Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITIONS OF ARGLABIN
AND ARGLABIN DERIVATIVES
Background of the Invention
Cancer is a leading cause of death in the United
States and affects people wor=~dwide. Surgery, radiation
and chemotherapy are the most widely used therapeutic
modalities. Chemotherapy agents create conditions within
the cel2 that limit cell growth and replication. DNA
synthesis may be inhibited by preventing purine
biosynthesis, pyrimidine biosynthesis, the conversion of
ribonucieotides to deoxyribonucleotides, antimetabolites,
intercalation, or cross-links. RNA synthesis, for
example, may be inhibited by antimetabolites. Protein
synthesis may be inhibited, for example, by agents that
deaminate asparagine. Additic>nally, agents that inhibit
the function of microtubules can be used as chemotherapy
agents.
Chemotherapy agents typically affect both
neoplastic and rapidly proliferating cells of normal
tissue such as bone marrow, hair follicles and intestinal
epithelium. Anorexia, nausea, vomiting, diarrhea,
suppression of bone marrow function and hair loss are
some of the negative effects commonly associated with
chemotherapy. Development of a chemotherapy agent that
is an effective antitumor agent with minimal toxicity
would be advantageous.
Summary of the Invention
It has been discovered that arglabin and various
derivatives of arglabin can function as effective
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chemotherapeutic agents, with fewer side-effects than
typically follow from use of other chemotherapeutic
agents.
In one aspect, the invention features a
pharmaceutical composition in unit dosage form suitable
for the treatment of a human cancer. The composition
consists essentially of about 40 mg to about 480 mg of
arglabin or a derivative thereof. The unit dosage of the
composition may be, for example, from about 175 mg to
about 315 mg or from about 240 mg to about 280 mg of
arglabin or a derivative thereof. Arglabin or a
derivative thereof may be used in the manufacture of a
medicament for the treatment of cancer. The compositions
can be used in the manufacture of a medicament for the
treatment of cancer. The compositions are useful for the
treatment of a wide variety of cancers, including, for
example, breast, colon, rectal, stomach, pancreatic,
lung, liver, ovarian, pancreatic and esophageal cancer,
leukemia, and lymphoma. The composition is particularly
useful for the treatment of lung, liver, and ovarian
cancer.
Dimethylaminoarglabin or a pharmaceutically
acceptable salt thereof is a particularly useful arglabin
derivative that may be used in the pharmaceutical
composition. Dimethylaminoarglabin or a pharmaceutically
acceptable salt thereof may be lyophilized.
The invention also features a composition
including a first chemotherapeutic agent that includes
arglabin or a derivative thereof and a second
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chemotherapeutic agent. The ~~econd chemotherapeutic
agent is not arglabin or a derivative thereof. The
composition is effective to suppress tumor growth in a
human. A particularly useful arglabin derivative is
dimethylaminoarglabin or a pharmaceutically acceptable
salt thereof. The second chemotherapeutic agent may be,
for example, an alkylating agent such as
cyclophosphamide, sarcolysin, or methylnitrosourea, an
antimetabolite such as methotrexate or fluorouracil, a
vinca alkaloid such as vinblastine or vincristine, an
antibiotic such as rubidomycin. or a platinum coordination
complex such as cisplatin. Two or more chemotherapeutic
agents may be in the composition with arglabin or a
derivative thereof. The compositions can be used in the
manufacture of a medicament for the treatment of cancer.
The invention also features compounds that
suppress tumor growth in a mammal. These compounds are
selected from the group represented by the following
Formulas I, II, III, IV, V and VI:
I
L y
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wherein RR1 is NHCHZPh or N (CHZCHz) 20, RR2 is NHCHzPh,
N (CHzCH2) 20, N (CH3) 2, or a pharmaceutically acceptable salt
thereof; and X is OH or C1. These arglabin derivatives
include dimethylaminoepoxyarglabin, dibromoarglabin,
arglabin chlorohydrin, 11,13 dihydroarglabin,
benzylaminoarglabin, morpholine-aminoarglabin,
benzylaminoepoxyarglabin, morpholine-aminoepoxyarglabin,
epoxyarglabinchlorohydrin or pharmaceutically acceptable
salts thereof. Compounds of Formula I, II, III, IV, V or
VI can be used in the manufacture of a medicament that
suppresses tumor growth in a mammal.
Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art
to which this invention belongs. Although methods and
materials similar or equivalent to those described herein
can be used in the practice or testing of the present
invention, suitable methods and materials are described
below. All publications, patent applications, patents,
and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the
present specification, including definitions, will
control. In addition, the materials, methods, and
examples are illustrative only and not intended to be
limiting.
Other features and advantages of the invention
will be apparent from the following detailed description,
and from the claims.
Brief Description of the Drawinct
Figure 1 depicts the synthesis of arglabin
derivatives 2 through 9.
Figure 2 depicts the synthesis of arglabin
derivatives 10 through 13.
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Figure 3 depicts the synthesis of arglabin
derivatives 14a-14d, 15a-15d and 16.
Figure 4 depicts the structure of compounds 17
through 21.
Figure 5 depicts the effect of increasing
concentrations of dimethylaminoarglabin hydrochloride on
the viability of transformed cells.
Figure 6 depicts the effect of increasing
concentrations of dimethylamir.:oarglabin hydrochloride on
the proliferation of transformed cells.
Figure 7 depicts the e:Efect of increasing
concentrations of dimethylamin.oarglabin hydrochloride on
the viability of normal cells.
Figure 8 is a spectrum index plot of naphthol
cleavage products. Figure 8A is in the absence of drug.
Figure 8B is in the presence of drug.
Description of the Preferred Embodiments
The invention provides novel compounds that
suppress tumor growth in humans. These compounds may be
synthesized from the parent compound arglabin (Figure 1),
which is isolated from Artemisia glabella. Various
arglabin derivatives may be made using a range of
chemistries. For example, epoxyarglabin may be produced
by epoxidation of the tri-substituted olefin double bond
with peracetic acid. Dichlorohydrins may be produced by
treatment of epoxyarglabin with an ether-acetone HC1
solution. Dibromoarglabin may be produced by reacting
arglabin with Br2 and carbontet:rachloride. Arglabin
chlorohydrins may be produced from arglabin by reaction
with a methanol hydrochloride solution. Epoxidation of
arglabin chlorohydrins with peracetic acid and chloroform
results in chromatographically separable epoxyarglabin
chlorohydrins. Arglabin diol, its isomer and dime may
be produced by hydrolyzing arglabin. The 1, 10 epimer of
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arglabin, epiarglabin may be produced by treatment of
arglabin diol with POCl,. Benzylaminoarglabin and
benzylaminoepoxyarglabin may be produced by treatment of
arglabin and epoxyarglabin with benzeneamine.
Dimethylaminoarglabin and dimethylaminoepoxyarglabin may
be produced by treatment of arglabin and epoxyarglabin
with dimethylamine. Morpholine-aminoarglabin and
morpholine-aminoepoxyarglabin may be produced by
amination of arglabin with morpholine. Pharmaceutically
acceptable salts of these compounds may be produced with
standard methods and used as antitumor agents. For
example, dimethylaminoarglabin hydrochloride and
dimethylaminoepoxyarglabin hydrochloride may be produced
by hydrochlorination. Dihydroarglabin may be produced by
treating arglabin with ethanol and H2/Ni. The various
arglabin derivatives set out above are depicted in
Figures 1-4.
The invention also relates to a method of
suppressing tumor growth in a human patient diagnosed
with cancer comprising administering arglabin or a
derivative thereof to the patient. While this method may
be used generally for the treatment of cancers such as
breast, colon, rectal, stomach, pancreatic, lung, liver,
ovarian, pancreatic and esophageal cancer, leukemia and
lymphomas, certain types of cancers, such as lung, liver
and ovarian cancer, are particularly amenable to this
therapeutic regimen. The compounds can be administered
topically, orally, intravenously, intraperitoneally,
intrapleurally, intrathecally, subcutaneously,
intramuscularly, intranasally, through inhalation or by
suppository, depending on the type of cancer and on
various patient indications. For example,
intraperitoneal administration may be used for some
patients with ascites. Intrapleural administration may
be used for certain patients with lung cancer.
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Suppositories may be used for patients with rectal
cancer. Arglabin or a derivative thereof may be
administered in a daily amount from about 40 mg to about
480 mg, preferably from about 175 mg to about 315 mg,
more preferably from about 240 mg to about 280 mg.
Typically the dosage ranges from about 0.5 rng/kg to about
7 mg/kg. In extreme conditions, up to about 20 mg/kg of
arglabin or a derivative thereof may be administered.
Once administered, these compounds act as antitumor
agents and may inhibit the growth of the tumor or may
cause the tumor to regress.
Without being bound by any particular biochemical
mechanism, these compounds may eliminate or inhibit the
growth of cancer cells by impeding farnesylation of
35 proteins such as the ras protein. The ras gene is a
protooncogene that plays a role in many types of human
cancers, including colorectal carcinoma, exocrine
pancreatic carcinoma, and myeloid leukemias (Barbacid,
1987, Ann. Rev. Biochem. 56:779). Approximately 20 to
30% of all human tumors can be attributed to the
activation of the ras protooncogene. Ras genes
constitute a multi-gene family that transform cells
through the action of a 21 kDa protein termed ras p21
(also referred to herein as "ras"). Ras functions as a
G-regulatory protein, hydrolyzing GTP to GDP. In its
inactive state, ras binds GDP. Upon activation of growth
factor receptors, ras exchanges GDP for GTP and undergoes
a conformational change. In its GTP-bound state, the
wild-type ras couples the signals of activated growth
factor receptors to downstream mitogenic effectors. The
intrinsic GTP-ase activity of ras eventually returns the
protein to its inactive GDP-bound state. In tumor cells,
a mutation in the ras gene results in a loss of
regulatory function, resulting in constitutive
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transmission of growth stimulatory signals and oncogenic
activation.
For both normal and oncogenic functions, ras must
be localized at the plasma membrane, a process that is
dependent upon proper post-translational processing of
the ras (Hancock, 1989, Cell 57:1167). In the first step
in the post-translational processing of ras, a farnesyl
group is attached to a cysteine residue at position 186
of the protein by reaction with farnesyl pyrophosphate.
Second, the carboxy-terminal three amino acids of the
protein are cleaved by the action of a specific protease.
Third, the carboxylic acid terminus is converted to a
methyl ester by alkylation with a methyl group.
Post-translational modification of ras is mediated
by an amino acid sequence motif frequently referred to as
a "CAAX box." In this sequence motif, C represents
Cysteine, A represents an aliphatic amino acid, and X is
another amino acid such as Methionine, Serine, or
Glutamine. Depending on the specific sequence of the
CAAX box, this motif serves as a signal sequence for
farnesyl-protein transferase or geranylgeranyl-protein
transferase, which catalyze the alkylation of the
cysteine residue of the CAAX sequence. Farnesylation of
ras is required for proteolytic processing,
palmitoylation, and tight binding of the ras protein to
cellular membranes.
In the absence of farnesylation, oncogenic forms
of ras cannot oncogenically transform cells. Indeed,
inhibitors of farnesyl-protein transferase have been
shown to block the growth of ras-transformed cells in
soft agar. Accordingly, inhibitors of farnesyl-protein
transferase, and of ras activity in general, are thought
to be useful anti-cancer therapeutics for many types of
cancers (Gibbs et al., 1984, Proc. Natl. Acad. Sci. USA
81:5704-5708; Jung et al., 1994, Mol. Cell. Biol.
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14:3707-3718; Predergast et al., 1994, Mol. Cell. Biol.
14: 4193-4202; Vogt et al., 19:95, J. Biol. Chem. 270:
660-664; and Maron et al., 1995, J. Biol. Chem. 270:
22263-22270).
As described below, arg~labin and derivatives
thereof appear to inhibit protc=in farnesylation.
In an alternative embodiment, a pharmaceutical
composition containing from about 40 mg to about 480 mg,
preferably from about 175 mg to about 315 mg, more
preferably from about 240 to about 280 mg of arglabin or
a derivative thereof is provided in unit dosage form.
The dose may be divided into 2~-4 daily doses. Typical
dosages of these pharmaceutical composition range from
about 0.5 mg/kg to about 7 mg/)cg. In extreme conditions,
up to about 20 mg/kg may be administered. Lyophilized
dimethylaminoarglabin and lyophilized pharmaceutically
acceptable salts such as dimethylaminoarglabin
hydrochloride are particularly useful as pharmaceutical
compositions. The optimal concentration of arglabin or a
derivative thereof in a pharmaceutically acceptable
composition may vary, depending on a number of factors,
including the preferred dosage of the compound to be
administered, the chemical characteristics of the
compounds employed, the formulation of the compound
excipients and the route of adrninistration. The optimal
dosage of a pharmaceutical composition to be administered
may also depend on such variables as the type and extent
of cancer metastases, the overall health status of the
particular patient and the relative biological efficacy
of the compound selected. The~~e compositions may be used
for the treatment of cancer, e;~pecially lung, liver and
ovarian cancer, although other cancers such as breast,
rectal, colon, stomach, pancreatic or esophageal cancer
are also beneficially treated with the compositions. In
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addition, hematopoietic cancers such as leukemias and
lymphomas may also be beneficially treated.
Compounds of the invention may be formulated into
pharmaceutical compositions by admixture with
pharmaceutically acceptable non-toxic excipients or
carriers. Such compounds and compositions may be
prepared for parenteral administration, particularly in
the form of liquid solutions or suspensions in aqueous
physiological buffer solutions; for oral administration,
particularly in the form of tablets or capsules; or for
intranasal administration, particularly in the form of
powders, nasal drops, or aerosols. Compositions for
other routes of administration may be prepared as desired
using standard methods.
A compound of the invention may be conveniently
administered in unit dosage form, and may be prepared by
any of the methods well known in the pharmaceutical art,
for example, as described in Remington's Pharmaceutical
Sciences {Mack Pub. Co., Easton, PA, 1980). Formulations
for parenteral administration may contain as common
excipients sterile water or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin,
hydrogenated naphtalenes, and the like. In particular,
biocompatible, biodegradable lactide polymer,
lactide/glycolide copolymer, or polyoxethylene-
polyoxypropylene copolymers are examples of excipients
for controlling the release of a compound of the
invention in vivo. Other suitable parenteral delivery
systems include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems,
and liposomes. Formulations for inhalation
administration may contain excipients such as lactose, if
desired. Inhalation formulations may be aqueous
solutions containing, for example, polyoxyethylene-9-
lauryl ether, glycocholate and deoxycholate, or they may
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be oily solutions for administration in the form of nasal
drops. If desired, the compounds can be formulated as a
gel to be applied intranasally. Formulations for
parenteral administration may .also include glycocholate
for buccal administration.
The invention also relates to an article of
manufacturing containing packaging material and arglabin
or a derivative thereof contained within the packaging
material. Arglabin or derivatives thereof are
therapeutically effective for ;suppressing tumor growth in
a human: The packaging material contains a label or
package insert indicating that arglabin or a derivative
thereof may be used for suppressing tumor growth in a
human. Dimethylaminoarglabin and pharmaceutically
acceptable salts thereof are a:rglabin derivatives that
are particularly useful in the article of manufacture.
In an alternate embodiment, the invention relates
to compositions and kits comprising a first
chemotherapeutic agent including arglabin or a derivative
thereof and a second chemotherapeutic agent. The second
chemotherapeutic agent is not ;~rglabin or a derivative
thereof. These compositions a:re effective to suppress
tumor growth in a human. Dimethylaminoarglabin or a
pharmaceutically acceptable sa:Lt thereof is a
particularly useful derivative of arglabin. Various
classes of chemotherapeutic agc=nts, including alkylating
agents, antimetabolites, vinca alkaloids, antibiotics or
platinum coordination complexes may be used in the
composition. For example, alk~~rlating agents such as the
nitrogen mustards cyclophosphamide and sarcolysin may be
used, although other alkylating agents such as
methylnitrosourea are also appropriate. Antimetabolites
such as the folic acid analog methotrexate or pyrimidine
analogs such as fluorouracil o:r 5-fluorouracil may be
used, as well as vinca alkaloids such as vinblastine or
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vincristine. An antibiotic such as rubidomycin can be an
appropriate chemotherapeutic agent, as well as platinum
coordination complexes such as cisplatin. Multiple
chemotherapeutic agents may be combined with arglabin or
a derivative thereof. For example, vincristine and
cyclophosphamide or vincristine and vinblastine may be
combined with arglabin or a derivative thereof.
The invention also relates to a method of
suppressing tumor growth in a human patient by
administering to the patient an amount of a composition
including a first chemotherapeutic agent including
arglabin or a derivative thereof and a second
chemotherapeutic agent effective to suppress tumor growth
in the human. The second chemotherapeutic agent is not
arglabin or a derivative thereof. These compositions
provide an enhanced antitumor effect and may also prevent
development of metastases. In particular, these
compositions are useful for overcoming tumors that are
drug-resistant. The agents may be administered
separately or as a cocktail. Toxicity may be reduced by
administering arglabin or a derivative thereof several
hours prior to administering the chemotherapy agent. The
compositions may be administered by any route.
The invention also relates to a method for
reducing the immundepressive effect of a chemotherapy
agent in a human patient by administering to the patient
an amount of arglabin or a derivative thereof effective
to augment the immune system of the patient upon
treatment of the patient with the chemotherapy agent.
The immune system may be augmented, for example, by
increasing the total number of leukocytes, T-lymphocytes,
B-lymphocytes, or immunoglobulins.
The invention will be further described in the
following examples, which do not limit the scope of the
invention described in the claims.
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EX2mplas
Examt~le l: Isolation of Arglabin - The smooth wormwood
Artem.isia glabella Kar. et Kir. is a perennial plant that
is widespread on the Kazakhstan dry steppe hills. The
aerial parts of A. glabella, including the leaves, buds,
flower buds and stems, contain sesquiterpene lactones
including arglabin throughout the vegetation stage of the
plant (Table I).
TABLE; I
1 Vegetation phase Plant Dry plant Isolated Arglabin
0 in
organ !g) arglabin dry plant
(g) !
Rosette leaves 1900 6.4 0.34
Buttonization leaves 1000 6.1 0.61
stems 1700 1.28 0.08
buds 1000 6.0 0.60
A variety of solvents were used to extract
sesquiterpene lactones from the dry plant material (Table
II). It was found that extracting the lactones from the
flowering stage of the plant with chloroform three times
at 45-50°C produced the highest yield.
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TABLE II
Solvent Extracted MaterialIsolated LactonesLactones
~
(g) in Dry plant
Water 1.5 arglabin 0.002
argolide traces
dihydrogarglolidetraces
Petrol-diethyl 3.2 arglabin 0.002
ether 1:1 argolide traces
dihydrogargiolidetraces
Benzene 3.4 arglabin traces
Diethyl ether 4.8 arglabin 0.11
argolide 0.007
dihydrogarglolidetraces
Chloroform 6.7 arglabin 0.150
argolide 0.0075
dihydrogarglolide0.0006
Ethanol 5.1 arglabin 0.08
argolide 0.01
An extraction device consisting of a counter-flow
continuous extractor, loading device and three vessels
isolated from the exterior environment was used for the
extractions. The solvent vessel has a filter,
distillator with an evaporator and condenser, and a
buffer capacity. The drying agent vessel consists of a
dryer, cyclone, cooler, ventilator and heater. The
cooling water vessel includes a saltpan with ventilation.
The extraction device also has a deodorizer with
ventilator, a waste tank and an extract collector.
Approximately 7.7 kg of dry material from
Artemisia glabella Kar. et Kir. was placed in the
extraction device and continuously mixed with solvent as
the material was moved through the extractor column. The
solvent moves in the opposite direction of the dry plant
material and gradually becomes saturated with extracted
substances. As the saturated solvent was discharged, it
was first filtered to remove plant material particles,
then evaporated. The filtered plant particles were
recirculated through the extractor for re-extraction.
Vapors from the evaporation were sent to the condenser.
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From the condenser, pure solvent was recovered and
recirculated to the extraction. device. Condensation
surfaces in the condenser were cooled with water pumped
from the salt pan where the water was previously cooled
with exterior air blown in the ventilator. Due to air-
vaporized cooling in the salt pan, the water may be
cooled down to temperatures considerably lower than the
ambient temperature.
The extracted substances refined from the solvent
are in the form of a tar. During this process,
approximately 7% of the plant material (539 grams) was
recovered.
The tar was further refined by addition of two
volumes (approximately 1.08 L) of 60°C ethanol with
continuous stirring to dissolve the tar. Distilled
water, heated to approximately 70°C, was added in a ratio
of about 2:1 alcohol to water. The tar-alcohol-water
solution was thoroughly stirred for 30 minutes, then left
at room temperature for approximately 24 hours or until a
precipitate was formed. The water alcohol solution was
filtered through a ceramic filter under vacuum. The
procedure was repeated with any precipitate remaining
after filtration.
The filtrate was rotary evaporated and the alcohol
was vacuum distilled in the form of an azeotropic mixture
with water containing 68-70% alcohol. After distillation
of the alcohol, the water solution yielded approximately
286 grams of refined tar.
The refined tar was separated into individual
components over a KCK silicagel column, with pressure,
using benzene as the eluant. Benzene fractions were
collected and analyzed for arglabin using thin-layer
chromatography (TLC) (silufol, benzene-ethanol, 9:1).
Arglabin-containing fractions 'were distilled to remove
benzene. Arglabin at this stage has a yellow color.
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Approximately 33 g of arglabin was produced, with a yield
of about 11.70.
Arglabin was recrystallized by dissolving in
hexane in a 1:10 ratio of product to hexane (w/v) and
heating. After arglabin was in solution, the product was
vacuum filtered. Crystals of arglabin were isolated from
the filtrate at room temperature. Approximately 21 g of
arglabin was recovered from this step. Arglabin has a
structure of 1 (R) , 10 (S) -epoxy-5 (S) , 6 (S) , 7 (S) -guaia-
3(4), 11(13)-dime-6,12-olide. The stereochemistry of
arglabin was determined through x-ray analysis.
The joining of the pentene and heptane ring and
heptane and y-lactone rings into two crystallographically
independent molecules of arglabin is transoid. Torsion
angles of 03C1CSH5 are -142 (1) and -136 (2) °, and H6C6C.,H~
are -167(2) and -159(3)°, respectively. The pentene
ring accepts the conformation of the la-envelope (~Cls=2.'9
and 1.5°) and the heptane ring is 7a,1, 10~i-chain
(DC'S=2.7 and 4.7°). The methyl group by the C-10 atom
has an equatorial a-orientation. Conformation of 'y-
lactone ring was between 7a-envelope and 6~3, 7a-semichair
but was closer to the latter (~Cl2z=2.0 and 6.1°).
The NMR spectrum of arglabin was recorded on a
Varian HA-100D apparatus in CDCI. Chemical shifts are
given in b-scale from signal TMC accepted for 0. There
are two three-proton singlets at 1.34 (methyl at epoxide)
and at 1.94 pprn (methyl at double bond). A single-proton
doublet was registered at 2.95 ppm with J=10 Hz (proton
at C). A single-proton triplet was detected with the
center at 3.97 ppm with J=10 Hz (lactone proton). Two
single-proton doublets were obtained at 5.42 ppm with
J=3Hz and 6.1 ppm with J=3Hz (exomethylene at lactone
cycle) and a single-proton signal at 5.56 (vinyl
protons). The structure of arglabin (Figure 1) was
confirmed on the basis of the NMR spectrum of the
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isolated compound and that of related sesquiterpene
lactones arborescien and ludartine.
Summary of arglabin characteristics: Colorless,
Melting Point of approximately 100-102°C (hexane); [a]2°D
+45.6° (c 0.3, CHC13) ; IR band~~ (KBr) 1760, 1660, 1150,
1125 cm-1; 1H-NMR (400 MHz, CDC:13) b 1.34 (3H, s, H-14) ,
1. 94 (3H, s, H-15) , 2.95 (1H, d, J lOHz, H-5) , 3.97 (1H,
t, J lOHz, H-6), 5.56 (1H, br s, H-3), 5.42 (1H, d, J
3Hz, H-13a) , 6.10 (1H, d, J 3Hz, H-13b) .
Example 2: Aralabin Derivatives - To assist the reader,
the names of the various compounds set out below are
followed with numerals to facilitate identification with
the compounds depicted in the :figures.
Reagents affecting the epoxide or olefin group of
arglabin were used to derivati:ae arglabin. Epoxidation
of the tri-substituted olefin double bond of arglabin 1
with peracetic acid (Figure 1) proceeded with high yield
and 95% stereoselectivity, forming 3R, 4(3-epoxyarglabin 2
(1 (IO) , 3 (4) -diepoxy-guai-11 (1:3) -en-6, 12-olid) . Silica
gel column chromatography with ethylether was used to
recover epoxyarglabin 2 with an approximate 65% yield.
IR and NMR spectra were used to confirm the structure of
epoxyarglabin 2.
Summary of epoxyarglabin 2 characteristics:
Melting point of 149-151°C (Et~O-CHZC12) ; [a] zzD + 94 .
0° (c
1.7, CHC13) ; IR bands (KBr) 1760, 1670 cm-1; 1H-NMR (400
MHz, py-ds) b 1.30 (3H, s, H-14), 1.68 (3H, s, H-15), 3.31
(1H, s, H-3, 4.11 (1H, t, J lOHz, H-6) , 5.43 (1H, d, J
3Hz, H-13a) , 6.16 (1H, d, J 3Hz , H-13b) .
Treatment of epoxyarglabin 2 with an ether-
acetone HCl solution produced dichlorohydrines 3 and 4
(Figure 1). An opening of both epoxygroups with a yield
of 60% and 95% regioselectivity was observed.
Dichlorohydrines 3 and 4 were diluted with water, washed
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with NaHC03, and purified by silica gel column
chromatography using Et20-iPr20 in a 1:1 ratio. IR and
NMR spectral data were used to confirm the structure.
Summary of dichlorohydrines 3 and 4
characteristics: 3 (60%) melting point 190-191°C; (a]2°n
-90.1° (c 0.34 acetone); IR bands (KBr) 3465, 1750, 1670
cm-l; 1H-NMR (400 MHz, py-ds) b 1.61 (3Hz, s, H-14) , 1.62
{3H, s H-15) , 4.42 (1H, dd, J10, 7Hz, H-3) , 4.48 (1H, t,
J lOHz, H-6) , 5.52 (1H, d, J 3Hz, H-13a) , 6.04 (1H, d, J
3Hz, H-13b) . 4 (5 0) melting point 176-178°C (CHZC12-
Et20) ; (a] 24D + 23.25° (c 0.43, CHC13) ; IR bands (KBr) 3680,
1770, 1670 cm-1; 1H-NMR (400 MHz, py-ds) b 1.41 {3H, s, H-
14); 1.57 (3H, s, H-15), 3.39 (1H, d, J lOHz, H-5), 3.42
( 1H, s, 10-OH) , 4 . 05 ( 1H, d, J 5 . 5Hz, H-3 ) , 4 . 40 ( 1H, s,
4-OH) , 4 .55 (1H, t, J lOHz, H-6) , 5.54 (1H, d, J 3Hz, H
13a) , 6.21 (1H, d, J 3Hz, H-13b) .
Other derivatives were generated by bromination
and interaction with N-bromosuccinimide in aqueous
acetone, resulting in the formation of mobile
bromohydrines on trisubstituted double bonds and partial
bromination of the exomethylene group. 3,4
Dibromoarglabin 5 was produced by treating arglabin I
with Br2 and carbontetrachloride at 0°C.
Treatment of arglabin 1 with a methanol HCl
solution gave a chromatographically separable mixture of
chlorohydrines 6/7 in an approximate 6:1 ratio with high
yield (Figure 1). Simultaneously, partial attachment of
HC1 elements to the exomethylene double bond was observed
by a Michael type reaction. Epoxidation of the
prevailing regioisomer 6 with peracetic acid in
chloroform resulted in a mixture of chromatographically
separable isomer epoxyarglabin chlorohydrins 8/9 in a 1:1
ratio. Structures of previously unknown chlorohydrines
6-9 were established on the basis of elemental and
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spectral analyses, taking into consideration the results
of epoxide 8 by x-ray.
Reflux of about 550 mg of arglabin 1 with
approximately 15 ml of acetonitrile and a drop of HBF4 for
1.5 hour resulted in diol 10 as the major product, and
its isomer 11 and the dime 12 in lower yield (Figure 2).
The reactions were neutralized, diluted with water,
extracted with chloroform, t:he:n purified by column
chromatography (10, petrol-ethyl ether 2:1; 11, petrol-
ethyl ether 1:1; 12, petrol--ethyl ether 1:3).
Summary of diol 10, isomer 11 and dime 12
characteristics: 10 melting point 184-185°C (ethyl
ether) ; [a]21D +72.3° {c 0.3, CHC13) ; IR bands (KBr) 3440,
1770, 1680 cm-'; 1H-NMR (400 MHz, py-ds) b 1.30 (3H, s, H-
14) , 1.92 (3H, br s, H-15) , 4.:L8 (1H, dd, J 10, lHz, H-
6), 5.43 (1H, br s, H-3), 5.38 (1H, d, J 3.5Hz, H-13a),
6.14 (1H, d, J 3.5Hz, H-13b) . 11: mp 149-15I°C (CHC13-
EtzO) ; [a]25D + 108.6° (c 0.3, CHC13; IR bands (KBr) 3460,
1770, 1670 cm-1; 'H-NMR (400 MHz, py-ds) b 1.35 (3H, s, H-
14) , 1.92 (3H, br s, H-15) , 3.:L0 (1H, d, J lOHz, H-5) ,
4.39 {1H, t, J lOHz, H-6), 5.48 (1H, br s, H-3), 5.44
(1H, d, J 3.5Hz, H-13a) , 6.14 (1H, d, J 3.5Hz, H-13b) .
12 : melting point 220-222°C (EtOH) ; [a] 22D + 80.6° (c
0.57, CHC13) ; IR bands (KBr) 3350, 1770, 1680, 1550 cm~l;
1H-NMR (400 MHz, py-ds) b 1.47 (3H, s, H-14) , 1. 92 (3H, br
s, H-15) , 4.32 (1H, t, J 10.5Hz, H-6) , 5.24 (1H, br s, H-
2), 5.50 (1H, br s, H-3), 5.41 (1H, d, J 3.5Hz, H-13a),
6.15 (1H, d, J 3.5Hz, H-13b) .
The 1,10-epimer of arglabin, epiarglabin 13 was
synthesized by adding approximately 0.1 ml of POC13 to a
cooled solution (approximately 0°C) of 120 mg of diol 10
in pyridine. (Figure 2) After stirring for 24 hours at
approximately -5°C, the reaction was worked up by
extraction with ethyl ether. After washing with 5% HC1
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and water, the residue was crystallized from petroleum
ethyl ether to give 40 mg of 1,10-epiarglabin 13.
Summary of 1, 10-epiarglabin 13 characteristics:
melting point 193-194°C (EtOH); [aJz°D +78.4° (c 0.43,
CHC13) ; IR bands (KBr) 1760, 1665, 1650, 1150 cm-'; 1H-NMR
(400 MHz, py-ds) b 1.30 (3H, s, H-14) , 1.90 (3H, br s, H-
15), 2.66 (1H, m, H-5), 4.18 (1H, dd, J 14.5, 12.5Hz, H-
6 ) , 5 . 43 ( 1H, m, H-3 ) , 5 . 3 8 ( 1H, m, H-3 ) , 6 . 14 ( 1H, d, J
3.5Hz, H-13b).
Interaction of arglabin 1 and epoxyarglabin 2 with
benzeneamine, dimethylamine and morpholine in an alcohol
medium proceeds chemoselectively as a Michael reaction on
the activated double bond of these molecules, resulting
in 56-850 of corresponding derivatives 14a-d and 15a-d
(Figure 3). The a configuration of the aminomethyl
residue was proved spectrally.
Synthesis of dimethylaminoarglabin 14b: Arglabiri
1 was mixed with 0.21 L of alcohol and heated to 40°C
until arglabin was fully dissolved. After filtering, a
33% solution of dimethylamine (0.023 L) was added
dropwise with stirring. The mixture was left for 24
hours at room temperature. The reaction was monitored
with TLC on silufol plates. After the amination reaction
was complete, the mixture was heated to 52°C and the
alcohol was vacuum distilled. Approximately 0.63 L of
chloroform was added to the remaining solvent and stirred
for 30 minutes. The mixture was poured into a separatory
funnel where the chloroform found in the lower part of
the funnel was collected. The chloroform extraction was
repeated two additional times with the aqueous layer.
Magnesium sulfate was used to dry the collected
chloroform. The chloroform - magnesium sulfate mixture
was stirred for 30 minutes, then vacuum filtered to
remove the chloroform. Approximately 22 g of
dimethylaminoarglabin 14b was produced.
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Dimethylaminoarglabin 14b was purified by first
dissolving in 5 volumes (w/v) of chloroform then mixing
with about 3 volumes (w/w) of ~;CK silica gel. After
evaporation of the solvent, the dry material was
chromatographically separated on a KCK silica gel column
made with a 1:22 ratio of adduct to sorbent. The column
was eluted by a mixture of petroleum diether and sulfuric
ether (1:1, 1:2). Fractions of approximately 14-17 mls
were collected and monitored with TLC.
Dimethylaminoarglabin 14b was recrystallized from the
fraction with chloroform and ether (1:1).
Summary of dimethylaminoarglabin 14b
characteristics: melting point 94.5 - 95.5°C, [a)21D +
47° (c 1.7, CHC13); elemental analysis 70.41% C, 8.7o H,
4.82 o N (C12H2503N) ; IR (z CHCl m.ax) 3050-3000 (shoulder) ,
2940, 2860, 2835, 2780, 2410, 1770 (carbonyl lactone),
1650 (double bond}, 1550-1530 (broad band), 1470, 1450,
1385, 1335, 1180, 1150, 1140, 1125 cm-1 (epoxy group); MS
(m/z, intensity in %) M+ HC1 291 (5.07, HCl), 247 (0.5},
188 (I, 2) , 115 (2, 19) , 105 (1, 6) , 97 (3, 2) , 77 (3, 5) , 70
(6, 2) , 67 (2, 9} , 58 (100) ; NMR (200 MHz, CDC13, b scale;
multiplety, P.P.M. KCCB) 1.90 (3H), 2.27 (6H), 4.00 (1H,)
- 9.5), broadened singlet 5.53 (1H), d.m. 2.66 (2H, J4 -
J2 - 5.5) .
Dimethylaminoarglabin hydrochloride 14d was
produced by dissolving dimethylaminoarglabin 14b with
0.22 L of alcohol and heating to 40°C. After vacuum
filtration, hydrogen chloride gas was produced by
addition of 0.2 kg of sodium chloride and drops of
concentrated sulfuric acid. The reaction was monitored
by TLC. When the reaction was complete, the mixture was
heated to 52°C and the ethanol was vacuum distilled.
Approximately 0.9 L of ethylacetate was added to the
remaining tar with intensive stirring. The resulting
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precipitate yielded approximately 21 g of
dimethylaminoarglabin hydrochloride 14d.
Approximately 0.1 L of chloroform was added to
dissolve dimethylaminoarglabin hydrochloride 14d, then
distilled to remove the chloroform. The remaining tar
was mixed with 0.83 L of ethylacetate with intensive
stirring. The mixture was cooled to insure complete
precipitation of the product. The resulting precipitate
was vacuum filtered to remove all solvent. The end
product was vacuum dried over anhydrone and dissolved
with apyretic distilled water at a ratio of 2 grams of
dry material to 100 ml of water. Yield of
dimethylaminoarglabin hydrochloride 14d was approximately
grams (950 of the estimated amount on this stage).
15 Summary of dimethylaminoarglabin hydrochloride 14d
characteristics: melting point 203-204°C (ethanol-
ether) ; Ca]Z1D + 61.53° (c 0.52, CHC13) ; IR 33050-3000
(broad band), 2980, 2970 (intensive broad band, N-H);
2890, 2970, 2360-2300 (broad band), 1775 (carbonyl of
20 lactone), 1650 (weak band), 1480, 1450, 1385, 1345, 1185,
1140-1120, 1100, 1065, 1040, 1010 cm-'; MS (m/z, intensity
in %) 291 (3.01, M+ HC1) , 115 (2.19) , 105 (1.5) , 97 (3.2) ,
91 (4.0), 77 (3.5), 70 (16.2), 67 (2.9), 58 (100); NMR
(200 MHz, CDC13, b-scale, multiplety, p.p.m. KCCB) c. 1.30
(3H) , c. 1.87 (3H) , c. 2.87 (6H) , d.m. 4.17 (1H, J1 - .72=
lOHz), broadened singlet 5.55 (1H).
11, 13 dihydroarglabin 16 was produced by treating
arglabin 1 with ethanol and H2/Ni.
Example 3: Lyophilization - The water solution of
dimethylaminoarglabin hydrochloride was filtered through
a cotton-gauze plug or 8 layers of gauze, and a sterile
Millipor filter to a sterile glass jar. The solution was
vacuum pumped out of the jar into a measuring buret and
aliquoted into 2 ml vials or ampules. The filled vials
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or ampules were maintained at ~-40°C on sterile shelves
for 24 hours prior to drying in a KC-30 lyophilizer or a
LS-45 lyophilizer. After this tempering period, the
drying process was started. The temperature was
maintained at -40°C for 2 hour;, then was gradually
increased to approximately 50°C, (plus or minus about
5°C?. The transition to approximately 50°C occurred over
about 12-13 hours of drying. 7"he final temperature did
not exceed +60°C. The total duration of drying time was
24 hours. After this, the vials with dry compound were
immediately covered with caps and rolled. Ampules were
soldered. Each vial or ampule contained about 0.04 g of
the preparation.
Vials or ampules that were not sterile filtered
were sterilized by autoclaving for 20 minutes at 120°C,
with pressure of 1.2 Atm.
Alternatively, the prepared dimethylaminoarglabin
hydrochloride water solution was filtered through a
cotton-gauze plug or 8 layers c>f gauze. Approximately
200 ml of the solution were poured into 500m1 bottles,
covered with cotton-gauze plug; and wrapped with oil-
paper. The filled bottles were sterilized by autoclaving
for 30 minutes at 120°C with 1.2 Atm of pressure. The
sterile solution was cooled to room temperature. Using
sterile technique, 2 ml of the solution was poured into
sterile lOml vials. The vials were then lyophilized as
described above. After lyophilization each vial
contained about 0.048 of the compound.
Yield of the compound was 17 g, equaling 88.2% for
this stage and 0.22% overall of dry natural material. The
lyophilized material had a white-straw color and a bitter
taste. Authenticity of the preparation was verified by
determining its melting point and recording IR-, mass-,
and NMR-spectra. The quality of the preparation was
controlled by diluting 1 mg of the preparation with 0.2
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ml of water. Addition of one drop of a saturated
vanillin solution in concentrated sulfuric acid turned
the mixture a violet color, indicating the presence of
terpenes. Lyophilized material may be stored for three
years.
Example 4: Isolation of Other Ses4uiterpene Lactones -
Structures for compounds 17 through 21 are shown in
Figure 4. Glabellin 17 was also isolated from Artemisia
glabella Kar. et Kir. The yield of the compound from dry
raw material was approximately 0.016%. The structure of
glabella was determined through IR-, UN-, NMR, C13 NMR-,
mass-spectra and chemical transitions.
Summary of Glabellin Characteristics: melting
point 130-131°C (petrol-diether); [a]2°D +90.9° (SO, 17,
chloroform) .
3-keto-eudesm-1 (2) , 4 (5) , 11 (13) -trim-6, 12-
olid(1) 18 was prepared by selective dehydration of a-
sautonine with a yield of 45% and may be produced from
more than 20 species of wormwood. The structure was
determined by IR-, UV- and NMR-spectra.
Summary of 18 characteristics: melting point 145-
147°C (methanol) ; [a] 18D -10.4° (with 1, 12; chloroform) .
Anobin 19 was extracted from Achilles nobilis L.
The 2a, 3a-epoxy-4a, l0a-dioxy-5,7a(H), 6(3(H)-guai-
11(13)-en-6,12-olide structure of anobin 19 was
established by IR-, NMR- and mass-spectra and chemical
transitions.
Epoxy estafiaton 20 was produced by isomerizing an
available terpene lactone such as estafiatine through
isomerization with etherate of trifluoride boron then
epoxidizing with re-chlorbenzoil acid. The 3-keto-
l0a(14)-epoxy-1,5,7a(H) 4,6(3(H)-guai-11(13)-en-6,12-olid
structure was determined by IR-, NMR- and mass-spectra.
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Gaigranin 21 was produced by extraction of the
aerial part of Gaillardio grandiflora with chloroform
then chromatographically separating on a silica gel
column. The structure of gaigranin 21 was confirmed by
IR-, UV-, NMR and Cesy-spectra.
Example 5: In-vitro Activity of ArQlabin and Derivatives
-Viability of Cells - Transformed cells and primary
cultures of normal cells were incubated with varying
concentrations of dimethylaminoarglabin hydrochloride to
determine its effect on the viability of the cells.
Mouse mastocytoma (P-815), myeloma (Z-P3x63Ag8.653
and Pai) and human erthyroleukemia (K-562) cell lines
were used. Primary cultures of normal mouse hepatocytes
were isolated from mouse liver using collagenase. Mouse
splenocytes were isolated using a glass homogenizer.
Marrow cells were obtained by washing the bone marrow.
See, for example, Shears, S.B. and Kirk, C.J. (1984),
Biochem. J. 219:375-382.
Cells were cultured in RPMI-1640 medium
supplemented with 10% fetal calf serum, 100 mM L-
glutamine and 50 ~.g/ml gentamycin at 37°C under 5% COZ.
Cells were seeded into 24-well plates at a density of
50,000 cells/well and grown until near confluency,
approximately 2 days, then transferred to 96-well plates
at the same density. Transformed cell lines were
incubated for 18 hours with dimethylaminoarglabin
hydrochloride, in concentrations ranging from 1.5 ~.g/ml
to 100 ~cg/ml. Viability of the cells was determined by
trypan blue exclusion. As shown in Figure 5, a two fold
reduction in viability was observed at 6 ~.g/ml for X-653
and K-562 cells and at 12 ~.g/ml for P-815 cells.
Approximately 25% of K-562 and X-653 cells survived at a
concentration of 12 ~g/ml, and the same proportion of P-
815 cells survived at a concentration of about 25 ~Cg/ml.
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Higher concentrations of dimethylaminoarglabin
hydrochloride further reduced the viability of all the
transformed cells.
The proliferation of the transformed cells was
assessed by incubating 3H-labeled thymidine in the media
for 18 hours. At the end.of the specified time period,
the proliferation was measured by counting the amount of
3H-thymidine incorporated. Thymidine incorporation
provides a quantitative measure of the rate of DNA
synthesis, which is typically directly proportional to
the rate of cell division. Figure 6 shows that
proliferation of X-653 and P-815 cells was effectively
blocked at concentrations of 6 ~,g/ml and 12 ~g/ml,
respectively.
Primary cultures of normal cells were incubated
for 18 hours with a concentration of
dimethylaminoarglabin hydrochloride ranging from 10 ~.g/ml
to 2560 ~,g/ml. Viability was measured by trypan blue
exclusion. Figure 7 shows that an increase in the
concentration of dimethylaminoarglabin hydrochloride
reduced the viability of the normal cells, but a much
higher concentration was necessary to kill the normal
cells, as compared to the transformed cells. At a
concentration of 320 ~.g/ml, the number of viable
splenocytes was reduced by 50% in comparison to the
control. At concentrations of 640 ~.g/ml and 1280 ~g/ml,
40% and 10%, respectively, of the splenocytes were still
viable. At these same concentrations, approximately 50%
and 25% of hepatocytes were still viable. Marrow cells
were more sensitive to dimethylaminoarglabin
hydrochloride. At a concentration of 160 ~.g/ml, only
about 50% of the marrow cells were still viable.
Increasing the concentration to 320 ~g/ml reduced the
viability to about 25%.
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Protein Prenylation - Mouse myeloma Pai cells were
cultured in the presence of 60 ~M dimethylaminoarglabin
hydrochloride. The cells were collected by
centrifugation at 600 x g for 10 minutes and then washed
twice in PBS. Control cell: were grown in the absence of
drug. The cells were solubilized in lysis buffer (50mM
Tris, pH 7.4, 25 mM EDTA, 0.050 Tween, 0.01 M NaCl) for
30 minutes on ice. Lysates were made by homogenization
for 5 minutes at 4°C and precipitated by centrifugation
at 12,000 x g for 10 minutes. The supernatant was
collected. Proteins were precipitated with
trichloroacetic acid and then ;successively washed with
ethanol and ethyl ether. A se:Lective naphthol cleavage
of the bond between isoprenoides and proteins was
performed as described by Epstein, W.W et al., (1991)
Proc. Natl. Acad. Sci. USA 88:9668-9670. In general, 5
mg of a potassium naphthoxide <~nd naphthol 4:1 mixture
was added to approximately 10 mg of precipitated protein.
After addition of 50 ~1 of dimEahylformamide, the tubes
were gassed with argon, capped and heated to 100°C for
eight to 15 hours. Reaction products were extracted with
hexane and analyzed by HPLC (Waters System) using a 0.4 x
15 cm reverse-phase Nova-Pac C,8 column. The column was
eluted with 20% water in acetonitrile at a flow of 1.0
ml/minute. Napthol cleavage products were detected at
360 nm (Figure 8) with a full-:kale deflection of 0.01 A
unit. In the control (Figure F3A), the farnesylcysteine
derivative eluted at 4.5 minutes and the
geranylgeranylcysteine derivat~~ve at 6 minutes. The
molar ratio of geranylgeranyl t:o farnesylcysteine was 6.
The influence of dimethylaminoarglabin
hydrochloride on cellular prenylation is shown in Figure
8B. Using 60 ~.M of dimethylam~_noarglabin hydrochloride,
the peak corresponding to the f=arnesylcysteine derivative
does not appear on the chromatogram, while the
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geranylgeranyl peak appeared as in the control. This
indicates that dimethyl-aminoarglabin hydrochloride can
prevent farnesylation of proteins without significant
effects on geranylgeranylation.
Examt~le 6: Ia-vivo Activity of Arglabin and Derivatives
Overall, the compounds in this family have low
toxicity and are tolerated at dosages exceeding the
therapeutic dosage. Conventional toxicology methods were
used to determine the LDso for an intraperitoneal
injection of a 2% solution of dimethylaminoarglabin
hydrochloride in dimethylsulfoxide (DMSO) in mice (weight
20-22 g) and rats (120-130 g). The LDso was 190-220 mg/kg
in mice and 280-310 mg/kg in rats. An autopsy of the
animals revealed plethoricy of internal organs,
vasodilatation of the mesentery and intestines.
Tolerant single doses in rats and rabbits did not
disturb the function of the liver, kidneys,
cardiovascular system, respiration or peripheral nervous
system. Blood pressure was maintained. In addition, no
pyrogenic, allergenic, teratogenic or embryo toxic
effects were observed in animals.
Maximum tolerable doses (MTD) of arglabin and
derivatives were determined by daily intraperitoneal
administration to rats, guinea pigs or mice and daily
intravenous administration to rabbits over a period of
five to 20 days. In general, the MTD ranged from about
20 mg/kg to about 50 mg/kg for all compounds tested. For
example, the maximum dosage of dimethylaminoarglabin
hydrochloride in a solution of DMSO ranged from 20 mg/kg
in rabbits, 30 mg/kg in mice, 45 mg/kg in guinea pigs to
50 mg/kg in rats. Reversible changes in glycolysis and
tissue respiration were observed in blood serum and
hepatic tissue, changes of hormonal balance and elevation
of protein in urine were observed after prolonged daily
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intraperitoneal administration of a 2% aqueous solution
of dimethylaminoarglabin hydrochloride.
Inhibition of Tumor Growth in Rats - Human tumors
were implanted into mice and rats (sarcoma M-l;
Lymphosarcoma of Pliss; carc:inosarcoma of worker;
carcinoma of Geren; Sarcoma 45,; Sarcoma 180; Sarcoma 37;
Alveolar Cancer of liver PC-l; solid adenocarcinoma of
Erlich; breast cancer (PMK}; L~rntphocytic leukemia P-388;
lymphoidleukemia L-1210; variants of lymphosarcoma of
Pliss resistant to rubidomycin, prospidine and
leukoeffdine; and variants of :sarcoma 45 resistant to
sarcolysin, 5-fluorouracil, prospidine, and rubidomycin).
Treatment was started 24 hours after implantation in mice
and from the time measurable tumor nodes were detected in
rats. Animals for the controls were formed into groups
of 10-15. For estimating the anti-tumor activity of the
compound, the percent tumor growth inhibition was
determined after the end of trE:atment. The results were
statistically analyzed using the t-test. Histologically,
regression of the tumors was accompanied by dystrophy,
necrosis of tumor cells, disturbance of blood supply to
tumor tissue, and replacement with connective tissue.
Tables III and IV summarize the percent tumor
growth inhibition activities of: arglabin and various
derivatives against both non-drug resistant and resistant
tumors. For comparison, Table III also contains the
percent tumor growth inhibition for colchicine, a
compound with known anti-tumor activity. Introduction of
haloids such as bromine and chlorine appears to increase
the anti-tumor activity. Epoxidation of arglabin on the
C3-C4 double bond also increases the anti-tumor activity.
Dimethylaminoarglabin and dimethylaminoarglabin
hydrochloride were effective agrainst a wide range of
tumors. One advantage of dimethylaminoarglabin
hydrochloride is that it is soluble in water.
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-30-
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
- 32 -
Combination Therapy - Results of the animal trials
permitted design of the most rational scheme of treatment
with dimethylaminoarglabin hydrochloride and other
antitumor drugs.
A complete disappearance of tumors resistant to
prospidin and rubidomycin was observed in 600 of rats
treated with the combination of dimethylaminoarglabin
hydrochloride, cisplatin and methotrexate. In addition,
this combination overcame the cross-resistance of
sarcoma-45 to methotrexate, sarcoma-45 to 5-Fluorouracil,
and Pliss' lymphosarcoma to rubidomycin. No animals
deaths were observed with this treatment.
The collateral sensitivity of leukofedin resistant
Pliss' lymphosarcoma after administration of sarcolysin
was accompanied by the complete disappearance of the
tumor in 60% of the rats. The combination of
dimethylaminoarglabin hydrochloride and sarcolysin, at
half of the MTD, caused a block in DNA synthesis
(synthesis inhibition index 94.1 - 97.10). This
combination did not decrease the blood cell level.
The combination of dimethylaminoarglabin
hydrochloride and methylnitrosourea was administered at
intervals of 2, 4 or 24 hours between the two drugs. It
was determined that it was optimal to administer
dimethylaminoarglabin hydrochloride two hours prior to
administration of methylnitrosourea. The cross
resistance of sarcoma-45 to prospidin and sarcoma-45 to
5-fluorouracil, Pliss' lymphosarcoma to rubidomycin and
Pliss' lymphosarcoma to prospidin was overcome with the
combination of dimethylaminoarglabin hydrochloride and
methylnitrosourea. Approximately 60% of the tumors
disappeared in the rats without adverse drug reactions.
Administration of methylnitrosourea prior to
administration of dimethylaminoarglabin hydrochloride
decreased the antitumor activity and increased toxicity.
CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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Histologically, fewer svmall pyknotic polymorphic
cells without clear structure were seen after treatment
with the combination dimethylaminoarglabin hydrochloride
and methylnitrosourea in comparison to the control group.
It was found that dimethylaminc>arglabin hydrochloride
administered 2 hours prior to methylnitrosourea reduced
toxicity.
The same results were seen when
dimethylaminoarglabin hydrochloride was administered two
hours prior to a mixture of vir.~cristine and vinblastine
for Geren's carcinoma and for Worker's carcinosarcoma.
Dimethylaminoarglabin hydrochloride moderately
increased the duration of life in the animals with non-
resistant and drug resistant tumors. The combination of
dimethylaminoarglabin hydrochloride and other antitumor
drugs further prolonged the duration of life. For
example, the combination of dimethylaminoarglabin
hydrochloride and vincristine prolonged life 114% in
animals with methylnitrosourea resistant tumors. The
combination of dimethylaminoarglabin hydrochloride and
cisplatin, at half of MTD, prolonged life 117% in animals
with methotrexate resistant L1210. A good therapeutic
effect was seen from the triple combination of
dimethylaminoarglabin hydrochloride, vincristine and
cyclophosphamide at half of MTD as compared with the
double combination of dimethylaminoarglabin hydrochloride
and vincristine or dimethylamin.oarglabin hydrochloride
and cyclophosphamide. The triple combination prolonged
duration of life by 209%. The quadruple combination of
dimethylaminoarglabin hydrochloride, vincristine,
cyclophosphamide and cisplatin was less effective than
the triple combination. This may be due to increased
toxicity of the antitumor drugs.
The effect of dimethylaminoarglabin hydrochloride
alone and in combination with other drugs was studied in
CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
- 34 -
the model of drug resistant metastasis of Pliss'
lymphosarcoma. The metastases in the inguinal lymphoid
nodes were the most sensitive among the initial and drug
resistant nodes. They did not develop in 10% of the
cases. The duration of life for dimethylaminoarglabin
hydrochloride alone was 128% in comparison with the
control group. The combination of dimethylaminoarglabin
hydrochloride and vincristine caused inhibition of tumor
growth, with tumor dissolution, in 30% of the rats.
Duration of life was increased 174% with the absence of
any new metastases in the inguinal lymphoid nodes.
The combination of dimethylaminoarglabin
hydrochloride and methotrexate prolonged the duration of
life 300% in animals with prospidin resistant Pliss'
lymphosarcoma. This combination led to an eight-fold
decrease in the frequency of metastasis.
In order to reveal the possible mechanisms of
dimethylaminoarglabin hydrochloride action against the
initial and drug resistant tumors and their metastases,
dimethylaminoarglabin hydrochloride and sarcolysin, alone
and in combination, were used for the treatment of
sarcoma 45 to investigate the disturbance of DNA
synthesis. Beneficial results were observed with
sarcolysin and with the combination of sarcolysin and
dimethylaminoarglabin hydrochloride in the case of the
non-drug resistant sarcoma 45. In the case of drug
resistant sarcoma 45, dimethylaminoarglabin hydrochloride
alone was very effective (DNA inhibition index 99%).
Moreover, DNA inhibition increased after 24 hours upon
daily administration over the subsequent 5 and 10 days.
This indicated that repeated administration of
dimethylaminoarglabin hydrochloride, rather than a single
administration of the MTD, had a cumulative antitumor
effect.
CA 02287318 1999-10-21
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The immunity of rats with initial and prospidin
resistant tumors with metastases was studied after
treatment with dimethylaminoarglabin hydrochloride alone
and in combination with other cytostatics. An
improvement of the immunodepression found after treatment
with sarcolysin and cisplatin was observed after
treatment of the animal tumors with dimethylaminoarglabin
hydrochloride. The combination of dimethylaminoarglabin
hydrochloride and sarcolysin or cisplatin increased
immunological indices, particularly if
dimethylaminoarglabin hydrochloride was administered two
hours before the cytostatic drugs. These results
suggested that dimethylaminoarglabin hydrochloride
softened the immunodepressive effect of cytostatics and
normalized the immune balance of the body. These data
show that dimethylaminoarglabin hydrochloride decreased
cytotoxicity and increased the efficacy against drug
resistant tumors alone and in combination.
Immune System Modulation - The effect of
dimethylaminoarglabin hydrochloride was determined in
intact and immunodepressed mice. Mice were
immunodepressed by administration of 200 mg/kg of
cyclophosphamide. Injection of cyclophosphamide resulted
in considerable leukopenia related primarily to
lymphopenia. The humoral immunity of the animals was
considerably suppressed, as was cell-mediated immunity
although to a lesser extent. Two dosages of a 2%
dimethylaminoarglabin hydrochloride solution, 50 and 100
mg/kg, were injected IP into white mongrel mice. The
Hemagglutination test and delayed-type hypersensitivity
reaction were determined before and after administration
of drug. It was determined that a single 50 mg/kg dosage
of dimethylaminoarglabin hydr_oc:hloride did not alter the
hemagglutination titer or the delayed-type
CA 02287318 1999-10-21
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hypersensitivity reaction. Dosages of 100 mg/kg resulted
in a slight decrease in hemagglutination titer.
Repeated IP injections of 10 to 50 mg/kg
dimethylaminoarglabin hydrochloride were administered
over five to 10 days to determine the effect of prolonged
administration. It was found that lower dosages such as
and 20 mg/kg, increased hemagglutination titers by
days 5 and 10. Administration of higher dosages, such as
30 mg/kg, resulted in a decreased hemagglutination test
10 index by day 10. No effect on delayed-type
hypersensitivity reaction was seen at day 5, but was
increased by day 10.
In intact mice, injection of 10 mg/kg increased
the total number of leukocytes through an increase in the
absolute number of lymphocytes. The relative number of
neutrophils was slightly reduced. Increasing the dosage
to 20 mg/kg did not result in an overall increase in
leukocyte number. The nitroblue tetrazolium assay was
used to assess the function of the neutrophils. It was
found that while the number of neutrophils was decreased,
activity of the neutrophils was not altered at 10 mg/kg.
With a higher dosage, 20 mg/kg, a decrease in nitroblue
tetrazolium positive neutrophils was seen, indicating a
decrease in function.
Daily IP administration of 10 and 20 mg/kg
dimethylaminoarglabin hydrochloride for 10 days to intact
mice resulted in a dramatic change in T-lymphocytes, B-
lymphocytes and natural killer cells. At 10 mg/kg, the
overall leukocyte count was increased through an increase
in natural killer cells and T-lymphocytes while B-
lymphocytes remained stable. It was found that the
increase in T-lymphocyte number was a result of an
increase in the level of the T-helper subpopulation. The
level of the T-suppressor subpopulation was not altered.
At a higher dosage (20 mg/kg), overall leukocyte number
CA 02287318 1999-10-21
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was not altered, even though B-lymphocyte number
decreased and T-lymphocyte decreased to a lesser extent.
Natural killer cell number was increased. The nitroblue-
tetrazolium assay was also decreased.
Overall, the effect of dimethylaminoarglabin
hydrochloride on the immune system depended on the
administrated dose. At a low dosage (lOmg/kg),
dimethylaminoarglabin hydrochloride increased T and B-
lymphocytes and natural killer cell levels. The increase
in T-lymphocytes was accompanied by an increase in the
level of the T-helper subpopul<~tion of T-lymphocytes.
Higher dosages of dimethylaminoarglabin hydrochloride
(20mg/kg) decreased T and B-lymphocyte number, but
increased certain other cell populations, such as natural
killer cells.
Injection of 10 mg/kg dimethylaminoarglabin
hydrochloride to immunodepressE:d mice for 10 days reduced
the leukopenia and lymphopenia observed in the mice. By
the tenth day of treatment, thE: total number of
lymphocytes in immunodepressed animals did not differ
from the values obtained from intact animals. The
increased number of T-lymphocytes was accompanied by an
elevation in the T-helper subpopulation as well as the T-
suppressor subpopulation, although to a lesser extent.
The numbers of B-lymphocytes we're not completely restored
to normal values.
Additional immunological data were obtained from
an "August" line of rats, weighing 140-160 g, with and
without Pliss lymphosarcoma. four indices, spontaneous
rosette forming of erythrocyte;, nitroblue tetrazolium
assay, delayed-type hypersensitivity and hemagglutination
were examined before treatment, during treatment (5 and
10 days) and 5 days after treatment with
dimethylaminoarglabin hydrochloride. Approximately 50
mg/kg of a 2% water solution of lyophilized-
CA 02287318 1999-10-21
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dimethylaminoarglabin hydrochloride was injected IP,
daily, for 10 days. Results are summarized in Table V.
Overall, lyophilized-dimethylaminoarglabin hydrochloride
stimulated delayed type hypersensitivity, but reduced all
other studied indices in intact rats. In rats with Pliss
Lymphosarcoma, the immune system was stimulated, as all
studied indices increased. An advantage of lyophilized-
dimethylaminoarglabin hydrochloride is that it
ameliorates the immunodepressive effect of known
cytostatics such as 5-fluorouracil and sarcolysin.
TABLE V
Rats with Pliss
Intact Rats Lymphosarcoma
INDEX After TreatmentAfter Treatment
Spontaneous- Decreased Increased
erythrocyte rosette 64-84% 94.8-162.5%
forming (E-ROK)
Nitroblue Decreased Increased
tetrazolium (NBT) 67-91% 79.2-175.4%
Delayed type Increased Increased
hypersensitivity 153-207% 89.9-132.2%
Hemagglutination Decreased Increased
(RHG) 43-53% 82.2-142.6%
Pharmacokinetics - Experimental pharmacokinetic
data were obtained for dimethylaminoarglabin
hydrochloride using 30 random bred rats of both sexes.
The rats weighed from 200 - 220 grams. Gas
chromatography and a FARM modelling program was used in
the analysis of all pharmacokinetic data. Intravenous 2
mg/kg dimethylaminoarglabin hydrochloride showed a
maximal level of 30 ~,g/ml in the blood serum within one
hour. Dimethylaminoarglabin hydrochloride spread quickly
throughout the organism from the blood to peripheral
tissues. The obvious volume of distribution was large,
indicating that it could pass through cell membranes and
tissue barriers. The highest concentration of drug was
CA 02287318 1999-10-21
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- 39 -
accumulated in the lungs and spleen during the first hour
after administration. Maximum lung and spleen
concentrations were 149.4 and 159 ~g/g, respectively.
Within three hours after administration, the
concentration in the liver and skeletal muscle was 228.6
and 176.4 ~,g/g, respectively. It was found that the
preparation accumulated in the liver and was retained for
a more extended period in comparison to other tissues.
Dimethylaminoarglabin hydrochloride was able to penetrate
through the blood-brain barrier. Brain tissue
concentration was 23.9 ~.g/g after one hour and stabilized
at 15.6 ~,g/g in 24 hours.
Dimethylaminoarglabin hydrochloride was excreted
fairly slowly. The biological half-life was about 46.8
hours in rats, with the average time of retention about
67 hours. Renal excretion proceeded slowly. The kidney
concentration was maximal after three hours. By 24
hours, the kidneys had the highest concentration, 56.6
~g/g. Total clearance was 0.05 ml/minute at a low
transportation rate of the preparation from peripheral
tissues into the blood.
Clinical Data on Dimethvlaminoaralabin Hydrochloride
A first clinical trial of dimethylaminoarglabin
hydrochloride was performed on 51 patients with end-stage
(III-IV) cancer. 20.7% of the patients in the first
clinical trial had lung cancer,. 17% had liver cancer, 17%
had stomach cancer, 9.4% had rectal cancer, 5.7% had
ovarian cancer, 5.7% had esophageal cancer, and the
remaining had either salivary eland, lymphosarcoma,
breast or large intestinal cant:er. The patient
population was 67.9% male and 32.1% female. Generally,
patients were intravenously given reconstituted
dimethylaminoarglabin hydrochloride in an aqueous
solution. In patients with asc:ites,
dimethylaminoarglabin hydrochloride was administered
CA 02287318 1999-10-21
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- 40 -
intraperitoneally. Intrapleural administration was used
for patients with pleurisy. Patients were given a very
small dose and monitored for any signs of allergic
reaction before proceeding. Initially, 80 mg of the
compound was given per day as a single dose, then
gradually increased to a maximum level. After 30-35
days, the dose was increased to 480 mg per day. At this
high dose, patients complained of nausea and vomiting.
It was estimated that the daily dose should be about 240-
280 mg for typical cases. Total dose over the course of
treatment was typically five to six grams of
dimethylaminoarglabin hydrochloride, but was as high as
grams. Immediately after administration of the
compound, patients reported a bitter taste that quickly
15 dissipated. Additional courses of treatment were
administered to some patients. A summary of the data
from the first clinical trial is shown in Table VI.
Patient condition before treatment was rated on a scale
of 1 to 3, where 1 was bedridden, 2 was a significant
20 restriction on activity and 3 retained full activity.
Therapeutic result was measured on a scale from 0 to 3,
where 0 was no improvement, 1 was insignificant or-
improvement for less than a month, 2 was considerable
improvement (25-50% reduction in tumor size) and 3 was
sharp improvement (50-100% reduction in tumor size).
CA 02287318 1999-10-21
WO 98/48789 PCT/pS98/07989
41
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
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CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
- 47 -
Overall, approximately 30% of the patients had a
considerable improvement. 55.6% of patients with liver
cancer had a considerable improvement after
dimethylaminoarglabin hydrochloride treatment. Patients
with lung and ovarian cancer responded particularly well
to dimethylaminoarglabin hydrochloride treatment as about
64% of lung cancer patients and 66% of ovarian cancer
patients had considerable improvement.
Dimethylaminoarglabin hydrochlc>ride had little toxicity
and did not suppress hematopoieais. During the trial, no
negative responses of the gastrointestinal tract or hair
follicles were registered.
In patients with primary liver cell carcinoma, the
size of the liver was reduced over 50% in two patients
and approximately 50% in another patient after treatment
with lyophilized-dimethylaminoarglabin hydrochloride.
Patients reported an improved state of mind and appetite.
Pain in the right hypochondrium disappeared.
Immunologic status of the patients was evaluated
using standard methods of rozette-formation and
phagocytosis. These indices were studied prior to
treatment, during treatment and after treatment. Blood
samples were taken from a finger. Analysis of the
average immunological values for this patient group
revealed a positive response to treatment. On days 3-5
of treatment, the number of T-lymphocytes was reduced
from 57% to 40.1%, the number of T-helper lymphocytes was
reduced from 50% to 37.3% and t:he neutrophil adhesiveness
decreased from 42% to 28.5%. Undifferentiated
lymphocytes increased from 21.5's to 42.2%. A general
change in the ratio of T-helper lymphocytes to T-
suppressor lymphocytes was due to an increase of T-
suppressor lymphocytes. The number of B-lymphocytes and
phagocytic activity remained stable. Total number of
leukocytes increased up to 9.4x:L09/L and the total number
CA 02287318 1999-10-21
WO 98/48789 PCT/US98/07989
- 48 -
of lymphocytes increased as well. The levels of all
types of immunoglobulins increased.
By day 20 of treatment, all indices returned to
normal. In some patients, indices returned to normal by
day 14. In patients that were analyzed 30 days after
treatment, a significant increase in the number of T-
lymphocytes and adhesiveness of neutrophils was observed.
A three to six month lag in dimethylaminoarglabin
hydrochloride production halted the first clinical trial.
In a second clinical trial, dimethylaminoarglabin
hydrochloride was given to 72 patients (61.1% male and
38.9% female) with stage IV cancer from different
localizations. Among the patients, 25% had carcinoma of
the stomach, 16.7% had liver cancer, 18.1% had lung
cancer and the remaining 40.2% had esophageal, breast,
ovarian, pancreatic, brain or lymphosarcoma. Patients
with poorer states had metastases to the liver (25%),
retroperitoneal lymph nodes (25), ascites (22.2%) and
exudative pleuritis (11.1%). Some of these patients had
been previously treated with dimethylaminoarglabin
hydrochloride in the first clinical trial. Results from
the second clinical trial are summarized in Table VII.
Table VII
RESPONSE ~ of Patients
2 Total Regression -
5
Partial regression over or 61.1
equal to
50$ decrease of tumor or metastasis
Absence of dynamics or 31.9
stabilization
3 Progression 7.0
0
Use of dimethylaminoarglabin hydrochloride as an
anti-tumor cytostatic in solid tumors has a number of
advantages. The preparation has no side effects, it does
not suppress hematopoiesis, it normalizes the functional
CA 02287318 1999-10-21
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condition of immune system, and has no allergenic effect.
As a cytostatic, it is particularly efficient for primary
cancer of the liver and other ~~olid tumors complicated
with polyserositis. Partial regression of tumor was
observed in 61.1% cases; stabilization of process - in
31.9% cases and recurrence was observed in only 7.0%.
88.9% of patients (64 of 72) responded to therapy: no
response was observed in 11.1% (8 of 72).
The following are abstracts from the case records
of selected patients, who received dimethylaminoarglabin
hydrochloride monochemotherapy.
Patient M, age 55, case number 305, entered the
hospital with multiple nodes on the skin of thorax and
abdomen, ulcer on the place of extripated breast, and
induration on the right breast. In a previous hospital
stay, a radical mastectomy had been performed at
Sakhalinsk Oncology Center due to breast cancer. After
surgery, she received 6 courses of polychemotherapy with
cyclophosphamide and methotrexate.
Symptomatic therapy was recommended because of
recurrence of the process. Before treatment, the abdomen
and thorax skin had multiple metastatic nodes with sizes
ranging from 0.5 to 1 cm. On the left side of thorax, an
ulcerous surface, approximately 10x12 cm, was present.
The right breast was deformed because of infiltrative
metastases. Edema was present in the lower extremities.
A blood analysis before treatment revealed the
following parameters: Hb-89, ESR-6 mm/hy, L-3.3, Er-3.8
ml, juv.ne-4, seg. ne-78, mon-1. The patient received 5
courses of dimethylaminoarglabin hydrochloride treatment
at a total dose from 6.0 to 7.3 grams. A blood analysis,
repeated after chemotherapy, revealed the following
parameters: Hb-122, ESR-20 mm/h, L-10.9, Er-3.8 ml, eos-
1, stab ne-3, seg, ne-64, lym-34, mon-2.
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During the treatment, the ulcer was epithelized,
the metastases nodes were resolved, and infiltration of
the right breast decreased 50a. Edema on the lower
extremities was gone.
In a 4 month old patient, case number N2225, a
complete recovery from liver cancer was observed. The
young patient was admitted to the surgical department of
the Karaganda Cancer Treatment Center in extremely poor
condition and diagnosed with embryonal carcinoma of the
liver. The cutaneous integuments were of yellowish
color. The patient had labored breathing. Cardiac
sounds were clear, rhythmical. Ps-150 per minute. The
tongue was moist, clear. The abdomen was enlarged. The
liver was enlarged and indured with a smooth surface, the
lower margin at the upper flaring portion of the ilium.
Ultrasonic tomography (UST) of the liver indicated
that the liver was enlarged and occupied the whole
abdominal cavity. The structure was dissimilar because
of the foci of dissimilar structure with hydrophilic rim
up to 5-6 cm, indicating a liver tumor.
Blood analysis before treatment revealed the
fol-lowing parameters: Hb-84, ESR-4 mm/h, L-10.9, Er-3.3
ml, eos-1, juv, ne-55, stab ne-45, seg, ne-14, lym-30,
mon-5. Paracentesis of the liver was performed under the
control on UST. Bare nuclei of tumor cells were observed
against a background of hepatic cells with degenerative
changes. The patient was diagnosed with embryonal liver
cancer.
A course of dimethylaminoarglabin hydrochloride
treatment, at a daily dose of 120 mg IV was started. The
total dose for the course of treatment was 2040 mg.
During treatment, a significant improvement was observed.
The abdomen became symmetric and smaller due to the
decrease in liver size.
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A UST indicated that the=_ liver projected from
under the coastal arch along midclavicular line on 4 em,
the outlines are even, the structure is dissimilar
because of the foci of dissimilar structure with
hydrophylic rim and foci of high echogenity 2.0-2.5 cm in
diameter. Conclusion: tumor with focal changes.
Blood analysis, repeated after treatment revealed
the following parameters: Hb-177, ESR-4 mm/h, er.-4.Oml,
Z-9.8, eos-3, seg. ne-28, lym-53, mon-6. The child was
discharged in a satisfactory condition. Two weeks later
a repeated course of treatment was given, which was well
tolerated by the patient.
As of early 1997, the baby's condition is
satisfactory. Her mother has not reported any sign of
recurrence. The palpation of the abdomen showed the
liver was smooth, projects from under the margin of the
coastal arch on 2 cm. The baby is believed to be cured.
Patient A, age 27, case number 543 was diagnosed
with a brain tumor. The neurosurgeon excluded the
possibility of operation because of the poor condition of
the patient. The patient was very weak and had expressed
bradykinesia of akineticorigid ayndrome type. Bilateral
exopthalm was reported. After 'the first course of
dimethylaminoarglabin hydrochloride treatment, his
condition was stabilized and no headaches were reported.
After the second course, the condition was stable. No
headaches were reported and the appetite was preserved.
This case confirmed the experim~=_ntal findings regarding
the compound's capability to get through the blood-brain
barrier.
The efficiency of dimeth.ylaminoarglabin
hydrochloride monochemotherapy was estimated according to
Karnofsky scale, 1997 (Table VI:II). No side effects of
dimethylaminoarglabin hydrochloride therapy were
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reported. Mean indices of peripheral blood are shown in
Table IX.
Table VIII
Karnofsky scale
Estimation of Description of the effect Number
of Il
i
the effect, Patients
in
abs,
N 0
90 Ability to keep normal 30 41.7
activities, minimum signs
of the disease.
80 Normal activities are 10 13.9
hardly performable, there
are some signs of the
disease.
70 Maintains himself, but is 24 33.3
not able to work.
60 Needs occasional 5 6.9
assistance, but is able to
maintain himself.
30 Extreme invalidism 3 4.2
Table IX
Lab. indices Before Treatment During After
1012/1 - 109/1 Treatment Treatment
Erythrocytes 2.3-3.0 2.4-3.0 3.0
Leukocytes 4.5-5.5 3.5-4.0 4.0
Lymphocytes 8-I5 28-35 15-20
Thrombocytes 2.0 (thsd) 2.3 2.8
The indices of immune system were determined using
rozette forming and phagocytosis methods. 57 patients
that had received dimethylaminoarglabin hydrochloride
were examined. Eleven indices of cellular and humoral
immunity were measured from each patient in order to
evaluate immune status. The following indices were
determined on 0.05 ml of peripheral blood: absolute and
relative amount of T- and B-lymphocytes, amount of non-
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differentiated "zero" cells, adhesion and phagocyte
activity of neutrophils, hemogram, level of serum
immunoglobulins. Indices were determined before
treatment, on days 2, 5 and 14 of treatment, and after
treatment. Table X summarizes the results before and
after treatment.
On the 2nd-5th day of treatment, the percentage of
T-lymphocytes and T-helper lymphocytes was reduced
considerably. The level of non-differentiated "zero"
cells increased. This population of non-differentiated
cells consisted of both aged and immature B- and T-
lymphocytes, and natural killer cells. No significant
shifts in hemogram were noted.
Beginning with the 6th-:10th day of treatment,
almost all indices returned to their initial values. In
two weeks, an increased ratio of T-lymphocytes and their
T-helper population was registered, while the number of
B-lymphocytes decreased. No changes in serum
immunoglobulin levels were registered at that time.
After the treatment, a statistically insignificant
elevation of T-lymphocytes percentage content was seen.
Absolute numbers of T-lymphocytes increased as well as
the number of T-helper lymphocytes enhancing phagocyte
activity of neutrophils. There was an increased number
of B-lymphocytes and also an increased amount of
immunoglobulins A, M and G. The number of
nondifferentiated cells was reduced.
The total number of lymphocytes in the peripheral
blood was elevated. As tumors can cause both
quantitative and qualitative changes in blood cells,
these parameters were checked after dimethylaminoarglabin
hydrochloride treatment. No shift in qualitative
(morphological) composition of blood cells was
identified, although some quantitative changes such as a
decreased number of neutrophils and an increased number
CA 02287318 1999-10-21
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of lymphocytes was observed. This suggests a reduction
lymphotoxic effects caused by the tumor. The immunologic
indices correlated with the clinical findings in most
cases.
Table X
Indices of patients' immunity before and after arglabin
treatment
Indices Before After
Treatment Treatment
(range) (range)
I
T-lymphocytes, ~ 48.001.74(28-72) 52.001.42(36-72)
1 T-lymphocytes, absolute0.720.08(0.13-1.67) 1.140.17 (0.18-2.99)
0
B-lymphocytes, ~ 19.121.02(8-12) 18.5010.0(4-32)
B-lymphocytes, absolute0.290.05(0.09-0.73) 0.450.07 (0.05-1.16)
T helper cells, ~ 39.121.25(24-52) 45.3011.26(28-60)
T suppressor, ~ 10.481.60(00-60) 10.500.95(00-24)
1 Non differentiated 32.641.74(8-52) 26.002.21(4-60)
5 lymphocytes, $
D-phag., ~ 41.360.95(28-52) 48.10.93 (36-85)
D-phag, abs 1.690.26(0.64-7.45) 2.320.19 (0.66-5.69)
Adhesion, N/ph 39.681.74(24-68) 42.401.72(24-66)
2 Immunoglobulins A, 1.480.02(1.06-2.0) 1.970.08 (0.90-2.84
0 g/1
G 14.3310.43(11.0-22.0) 17.910.41(11.6-22.0)
M 1.360.03(1.18-1.84) 1.530.03 (1.20-1.84)
Leukocytes, 10'/1 5.970.60(3.40-18.6) 9.140.61 (3.40-18.9)
Neutrophils, ~ 4.720.91(0.0-23.0) 5.450.79 (0-20)
2 Segmentation nucleus 61.6845.0(23-85) 63.051.79(38-88)
5
Eosinophil 2.720.71(0-18) 3.35f1.30(0-33)
Monocytes 5.282.48(0-12) 4.450.44 (1-12)
Lymphocytes 25.522.02(6-57) 23.1511.7(4.0-47)
Mean values of immunological indices were
30 evaluated with regression analysis. Functional
conditions of the immune system were estimated using
integral indices such as a mean intensity (correlation)
expressed in relative units. That the index-intensity of
the immune system increased during treatment indicates
35 that the immune system responded to treatment.
CA 02287318 1999-10-21
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- 55 -
Correlation analysis of these data indicate that
during treatment, the total amount of true bound
parameters increased (the number of bonds with r> 0.7
increased, and the number of negative bonds was reduced}.
The number of interrelations between the elements
of immunity increased, namely between the lymphocyte and
neutrophil elements.
Thus, the statistical data analyzed with different
methods of statistical assay, indicate that
dimethylaminoarglabin hydrochloride is active as an agent
stimulating some immunity factors and improving the
functional condition of the immune system. This
indicates the immunostimulative effect of the
preparation.
Other Embodiments
It is to be understood i~hat while the invention
has been described in conjunction with the detailed
description thereof, the foregoing description is
intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended
claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
