Note: Descriptions are shown in the official language in which they were submitted.
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POLYMERIC COUPLING AGENTS AND
PHARMACEUTICALLY-ACTIVE POLYMERS MADE THEREFROM
FIELD OF THE INVENTION
This invention relates to polymeric coupling agents as intermediates,
pharmaceutically-
active polymers made therefrom, composition comprising said polymers and
shaped articles
made therefrom.
BACKGROUND TO THE INVENTION
It has become common to utilize implantable medical devices for a wide variety
of
medical conditions, e.g., drug infusion and haemodialysis access. However,
medical device
implantation often comes along with the risk of infections (1), inflammation
(2), hyperplasia
(3), coagulation (4). It is therefore important to design such materials to
provide enhanced
biocompatibility. Biocompatibility is defined as the ability of a material to
perform with an
appropriate host response in a specific application. The host relates to the
environment in which
the biomaterial is placed and will vary from being blood, bone, cartilage,
heart, brain, etc.
Despite the unique biomedical related benefits that any particular group of
polymers may
possess, the materials themselves, once incorporated into the biomedical
device, may be
inherently limited in their performance because of their inability to satisfy
all the critical
biocompatibility issues associated with the specific application intended. For
instance while
one material may have certain anticoagulant features related to platelets it
may not address key
features of the coagulation cascade, nor be able to resist the colonization of
bacteria. Another
material may exhibit anti-microbial function but may not be biostable for
longterm
applications. The incorporation of multi-functional character in a biomedical
device is often a
complicated and costly process which almost always compromises one polymer
property or
biological function over another, yet all blood and tissue contacting devices
can benefit from
improved biocompatibility character. Clotting, toxicity, inflammation,
infection, immune
response in even the simplest devices can result in death or irreversible
damage to the patient.
Since most blood and tissue material interactions occur at the interface
between the biological
environment and the medical device, the make-up of the outer molecular
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layer (at most the sub-micron layer) of the polymeric material is relevant to
the biological
interactions at the interface. This is a particularly challenging problem for
biodegradable
polymer systems when a continuous exposure of new surfaces through erosion of
the bulk
polymer requires a continuous renewal of biocompatible moieties at the
surface.
Bioactive agents containing polymer coatings have been developed to improve
the
biocompatibility of medical device surfaces. Patnaik et al. (5) described a
method of
attaching bioactive agents, such as heparin (an anti-coagulant) to polymeric
substrates via
a hydrophilic, isocyanate/amine-terminated spacer in order to provide a
coating of the
bio-active material on the medical device. The investigator found that the
bioactive
agent's activity was achieved when the spacer group had a molecular weight of
about
100-10,000 daltons. But most preferably that is of 4000 daltons.
Unfortunately, such a
material would only be applicable for substrates which were not intended to
under go
biodegradation and exchange with new tissue integration since the heparin in
limited to
surface and does not form the bulk structure of the polymer chains.
Another example of biomaterial design relates to infection control. In the
last
decade, a number of strategies have been used in attempts to solve problems
such as
those associated with medical device infection. One approach is to provide a
more
biocompatible implantable device to reduce the adhesion of bacteria. Silver
coated
catheters have been used to prevent exit site infections associated with
chronic venous
access (6) and peritoneal dialysis (7). However, longterm studies have failed
to
demonstrate a significant reduction in the number or severity of exit site
infections. In
addition, bacterial resistance to silver can develop over time and carries
with it the risk of
multiple antibiotic resistances (8).
Since bacteria adhesion is a very complex process, complete prevention of
bacteria adhesion is difficult to achieve with only a passive approach. There
remains a
need for local controlled drug delivery. The advantages for the latter
approach include 1)
a high and sustained local drug concentration can be achieved without the
systemic
toxicity or side effects which would be experienced from systemic doses
sufficient to
obtain similar local drug concentration; 2) high local drug concentration can
be attained,
even for agents that are rapidly metabolized or unstable when employed
systemically; 3)
some forms of site-specific delivery have the potential to establish and
maintain local
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drug action, either by preventing its efflux from the arterial wall or by
using vehicles or
agents that have a prolonged duration of action; 4) it gives the potential for
designing a
smart drug delivery system, which can be triggered to start the release and/or
modulate
the rate of release according to the infection status.
Methods for obtaining compositions which contain drugs and polymers in a
composite form to yield bioactive agent release coatings are known. For
example,
Chuodzik et al. (9) formulated a coating composite that contained a bioactive
agent (e.g. a
drug) and two polymers, i.e., poly(butyl methacrylate) and poly(ethylene-co-
vinyl
acetate). The coating formed from the above formulation provided good
durability and
flexibility as well as significant drug release, which could be particularly
adapted for use
= with devices that undergo significant flexion and/or expansion in the
course of their
delivery and/or use, such as stents and catheters. These approaches have the
benefit of
localized delivery at high drug concentration, but are unable to keep a
sustained and
controlled release of drug for long periods. Ragheb et al.(10) found a method
for the
controlled release of a bioactive agent from polymer coatings. Wherein, two
coating
layers of polymer were applied to a medical device. The first layer of the
device is an
absorbent material such as parylene derivatives. Drug or bioactive agent is
deposited
over at least a portion of this layer. The second biocompatible polymer layer
on top of
the drug and the first layer must be porous. The polymer is applied by vapor
deposition
or by plasma deposition. Since the drug release mechanism is totally
controlled by
porous sizes, making a suitable porous size distribution in the second layer
in order to
satisfy the required release model is often a technical challenge. As well,
this type of
system requires multiple processing steps which increases production cost and
adds to the
need for QA/QC steps.
In addition to the traditional diffusion-controlled delivery systems described
in the
above references, there exist several more sophisticated in situ drug delivery
polymers
which can alter the efficacy of drugs by improving target delivery and
changing the
control parameters of the delivery rate. These include biodegradable hydrogels
(11),
polymeric liposomes (12), bioresorbable polymers ( 13) and polymer drugs (14-
16).
Polymer drugs contain covalently attached pharmaceutical agents on the polymer
chain
as pendent groups, or even incorporated into the polymer backbone. For
example,
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Nathan et al (17) conjugated penicillin V and cephradine as pendant
antibiotics to
polyurethanes. Their work showed that hydrolytically labile pendant drugs were
cleaved
and exhibited antimicrobial activities against S. aureus, E. faecalis and S.
pyogenes.
Ghosh et al. (18) coupled nalidixic acid, a quinolone antibiotic, in a pendant
manner to an active vinyl molecule. These vinyl groups can then be polymerized
to
generate a polymer with pendent antibiotics on each monomer. However, having
such
pendant groups will dramatically alter the physical structure of the polymer.
A better
strategy would be to have the drugs within the linear backbone portion of the
polymer.
In in-vivo hydrolysis studies they reported a 50% release of drug moieties
over the first
100 hours. This quinolone drug has been shown to be effective against gram
negative
bacteria in the treatment of urinary track infections, however chemical
modifications of
the latter (e.g. ciprofloxacin, norfloxacin and others) have a wider spectrum
of activity.
More recent work on the conjugation of norfloxacin to mannosylated dextran has
been
reported. This was driven in an effort to increase the drug's uptake by cells,
enabling
them to gain faster access to micro-organisms (19). The studies showed that
norfloxacin
could be released from a drug/polymer conjugate by enzyme media and in vivo
studies,
the drug/polymer conjugate was effective against Mycobacterium tuberculosis
residing
in liver (20). In the system, norfloxacin was attached pendant to sequences of
amino-
acids which permitted its cleavage by the lysosomal enzyme, cathepsin B.
Santerre (13a) describes the synthesis and use of novel materials to which
when
added to polymers converts the surface to have bioactive properties, while
leaving the
bulk properties of the polymer virtually intact. Applications are targeted for
the
biomedical field. These materials are oligomeric fluorinated additives with
pendant
drugs that are delivered to the surface of bulk polymers during processing by
the
migration of the fluorine groups to the air/polymer interface. These materials
can deliver
a large array of drugs, including anti-microbials, anti-coagulants and anti-
inflammatory
agents, to the surface. However modification is limited to the surface. This
becomes a
limitation in a biodegradable polymer which may require sustained activity
throughout
the bio-erosion process of the polymer.
Santerre and Mittleman (14) teach the synthesis of polymeric materials using
pharmacologically-active agents as one of the co-monomers for polymers.
Wherein, 1,6-
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diisocynatohexane and/or 1,12-diisocyanatododecane monomers or their
oligomeric
molecules are reacted with the antimicrobial agent, ciprofloxacin, to form
drug polymers.
The pharmacologically-active compounds provide enhanced long term anti-
inflammatory, anti-bacterial, anti-microbial and/or anti-fungal activity.
However, since
the reactivities of the carboxylic acid group and the secondary amine group of
ciprofloxacin with the isocyanate groups are different, the reaction kinetics
become
challenging. As well, formulations must be selective in order to minimize
strong van der
Waals interactions between the drug components and hydrogen bonding moieties
of the
polymer chains since this can delay the effective release of drug. Hence, an
improvement
over the latter system are biomonomers made up of the drugs and agents which,
without
being bound by theory, would ensure a less restricted access of the drug
during hydrolysis
of the polymer, as well as providing more uniform chemical function for
reaction with the
isocyanate groups or other monomer reagents.
PUBLICATIONS
(1) Mittelman, MW, "Adhesion to biomaterials" in Bacterial Adhesion: Molecular
and Ecological diversity, M Fletcher(ed) 89-127, 1996)
(2) John F. Burke, et al., "Applications of materials in medicine and
dentistry", in
Biomaterials Science, 1996, Ch. 7, pp 283-297.
(3) Martin R. Bennett, Michael O'Sullivan, "Mechanism of angioplasty and stent
restenosis: implications for design of rational therapy", Pharmacology &
Therapeutics 91(2001) pp 149-166.
(4) Eberhart, R.C., and C.P. Clagett, "Platelets, catheters, and the vessel
wall; catheter
coatings, blood flow, and biocompatibility", Seminars in Hematology, Vol. 28,
No. 4, Suppl. 7, pp 42-48 (1991).
(5) US Patent No. 6,096,525 - Patnaik, BK. Aug. 1,2000
(6) Groeger J. S. et al., 1993, Ann. Surg. 218:206-210.
(7) Mittelman M. W., et.al., 1994. Ann. Conf. Peritoneal Dialysis, Orlando.
Fla.
(8) Silver S. et al., 1988, Ann. Rev. Microbiol. 42:717-743
(9) U.S. Patent. No.6,344,035 - Chudzik, et al. Feb. 5, 2002
(10) US. Patent No. 6,299,604 - Ragheb, et al. Oct. 9, 2001
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(11) US. Patent No. 6,703,037 - Hubbell et al. Mar. 9, 2004
(12) Valerio D. et al. Biomaterials, 19:1877-1884 (1998)
(13) U.S. Patent No. 4,916,193 - Tang et al. and U.S. Pat. No. 4,994,071 -
MacGrego
(13a)U.S. Patent filed on June 7, 2002, Application # 10/162,084, Santerre,
Paul J.
(14) US. Patent No. 5,798,115 - Santerre, Paul J. and Mittleman, Marc W. Aug.
25, 1998.
(15) Modak S. M., Sampath, L., Fox, C. L., Benvenisty A., Nowygrod, R.,
Reemstmau, K. Surgery, Gynecology & Obstetrics ,164, 143-147 (1987).
(16) Bach, A.; Schmidt, H.; Bottiger, B.; Schreiber B.; Bohrer, H.; Motsch,
J.; Martin, E.;
Sonntag, H. G., J Antimicrob. Chemother. , 37, 315, (1996)
(17) Nathan, A.; Zalipsky, S.; Ertel, S. I.; Agarthos, S. N.; Yarmush, M. L.;
Kohn. J.
Bioconjugate Chem. 1993, 4, 54-62.)
(18) Ghosh M. Progress in Biomedical polymers, Gebekin CG. Et al (ed), Plenum
press, New York, 1990, 335-345; Ghosh M. Polymeric Materials,
Science&Engineering 1988, 59: 790-793
(19) Coessens, V.; Schacht,E., Domurado, D. J. Controlled Release 1997, 47 283-
291
(20) Roseeuw, E.; Coessens V.; Schacht E., Vrooman B.; Domurado, D.; Marchal
G. J Mater.
Sci: Mater. Med. 1999, 10, 743-746
(21) Hemmerich, K. J. Polymer materials selection for radiation sterilized
products,
Medical Device & Diagnostic Industry, February, 2000
(22) ISO 11137: Sterilization of health care products-Requirements for
validation and routine
control-Radiation sterilization.
SUMMARY
Since the availability of drugs that can serve as commercial monomers,
specifically
designed for the synthesis of the above drug polymers or polymers to be used
in composites are
limited, there is a need for custom synthesis methods of the drug precursors.
Rather than
depending on the chemical function that common commercial drugs inherently
provide, it
would be better provide monomers that have similar multi-functional groups and
preferably
similar di-functional groups for the synthesis of hydrolysable type polymers.
This disclosure
sets forth a group of novel diamine or diol monomers that simultaneously
incorporate the
following features: 1) they are synthesized under mild conditions for coupling
biological or
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pharmaceuticals or biocompatible components together via a hydrolysable bond;
2) they
contain selectively reactive groups (di-functional or greater) (including
amines (secondary or
primary) and hydroxyls) that could be used for subsequent polymerization of
polyesters,
polyamides, polyurethanes, polysulfonamides and many other classical step
growth polymers;
3) they contain selectively hydrolysable groups that permit the release of
defined degradation
products consisting of biological, pharmaceutical or biocompatible components;
4) their
molecular weights may vary depending on the molecular weight of the
pharmaceutical or
biocompatible reagents to be as high as 4000, but typically the molecular
weights of the
molecules will be preferably less than 2000 in order for them to have good
mobility of the
molecular segment once incorporated within the polymer, and have good
reactivity in the
reaction polymerization solution; 5) they provide a strategy for enhancing the
introduction of
important biological, pharmaceutical or biocompatible reagents which otherwise
contain
functional groups (such as shielded esters, sulphonamides, amides and
anhydrides) that would
have poor reactivity in hydrolytic reactions due to strong van der Waals or
hydrogen bonding
between drug polymer backbones. 6) Since, these molecules will have similar
functional groups
they will provide consistent and more predictable reactivity in a classical
step growth
polymerization. This invention describes the unique synthesis pathways for the
biomonomers,
provides examples of their use in the synthesis of polymers and defines
methods of processing
said polymers for applications as biodegradable materials ranging from
biomedical to
environmental related products.
It is an object of the present disclosure to provide synthetic pathways of
biological
coupling agents/biomonomers comprising, such as, anti-inflammatory, anti-
bacterial, anti-
microbial and/or anti-fungal pharmaceuticals as biomonomer precursors with
good reactivity
for step growth polymer synthesis.
It is a further object of the present disclosure to provide biological
polymers comprising
said biological coupling compounds/monomers with pharmaceutically active
properties.
It is a further object of the present disclosure to provide said polymer
compounds alone
or in admixture with a compatible polymeric biomaterial or polymer composite
biomaterials for
providing a shaped article having pharmaceutically active properties.
It is a further object of the present disclosure to provide said shaped
article for use as a
medical device, comprising a body fluid and tissue contacting device in the
biomedical sector,
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or for use in the biotechnology sector to provide anti-infection, anti-
inflammatory properties.
It is a further object of the present disclosure to provide said polymer
compounds alone
as a coating or in admixture with either a base polyurethane, polysilicone,
polyester,
polyethersulfone, polycarbonate, polyolefin or polyamide for use as said
medical devices in the
biomedical sector, for improving anti-infection, anti-inflammatory,
antimicrobials, anti-
coagulation, anti-oxidation, anti-proliferation function.
It is a further object of this disclosure to provide processes of manufacture
of said
biomonomers, polymers containing said biomonomers, said admixtures and said
shaped
articles.
This disclosure, generally, provides the unique synthesis pathways for
covalently
coupling biologicals or pharmaceuticals or biocompatible components to both
sides of a
flexible diol or diamine, such as but not limited to triethylene glycol or any
other kind of linear
diol or diamine under mild conditions. Bioactive agents must possess a
reactive group such as a
carboxylic acid, sulfonate or phosphate group which can be conjugated to the
flexible diols or
diamines by using a carbodiimide-mediated reaction. Bioactive agents used in
the coupling
reaction must also contain selectively reactive multifunctional and preferably
di-functional
groups (including amines (secondary or primary) and hydroxyls) that could be
used later on for
subsequent polymerization of polyesters, polyamides, polyurethanes,
polysulfonamides and any
other classical step growth polymer pharmaceutic containing coupling
agents/monomers.
This disclosure provides in one aspect, a biological coupling agent
(biomonomer)
having a central portion comprising of flexible i.e. not limiting chain
dynamic movement such
as do aromatic rings, linear or aliphatic (saturated) segments of < 2000
theoretical molecular
weight and hydrolysable linkages.
This disclosure provides a biological coupling agent of the general formula
(III)
PBio-LINK A-PBio (III)
wherein PBio is a biologically active agent fragment or precursor thereof
linked to LINK A
through a hydrolysable covalent bond and having at least one functional group
to permit step
growth polymerization; and LINK A is a coupled central flexible linear first
segment of <2000
theoretical molecular weight linked to each of said PBio fragments.
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By the term "biomonomers" in this specification, is meant compounds of the
formulae
(III) used in the synthesis of the compounds of formula (I) through the use of
the functional
group for step growth polymerization.
Most preferably each of the PBio fragments is limited to a single functional
group for
use in step growth polymerization.
Thus, in a further aspect, this disclosure provides a pharmaceutically-active
polymeric
compound of the general formula (I),
Y- [Yõ - LINK B - X]m - LINK B (I)
wherein (i) X is a coupled biological coupling agent ofthe general formula
(II)
Bio - LINK A - Bio (II)
wherein Bio is a biologically active agent fragment or precursor thereof
linked to LINK A
through a hydrolysable covalent bond; and LINK A is a coupled central flexible
linear first
segment of <2000 theoretical molecular weight linked to each of said Bio
fragments;
(ii) Y is LINK B-OLIGO; wherein
(a) LINK B is a coupled second segment linking one OLIGO to another OLIGO
and an OLIGO to X or precursor thereof; and
(b) OLIGO is a short length of polymer segment having a molecular weight of
less than
5,000 and comprising less than 100 monomeric repeating units;
(iii) m is 1- 40; and
(iv) n is selected from 2 - 50.
Yn may have a molecular weight of less than 15,000. The molecular weight may
be less
than 10,000. This molecular weight may also be less than 5,000.
This disclosure provides in another aspect, a pharmaceutically-active
polymeric
material having a backbone made from said biomonomer. Such polymers comprise
oligomeric
segments of <5,000 theoretical molecular weight and optional link segments,
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herein denoted [link 13] covalently coupled to the oligomeric segment denoted
herein
[oligo] and the said biomonomer.
By the term "oligomeric segment" is meant a relatively short length of a
repeating
unit or units, generally less than about 50 monomeric units and molecular
weights less
than 10,000 but preferably <5000. Preferably, [oligo] is selected from the
group
consisting of polyurethane, polyurea, polyamides, polyallcylene oxide,
polycarbonate,
polyester, polylactone, polysilicone, polyethersulfone, polyolefin, polyvinyl,
polypeptide,
polysaccharide; and ether and amine linked segments thereof.
By the term "LINK A molecule" is meant a molecule covalently coupling
bioactive agents together in said biomonomer. Typically, LINK A molecules can
have
molecular weights ranging from 60 to 2000 and preferably between 60 to 700,
and have
multi-functionality but preferably di-functionality to permit coupling of two
bioactive
agents. Preferably the LINK A molecules are synthesized from the groups of
precursor
monomers selected from diols, diamines and/or compounds containing both amine
and
hydroxyl groups, with or without water solubility. Examples of typical LINK A
precursors are given in Table 1 but they are not limited to this list.
Table 1
- Ethylene glycol
-Butane diol
-Hexane diol
-Hexamethylene diol
-1,5 pentanediol
-2,2-dimethy1-1,3 propanediol
-1,4-cyclohexane diol
-1,4-cyclohexanedimethanol
=
-Tri(ethylene glycol)
-Poly(ethylene glycol), Mn: 100-2000
-Poly(ethylene oxide) diamine, Mn: 100-2000
-Lysine esters
-Silicone diols and diamines
-Polyether diols and diamines
-Carbonate diols and diamines
-Dihydroxy vinyl derivatives
-Dihydroxy diphenylsulfone
-Ethylene diamine
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-Hexamethylene diamine
-1,2-diamino-2 methylpropane
-3,3,-diamino-N-methyldipropylamine
-1,4 diaminobutane
-1,7 diaminoheptane
-1,8 diaminooctane
By the term "LINK B molecule" is meant a molecule covalently coupling oligo
units together to form the second coupling segments within the central
portion.
Typically, LINK B molecules can have molecular weights ranging from 60 to 2000
and
preferably 60-700, and have difunctionality to permit coupling of two oligo
units.
Preferably the LINK B molecules are synthesized from diamines, diisocyanates,
disulfonic acids, dicarboxylic acids, diacid chlorides and dialdehydes.
Terminal
hydroxyls, amines or carboxylic acids on the oligo molecules can react with
diamines to
form oligo-amides; react with diisocyanates to form oligo-urethanes, oligo-
ureas, oligo-
amides; react with disulfonic acids to form oligo-sulfonates, oligo-
sulfonamides; react
with dicarboxylic acids to form oligo-esters, oligo-amides; react with diacid
chlorides to
form oligo-esters, oligo-amides; and react with dialdehydes to form oligo-
acetal, oligo-
imines.
By the term "pharmaceutical or biologically active agent", or precursor
thereof, is
meant a molecule that can be coupled to LINK A segment via hydrolysable
covalent
bonding. The molecule must have some specific and intended pharmaceutical or
biological action. Typically the [Bio] unit has a molecular weight ranging
from 40 to
2000 for pharmaceuticals but may be higher for biopharmaceuticals depending on
the
structure of the molecule. Preferably, the Bio unit is selected from the group
of anti-
inflammatory, anti-oxidant, anti-coagulant, anti-microbial (including
fluoroquinolones),
cell receptor ligands and bio-adhesive molecules, specifically oligo-peptides
and oligo-
saccharides, oligonucleic acid sequences for DNA and gene sequence bonding,
and
phospholipid head groups to provide cell membrane mimics. The Bio component
must
have difunctional groups selected from hydroxyl, amine, carboxylic acid or
sulfonic acid
so that after coupling with Link A molecule, said biomonomer can react with
the
secondary groups of oligomeric segment to form LINK B linkage. The said
secondary
group may be protected during the reaction of primary groups with the LINK A.
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Table 2. Typical Pharmaceutical Molecules Used For The Synthesis Of
BiomonomerCoupling Agents
Pharmaceuticals Function Chemical structures
. 0 0
Norfloxacin Antimicrobial F
COON
Ciprofloxacin Antimicrobial 0 0
F 0C
C
0OON 1
HN\ ......7 11
Amfenac Antiinflammatory
c112¨ CO 2H
ll m,
0 ¨2
Aceclofenac Antiinflammatory
Cl. 9 r,
40 ,TH cii 2¨ -
-'0¨cH 2¨Co 2H
Cl.
o
Oxaceproll Antiinflammatory
OH
>----
0
Enoxolonel Antiinflammatory 002H
0 H op
00
O
HOS 111
Bromofenac Antithronibic
Ir .2,02.
B. ..2
Tirofibanl Antithrombic HN
CcyCOIN40
0 A ,
0' '0
0
Lotrafibanl Antithrombic /
N
HN,...
r.....õ0.,
H s---002H
12 .
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Acivicin Antiproliferation ci 0
-0 ,fliz
Alkeren Antiproliferation COON
NH2
This disclosure is of particular value to those pharmacologically active
compounds
which are bioresponsive as hereinabove defined to provide in vivo a
pharmacological active
ingredient which has at least two functional groups but one of the functional
groups has low
reactivity with diisocyanates to form oligo-urethanes, or oligo-ureas, oligo-
amides; react with
disulfonic acids to form oligo-sulfonates, oligo-sulfonamides; react with
dicarboxylic acids to
form oligo-esters, oligo-amides; react with diacid chlorides to form oligo-
esters, oligo-amides;
and react with dialdehydes to form oligo-acetal, oligo-imines. Such a
pharmacological agent
would include the fluoroquinolone family of antibiotics, or anti-coagulants,
anti-inflammatory
or anti-proliferative agents of the type listed in Table 2 above.
The present disclosure is of particular use wherein the pharmacologically-
active
fragment is formed from the antibacterial 7-amino-l-cyclopropy1-4-oxo-1,4-
dihydroquinoline
and naphthyridine-3-carboxylic acids described in U.S. Pat. No. 4,670,444. The
most preferred
antibacterial members of these classes of compounds is 1-cyclopropy1-6-fluoro-
1,4-dihyro-4-
oxo-7-piperazine-quinoline-3-carboxylic acid and 1-ethy1-6-fluoro-1,4-dihyro-4-
oxo-7-
piperazine-quinoline-3-carboxylic acid having the generic name ciprofloxacin
and norfloxacin,
respectively. Others of this class include sparfloxacin and trovafloxacin.
Without being bound by theory, it is believed that the presence of LINK A as
hereindefmed, allows of a satisfactory "inter-bio distance" in the
biologically-active polymer
according to the invention, which inter-bio distance facilitates hydrolysis in
vivo to release the
biologically-active ingredient. LINK A offers a range of hydrolysis rates by
reason of chain
length variation and possibly, also, due to steric and conformational
variations resulting from
the variations in chain length.
Prior art compounds not having LINK A chain length variations but having LINK
B
chain lengths between the two biological entities cannot provide this
advantageous variations in
hydrolysis rates.
The present disclosure is of particular use wherein the pharmacologically-
active
fragment is formed from the anti-inflammatory (2S,35)-1-Acety1-4-hydroxy-
pyrrolidine-2-
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carboxylic acid having generic name Oxaceprol and
(2S4aS,6aS,6bR,8aR,10S,12aS,12bR,
14bR)10-hydroxy-2,4a,6a,6b,9,9,12 a-heptamethyl-13 -oxo-
1,2,3 ,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydro-picene-2-
carboxylic
acid having the generic name Enoxolone.
The present disclosure is of particular use wherein the pharmacologically-
active
fragment is formed from the anti-thrombic (S)-2-(butane-l-sulfonylamino)-314-
(4-piperidin-4-
yl-butoxy)phenyl]-propionic acid having the generic name Tirofibanc and [(S)-7-
([4,4lbipiperidiny1-1-carbony1)-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-benzo [e]
[1,4] diazepin-2 -
yl] -acetic acid having the generic name Lotrafiban.
The present disclosure is of particular use wherein the pharmacologically-
active
fragment is formed from the anti-neuplastic (aS, 5S)-a-amino-3-chloro-2-
isoxazoleacetic-5-
acetic acid having the generic name Acivicin and 4-[Bis(2-chloroethyl)amino+L-
phenylalanine having the generic name Alkeren.
The oligomeric polymeric segment preferably has a molecular weight of <10,000;
and
more preferably, <5,000.
The term "theoretical molecular weight" in this specification is the term
given to the
absolute molecular weight that would result from the reaction of the reagents
utilized to
synthesize any given bioactive polymers. As is well known in the art, the
actual measurement
of the absolute molecular weight is complicated by physical limitations in the
molecular weight
analysis of polymers using gel permeation chromatography methods. Hence, a
polystyrene
equivalent molecular weight is reported for gel permeation chromatography
measurements.
Since many pharmaceutically active compounds absorb light in the UV region,
the gel
permeation chromatography technique also provides a method to detect the
distribution of
pharmaceutically active compound coupled within polymer chains.
The polymeric materials of use herein have polystyrene equivalent molecular
weights of
chains ranging from 2x103 to 1x106, and preferably in the range of 2x103 to
2x105.
In a further aspect, this disclosure provides compositions of polymers
containing
biomonomers alone or a base polymer in admixture with polymers containing
biomonomers, as
hereinabove defined, preferably in the form of a shaped article.
Examples of typical base polymers of use in admixture with aforesaid bioactive
polymers includes polyurethanes, polysulfones, polycarbonates, polyesters,
polyethylene,
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polypropylene, polystyrene, polysilicone, poly(acrylonitrile-
butadienestyrene), polyamide,
polybutadiene, polyisoprene, polymethylmethacrylate, polyvinylacetate,
polyacrylonitrile,
polyvinyl chloride, polyethylene terephtahate, cellulose and other
polysacharides. Preferred
polymers include polyamides, polyurethanes, polysilicones, polysulfones,
polyolefins,
polyesters, polyvinyl derivatives, polypeptide derivatives and polysaccharide
derivatives. More
preferably, in the case of biodegradable base polymers these would include
segmented
polyurethanes, polyesters, polycarbonates, polysaccharides or polyamides.
The polymers containing said biomonomers, or the admixed compositions may be
used
as a surface covering for an article, or, most preferably, where the polymers
or admixtures are
of a type capable of being formed into 1) a self-supporting structural body,
2) a film; or 3) a
fiber, preferably woven or knit. The composition may comprise a surface or in
whole or in part
of the article, preferably, a biomedical device or device of general
biotechnological use. In the
case of the former, the applications may include cardiac assist devices,
tissue engineering
polymeric scaffolds and related devices, cardiac replacement devices, cardiac
septal patches,
intra aortic balloons, percutaneous cardiac assist devices, extra-corporeal
circuits, A-V fistual,
dialysis components (tubing, filters, membranes, etc.), aphoresis units,
membrane oxygenator,
cardiac by-pass components (tubing, filters, etc.), pericardial sacs, contact
lens, cochlear ear
implants, sutures, sewing rings, cannulas, contraceptives, syringes, o-rings,
bladders, penile
implants, drug delivery systems, drainage tubes , pacemaker lead insulators,
heart valves, blood
bags, coatings for implantable wires, catheters, vascular stents, angioplasty
balloons and
devices, bandages, heart massage cups, tracheal tubes, mammary implant
coatings, artificial
ducts, craniofacial and maxillofacial reconstruction applications, ligaments,
fallopian tubes.
The applications of the latter include the synthesis of bioresorbable polymers
used in products
that are environmentally friendly (including but not limited to garbage bags,
bottles, containers,
storage bags and devices, products which could release reagents into the
environment to control
various biological systems including control of insects, biologically active
pollutants,
elimination of bacterial or viral agents, promoting health related factors
including enhancing
the nutritional value of drinking fluids and foods, or various ointments and
creams that are
applied to biological systems (including humans, animals and other).
In a preferred aspect, this disclosure provides an admixed composition, as
hereinabove
defined, comprising in admixture either a segmented polyurethane, a polyester,
a
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polycarbonate, polysaccharide, polyamide or polysilicone with a compatible
polymer
containing said biomonomer.
The polymers containing said biomonomer are synthesized in a manner that they
contain a polymer segment, i.e. the [oligo] segments and said biomonomer in
the backbone of
polymer containing biochemical function with either inherent anti-coagulant,
anti-
inflammatory, anti-proliferation, anti-oxidant, anti-microbial potential, cell
receptor ligands,
e.g. peptide ligands and bio-adhesive molecules, e.g. oligosaccharides,
oligonucleic acid
sequences for DNA and gene sequence bonding, or a precursor of the bioactive
component.
The in vivo pharmacological activity generated may be, for example, anti-
inflammatory, anti-bacterial, anti-microbial, anti-proliferation, anti-fungal,
but this disclosure is
not limited to such biological activities.
The claimed invention relates to a compound of the general formula (III):
PBio-LINK A-PBio (III),
wherein PBio is formed from a fluoroquinolone comprising at least two
functional groups
selected from hydroxyl, amine, carboxylic acid, and sulfonic acid and wherein
one of said
functional groups is functional to permit step growth polymerization, and
wherein PBio is
linked to LINK A through a hydrolysable covalent bond; and LINK A is a coupled
central
flexible linear first segment of <2000 Daltons of theoretical molecular weight
linked to each of
said PBio fragments, wherein LINK A comprises polyalkyl, polyethylene oxide,
polyalkylene
oxide, polyamides, polyester, polyvinyl, or polysiloxanes. The claimed
compound may be (a)
NORF-TEG-NORF, wherein PBio is Norfloxacin (NORF) and LINK A is tri(ethylene
glycol)
(TEG); (b) CIPRO-TEG-CIPRO, wherein PBio is Ciprofloxacin (CIPRO) and LINK A
is
tri(ethylene glycol) (TEG); (c) CIPRO-HDL-CIPRO, wherein PBio is Ciprofloxacin
(CIPRO)
and LINK A is hexane diol (HDL); or (d) NORF-HDA-NORF, wherein PBio is
Norfloxacin
(NORF) and LINK A is hexamethylene diamine.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the invention may be better understood, preferred embodiments
will now
be described by way of example only, with reference to the accompanying
drawings wherein:
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Figure 1. is a proton nuclear magnetic resonance spectrum of biomononer
(coupling
agent) NORF-TEG-NORF
Figure 2. is the carbon nuclear magnetic resonanece spectrum of biomonomer NOF-
TEG-
NORF
Figure 3. is a positive electrospray mass spectum of biomonomer NORF-TEG-NORF
Figure 4. is a proton nuclear magnetic resonance spectrum of biomononer CIPRO-
TEG-
CIPRO
Figure 5. is a carbon nuclear magnetic resonanece spectrum of biomonomer of
CIPRO-
TEG-CIPRO
Figure 6. is a positive electrospray mass spectum of biomonomer CIPRO-TEG-
CIPRO
Figure 7. is a proton nuclear magnetic resonance spectrum of POC
Figure 8. is a carbon nuclear magnetic resonanece spectrum of POC
Figure 9. is a positive electrospray mass spectum of POC
Figure 10. is a proton nuclear magnetic resonance spectrum of biomononerPOC-
TEG-
POC
Figure 11. is a carbon nuclear magnetic resonance spectrum of biomononerof POC-
TEG-
POC
Figure 12. is a positive electrospray mass spectum of POC-TEG-POC
Figure 13. is a proton nuclear magnetic resonance spectrum of PAK
Figure 14. is a carbon nuclear magnetic resonance spectrum of PAK
Figure 15. is a positive electrospray mass spectum of PAK
Figure 16. is a proton nuclear magnetic resonance spectrum of biomononer PAK-
TEG-
PAK
Figure 17. is a proton nuclear magnetic resonance spectrum of biomononer PAK-
TEG-
PAK
Figure 18. is a positive electrospray mass spectum of biomononer PAK-TEG-PAK
Figure 19. is a gel permeation chromatography analysis of THDI/PCL/NORF
Figure 20. is a gel permeation chromatography analysis of THDI/PCL/CIPRO
Figure 21. is a cytotoxicity test of control polymer and drug polymers with
mammalian
cells
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Figure 22. is a graph of the released norfloxacin from NF polymer in the
presence and
absence of cholesterol esterase; and
Figure 23. is a graph of Bacteria Counts from Implanted Coupons
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Synthesis of Biomonomers.
A description of the novel process for preparing the biological coupling
agents/biomonomers of production D is set forth in Scheme A, where, R is
CH2CH3 or
cyclopropyl for norfloxacin and ciprofloxacin, respectively. Typically, linkA
molecules
have molecular weights ranging from 60 to 2000 and preferably 60 to 700, and
must have
at least di-functionality to permit coupling of at least two [Bio] units. The
[Bio] unit has
a molecular weight < 2000 but may be higher depending on the structure of the
molecule.
Preferred [Bio] components include but are not limited to the following
categries and
examples: Anti-inflammatory: non-steroidal- Oxaceprol, steroidal Enoxolone;
antithrombotic: Tirofiban, Lotrafiban; anti-coagulant: heparin; anti-
proliferation: acivicin
and alkeren; anti-microbial: fluoroquinolones such as norfloxancin,
ciprofloxacin,
sparfloxacin and trovafloxacin and other fluoroquinolones.
Scheme A provides a general synthetic procedure for preparing the compounds of
product D with formula (I).
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OH
el el
lel
F ell
/----\ p p
4- Ste A Ste B
. -CI 0- Product A
).-
HN \ 7
r)I CHC13 4hrs
Me0H, 50 C
R
1101
F 0 . .
,---, , 1 OH Step C o
lik ¨ N \ 7 ='- (11 Tri(ethylene glycol)
DMAP
0 R EDAC
in DCM
Rm temp. I week
product B
1010 F= =
I 1
0(CH2CH20) 2CH2CH20 el el
Oil
N \ 7
111 1)1 EV\ t - 11
111101 R
1%H20 + ' R
1%CF3COOH
Step D Product C
in DCM
V
I 7
Fyl,.. F
0(CH2CH20) 2CH2CH20
r---\ I
m
HI\ 7
'? N NH
\_____/
R R
Formula (I) Product D =
R: CH2C H3: Norfloxacin
/c : Ciprofloxacin
DMAP: 4-(dimethylamino)pyridine
EDAC: 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide
DCM: Dichloromethane
Scheme 1: Synthetic route for bioactive monomers
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In step A, a pharmaceutically active drug, such as norfloxacin or
ciprofloxacin (in
the form of hydrochloride salt) is reacted with protecting groups such as
trityl halides in
the presence of triethylene amine to provide an intermediate with both amine
and
carboxylic acid groups protected with a trityl group. It is understood by
those skilled in
the art that other protecting groups can be used as exemplified in this
document's
examples.
A suitable trityl halide is reacted with norfloxacin or ciprofloxacin
hydrochloride
=
salt in a suitable solvent, such as chloroform: Many other solvents may be
needed
depending on the solubility of the selected protecting groups and the agents
forming the
biomonomer. Suitable trityl halides include trityl chloride and trityl
bromide. A
preferred trityl halide is trityl chloride. The amount of trityl halide ranges
from 2 to 4
molar equivalent of norfloxacin/ciprofloxacin, a preferred amount is 2.2 molar
equivalents. Triethylamine is added to scavenge free HC1 which is generated as
a by-
product. A little excess amount of triethylamine will avoid the deprotection
of the N-
triethylamine group in the following selective hyrolyzation step. In the case
of
ciprofloxacin, an excess molar amount of triethylene amine such as 2 to 4
times was
added into reaction mixture. A preferred amount is 3 times. The reaction
mixture is
stirred for a period of time ranging from 2-24 hours in a temperature range of
0 C to 60
C. A preferred stirring time is 4 hours and a preferred temperature is 25 C.
A
homogenous solution is obtained. Following this step, product A is left in the
reaction
solution for the next step of the in-situ reaction. No isolation of the
product A is required
during processing.
In step B, the reaction product of step A, such as norfloxacin/ciprofloxacin
with
both amine and carboxylic acid groups protected with trityl group, is
selectively
deprotected to yield product B containing free carboxylic acid and N-
triethylamine
groups.
For example, in step B, a large amount of methanol was added into the reaction
mixture of step A. The volume of methanol ranges from equivalent to two times
that of
the solvent used in step A. A preferred volume is 1.5 times that of the
solvent volume.
The reaction mixture is stirred for 1-24 hrs in a temperature range from 25 C
to 60 C. A
preferred stirring time is 2 hrs and a preferred temperature is 50 C. The
selectively
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deprotected fluroquinolone material is precipitated from the reaction
solution. Product B
is recovered from the reaction zone by filtration after the reaction mixture
is cooled down
to room temperature. Product B is further purified from CHC13/Methanol (9:1)
by
standard recrystallization method.
In step C, the purified amine-protected fluroquinolone is coupled to both
sides of
a diol or diamine (in this example, triethylene glycol is used) containing a
flexible and/or
water-soluble central portion.
For example, the purified amine-protected fluroquinolone (Product B) is
coupled
to a tri(ethylene glycol) in the presence of a suitable coupling agent such as
1-ethy1-3-(3-
dimethylamino-propyl)carbodiimide herein denoted as EDAC and an appropriate
base
such as 4-(dimethylamino)pyridine herein denoted as DMAP as a catalyst. Other
coupling reagents may include various carbodiimides such as CMC (1-cyclohexy1-
3-(2-
morpholinoethyl)carbodiimide), DCC (N,N'-dicyclohexyl- carbodiimide), DIC
(Diisopropyl carbodiimide) etc, but are not limited to these. The amount of
diol ranges
from 0.3 to 0.5 molar equivalent of product B. A preferred amount of diol is
0.475 molar
equivalent of product B. The amount of coupling agent EDAC ranges from 2 to10
times
molar equivalent of product B. A preferred amount of EDAC is 8 times molar
equivalent. The amount of base DMAP can range from 0.1 to equal molar amount
of
product B. A preferred amount is 0.5 molar equivalents. The reaction was
carried out in
a suitable solvent such as dichloromethan.e under a noble atmosphere such as
nitrogen,
argon. Other solvents may be appropriate depending on their solubility
properties with
product B and their potential reactivity with the reagents. The reactants are
typically
stirred together for a period of time ranging from 24 hours to 2 weeks at a
temperature
range from 0 C to 50 C. A preferred stirring time is one week and a
preferred
temperature is 25 C.
After the reaction is finished, solvent is removed by rotary evaporator. The
residues are washed with water several times to remove soluble reagents such
as EDAC.
The solids are then dissolved in chloroform. Product C in Scheme 1 is
recovered from
the solution by standard extractive methods using chloroform as the extraction
solvent.
Product C was isolated by column chromatography using a developer made up of
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chloroform/methanol/ammonia hydroxide aqueous solution (9.2:0.6:0.2). Product
C is
further purified with recrystallization techniques from chloroform and
methanol.
In step D, the N-trietylamine groups of the purified product C are deprotected
to
yield the corresponding desired pharmaceutical coupling agent/biomonomer.
For example, the appropriate product C is reacted with a small amount of water
in
the presence of a small amount of weak acid, such as trifluoroacetic acid, in
a suitable
organic solvent such as dichloromethane. The amount of water can range from 1%
to
10% volume percentage and a preferred amount is 1%. The amount of
trifluoroacetic
acid is between 1% to 10% volume percent, with a preferred amount being 2%.
The
reaction mixture is stirred within a temperature range of 0 C to 50 C over a
time period
of 2 to 24 hours. A preferred temperature is 25 C and a preferred time period
is 4 hours.
Product D is precipitated from reaction solution and collected by filtration.
The product
is further purified by washing with CHC13.
Use of Biomonomers in a Polymer Synthesis.
The pharmaceutically active polymers are synthesized in a traditional stepwise
polymerization manner as are well known in the art. A multi-functional LINK B
molecule and a multi-functional oligo molecule are reacted to form a
prepolymer. The
prepolymer chain is extended with said biomonomer to yield a polymer
containing the
biomonomers. Non- biological extenders such as an ethylene diarnine, butane
diol,
ethylene glycol and others may also be used. The linkB molecule is preferably,
but not
so limited, to be di-functional in nature, in order to favour the formation of
a linear,
polymer containing biomonomers. Preferred linkB molecules for biomedical and
biotechnology applications are diisocyanates: for example, 2,4 toluene
diisocyanate; 2,6
toluene diisocyanate; methylene bis(p-phenyl)diisocyanate; lysine diisocyanato
esters;
1,6 hexane diisocyanate; 1,12 dodecane diisocyanate; bis-methylene
di(cyclohexyl
isocyanate); trimethyl-1,6 diisocyanatohexane, dicarboxylic acids,. di-acid
chlorides,
disulfonyl chlorides or others. The oligo component is preferably, but not so
limited,
difunctional, in order to favor the formation of a linear polymer containing
said
biomonomers. Preferred oligo components are terminal diamine and diol reagents
of: for
example, polycarbonate, polysiloxanes, polydimethylsiloxanes; polyethylene-
butylene
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co-polymers; polybutadienes; polyesters including polycaprolactones,
polylactic acid, and
other polyesters; polyurethane/sulfone co-polymer; polyurethanes; polyamides;
including
oligopeptides (polyalanine, polyglycine or copolymers of amino-acids) and
polyureas;
polyalkylene oxides and specifically polypropylene oxide, polyethylene oxide
and
polytetramethylene oxide. The molecular weights of the [oligo] groups are less
than
10,000, but preferably have molecular weights of less than 5000. Synthesis of
the
prepolymers to the bioactive polymer can be carried out by classical
urethane/urea
reactions using the desired combination of reagents but with the excess amount
of linkB
molecules in order to end-cap the prepolymer with linkB molecule. When the
prepolymer with desired chain length is reached, said biomonomer is added to
extend the
prepolymer chain giving a final bioactive polymer. Alternatively the
biomonomers may
be substituted for inclusion as the oligo groups.
Bioactive polymers can be synthesized with different components and
stoichometry. Prior to synthesis, the LINK B molecules are, preferably, vacuum
distilled
to remove residual moisture. The biomonomers are desiccated to remove all
moisture.
Oligo components are degassed overnight to remove residual moisture and low
molecular
weight organics.
While reactants can be reacted in the absence of solvents if practical, it is
preferable to use organic solvents compatible with the chemical nature of the
reagents, in
order to have good control over the characteristics of the final product.
Typical organic
solvents include, for example, dimethylacetamide, acetone, tetrahydrofuran,
ether,
chloroform, dimethylsulfoxide and dimethylformamide. A preferred reaction
solvent is
dimethylsulfoxide (DMSO, Aldrich Chemical Company, Milwaukee, Wis.).
In view of the low reaction activity of some diisocyanates, e.g. DDI and THDI,
with oligo precursor diols, a catalyst is preferred for the synthesis. Typical
catalysts are
similar to those used in the synthesis of urethane chemistry and, include,
dibutyltin
dilaurate, stannous octoate, N,N' diethylcyclohexylamine, N-methylmorpholine,
1,4 diazo
(2,2,2) bicyclo-octane and zirconium complexes such as Zr tetrakis (2,4-
pentanedionato)
complex.
In the first step of the preparation of a prepolymer, for example, the linkB
molecules are added to the oligo component and, optionally, catalyst to
provide the
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prepolymer of the bioactive polymer. The reaction mixture is stirred at a
temperature of
60 C for a suitable time period, which depends on the reaction components and
the
stoichiometry. Alternate temperatures can range between 25 C to 110 C.
Subsequently,
said biomonomer is added to the prepolymer and, generally, the mixture is
allowed to
react overnight. The reaction is terminated with methanol and the product is
precipitated
in ether or a mixture of distilled water with ether or other suitable
solvents. The
precipitate is dissolved in a suitable solvent, such as acetone and
precipitated in ether or a
mixture of distilled water with ether again. This process was repeated 3 times
in order to
remove any residual catalyst compound. Following washing, the product is dried
under
vacuum at 40 C. =
Alternatively, the biomonomers can be used to make polyamides using classical
reactions such as those described below.
Fabrication of product:
The pharmaceutical polymers containing biomonomers are either used alone or
admixed
with suitable amounts of base polymers in the fabrication of article products.
If admixed
in a blend, then suitable polymers may include polyurethane, polyester or
other base
polymers. Product may be formed by; 1) compounding methods for subsequent
extrusion
or injection molding or articles; 2) co-dissolving of base polymer with
bioactive polymer
into a solvent of common compatibility for subsequent casting of an article in
a mold or
for spinning fibers to fabricate an article; 3) wetting the surface of an
article with a
solution of bioactive polymer or a blend in solvent of common compatibility
with a
polyurethane or other polymer to which the bioactive polymer solution is being
applied;
or 4) in admixture with a curable polyurethane, for example, 2 part curing
system such as
a veneer. All of the above processes can be used with the pure polymer,
containing the
biomonomer groups or with blends of said polymer and common biomedical
polymers.
The invention, thus, provides the ability to synthesize a range of novel
polymeric
materials possessing intramolecular properties of pharmaceutical or biological
nature.
When said polymers are used alone or in admixture with, for example, a
polyurethane,
the bioactive polymer provides the composite having better pharmaceutical
function,
particularly for use in medical devices, promoting cell function and
regulation, tissue
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integration, pro-active blood compatibility and specifically anti-
coagulant/platelet
function, biostability function, anti-microbial function and anti-inflammatory
function, or
for use in the biotechnology sector for biological activity.
The application for these materials include the synthesis of bioresorbable
polymers
used in medical device products that require the delivery of biologicals,
pharmaceuticals or
the release of biocompatible materials upon biodegradation within or in
contact with a
biological body (human or animal). This includes the manufacturing of products
in the
form of films (cast or heat formed), fibres (solvent or melt spun), formed
into composite
materials (polymers combined in any form with ceramics, metals or other
polymers) of any
shape, injection molded, compression molded, extruded products. Such product
can
include but are not limited to: cardiac assist devices, tissue engineering
polymeric
scaffolds and related devices, cardiac replacement devices, cardiac septal
patches, intra
aortic balloons, percutaneous cardiac assist devices, extra-corporeal
circuits, A-V fistual,
dialysis components (tubing, filters, membranes, etc.), aphoresis units,
membrane
oxygenator, cardiac by-pass components(tubing, filters, etc.), pericardial
sacs, contact
lens, cochlear ear implants, sutures, sewing rings, cannulas, contraceptives,
syringes, o-
rings, bladders, penile implants, drug delivery systems, drainage tubes ,
pacemaker lead
insulators, heart valves, blood bags, coatings for implantable wires,
catheters, vascular
stents, angioplasty balloons and devices, bandages, heart massage cups,
tracheal tubes,
mammary implant coatings, artificial ducts, craniofacial and maxillofacial
reconstruction
applications, ligaments, fallopian tubes.
Other non-medical applications may include of bioresorbable polymers used in
products that are environmentally friendly (including but not limited to
garbage bags,
bottles, containers, storage bags and devices, products which could release
reagents into the
environment to control various biological systems including control of
insects, biologically
active pollutants, elimination of bacterial or viral agents, promoting health
related factors
including enhancing the nutritional value of drinking fluids and foods, or
various ointments
and creams that are applied to biological systems (including humans, animals
and other).
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In these examples, the following acronyms are used.
NORF (Norfloxacin)
CIPRO (Ciprofloxacin)
OC (Oxaceprol)
POC (Protected Oxaceprol)
TF (Tirofiban)
PTF (Protected Tirofiban)
AK (Alkeren)
PAK (Protected Alkeren)
AF (Amfenac)
AV (Acivicin)
BF (Bromfenac)
TEG (Triethylene glycol)
HDL (1,6-Hexanediol)
HDA (1,6-Hexanediamine)
TrC1 (Trityl Chloride)
* DMAP (4-(dimethylamino)pyridine)
EDAC (1-ethy1-3-(3-dimethylamino-propyl)carbodiimide)
TEA (Triethylene amine)
TFA (Trifluoroacetic acid)
THDI (trimethyl- 1,6 diisocyanatohexane)
PCL polycarprolactone diol
AC (Adipoyl Chloride)
THDI/PCL/TEG (segmented polyurethane)
DBTL (dibutyltin dilaurate)
DCM (Dichloromethane)
DMF (dimethylformamide)
TLC (thin layer chromatography)
CC (Column chromatography)
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Where appropriate all isocyanate reactions were catalysed with DBTL
(dibutyltin
dilaurate).
Nuclear magnetic resonance was used to identify the structure of the
biomonomer.
Mass spectroscopy was used to confirm the molar mass of the synthesized
biomonomer.
Gel permeation chromatography was used to define the distribution of [Bio] the
moiety within the drug polymer and to estimate relative molecular weights of
the
polymer.
Characterization of tin residues located at the surface of the drug polymer
coatings was demonstrated using X-ray photoelectron spectroscopy (measuring
chemical
composition) at 90 degree. Elimination of tin residues is important for
biological
applications since the latter is toxic.
In vitro evaluation of antimicrobial release and biodegradation were performed
in
order to assess the rates of degradation for the different antimicrobial
polymer
formulations and determines periods of efficacy. In these studies the polymers
are
incubated with enzyme and the solution is recovered for separation of
degradation
products. Hydrolytic enzymes related to monocyte macrophages, specifically
cholesterol
esterase, and neutrophils (elastase), with in a pH 7 phosphate buffered saline
solution
may be used for in vitro tests over a 10-week time frame. Degradation products
may be
characterized using High Performance Liquid Chromatography (HPLC), combined
with
mass spectroscopy.
Minimum inhibitory concentration (MIC) assays were used to evaluate the
antimicrobial activity of incubating solutions obtained from drug polymer
biodegradation
studies against P. aeruginosa. Turbidity of each culture was recorded to
evaluate the
inhibitory properties of degradation solution of drug polymers.
Sterilization stability of drug polymers was estimated after drug polymers
were
sterilized by 7-radiation sterilization (radiation dose: 25 Kgy), a standard
method in the
medical device field. GPC measurements were carried on with these samples
before and
after they were radiated and after a time period of 1 to 4 weeks.
Biocompatibility study of the drug polymers was also performed in order to
assess
the biocompatibility of control and drug polymers with mammalian cells. In
this study,
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HeLa cells were cultured directly onto the polyurethane polymers films and
incubated at
37 C for 24 hours. Cell viability was measured by staining for succinate
dehydrogenase.
In vivo animal studies are performed on substrates, devices or articles
according
to the invention formed in whole or in part of bioactive polymers. The
articles containing
either bioactive polymer or non-bioactive control polymer were implanted in
the
peritonitis of male rats accompanied with an inoculation of P. aeurogniosa
bacteria. The
articles were explanted after rats were housed for 1 week. The effect of the
antimicrobial
polymer was evaluated.
EXAMPLES
The following examples illustrate the preparation of biomonomers and
bioresponsive pharmacologically active polymers according to the invention.
Example 1:
NORF-TEG-NORF and CIPRO-TEG-CIPRO are examples of antimicrobial drug
containing biomonomers according to the invention. The example shows the use
of a
single drug or combination of drugs. The conditions of synthesis for this
reaction are as
follows.
In step A, of NORF(1.3g, 4 mmol) / or CIPRO hydrochloride salt (4 mmol) were
reacted with trityl chloride (2.7g, 8.8 mmol) and TEA(0.6m1, 8 mmol) (Aldrich,
99%)/or
12 mmol of TEA in the case of CIPRO in 40 ml of CHC13 for four hours at room
temperature. A clear solution was obtained.
In step B, 40 ml of methanol was added into the above clear solution. The
mixture was heated to 50 C and stirred for one hour; a precipitate appeared
in the
solution. After the reaction mixture was cooled down to room temperature,
precipitates
were collected by filtration. The precipitate was further purified from
CHC13/methanol.
3.4 mmol of Product B were obtained. Yield was usually greater than 85%.
In step C, Product B (20 mmol), TEG (1.44g, 9.5 mmol), DMAP (1.24g, lOmmol)
were dissolved in 100 ml DCM. EDAC (31g, 160 mmol) was then added into the
reaction system. The reaction mixture was stirred at room temperature under a
nitrogen
atmosphere for one week. After reaction was finished, DCM was removed by
rotary
evaporator. The residues were washed with de-ionized water several times to
remove
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soluble reagents such as the by-product of urea. The solids were then
dissolved in
chloroform and washed with de-ionized water again. The crude product of the
reaction
was recovered from the solution by extraction. Product C was isolated by
column
chromatograph using the developer of chloroform/methanol/ammonia hydroxide
aqueous
solution (9.2:0.6:0.2). Product C is further purified with recrystallization
technique from
chloroform and methanol. Product C can be obtained with a yield of 85%.
In step D, the purified product C (5.4g, 4.4 mmol) was dissolved in chloroform
containing one volume percent of water and 1 volume percent of trifluoroacetic
acid. The
reaction solution was stirred at room temperature for 4 hrs. White
precipitates that were
produced in the reaction were collected by filtration and purified by washing
with
chloroform. Following washing Product D, i.e. the biomonomer was dried in
vacuum
oven for 24 hours at a temperature of 40 C. The pure Product D i.e. said
biomonomer
can be obtained with a yield of 95%.
1H NMR of NORF-TEG-NORF: (400 MHz, DMSO). 8: 9.33 (bs, 2H, NR), 8.52
(s, 2H, H2, ar), 7.66 (d, 2H, J= 13.6 Hz, H5, ar), 7.01 (d, 2H, J= 7.2Hz, 1/8,
ar), 4.33 (q,
4H, J = 6.8 Hz, N-CH2-CH3), 4.26 (t, 4H, J = 4.8 Hz, CO2CH2), 3.71 (t, 4H, J
=4.8 Hz,
CO2CH2CH2) 3.48-3.28 (m, 16H, piperazine), 1.33 (t, 6H, J = 6.8 Hz, NCH2CH3).
[FIGURE 1]
13C NMR of NORF-TEG-NORF: (400 MHz, DMSO). 8: 171.9, 164,7, 159.3,
159.0, 153.8, 151.4, 149.0, 143.4, 143.3, 136.4, 123.4, 122.0, 119.0, 116.0,
112.4, 109.4,
106.6, 70.4, 68.9, 63.6, 48.6, 47.1, 43.1, 43.0, 14.6, [FIGURE 2]
ES-MS of NORF-TEG-NORF (m/z, %) (Positive mode): Calculated for mass
C38H46F2N608: 752 amu, found 753, 377 (M+2H)+.[FIGURE 3]
1H NMR of CIPRO-TEG-CIPRO: (400 MHz, DMSO). 8: 9.16 (bs, 2H, NH-R),
8.30 (s, 2H, H2, ar), 7.49 (d, 2H, J = 13.2 Hz, H5, ar), 7.34 (d, 2H, J = 7.6
Hz, H8, ar),
4.25 (t, 4H, J = 5.2 Hz, N-CH(CH2)2); 3.73 (t, 411, J = 4.4 Hz, CO2CH2), 3.46-
3.30(m,16H, piperazine), 1,22 (q, 4H, J = 6.4 Hz, CH(CH2 CH2)), 1.07 (m,
4H,CH(CH2CH2)). [FIGURE 4]
13C NMR of CIPRO-TEG-CIPRO: (400 MHz, DMSO). 8: 171.9, 164.1, 158.7,
153.9, 151.5, 148.4, 143.0, 142.9, 138.1, 122.6, 122.5, 111.9, 111.7, 109.2,
107.0, 79.6,
70.5, 70.4, 68.9, 63.7, 47.0, 43.2, 35.3, 7.9. [FIGURE 5]
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ES-MS of CIPRO-TEG-CIPRO (rn/z, %) (Positive mode): Calculated for mass
C40R46F2N608: 776 amu, found 777 (M+H+); 389 (M+2H)+. [FIGURE 61
, Example 2:
CIPRO-HDL-CIPRO is example of biomonomer according to the invention and
different from example 1 by the introduction of a hydrophobic link A molecule
rather
than hydrophilic link A molecule. The conditions of synthesis for this
reaction are as
follows.
The reaction conditions for selectively protecting amine groups of CIPRO are
the
same as the step A and B in Example1.
In step C, Product B (20 mmol), HDL (9.5 mmol), DMAP (1.24g, 1 Ommol) were
dissolved in 100 ml DCM. EDAC (31g, 160 mmol) was then added into reaction
system.
The reaction mixture was stirred at room temperature under a nitrogen
atmosphere for
one week. After the reaction was finished, DCM was removed by rotary
evaporator. The
residues were washed with de-ionized water several times to remove soluble
reagents
such as the by-product of urea. The solids were then dissolved in chloroform
and washed
with de-ionized water again. The crude product of the reaction was recovered
from the
solution by extraction. Product C was isolated by column chromatography using
the
developer of chloroform/methanol/ammonia hydroxide aqueous solution
(9.2:0.6:0.2).
Product C is further purified with a recrystallization technique from
chloroform and
methanol.
In step D, the purified product C (4 mmol) was dissolved in chloroform
containing one volume percent of water and 1 volume percent of trifluoroacetic
acid. The
reaction solution was stirred at room temperature for 4 hrs. White
precipitates produced
in the reaction were collected by filtration and purified by washing with
chloroform.
Following washing Product D, i.e. the biomonomer was dried in vacuum oven for
24
hours at a temperature of 40 C.
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Example 3:
NORF-HDA-NORF is example of biomonomer according to the invention and
different from example 1 in that a diamine is used to generate an amide rather
than ester
linkage in the biomonomer. The conditions of synthesis for this reaction are
as follows.
The reaction conditions for selectively protecting amine groups of NORF are
the
same as the step A and B in Example 1.
In step C, Product B (20 mmol), HDA (9.5 mmol), DMAP (1.24g, lOmmol) were
dissolved in 100 ml DCM. EDAC (31g, 160 mmol) was then added into reaction
system.
The reaction mixture was stirred at room temperature under a nitrogen
atmosphere for
one week. After the reaction was finished, DCM was removed by rotary
evaporator. The
residues were washed with de-ionized water several times to remove soluble
reagents
such as the by-product of urea. The solids were then dissolved in chloroform
and washed
with de-ionized water again. The crude product of the reaction was recovered
from the
solution by extraction. Product C was isolated by column chromatography using
the
developer of chloroform/methanol/ammonia hydroxyl aqueous solution
(9.2:0.6:0.2).
Product C is further purified with recrystallization technique from chloroform
and
methanol.
In step D, the purified product C (4 mmol) was dissolved in chloroform
containing one volume percent of water and 1 volume percent of trifluoroacetic
acid. The
reaction solution was stirred at room temperature for 4 hrs. White
precipitates produced
in the reaction were collected by filtration and purified by washing with
chloroform.
Following washing Product D, i.e. the biomonomer was dried in vacuum oven for
24
hours at a temperature of 40 C.
Example 4:
0C-TEG-0C is an example of anti-inflammatory drug containing biomonomer
according to the invention. The biomonomer was synthesized using Oxaceprol
(OC), by
reacting the carboxylic acid with the hydroxyl of TEG and leaving the hydroxyl
for
subsequent use in the polymerization. The conditions of synthesis for this
reaction are as
follows.
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In step A, OC (11.55 mmol) was reacted with t-butyldimethylsilyl chloride
(28.87
mmol) and 1,8-diazabicylco[5.4.0]undec-7-ene (30.03 mmol) in 4 ml of
acetonitrile at 0
C during the addition of the base and then overnight at ambient temperature. A
precipitate developed during the progress of the reaction The precipitate was
filtered.
In step B, the filtrate was treated with water (10 ml) and extracted with n-
pentane
(2 x 5 m1). The solvent for the aqueous portion was removed under reduced
atmosphere.
The residue was dissolved in methanol (10 mL), tetrahydrofuran (5 mL), water
(5 mL)
and then treated with 2N aqueous sodium hydroxide (8mL). The reaction mixture
was
stirred for 1.5 h at room temperature, adjusted to a pH = 3 with 1N HCI,
concentrated and
filtered. The precipitate obtained was recrystallized from water and afforded
pure 4 (2.79
g, 84%).
NMR: (400 MHz, CDC13) 8: 4.87 (bs, 1H, CO2H), 4.61 (dd, 1H, J = 8.0 Hz,
6.4Hz, CHCO2H), 4.48 (p, 1H, J = 4.4 Hz, CHOSi), 3.67 (dd, 1H, J = 10.4 Hz,
4.8Hz,
CHHN), 3.36 (dd, 1H, J=10.4 Hz, 6.0Hz ,CHHN), 2.36 (dt, 2H, J =13.2 Hz, 5.2
Hz, 2H,
CH2CHCO2H), 2.12 (s, 3H, COCH3), 0.86 (s, 9H, C(CH3)3), 0.08 (s, 3H, SiCH3),
0.07 (s,
3H, SiCH3). [FIGURE 7]
13C NMR: (400 MHz, CDC13) 8:172.7, 172.3, 70.0, 58.3, 56.2, 37.1, 25.6õ 22.2,
17.9, -4.8, -5Ø [FIGURE 8]
ES-MS (m/z, %) (Negative 'mode): Calculated for mass Ci3H25NO4Si: 287 amu,
found 286.1. [FIGURE 9]
In step C, Product B (3.48 mmol), TEG (1.58 mmol), DMAP (0.16 mmol) were
dissolved in DCM (5 ml). EDAC (3.95 mmol) was then added into the reaction
solution
cooled to 0 C.. The resulting solution was stirred for 1 h at 0 C, the
cooling was
removed, and the mixture was stirred for 5 days at ambient temperature. The
solvent was
removed under reduced pressure. Water (20 mL) and the system was extracted
with
pentane (3 x 10 mL). The combined pentane extracts where dried using sodium
sulphate,
filtered, and the solvent removed under reduced pressure. This produced 0.74 g
(67%) of
the desired product.
= 11-1 NMR: (400 MHz, CDC13) 8: 4.87 (m, 2H, CHCO2), 4.26 (m, 2H, CHOSO,
4.05 (m, 2H, CHHN), 3.74-3.31 (m,4H), 3.31 (m, 2H, CHHN), 2.13 (m, 4H,
CH2CHCO2), 2.12 (s, 6H, COCH3), 2.0 (m, 4H), 1.18 (m, 4H), 0.81 (s, 18H,
C(CH3)3), -
0.003 (s, 6H, SiCH3), -0.03 (s, 6H, SiCH3). [FIGURE 10]
13C NMR: (400 MHz, CDC13) 8: 172.3, 171.0, 72.5, 70.6, 70.4, 64.0, 60.3, 57.5,
57.3, 55.9, 54.3, 52.1, 40.4, 38.3, 34.0, 26.6, 22.1, 20.9, 17.8, 14.1, -4.8, -
5Ø [FIGURE
11]
=
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ES-MS (m/z, %) (Positive mode): Calculated for mass C32H60N2010Si2: 688 amu,
found 689.3. [FIGURE 12] =
In step D, the purified product C (0.7 mmol) was dissolved in THF (5m1). The
resulting solution was cooled to 0 C before the addition of tetra n-butyl
ammonium
fluoride (x ml, 1.4 mmol). The resulting solution was stirred at 0 C for 5
min before the
removal of the ice bath and continued stirring for an additional 40 min at
ambient
temperature. The solvent was removed at reduced atmosphere and the residue was
treated with water and the pH of the solution was adjusted to 3 upon which a
precipitate
resulted. The precipitate was filtered to produce the desired product.
Example 5:
TF-TEG-TF is an example of anti-thrombic drug containing biomonomer
according to the invention. The biomonomer is synthesized using tirofiban
(TF), reacting
the carboxylic acid with the hydroxyl of TEG and leaving the amines for
subsequent use
in the polymerization. The conditions for synthesis for this reaction are as
follows.
In step A, TF(4 mmol) is reacted with trityl chloride (8.8 mmol) and TEA (8
mmol) (Aldrich, 99%) in 40 ml of CHC13 for four hours at room temperature. A
clear
solution is obtained.
In step B, 40 ml of methanol is added into the above clear solution. The
mixture
is heated to 50 C and stirred for one hour, a lot of precipitates appeared in
the solution.
After the reaction mixture is cooled down to room temperature, precipitates
were
collected by filtration. They were further purified from CHC13/methanol. 3.4
mmol of
Product B were obtained.
In step C, Product B (20 mmol), TEG (9.5 mmol), DMAP (1.24g, lOmmol) were
dissolved in 100 ml DCM. EDAC (31g, 160 mmol) is added into the reaction
system.
The reaction mixture is stirred at room temperature under a nitrogen
atmosphere for one
week. After reaction is finished, DCM was removed by rotary evaporator. The
residues
were washed with de-ionized water several times to remove soluble reagents
such as the
by-product of urea. The solids were then dissolved in chloroform and washed
with de-
ionized water again. The crude product of the reaction is recovered from the
solution by
extraction. Product C was isolated by column chromatography using the
developer of
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chloroform/methanol/ammonia hydroxide aqueous solution (9.2:0.6:0.2). Product
C is
further purified with recrystallization technique from chloroform and
methanol.
In step D, the purified product C (4 mmol) is dissolved in chloroform
containing
one volume percent of water and 1 volume percent of trifiuoroacetic acid. The
reaction
solution is stirred at room temperature for 4 hrs. White precipitates produced
in the
reaction were collected by filtration and purified by washing with chloroform.
Following
washing Product D, i.e. the biomonomer is dried in vacuum oven for 24 hours at
a
temperature of 40 C.
Example 6:
AK-TEG-AK is an example of anti -proliferation drug containing biomonomer
according to the invention. The biomonomer was synthesized using Alkeren (AK),
reacting the carboxylic acid with the hydroxyl of TEG and leaving the amines
for
subsequent use in the polymerization. The conditions for synthesis for this
reaction are as
follows,
In step A, AK (0.32 mmol) was reacted with di-tert-butyl carbonate (0.5 mmol)
and TEA(0.32 mmol) (Aldrich, 99%) in THF (4 m1). The suspension was cooled to
0 C
before the addition of the anhydride. Dimethylformamide (0.9 ml) was added to
homogenize the reaction mixture. The solution was stirred for 2 hours at 0 C,
and
thereafter overnight at ambient temperature. The solution is then evaporated
under
reduced pressure and the yellowish oily residue obtained is redissolved in a 5
% aqueous
solution of sodium bicarbonate (3m1). The solution is washed with petroleum
ether ( 3 x
3 ml) and the aqueous phase was acidified to a pH of 3 with a 1N hydrochloric
acid
solution. The mixture was extracted with ethyl acetate (3 x 3 m1). The organic
phases
were dried over anhydrous sodium sulfate, filtered and then evaporated under
reduced
pressure. The residue is dissolved in a mixture of hexane, ethyl acetate, and
acetic acid
(20:10:1) (3 ml). It is subsequently purified by chromatography on a silica
column. This
produced 115 g (86%) of the desired product (Rf = 0.49).
11-1 NMR: (400 MHz, CDC13) 8: 11.02 (bs, 1H, CO21/), 7.08 (d, .2H, J= 5.4 Hz,
Ar-B), 6.64 (d, 2H, J= 5.4 Hz, Ar-B), 4.97 (d, 1H, J = 5 Hz, NH), 4.57 (m, 1H,
CHCO2H), 3.72-3.59 (m, 8H, CH2CH2C1) , 3.12-2.98 (m, 2H, CH2CH), 1.42 (s, 9H,
C(CH3)3). [FIGURE 13]
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13C NMR: (400 MHz, CDC13) 5:177.3, 176.6155.4, 144.9, 130.7, 112.3, 80.2,
54.4, 53.6, 40.3, 28.3, 20.8. [FIGURE 14]
ES-MS (m/z, %) (Positive mode): Calculated for mass C18H2602N204: 404 amu,
found 405.1. [FIGURE 15]
In step C, Product B (0.20 mmol), TEG (0.09 mmol), DMAP (0.009 mmol) were
dissolved in DCM (2m1). To this stirring solution at 0 C was added EDC-HC1
(0.22
mmol) in dichloromethane (1 ml) dropwise over 10 min. The resulting solution
was
stirred for 1 h at 0 C, the cooling was removed, and the mixture was stirred
for 3 days at
ambient temperature. The progress of the reaction was monitored by thin layer
chromatography. Once complete disappearance of starting material was observed,
the
reaction solvent was removed under reduced atmosphere. The product was
purified by
column chromatography (Rf = 0.93) eluting with chloroform: methanol (9: 1).
This
produced 0.8 g (73%) of the desired product.
NMR: (400 MHz, CDC13) 5: 7.03 (d, 4H, J= 5.4 Hz, Ar-H), 6.64 (d, 4H, J= 5.4
Hz, Ar-H), 4.98 (d, 2H, J = 5.0 Hz, NH), 4.55 (m, 2H, CHCO2H), 4.28 (m, 4H,
CO2CH2)
3.72-3.59 (m, 24H, CH2CH2C1, OCH2CH2OCH2) , 3.06-2.95 (m, 4H, CH2CH), 1.42 (s,
18H, C(CH3)3). [FIGURE 16]
13C NMR: (400 MHz, CDC13) 5:177.3, 171.9, 155.0, 144.6, 130.7, 112.5, 79.8,
70.6, 69.0, 64.3, 54.4, 53.7, 40.2, 37.1, 28.3. [FIGURE 17]
ES-MS (m/z, %) (Positive mode): Calculated for mass C42H62C14N40113: 922 amu,
found 923.2. [FIGURE 18]
In step D, the purified product C (2 mmol) was dissolved in chloroform
containing one volume percent of water and 1 volume percent of trifluoroacetic
acid. The
reaction solution was stirred at room temperature for 2 hrs. White
precipitates produced
in the reaction were collected by filtration and purified by washing with
chloroform.
Following washing Product D, i.e. the biomonomer was dried in vacuum oven for
24
hours at a temperature of 40 C.
Example 7:
THDI/PCL/NORF is an example of pharmaceutically active polyurethane
containing 15% of drugs according to the invention. The conditions of
synthesis for this
reaction are as follows.
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1.5 grams of PCL are reacted with 0.27 grams of THDI in the presence of 0.06
ml
of the catalyst, dibutyltin dilaurate, in a nitrogen atmosphere with in
dimethylsulfoxide
(DMSO) (10 mL) for one hour. The reaction temperature is maintained between 60-
70 C. 0.32 grams of NORF-TEG-NORF is dissolved in 5 ml DMSO was then added
into
reaction system. The reaction is keep at 60-70 C for 5 hours and then at room
temperature for overnight. Reaction is finally stopped with 1 ml of methanol.
The final
drug polymer is precipitated in a mixture of ether/water (50 v/v%). The
precipitated
polymer is then dissolved in acetone and precipitated in ether again. This
washing
procedure is repeated three times.
Norfloxacin is the only component in the drug polymer which has a strong
detectable absorbance at 280nm in the UV range. Hence, its presence can be
detected
using a UV detector. Figure 5 super-imposes the UV chromatogram for the drug
polymer
with its universal gel permeation chromatography (GPC) curves using a
universal
refractive index detector. Similar data is shown for a Ciprofloxacin polymer
in Figure 6.
The latter detects the presence of all molecules because it has a dependence
on mass of
material present, eluting out of the GPC column at a specific time. Hence, a
comparison
of the two signals shows that the distribution of norfloxacin is identical to
the distribution
of actual molecular weight chains, meaning that there was no preferential
coupling of
norfloxacin/ciprofloxacin to low versus high molecular weight chains or vice-
versa;
implies that the coupling of norfloxacin/ciprofloxacin was uniform.
Example 8:
AC/CIPRO is an example of pharmaceutically active polyamide containing
antimicrobial drug Ciprofloxacin according to the invention. It differs from
example 1 in
that it is not a polyurethane and shows the versatility for the use of the
biomonomers in a
range of step growth polymerizations. The conditions for this synthesis are a
common
polyamide interfacial polycondensation reaction. They are described as
follows:
A solution of 3.88 g (5 mmol) of CIPRO-TEG-CIPRO and 1.06 g (10 mmol) of
sodium carbonate in 30 ml of water was cooled in an ice bath for 15 min before
addition
of as the water phase to a 150 ml flask containing a stir bar. A organic
solution
containing 0.915 g of adipoyl chloride (AC, 5mmol) in 20 ml of methylene
chloride was
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added slowly into the water phase under vigorously stirring. The organic
solution has
been previously cooled in an ice bath for 15 min. Immediately after addition
of the
organic phase, an additional 5 ml of methylene chloride was used to rinse the
original
acid chloride container and transfer the solvent to reaction flask. The
polymerization
medium was stirred at maximum speed for an additional 5 min. The resulting
polymer
was collected by filtration. The polymer was then washed with water for at
least 3 times.
It was then washed with acetone twice. The product was vacuum-dried at 40 C
for 24
hours.
Example 9:
Gamma irradiation is a popular and well-established process for sterilizing
polymer-based medical devices (21). It has been known, however, that this
technique can
lead to significant alterations in the materials being treated. High-energy
radiation
produces ionization and excitation in polymer molecules. The stabilization
process of the
irradiated polymer results in physical and chemical cross-linking or chain
scission, which
occurs during, immediately after, or even days, weeks after irradiation. In
this example,
NF and CP polymers are dissolved in a suitable solvent such as chloroform at
10%. The
films are cast in a suitable holder such as Teflon mold and placed in a 60 C
air flowing
oven to dry. The dried films are sterilized by gamma radiation. The dose shall
be
capable of achieving the pre-selected sterility assurance level (22). One of
two
approaches shall be taken in selecting the sterilization dose: (a) selection
of sterilization
dose using either 1)bioburden information, or 2)information obtained by
incremental
dosing; b) Selection of a sterilization dose of 25 Kgy following
substantiation of the
appropriateness of this dose. Each sample had twelve films (N=3) to be
sterilized by
Gamma radiation. Resultant chemical changes can be detected at different time
points as
follow: a) No sterile (3); b)Immediately after irradiation (3); c) Two weeks
after
irradiation (3); d)1 month after irradiation (3). After Gamma sterilization,
the films are
analyzed by GPC to detect the change in the number-averaged molecular weight
(Mn),
weight-averaged molecular weight (Mw), and polydispersity (Mw/Mn) of polymer
chains
before and after radiation. The results are listed in Table 3. It shows that
no obvious
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physical and chemical changes happened to the drug polymers after radiation
sterilization.
Table 3, Mn, Mw and Polydispersity of drug polymers before and after Radiation
Samples Mn gjmol Mw g/mol
PI
THDI/PCLINF:
A: 3.2x104
6.9x104 2.1
B: 3.2x104
6.2x104 2.1
C: 3.0x104
6.4x104 2.1
Right after radiation
A: 3.0x104
6.2x104 2.0
B: 2.9x104
6.2x104 2.0
3.2x104 6.3x104
C: 2.0
1 week after radiation
A: 2.9x104
6.0x104 2.1
B: 3.1x104
6.7x104 2.2
C: 2.8x104
6.0x104 2.1
2 weeks after radiation
A: 2.9x104
6.0x104 2.1
B: 3.0x104
6.4x104 2.1
C: 3.0x104
6.3x104 2.1
1 month after radiation
A: 2.8x104
6.1x104 2.1
B: 2.8x104
5.8x104 2.1
C: 2.8x104
5.9x104 2.1
THDI/PCLICP:
A: 2.1x104
3.4x104 1.6
B: 2.1x104
3.3x104 1.6
C: 2.1x104
3.3x104 1.6
Right after radiation
A: 2.1x104
3.4x104 1.6
B: 2.3x104
3.6x104 1.6
C: 2.3x104
3.7x104 1.6
1 week after radiation
A: 2.3x104
4.0x104 1.6
B: 2.2x104
3.6x104 1.6
C: 2.2x104
3.7x104 = 1.6
2 weeks after radiation
A: 2.2x104
3.7x104 1.7
B: 2.2x104
3.6x104 1.7
C: 2.2x104
3.9x104 1.7
1 month after radiation
A: 2.1x104
3.4x104 1.6
B: 2.1x104
3.6x104 1.7
C: 2.1x104
3.5x104 1.7
Example 10:
This example shows the in vitro cytotoxicity of a non-bioactive control
polymer,
NF and CP polymers with mammalian cell lines using a direct contact method. In
this
method, 1 ml of polymer DMSO solutions containing 1 mg/ml, 3 mg/ml and 5
mg,/ml,
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respectively, of control or drug polymer is loaded on each Millipore 0.451im
filter that is
set on top of agar in a Petri dish. These dishes are then incubated at 37 C
in a humidified
atmosphere of 5% CO2 for 24 hours. After the solvent is diffused into agar,
these filters
with polymers loaded on it are transferred into a new Petri dish containing
solidified agar.
HeLa cells are seeded onto these filters. The dishes are incubated at 37 C in
a humidified
atmosphere of 5% CO2 for 48 hours. Cells are stained with succinic
dehydrogenase
staining buffer. The stained areas on the filters show the cytotoxicity of
materials.
Figure 7 show the scanned pictures of stained cells that are seeded on the
filters loaded
with different amounts of control, NF and CP polymers. There are no unstained
areas in
each filter. The results show that the control polymer and bioactive polymers
have good
biocompatibility with mammalian cells.
Example 11:
NF polymer was used to evaluate the ability of a hydrolytic enzyme to degrade
the material and preferentially release drug. NF polymer was coated onto small
glass
cylinders, and then incubated in the presence and absence of hydrolytic enzyme
(i.e.
cholesterol esterase) for up to 10 weeks at 37 C. At each week interval the
incubation
solution was removed from NF polymer and fresh enzyme solution was added. The
incubation solutions were assayed via high pressure liquid chromatography
(HPLC).
Standard solutions of pure norfloxacin were run through an HPLC system to get
calibration curve of this system. Norfloxacin concentration in the incubated
solution was
determined by comparison of drug peak area of incubation solution to
calibration curve.
Figure 8 shows the released norfloxacin from NF polymer in the presence and
absence of
cholesterol esterase. In the presence of CE, there is an obvious release of
Norfloxacin 10
weeks. However in the absence of CE, there only is some release of drug in the
first 6
weeks and it is lower than that of the enzyme incubated samples throughout the
experiment.
The same NF polymer incubation solutions assayed via HPLC were also
evaluated for antimicrobial activity using a biological assay. A macro-
dilution minimum
inhibitory concentration (MIC) assay was employed to determine the
concentration of
antimicrobial (norfloxacin) that would inhibit the growth of a pathogen often
associated
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with device-related infections, Pseudomonas aeruginosa. The MIC for this
organism and
norfloxacin was determined to be 0.8 mu g/mL. Incubation solutions from both
enzyme
and buffer control treatment of NF polymer were used in a biological assay
matrix that
was designed to estimate the concentration of norfloxacin as a function of
incubation time
and treatment. The data are presented in Table 4. Anti-microbial activity was
not
detected in the NF polymer exposed to buffer (control) incubation solution
after 2 weeks.
However, the enzyme-treated NF polymers released clinically significant levels
(>MIC
levels) of antibiotic over a 10 week incubation period. These biological assay
data show
a significant correlation with the HPLC data described above. The results of
these
experiments demonstrate that the antibiotic agent is released from NF polymer
under
enzymatic activation, and that the antibiotic has antimicrobial activity
against a clinically
significant bacterium. Furthermore, clinically significant concentrations
(i.e., MIC level)
of the antibiotic are released over an extended period of time, 10 weeks.
Table 4... MIC Assay for antibacterial activity of degraded NF polymer
solutions
Samples containing drugs greater than or less than MIC level
: go Aro 1 2 3 4 5 6 7 8
o
4*0
'40 4 CE CE CE CE buffer buffer buffer buffer
I week : . - , 7' l' '..- i -
' __________________________________ k ...4. . 5
2 weeks i = -' !. - !,, 4,, 1,... '
',-;;0,.., ----.4,----r- --ii...i..,=aa,...`:-.'õ,ukL',.',n-----4.- --1 -
4,4¨.4+
= 1, . 4, 1 .y,1^,,.:,,,!.* ' <
., i < <
3 weeks 1,`,..i. =' i: ' ,1 ' !7-'t ' 01'' -'' : ' j
MEL, ..b1d.......;-,..41%..,
4 weeks ::: ' '; ' ----: 1r-7;::i 11- ,
<
1 <
6 weeks -," ' 11 ' ' ,;= F .-.,i:,. , ,!:õ.,,,
1 < < < <
-...........,..;,,...,....,....,L.....õ,..
8 weeks < < < . <
lOweeks . l' , -- 1 < < < <
. ] , .1 ...... , i
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Example 13:
In vivo animal studies are performed on formed coupons made of control and CP
polymer with a dimension of 1x2 cm2. The coupons were implanted in the
peritoneal
cavity of male rats. The coupons were explanted after rats were housed for 1
week. The
experimental conditions according to the invention are as follows:
For implantation, 5 male Sprague-Dawley rats (250-300 g) were used for every
group of experiment. After they were anesthetised,. a 2 cm laparotomy incision
was made
in the abdomen. The omentum and gubernaculum tissues were resected as they
tend to
envelop the coupon. Then either a control coupon or a CP coupon (1x2 cm2) was
implanted in the abdominal cavity. The incision was closed in two layers.
After animals
were housed for 1 week (rats were monitored daily), coupons were explanted
from rats.
Gross observations were made including adhesion, abscess, inflammation, and
encapsulation. It was found that no adhesion, abscess and inflammation
associated with
CP polymer coupons, but there was obvious adhesion, abscess and serious
inflammation
associated with implanted control polymer coupons. Coupons were retrieved with
sterile
surgical instruments. A swab was taken of the peritoneal cavity. Coupons were
rinsed in
PBS buffer to remove non-adherent cells and placed in sterile tubes for
further bacteria
culture. Bacteria counts obtained from cultures of control and CP coupons are
shown in
Figure 9. Clearly, CP coupons show an antimicrobial effect, yielding
significantly lower
colony forming units (CFUs).
Example 12:
Examples of biomedical articles that integrate the bioactive polymers to the
polymers using described methods 1, 2, 3 below include, for example, the
following
articles that are in whole or in part made of polyurethane components, namely,
cardiac
assist devices, tissue engineering polymeric scaffolds and related devices,
cardiac
replacement devices, cardiac septal patches, intra aortic balloons,
percutaneous cardiac
assist devices, extra-corporeal circuits, A-V fistual, dialysis components
(tubing, filters,
membranes, etc.), aphoresis units, membrane oxygenator, cardiac by-pass
components(tubing, filters, etc.), pericardial sacs, contact lens, cochlear
ear implants,
sutures, sewing rings, cannulas, contraceptives, syringes, o-rings, bladders,
penile
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implants, drug delivery systems, drainage tubes , pacemaker lead insulators,
heart valves,
blood bags, coatings for implantable wires, catheters, vascular stents,
angioplasty
balloons and devices, bandages, heart massage cups, tracheal tubes, mammary
implant
coatings, artificial ducts, craniofacial and maxillofacial reconstruction
applications,
ligaments, fallopian tubes, biosensors and bio-diagnostic substrates.
Non-biomedical articles fabricated by hereinbefore method 1) include, for
example, extruded health care products, bio-reactor catalysis beds or affinity
chromatography column packings, or a biosensor and bio-diagnostic substrates.
Non-medical applications that are exemplified by method 2) include fibre
membranes for water purification.
Non-medical applications of the type exemplified by method 3) include
varnishes
with biological function for aseptic surfaces.
Although this disclosure has described and illustrated certain preferred
embodiments of the invention, it is to be understood that the invention is not
restricted to
those particular embodiments. Rather, the invention includes all embodiments
which are
functional or mechanical equivalence of the specific embodiments and features
that have
been described and illustrated.
42
