Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF IIN~VIUNE RESPONSES
FIFZD OF THE INVENTION
THIS invention relates to the treatment of conditions in the human body
which are associated with inappropriate immune responses. More
particularly, this invention is concerned with the treatment of conditions in
which the inappropriate immune response is associated with inappropriate-
immune cell metabolic activity which, in turn, is mediated, or at least
thought to be associated with the Tyrosine Kinase Cascade in which Protein
Tyrosine Kinase, hereinafter referred to as "PTK", plays an important role,
or with Cycline Dependent Kinase, hereinafter referred to as "CDK" in the
immune cell. The invention consequently provides for the treatment of and
for medicaments for use in the treatment of, a large variety of ailments
associated with inappropriate immune responses in the animal body, which
term is intended herein to include the human body.
BACKGROUND TO THE INVENTION
It is well known that immune cells play an important role in the immune
system of the animal body. A variety of such cells have been identified.
Amongst these the T lymphocytes, also known as T-cells, and the B-
lymphocytes, also known as B-cells, have been recognized for their
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important role in the immune response of the animal body against foreign
infections. Additionally, other cells capable of producing immunological
mediators have an immune function. The normal, or appropriate response
- of such cells to an extraneous stimulant, for example a viral or bacterial
infection of the animal body or to endogenous immune stimulants, for
example oncogenic transformation, is to cause the activation of the
metabolic pathways of the cells in issue resulting in the production of
immunological mediators including cytokines, lymphokines, chemokines,
growth factors and cytotoxic cells and usually also gives rise to the
proliferation of such immune cells. The immunological mediators, in turn,
perform or complement the function of neutralizing the infective agent, or
toxins released by it, as part of the natural infection combating or healing
process of the body. These pathways involve complex biochemical and
signalling systems to which further reference is made below.
It is also well-known that, for reasons which are not fully understood at
present, the response of the immune system is sometimes abnormal and
gives rise to the expression by, and secretion from, the immune cells of
products considered to be inappropriate. While such abnormality may be
associated with an abnormal proliferation of such cells, it is not necessarily
the case. Such abnormality in immune cell activity may simply be
manifested by the secretion of an inappropriate quantity of immune cell
products without necessarily being associated with an increase in the
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proliferative activity of such cells.
Inappropriate immune response has been implicated in a large variety of
ailments affecting the human and animal body. The present invention is
directed at the treatment of such conditions as will appear below. In the
conditions in question the manifestation of an inappropriate immune
response, including the presence of cytotoxic cells, and/or of an
inappropriate quantity of the immune cell products required to combat a
particular infection or condition, give rise to what may be thought of as an
attack by the immune system on specific organs of the body itself, or even
to a non-specific immunological attack on the body generally, rather than
such attack being appropriately directed at the extraneous or endogenous
antigen or agent or metabolites such as toxins produced thereby. The target
organs of such inappropriate attack are diverse and, as will be seen from the
list of conditions enunciated below, encompass the skin, the muscles, the
skeletal structure, the joints, the blood, the brain and nervous system, the
internal organs including the bowel, kidneys, lungs, liver, and even the
sensory organs including the eyes.
In some of the conditions or syndromes with which this invention is
concerned a primary or initiating causative agent or stimulant for the
immune response has been identified. It is however a feature of such
conditions or syndromes that the immune response may persist even after
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the causative agent or stimulant, such as, for example, a viral or microbial
infection or other form of antigen, has been eliminated by the body's own
defense mechanism, namely the immune system, or by means of
- pharmaceutical intervention, or by a combination thereof. In other cases
no, or at least no as yet identified causative agents are known. All these
conditions are considered to be immune mediated ailments.
For a clear understanding of the nature and impact of the present invention
it is necessary briefly to explain the current understanding of the nature of
immune response activity in the body. Immune cells, and in particular T
lymphocytes are dependent for their biological function on signal
transduction through the T cell receptor which is unique to, and present
only on T lymphocyte surfaces. The T cell receptor is a complex group of
surface molecules including CD4 or CDe surface molecules. The CD4, CD8
and certain other surface molecules are capable of transmitting a signal from
the external surface of the cell membrane to the internal environment of the
cell i.e. to the cytoplasma of the cell. These signals are transmitted through
an enzyme pathway known as the Tyrosine Kinase Cascade. Activated
Tyrosine Kinases phosphorilate certain proteins leading to the development
of new signal intermediates. In this cascade the enzyme known as Protein
Tyrosine Kinase [PTK] plays an important role. This enzyme is a protein
featuring free sulfhydryl groups. The Tyrosine Kinase cascade ultimately
terminates in the expression of specific genes in the T cell. These genes
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respectively code for a variety of immunological mediators including
cytokines and for structural proteins required for cell proliferation. The
immune response mediated ailments with which this invention is concerned
are, as mentioned before, associated with uncontrolled expression of
immunological mediators by the immune cells and uncontrolled cytotoxic
cell activity.
DISCUSSION OF PRIOR ART
Dimethylformamide or DMF is a volatile, non-ionic liquid at room
temperature and is well known for its use as an industrial solvent. It is
used, amongst others, in the manufacturing process of various products for
human consumption, including in the manufacturing processes of
pharmaceutical products. Its toxicity is well studied. It is known to be non-
toxic at levels many times higher than the levels with which the present
invention is concerned. Such toxicity as DMF has at high levels of
concentration in the human body is attributed to its depletion of
glutathione in hepatocytes in the liver resulting in hepatocyte necrosis.
It has been claimed by Cryopreservation Technologies CC in PCT
application PCT/US96/19697 published on 26 June 1997 under WO
97/22248 that dimethylformamide and related compounds may be used in
the treatment of viral and/or microbial infections. That publication cites
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examples showing that DMF may be administered transdermally to a patient
to obtain a DMF in blood level aimed to be 100 ppm, but the levels
reported as actually achieved varied from this target, in HIV positive
- patients. It further reports that after such treatment with DMF for a period
of less than three weeks, the viral load in the patient had dropped from 120
000 to 500/ml. It also reports an improvement in the symptoms of acne and
German measles in patients treated with DMF to achieve a DMF/blood
level of 50 - 100 ppm. That disclosure relates to the use of DMF as an anti-
viral arid anti-microbial agent and is silent on the immune response
regulatory properties of the compounds with which this invention is
concerned, and on the use of such compounds in the treatment of immune
cell related illnesses or the use of such compounds in the manufacture of
medicaments for use in the treatment of such conditions.
OBJECT OF THE INVENTION
It is an object of the invention to provide a method of modulating an
immune response in the animal or human body and of treating ailments
associated with inappropriate immune responses in the animal, including
human, body.
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DESCRIPTION OF 'THE INVENTION
It has now surprisingly been found, in contradistinction with the prior art
- knowledge regarding the toxicity and anti-viral and anti-bacterial
properties
of DMF that such product may be used in sub-toxic dosages to modulate the
immune responsive metabolic processes in cells having an immunological
function by affecting such processes to stimulate or suppress the expression
or secretion of immunological mediators by such cells without destruction
of the cells through the administration of DMF to the body.
According to the present invention there is thus provided a method of
affecting an immune response in an animal comprising the steps of
administering to such animal a non-toxic immune modulating effective
quantity of a compound selected from the group consisting of compounds
of the general chemical formula (I)
R,-CO-NR=R, (I)
wherein
R, is selected from the group consisting of H and lower [i.e. C, to C,]
alkyl and CI to C3 alkenyl groups;
R2 and R3 are the same or different and each is selected from the
group consisting of H, lower [i.e. C, to C,] alkyl and CZ to C3 alkenyl
groups which may optionally be halogenated or hydroxylated, or
which may in combination with one another form a -(CH2)" group
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wherein n is any number from 2 to 5, or the group -(CH=)I-O-(CH~)~
and metabolites and prodrugs thereof.
- The immune response to be affected by the above method may be an
immune response of the immune cells forming part of the immune system
of the body.
The immune cells may be T lymphocytes and or B lymphocytes.
The method may be performed to reduce the expression or secretion of
immune cell products in the body.
According to a further aspect of the present invention there is provided a
method of treatment of an animal afflicted with an ailment associated with
inappropriate immune responses in that animal, comprising the steps of
administering to such animal a non-toxic therapeutically effective quantity
of a compound selected from the group consisting of compounds of the
general chemical formula (I)
R,-CO-NRZR3 (I)
wherein
R, is selected from the group consisting of H and Iower [i.e. C, to C3]
alkyl and Cz to C3 alkenyl groups;
R~ and R, are the same or different and each is selected from the
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group consisting of H, lower [i.e. C, to C3] alkyl and Cz to C3 alkenyl
groups which may optionally be halogenated or hydroxylated, or
which may in combination with one another form a -(CHI),; group
wherein n is any number from 2 to 5, or the group -(CHz)~-O-(CHI)z
and metabolites and prodrugs thereof.
According to another aspect of the invention it relates to use of a compound
selected from the group consisting of compounds of the general chemical
formula (I)
R,-CO-NRzR3 (I)
wherein
R, is selected from the group consisting of H and lower [i.e. C, to C3]
alkyl and CZ to C3 alkenyl groups;
R~ and R3 are the same or different and each is selected from the
group consisting of H, lower [i.e. C, to C3] alkyl and C2 to C, alkenyl
groups which may optionally be halogenated or hydroxylated, or
which may in combination with one another form a -(CH~)o group
wherein n is any number from 2 to 5, or the group -(CH2)~-O-(CH2)z
and metabolites and prodrugs thereof
in the manufacture of a medicament for use in a method of treatment of an
animal afflicted with an ailment associated with inappropriate immune
responses in that animal.
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Further according to the present invention it relates to a pharmaceutical
composition comprising a compound selected from the group consisting of
compounds of the general chemical formula (I)
Rl-CG-~~R3 CI)
wherein
R, is selected from the group consisting of H and lower [i.e. C, to C3]
alkyl and CZ to C3 alkenyl groups;
RZ and R3 are the same or different and each is selected from the
group consisting of H, lower [i.e. C, to C3] alkyl and Cz to C, alkenyl
groups which may optionally be halogenated or hydroxylated, or
which may in combination with one another form a -(CHz)n group
wherein n is any number from 2 to 5, or the group -(CHZ)z-O-(CHZ)1
and metabolites and prodrugs thereof
in a pharmaceutically acceptable dosage form for use in the treatment of an
animal afflicted with an ailment associated with inappropriate immune
responses in that animal.
In all the treatment related aspects of the invention the ailment to be
treated may be any one of the following:
Systemic Lupus Erythematosus [SLE]
Scleroderma [Systemic sclerosis]
Vasculitis Syndrome [including Wagener's thrombosis and all forms of
Giant cell arthritis]
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Dermatomiositis
Asthma
Adult Respiratory Distress Syndrome [ARDS]
Systemic Inflammatory Response Syndrome [SIRS]
Inflammatory Bowel Disease
Chronic Hepatitis
Rheumatoid Arthritis
Rheumatic fever
Myasthenia Gravis
Multiple Sclerosis
Psoriasis
Eczema
Multiple myeloma
Reiter's Syndrome
Glomerulonephritis
Polymyalgia Rheumatics
Ankylosing spondylitis
Polyarteritis Nodosa
Allergic Rhinitis
Diabetes mellitus
Optical Neuritis
Acute Transversmielitis
Head Injuries
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Spinal Cord injuries
Post sub-Arachnoidal Bleeding Vasospasma
In all the aforementioned aspects of the invention the compounds of choice
is dimethylformamide and its metabolites namely N-methylformamide, N-
methyl-isocyanite and its carbamates, and N-acetyl-S-(N-
methylcarbamoyl)cysteine and any prodrugs of such metabolites.
Further according to the invention the method is performed by
administering to the patient to be treated a quantity of DMF sufficient to
maintain a DMF-plasma level of between 0.001% and 0.1% and most
preferably between 0.01% and 0.05%. Likewise, the medicament according
to the invention is adapted in use to administer to the patient a quantity of
DMF at such rate as to achieve and maintain a DMF-plasma level of
between 0.001 % and 0.1% and most preferably between 0.01% and 0.05%.
Also, in all aspects of the invention the compound may be administered by
any route of administration, such as orally, nasally, rectally, intravenously,
intramuscularly, subcutaneously or transdermally. The preferred route of
administration of the compound is transdermally. Preferably, however, it
is administered by a transdermal patch.
As will appear from the examples below, mixed lymphocytes were exposed
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to a non-specific stimulator of lymphocytes, such as PHA at 1 to 10 ~cg per
200 ~L. When these lymphocytes were concurrently also exposed to very
low concentrations of DMF a decreased metabolic activity was demonstrated
- as evidenced by the fact that these cells were less capable of reducing a
patented [U.S. Patent No. 5,501,959] redox indicator. This inhibition of
metabolic activity is not the result of direct cell toxicity as is evidenced
by
the fact that the therapeutic concentration to effect inhibition of metabolic
activity and therefor proliferation, is lower than a concentration which
causes activation of cell metabolism and therefor proliferation.
By bringing the immune active cells in need of modulation into contact
with DMF or any of its metabolites in a therapeutic concentration, it is
possible to diminish the metabolic activity of cells with immune function.
By decreasing the metabolic activity of T lymphocytes the responsiveness of
these T lymphocytes to certain T lymphocyte specific antigens and antigen,
major histocompatibility complex molecule combinations will be severely
inhibited. It is the antigen specific inhibition of T lymphocyte responses
that forms the basis of the clinical application of DMF to modulate immune
cell mediated diseases proposed by the present invention.
For the transdermal administration of DMF to a patient in need of
treatment according to the invention it is proposed to use a mufti-purpose
transdermal administration system that is able to deliver a variety of drugs
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including DMF and the above-mentioned related compounds
therapeutically.
The patch design parameters considered important include the following:
1. The patch must have pre-determined dimensions as it determines the
amount of active ingredient [drug] absorbed over a certain time.
2. Drug administration must not be time dependent.
3. Drug concentration should be variable according to patient profile.
4. The patch must be stable, and deliver repeatable therapeutic
concentrations of agent [drug] that is administered.
5. The tempo of drug [agent] administration is determined by the skin,
thus the desorption of the drug is through the membrane should be
the same or very close to the absorption tempo of the skin.
6. The patch and the drug must have a relative long shelf life.
To achieve the above requirements a patch has been designed with the
following features:
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1. High density nylon backing material.
2. Safety border consisting of nylon of PATFE [TeflonJ.
3. Low density septa NYLON or PTFE.
4. Hydrophilic or hydrophobic NYLON or PTFE membrane
[Depending on which drug is administered].
5. Membrane pore size. of 0.05-0.45 micron depending on which drug is
administered.
6. Diatomaceous earth [SiOz] adsorbent material.
7. Stabilisation agent [Salting agent to decrease evaporation as well as
homogenic weight distribution through the membrane].
8. Suitable skin adhesive which is not reactive with the drug.
9. Anti-irritation agent - Vitamin E [This agent can be applied before,
during or after application of the agent to protect the skin against
side effects].
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More particularly a patch was made up as follows:
1. Backing: material and septa
Description Round semi-transparent nylon disk
0.1-0.4
mm thick
Diameter 20 - 100 mm
Average mass 100 mg - 600 mg
Septa description Round soft polypropylene/polyethylene
with nylon or PTFE backing
Septa diameter 5 - 25 mm
Septa thickness 0.2 - 1 mm
2. Teflon membrane
Description Round white membrane
Diameter 20 - 100 mm
Average Mass 50 - 500 mg
Pore Size 0.2 - 0.8 micron
3. Teflon border ring
Description Round semi-transparent 0.1 - 0.4
mm thick
Inner diameter 10 - 90 mm
Outer diameter 20 - 100 mm
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I~Average mass ~ 20 - 200 mg
- 4. Absorbent material
Description Silicon dioxide [diatomaceous earth]
Mass / patch 1 - 10 g
Stabilisation agent Sodium chloride and magnesium /
calcium
description carbonate
APPLICATION TECHNICZUE
Indirect administration of the drug such as DMF may be done by
introducing a known amount of the active agent with a syringe into the
Silicon dioxide adsorbent after the patch had been applied to the skin.
The following advantages are obtained by using this technique:
1. Controlled agent administration from an ampoule/container onto a
patch.
2. Therapeutic agent dosage can be predetermined according to patient's
profile.
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3. By making the agent concentration dependent, the time of treatment
and area of skin exposure stays constant during treatment.
- 4. Patch and agent can be applied with confidence without overdosing
the patient.
5. Patch and agent are easy administered and the patient can be
discharged after administration.
6. Patch is very stable, has an unlimited shelf Iife, and agent
administered from an ampoule has at least a two year expiry date,
unless otherwise stipulated.
EXAMPLES OF THE INVENTION
The invention will now be demonstrated with reference to the following
examples without thereby limiting the scope of the invention to the
illustrative embodiments.
EXAMPLE 1
Peripheral human lymphocytes were isolated from whole blood of healthy
volunteers by using a density gradient separation technique well described
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and known in the immunology research literature. Briefly described, by
this technique peripheral whole blood is diluted 1:2 with phosphate buffered
saline or cell culture media such as RPMI1640. A density gradient such as
- Histopaque 1077 [Sigma Cat # 1077] is then layered underneath the diluted
peripheral blood, taking care to create a sharp interface. The density
gradient is then centrifuged at 600 g for 20 to 25 minutes. After
centrifugation a clear buffy coat layer containing mostly lymphocytes is
easily visible. This layer is then removed and further processed by placing
the buffy coat in an additional tube and washing the cells washed 3 times
in PBS or cell culture media. After each of the wash steps the cells are
centrifuged at 400 g for 9 minutes. After the third wash the Lymphocytes
are collected from the pellet. The cells are counted and diluted to the
desired cell concentration.
A variety of DMF concentrations were prepared using complete cell culture
medium as the diluent. The following concentrations were prepared: 1%,
0.1%, 0.05%, 0.01% and 0.001%.
The isolated lymphocytes were diluted to the required concentration using
a complete cell culture medium.
The complete cell culture medium used in the above steps contained
Rl'MI1640 with HEI'ES 20-25 mM, L-glutamine 1 mM, dimercapto-ethanol
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2x10-5 mM and 10% heat inactivated fetal calf serum.
The prepared lymphocytes were placed in 96 well cell culture plates at a
- concentration of 150,000 cells per well. PHA [Phyto-Heame-Agglutinin] was
added to each test well at a concentration of 1 to 10 ~cg per 200 ~cL. At the
same time DMF was added to each of the test wells at the predetermined
concentration and the culture wells were incubated at 37°C in 5% CO2,
95%
OZ atmosphere for I to 4 days.
A redox indicator sold under the trademark AlamarBlue was added to each
of the culture wells at 10% volume:volume. The cultures were incubated
for 18 hours with AlamarBlue prior to determining the absorbance at
570nm with 630nm as a reference.
The results may be summarised as follows:
The culture in which peripheral lymphocytes from healthy volunteers were
exposed to PHA and DMF at 1% concentration for 24 hours reduced 102%
of the quantity of AlamarBlue reduced by the culture in the normal PHA
growth well. However, for the cultures in which DMF was added at a
concentration of only 0.05% reduced only 52% of the quantity of
AlamarBlue reduced by the culture containing PHA stimulated
lymphocytes. This demonstrates a significant inhibition of metabolic
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activity in the culture well which correlates very closely with the level of
cell proliferation in the particular well.
When comparing the metabolic activity as indicated by AlamarBlue
reduction of unstimulated lymphocytes to unstimulated lymphocytes
exposed to low concentration of DMF, a 40% decrease in reduction of
AlamarBlue is observed for lymphocytes exposed to 0.05% DMF
concentration. [See Figures 1 and 2 which represent typical results of a
number of repetitions of the experiment]
EXA1V1PLE 2
To demonstrate the postulated mechanism of action of the DMF in causing
depression of lymphocyte metabolic activity a further series of experiment
was performed. In these experiments the lymphocytes of healthy volunteers
were isolated using the technique described in Example 1. After the
lymphocytes were isolated the lymphocytes were exposed to DMF at various
concentrations for varying lengths of incubation time. The DMF
concentrations used were 0.1%, 0.01%, and 0.001% and the incubation times
were: 5, 15, 30, 75, 120 and 180 minutes. The Tyrosine kinase activity was
determined using a commercially available Tyrosine kinase assay kit
available from Boehringer Mannheim [Catalogue Number 1534505]. The
results were as presented in the graphs of Figures 3 and 4. Over a. 3 hour
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exposure to a 0.01% concentration of DMF the tyrosine kinase activity of
the tested lymphocytes were inhibited by 71%. It is pointed out that in this
graph the lower the absorbance reading, the higher the degree of tyrosine
kinase activity inhibition. The general shape of the graphs remained the
same regardless of the time allowed for development of the colour reaction.
The results support the postulate on which the present invention is based
that DMF at certain concentrations, when exposed to lymphocytes for a
certain period of time serves to block Tyrosine kinase activity. There is the
possibility that the observed decreased activity in tyrosine kinases in the
tested lymphocytes might be the result of stimulation of certain tyrosine
phosphatases. The presented graphs are typical examples of many graphs
obtained.
EXAMPLE 3
The PTK inhibitory activity of DMF and two of its metabolites was further
demonstrated by comparing it to a commercially available PTK inhibitor
Piceattanol marketed by Boehringer Mannheim under # 1534505 on two cells
lines named HELA, a Cervix cancer cell line and HEP3B a liver cancer cell
line. The following results were obtained:
The inhibitor was used in 10% DMSO solution.
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The test was carned under the following conditions and yielded the results
shown below:
Stank: 0,043
Funct: Cntrl
Endogenous phosphatase: 20.3%
Endogenous phosphorylation: 0.14 [0,208]
A~ Av. Inh~ition
HELA
Inhibitor . 0,076 [0,132]
[Pilleattanol
+ 10% DMSO] 66% 75% 29,5%
Clean Cells 0,115 [0,176]100% 0%
2% Metabolite I 0,099 [0,167]86% 95% 9,5%
0,2% Metabolite II 0,074 [0,140]64% 80% 28
5%
2% Metabolite 0,055 [0,091]48% 55% ,
II 48,5%
0,2% DMF 0,073 [0,146]63% 83% 27%
2% DMF 0,042 [0,082]37% 47% 58%
HEP3B
Inhibitor . 0,055 [0,111]60% 60% 40%
[Pilleattanol
+ 10% DMSO] 66% 75% 29,5%
Clean Cells 0,091 [0,186]100% 0%
2% Metabolite I 0,090 [0,150]99% 81% 10%
0,2% Metabolite II 0,036 [0,060]40% 32% 64%
2% Metabolite 0,095 [0,095]60% 51% 44
II 5%
0,2% DMF 0,080 [0,123]88% 66% ,
33,3%
2% DMF 0,040 [0,064]44% 34% 61%
IC50 1,7 - 17~M was established
IC50 1,7 - 17~.M was established
CODE
Metabolite I - N-methylformamide
Metabolite II - N-methylisocyanite
DMF - Dimethylformamide
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