to normalize Thus, the post-
operative patient may be in need of administration of the modified ITPs used
in the present invention for a period of time following the trauma of surgery
that will depend on factors that the physician of ordinary skill will
comprehend
and determine. Among these factors are whether the patient is fasted or
supplied with a glucose infusion or beverage, or some other form of
sustenance following surgery, and also, without limitation, the patient's sex;
weight and age, the severity of any inability to regulate blood glucose, the
underlying causes of any inability to regulate blood glucose, the actual
severity of the trauma caused by the surgery, the route of administration and
bioavailability, the persistence in the body, the formulation, and the potency
of
the compound administered. The preferred duration of administration of the
compounds used in the present invention is not more than five days following
surgery.
4. Insulin Resistance Treatment
The modified ITPs of the invention may be utilized to treat insulin
resistance independently from their use in post surgery treatment. Insulin
resistance may be due to a decrease in binding of insulin to cell-surface
receptors, or to alterations in intracellular metabolism. The first type,
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characterized as a decrease in insulin sensitivity, can typically be overcome
by increased insulin concentration. The second type, characterized as a
decrease in insulin responsiveness, cannot be overcome by large quantities
of insulin. Insulin resistance following trauma can be overcome by doses of
insulin that are proportional to the degree of insulin resistance, and thus is
apparently caused by a decrease in insulin sensitivity.
The dose of modified ITPs effective to normalize a patient's blood
glucose level will depend on a number of factors, among which are included,
without limitation, the patient's sex, weight and age, the severity of
inability to
regulate blood glucose, the underlying causes of inability to regulate blood
glucose, whether glucose, or another carbohydrate source, is simultaneously
administered, the route of administration and bioavailability, the persistence
in
the body, the formulation, and the potency.
K. Therapeutic Uses of Modified Kring~le 5 Peptides
As described earlier, angiogenesis includes a variety of processes
involving neovascularization of a tissue including "sprouting",
vasculogenesis,
or vessel enlargement. With the exception of traumatic wound healing,
corpus leuteum formation and embryogenesis, it is believed that the majority
of angiogenesis processes are associated with disease processes and
therefore the use of the present therapeutic methods are selective for the
disease and do not have deleterious side effects.
There are a variety of diseases in which angiogenesis is believed to be
important, which may be treatable with the modified peptides of the invention.
These diseases include, but not limited to, inflammatory disorders such as
immune and non-immune inflammation, chronic articular rheumatism and
psoriasis, disorders associated with inappropriate or inopportune invasion of
vessels such as diabetic retinopathy, neovascular glaucoma, restenosis,
capillary proliferation in atherosclerotic plaques and osteoporosis, and
cancer
associated disorders, such as solid tumors, solid tumor metastases,
angiofibromas, retrolental fibroplasia, hemangiomas, Kaposi sarcoma and the
like cancers which require neovascularization to support tumor growth.
The modified kringle 5 peptides of the invention find use in methods
which inhibit angiogenesis in a diseased tissue ameliorates symptoms of the
disease and, depending upon the disease, can contribute to cure of the
disease. The modified peptides of the invention are more stable in vivo and,
as such, smaller amounts of the modified peptide can be administered for
effective treatment In one embodiment, the invention contemplates inhibition
of angiogenesis, per se, in a tissue. The extent of angiogenesis in a tissue,
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and therefore the extent of inhibition achieved by the present methods, can
be evaluated by a variety of method, for detecting b'5#3 immunopositive
immature and nascent vessel structures by immunohistochemistry.
As described herein, any of a variety of tissues, or organs comprised of
organized tissues, can support angiogenesis in disease conditions including
skin, muscle, gut, connective tissue, joints, bones and the like tissue in
which
blood vessels can invade upon angiogenic stimuli.
In one related embodiment, a tissue to be treated with the modified
kringle 5 peptides of the invention is an inflamed tissue and the angiogenesis
to be inhibited is inflamed tissue angiogenesis where there is
neovascularization of inflamed tissue. In this class the method contemplates
inhibition of angiogenesis in arthritic tissues, such as in a patient with
chronic
articular rheumatism, in immune or non-immune inflamed tissues, in psoriatic
tissue and the like.
The patient treated in the present invention in its many embodiments is
desirably a human patient, although it is to be understood that the principles
of the invention indicate that the invention is effective with respect to all
mammals, which are intended to be included in the term "patient." In this
context, a mammal is understood to include any mammalian species in which
treatment of diseases associated with angiogenesis is desirable, particularly
agricultural and domestic mammalian species.
In another related embodiment, a tissue to be treated with the modified
kringle 5 peptides of the invention is a retinal tissue of a patient with
diabetic
retinopathy, macular degeneration or neovascular glaucoma and the
angiogenesis to be inhibited is retinal tissue angiogenesis where there is
neovascularization of retinal tissue.
In an additional related embodiment, a tissue to be treated with the
modified kringle 5 peptides of the invention is a tumor tissue of a patient
with
a solid tumor, a metastases, a skin cancer, a breast cancer, a hemangioma or
angiofibroma and the like cancer, and the angiogenesis to be inhibited is
tumor tissue angiogenesis where there is neovascularization of a tumor
tissue. Typical solid tumor tissues treatable by the present methods include
lung, pancreas, breast, colon, laryngeal, ovarian, and the like tissues.
Inhibition of tumor tissue angiogenesis is a particularly preferred
embodiment because of the important role neovascularization plays in tumor
growth. In the absence of neovascularization of tumor tissue, the tumor tissue
does not obtain the required nutrients, slows in growth, ceases additional
growth, regresses and ultimately becomes necrotic resulting in killing of the
tumor.
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The present invention thus provides for a method of inhibiting tumor
neovascularization by inhibiting tumor angiogenesis according to the present
methods using the modified kringle 5 peptides of the invention. Similarly, the
invention provides a method of inhibiting tumor growth by practicing the
angiogenesis-inhibiting methods. The methods are also particularly effective
against the formation of metastases because (1 ) their formation requires
vascularization of a primary tumor so that the metastatic cancer cells can
exit
the primary tumor and (2) their establishment in a secondary site requires
neovascularization to support growth of the metastases.
In a related embodiment, the invention contemplates the practice of the
method in conjunction with other therapies such as conventional
chemotherapy directed against solid tumors and for control of establishment
of metastases. The administration of the modified kringle 5 peptides of the
invention is typically conducted during or after chemotherapy, although it is
preferably to inhibit angiogenesis after a regimen of chemotherapy at times
where the tumor tissue will be responding to the toxic assault by inducing
angiogenesis to recover by the provision of a blood supply and nutrients to
the tumor tissue. In addition, it is preferred to administer the modified
kringle 5
peptides after surgery where solid tumors have been removed as a
prophylaxis against metastases. Insofar as the present methods apply to
inhibition of tumor neovascularization, the methods can also apply to
inhibition of tumor tissue growth, to inhibition of tumor metastases
formation,
and to regression of established tumors using the modified kringle 5 peptides
of the invention.
Restenosis is a process of smooth muscle cell (SMC) migration and
proliferation at the site of percutaneous transluminal coronary angioplasty
which hampers the success of angioplasty. The migration and proliferation of
SMC's during restenosis can be considered a process of angiogenesis which
is inhibited by the modified kringle 5 peptides of the present invention.
Therefore, the invention also contemplates inhibition of restenosis by
inhibiting angiogenesis in a patient following angioplasty procedures. For
inhibition of restenosis, the modified kringle 5 peptide is typically
administered
after the angioplasty procedure for from about 2 to about 28 days, and more
typically for about the first 14 days following the procedure.
The present method for inhibiting angiogenesis in a tissue comprises
contacting a tissue in which angiogenesis is occurring, or is at risk for
occurring, with a composition comprising a therapeuticlly effective amount of
a modified kringle 5 peptide. The dosage ranges for the administration of the
modified kringle 5 peptide depend upon the form of the peptide, and its
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potency, as described further herein, and are amounts large enough to
produce the desired effect in which angiogenesis and the disease symptoms
mediated by angiogenesis are ameliorated. The dosage should not be so
large as to cause adverse side effects, such as hyperviscosity syndromes,
pulmonary edema, congestive heart failure, and the like. Generally, the
dosage will vary with the age, condition, sex and extent of the disease in the
patient and can be determined by one of skill in the art. The dosage can also
be adjusted by the individual physician in the event of any complication.
As angiogenesis inhibitors, such modified kringle 5 peptides are useful
in the treatment of both primary and metastatic solid tumors and carcinomas
of the breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus;
stomach; pancreas; liver; gallbladder; bile ducts; small intestine; urinary
tract
including kidney, bladder and urothelium; female genital tract including
cervix,
uterus, ovaries, choriocarcinoma and gestational trophoblastic disease; male
genital tract including prostate, seminal vesicles, testes and germ cell
tumors;
endocrine glands including thyroid, adrenal, and pituitary; skin including
hemangiomas, melanomas, sarcomas arising from bone or soft tissues and
Kaposi's sarcoma; tumors of the brain, nerves, eyes, and meninges including
astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas, Schwannomas and meningiomas; solid tumors arising from
hematopoietic malignancies such as leukemias and including chloromas,
plasmacytomas, plaques and tumors of mycosis fungoides and cutaneous 'T-
cell lymphoma/leukemia; lymphomas including both Hodgkin's and non-
Hodgkin's lymphomas; prophylaxis of autoimmune diseases including
rheumatoid, immune and degenerative arthritis; ocular diseases including
diabetic retinopathy, retinopathy of prematurity, corneal graft rejection,
retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal
neovascularization due to macular degeneration and hypoxia; abnormal
neovascularization conditions of the eye; skin diseases including psoriasis;
blood vessel diseases including hemagiomas and capillary proliferation within
atherosclerotic plaques; Osler-Webber Syndrome; myocardial angiogenesis;
plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
wound granulation; diseases characterized by excessive or abnormal
stimulation of endothelial cells including intestinal adhesions, Crohn's
disease, atherosclerosis, scleroderma and hypertrophic scars (i.e. keloids)
and diseases which have angiogenesis as a pathologic consequence
including cat scratch disease (Rochele minalia quintosa) and ulcers
(Helicobacter pylori). Another use is as a birth control agent which inhibits
ovulation and establishment of the placenta.
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The modified kringle 5 peptides of the present invention may also be
useful for the prevention of metastases from the tumors described above
either when used alone or in combination with radiotherapy and/or other
chemotherapeutic treatments conventionally administered to patients for
treating angiogenic diseases. For example, when used in the treatment of
solid tumors, the modified kringle 5 peptides of the present invention may be
administered with chemotherapeutic agents such as alpha inteferon, COMP
(cyclophosphamide, vincristine, methotrexate and prednisone), etoposide,
mBACOD (methortrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine and dexamethasone), PRO-MACE/MOPP (prednisone,
methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol,
etoposide/mechlorethamine, vincristine, prednisone and procarbazine),
vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,
platelet
factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-
PG and the like. Other chemotherapeutic agents include alkylating agents
such as nitrogen mustards including mechloethamine, melphan, chlorambucil,
cyclophosphamide and ifosfamide; nitrosoureas including carmustine,
lomustine, semustine and streptozocin; alkyl sulfonates including busulfan;
triazines including dacarbazine; ethyenimines including thiotepa and
hexamethylmelamine; folic acid analogs including methotrexate; pyrimidine
analogues including 5-fluorouracil, cytosine arabinoside; purine analogs
including 6-mercaptopurine and 6-thioguanine; antitumor antibiotics including
actinomycin D; the anthracyclines including doxorubicin, bleomycin,
mitomycin C and methramycin; hormones and hormone antagonists including
tamoxifen and cortiosteroids and miscellaneous agents including cisplatin and
brequinar. For example, a tumor may be treated conventionally with surgery,
radiation or chemotherapy and kringle 5 administration with subsequent
kringle 5 administration to extend the dormancy of micrometastases and to
stabilize and inhibit the growth of any residual primary tumor.
L. The
a derivatives and conjugates of the opioid molecules and analgesic
agents may be used in several different ways and to achieve several different
ends. These materials may be used in place of typical antinociceptive agents
for alleviating pain. As compared with drugs currently available, the
materials
of this invention can alleviate pain without central mediated side effects or
potential of addiction or loss of efficacy, and are available for alleviating
pain
for a substantially longer time than conventionally administered drugs. Opioid
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derivatives and conjugates of this invention also may be utilized (in
accordance with U.S. Patent 5,482,930) as anti-inflammatory and/or anti-
irritation agents or in general to inhibit vascular leakage from tissues. In
addition, as is known in the art, these materials may be used to treat hosts
which are or have become tolerant to morphine (or to treat patients
undergoing methadone treatment programs), as well as treatment of narcotics
withdrawal in general.
M. Therapeutic Uses of Modified Immuno-suppressants
A variety of immuno-suppressant agents such as cyclosporin and
derivatives, corticosteroids, sulfasalazine, thalidomide, methotrexate, OKT3,
peptide-T, or agents that inhibit T-cell activation or adhesion would be
useful
to prior to transplantation to mask immune responsiveness and organ
rejection. Such agents could be applied at the time of tissue harvest (e.g.
heart, lung, liver harvest) or immediately prior to restitution of blood flow
in the
recipient. Such immuno-suppressant agents would prevent the recognition of
foreign antigen from the donor tissue that would facilitate short term
acceptance and facilitate longer term ability for the host to accommodate the
transplanted organ.
N. Therapeutic Uses of Modified Antibiotics:
The modified antibiotics of the invention find use in treating infections.
O. Therapeutic Uses of Modified Antidepressants
The modified antidepressants of the invention are useful for
treating depression.
P. Therapeutic Uses of Modified Anti-
The human immunodeficiency virus (HIV), which is responsible for
acquired immune deficiency syndrome (AIDS), is a member of the lentivirus
family of retroviruses. There are two prevalent types of HIV, HIV-1 and HIV-
2, with various strain of each having been identified. HIV targets CD-4+
cells,
and viral entry depends on binding of the HIV protein gp41 to CD-4+ cell
surface receptors.
Modified anti-viral or anti-fusogenic peptides of the invention may be
used as a therapeutic agent in the treatment of patients who are suffering
from HIV infection, and can be administered to patients according to the
methods described below and other methods known in the art. Effective
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therapeutic dosages of the modified peptides may be determined through
procedures well known by those in the art and will take into consideration any
concerns over potential toxicity of the peptide.
The modified peptides can also be administered prophylactically to
previously uninfected individuals. This can be advantageous in cases where
an individual has been subjected to a high risk of exposure to a virus, as can
occur when individual has been in contact with an infected individual where
there is a high risk of viral transmission. This can be expecially
advantageous
where there is no known cure for the virus, such as the HIV virus. As a
example, prophylactic administration of a modified anti-HIV peptide would be
advantageous in a situation where a health care worker has been exposed to
blood from an HIV-infected individual, or in other situations where an
individual engaged in high-risk activities that potentially expose that
individual
to the HIV virus.
1. SIV and anti-SIV ueptides: Simian immunodeficiency viruses
(SIV) are lentiviruses that cause acquired immunodeficiency syndrome
(AIDS)-like illnesses in susceptible monkeys. Modified anti-viral peptides
according to the invention can be used for the treatment of infected animals
or as a prophylactic in a similar fashion as for HIV.
2. RSV: Respiratory syncytial virus (RSV) is a respiratory
pathogen, especially dangerous in infants and small children where it can
cause bronchiolitis (inflammation of the small air passages) and pneumonia.
RSVs are negative sense, single stranded RNA viruses and are members of
the Paramyxoviridae family of viruses. The route of infection of RSV is
typically through the mucous membranes by the respiratory tract, i.e., nose,
throat, windpipe and bronchi and bronchioles. Antiviral peptides according to
the invention can be used for prevention and treatment of RSV related
diseases.
3. HPV: Human parainfluenza virus (HPIV or HPV), like RSV, is
another leading cause of respiratory tract disease, and like RSVs, are
negative sense, single stranded RNA viruses that are members of the
Paramyxoviridae family of viruses. There are four recognized serotypes of
HPIV -- HPIV-1, HPIV-2, HPIV-3 and HPIV-4. HPIV-1 is the leading cause of
croup in children, and both HPIV-1 and HPIV-2 cause upper and lower
respiratory tract illnesses. HPIV-3 is more often associated with
bronchiolitis
and pneumonia. Antiviral peptides according to the invention can be used for
treatment of HPV related diseases.
4. MeV: Measles virus (MV or MeV) is an enveloped negative,
single-stranded RNA virus belonging to the Paramyxoviridae family of viruses.
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Like RSV and HPV, MeV causes respiratory disease, and also produces an
immuno-suppression responsible for additional, opportunistic infections. In
some cases, MeV can establish infection of the brain leading to severe
neurlogical complications. Antiviral peptides according to the invention can
be used for treatment of RSV related diseases.
Q. Therapeutic Uses of Modified Antihistamine Agents
Modified anthistamine agents find use in treating excess histamine
formed in body tissues including allergic reactions.
R. Therapeutic Uses of Modified Anti-angina Agents
Modified anthi-angina agents find use in treating angina
including treatment of choking and suffocating sensations.
Angina results from insufficient blood supply to the heart, and is often
caused by blockages in the arteries that feed the heart muscle with blood
(coronary artery stenoses due to atherosclerosis). "Unstable" angina
conditions, can develop into acute coronary syndromes (ACS), including
myocardial infarction. Antianginal therapies include treatment with
nitroglycerin and the use of aspirin and heparin.
Platelet activation and aggregation play an important and essential role
in the formation of intracoronary thrombus in acute coronary syndromes
(ACS).. Glycoprotein Ilb/Illa receptor inhibitors are currently used in
connection with heparin and aspirin in ACS. Glycoprotein Ilb/Illa receptor
inhibitors block the final step for platelet aggregation and fibrinogen
binding,
thus preventing thrombus formation. Tirofiban is a potent, synthetic, non-
peptide and specific glycoprotein Ilb/Illa receptor inhibitor and has shown to
be well tolerated and to reduce the risk of ischaemic complications in
patients
with unstable angina, non-Q-wave myocardial infarction and high-risk patients
undergoing revascularisation when used in combination with aspirin and
heparin. Other GP Ilb/Illa receptor inhibitors include abciximab and
eptifibatide.
S. Use of Modified Thyroxine Molecules
Thyroxine, an amino acid of the thyroid gland (Merck Index, 1989,
9348:1483) and thyroxine analogues are well-known in the art. It is well
established in the literature that thyroid hormones, specifically thyroxines
T3
and T4, have two distinct types of biological actions: one on cell metabolism,
the second on cell differentiation and development (Jorgensen, 1978,
"Thyroid Hormones and Analogues II. Structure-Activity Relationships," In:
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Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, C. H. Li, ed.,
Academic Press, New York). For example, thyroxine suppresses uptake of
iodine by the thyroid (Money et al., 1959, "The Effect of Various Thyroxine
Analogues on Suppression of locline-131 Uptake by the Rat Thyroid,"
Endocrinology 64:123-125) and induces cell differentiation as studied by
tadpole metamorphosis (Money et al., 1958, "The Effect of Change in
Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of
Rana Pipiens Tadpoles," Endocrinology 63:20-28). Additionally, thyroxine and
certain thyroxine analogues depress growth of non-malignant mouse pituitary
thyrotropic tumors (Kumaoka et al., 1960, "The Effect of Thyroxine Analogues
on a Transplantable Mouse Pituitary Tumor," Endocrinology 66:32-38;
Grinberg et al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V.
Effect of Various Thyroxine Analogs on Growth and Secretion," Cancer
Research 22:835-841 ).
The structural requirements of thyroxine and thyroxine analogues for
metabolic stimulation and induction of cell differentiation are not identical
(see
Jorgensen, 1978, "Thyroid Hormones and Analogues II. Structure-Activity
Relationships," In: Hormonal Proteins and Peptides, Vol. VI, p. 150, C. H. Li,
ed., Academic Press, New York). For example, Money et al. have found that
there is no correlation between suppression of thyroid iodine uptake and
induction of tadpole metamorphosis (Money et al., 1958, "The Effect of
Change in Chemical Structure of Some Thyroxine Analogues on the
Metamorphosis of Rana Pipiens Tadpoles," Endocrinology 63:20-28). Based
on these observations, it was conceived that as yet unidentified cellular
responses may be altered or induced by certain thyroxine analogues which
do not exhibit either mode of action (metabolic or differentiating) exhibited
by
thyroxine T3 and T4.
Deficiency of thyroid activity, whether occurring spontaneously or
resulting from surgical removal of thyroid gland, thyroiditis, or decreased
function secondary to pituitary degeneration results in clinical
hypothyroidism.
Whatever the cause, the symptom is treated by replacement therapy using
the modified thyroxine molecules of the invention.
The present invention also relates to a method for the treatment of
anemia which is associated with rheumatoid arthritis and of the anemia
present in patients having a viral or bacterial infection wherein symptoms of
rheumatoid arthritis are additionally present using the modified thyroxine
molecules of the invention.
According to the invention, the associated anemia which is characterized as
being moderately hypochromic and normocytic is treated by administering to
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a patient in need of treatment a composition for increasing the thyroxine in
the
blood stream and thereby increasing the ceiling on the number of red cells
maturing from the stem cells in the blood stream. The composition can
include the presence of an anti-inflammatory agent so as to treat the
inflammation present and reduce any pain.
T. Use of Modified Bronchodialators and Anti-Asthmatic A,q~ents
Anti-asthmatic agents find use in the treatment of asthma and other
lung diseases. Such anti-asthmatic agents include bronchodialators like
albuterol (Proventil or Ventolin) and maleimidopropionamyl-1-
theobromineacetamide.
U. Uses of Modified Diaginostic Agients
The diagnostic agent employed and the vascular protein or proteins
targeted will depend upon whether one wishes to diagnostically image the
anatomic compartment over an extended period of time, whether one wishes
to preferentially image only a specific cell type or compartment, or both.
Applications for covalently bonding a diagnostic agent of interest to a long-
lived vascular protein for diagnostic imaging of the vascular space over an
extended period of time are numerous and include enhancing the ability to
detect abnormalities in blood flow throughout the entire mammalian vascular
system, including the detection of internal injury causing abnormal bleeding
or, alternatively, the presence of thromboses. For example, one may wish to
image the vascular space over an extended period of time to detect the
effects of a particular treatment while they occur, i.e., detecting the
disappearance of an embolism, the stoppage of internal bleeding, or the like.
Diagnostically imaging the vascular space over an extended
period of time also allows for the detection of various diseases associated
with the vascular system, i.e., such as arterial blockage in the heart. Thus,
diagnostically imaging the vascular system over an extended period of time
may be employed to non-invasively detect a consistently reduced blood flow
to the heart. Such a method also provides a means for quantitatively
measuring cardiac efficiency and ventricular output volume over an extended
period of time, i.e., during extended periods of exercise, or the like.
Other applications for such a method arise from the ability to non-
invasively visualize anatomical structures of the mammalian vascular system
and the effects on those anatomical structures over time of the administration
of various drugs, such as vasodilators, vasoconstrictors, or the like. Such
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may allow for the early detection of developmental vascular abnormalities,
injuries, or the like.
Additional applications arising from the ability to diagnostically
image the vascular space over an extended period of time include functional
assessment of the cardiovascular system as routinely utilized in nuclear
medicine for single measurements.
Applications for preferentially bonding a diagnostic agent of
interest to a specific protein or proteins present in the vascular system so
as
to diagnostically image only a specific cell type or compartment are also
numerous. For example, having the ability to preferentially direct a
diagnostic
agent of interest to a specific cell type in the vascular system can allow for
the
non-invasive and early detection of lesions or various tumors associated with
the mammalian vascular system by directing the bifunctional anchor molecule
to a tumor specific cell surface protein.
Additionally, diagnostic agents can be directed to cell surface
proteins of specific cell types predominantly associated with specific
anatomic
compartments, allowing one to preferentially diagnostically image such
compartments as lymph nodes, Peyer's patches, kidney glomeruli, liver,
pancreas, tonsil, or any other organ to which mobile cells in the vasculature
will migrate.
Other applications for preferentially diagnostic imaging a specific
cell type or compartment of the vascular system include diagnosis and
treatment of stenosis or plaque, vascular shunt reendothelialization or shunt
failure due to tissue growths, or organ rejection due to tissue migration.
The diagnostic agents of the invetion may be delivered to a local site
via a local delivery device. Delivery devices include catheters, syringes,
trocars and endoscopes. Delivery of the agent to a local site allows imaging
of the specific area of delivery. The agents that find particular use in
localized
delivery are the non-specific diagnostic agents such as NHS-derivatives.
The invention can be more clearly illustrated by the following non-
limiting examples.
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Example 1
Preparation of Modified RGD Peptide AGYKPEGKRGDAK
RGD peptide AGYKPEGKRGDAK (SEQ ID N0:1) was synthesized
and modified to include a linking group and a maleimide group according to
the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Pro-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of
the Fmoc protecting group was achieved using a solution of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). In last
elongation step, the synthesis was then re-automated for the addition of the
3-maleimidopropionic acid (Step 2). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 3). The product was purified by preparative reversed phased
HPLC using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B))
over 180 min at 9.5 mL/min using a Phenomenex Luna 10 N phenyl-hexyl, 21
mm x 25 cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and
254 nm to afford the desired molecule in >95% purity, as determined by RP-
HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
2HN-AGYKPEGKRGDAK
Step 2 3-maleimidopropionic acid
0~ ~ 0J~ ~
/,N' v 'NH-AGYKPEGKRGDAK-[,_,;3)
'n~
O
Step 3 85% TFA/5% TIS/5% thioanisole/5% phenol
O O
N~NH-AGYKPEGKRGDAKNH 2
O
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Example 2
Preparation of Modified RGD Peptide KRGDACEGDSGGPFC
RGD peptide KRGDACEGDSGGPFC (SEQ ID N0:2) was synthesized
and modified to include a linking group and a maleimide group according to
the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 pmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH, Fmoc-
Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-
OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C), Fmoc-Ala-.
OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-
OH They were dissolved in N,N-dimethylformamide (DMF) and, according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). C
are cyclized cysteine. The cyclisation was achieved by ,cyclization by
treatment with TI(TFA)3 (3 equiv. on 175 ummol scale) when the coupling was
paused at last lysine residue (step 2). After cyclization, In last elongation
step,
the synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 4). The product was purified by preparative reversed phased
HPLC using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B))
over 180 min at 9.5 mL/min using a Phenomenex Luna 10 N phenyl-hexyl, 21
mm x 25 cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and
254 nm to afford the desired protein in >95% purity, as determined by RP-
HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
Fmoc-KRGDAC*EGDSGGPFC*
Step 2
i
Fmoc-KRGDA *EGDSGGPFC* a:~
Step 3 a)20% piperidine in DMF
~ b) 3-maleimidopropionic acid
O
N NH -KRGDAC EGDSGGPFC
O
Step 4 I 85% TFA/5% TIS/5% thioanisole/5% phenol
O O
N~NH -KRGDAC*EGDSGGPFC*-CONH 2
\ O
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Example 3
Preparation of Modified RGD Peptide KRGDACEGDSFFPFC
RGD peptide KRGDACEGDSFFPFC (SEQ ID N0:3) was synthesized
and modified to include a linking group and a maleimide group according to
the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 pmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH, Fmoc-
Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-
OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C), Fmoc-Ala-
OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-
OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1 ). C are cyclized cysteine.
The cyclisation was achieved by, cyclization by treatment with TI(TFA)3 (3
equiv. on 175 ummol scale) when the coupling was paused at last lysine
residue (step 2). After cyclization, In last elongation step, the synthesis
was
then re-automated for the addition of the linking group s and the 3-
maleimidopropionic acid (Step 3 ). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
EtzO (Step 4). The product was purified by preparative reversed phased
HPLC using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B))
over 180 min at 9.5 mUmin using a Phenomenex Luna 10 N phenyl-hexyl, 21
mm x 25 cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and
254 nm to afford the desired peptidein >95% purity, as determined by RP-
HPLC.
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Fmoc-Ramage Resin
Step 1 SP PS
Fmoc-KRGDAC*EGDSGGPFC*-
Step 2
Fmoc-KRGDA *EGDSGGPF
a) 20% piperidine in DMF
Step 3 b) Fmoc-AEEA-OH (1 )
c) Fmoc-AEEA-OH (2)
c) 3-maleimidopropionic acid
O O
N~NH-(AEEA)2-KRGDAC*EGDSGGPFC~-- -~;
O
Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol
O O
N~ NH-(AEEA)2-KRGDAC*EGDSGGPFC*
O
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Example 4
Preparation of Modified GP-41A peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
GP-41A peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
(SEQ ID N0:4) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-
OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-
Ser(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH. They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence, activated
using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of
the Fmoc protecting group was achieved using a solution of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). In the last
elongation step, the synthesis was automated for the addition of the 3-
maleimidopropionic acid (Step 2). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 3). The product was purified by preparative reversed phased
HPLC using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B))
over 180 min at 9.5 mL/min using a Phenomenex Luna 10 N phenyl-hexyl, 21
mm x 25 cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and
254 nm to afford the desired molecule in >95% purity, as determined by RP-
HPLC.
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Fmoc-Ramage Resin
Step 1 I SPPS
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-
Step 2 ~ 3-maleimidopropionic acid
O O
N~ NH -YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW
O
Step 3 g5% TFA/5% TIS/5% thioanisole/5% pheno
O O
N~NH-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFCONI-~
O
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Example 5
Preparation of Modified GP-41 B peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
GP-41 B peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
(SEQ ID N0:5) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH, Fmoc
Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)
OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-
OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,
Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH.
They were dissolved in N,N-dimethylformamide (DMF) and, according
to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The
selective deprotection of the Lys (Aloc) group is performed manually and
accomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4
dissolved in 5 mL of C6H6 CHC13 (1:1) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHC13 (6 x 5 mL), 20% AcOH in
DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
In the last elongation step, the synthesis was automated for the
addition of the 3-maleimidopropionic acid (Step 3). Between every coupling,
the resin was washed 3 times with N,N-dimethylformamide (DMF) and 3
times with isopropanol. The peptide was cleaved from the resin using 85%
TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-
ice cold Et20 (Step 4). The product was purified by preparative reversed
phased HPLC using a Varian (Rainin) preparative binary HPLC system:
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gradient elution of 30-55% B (0.045% TFA in HZO (A) and 0.045% TFA in
CH3CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10 N
phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD
II) at ~, 214 and 254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC.
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Fmoc-Ramage Resin
Step 1 SP PS
spa
YTSLIHSLIEESQNQQEK NEQELLELDK (Aloc)WASLWNWF-
Step 2 IPd(PPh3)4/NMM/HOAc/CHCI 3:C6H6
NH2
YTSLIHSLIEESQNQQEK NEQELLELDNH CO-WASLWNWF - ~'~
Step 3 3-maleimidopropionic acid
O
O
NH~N
O
YTSLIHSLIEESQNQQEK NEQELLELDNH CO-WASLWNWF - ~ry
Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol
O O
NH~ N
O
YTSLIHSLIEESQNQQEK NEQELLELDNH CO-WASLWNWFCONH 2
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Example 6
Preparation of Modified GP-41C peptide
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
GP-41C peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
(SEQ ID N0:6) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-
Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-
OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,
Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1).
The selective deprotection of the Lys (Aloc) group is performed
manually and accomplished by treating the resin with a solution of 3 eq of
Pd(PPh3)4 dissolved in 5 mL of C6H6 CHC13 (1:1) : 2.5% NMM (v:v): 5% AcOH
(v:v) for 2 h (Step 2). The resin is then washed with CHC13 (6 x 5 mL), 20%
AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
In the last elongation step, the synthesis was automated for the
addition of the Fmoc-AEEA-OH and finally 3-maleimidopropionic acid (Step
2). Between every coupling, the resin was washed 3 times with N,N-
dimethylformamide (DMF) and 3 times with isopropanol. The peptide was
cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%
phenol, followed by precipitation by dry-ice cold Et20 (Step 3). The product
was purified by preparative reversed phased HPLC using a Varian (Rainin)
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preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA
in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mUmin using
a Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25 cm column and UV
detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to afford the desired
molecule in >95% purity, as determined by RP-HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
YTSLIHSLIEESQNQQEK Aloc)NEQELLELDKWASLWNW~ ~~
Step 2 IPd(PPh3)4/NMM/HOAc/CHC13:C6H6
NH2
YTSLIHSLIEESQNQQEN NEQELLELDKWASLWNWF-
Step 3 3-maleimidopropionic acid
O O
HN~N
O
YTSLIHSLIEESQNQQEN NEQELLELDKWASLWNWF- -
Step 4 85% TFA/5% TIS/5% thioanisole/5% pheno
O O
HN~ N
O
YTSLIHSLIEESQNQQEN NEQELLELDKWASLWNWFCONH2
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Example 7
Preparation of Modified RSV peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ
ID N0:7) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale is performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Rink Amide MBHA. The following protected amino acids are
sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH,
Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-
Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-
OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-
Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according
to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group is achieved using a solution of 20%
(V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).
The amino group of the final amino acid is acetylated using Acetic Acid
activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The
selective deprotection of the Lys (Aloc) group is performed manually and
accomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4
dissolved in 5 mL of CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin
is then washed with CHC13 (6 x 5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6
x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the
addition of the 3-maleimidopropionic acid (Step 3).
Between every coupling, the resin is washed 3 times with N,N-
dimethylformamide (DMF) and 3 times with isopropanol. The peptide is
cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%
phenol, followed by precipitation by dry-ice cold Et20 (Step 4). The product
is
purified by preparative reversed phased HPLC using a Varian (Rainin)
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preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA
in H20 (A) and 0.045% TFA in CH3CN (B) over 180 min at 9.5 mL/min using a
Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25 cm column and UV
detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to afford the desired
molecule in >95% purity, as determined by RP-HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV-Aloc) -
Lys
Step 2 IPd(PPh3)4/NMM/HOAc/CHC3:C6H6
i I
Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV -Lys -PS
Step 3 3-maleimidopropionic acid
OII O
HN~N
O
Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV-HN
Step 4 I 85% TFA/5% TIS/5% thioanisole/5% phenol
YPSDEYDASISQVNEEINQALAYIRkADELLENV
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Example 8
Preparation of Modified RSV peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ
ID N0:8) was synthesized and modified to include a linking group and a
maleimide group to produce the modified peptide depicted below.
Solid phase peptide synthesis on a 100 Nmole scale is performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Rink Amide MBHA. The following protected amino acids are
sequentially added to resin: Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-
Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,
according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group is achieved using a solution of
20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step
1). The amino group of the final amino acid is acetylated using Acetic Acid,
activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The selective
deprotection of the Lys (Aloc) group is performed manually and accomplished
by treating the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in 5 mL
of
CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with
CHC13 (6 x 5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF
(6 x 5 mL). The synthesis is then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin is
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide is cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
EtzO (Step 4). The product is purified by preparative reversed phased HPLC
using a Varian (Rainin) preparative binary HPLC system: gradient elution of
30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B) over 180
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min at 9.5 mUmin using a Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25
cm column and UV detector (Varian Dynamax UVD II) at x,214 and 254 nm to
afford the desired molecule in >95% purity, as determined by RP-HPLC.
O
l~H i -NH-VYPSDEYDASISQVNEEINQALAYIRKADELLENVCONH2
O
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Example 9
Preparation of Modified RSV peptide
VYPSDEYDASISQVNEEINQALAYIRKADELLENV
RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ
ID N0:9) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale is performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Rink Amide MBHA. The following protected amino acids are
sequentially added to resin: Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, '
Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH. They
are dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).
Removal of the Fmoc protecting group is achieved using a solution of 20%
(V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The
amino group of the final amino acid is acetylated using Acetic Acid activated
using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).The selective
deprotection of the Lys (Aloc) group is performed manually and accomplished
by treating the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in 5 mL
of
CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with
CHC13 (6 x 5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF
(6 x 5 mL). The synthesis is then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin is
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide is cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 4). The product is purified by preparative reversed phased HPLC
using a Varian (Rainin) preparative binary HPLC system: gradient elution of
30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B) over 180
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min at 9.5 mUmin using a Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25
cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to
afford the desired molecule in >95% purity, as determined by RP-HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
Ac-VYPSDEYDASISQVNEEINQALAYIRK(Aloc~qpELLENV-PS
Step 2 I Pd(PPh3)4/NMM/HOAc/CHC~:CsHs
Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV
Step 3 3-maleimidopropionic acid
HNr v 'N
0
Ac-VYPSDEYDASISQVNEEINQALAYIR-HN~ -ADELLENV ~'
n
Step 4 ~ 85% TFA/5% TIS/5% thioanisole/5% phenol
HN
Ac-VYPSDEYDASISQVNEEINQALAYIR- HN'~ -NH-ADELLENV-COzNH
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Example 10
Preparation of Modified GLP-1 peptide
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK
GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID
NO:10) was synthesized and modified to include a linking group and a
maleimide group according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-
Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-
OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-
Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-His(Boc)-OH, The following
protected amino acids were sequentially added to resin: They were dissolved
in N,N-dimethylformamide (DMF) and, according to the sequence, activated
using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of
the Fmoc protecting group was achieved using a solution of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The
selective deprotection of the Lys (Aloc) group is performed manually and
accomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4
dissolved in 5 mL of C6H6 CHC13 (1:1 ) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2
h (Step 2). The resin is then washed with CHC13 (6 x 5 mL), 20% AcOH in
DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).The synthesis was
then re-automated for the addition of the 3-maleimidopropionic acid (Step 3).
Between every coupling, the resin was washed 3 times with N,N-
dimethylformamide (DMF) and 3 times with isopropanol. The peptide was
cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%
phenol, followed by precipitation by dry-ice cold Et20 (Step 4). The product
was purified by preparative reversed phased HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA
in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mL/min using
a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm column and UV
detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to afford the desired
molecule in >95% purity, as determined by RP-HPLC.
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Ramag resin
Step 1 SP PS
Boc -HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK(Aloe -
Step 2 Pd(PPh3)4 /NMM/AcOH/Benzene/DMF
NH2
H
Boc-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR .N N
H O
Step 3 3-maleimidoproprionic acid
O O
HN~ N
O
H
Boc-HAEGTFTSDVSSYLEGQAA KEFIAWLVKGR ~ N N-
H O
Step 4 85% TFA/5% TIS/5% thioanisol/5% phenol
O O
HN~ N
O
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR ~N NH2
H O
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Example 11
Preparation of Modified GLP-1 peptide
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK
GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID
N0:1 1 ) was synthesized and modified to include a linking group and a
maleimide group, as described below.
Solid phase peptide synthesis on a 100 pmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-
OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Aloc)-OH,
Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-
Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-His(Boc)-OH, The following protected
amino acids were sequentially added to resin: They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection
of the Lys (Aloc) group is performed manually and accomplished by treating
the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in 5 mL of CsHs CHC13
(1:1 ) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h (Step 2). The resin is then
washed with CHC13 (6 x 5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5
mL), and DMF (6 x 5 mL). The linking group Fmoc-AEEA-OH was added and
then the Fmoc was removed in the usual fashon. This procedure was redone
to add a second AEEA linking group.
The synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 4).
The product was purified by preparative reversed phased HPLC using
a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55%
B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at
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9.5 mUmin using a Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to
afford the desired molecule in >95% purity, as determined by RP-HPLC.
Example 12
Preparation of Modified K5 peptide PRKLYDYK
K5 peptide PRKLYDYK (SEQ ID N0:12) was synthesized and
modified to include a linking group and a maleimide group according to the
synthesis scheme set forth below.
Using automated peptide synthesis, the following protected amino
acids were sequentially added to Rink Amide MBHA resin (0.48 mmol/mg)
(250 ~mol scale): Fmoc-Lys(Aloc)OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(tBu)-OH,
Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH. Each coupling was accomplished using 2 equivalents of amino
acid, 1 equivalent HBTU, and 2 equivalents DIEA and performed twice for 30
min. The Fmoc group of the N-terminal amino acid (Pro) was removed using
20% piperidine/DMF (3 x 10min).
The resin was subsequently washed with 6 x 4 mL DMF, 3 x 3 mL
EtOH and 6 x 4 mL DMF. Acetylation of the N-terminus was accomplished
manually on the Symphony by adding 4 mL of 15 equivalents HOAc, 2
equivalents DIEA and 4 equivalents HBTU in DMF. Acetic capping was
performed 2 x 30 min. The resin was subsequently washed with 3 x 4 mL
CHZCI2, 6 x 4 mL 0.5% DIEA/CHZC12, 3 x 4 mL EtOH and 6 x 4 mL
DMF.Selective deprotection of the Lys(Aloc) group was performed manually .
on the Symphony by treating the resin with a solution of 3 equivalents of
Pd(PPh3)4 dissolved in 5 mL of CHCI3:Benzene (1:1) with 2.5% NMM (v/v) and
5% HOAc for 2 h. The resin was then washed with CHC13 (6 x 5 mL), 0.5%
DIEA in CH2C12 (6 x 5 mL), 0.02 M sodium diethylthiocarbamate in DMF (6 x 5
mL), EtOH (3 x 4 mL) and DMF (6 x 5 mL).
Coupling of 3-maleimidoproprionic acid (MPA) was performed by
resuming automation on the Symphony, which involves delivery of 2
equivalents of MPA, 2 equivalents DIEA and 1 equivalent HBTU to the
reaction vessel. The coupling was carried out twice at 30 min. Washing was
conducted using 6 x 4 mL DMF, 3 x 3 mL EtOH and 6 x 4 mL DMF. Cleavage
from the resin was performed by automation using 10 mL of the following
cleavage mixture: 85% TFA/5% triisopropyl silane/5% thioanisol/5% phenol.
After the peptide was cleaved from the resin for 2 hrs, the resin was washed
with TFA and CH2C12. The combined cleavage and washing liquors
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concentrated to 1-2 mL using a rotovap with mild heating (30 °C) and
the
peptide was precipitated with EtzO. The precipitate was collected by
filtration
using a SPPS manifold and washed with 10 mL of ethyl acetate and 30 mL of
Et20. The precipitate was subsequently dissolved in 10 mL of water
containing 5% acetonitrile (0.04% TFA) in water (0.04% TFA) for
chromatographic purification.
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Tricyclic amide resin or Fmoc-MBHA
Step I SPPS
NHzPRKLYDYK(Aloc~ ;~
Step 2 AcOH/NMM/HBtU/DMF
Ac-PRKLYDYK(~lloc~ «-
Step 3 Pd(PPh~4/NMM/AcOH/Benzene/DMF
NH2
Ac-PRKLY DYE N
H O
Step 4 3-maleimidoproprionic acid/NMM/HBTU
'I O O
HN~ N
O
Ac-PRKLYDY~N
H O
Step 5 85% TFA/5% TIS/S% thioanisol/5%
O O
HN~ N
O
Ac-PRKLYDY~N NH2
H O
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Example 13
Preparation of Modified K5 peptide RNPDGDVGGPWAWTTAPRKLYDY
K5 peptide RNPDGDVGGPWAWTTAPRKLYDY (SEQ ID N0:13) was
synthesized and modified to include a linking group and a maleimide group
according to the synthesis scheme set forth below.
Using automated peptide synthesis, the following protected amino
acids were sequentially added to Rink Amide MBHA resin (0.48 mmol/mg)
(100 pmol scale): Fmoc-Tyr(tBu)OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,
Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(tBu)-OH, Fmoc-
Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, MPA.
Each coupling was accomplished using 5 equivalents of amino acid, 1
equivalent HBTU, and 2 equivalents DIEA and performed twice for 30 min.
Cleavage from the resin was performed by automation using 10 mL of the
following cleavage mixture: 85% TFA/5% triisopropyl silane/5% thioanisol/5%
phenol. After the peptide was cleaved from the resin for 2 hrs, the resin was
washed with TFA and CHZC12.
The combined cleavage and washing liquors concentrated to 1-2 mL
using a rotovap with mild heating (30 °C) and the peptide was
precipitated .
with Et20. The precipitate was collected by filtration using a SPPS manifold
and washed with 10 mL of ethyl acetate and 30 mL of Et20. The precipitate
was subsequently dissolved in 10 mL of water containing 5% acetonitrile
(0.04% TFA) in water (0.04% TFA) for chromatographic purification.
Purification of all the peptides was performed using a Phenomenex Luna 10 N
phenyl-hexyl, 21 mm x 250 mm column equilibrated with a water/TFA mixture
(0.045% TFA in H20; Solvent A).
Elution was achieved at 18 mL./min by running a 10-30% acetonitrile
gradient over 60 min (0.045% TFA in CH3CN; Solvent B). Peptides were
detected by UV absorbance (Varian Dynamax UVD II) at 214 and 254 nm.
Fractions were collected in 9 mL aliquots. Fractions containing the desired
product were identified by mass after direct injection onto LC/MS. The
selected fractions were subsequently analyzed by analytical HPLC (10-40%
solvent B over 20 min; Phenomenex Luna 5 N phenyl-hexyl, 10 mm x 250 mm
column, 0.5 mUmin) to identify fractions with >_ 95% purity for pooling. The
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pool was freeze-dried using dry ice and acetone and subsequently lyophilized
for at least 2 days to yield a white powder.
Ramag resin
Step 1 SPPS
2HN-RNPDGDVGGPWAWTTAPRKLYDY-
Step 2 3-maleimidoproprionic
acid
O O
N.~ H. RNPDGDVGGPWAYTTAPRKLYDI!
O ,
Step 3 85% TFA/5% TIS/5% thioanisol/5%
phenol
O O
N~ N' RNPDGDVGGPWAYTTAPRKLYDY-CONH2
H
O
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Example 14
Preparation of Modified BBB peptide YGRKKRRQRRRL
BBB peptide YGRKKRRQRRRL (SEQ ID N0:14) was synthesized and
modified to include a linking group and a maleimide group according to the
synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1). After the tyrosine
deprotection, Biotin was anchored at the N-terminus via regular activation and
coupling conditions. The selective deprotection of the Lys (Aloc) group is
performed manually and accomplished by treating the resin with a solution of
3 eq of Pd(PPh3)4 dissolved in 5 mL of C6H6 CHC13 (1:1) : 2.5% NMM (v:v):
5% AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHC13 (6 x 5
mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5
mL).The synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin was
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide was cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 4). The product was purified by preparative reversed phased
HPLC using a Varian (Rainin) preparative binary HPLC system: gradient
elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B))
over 180 min at 9.5 mlJmin using a Phenomenex Luna 10 N phenyl-hexyl, 21
mm x 25 cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and
254 nm to afford the desired molecule in >95% purity, as determined by RP-
HPLC.
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Fmoc-Ramage Resin
Step 1 SPPS
YGRKKRRQRRRLys(Aloc)
Step 2 ~ Biotin
Biotin-NH-YGRKKRRQRRRLys(Aloc
Step 3 Pd(PPh~4/NMM/HOAc/CHC13:C~-16
NH2
Biotin-NH-YGRKKRRQRRR-HN
U
Step 4 I 3-maleimidopropionic acid
Biotin-NH-YGRKKRRQRRR-
O O
HN~N
O
H
Step 5 85% TFA/5% TIS/5% thioanisole/5% pheno
v
O OII O
HN~ H HN~N
O
S
NH-YGRKKRRQRRR~-IN NH2
U
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Example 15
Preparation of Modified BBB peptide YGRKKRRQRRRL
BBB peptide YGRKKRRQRRRL (SEQ ID N0:15) was synthesized and
modified to include a linking group and a maleimide group according to the
synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 Nmole scale was performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids were
sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection
of the Lys (Aloc) group is performed manually and accomplished by treating
the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in 5 mL of C6H6 CHC13
(1:1 ) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h (Step 2). The resin is then
washed with CHC13 (6 x 5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5
mL), and DMF (6 x 5 mL). After the aloc deprotection, Biotin was anchored at
the s-N terminal of the deprotected lysine via regular activation and coupling
conditions. The Fmoc removal of the N-terminus was then achieved with
standard conditions. The synthesis was then re-automated for the addition of
the 3-maleimid~propionic acid at the end terminus (Step 3). Between every
coupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)
and 3 times with isopropanol. The peptide was cleaved from the resin using
85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by
dry-ice cold Et20 (Step 4). The product was purified by preparative reversed
phased HPLC using a Varian (Rainin) preparative binary HPLC system:
gradient elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in
CH3CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10 N
phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD
II) at ~, 214 and 254 nm to afford the desired molecule in >95% purity, as
determined by RP-HPLC.
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Example 16
Preparation of Modified dyrnorphin peptide YGGFLRRIRPKLK
Dynorphin peptide YGGFLRRIRPKLK (SEQ ID N0:16) was
synthesized and modified to include a linking group and a maleimide group
according to the synthesis scheme set forth below.
Solid phase peptide synthesis on a 100 pmole scale is performed
using manual solid-phase synthesis, a Symphony Peptide Synthesizer and
Fmoc protected Ramage Resin. The following protected amino acids are
sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-
OH, Fmoc-Gly-OH, Boc-Tyr(tBu)-OH. They are dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting
group is achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes (step 1). The amino group of the
final amino acid is acetylated using Acetic Acid activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA).The selective deprotection of the
Lys (Aloc) group is performed manually and accomplished by treating the
resin with a solution of 3 eq of Pd(PPh3)4 dissolved in 5 mL of
CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with
CHC13 (6 x 5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF
(6 x 5 mL). The synthesis is then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Between every coupling, the resin is
washed 3 times with N,N-dimethylformamide (DMF) and 3 times with
isopropanol. The peptide is cleaved from the resin using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold
Et20 (Step 4). The product is purified by preparative reversed phased HPLC
using a Varian (Rainin) preparative binary HPLC system: gradient elution of
30-55% B (0.045% TFA in H20 (A) and 0.045% TFA in CH3CN (B) over 180
min at 9.5 mL/min using a Phenomenex Luna 10 N phenyl-hexyl, 21 mm x 25
cm column and UV detector (Varian Dynamax UVD II) at ~, 214 and 254 nm to
afford the desired molecule in >95% purity, as determined by RP-HPLC.
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Example 17
2-(2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyrl]ethoxy]-
maleimido~ropionyrlacetamide. i(Modified Cetirizine]
A mixture of 1-[(4-chlorophenylmethyl]-piperazine 1, methyl (2-
chloroethoxy)-acetate 2 and sodium carbonate in anydrous xylene is heated
under reflux with good stirring as indicated in the schematic below. The
reaction mixture is then cooled and filtered and the solid is washed with
benzene, the washed solid being discarded. The filtrate is evaporated to
dryness and the evaporation residue is purified by chromatography on a
column of silica (eluent: chloroform:methanol 97:3 v/v). This generated methyl
2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]-acetate 3. The
compound is dissolved in of absolute ethanol. 1 N ethanolic solution of
potassium hydroxide is then added thereto and the reaction mixture is heated
under reflux for 4 hours. It is cooled and the precipitate removed by
filitration,
after washing with diethyl ether.~The filtrate is evaporated to dryness and
the
evaporation residue is triturated with diethyl ether and left to crystallize.
The
compound. potassium 2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-
piperazinyl]ethoxy]-acetate is then obtained. The potassium salt is dissolved
in water and adjusted with 10% hydrochloric acid to a pH of 4. The solution is
extracted with chloroform and the organic phase is dried over anhydrous
magnesium sulfate, whereafter it is evaporated to dryness. The evaporation
residue is triturated with diethyl ether and left to crystallize to produce 2-
[2-[4-
[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-acetic acid 4. 2-[2-[4-[(4-
chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-acetic acid 4 is then placed
in DMF and activated with and O-(benzotriazol-1-yl)-N,N',N',N',-
tetramethyluronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). To the reaction mixture is added 3-maleimidopropylamine. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, triturated with cold ether and left to
crystallize.
This last step generated 5 2-[2-[4-[(4-chloropheny)phenylmethyl[-1-
piperazinyl]ethoxy]-maleimidopropionylacetamide, a modified antihistamine
molecule.
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CI C
uNH CI~~~OEt ~ i ~~O OEt
a
i i
NazC03, Xylene ~ 3
KOH, EtOH
O
CI ~ O \ H NON \ C ~ OI~
i ~~0~ ~ ~ i ~~O~OH
U ~ U
HBTU DIEA,
CHZCIz:DMF ~
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Example 18
11-(N-maleimidouropionyl-4-piperidylideneJl-8-chloro-6.11-dihyrdro-5H-
benzo-[5,6]-c lrclohepta-(1,2-b~-pyridine i(Modified LoratidineL
11-(N-8-chloro-4-piperidylidene)-6,11-dihydro-5H-benzo-[5,6]-
cyclohepta-[1,2 -b]-pyridine 1 is placed is placed in DMF and activated with
and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as indicated
in the schematic below. To the reaction mixture is added 3-
maleimidopropionic acid. The reaction is stirred for 3 hours. The organic
phase is then washed with water and brine, dried over MgS04,
chromatographied triturated with cold ether and left to crystallize to
generate
2, 11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6,11-dihydro-5H-
benzo-[5,6]-cyclohepta-[1,2-b]-pyridine, a modified antihistamine molecule.
O
HON O
O
J
C HBTU, DIEA,
CH2CI2:DMF
1 2
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Example 19
Modified Tirofiban
A four-neck round bottom flask equipped with a mechanical stirrer,
condenser, nitrogen inlet, HCI trap, heating unit and a thermometer probe is
purged with nitrogen overnight and then charged with L-tyrosine 1, CH3CN,
N,O-bis-trimethylsilyl-trifluoromethyl-acetamide. The suspension is heated to
gentle reflux for 2 h. The resulting clear solution O,O'-bis-trimethylsilyl-
(L)-
tyrosine 2, is cooled pyridine and n-BuS02Cl are slowly added over 30
minutes as indicated in the schematic below. The reaction mixture is then
stirred at room temperature. Almost all the solvent is removed in a batch
concentrator, and the resulting oily residue is treated with 15%/KHS04 and
stirred vigorously for 1 hour. The mixture is extracted with i-propyl acetate.
The combined organic layer is treated with Ecosorb TM S-402 and stirred at
room temperature overnight. Ecosorb TM is removed by filtration and the filter
cake is washed with i-propyl acetate. The filtrate is evaporated to dryness
and
the resulting yellow oil is dissolved in hot EtOAc. Hexane is added slowly to
the stirring solution and the resulting slurry is stirred at room temperature
overnight. The solid is collected by filtration and the filter cake is washed
with
EtOAc/hexane. After drying under vacuum is obtained as a white solid.
To a four-neck round bottom flask equipped with a mechanical stirrer,
condenser, nitrogen inlet and a thermometer probe is charged N-n-
butanesulfonyl-(L)-tyrosine 3, 4-(4-pyridinyl)-butyl chloride.HCl 4 and DMSO.
With vigorous stirring, 3 N aq. KOH is added over 15 min.
The temperature is maintained in the 30-40°C range for this
operation
using cooling water. Potassium iodide is added, and the mixture is heated for
36h. After cooling to room temperature, the mixture is diluted with 0.25 N
NaOH and extracted once with t-butyl methyl ether. The aqueous layer is
treated with Ecosorb S-402 and Nuchar SA and the resulting mixture is
mechanically stirred for 1 h. The mixture is filtered through a coarse-
porosity
sintered funnel and the filtered cake is washed with water. The combined
filtrate is placed in a vessel equipped with a pH meter probe and a
mechanical stirrer. With vigorous stirring, NaCI is added, stirred for 30 min,
and then 50% aq. acetic acid wash added until pH 4.80, and stirring
continued for 2-3 h. The resulting slurry is filtered through a coarse-
porosity
sintered funnel, and the cake is washed with water. The crude product is
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dried under house-vacuum under a positive nitrogen pressure to give beige
solid 5 having a wt % purity of 95%.
Selective hydrogenation of the pyridine ring to piperidine ring is
accomplished by using 5 wt% of 10% Pd/C in AcOH at 60C to give the target
product cleanly without reduction of the phenolic ring. Filtration of the
reaction
mixture, evaporation of acetic acid followed by crystallizing the product 6
from 6% AcOH/water.
To a RB flask equipped with a thermometer probe and addition funnel
is charged the crude and 0.25 N NaOH. After complete dissolution, the
solution is cooled to room temperature, and adjusted to pH 7 by slow additon
of 1 N HCI .The solution is further brought down to pH 5.5 by slow addition of
0.5 N HCI. Stirring is continued for 1 h, then the slurry is filtered through
a
coarse funnel padded with a sheet of shark-skin paper and a polypropylene
pad (10 mu m) and the cake is washed with water. The solid is dried under
house vacuum with nitrogen sweep to give a beige solid. The compound is
then placed in DMF and activated with and O-(benzotriazol-1-yl)-N,N',N',N',-
tetramethyluronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). To the reaction mixture is added 3-maleimidopropylamine. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, triturated with cold ether and left to
crystallize to
generate the modifed tirofiban 7. Tirofiban is an anti-angina agent.
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0 0
HO NH2 N,O bis-trimethylsilyltrifluoro NH
methylacetonide (Me)~i-O
O H Acetonitrile I
O-Si(Me)3
2
~SO~I
Pyridine
O H CI
HO N'S~ ~ ~ - O H
I O ~.~ N' 4 HO N'SO~./~
O ~ KOH, KI, I
DMSO:H~ ~ OH
_ G5
3
5wt
10% Pd/C
AcOH
V
HO O N-~~ O /'~O O H
H ~ NON N-g~
r~,NH O O H O
I ~NH
O
HBTU, DIE4, ~ O
CH~12DMF
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Example 20
N-~(1 ~(SJ~-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-
prolinylmaleimidopropionilamide ~(modifed-EnalaprilZ
Ethyl 2-oxo-4-phenylbutyrate 1 and L-alanyl-L-proline 2 are dissolved
in a 1:1 ethanol-water solvent as indicated in the schematic below. A solution
of sodium cyanoborohydride in ethanol-water is added dropwise at room
temperature over the course of two hours. When reaction is complete, the
product is absorbed on strong acid ion-exchange resin and eluted with 2%
pyridine in water. The product-rich cuts are freeze dried to give crude N-(1-
ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline 4 and the compound is
purified by chromatography to yield the desired isomer. The compound 4 is
then placed in DMF and activated with and O-(benzotriazol-1-yl)-N,N',N',N',-
tetramethyluronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). To the reaction mixture is added 3-maleimidopropylamine. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, triturated with cold ether and left to
crystallize to
produce 5 N-(1 (S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-
prolinylmaleimidopropionilamide, a modified anti-hypertensive agent.
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O
Et0 O O OH H
O HZN~N
D
NaH(CN)~ MeOH
\ /
4
0
N
H 2N'~
O
HBTU, DIEA,
CHZCI2DMF
O
Et0 N O HEN
O O
O
\ ~ 5
Example 21
Maleimidopropyrnamyl- E-I(3,4,5-trimethoxybenz-amido)-caproicamide
Modified-Capobenic AcidJi
3,4,5-trimethoxybenzoyl chloride 1 is added along with amino-hexanoic
acid 2 in a solution of 1 N NaOH as indicated in the schematic below. The
resulting solution is preferably treated with char to decolorize it, the char
is
filtered, and the filtrate neutralized with dilute HCI to Congo red indicator
end-
point. The resulting precipitate is separated by filtration washed with water,
dried, then recrystallized from ethanol to genarate 3. The compound is then
placed in DMF and activated with and O-(benzotriazol-1-yl)-N,N',N',N',-
tetramethyluronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). To the reaction mixture is added 3-maleimidopropylamine. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, triturated with cold ether and left to
crystallize in
order to produce 4 Maleimidopropynamyl- s-(3,4,5-trimethoxybenz-amido)-
caproicamide, an anti-arrhthymetic agent
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O
Me0 I ~ CI H~~OH D Me0 ~ H~OH
Me0 OMe NaOH, H~J Me0 OMe
3
0
N
H ~I'~
O
HBTU, DIEA,
CHZCI2DMF
d
O O
II N
Me0 I w
O
Me0
OMe
Example 22
MaleimidopropionamYl-1-theobromineacetamide i(Modified
1-theobromineacetic acid)
1-theobromineacetic acid 1 is placed in DMF and activated with O-
(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium hexafluorophosphate
(HBTU) and Diisopropylethylamine (DIEA) as indicated in the schematic
below. To the reaction mixture is added 3-maleimidopropylamine. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, chromatographied, triturated with cold ether and
left to crystallize to produce 2 Maleimidopropionamyl-1-
theobromineacetamide, a modifed bronchodilator.
~3
O
CH3
2
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Example 23
4-anilino-1-I(2-phenethyrl)piperidine ((Modified-Fentanyf~
1-phenylethyl-4-piperidone 1 was placed in 1,2, dichloroethane along
with aniline 2, sodiumcyanoborohydride and it is refluxed for 18 hours. The
reaction is then cooled to RT and the reaction is extracted with brine to
generate 3 as indicated in the schematic below. Finally The compound is then
placed in DMF and activated with and O-(benzotriazol-1-yl)-N,N',N',N',-
tetramethyluronium hexafluorophosphate (HBTU) and Diisopropylethylamine
(DIEA). To the reaction mixture is added 3-maleimidopropionic acid. The
reaction is stirred for 3 hours. The organic phase is then washed with water
and brine, dried over MgS04, triturated with cold ether and left to
crystallize to
generate 4, a modified pain killer (opioid molecule).
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I
w
~ 2
NAH(CN)3, 1,2
i
~I
1
HO-
O
O
'I
4
Example 24
Maleimido~ro~amvl2-f4-(2-oxocvclouentan-1-
Ethyl 2 cyclopentanonecarboxylate 1 and ethyl 2-(4-
iodomethylphenyl)propionate 2 are placed in N,N,dimethylformamide along
with potassium hydroxyde as indicated in the schematic below. The solution
is stirred at room temperature for 5 hours and at 50°C for 1 hour. The
reaction
is cooled and acidified with acetic acid and N,N,dimethylformamide is
removed by vacuum. The residue is extracted with ether and the organic
phase is washed with water and dried on Mg2S04 to afford 3 Finally, the
compound 3 is then placed in DMF and activated with and O-(benzotriazol-1
yl)-N,N',N',N',-tetramethyluronium hexafluorophosphate (HBTU) and
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Diisopropylethylamine (DIEA). To the reaction mixture is added 3-
maleimidopropylamine. The reaction is stirred for 3 hours. The organic phase
is then washed with water and brine, dried over MgS04, chromatographied,
triturated with cold ether and left to crystallize to generate 4
Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide
to produce the modified anti-inflammatory agent.
0 0
° °
OEt O ~ \ OH
~OEt I a) KOH
b) AcOH
O\
Nr~\
H ~I'~
O
HBTU, DIEA,
CH ~12DMF
O O
°
~/. ~ O
4
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Example 25
N-maleimidopropionyl-N-methyl 3-i(p-trifluoromethyrlphenoxyy-3-
phenylpropyrlamine i(Modified Fluoxetine)
~i-dimethylaminopropiophenone hydrochloride 1 is converted to the
corresponding free base by the action of aqueous sodium hydroxide. The
liberated free base is taken up in ether, the ether layer separated and dried,
and the ether removed therefrom in vacuo. The residual oil comprising ~i-
dimethylaminopropiophenone is dissolved in tetrahydrofuran, and the
resulting solution added in dropwise fashion with stirring to a solution of
diborane in tetrahydrofuran. The reaction mixture is stirred overnight at room
temperature. Next, aqueous hydrochloric acid is added to decompose any
excess diborane present. The tetrahydrofuran is removed by evaporation.
The acidic solution is extracted twice with benzene, and the benzene extracts
are discarded. The acidic solution is then made basic with an excess of 5 N
aqueous sodium hydroxide. The basic solution is extracted three times with
benzene. The benzene extracts are separated and combined, and the
combined extracts washed with a saturated aqueous sodium chloride and
then dried to produce 2. A solution containing N,N,-dimethyl 3-phenyl-3-
hydroxypropylamine 2 in chloroform is saturated with dry gaseous hydrogen
chloride. Thionyl chloride is then added to the chloroform solution at a rate
sufficient to maintain reflux. The solution is refluxed an additional 5 hours.
Evaporation of the chloroform and other volatile constituents in vacuo yielded
N,N-dimethyl 3-phenyl-3-chloropropylamine hydrochloride 3 which is collected
by filtration, and the filter cake washed twice with acetone. P-
trifluoromethylphenol 4 solid sodium hyroxide and methanol are placed in a
round-bottom flask equipped with magnetic stirrer, condenser and drying
tube. The reaction mixture is stirred until the sodium hydroxide had
dissolved.
Next, N,N-dimethyl 3-phenyl-3-chloropropylamine hydrochloride is added. The
resulting reaction mixture is refluxed for about 5 days and then cooled. The
methanol was then removed by evaporation, and the resulting residue taken
up in a mixture of ether and 5 N aqueous sodium hydroxide. The ether layer
is separated and washed twice with 5 N aqueous sodium hydroxide and three
times with water. The ether layer is dried, and the ether removed by
evaporation in vacuo to yield as a residue N,N-dimethyl 3-(p-
trifluoromethylphenoxy)-3-phenylpropylamine 5. A solution containing
cyanogen bromide in benzene and toluene is placed in a three-neck round-
bottom flask equipped with thermometer, addition funnel, drying tube and inlet
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tube for nitrogen. The solution is cooled and nitrogen gas is bubbled thru the
solution. Next, a solution of N,N-dimethyl 3-(p-trifluoromethylphenoxy)-3-
phenylpropylamine 5 dissolved in benzene is added in dropwise fashion. The
temperature of the reaction mixture is allowed to rise slowly to room
temperature, at which temperature stirring is continued overnight while still
maintaining a nitrogen atmosphere.The reaction mixture is washed twice with
water, once with 2 N aqueous sulfuric acid and then with water until neutral.
The organic layer is dried, and the solvents removed therefrom by
evaporation in vacuo to yield N-methyl-N-cyano 3-(p-trifluoromethylphenoxy)-
3-phenylpropylamine 6. A solution of potassium hydroxide, water, ethylene
glycol and of N-methyl-N-cyano 3-(p-trifluoromethylphenoxy)-3-
phenylpropylamine is placed in a three-neck, round-bottom flask equipped
with magnetic stirrer and condenser. The reaction mixture is heated to
refluxing temperature for 20 hours, and is then cooled. The reaction mixture
is
extracted with ether. The ether extracts are combined, and the combined
extracts washed with water. The water wash is discarded. The ether solution
is next contacted with 2 N aqueous hydrochloric acid. The acidic aqueous
layer is separated. A second aqueous acidic extract with 2 N hydrochloric
acid is made followed by three aqueous extracts and an extract with saturated
aqueous sodium chloride. The aqueous layers are all combined and made
basic with 5 N aqueous sodium hydroxide. N-methyl 3-(p-
trifluoromethylphenoxy)-3-phenylpropylamine 7, formed in the above reaction,
is insoluble in the basic solution and separated. The amine is extracted into
ether. The ether extracts are combined, and the combined extracts washed
with saturated. aqueous sodium chloride and then dried. Evaporation of the
ether in vacuo yielded N-methyl 3-(p-trifluoromethylphenoxy)-3-
phenylpropylamine 7. Finally, the compound 7 is then placed in DMF and
activated with and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To the
reaction mixture is added 3-maleimidopropionic acid. The reaction is stirred
for 3 hours. The organic phase is then washed with water and brine, dried
over MgS04, chromatographied, triturated with cold ether and left to
crystallizeto produce 8 to produce the modified anti-depressant molecule
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O N, HO N'
I I
I A) B-~Gin THF
1 B) H~ 2
HCI(gaz),
Thionylchloride
d
F~ / \ O N~ F~ / \ OH4 CI
~I
NaOH, MeOH
3
BrcN
Toluene:Benzene
F~ / \ O N~ ~ F~ / \ O _ N~
H
CN KOH, Water ethelyne glycol
w 7
6 0
HO~
O 11110
HBTU, DIEA
CHZCI2DMF
F~ / \ O N~ O
I O O
g
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Example 26
Maleimidoproluionamyl-3,5-3',5' tetraiodothyroninamide QModifed-
Thyroxine)
N-t-Boc-3,5-3',5' tetraiodothyronine 1 is placed in DMF and activated
with and O-(benzotriazol-1-yl)-N,N',N',N',-tetramethyluronium
hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as indicated
in the schematic below. To the reaction mixture is added 3-
maleimidopropylamine. The reaction is stirred for 3 hours. The organic phase
is then washed with water and brine, dried over MgS04, triturated with cold
ether and left to crystallize to produce 2. Finally the compound is placed in
a
25% solution of TFA in CH2C12 for 15 minutes and the CH2C12 is removed
invacuo. The oily residue is then lyophilized to yield the desired compound 3
a modified thyroxine for treament of thyroid deficiency, i.e., an anti-thyroid
deficiency agent.
o~
H
HO ~ ~ O ~ ~ NHBoc ~ O HO ~ ~ O
I I ,~ I
HO"O HBTU, DIEA,
CH~12DMF
2
25%TFA in CH~12
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Example 27
2S-hyrdroxy-3R-[1 S-~(MEEA-EDA-carbonyl)-2,2-dimeth~-
propylcarbamoyl]-5-methylhexanohydroxamic acid i( Modified MMPI]
The compound 1 and 3,4-dihydro-2H-pyran in CHZC12 and pyridinium
p-toluenesulfornate are stirred at room temperature for 12 h as indicated in
the schematic below. Then the solution is diluted with EtOAc and washed with
half-saturated brine to remove the catalyst. The solvent is evaporated and the
residue is treated with NaOH(1 N) and EtOH for 30 min. The solution is
acidified with AcOH, and the product is extracted with EtOAc. The EtOAc
solution is dried, evaporated to give the THP ether 2. The compound 2, DCC
and HOBT in CH2C12 are stirred at room temperature for 60 min. Then MEEA-
EDA HCI (N-(2-aminoethyl) [2-(2-maleiimidoethoxy)ethoxy]acetamide) and N-
methylmorpholine are added. The reaction is stirred for 2 h., and then
quenched by addition of AcOH. The precipitate is removed by filtration. The
filtrate is washed with diluted HCI, NaHC03 and dried. The crude product is
used for the next step.The crude product is treated with 2N HCI HZO/EtOH
1:1) for 30 min. EtOH is evaporated. The product is extracted with CHZCIZ..
The combined CH2Clz layers are washed with NaHC03 and dried.
Evaporation of the solvent gives a residue, which is purified by flash column
chromatography to afford 3. This compound can be purified further by HPLC
on a reverse phase column and lyophilized to produce the modified MMPI.
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O
O 1. I py TsOH O H O
HOHN Y 'OH THPOHN N Y 'OH
~H ~ 2. NaOH ZSTHP
1 2
1.DCC/HOBT
2. MESA-EDA HCI
3. NCI
O
O O
HOHN N~N~N~O~O~IV
H O
aH ~ _ O
EXAMPLE 28
Preparation of rhodamine NHS ester
Rhodamine GreenT"''-X, succinimidyl ester, hydrochloride mixed isomers is
commercially available from Molecular Probes (Eugene Oregon) as illustrated
below:
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Example 29
In vivo addition of NHS-rhodamine
New Zealand rabbits (2 Kg), male or female, were intramuscularly
anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) and
Acepromazine (0.75 mg/kg) prior to surgical exposure of left carotid artery.
Both carotid arteries were isolated and blood flows were measured. A
catheter (22G) was inserted in the arterial segment and rinsed with 0.9%
sodium chloride via catheter until there was no more visible evidence of
blood in the segment.
A 1-cm incubation chamber was created by ligatures in the segment
area. The incubation chamber was flushed three times with 1 mL of 0.9%
sodium chloride. A solution of 1001 of 500~M NHS-Rhodamine was
prepared and incubated in the incubation chamber for 3 minutes. The excess
of rhodamine was withdrawn with a 1 mL syringue. The incubation chamber
was washed once again with 3 times 100 mL of 0.9% sodium chloride. The
incubation chamber was then removed from the rabbit, cut in three pieces
and dipped in 10% formalin for further evaluation. The NHS-Rhodamine
treated arteries exhibited dramatic levels of fluorescence whereas those
arteries treated solely with Rhodamine exhibited little fluorescence over
background. These results demonstrate that Rhodamine was covalently
bonded to a local delivery site.
Example 30
Preparation of ['H ]!-NHS-propionate
[3H ]-NHS-propionate is available from Amersham Canada Ltd.
(Oakville, Ontario, Canada) and can be prepared from the tritiated propionic
acid through known to the art condensation of N-hydrosuccinimide in
presence of EDC in DMF or methylene chloride.
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Example 31
In vivo pharmacokinetics studies of ['H 1-NHS-propionate
New Zealand rabbits (2 kg), male or female, were intramuscularly
anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) et Acepromazine
(0.75 mg/kg) prior to surgical exposure of left carotid artery. Segments of 10
mm of carotids, were transiently isolated by temporary ligatures and rinsed
with 0.9% sodium chloride via a cannula until there was no more visible
evidence of blood components.
A catheter (18G) was inserted in the arterial segment and served to
introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston
Scientific Inc.). A vascular damage (angioplasty) was performed on the
isolated segment in order to eliminate the layer of endothelial cells. The
angioplasty balloon was serially inflated at different atmospheres (4, 6, 8
and
10) during 1 minute, with 45 seconds of delay between inflations. At 4
atmospheres a balloon traction was performed 5 times and 1000 U/kg of
heparin were infused in the blood circulation.
The angioplasty balloon was then retrieved from the artery and the
catheter was reintroduced. The arterial segment was rinsed 3 times with
saline, and 100 pM of [3H ]-NHS-propionate was incubated within the isolated
segment of the artery for either 30 seconds, 3 minutes or 30 minutes. At the
end, the excess of incubation liquid was withdrawn from the artery, and the
segment was rinsed 5 times with saline. The treated artery was immediately
harvested, and incorporation of [3H] -labeled compounds within the artery was
evaluated by scintillation counting. After 30 seconds of incubation, we
recorded an association efficiency of 2.55%. At 3 min and 30 min, we
recorded an association efficiency of 5.5 and 6.5%, respectively. We decided
that a 3 min incubation time was sufficient to treat the artery in an
efficient
way.
When evaluating the retention levels, 100 ~M of [3H ]-NHS-propionate
or [3H ]-propionate were incubated with the artery for a period of 3 minutes,
after which the segment has been rinsed 5 times with saline. The catheter
was then removed and the arteriotomy site was closed with microsutures,
thus reestablishing the blood flow within the carotid. Finally, the neck wound
was closed with sutures, and animals are allowed to recuperate. Three days
following the treatment, the animals are sacrificed with an overdose of sodium
pentobarbital, the carotid segments are removed and examined for
compound's presence by scintillation counting. 10.94% retention of [3H ]-NHS-
propionate was monitored after three days following a 3 minute incubation
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period based on residual radioactivity in the artery. The difference in
retention efficiency between covalently and non covalently bound propionate
after a 3 minutes incubation period was determined. An outstanding 12 fold
enhancement in retention was recorded (0.6% of total amount incubated
against 0.046% for the non covalently bound) in favor of the NHS-propionate.
This indicates that the tissue association of a compound is dramatically
enhanced by the covalent attachment in vivo. Subsequent restitution of blood
flow demonstrated retention [3H ]-NHS-propionate of approximately 10% of
the material 72 hours after injury. This represents excessive tissue retention
using the embodied technology of agents markedly beyond that seen with all
drug delivery technologies as exemplified in the literature for standard non
covalent agents (Circulation 1994 89 (4) 1518-1524).
Example 32
Synthesis of [3zP] NHS derivative
To a solution of protected R and R' (both R and R' can be alkyl, phenyl
or alkoxy groups, and X is either O or S, alkoxy, alkyl and any other
functionality stable under. these conditions) phosphodiester (0.1 mmol) and N-
hydroxysuccinimide (0.2 mmol) is added diisopropylethylamine 0.11 mmol),
followed by addition of HBTU (0.22 mmol). The reaction mixture is stirred at
room temperature for 36 hours. DMF is removed by vacuum distillation and
the residue is dissolved in MeOH (10 mL). The MeOH solution is filtered to
remove the insolubles, the filtrate is concentrated in vacuo, and the residue
is
dissolved in a minimum amount of MeOH. Water is then added to induce
precipitation and the precipitate is dried on vacuum to give the desired
compound.
32
~R_''~~~~IOR
OR'
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The yield of the reaction can usually be improved by using EDC as the
coupling reagent, as exemplified below. To a solution of R and R'
phosphodiester (0.054 mmol) and N-hydroxysuccinimide (0.115 mmol) in
anhydrous DMF (3 mL), is added EDC (31 mg, 0.162 mmol). The solution is
stirred at room temperature for 24 hours. DMF is removed by vacuum
distillation and the residue is further dried on high vacuum. The residue is
then dissolved in a minimum amount of MeOH (0.12 mL) and H20 (3.2 mL) is
added to induce precipitation. The precipitates are washed with Hz0 (3 x 0.8
mL) and dried on vacuum to give a solid product.
Any protected phosphonate derivatives may undergo similar
transformation.
Example 33
New Zealand rabbits (2 kg), male or female, were anesthetized with
xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75 mg/kg)
intramuscularly prior to surgical exposure of left carotid artery. Carotid
arteries
were surgically dissected and segments of approximately 10 mm length were
isolated. The vessels were cannulated and rinsed with 0.9% sodium chloride
until there was no more visible evidence of blood components.
A catheter (18G) was inserted in the arterial segment and served to
introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston
Scientific Inc.). Vascular damage (angioplasty) was performed on the isolated
segment in order to eliminate the layer of endothelial cells. The angioplasty
balloon was serially inflated at different atmospheres (4, 6, 8 and 10) for 1
minute, with 45 seconds of delay between inflations. At 4 atmospheres a
balloon traction was performed 5 times and 1000 U/kg of heparin were
infused in the blood circulation.
The angioplasty balloon was then retrieved from the artery and the
catheter was reintroduced. The arterial segment was rinsed 3 times with
saline, and 100 NM of [32P]- NHS-[linking group] was incubated within the
isolated segment of the artery for 3 minutes. At the end, the excess of
incubation liquid was withdrawn from the artery, and the segment was rinsed
5 times with saline. The vessel was sutured closed, blood flow restored and
surgical wounds repaired. Animals were returned to the vivarium for periods
up to four weeks. Tissue retention of [32P]-NHS-[linking group] was
evaluating using whole animal radiography at selected periods of time after
injury.
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Example 34
Synthesis of ['3'I]- NHS derivative
To a solution of protected amino protected ['3' I]-iodotyrosine (0.1
mmol) and N-hydroxysuccinimide (0.2 mmol) is added diisopropylethylamine
(0.11 mmol), followed by addition of HBTU (0.22 mmol). The reaction mixture
is stirred at room temperature for 12 hours. DMF is removed by vacuum
distillation and the residue is dissolved in MeOH (10 mL). The MeOH solution
is filtered to remove the insolubles, the filtrate is concentrated in vacuo,
and
the residue is dissolved in a minimum amount of MeOH. Water is then added
to induce precipitation and the precipitate is dried on vacuum to give the
desired compound.
The yield of the reaction can usually be improved by using EDC as the
coupling reagent, as exemplified below. To a solution of ['3' I]-iodotyrosine
(0.054 mmol) and N-hydroxysuccinimide (0.115 mmol) in anhydrous DMF (3
mL), is added EDC (31 mg, 0.162 mmol). The solution is stirred at room
temperature for 24 hours. DMF is removed by vacuum distillation and the
residue is further dried on high vacuum. The residue is then dissolved in a
minimum amount of MeOH (0.12 mL) and water (3.2 mL) is added to induce
precipitation. The precipitates are washed with H20 (3 x 0.8 mL) and dried on
vacuum to give a solid product.
H
Example 35
In vivo pharmacology of'3'1 derivative
New Zealand rabbits (2 Kg), male or female, were anesthetized with
xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75 mg/kg)
intramuscularly prior to surgical exposure of left carotid artery. Carotid
arteries
were surgically dissected and segments of approximately 10 mm length were
isolated. The vessels were cannulated and rinsed with 0.9% sodium chloride
until there was no more visible evidence of blood components.
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A catheter (18G) was inserted in the arterial segment and served to
introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston
Scientific Inc.). Vascular damage (angioplasty) was performed on the isolated
segment in order to eliminate the layer of endothelial cells. The angioplasty
balloon was serially inflated at different atmospheres (4, 6, 8 and 10) for 1
minute, with 45 seconds of delay between inflations. At 4 atmospheres a
balloon traction was performed 5 times and 1000 U/kg of heparin were
infused in the blood circulation.
The angioplasty balloon was then retrieved from the artery and the
catheter was reintroduced. The arterial segment was rinsed 3 times with
saline, and 100 NM of ['3'I]-NHS-[linking group] was incubated within the
isolated segment of the artery for 3 minutes. At the end, the excess of
incubation liquid was withdrawn from the artery, and the segment was rinsed
5 times with saline. The vessel was sutured closed, blood flow restored and
surgical wounds repaired. Animals were returned to the vivarium for periods
up to four weeks. Tissue retention of ['3'I]-NHS-[linking group] was evaluated
using whole animal radiography at selected periods of time after injury
Example 36
Intrapulmonary Delivery of 2-[2-[4-[(4-chloropheny)phenylmethyl[-1-
piperazinyl]ethoxy]-maleimidopropionylacetamide. (Modified-Cetirizine)
A Bird Micronebulizer in line with a Bird Mark 7 respirator may be charged
with 5-10 ml of a solution of 12 mg/ml 2-[2-[4-[(4-chloropheny)phenylmethyl[-
1-piperazinyl]ethoxy]-maleimidopropionylacetamide in mannitol/phosphate
buffer. The Micronebulizer may then bes used to simultaneously ventilate and
dose a patient at 22 cm HZ O at a rate of 1.8 mg/min for 30 min. At this
pressure the patient shouldl ventilate at approximately normal inspiratory
volume. The patient should be allowed to exhale normally after each
ventilated breath. In addition, the patient should be positioned supine for
dosing. After the first dosing period the pateint should be allowed to breathe
normally for another 20 minutes. After the 20 minute period, a second
dosing should be performed in the same way as the first. Blood plasma
samples should be taken at the initiation time of the first dose and
thereafter
to monitor the levels of 2-[2-[4-[(4-chloropheny)phenylmethyl[-1-
piperazinyl]ethoxy]-maleimidopropionylacetamide .
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EXAMPLE 37
Intrapulmonary Delivery of 2-[2-[4-[(4-chloropheny)phenylmethyl[-1-
piperazinyl]ethoxy]-maleimidopropionylacetamide (Modified-Cetirizine)
Using the Spiros DPI System
The Spiros DPI is an aerosol generation system that is largely
independent of the inspiratory flow rate and its use is described in US Patent
No. 6, 060, 069.
A modified beclomethasone dipropionate (BDP) formulation may be prepared
by first micronizing through conventional means (e.g., a jet mill) to produce
a
range of particle sizes that are likely to undergo sedimentation in the human
airway. Generally, fine particles in the range of 0.5 to 5.8 microns in
diameter
are thought to undergo sedimentation between the oropharynx and small
bronchioles. Particles within this general size category are thought to be in
the "respirable range." Such micronized materials have excessive surface
free energy, and as a result have a tendency to adhere strongly to many
surfaces, most especially to themselves.
Lactose particles in the size range of 20 to 100 microns may be mixed with
the smaller diameter micronized drug particles to create a homogenous
blend. Each lactose particle will generally bind to a number of smaller drug
particles in the blend. The blend flows more easily during the packaging and
dose metering process.
The formulation may be then filled into cassettes, each containing 30
individual doses. The cassettes may then packaged in sealed foil pouches.
The following steps using the Spiros BPI system may be used to deliver a
dose of inhaled drug: 1. The Spiros DPI does not need to be primed; 2. The
blue plastic cap is removed from the mouthpiece; 3. The inhaler is held level;
4. The lid of the DPI is opened as far back as possible (The lid will click
when
it has reached the correct angle); 5. The lid is then closed completely; 6.
Before bringing the inhaler up to the mouth, the patient breathes out, making
sure not to breathe into the inhaler.;
7. The inhaler is brought up to the mouth in a level position; 8. The lips are
sealed fully around the mouthpiece, making sure there is no gap between the
mouthpiece and the lips; 9. The patient breathes in through the mouth for
about 4 seconds, preferably at a flow rate of about 20 LPM. The motor will
turn on and the patient may taste/feel the drug as it is inhaled; 10. The
patient
holds their breath for as long as possible, up to 10 seconds. 11. The Spiros
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DPI is held in a level position during loading and dosing.
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SEQUENCE LISTING
<110> CONJUCHEM, INC.
EZRIN, ALAN M.
FLESER, ANGELICA
ROBITAILLE, MARTIN
MILNER, PETER G.
BRIDON, DOMINQUE P.
<120> PULMONARY DELIVERY FOR BIOCONJUGATION
<130> REDC-1810
<140>
<141>
<150> 60/152,681
<151> 1999-09-07
<160> 16
<170> PatentIn Ver. 2.1
<210> 1
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 1
Ala Gly Tyr Lys Pro Glu Gly Lys Arg Gly Asp Ala Lys
1 5 10
<210> 2
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 2
Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Cys
1
CA 02383798 2002-03-04
WO 01/17568 PCT/IB00/01429
1 5 10 15
<210> 3
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 3
Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Cys
1 5 10 15
<210> 4
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 4
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
<210> 5
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 5
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
2
CA 02383798 2002-03-04
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1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
<210> 6
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 6
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
<210> 7
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 7
Val Tyr Pro Ser Asp Glu Tyr Asp Ala Ser Ile Ser Gln Val Asn Glu
1 5 10 15
Glu Ile Asn Gln Ala Leu Ala Tyr Ile Arg Lys Ala Asp Glu Leu Leu
20 25 30
Glu Asn Val Lys
3
CA 02383798 2002-03-04
WO 01/17568 PCT/IB00/01429
<210> 8
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 8
Lys Val Tyr Pro Ser Asp Glu Tyr Asp Ala Ser Ile Ser Gln Val Asn
1 5 10 15
Glu Glu Ile Asn Gln Ala Leu Ala Tyr Ile Arg Lys Ala Asp Glu Leu
20 25 30
Leu Glu Asn Val
<210> 9
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 9
Val Tyr Pro Ser Asp Glu Tyr Asp Ala Ser Ile Ser Gln Val Asn Glu
1 5 10 15
Glu Ile Asn Gln Ala Leu Ala Tyr Ile Arg Lys Ala Asp Glu Leu Leu
20 25 30
Glu Asn Val
<210> 10
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
4
CA 02383798 2002-03-04
WO 01/17568 PCT/IB00/01429
Peptide
<400> 10
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys
20 25 30
<210> 11
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 11
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 12
Pro Arg Lys Leu Tyr Asp Tyr Lys
1 5
<210> 13
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
CA 02383798 2002-03-04
WO 01/17568 PCT/IB00/01429
Peptide
<400> 13
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Ala Trp Thr Thr Ala
1 5 10 15
Pro Arg Lys Leu Tyr Asp Tyr
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 14
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 15
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys
1 5 10
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 16
6
CA 02383798 2002-03-04
WO 01/17568 PCT/IB00/01429
Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys
