U.S. patent application number 13/360778 was filed with the patent office on 2012-05-24 for aminopterin dosage forms and methods for inflammatory disorders.
Invention is credited to Barton A. Kamen, John Zebala.
Application Number | 20120128703 13/360778 |
Document ID | / |
Family ID | 36971856 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120128703 |
Kind Code |
A1 |
Kamen; Barton A. ; et
al. |
May 24, 2012 |
Aminopterin Dosage Forms and Methods for Inflammatory Disorders
Abstract
There is disclosed dosage forms and methods for treating a
patient with an inflammatory disorder with a therapeutically
effective amount of aminopterin, or a pharmaceutically acceptable
salt thereof, that achieve efficacy without concomitant toxicity.
Specifically, there is disclosed a method for treating an
inflammatory disorder in a patient with uninterrupted doses of
aminopterin.
Inventors: |
Kamen; Barton A.; (US)
; Zebala; John; (US) |
Family ID: |
36971856 |
Appl. No.: |
13/360778 |
Filed: |
January 29, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11928890 |
Oct 30, 2007 |
8129383 |
|
|
13360778 |
|
|
|
|
11078271 |
Mar 11, 2005 |
7312217 |
|
|
11928890 |
|
|
|
|
Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
Y10S 514/825 20130101;
Y10S 514/863 20130101; A61P 37/00 20180101; A61P 11/00 20180101;
A61P 29/00 20180101; A61P 37/08 20180101; Y10S 514/886 20130101;
A61P 19/02 20180101; A61P 1/04 20180101; A61P 37/06 20180101; Y10S
514/903 20130101; A61P 17/02 20180101; A61K 31/525 20130101; A61P
43/00 20180101; A61P 17/06 20180101; A61K 31/519 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61P 37/06 20060101 A61P037/06; A61P 29/00 20060101
A61P029/00 |
Claims
1-16. (canceled)
17. A method for treating systemic lupus erythematosus,
graft-versus-host-disease (GVHD) and polymyositis comprising
administering an effective amount of aminopterin or a
pharmaceutically acceptable salt thereof.
18. The method for treating systemic lupus erythematosus,
graft-versus-host-disease and polymyositis of claim 17 wherein the
therapeutically effective amount of aminopterin is not greater than
0.07 mg aminopterin per kilogram of patient body weight.
Description
[0001] This is a continuation-in-part of pending application Ser.
No. 11/078,271, filed 11 Mar. 2005.
BACKGROUND
[0002] Aminopterin, or 4-amino-pteroyl-L-glutamic acid, is a potent
antifolate [see Franklin, U.S. Pat. No. 2,575,168]. Aminopterin
binds and inhibits dihydrofolate reductase (DHFR) from a variety of
species, including humans. Synthesized in 1946 by the Lederle
Laboratories, a division of American Cyanamid Co., aminopterin was
marketed in 0.5 mg tablets in 1953 for the treatment of childhood
leukemia. In 1965, the marketing of aminopterin as a pharmaceutical
in the United States ceased.
[0003] In 1951, Gubner treated patients with rheumatoid arthritis,
psoriatic arthritis, uncomplicated psoriasis, and atopic dermatitis
with 0.75-2 mg/day for durations of approximately 1 week and
greater, or cumulative weekly doses of greater than 5.25 mg [Gubner
et al., Am. J. Med. Sci. 22:176, 1951; and Gubner, Arch. Derm.,
Chicago 64:688, 1951]. Although some patients improved, the
majority of patients developed toxic reactions including
stomatitis, nausea, diarrhea, and alopecia that necessitated
discontinuation of the drug. Gubner concluded that "the toxic
effects of aminopterin place practical limitations on its use as a
therapeutic agent" [Gubner et al., Am. J. Med. Sci. 22:176,
1951].
[0004] In 1955, Rees et al. treated 171 patients with psoriasis
with five dosage schedules using the 0.5 mg tablet that comprised:
(i) 1 tablet daily for six days; (ii) 1 tablet daily for six days,
one week rest, then 1 tablet daily for six days; (iii) 1 tablet
daily for 12 days; (iv) 2 tablets daily for six days; and (v) 2
tablets daily for three days, then 1 tablet daily for six days
[Rees et al., AMA Arch. Derm. 72(2):133-43, August 1955]. A rapid
clearing of psoriatic lesions were noted, with toxic reactions
occurring with a frequency of 0%, 2.5%, 13%, and 30% on schedules
(i), (ii), (iii) and (iv), respectively. Too few patients were
treated with schedule (v) to assess. Toxic reactions included
stomatitis, alopecia, and leucopenia. When the above schedules were
concluded, the psoriatic lesions invariably recurred, usually
within weeks. Some patients were given multiple courses of the
above schedules, with rest periods between courses.
[0005] In 1958, Edmundson and Guy treated patients with a schedule
comprising 1 tablet daily for six days, withdrawal for three days,
and again daily for six days, for a total of 6 mg in 12 doses
[Edmundson and Guy, AMA Arch. Derm. 78(2):200-3, August 1958].
Improvement was noted, and remissions typically lasted several
months.
[0006] In 1959, Rees and Bennett report the treatment of 329
patients with psoriasis using the same schedules from their 1955
study [Rees and Bennett, J. Invest. Dermatol. 32(1):61-66, January
1959). Courses of the schedules were repeated in some patients
every 3 weeks to once every 3 years, although it is not disclosed
which schedules were repeated. The overall incidence of toxicity
was 21% and the most common toxic reactions were stomatitis and
intensification of the lesions, followed by alopecia, GI
disturbances, and leucopenia. The authors noted that treatment
should be discontinued at the slightest hint of a toxic
reaction.
[0007] In 1961, Rees and Bennett compared the effect of daily doses
of 0.5 mg aminopterin against daily doses of 2.5 mg methotrexate
using the same schedules from their 1955 study [Rees and Bennett,
Arch. Dermatol. 83:970-72, June 1961]. In 1963, Strakosh also
compared the effect of daily doses of 0.5 mg aminopterin against
daily doses of 2.5 mg methotrexate according to schedules
comprising: (i) 1 tablet daily for 12 days, followed by one-week's
rest, and repeated as often as deemed advisable; (ii) 1 tablet
daily for 3 days followed by three-days' rest period and repeated
until 12 tablets in all were given, and then followed by one-week's
rest period and repeated as often as deemed advisable (i.e., four
cycles interrupted by a week rest each); and (iii) any variation of
(i) and (ii) above [Strakosch, Dermatologica 126:259-267, 1963].
Both concluded that methotrexate is less toxic and less efficacious
than aminopterin in treating psoriasis. However, the doses compared
were not equipotent to one another, with methotrexate being used in
an amount 4-fold less than would be required to be equipotent with
aminopterin. Thus, by modern standards no conclusions could be
drawn regarding the actual relative efficacy, toxicity, or
therapeutic index of these antifolates. Later, Rees et al. suggest
the opposite, that methotrexate may be more safe and efficacious
than aminopterin in treating psoriasis [Rees et al., Arch.
Dermatol. 90:544-52, December 1964].
[0008] In 1964, Rees et al. review the literature on the standard
of practice with aminopterin in treating psoriasis, describing all
known schedules of administration [Rees et al., Arch. Dermatol.
90:544-52, December 1964]. In all cases, dosing schedules were
similar, being comprised of daily dosings of tablets for periods
greater than 1 week until efficacy or toxicity was observed, at
which time dosing was interrupted by rest periods of varying
duration.
[0009] The prior art are also contains several reports of using
aminopterin to reduce inflammation in animal models. In 1952,
Gubner et al. demonstrated the efficacy of cumulative weekly doses
of 0.3 mg/kg aminopterin in the rat formaldehyde arthritis model
[Gubner et al., J. Invest. Dermatol. 19(4):297-305, October 1952].
In 1964, Page demonstrated the efficacy of cumulative weekly doses
of 0.35 mg/kg aminopterin in the rabbit dermal inflammation model,
but in the process the animals became severely leucopenic and one
subject died [Page, Ann. N.Y. Acad. Sci. 116:950-63, Aug. 27,
1964]. In 1986, Galivan et al. demonstrated the efficacy of
cumulative weekly doses of 0.12 mg/kg aminopterin in the rat
adjuvant arthritis model, but the animals suffered from severe
toxicity [Galivan, et al., Methotrexate in adjuvant arthritis, in
Chemistry and Biology of Pteridines 1986. Pteridines and Folic Acid
Derivatives, B. A. Cooper and V. M. Whitehead, Editors. 1986,
Walter de Gruyter & Co.: Berlin. p. 847-49]. Similarly, in 2000
Andersson et al. demonstrated the efficacy of cumulative weekly
doses of >1.5 mg/kg aminopterin in the rat antigen-induced
arthritis model, but again the animals suffered from severe
toxicity [Andersson, et al., Eur. J. Pharm. Sci. 9(4):333-43,
2000].
[0010] Inflammatory diseases, whether of a chronic or acute nature,
represent a substantial problem in the healthcare industry. Chronic
inflammation is considered to be inflammation of a prolonged
duration (weeks or months) in which active inflammation, tissue
destruction and attempts at healing are proceeding simultaneously
(Cotran, R. S., Kumar, V., and Robbins, S. L., Robbins Pathological
Basis of Disease. W.B. Saunders Co., p. 75, 1989). Although chronic
inflammation can follow an acute inflammatory episode, it can also
begin as an insidious process that progresses with time, for
example, as a result of a persistent infection (e.g., tuberculosis,
syphilis, fungal infection) which causes a delayed hypersensitivity
reaction, prolonged exposure to endogenous (e.g., elevated plasma
lipids) or exogenous (e.g., silica, asbestos, cigarette tar,
surgical sutures) toxins, or autoimmune reactions against the
body's own tissues (e.g., rheumatoid arthritis, systemic lupus
erythematosus, multiple sclerosis, psoriasis). Chronic inflammatory
diseases include many common medical conditions such as rheumatoid
arthritis, restenosis, psoriasis, multiple sclerosis, surgical
adhesions, tuberculosis, chronic inflammatory lung diseases (e.g.
asthma, pneumoconiosis, chronic obstructive pulmonary disease,
nasal polyps and pulmonary fibrosis), periodontal disease (i.e.
periodontitis) and polycystic kidney disease.
[0011] Multiple sclerosis (MS), affecting 350,000 people
(women:men=2:1) in the United States with 8,000 new cases reported
each year, is the most common chronic inflammatory disease
involving the nervous system. The predominant pathologic findings
are demyelination accompanied by disruption of underlying axons
(Trapp et al., New Engl. J. Med. 338:278-85, 1998; Prineas, J. W.,
"Pathology of Multiple Sclerosis" in: Cook, S. D., ed. Handbook of
Multiple Sclerosis. New York: Marcel Dekker, Inc., 1990:187-215).
The disease affects young adults who usually present with a
relapsing, remitting pattern of neurologic involvement and progress
to a chronic phase with increasing difficulty in ambulation and
coordination. Roughly half of MS cases progress to a more chronic
phase. Although the disease does not result in early death or
impairment of cognitive functions, it cripples the patient by
disturbing visual acuity; simulating double vision; disturbing
motor functions affecting walking and use of the hands; producing
bowel and bladder incontinence; spasticity; and sensory deficits
(touch, pain and temperature sensitivity).
[0012] MS is a demyelinating disease of the CNS with unknown cause
and no known cure. Though single episodes of demyelination can
occur, once the disease is established in multiple sites in the
brain, spinal cord, and optic nerves, MS frequently follows a
clinically relapsing-remitting course while lesions in the CNS
continue to progress. During this phase, an immune mediated
inflammatory response to myelin antigens is thought to play a major
role in the pathogenesis of developing lesions. Then, in a
clinically progressive phase, at least fifty-five percent of
patients worsen, with clinical relapses sometimes punctuating their
clinical decline. The mechanism of tissue damage to the CNS is not
known with certainty in the progressive phase of MS. It is thought,
however, that axonal damage, perhaps through some type of immune
mediation, is important in this phase of the disease, though some
axonal damage certainly occurs during the inflammatory phase.
Perivascular infiltration of T lympocytes and macrophages in brain
lesions is one of the characteristics of MS. Activation of
myelin-reactive T cells in the periphery is an early event in the
MS process. These activated T cells facilitate the production of B
cells of antibodies against myelin, and activate macrophages to
attack oligodendrocytes in the CNS. Treatment options for patients
with MS are limited. Currently, the primary drugs used to treat MS
are interferons and glatiramer acetate. Such treatments have been
shown to slow, but not arrest, the clinical course of progression
in progressive MS.
[0013] Experimental Autoimmune Encephlomyelitis (EAS) is an
experimental model for human MS (Mackay, et al., Clin. Exp.
Immunol. (1973) 15:471-82). The use of this model can be
demonstrated in guinea pigs (Lisak, et al., J. Immunol. (1970)
104(6):1435-47); hens (Blaszczyk, et al., Folia biologica (Praha)
(1977) 23:299; female rats (Levine, and Sowinski, Arch. Int.
Pharmacodyn. (1977) 230:309-318; Badger, et al., Agents and Actions
(1989) 27 (3/4):335-7; Desai, and Burton, Agents and Actions (1989)
27 (3/4): 351-355; Vogel, et al., Can. Res. (1969) 29:2249-2253;
Przuntek, et al., Neuropharma. (1987) 26(2/3):255-260); male rats
(Rosenthale, et al., Arch Int. Pharmacodyn. (1969) 179(2):251-275);
rabbits (Brandiss, et al., Ann. NY Acad. Sci. 356-368); and male
and female rats (Levy, and Whitehouse, Agents and Actions (1974)
(4/2):113-116; Martel, et al., Can. J. Physiol. Pharmacol. (1977)
55:48-51).
[0014] Chronic inflammatory lung disease, including for example,
asthma, pneumoconiosis, chronic obstructive pulmonary disease,
nasal polyps, and pulmonary fibrosis, affect many people worldwide.
Typically such diseases are characterized by an invasive
inflammatory process, and thickening of the affected tissues.
[0015] Asthma is an inflammatory disease that is associated with
widespread but variable airflow obstruction. Asthma is defined as a
"disease characterized by an increased responsiveness of the
trachea and bronchi to various stimuli, and manifested by
widespread narrowing of the airways that changes in severity either
spontaneously or as a result of treatment" (American Thoracic
Society). About 5% of the population suffers from asthma. The
pathogenesis of asthma is poorly understood. Multiple complex
immune system mechanisms probably are involved. Numerous cytokines
derived from tissue mast cells, eosinophils, T lymphocytes,
macrophages, neutrophils, and other lung cells are critical in
initiating and perpetuating the asthmatic response and play
important roles in the immunopathogenesis of airways in
inflammation (Current Medical Diagnosis & Treatment, 36.sup.th
ed.). In persons with asthma, the airways narrow
(bronchoconstriction) in response to stimuli that don't affect the
airways of normal lungs. The narrowing can be triggered by many
stimuli (an allergen), such as pollens, dust mites, animal dander,
smoke, cold air, and exercise. Bronchodilator drugs that relieve
attacks of asthma through non-specific blockade of
beta-adrenergenic receptors cause side effects such as rapid
heartbeat, restlessness, headache, and muscle tremors. Theophylline
is another bronchodilator drug whose administration must be closely
monitored by a doctor, because too little drug in the blood may
give little benefit, and too much drug may cause life-threatening
abnormal heart rhythms or seizures and patients may also experience
insomnia, agitation, and vomiting. Corticosteriods drugs are
exceptionally effective at reducing asthma symptoms by blocking the
body's inflammatory response but long term use may result in poor
wound healing, loss of calcium from the bones, stomach bleeding,
premature cataracts, elevated blood sugar levels, hunger, weight
gain, mental problems, and stunted growth in children.
[0016] The benefits of asthma treatments have been investigated in
animals (Nagao, et al., J. Pharma. Pharmacol. (2004), 56:187-196)
and humans (Comet, et al., Resp. Med. (2006), 100:411-19; Rabe, et
al., Lancet. 368:744-53; Busse, et al., Ann. All. Asth. Immunol.
96:60-68; Johnston, et al., New Engl. J. Med. 354 (15):1589-1600;
Bateman, et al., Ann. All. Asth. Immunol. 96:679-686).
[0017] Systemic lupus erythematosus (SLE) is a disorder of the
immune system. Lupus can affect many parts of the body, including
the joints, skin, kidneys, heart, lungs, blood vessels and brain.
Although patients with the disease may have many different
symptoms, some of the most common ones include extreme fatigue,
painful or swollen joints (arthritis), unexplained fever, skin
rashes, and kidney problems. Other symptoms include chest pain upon
deep breathing, unusual loss of hair, pale or purple fingers from
cold or stress (Raynaud's phenomenon), sensitivity to sun, swelling
(edema) in legs or around eyes, mouth ulcers and swollen glands.
New symptoms may continue to appear years after the initial
diagnosis, and different symptoms can occur at different times.
Several systems of the body may be affected including the kidneys,
lungs, CNS, blood vessels, blood and heart.
[0018] Several methods of treatment are currently employed to treat
SLE. Nonsteroidal anti-inflammatory drugs (NSAIDS) are often used,
either alone or in combination, including ibuprofen and naproxen.
Common side effects include stomach upset, heartburn, diarrhea and
fluid retention. Antimalarials, such as hydrochloroquinone, are
also commonly used to treat lupus. They are generally used to treat
fatigue, joint pain, skin rashes, and inflammation of the lungs.
Side effects include stomach upset, and, rarely, damage to the
retina of the eye. Corticosteroids are also used to treat lupus.
Examples include prednisone, hydrocortisone, methylprednisone and
dexamethasone. Short-term side effects include swelling, increased
appetite and weight gain. These side effects generally stop when
the drug is stopped. Long-term side effects can include stretch
marks of the skin, weakened or damaged bones (osteoporosis and
osteonecrosis), high blood pressure, damage to the arteries,
diabetes, infections and cataracts. Typically, the higher the dose
and the longer they are taken, the greater the risk and severity of
side effects. For some patients whose kidneys or CNS are affected
by lupus, immunosuppressives may be used. These include
cyclophosamide and mycophenolate mofetil. Side effects may include
nausea, vomiting, hair loss, bladder problems, decreased fertility,
and increased risk of cancer and infection. The risk for side
effects increases with the length of treatment.
[0019] Polymyositis is a disease of muscle featuring inflammation
of the muscle fibers. The muscles typically affected are those
closest to the trunk or torso. Polymyositis can be associated with
skin rash and is then referred to as dermatomyositis. It also can
affect other areas of the body. Weakness of muscles is the most
common symptom of polymyositis. The onset can be gradual or rapid.
This results in varying degrees of loss of muscle power and
atrophy. Patients can also feel fatigue, a general feeling of
discomfort and have weight loss and/or low grade fever. Heart and
lung involvement can lead to irregular heart rhythm, heart failure,
and shortness of breath. Initially, polymyositis is treated with
high doses of corticosteroids and are usually required for years.
Corticosteroids have many predictable and unpredictable side
effects. In high doses they commonly cause increase in appetite and
weight, puffiness of the face and easy bruising, sweats, facial
hair growth, upset stomach, sensitive emotions, leg swelling, acne,
cataracts, osteoporosis, high blood pressure, worsening of
diabetes, increased risk of infection, and rarely, avascular
necrosis. Further, abruptly stopping corticosteroids can cause
flares of the disease and result in other side effects including
nausea, vomiting and decreased blood pressure. Immunosupressives
may used in the cases where corticosteroids do not adequately
improve polymyositis. These include the use of methotrexate and
azathioprine. These medications can cause liver and bone marrow
side effects and require blood monitoring. Cyclophosphamide,
chlorambucil and cyclosporine may also be used, however these
compounds also have serious side-effects.
[0020] Graft-versus-host-disease (GVHD), an immune effect, is the
most common complication among patients who survive beyond 100 days
after allogenic hematopoietic cell transplantation. The incidences
of acute GVHD (aGVHD) and chronic GVHD (cGVHD) are 30-80% and
30-80%, respectively. GVHD itself and treatment-related infections
are the principal causes of nonrelapse mortality. For aGVHD after
methotrexate alone or methotrexate in combination with cyclosporine
A as prophylaxis regimens, methylprednisone is the best initial
therapy. Prednisone and cyclosporine A are considered as the
first-line therapy for patients with cGVHD. Other therapeutic
options for aGVHD or cGVHD are anti-thymocyte globulin,
mycophenolate mofetil, tacrolimus, azathioprine, thalidomide,
monoclonal antibodies directly against CD3, CD25, CD52, cytotoxic
T-lymphocyte antigen (CTLA)-4 or tumor necrosis factor alpha,
extracorporeal photochemotherapy and PUVA therapy. Most
immunosuppressive agents require continuous administration of the
drug to be effective. Consequently, infectious complications are
frequently observed and tremendously increase the treatment cost.
Although treatment with cyclosporine and gluccocorticoids can
control manifestations of cGVHD in some patients, the therapeutic
options are limited for those with steroid-refractory cGVHD.
SUMMARY
[0021] The present disclosure provides dosages and methods for
treating a patient with an inflammatory disorder with a
therapeutically effective amount of aminopterin, or a
pharmaceutically acceptable salt thereof. The present disclosure
provides unexpected advantages of patient convenience without
creating concomitant toxicity manifestations. In particular, it has
been discovered that aminopterin can be used in uninterrupted
cycles of doses as an anti-inflammatory agent, and in some
embodiments at doses lower than previously taught in the art.
[0022] There is disclosed a method for treating inflammatory
conditions with aminopterin without creating unacceptable toxicity
manifestations. Another related embodiment is a method of treating
bronchopulmonary dysplasia, canine atopic dermatitis, and bovine
acute pneumonic pasteurellosis with an antifolate, and aminopterin
in particular. Another related embodiment is a method of treating
inflammatory bowel disease, asthma, and multiple sclerosis (MS)
with aminopterin. Another related embodiment is a method of
treating systemic lupus erythematosus (SLE),
graft-versus-host-disease (GVHD) and polymyositis with aminopterin.
Further, it has been discovered that aminopterin doses can be
administered in dosage forms containing less aminopterin per tablet
than described by earlier publications. These lower doses are
achieved by the development of tablets containing less than 0.5 mg
aminopterin, which allow lower doses to be administered to patients
and also allow the ability to tailor these lower doses more
accurately to a particular patient's body weight.
[0023] Another aspect is treatment of rheumatoid arthritis,
psoriatic arthritis and atopic dermatitis in humans with a weekly
dose of aminopterin of 5.25 mg or less. A related embodiment is a
method of dosing a human with psoriasis with a cumulative weekly
dose of less than 2 mg aminopterin. A related embodiment is a
dosage form of aminopterin containing less than 0.5 mg per tablet.
It will be appreciated by one in the art that the above identified
aspects avoid the toxicity and irregular dosing schedules of
aminopterin that were a major inconvenience to patients with
inflammatory disorders. Another aspect of the disclosure are
tablets containing less than 0.5 mg aminopterin per tablet, which
make it possible to administer lower doses and also make it easier
to tailor lower doses to a particular patient's weight in order to
avoid toxicity.
[0024] Another aspect of the disclosure is treatment of MS,
polymyositis, SLE, GVHD or asthma in humans with a weekly dose of
aminopterin of 7.5 mg/week or less. A related embodiment is a
method of dosing a human with MS or asthma with a cumulative weekly
dose of less than 5.25 mg or less of aminopterin. A related
embodiment is a method of dosing a human with MS, polymyositis,
SLE, GVHD or asthma with a cumulative weekly dose of less than 2 mg
aminopterin. Another related embodiment is a method of dosing a
human with MS, polymyositis, SLE, GVHD or asthma with a cumulative
weekly dose of less than 1 mg/kg aminopterin.
[0025] Further still, antifolates and aminopterin in particular
have been discovered to be efficacious in the treatment of
bronchopulmonary dysplasia, and inflammatory disorders that occur
naturally in domestic and agricultural animals, and in some
embodiments with only a single dose of aminopterin. In particular,
embodiments provide:
[0026] A method for treating an inflammatory disorder in a patient,
comprising administering to said patient a therapeutically
effective amount of aminopterin, or a pharmaceutically acceptable
salt thereof, in uninterrupted cycles. In preferred embodiments,
the number of uninterrupted cycles is at least 24. In yet other
preferred embodiments, the periodicity of the uninterrupted cycles
is weekly, wherein the number of doses in each cycle is 2, and more
preferably 1. In some embodiments, a second drug is used in
combination therapy. Folic acid is particularly preferred as the
second drug.
[0027] Typically, each cycle consists of less than 0.07 mg
aminopterin per kilogram of patient body weight that can be
administered in less than 5 tablets. For example, a 0.25 kg patient
would be administered less than 0.0175 mg aminopterin, whereas a
1.0 kg patient would be administered less than 0.07 mg aminopterin,
whereas a 60 kg patient would be administered less than 4.2 mg
aminopterin, and whereas a 100.0 kg patient would be administered
less than 7 mg aminopterin; thus in a preferred embodiment a tablet
may contain between about 0.0175 mg and about 7 mg aminopterin,
preferably between about 0.0175 mg and about 0.5 mg, and most
preferably between 0.025 mg and about 0.5 mg. Optimal dosing
without toxicity manifestations is achieved in particularly
preferred embodiments utilizing one or more tablets containing less
than 0.5 mg aminopterin each.
[0028] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a single dose of a therapeutically effective amount of
an antifolate, or a pharmaceutically acceptable salt thereof. In
preferred embodiments, the antifolate is aminopterin. Preferred
inflammatory disorders that can be treated with a single dose of an
antifolate include human bronchopulmonary dysplasia, canine atopic
dermatitis, bovine acute pneumonic pasteurellosis, SLE, GVHD and
polymyositis. Canine atopic dermatitis is one of the most common
conditions seen by veterinarians, and bovine acute pneumonic
pasteurellosis, also known as `shipping fever` in transported
cattle costs the cattle industry $1 billion per year (Malazdrewich,
et al. Vet. Pathol. 38:297, 2001). In shipping fever, the ability
to treat affected animals with a minimum number of drug
administrations has critical economic advantages.
[0029] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a therapeutically effective amount of aminopterin, or
a pharmaceutically acceptable salt thereof, wherein the
inflammatory disorder is SLE, GVHD or polymyositis.
[0030] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a therapeutically effective amount of aminopterin, or
a pharmaceutically acceptable salt thereof wherein the inflammatory
disorder is selected from MS and asthma.
[0031] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a therapeutically effective amount of an antifolate,
or a pharmaceutically acceptable salt thereof, wherein the
inflammatory disorder is selected from the group consisting of
canine atopic dermatitis, bronchopulmonary dysplasia, SLE, GVHD and
polymyositis. When the antifolate is aminopterin, the
therapeutically effective amount administered per week is between
about 0.0005 and about 0.07 mg per kilogram of patient body weight.
For example, a 0.05 kg patient would be administered between about
0.000025 mg and about 0.0035 mg aminopterin; a 0.1 kg patient would
be administered between about 0.00005 mg and about 0.007 mg
aminopterin; a 0.25 kg patient would be administered between about
0.000125 mg and about 0.0175 mg aminopterin; a 1.0 kg patient would
be administered between about 0.0005 mg and about 0.07 mg
aminopterin; a 60 kg patient would be administered between about
0.03 and about 4.2 mg aminopterin; and a 100 kg patient would be
administered between about 0.05 mg and about 7 mg aminopterin. In
preferred embodiments, the above doses are in tablet form. In
another preferred embodiment, the amount of aminopterin or
pharmaceutically acceptable salt thereof, within the tablet is
between 0.000025 mg and about 0.5 mg, more preferably between about
0.00005 mg and about 0.5 mg, more preferably between about 0.000125
mg and about 0.5 mg. In another preferred embodiment, the amount of
aminopterin, or pharmaceutically acceptable salt thereof, within
the tablet is between about 0.000025 mg and about 0.5 mg, more
preferably between about 0.000025 mg and about 0.45 mg, and more
preferably between about 0.000025 mg and about 0.4 mg. One skilled
in the art will readily recognize tablets may contain aminopterin,
or a pharmaceutically acceptable salt thereof, in amounts of 0.05
mg, 0.075 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg,
0.4 mg, 0.45 mg and 0.5 mg per tablet. In another preferred
embodiment, the therapeutically effective amount of the antifolate
is administered in uninterrupted cycles. In another preferred
embodiment, the antifolate is methotrexate and the dosages and
tablet strengths of the aforementioned ranges for aminopterin are
increased about 10-fold.
[0032] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a therapeutically effective amount of aminopterin, or
a pharmaceutically acceptable salt thereof, wherein the
therapeutically effective amount of aminopterin administered per
week is less than 0.07 mg aminopterin per kilogram of patient body
weight. In this method, aminopterin is preferably administered in
one or more tablets, wherein at least one tablet contains less than
0.5 mg aminopterin. For example, a 0.25 kg patient would be
administered less than 0.0175 mg aminopterin; a 1.0 kg patient
would be administered less than 0.07 mg aminopterin; a 60 kg
patient would be administered less than 4.2 mg aminopterin; and a
100 kg patient would be administered less than 7.0 mg aminopterin;
thus in a preferred embodiment, a tablet would contain aminopterin,
or a pharmaceutically acceptable salt thereof, in the amount
between about 0.0175 mg and about 4.2 mg, more preferably between
about 0.0175 mg and about 2.0 mg, more preferably between about
0.0175 and about 1.0 mg, and most preferably between about 0.0175
mg and about 0.5 mg. One skilled in the art will readily recognize
tablets may contain aminopterin, or a pharmaceutically acceptable
salt thereof, in amounts of 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4
mg, and 0.5 mg per tablet.
[0033] In other embodiments, a method is provided for treating an
inflammatory disorder in a patient, comprising administering to
said patient a therapeutically effective amount of aminopterin, or
a pharmaceutically acceptable salt thereof, wherein the
inflammatory disorder is treated more rapidly than with
methotrexate. The rate of treatment is quantified with a disease
manifestation scoring system selected from the group consisting of
ACR20, ACR50, ACR70, ACR-N, JRA30% DOI, JRA50% DOI, JRA70% DOI,
PASI, pulmonary function testing, oxygen saturation, lesional
scores, Kurtzke's expanded disability status scale (EDSS), and
pruritis scores. In other embodiments, the rate of treatment is
quantified by the area under the efficacy-time curve.
[0034] In other embodiments, a pharmaceutical composition is
provided comprising a therapeutically effective amount of
aminopterin, or a pharmaceutically acceptable salt thereof, wherein
the pharmaceutical composition contains less than 0.5 mg
aminopterin. The pharmaceutical composition is preferably a tablet,
and in certain embodiments is a 0.05 mg, 0.075 mg, 0.1 mg, 0.15 mg,
0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, or 0.45 mg tablet. In a
particularly preferred embodiment, the active pharmaceutical
ingredient is substantially free of impurities.
[0035] These and other aspects will become apparent upon reference
to the detailed description and illustrative examples which are
intended to exemplify non-limiting embodiments. All references
disclosed herein are hereby incorporated by reference in their
entirety as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a dose-response plot of aminopterin and
methotrexate in the murine air-pouch model of inflammation.
[0037] FIG. 2 is a plot showing the efficacy of aminopterin and
methotrexate days after the last dose of each drug in the murine
air-pouch model of human inflammation.
[0038] FIG. 3 is a plot showing the relative efficacy of
aminopterin and methotrexate in the rat oxygen-toxicity model of
human bronchopulmonary dysplasia.
[0039] FIG. 4 is a plot showing the efficacy of aminopterin in
canine atopic dermatitis in a 4-week trial.
[0040] FIG. 5 is a plot showing adenosine release from ma104 cells
after 8 hours incubation with various concentrations of
aminopterin.
[0041] FIG. 6 is a plot showing the total amount of (aminopterin)
AMT and methotrexate (MTX) in the liver after the cessation of
dosing together with the amount of free dihydrofolate (DHFR).
GLOSSARY
[0042] Prior to setting forth detail, it may be helpful to an
understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0043] The term "active pharmaceutical ingredient" as used herein
means a mixture of an antifolate and impurities resulting from one
or more organic synthetic steps. An organic synthetic step will
comprise the partial or complete transformation of one or more
chemicals to one or more new chemicals, and will also usually
entail at least one or more purification steps to enrich the one or
more new chemicals. The one or more purification steps will consist
of methods known to those skilled in the art, such as, for example,
crystallization, extraction, and chromatography. Ideally,
purification steps are not required, or the one or more
purification steps will enrich a single new chemical
preferentially. For example, an active pharmaceutical ingredient
containing aminopterin is made by the transformation of
2,4-diamino-6-(bromomethyl)pteridine and
N-(4-aminobenzoyl)-L-glutamic acid in dimethylacetamide to
aminopterin plus several impurities, wherein the impurities
comprise at least folic acid and untransformed
2,4-diamino-6-(bromomethyl)pteridine and
N-(4-aminobenzoyl)-L-glutamic acid.
[0044] The term "aminopterin purity" as used herein means the
percentage of antifolate in an active pharmaceutical ingredient or
pharmaceutical composition, wherein the antifolate is aminopterin
(see glossary definition of "impurities").
[0045] The term "antifolate" as used herein means a molecule and/or
metabolites of the molecule that interfere with the normal
metabolism or utilization of folic acid (i.e. folate) and/or
metabolites of folic acid in a cell-free biochemical system or in
cells found in cell culture, tissue culture, leukemia, cancer, a
mammal, and a human. For example, aminopterin and methotrexate, as
well as their polyglutamated metabolites, are antifolates.
Typically, an antifolate and/or metabolite of the antifolate will
interfere with the normal metabolism or utilization of folic acid
and/or metabolites of folic acid by blocking their binding to one
or more enzymes or receptors that include, for example, the reduced
folate receptor, the folic acid receptor, folylpolyglutamate
synthase, dihyrofolate reductase, thymidylate synthase,
methylene-tetrahydrofolate reductase, amido
phosphoribosyltransferase, glycinamide ribonucleotide
transformylase, aminoimidazole carboxamide ribonucleotide
transformylase, and homocysteine methyltransferase. Examples of
folic acid metabolites that an antifolate and/or metabolites of the
antifolate will interfere with include, but are not limited to,
5-methyl-tetrahydrofolate-(glu).sub.n,
5,10-methylene-tetrahydrofolate-(glu).sub.n,
tetrahydrofolate-(glu).sub.n,
N-5-fomamino-tetrahydrofolate-(glu).sub.n,
5,10-methenyl-tetrahydrofolate-(glu).sub.n,
10-formyl-tetrahydrofolate-(glu).sub.n, and
5-formyl-tetrahydrofolate-(glu).sub.n, where -(glu).sub.n refers to
the glutamates attached to the metabolite and n is the number of
attached glutamates. When n=1, no glutamates have been added to
these folic acid metabolites beyond that found in the original
folic acid molecule. When n is greater than 1 these folic acid
metabolites are considered to be polyglutamates (see glossary term
below).
[0046] The term "AUC" as used herein means the area under the
plasma concentration-time curve for a single dose of a drug as
described more fully in Shargel and Yu, Applied Biopharmaceutics
and Pharmacokinetics, 4.sup.th Edition, 1999, Appleton & Lange,
Stamford, Conn., incorporated herein by reference. The AUC is
proportional to the amount of drug that reaches the plasma.
[0047] The term "combination therapy" as used herein refers to the
use of two or more therapeutics according to a therapeutic protocol
with the aim of providing a highly optimized treatment plan to most
effectively treat an inflammatory disorder in a patient.
[0048] The term "disease manifestation" as used herein refers to
any undesired result of an inflammatory disorder. Particular
disease manifestations include, but are not limited to lethargy,
joint pain, joint inflammation, joint damage, inflammatory cells in
joint fluid, and psoriatic skin lesions. Disease manifestations are
often quantified in the art using well known scoring systems, such
as for example, ACR20, ACR50, ACR70 or ACR-N in rheumatoid
arthritis (Felson et al., Arthritis Rheum. 38:727, 1995; Bathon et
al., N. Engl. J. Med. 343:1586, 2000; and William St. Clair et al.,
Arthritis Rheum. 50(11):3432-3443, 2004); JRA30% DOI, JRA50% DOI
and JRA70% DOI in juvenile rheumatoid arthritis (Giannini et al.,
Arthritis Rheum. 40(7):1202-1209, 1997; and Lovell et al.,
Arthritis Rheum. 48(1):218, 2003), the PASI (Psoriasis Activity and
Severity Index) score in psoriasis (Finlay et al., Br. J. Dermatol.
123:751, 1990); pulmonary function testing and oxygen saturation in
bronchopulmonary dysplasia and asthma; lesional and pruritis scores
in canine atopic dermatitis (Olivry and Mueller, Veterinary
Dermatol. 14:121-146, 2004); Kurtzke's expanded disability status
scale (EDSS) (Kurtzke J. F., Neurology. 33:1444-52, 1983) in
multiple sclerosis; the gradings of GVHD are according to accepted
criteria (Thomas et al., New Engl. J. Med. 292:832-843, 895-902,
1975); the group classifications of polymyositis (Holden et al.,
Can. Med. Assoc. J. 132:Mar. 15, 1985; Bohan A., and Peter, J. B.,
New Engl. J. Med. 292:344-347, 1975). Other ways of quantifying
disease manifestations in a patient with an inflammatory disorder
will be familiar to those skilled in the art.
[0049] The term "efficacy" as used herein means an antifolate is
therapeutically effective. Generally, a greater level of efficacy
will be achieved by increasing the dose and/or frequency of
administration of an antifolate given to a population, such that a
greater proportion of the population will receive a benefit and/or
there will be a greater magnitude of benefit in an individual
patient. If a first antifolate is more potent than a second
antifolate, it will reach a greater level of efficacy than the
second antifolate using identical amounts of each.
[0050] The term "impurities" as used herein refers to the
impurities found in the active pharmaceutical ingredient.
Impurities arise during the organic synthetic steps employed in the
preparation of the active pharmaceutical ingredient, and in the
case of aminopterin include, for example, folic acid, pterins, and
conjugates of p-aminobenzoic acid (i.e. pABAglu). Impurities may be
the result of incomplete transformation of chemicals during an
organic synthetic step, or one or more side-reactions that result
in chemicals being transformed into unintended new chemicals. As
defined herein, the pharmaceutically acceptable carriers and
optional therapeutic ingredients in a pharmaceutical composition do
not constitute impurities. Impurities in a pharmaceutical
composition pertain only to those impurities in the active
pharmaceutical ingredient used to make the pharmaceutical
composition. Impurities are quantitated using any measurable
property suitable for quantitating molecules. Such measurable
properties will be familiar to those in the art and include, for
example, HPLC peak area (i.e. "area"), mass and moles. Impurities
will typically be conveniently quantitated based on their area, but
may also be quantitated according to their weight or moles using,
for example, uv absorbance of collected HPLC peak fractions and the
known extinction coefficient and molecular weight of each impurity.
If the molecular weight of an impurity is unknown, mass
spectrometry may be used. The percentage of an impurity in an
active pharmaceutical ingredient or pharmaceutical composition is
the amount of the impurity divided by the total amount of
impurities plus antifolate multiplied by 100, wherein all
impurities and the antifolate are quantitated using the same
measurable property. For example, the percentage of an impurity in
an active pharmaceutical ingredient or pharmaceutical composition
may be expressed as an area %, weight %, or mole %. In a specific
example, if an impurity constitutes 0.1 micromole of an active
pharmaceutical ingredient and the aminopterin plus total impurities
in the active pharmaceutical ingredient together constitute 1
micromole, the percentage of the impurity in the active
pharmaceutical ingredient is 10 mole %. In another example, if an
impurity is 0.25 area units of a pharmaceutical composition and the
total impurities plus antifolate together are 1.0 area units of the
pharmaceutical composition, the percentage of the impurity in the
pharmaceutical composition (or the active pharmaceutical
ingredient) is 25 area %. In a further example, if an impurity is
0.04 mg of a pharmaceutical composition containing 2 mg of the
active pharmaceutical ingredient, the percentage of the impurity in
the pharmaceutical composition is 2 weight %. The percentage of
total impurities may be obtained by summing the percentages of all
individual impurities in the active pharmaceutical ingredient or
pharmaceutical composition, wherein all the individual impurities
are quantitated using the same measurable property. The percentage
of antifolate in an active pharmaceutical ingredient or
pharmaceutical composition is the amount of antifolate divided by
the total amount of impurities plus antifolate multiplied by 100,
wherein all impurities and the antifolate are quantitated using the
same measurable property. Thus, the percentage of total impurities
in the active pharmaceutical ingredient or pharmaceutical
composition may alternatively be obtained by subtracting the
percentage of antifolate from 100%.
[0051] The term "oral bioavailability" as used herein refers to the
fraction of an antifolate dose given orally that is absorbed into
the plasma after a single administration to a patient. A preferred
method for determining the oral bioavailability is by dividing the
AUC of an antifolate dose given orally by the AUC of the same
antifolate dose given intravenously to the same patient, and
expressing the ratio as a percent. Other methods for calculating
oral bioavailability will be familiar to those skilled in the art,
and are described in greater detail in Shargel and Yu, Applied
Biopharmaceutics and Pharmacokinetics, 4.sup.th Edition, 1999,
Appleton & Lange, Stamford, Conn., incorporated herein by
reference.
[0052] The term "patient" is an animal or a human.
[0053] The term "pharmaceutical composition" as used herein means
the active pharmaceutical ingredient combined with one or more
pharmaceutically acceptable carriers, and optionally other
therapeutic ingredients. Suitable pharmaceutically acceptable
carriers will be familiar to those skilled in the art, and will
comprise, for example, microcrystalline cellulose, lactose, silicon
dioxide, croscarmellose, sodium benzoate, sorbitol, magnesium
stearate and flavoring. The active pharmaceutical ingredient will
typically comprise only a small percentage of the total
pharmaceutical composition. For example, a "2 mg aminopterin
tablet" is a pharmaceutical composition that weighs about 100 mg,
and comprises about 98 grams of pharmaceutically acceptable
carriers and about 2 mg of the active pharmaceutical ingredient.
The 2 mg of the active pharmaceutical ingredient consists mostly of
aminopterin and a small fraction of impurities (see glossary
definition of "impurities" and "active pharmaceutical ingredient").
If this pharmaceutical composition is said to be "substantially
free of impurities", then the small fraction of impurities will be,
for example, less than 5 area %, less than 5 weight %, or less than
5 mole % percent of the active pharmaceutical ingredient (see
glossary definition of "substantially free of impurities").
[0054] The term "percent inhibition" as used herein refers to the
percent of DHFR molecules bound by antifolate; aminopterin or
methotrexate. The DHFR molecules on which the percentage is
calculated may be those from within the entire patient, or those
from within a particular representative sampled subfraction of the
patient (e.g. a particular organ such as the liver, blood, bone
marrow, etc.).
[0055] The term "polyglutamates" as used herein refers to folate
and antifolate metabolites that have attached two or more
glutamates. The enzyme folylpolyglutamate synthase attaches
additional glutamates to folate metabolites and some antifolates
beyond the glutamate that is on the original folate metabolite and
some antifolates to form a polyglutamate chain. Examples of
aminopterin and methotrexate polyglutamates include
aminopterin-(glu).sub.n and methotrexate-(glu).sub.n, where
-(glu).sub.n refers to the glutamates attached to the antifolate
and n is the number of attached glutamates. When n=1, no glutamates
have been added beyond that in the original antifolate molecule.
When n is greater than 1 these antifolates are considered to be
polyglutamates, and thus have a polyglutamate chain. A
polyglutamate chain is said to have a length, wherein a first
polyglutamate chain is said to have a longer length than a second
polyglutamate chain if the n of the first polyglutamate chain is
larger than the n of the second polyglutamate chain. Folate and
antifolate metabolites having polyglutamate chains are often
referred to as polyglutamated species or polyglutamates. A mixture
of polyglutamate chains having different lengths is said to
comprise polyglutamate chain lengths. Both aminopterin and
methotrexate are metabolized to polyglutamates having an n of from
2 to about 5, as described in greater detail in, Gangjee et al.,
Curr. Med. Chem. Anti-Canc. Agents. 2(3):331, 2002, incorporated
herein by reference.
[0056] The term "potency" as used herein means the effectiveness of
a dosage to achieve a particular level of efficacy. The dosage
takes into account the amount of drug given at each administration
(i.e. dose), the frequency of administration, and optionally, the
total number of doses to be given. The effectiveness of a dosage
can be quantitated by measuring the cumulative amount of drug
administered in a defined period (i.e. the "dose rate") of the
dosage and that results in a particular level of efficacy, where
effectiveness and potency are inversely related to the dose rate. A
first drug whose dosage has greater effectiveness or potency than
the dosage of a second drug is said to be more potent than the
second drug. For example, a first antifolate is more potent than a
second antifolate if both achieve the same level of efficacy using
a dose rate that is smaller in the first antifolate than the second
antifolate. In a more specific example, a first antifolate is more
potent than a second antifolate if both achieve the same level of
efficacy using a dosage that is identical except for the dose of
the first antifolate being smaller than the dose of the second
antifolate. In a further example, a first antifolate is more potent
than a second antifolate if both achieve the same level of efficacy
using a dosage that is identical except for the frequency of
administration of the first antifolate being smaller than the
frequency of administration of the second antifolate. Two different
antifolates may have different potencies, and will therefore
achieve the same level of efficacy at different therapeutically
effective amounts. For example, aminopterin is about 25 times more
potent than methotrexate, and therefore the therapeutically
effective amount of aminopterin required for a level of efficacy is
about 25-fold less than the therapeutically effective amount of
methotrexate required for the same level of efficacy. The
therapeutically effective amounts of two antifolates (i.e. either
as a dosage or as a single dose) that result generally in the same
level of efficacy are referred to herein as equi-potent. For
example, two oral doses of 2 mg/m.sup.2 aminopterin are
approximately equi-potent to four oral doses of 25 mg/m.sup.2
methotrexate.
[0057] The term "substantially free of impurities" as used herein
means that the percentage of total impurities in an active
pharmaceutical ingredient or in a pharmaceutical composition is
less than five percent (see definition of "impurities"). For
example, a pharmaceutical composition or active pharmaceutical
ingredient is said to be substantially free of impurities if the
percentage of total impurities is less than 5 area %, 5 weight % or
5 mole %. As used herein, "substantially free of impurities" also
means that the percentage of antifolate in an active pharmaceutical
ingredient or in a pharmaceutical composition is ninety-five
percent or greater (see definition of "impurities"). For example, a
pharmaceutical composition that has a percentage of antifolate with
an area %, weight %, or mole % equal to or greater than
ninety-five, is said to be substantially free of impurities. In a
more specific example, a pharmaceutical composition or active
pharmaceutical ingredient that has an aminopterin purity with an
area %, weight %, or mole % equal to or greater than ninety-five,
is said to be substantially free of impurities.
[0058] The term "therapeutic component" as used herein refers to a
therapeutically effective dosage (i.e. dose and frequency of
administration) that eliminates, reduces, or prevents the
progression of a particular disease manifestation in a percentage
of a population. An example of a commonly used therapeutic
component is the ED.sub.50, which describes the dose in a
particular dosage that is therapeutically effective for a
particular disease manifestation in 50% of a population.
[0059] The term "therapeutic index" as used herein refers to the
ratio of a particular toxicity component (i.e. the dose that is
toxic in a percentage of a population, for example, the TD.sub.50)
to a particular therapeutic component (i.e. the dose that is
effective in percentage of a population, for example, the
ED.sub.50). It will be understood by a skilled practitioner that
the determination of the therapeutic index of an antifolate, such
as aminopterin, need not be determined for every patient in a
population with a inflammatory disorder treated according to the
disclosure. It is sufficient to use a representative number of
patients with the inflammatory disorder to establish a therapeutic
index for the entire patient population with the inflammatory
disorder, where at least 5 patients with the inflammatory disorder
will be required in order to establish a therapeutic index for the
entire patient population with the inflammatory disorder.
[0060] The term "therapeutic protocol" as used herein refers to a
schedule of dosing, routes of administration, and schedule duration
for two or more drugs that are employed within combination therapy.
The schedule may be further divided into specific phases each of a
specified duration. For example, the phases of modern rheumatoid
arthritis treatment involving the treating with NSAIDs and various
other small-molecule or biologic DMARDS either sequentially or
together constitute specific phases of a therapeutic protocol.
During each phase, the type of drugs to be given, their dosing, and
routes of dosing are defined for all patients in general or may be
modified based on other disease factors such a laboratory or
imaging tests.
[0061] The term "therapeutically effective amount" as used herein
means the dosage (dose or amount, and frequency) of antifolate
which directly or indirectly kills inflammatory cells, arrests the
accumulation of inflammatory cells, or reduces the accumulation of
inflammatory cells in a human or other mammal afflicted with an
inflammatory disorder (i.e. a patient) such as, for example,
arthritis of undefined etiology, rheumatoid arthritis, juvenile
rheumatoid arthritis, atopic dermatitis, bronchopulmonary
dysplasia, asthma, multiple sclerosis, inflammatory bowel disease,
psoriatic arthritis, SLE, GVHD, polymyositis and psoriasis, or a
animal with, for example, canine atopic dermatitis or bovine acute
pneumonic pasteurellosis. The term as used herein shall also mean
the dosage of an antifolate which directly or indirectly reduces or
increases the activity of molecules secreted by inflammatory and/or
non-inflammatory cells participating in an inflammatory disorder in
a human or mammal, such that the amount of antifolate arrests,
reduces, or eliminates altogether the degree of pathologic
inflammation associated with the inflammatory inflammatory
disorder. Typically, a therapeutically effective amount will also
eliminate, reduce, or prevent the progression of, one or more
disease manifestations. A skilled artisan readily recognizes that
in many cases antifolates may not provide a cure, but may only
provide partial benefit. Furthermore, the skilled artisan
recognizes that because individual patients and disease states may
vary, some patients may receive little or no benefit at all. A
dosage of antifolate that "kills", "arrests", "reduces" or
"eliminates" as described above, in a least some patients, is
considered therapeutically effective. The dose magnitude of a
therapeutically effective amount of aminopterin in the acute or
chronic management of a inflammatory disorder will vary with the
severity of the inflammatory disorder to be treated and the route
of administration. The dosage and dose rate of aminopterin will
depend on a variety of factors, such as the weight and calculated
surface area of the patient, the specific pharmaceutical
composition used, the object of the treatment, i.e., therapy or
prophylaxis, the nature of the disease to be treated, the judgment
of the treating physician, and the response of the individual
patient. In general, a therapeutically effective amount of
aminopterin will be a dose of aminopterin from 0.001-0.07 mg/kg,
0.005-0.03 mg/kg, and most preferably 0.010-0.03 mg/kg, given as a
single or divided dose. Thus, for example, the therapeutically
effective amount of aminopterin, or pharmaceutically acceptable
salt thereof, given as a single or divided dose, for a 0.25 kg
patient would range between 0.00025-0.0175 mg, 0.00125-0.0075 mg,
and most preferably 0.0025-0.0075 mg; whereas for a 1.0 kg patient
the therapeutically effective amount of aminopterin would range
between 0.001-0.07 mg, 0.005-0.03 mg, and most preferably
0.010-0.03 mg; whereas the therapeutically effective amount of
aminopterin for a 60 kg patient would range between 0.06-4.2 mg,
0.3-1.8 mg, and most preferably 0.6-1.8 mg aminopterin; whereas the
therapeutically effective amount of aminopterin for a 100 kg
patient would range between 0.1-7 mg, 0.5-3 mg, and most preferably
1-3 mg aminopterin. Thus, the amount of aminopterin, or
pharmaceutically acceptable salt thereof, within the tablet could
thus range between 0.00025-7.0 mg, more preferably between
0.00025-2.0 mg, more preferably between 0.00025-0.5 mg, more
preferably between 0.0025-0.5 mg, more preferably between 0.025-0.5
mg. As encompassed by these ranges, one skilled in the art will
readily recognize tablets may contain aminopterin, or a
pharmaceutically acceptable salt thereof, in amounts of 0.05 mg,
0.075 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4
mg, 0.45 mg and 0.5 mg per tablet.
[0062] Patients may be upward titrated from below to within these
dose ranges to a satisfactory control of disease manifestations.
Once improvement in the patient's condition has occurred, a
maintenance dosage of a composition of this disclosure is
administered, if necessary. Subsequently, the dose rate may be
reduced by reducing the dose or frequency of administration, or a
combination of both, as a function of the symptoms, to a level at
which the improved condition is retained. When the symptoms have
been alleviated to the desired level, the practitioner may elect to
cease treatment. Patients may, however, require intermittent
treatment upon any recurrence of disease symptoms, or
prophylactically scheduled treatments as required. The
therapeutically effective amount of aminopterin may optionally be
administered prior to, contemporaneous with, or after at least one
therapeutically effective dose of leucovorin or folic acid.
[0063] The term "toxicity component" as used herein refers to the
dosage (i.e. dose and frequency of administration) required to
produce a toxicity manifestation in a percentage of a population.
An example of a commonly used toxicity component is the TD.sub.50,
which describes the dose in a particular dosage required to produce
a toxicity manifestation in 50% of a population. Generally, there
is an inverse relationship between dose and frequency necessary to
produce the same toxicity component, where less antifolate given
more often will produce the same toxicity component as more
antifolate given less often.
[0064] The term "toxicity manifestation" as used herein refers to
any undesired effect of a drug. A drug is said to be toxic or have
toxicity if it causes a toxicity manifestation in a percentage of a
population. For antifolates, particular toxicity manifestations
include, but are not limited to mucositis, alopecia, diarrhea,
myelosuppression, nephrotoxicity, hepatotoxicity, severe
hepatotoxicity, neurotoxicity, and death. Toxicity manifestations
also include indirect indications of toxicity such as
thrombocytopenia (e.g. <50,000/.mu.L), neutropenia (e.g.
<750/.mu.L), elevated liver enzymes (e.g. >5 times normal),
erythrocyte antifolate (e.g. eAMT and eMTX), the necessary
discontinuation of therapy by a patient because of the inability to
endure further treatment with the drug, or increased hospital
admissions due to the inability to continue therapy. Thus, a
practitioner can assess the presence and the magnitude of a
particular toxicity manifestation. Many, if not all, of the
toxicity manifestations of antifolates can be reversed by the
prior, contemporaneous or subsequent administration of leucovorin,
a reduced folate that is well known in the art to rescue antifolate
toxicity.
DETAILED DESCRIPTION
[0065] Embodiments described herein provide dosages and methods for
treating a patient with an inflammatory disorder with a
therapeutically effective amount of aminopterin, or a
pharmaceutically acceptable salt, that provide unexpected
advantages of patient convenience while also achieving efficacy
without concomitant toxicity. The inflammatory disorder may occur
in humans and comprise, for example, arthritis of undefined
etiology, rheumatoid arthritis, psoriatic arthritis, juvenile
rheumatoid arthritis, psoriasis, inflammatory bowel disease,
asthma, multiple sclerosis, atopic dermatitis, SLE, GVHD,
polymyositis and bronchopulmonary dysplasia. The animal
inflammatory disorder may occur in animals and comprise, for
example, arthritis, canine atopic dermatitis and bovine acute
pneumonic pasteurellosis.
[0066] Although not to be limited by theory, aminopterin inhibits
enzymes involved in purine and pyrimidine metabolism, including
DHFR, that leads indirectly to the extracellular release of
adenosine.
[0067] DHFR is said to be inhibited in a patient if the percent
inhibition comprises the range of 5% to 100%. In some embodiments,
the percent inhibition of DHFR in a patient during each dosing
cycle of a therapeutically effective amount of aminopterin is
greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%. In
other embodiments, the percent inhibition during each cycle of a
therapeutically effective amount of aminopterin will comprise a
range of DHFR inhibition, where the lower end of the range
comprises percent inhibition greater than 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80% and 90%; and the upper end of the range
comprises percent inhibition less than 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80% and 100%. In preferred embodiments, the cycle is
weekly, and the range of percent inhibition of a therapeutically
effective amount of aminopterin during each cycle comprises
50%-99%, 50%-95%, 50%-90%, 50%-85% and 50%-75%. In another
preferred embodiment, a method is provided for inhibiting DHFR in a
patient, comprising administering to said patient a therapeutically
effective amount of aminopterin, or a pharmaceutically acceptable
salt thereof, wherein the therapeutically effective amount of
aminopterin, or pharmaceutically acceptable salt thereof,
administered per week is between 0.0005 and 0.07 mg per kilogram of
patient body weight. In further embodiments, the aminopterin is
administered in uninterrupted cycles.
[0068] Adenosine is a powerful anti-inflammatory autocoid that
regulates innate immunity (reviewed by Hasko and Cronstein, (2004)
Adenosine: an endogenous regulator of innate immunity. Trends
Immunol., 25/1:33-39) and that decreases the activation of
antigen-stimulated cells, decreases the expression of adhesion
molecules, and possibly induces T cell apoptosis (Cronstein, et
al., Proc. Natl. Acad. Sci. USA (1991), 88: 2441; Cronstein, et
al., J. Clin. Invest. (1993), 92/6:2675; Genesteir, et al., J.
Clin. Invest. (1998), 102:322-328; Morabito, et al., J. Clin.
Invest. (1998), 101:295-300; Paillot et al., Transplant Proc.
(1998), 30/5:2348-50; Johnston et al., Clin. Immunol. (2005),
114/2:154-63). The pivotal role of adenosine in human inflammatory
disorders such as rheumatoid arthritis and psoriasis is now
appreciated. Adenosine can be measured directly in the synovial
fluid collected from patients with rheumatoid arthritis (Ottonello
et al., Rheumatology (2002), 41:1249-1260).
[0069] Thus, in one embodiment, a method is provided for treating a
patient having an inflammatory disorder inhibited by the release of
adenosine, wherein the method comprises administering to said
patient in need thereof a therapeutically effective amount of
aminopterin, or a pharmaceutically acceptable salt thereof.
Inflammatory disorders that may be inhibited by the release of
adenosine are readily identified by methods readily available to
those skilled in the art. In one method for identifying an
inflammatory disorder inhibited by the release adenosine, an
adenosine receptor knockout (e.g. A.sub.2A and/or A.sub.3 adeonsine
receptors) animal is prepared, wherein there exists a model of a
human or animal inflammatory disorder (Montesinos et al. (2003)
Arthritis Rheum., 48/1:240-247). Aminopterin is then administered
to control (e.g. wild type) animals and the knockout animals,
wherein both have the inflammatory disorder (e.g.
carrageenan-induced inflammation in the mouse air-pouch model of
inflammation).
[0070] The inflammatory disorder is said to be inhibited by the
release of adenosine if the `therapeutic component` (see Glossary
above) in the control animals is at least 2, 5, and more
preferably, at least 10 times less than in the knockout animals. In
many embodiments, no therapeutically effective amount will exist in
the knockout animals (i.e. they are completely insensitive to the
therapeutic effects of aminopterin). It will be appreciated that
this method allows the adenosine receptor isoform involvement in
the disorder to be characterized.
[0071] In another method for identifying an inflammatory disorder
inhibited by the release adenosine, control and test groups of
animals or humans with an inflammatory disorder are both
administered aminopterin as described herein. The test group is
then administered an inhibitor of adenosine binding to one or more
adenosine receptors. Some inhibitors will be selective for
particular adenosine receptor isoforms, and this will permit
isoform involvement in the disorder to be characterized. Suitable
inhibitors of adenosine receptor binding include, but are not
limited to, caffeine, DPCPX, PACPX, ZM241385, CSC, alloxane, and
MRS-1220 (as defined in Hasko and Cronstein, (2004) Adenosine: an
endogenous regulator of innate immunity. Trends Immunol.,
25/1:33-39).
[0072] The inflammatory disorder is said to be inhibited by the
release of adenosine if the `therapeutic component` (see Glossary
above) in the control animals is at least 2, 5, and more
preferably, at least 10 times less than in the test group
administered the inhibitor of adenosine receptor binding. In many
embodiments, no therapeutically effective amount will exist in the
group administered the inhibitor of adenosine receptor binding
(i.e. they are completely insensitive to the therapeutic effects of
aminopterin).
[0073] In still other methods for identifying an inflammatory
disorder inhibited by the release adenosine, control and test
groups of animals or humans with an inflammatory disorder are both
administered an agonist of one or adenosine receptors (i.e. a mimic
of adenosine). Some agonists will be selective for particular
adenosine receptor isoforms, and this will permit isoform
involvement in the disorder to be characterized. Suitable agonists
of adenosine receptors include, but are not limited to, CPA, CCPA,
CGS-21680, IB-MECA and 2Cl-IB-MECA (as defined in Hasko and
Cronstein, (2004) Adenosine: an endogenous regulator of innate
immunity. Trends Immunol., 25/1:33-39).
[0074] The inflammatory disorder is said to be inhibited by the
release of adenosine if there is a `therapeutic component` in the
test group of animals, relative to the disease manifestations in
the control group (see Glossary above for `therapeutic component).
In some embodiments, the therapeutic component will be a dose of
agonist that completely eliminates, reduces, or prevents the
progression of a particular disease manifestation in the test group
of animals.
[0075] Within certain embodiments, the present disclosure provides
a method for treating an inflammatory disorder in a patient with
uninterrupted cycles of aminopterin doses, wherein the doses are a
therapeutically effective amount. Uninterrupted means that
aminopterin doses are repetitively administered to a patient for at
least 4 cycles, 5 cycles, 10 cycles, 12 cycles, 24 cycles, 36
cycles, and most preferably greater than 52 cycles, wherein the
periodicity of the cycles is constant, and wherein the greatest
duration between the last dose of one cycle and the first dose of
the next cycle does not exceed 21 days, 14 days, and most
preferably 7 days. Within this definition, `periodicity of the
cycles is constant` means that the duration between corresponding
doses in consecutive cycles is constant to within a 12 hour range.
For example, if the periodicity is denoted to be 7 days (i.e. 168
hours), then according to the present disclosure the phrase
`periodicity of the cycles is constant` will be construed to mean
that the duration between corresponding doses in consecutive cycles
may range from 162 to 174 hours. Further within this definition,
the number of aminopterin doses in each cycle can range from 1 to
5, and each individual dose may comprise taking one or a plurality
of individual dosage forms.
[0076] Thus, in a preferred embodiment 1 dose of aminopterin is
administered to a patient every 7 days for at least 4 cycles, and
most preferably for at least 52 cycles (i.e. a year). In this case,
the number of doses per cycle is only a single dose, the
periodicity is 7 days, and the greatest duration between the last
dose of one cycle and the first dose of the next cycle is 6 days.
In another preferred embodiment one dose of aminopterin is
administered on Monday and one on Tuesday for at least 52 cycles.
In this case, the number of doses per cycle is 2, the periodicity
is 7 days, and the greatest duration between the last dose of one
cycle and the first dose of the next cycle is 5 days (i.e.
Wednesday through Sunday). In yet another preferred embodiment, a
dose of aminopterin is administered in the morning and another at
night on a particular day of the week by taking two tablets with
each dose, this cycle is then repeated for at least 52 cycles. In
this case, the number of doses per cycle is 2 where each dose
comprises taking 2 dosage forms, the periodicity is 7 days, and the
greatest duration between the last dose of one cycle and the first
dose of the next cycle is 6 days (i.e. the days between the day of
the week the doses is given). It will be understood that other
schedules are within the embodiments of the disclosure. For
example, in one embodiment, one dose of aminopterin is administered
on Monday and one on Wednesday for at least 52 cycles. In this
case, the number of doses per cycle is 2, the periodicity is 7
days, and the greatest duration between the last dose of one cycle
and the first dose of the next cycle is 4 days (i.e. Thursday
through Sunday). In the most preferred embodiment, the periodicity
is weekly (i.e. 7 days).
[0077] In other embodiments, methods are provided for treating an
inflammatory disorder in a patient comprising administering a
single cycle of aminopterin in a therapeutically effective amount.
Most preferably, SLE, GVHD and polymyositis in a human patient is
effectively treated with a single cycle of aminopterin, wherein the
number of doses in the cycle is 1.
[0078] In other embodiments, methods are provided for treating an
inflammatory disorder in a patient comprising administering a
single cycle of aminopterin in a therapeutically effective amount.
Most preferably, bronchopulmonary dysplasia in a human and acute
pneumonic pasteurellosis in a cow is effectively treated with a
single cycle of aminopterin, wherein the number of doses in the
cycle is 1.
[0079] In other embodiments, methods are provided for treating an
inflammatory disorder in a patient more rapidly than it can be
treated with methotrexate by administering to the patient one or
more doses of aminopterin in a therapeutically effective amount. In
particular, a therapeutically effective amount of aminopterin will
eliminate, reduce, or prevent the progression of, one or more
disease manifestations in the patient more rapidly than a
therapeutically effective amount of methotrexate. While the rate to
reach a particular efficacy can be achieved more rapidly with
aminopterin than with methotrexate in some embodiments, it will be
understood that the final maximum level of efficacy achieved may be
the same or different. In preferred embodiments, the final level of
efficacy achieved will be greater for aminopterin than for
methotrexate, in addition to this level being reached more rapidly
by aminopterin. The rate can be measured using any quantitative
endpoint of disease activity (see `disease manifestation` and
`therapeutically effective amount` in the Glossary). In preferred
embodiments, the rate is quantified as a function of time by
measuring any of the following scoring system: ACR20, ACR50, ACR70,
ACR-N, JRA30% DOI, JRA50% DOI, JRA70% DOI, PASI, pulmonary function
testing, oxygen saturation, lesional scores, and pruritis scores.
In other preferred embodiments, the rate is quantified by
documenting an increase in the area under the `efficacy-time
curve`, wherein the curve is established by plotting the output
parameter of the scoring system as a function of time {see for
example, Bathon et al., N. Engl. J. Med. 343:1586, 2000 for an
example of this approach). The area under the efficacy-time curve
is not to be confused with the `AUC` defined in the Glossary for
the area under the plasma concentration-time curve for a single
dose of a drug.
[0080] In still further embodiments the weekly dosage comprises a
cumulative dose of aminopterin ranging from 0.001-0.07 mg/kg,
0.005-0.03 mg/kg, and most preferably 0.010-0.03 mg/kg. For
example, a 0.25 kg patient would be administered between 0.00025 mg
and 0.0175 mg aminopterin, between 0.00125 mg and 0.0075 mg
aminopterin, and between 0.0025 mg and 0.0075 mg aminopterin;
whereas a 1.0 kg patient would be administered between 0.001 mg and
0.07 mg aminopterin, between 0.005 mg and 0.03 mg aminopterin, and
between 0.01 mg and 0.03 mg aminopterin; whereas a 100 kg patient
would be administered between 0.1 mg and 7.0 mg aminopterin,
between 0.5 mg and 3.0 mg aminopterin, and between 1.0 mg and 3.0
mg aminopterin. For a typical 60 kg adult, the weekly dosage thus
comprises a cumulative dose of aminopterin ranging from 0.06-4 mg,
0.3-1.8 mg, and most preferably 0.6-1.8 mg. In other preferred
embodiments, the dosage form contains 0.05 mg, 0.75 mg, 0.1 mg,
0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg,
0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg,
095 mg or 1.0 mg aminopterin or therapeutically acceptable salt
thereof. In still other embodiments the dosage form is a tablet. In
another embodiment, the weekly dosage comprises taking one to three
dosage forms by mouth of any dose combination thereof. The above
cumulative weekly dose of aminopterin can be given either in a
single administration at a particular time, or as a plurality of
administrations during a single day, or over multiple days. Using
the methods of the instant disclosure, it has been discovered that
aminopterin can be given to a patient with an inflammatory disorder
without toxicity manifestations, and in the most preferred
embodiments without interruption. In another preferred embodiment,
the amount of aminopterin, or pharmaceutically acceptable salt
thereof, within a dosage form is between 0.00025 mg and 7.0 mg
aminopterin, more preferably between 0.00025 mg and 0.5 mg
aminopterin, more preferably between 0.00025 mg and 0.4 mg
aminopterin, more preferably between 0.00025 mg and 0.3 mg
aminopterin, more preferably between 0.00025 mg and 0.2 mg
aminopterin, and more preferably between 0.00025 mg and 0.1 mg
aminopterin. In preferred embodiments the dosage form is a
tablet.
[0081] When aminopterin is administered at the above disclosed
cycles and dosages, DHFR is inhibited in a patient and an amount of
adenosine is released to inhibit an inflammatory disorder.
[0082] Embodiments of the present disclosure further provide
methods for treating an inflammatory disorder in a patient using
combination therapy, which comprises administering to said patient
a therapeutically effective amount of aminopterin, or a
pharmaceutically acceptable salt thereof, according to a
therapeutic protocol involving at least one other therapeutic. The
at least one other therapeutic may be administered prior to,
contemporaneous with, or after administering the aminopterin. The
at least one other therapeutic also includes a single dosage form
containing aminopterin and at least one other therapeutic, a
multiple dosage form, wherein the aminopterin and the at least one
other therapeutic are administered separately, but concurrently, or
a multiple dosage form wherein the two components are administered
separately, but sequentially. The at least one other therapeutic
can be, for example, folic acid, leucovorin, dextromethorphan,
memantine, prednisone, a cox-2 inhibitor, a non-steroidal
anti-inflammatory drug, vincristine, dexamethasone, asparaginase,
daunorubicin, mercaptopurine, etoposide, cytarabine, doxorubicin,
cisplatin, ifosfamide, paclitaxel, 5-fluoruracil,
diahydrogalacitol, tamoxifen, piperazinedione, mitoxantrone,
diaziquone, aminothiadiazole, methotrexate, tenoposide,
vincristine, echinomycin, 6-mercatopurine, dexamethasone,
cyclophosphamide, soluble TNF receptors, antibodies, and humanized
antibodies.
[0083] In preferred embodiments, a dose of aminopterin from
0.001-0.066 mg/kg, 0.005-0.03 mg/kg, and most preferably 0.010-0.03
mg/kg, is suitable for use in a therapeutic protocol employed
during a combination therapy. In some embodiments, aminopterin can
be directly substituted for methotrexate in a therapeutic protocol
employing methotrexate by administering aminopterin at about 4-8%
of the dose of methotrexate in the protocol. This substitution
yields at least the same level of efficacy and therapeutic index as
methotrexate, but with far fewer tablets taken by the patient.
[0084] In one embodiment, aminopterin is substituted for
methotrexate in the treatment of adult rheumatoid arthritis in a
therapeutic protocol employing another non-steroidal
anti-inflammatory drug by administering a single weekly oral dose
of 0.5 to 2 mg aminopterin instead of a single weekly dose of 7-25
mg methotrexate. In another embodiment, aminopterin is substituted
for methotrexate in the treatment of juvenile rheumatoid arthritis
in a therapeutic protocol employing another non-steroidal
anti-inflammatory drug by administering a single weekly oral dose
of 0.5 to 2 mg aminopterin instead of a single weekly dose of 7-25
mg methotrexate. In still another embodiment, psoriasis in an adult
is treated in a therapeutic protocol by administering a single
weekly oral dose of 1 to 4 mg aminopterin instead of a single
weekly dose of 15-25 mg methotrexate, or as two weekly oral doses
of 1 to 2 mg aminopterin instead of two weekly doses of 7-13 mg
methotrexate.
[0085] In another embodiment, it has been found that methotrexate,
or a pharmaceutically acceptable salt thereof, is effective in
treating canine atopic dermatitis and bronchopulmonary dysplasia.
In a preferred embodiment, these two disorders are effectively
treated with weekly doses of 7-25 mg/m.sup.2 or 0.01-2 mg/kg given
via oral or parenteral routes. In this embodiment of treating
canine atopic dermatitis and bronchopulmonary dysplasia,
methotrexate can substitute for aminopterin, as disclosed herein,
using 10 to 20-fold larger doses (by weight) than aminopterin. The
efficacy of methotrexate in bronchopulmonary dysplasia and canine
atopic dermatitis was observed to have a slower onset than
aminopterin.
[0086] An embodiment of the present disclosure further provides
methods of treating an inflammatory disorder in a human, which
comprises administering to said human an active pharmaceutical
ingredient substantially free of impurities, wherein the antifolate
in the active pharmaceutical ingredient is a therapeutically
effective amount of aminopterin, or a pharmaceutically acceptable
salt thereof. In some embodiments, the impurities may include, for
example, folic acid, pterins, and conjugates of p-aminobenzoic
acid. In preferred embodiments, the aminopterin purity in the
active pharmaceutical ingredient is equal to or greater than 95
area %, equal to or greater than 95 weight %, or equal to or
greater than 95 mole % (see "aminopterin purity" in glossary). In
other embodiments, the active pharmaceutical ingredient has less
than 5 area %, less than 5 weight %, or less than 5 mole %
impurities, and more preferably less than 3 area %, less than 3
weight %, or less than 3 mole % impurities. In preferred
embodiments, administering an active pharmaceutical ingredient
containing an aminopterin dose from 0.03 mg/m.sup.2 to about 2
mg/m.sup.2, or 0.001-0.066 mg/kg will provide a therapeutically
effective amount of aminopterin and will be substantially free of
impurities.
[0087] The chemical synthesis of an active pharmaceutical
ingredient substantially free of impurities and containing
aminopterin can be performed by several different sequences of
organic synthetic steps. It is understood that one of ordinary
skill in the art would be able to make an active pharmaceutical
ingredient substantially free of impurities and containing
aminopterin in light of the following disclosure, including the
Examples, and information known to those of ordinary skill in the
chemical synthesis field. For example, beginning with readily
available starting materials, an active pharmaceutical ingredient
substantially free of impurities and containing aminopterin may be
synthesized according to Scheme 1.
##STR00001##
[0088] As illustrated above, the commercially available
2,4,5,6-tetraminopyrimidine, compound 1, may be condensed with
.beta.-bromopyruvaldoxime to provide
2,4-diamino-6-(bromomethyl)pteridine, compound 2 [see
Taghavi-Moghadam and Pfleiderer, Tet. Lett. 38:6835, 1997 and
Taylor and Portnoy, J. Org. Chem. 38:806, 1973]. Alternatively,
compound 1 may be reacted with 1,3-dihydroxyacetone to provide
2,4-diamino-6-pteridinemethanol, compound 5 [see Baugh and Shaw, J.
Org. Chem. 29:3610, 1964]. Compound 5 is purified and reacted with
HBr and dibromotriphenylphosphorane (Ph.sub.3PBr.sub.2) in
dimethylacetamide to afford compound 2 [see Piper and Montgomery,
J. Org. Chem. 42:208, 1977; Piper and Montgomery, J. Heterocycl.
Chem. 11:279, 1974; Piper and Montgomery, U.S. Pat. No. 4,077,957;
and Piper and Montgomery, U.S. Pat. No. 4,079,056]. In still other
embodiments, compound 2 can be arrived at via the reaction of
compound 1 with 1,1-dichloroacetone to form
2,4-diamino-6-(methyl)pteridine, which is then reacted with bromide
[see Catalucci, U.S. Pat. No. 4,224,446].
[0089] Regardless of the route to its synthesis, compound 2 is
condensed with commercially available N-(p-aminobenzoyl)-L-glutamic
acid, compound 3, in dimethylacetamide to afford the active
pharmaceutical ingredient substantially free of impurities and
containing aminopterin, compound 4, as the antifolate [see Piper
and Montgomery, J. Org. Chem. 42:208, 1977; Piper and Montgomery,
U.S. Pat. No. 4,077,957; Piper and Montgomery, U.S. Pat. No.
4,079,056; and Catalucci, U.S. Pat. No. 4,224,446].
[0090] In other embodiments, an active pharmaceutical ingredient
substantially free of impurities and containing aminopterin as the
antifolate may be obtained by purification of aminopterin
preparations contaminated with impurities by, for example,
ion-exchange chromatography or by HPLC [see Heinrich et al., J. Am.
Chem. Soc. 75:5425, 1953 and Tong et al., Lancet 2:719, 1975]. In a
preferred embodiment, an active pharmaceutical ingredient
substantially free of impurities and containing aminopterin as the
antifolate may be obtained by the direct transformation (i.e.
amination) of folic acid to aminopterin [see Zebala, U.S. Pat. No.
7,235,660].
[0091] An embodiment of the present disclosure further provides
pharmaceutical compositions substantially free of impurities and
comprising an active pharmaceutical ingredient, wherein the
antifolate in the active pharmaceutical ingredient is a
therapeutically effective amount of aminopterin, or a
pharmaceutically acceptable salt thereof. In some embodiments, the
impurities may include, for example, folic acid, pterins, and
conjugates of p-aminobenzoic acid. In preferred embodiments, the
pharmaceutical composition has less than 5 area %, less than 5
weight %, or less than 5 mole % impurities, and more preferably
less than 3 area %, less than 3 weight %, or less than 3 mole %
impurities. In other embodiments, the aminopterin purity in the
pharmaceutical composition is equal to or greater than 95 area %,
equal to or greater than 95 weight %, or equal to or greater than
95 mole %. In preferred embodiments, a pharmaceutical composition
containing from 0.2 mg to about 2.0 mg aminopterin will provide a
therapeutically effective amount of aminopterin and will be
substantially free of impurities. In particularly preferred
embodiments, a pharmaceutical composition contains 0.5 mg
aminopterin will provide a therapeutically effective amount of
aminopterin and will be substantially free of impurities.
[0092] The aminopterin purity of the active pharmaceutical
ingredient is used to establish how much active pharmaceutical
ingredient is required in the pharmaceutical composition to obtain
a desired final dose of aminopterin, or a pharmaceutically
acceptable salt thereof, in the pharmaceutical composition. For
example, if a pharmaceutical composition is to contain about 1 mg
of aminopterin and the aminopterin purity in the active
pharmaceutical ingredient is 95 weight %, then about 1.0526 mg of
the active pharmaceutical ingredient will be required in the
pharmaceutical composition to provide about 1 mg of
aminopterin.
[0093] In addition to the active pharmaceutical ingredient,
pharmaceutical compositions substantially free of impurities
contain one or more pharmaceutically acceptable carriers (see
glossary definition of "pharmaceutical composition").
Pharmaceutical compositions substantially free of impurities are
most readily prepared by combining an active pharmaceutical
ingredient substantially free of impurities, wherein aminopterin is
the antifolate in the active pharmaceutical ingredient, in intimate
admixture with one or more pharmaceutical carriers according to
conventional pharmaceutical compounding techniques.
[0094] The carrier may take a wide variety of forms depending on
the form of the pharmaceutical composition (i.e. "preparation" or
"form") desired for administration, e.g., oral, parenteral
(including intravenous injections or infusions), or intrathecal. In
preparing the pharmaceutical composition in an oral dosage form any
of the usual pharmaceutical carriers may be employed. Usual
pharmaceutical carriers include, for example, water, glycols, oils,
alcohols, flavoring agents, preservatives, coloring agents, and the
like in the case of oral liquid preparations (such as for example,
suspensions, solutions, and elixirs); aerosols; or carriers such as
starches, sugars (e.g. lactose), microcrystalline cellulose,
diluents, granulating agents, lubricants, binders, disintegrating
agents and the like, in the case of oral solid preparations (such
as for example, powders, capsules, and tablets) with the oral solid
preparations generally being preferred over the oral liquid
preparations. For pediatric patients, however, it will be
appreciated to those skilled in the art that pleasant tasting oral
liquid preparations are preferred.
[0095] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage form in
adults, in which case solid pharmaceutical carriers are employed.
If desired, tablets may be coated by standard aqueous or nonaqueous
techniques. The parenteral dosage form can consist of a sterile
solution of the active ingredient, either in its free or salt form,
in physiological buffer or sterile water. In addition, parenteral
solutions can contain preservatives such as benzalkonium chloride,
methyl- or propyl-paraben and chlorobutanol. Suitable
pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences, a standard reference text in this field. In embodiments
employing parenteral, oral liquid, or other aqueous compositions,
care must be taken since electrophilic substitution by water
converts aminopterin to folic acid, and such preparations have been
noted to accumulate folic acid to undesirable levels over the
course of six months of storage. Accordingly, such aqueous
compositions are best stored desiccated and hydrated within several
hours to several days prior to patient administration.
[0096] In addition to the common dosage forms set out above, the
pharmaceutical compositions of the present disclosure may also be
administered by controlled release means and/or delivery devices
such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899;
3,536,809; 3,598,123; 3,630,200; 4,008,719; 4,687,660 and
4,769,207, the disclosures of which are hereby incorporated by
reference.
[0097] Optionally, the pharmaceutical composition contains other
therapeutic ingredients. Such therapeutic ingredients may be added
to ameliorate certain side-effects, particularly those of
aminopterin, or add to patient convenience by reducing the number
of dosage forms that must be taken. Suitable therapeutic
ingredients for combining with the pharmaceutical composition may
include, for example, folic acid, leucovorin, dextromethorphan,
memantine, prednisone, a cox-2 inhibitor, a non-steroidal
anti-inflammatory drug, vincristine, dexamethasone, asparaginase,
daunorubicin, mercaptopurine, etoposide, cytarabine, doxorubicin,
cisplatin, ifosfamide, paclitaxel, 5-fluoruracil,
diahydrogalacitol, tamoxifen, piperazinedione, mitoxantrone,
diaziquone, aminothiadiazole, methotrexate, tenoposide,
vincristine, echinomycin, 6-mercatopurine, dexamethasone,
cyclophosphamide, soluble TNF receptors, antibodies, and humanized
antibodies.
[0098] As used in the methods and compositions of the present
disclosure, the term "pharmaceutically acceptable salts" refers to
salts prepared from pharmaceutically acceptable non-toxic acids or
bases including inorganic acids and bases and organic acids and
bases. The sodium or di-sodium salts of aminopterin are
pharmaceutically acceptable salts of aminopterin. As used herein,
the term "aminopterin", unless specified otherwise, refers to the
acid form of aminopterin or a pharmaceutically acceptable salt of
aminopterin.
[0099] Since aminopterin is both basic and acidic, salts may be
prepared from pharmaceutically acceptable non-toxic acids or bases
including inorganic and organic acids or inorganic and organic
bases. Such salts may contain any of the following anions: acetate,
benzensulfonate, benzoate, camphorsulfonate, citrate, fumarate,
gluconate, hydrobromide, hydrochloride, lactate, maleate,
mandelate, mucate, nitrate, pamoate, phosphate, succinate, sulfate,
tartrate and the like. Such salts may also contain the following
cations: aluminum, calcium, lithium, magnesium, potassium, sodium,
zinc, benzathine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine, and procaine.
[0100] Unless the route of administration is specified in a method
disclosed herein, any suitable route of administration may be
employed for providing a human with a therapeutically effective
amount of aminopterin, or a pharmaceutically acceptable salt
thereof. For example, oral, intrathecal, rectal, parenteral,
transdermal, subcutaneous, intramuscular, and the like may be
employed as appropriate. Dosage forms include tablets, coated
tablets, troches, dispersions, suspensions, solutions, caplets,
capsules, patches, and, the like. Pharmaceutical compositions
include those suitable for oral, rectal, intrathecal, and
parenteral (including subcutaneous, intramuscular, and intravenous)
administration, although the most suitable route in any given case
will depend on the nature and severity of the inflammatory disorder
being treated. The most preferred route of the present disclosure
is the oral route. The pharmaceutical compositions may be
conveniently presented in unit dosage form, and prepared by any of
the methods well known in the art of pharmacy.
[0101] Pharmaceutical compositions of the present disclosure
suitable for oral administration may be presented as discrete units
such as capsules, cachets, or tablets, or aerosols sprays, each
containing a predetermined amount of the pharmaceutically active
ingredient, as a powder or granules, or as a solution or a
suspension in an aqueous liquid, a non-aqueous liquid, an
oil-in-water emulsion, or a water-in oil liquid emulsion. Such
pharmaceutical compositions may be prepared by any of the methods
of pharmacy, but all methods include the step of bringing into
association the active pharmaceutical ingredient with at least one
pharmaceutical carrier. In general, the pharmaceutical compositions
are prepared by uniformly and intimately admixing the active
pharmaceutical ingredient with liquid pharmaceutical carriers or
finely divided solid pharmaceutical carriers or both, and then, if
necessary, shaping the product into the desired presentation.
[0102] For example, a tablet may be prepared by compression or
molding, optionally, with one or more accessory ingredients.
Compressed tablets may be prepared by compressing in a suitable
machine the active pharmaceutical ingredient in a free-flowing form
such as powder or granules, optionally mixed with a binder,
lubricant, inert diluent, surface active or dispersing agent.
Molded tablets may be made by molding in a suitable machine, a
mixture of the powdered compound moistened with an inert liquid
diluent. Desirably, each tablet contains from about 0.5 mg to about
2 mg of aminopterin or a therapeutically acceptable salt thereof,
and each cachet or capsule contains from about 0.5 mg to about 2 mg
of aminopterin or a therapeutically acceptable salt thereof. Most
preferably, the tablet, cachet or capsule contains either one of
two dosages, about 0.5 mg or about 1 mg of aminopterin or a
therapeutically acceptable salt thereof.
[0103] In still other embodiments, pharmaceutical compositions
contain a therapeutically effective amount of aminopterin, or a
therapeutically acceptable salt thereof, with a standard deviation
of less than 5%, and more preferably a standard deviation of less
than 3%. The standard deviation is a measure of dose uniformity
(i.e. consistency) in the pharmaceutical compositions with a
smaller standard deviation being an indication of greater dose
uniformity. The pharmaceutical compositions may be different
portions of a single pharmaceutical composition from a single
formulation batch, or may be from multiple pharmaceutical
compositions of the same dosage form that are each the result of
different formulation batches. Thus, the standard deviation can be
a measure of dose uniformity both within the same batch and between
batches.
[0104] The standard deviation of an antifolate in pharmaceutical
compositions is determined by measuring the amount of antifolate in
a known amount of each pharmaceutical composition using methods
familiar to those in the art. For example, the amount of
aminopterin and methotrexate in a pharmaceutical composition can be
quantitated using scanning uv spectrophotometry, HPLC, or a
radioligand DHFR binding assay [see Kamen et al., Anal. Biochem.
70:54, 1976 and Ratliff et al., J. Clin. One. 16:1458, 1998]. As
defined herein, calculation of a standard deviation requires
measuring aminopterin from at least two randomly selected parts of
a single batch of a pharmaceutical composition, or from each of at
least two different batches of a pharmaceutical composition.
[0105] The disclosure is further defined by reference to the
following examples describing in detail, the methods, and use and
preparation of the pharmaceutical compositions. It will be apparent
to those skilled in the art, that many modifications, both to
materials, and methods, may be practiced without departing from the
purpose and interest of the invention.
EXAMPLES
[0106] In Examples 1-15 that follow, "aminopterin" is the
antifolate in an active pharmaceutical ingredient employed in a
pharmaceutical composition substantially free of impurities.
Example 1
Efficacy in the Murine Air-Pouch Model of Inflammation
[0107] To identify the optimum dose of aminopterin for treating
rheumatoid arthritis and to quantitate its potency relative to
methotrexate, we tested aminopterin and methotrexate in a murine
air-pouch model of inflammation whose dose-response relationship to
methotrexate has been shown by Cronstein et al. to correlate
exceedingly well with the dose-response relationship of
methotrexate in humans [Cronstein, B. N., D. Naime, and E. Ostad,
The anti-inflammatory mechanism of methotrexate. Increased
adenosine release at inflamed sites diminishes leukocyte
accumulation in an in vivo model of inflammation. J. Clin. Invest.,
1993. 92(6):2675-82].
[0108] Accordingly, groups of 5 animals each (10-15 week-old male
mice) were given a fixed dose (mg/kg) per group of aminopterin
(0.0005, 0.001, 0.002, 0.005, 0.008, 0.010, 0.050) or methotrexate
(0.01, 0.02, 0.05, 0.08, 0.10, 0.50, 1.00) by weekly ip injection
on days 1, 7, 14 and 21. One readily recognizes that the above
fixed doses for a 0.25 kg animal, for example, would represent
0.000125 mg, 0.00025 mg, 0.0005 mg, 0.00125 mg, 0.002 mg, 0.0025
mg, and 0.0125 mg aminopterin, respectfully, and 0.0025 mg, 0.005
mg, 0.0125 mg, 0.02 mg, 0.025 mg, 0.125 mg, and 0.25 mg
methotrexate, respectfully. A separate group of 5 animals was given
vehicle by ip injection on the same days (0.9% saline, control,
n=5). On day 16, air pouches were induced on the animals by
injecting 3 ml of air subcutaneously on the back. On day 22 (i.e.
one day after the last dose of drug or vehicle), inflammation was
induced by injection of 1 ml of a suspension of carrageenan (2%
weight/volume in calcium- and magnesium-free PBS). The mice were
put back in their cages and allowed to run free for 4 hours. The
mice exhibited no signs of toxicity at the end of the dosing
period. After 4 hours, the animals were sacrificed, the pouches
flushed with 2 ml of PBS and the exudates harvested. Aliquots were
diluted 1:1 with methylene blue (0.01% w/v in PBS), and the cells
were counted.
[0109] These studies demonstrated that aminopterin and methotrexate
diminished in a dose-dependent fashion the number of neutrophils
that accumulated in the carrageenan-treated air pouches with
EC.sub.50s of 0.0009 mg/kg/wk and 0.04 mg/kg/wk, respectively (FIG.
1). Thus, aminopterin was found to be 43-fold more potent than
methotrexate (P<0.001). That efficacy of aminopterin can be
obtained at such low weekly doses has not been reported in the
prior art. Weekly doses in the above range are known to be
many-fold below the maximally tolerated weekly dose of aminopterin,
where mucositis just starts to appear [Ratliff, et al., Phase I and
pharmacokinetic trial of aminopterin in patients with refractory
malignancies. J. Clin. Oncol., 1998. 16(4):1458-64].
Example 2
Duration of Efficacy in the Murine Air-Pouch Model of
Inflammation
[0110] Although the 43-fold difference in EC.sub.50s between
aminopterin and methotrexate in their anti-inflammatory activity
was felt to be most likely due to the preferential uptake
efficiency of aminopterin, we also considered the possibility that
a difference in the rate of drug excretion from cells could be
contributing to this difference by essentially increasing the
magnitude of the "trough" of one drug over another prior to each
weekly dose. We therefore examined the rate at which
anti-inflammatory activity was lost in the air-pouch model after
the cessation of 4 weeks of dosing at the maximally efficacious
dose (FIG. 2).
[0111] Accordingly, animals (10-15 week-old male mice) were given
weekly ip injections of vehicle (0.9% saline, control, n=60),
aminopterin (n=60, 0.05 mg/kg) or methotrexate (n=60, 1.0 mg/kg)
over the course of a month on days 1, 7, 14 and 21. On day 16, air
pouches were induced on the animals by injecting 3 ml of air
subcutaneously on the back. On day 25 the air pouches were
re-inflated with 1.5 ml of air. Pouches were re-inflated as needed
to maintain them. On days 22 (+1), 24 (+3), 26 (+5), 29 (+8), 33
(+12), and 43 (+22) inflammation was induced in a group of n=5
animals from the vehicle, aminopterin and methotrexate arms of the
study by injection of 1 ml of a suspension of carrageenan as a 2%
weight/volume solution in calcium-free and magnesium-free PBS
(numbers in parentheses indicate days after the fourth and last ip
injection of either vehicle or drug). After 4 hours, the mice were
sacrificed, the pouches flushed with 2 ml of PBS and the exudates
harvested. Aliquots were diluted 1:1 with methylene blue (0.01% w/v
in PBS), and the cells were counted.
[0112] The data showed no statistically significant difference
between the rate of recovery of inflammation at 8, 12 or 22 days
after the last antifolate dose, and revealed that the duration of
action of both aminopterin and methotrexate was quite long,
consistent with the long intracellular half-lives of polyglutamated
antifolates. This example shows that the greatest duration between
the last dose of one cycle and the first dose of the next cycle can
be up to 21 days for aminopterin.
Example 3
Efficacy in the Rat Oxygen-Toxicity Model of Inflammation
[0113] To identify the optimum dose of aminopterin for treating
bronchopulmonary dysplasia in humans and to quantitate its potency
relative to methotrexate, we tested aminopterin and methotrexate in
a murine oxygen-toxicity model of inflammation [Deng, H.; Mason, S,
N.; Richard L. Auten, J. Lung inflammation in hyperoxia can be
prevented by antichemokine treatment in newborn rats. American
Journal of Respiratory Critical Care Medicine 2000, 162,
2316-2323].
[0114] Briefly, timed pregnant rats were placed in air (two
litters) or 95% oxygen (one litter) the day of delivery after
recombining litters among four just delivered dams. Litter sizes
were the same. Aminopterin (0.2 mg/kg), methotrexate (0.35 mg/kg)
or vehicle were then introduced by the ip route on day 2 after
birth. Dams were rotated daily between air and oxygen cages to
avoid maternal oxygen toxicity. On day 8, the pups were weighed and
then euthanized, tracheas are cannulated and the pulmonary artery
is perfused with saline. Bronchoalveolar lavage fluid (BALF) was
then obtained for cell count and chemokine analysis (FIG. 3). The
lungs were removed and snap frozen for myeloperoxidase activity
assay (neutrophil accumulation within lung parenchyma) and
8-isoprostane, a marker of lipid oxidation. The pulmonary artery
was perfused and the lungs were inflation-fixed with buffered
formalin at 25 cm H.sub.2O.
[0115] As expected, the data showed that hyperoxia resulted in a
marked influx of inflammatory cells, predominantly comprised of
neutrophils (PMNs). The data showed a statistically significant
reduction in the number of neutrophils after only a single 0.2
mg/kg injection of aminopterin 6 days prior to sacrificing the
animals. In contrast, methotrexate at 17-times the dose of
aminopterin (0.35 mg/kg), failed to reduce the influx of
neutrophils. Efficacy of aminopterin can be obtained using a single
low dose. This example shows that bronchopulmonary dysplasia can be
treated with an antifolate, and that achieving efficacy occurs at a
much more rapid rate for aminopterin than for methotrexate. This
dose is many-fold lower than the maximally tolerated dose of
aminopterin, which produces mucositis [Ratliff, et al., Phase I and
pharmacokinetic trial of aminopterin in patients with refractory
malignancies. J. Clin. Oncol., 1998. 16(4):1458-64].
Example 4
2,4-Diamino-6-pteridinemethanol (5) hydrobromide salt
[0116] 2,4,5,6-Tetraminopyrimidine.H.sub.2SO.sub.4.H.sub.2O (75.0
g, 0.293 mole) was added to a stirred solution of
BaCl.sub.2.2H.sub.2O (71.5 g, 0.293 mole) in H.sub.2O (1.45 l.) at
85-90.degree. C. The mixture was stirred rapidly at about
90.degree. C. for 15 min, cooled to 40.degree. C., and filtered
from BaSO.sub.4, which was washed thoroughly on a funnel with
H.sub.2O. The clear, yellow filtrate was then diluted further with
H.sub.2O to give a volume of 4.35 l. This solution of the
tetraminopyrimidine.2HCl was then added to a solution of NaOAc
(4.35 l. of 4 N) in which dihydroxyacetone (79.3 g, 0.88 mole) and
cysteine.HCl.H.sub.2O (51.5 g, 0.293 mole) had just been dissolved.
The resulting solution was stirred mechanically at room temperature
while a slow stream of air was continuously passed through it for
26 hr. (Yellow-orange solid began separating after 2 hr.) The
mixture was then kept in a refrigerator for 16 hr before the solid
was collected, washed successively with cold H.sub.2O, EtOH, and
Et.sub.2O before it was dried to constant weight in vacuo over
P.sub.2O.sub.5 at 25.degree. C. [The crude product mixture (47 g)
was weighed in order to obtain an estimate of the volume of 48% HBr
required to form hydrobromide salts.] A mechanically stirred
mixture of the dried solid and EtOH (6.05 l.) was heated to
70.degree. C., and a solution of 48% HBr (28 ml) in EtOH (490 ml)
was added in a thin stream while the mixture was maintained at
70-75.degree. C. The mixture was then refluxed for about 5 min with
rapid stirring while nearly all of the solid was dissolved. The hot
solution was treated with Norit and filtered through a Celite mat.
The clear yellow filtrate was kept in a refrigerator overnight
while a first crop of orange-colored solid separated. The collected
solid was washed with EtOH, then dried in vacuo (56.degree. C. over
P.sub.2O.sub.5) to give 17.2 g of product. The filtrate was
concentrated by evaporation (rotary evaporator, H.sub.2O aspirator,
bath to 35.degree. C.) to about 2 l. and then refrigerated to give
a second crop, which was dried as before, of 10.2 g; total yield
27.4 g (34%). The .sup.1H NMR spectrum of this material in
CF.sub.3CO.sub.2D showed it to contain a barely detectable amount
of methyl substituted 2,4-diaminopteridine.HBr as evidenced by very
weak signals at .delta.2.83 (CH.sub.3) and .delta.8.85 (pteridine
ring H). Strong signals produced by the desired product occur at
.delta.5.28 (6-CH.sub.2O) and .delta.9.08 (C.sub.7--H). The
proportion of desired product to the methyl-substituted contaminant
was estimated from the .sup.1H NMR integrals to be 20:1. The
.sup.1H NMR spectrum also revealed retention of a small amount of
EtOH in the product dried as described but not enough to interfere
with the conversion of it to 2.
Example 5
2,4-Diamino-6-(bromomethyl)pteridine (2) hydrobromide salt from
(5)
[0117] Bromine (59.6 g, 0.373 mole) was added dropwise over a
30-min period to a stirred solution of triphenylphosphine (97.7 g,
0.373 mole) in anhydrous 486 ml of dimethylacetamide (DMAC) kept at
about 10.degree. C. (ice bath) and protected from atmospheric
moisture. (Bromine remaining in the funnel was rinsed with 10 ml of
DMAC). A smooth suspension containing finely divided, crystalline
triphenylphosphine dibromide resulted. The
2,4-diamino-6-pteridinemethanol.HBr (2) (25.4 g, 0.093 mole)
described above was added in one portion through a powder funnel
(with the aid of 10 ml DMAC). The ice bath was removed, and the
stirred mixture was allowed to warm to 20-25.degree. C. After about
1 hr, complete solution had occurred. The solution, which gradually
developed a dark-red color, was kept at 20-25.degree. C. for 1 hr
longer and was then chilled (ice bath) before it was treated with
EtOH (72 ml). After overnight refrigeration, the solvents were
removed by evaporation in vacuo. The dark, semisolid residue was
stirred with two 300-ml portions of benzene (to remove
triphenylphosphine oxide), and each portion was removed from the
benzene-insoluble product by decantation. The solid that remained
was dissolved with stirring in glacial AcOH (660 ml) which had been
preheated to 80.degree. C. The mixture was kept in a bath at
80.degree. C. until solution was complete. A tan crystalline solid
separated as the dark solution was allowed to cool. Overnight
refrigeration caused the AcOH to partially freeze. When it had
thawed, the solid was collected, washed with chilled AcOH followed
by Et.sub.2O, and dried in vacuo (over P.sub.2O.sub.5 and NaOH
pellets) at successive temperatures of 25.degree. C., 56.degree.
C., and 110.degree. C. (The higher temperature was necessary for
complete removal of AcOH). The yield was 15.3 g (49%). (Some runs
afforded 60% yield). This sample was further purified by
reprecipitation from MeOH solution (Norit) by addition of Et.sub.2O
followed by drying in vacuo (25.degree. C., P.sub.2O.sub.5), yield
13.0 g (42%) of pale-yellow solid. Spectral data: .lamda.max, nm
(.epsilon..times.10.sup.-3), 0.1 N HCl, 249 (17.3), 339 (10.5), 353
(sh); pH 7, 258 (21.2), 370 (6.87); 0.1 N NaOH, 258 (21.5), 370
(6.94); .sup.1H NMR(CF.sub.3CO.sub.2D), .delta. 4.70 (s, 2,
CH.sub.2) and .delta. 9.08 (s, 1, C.sub.7--H); estimated proportion
relative to the methyl-substituted contaminant, 25:1. The
preparation of 2 described above is typical of several runs that
gave similar yields of material whose .sup.1H NMR spectra differed
only slightly in the estimated proportion of 2 with respect to the
methyl-substituted contaminant. The proportions usually ranged from
16:1 to 25:1, which corresponds to a percentage of 2 of 94 to
96%.
Example 6
2,4-Diamino-6-(bromomethyl)pteridine (2) hydrobromide salt from
(1)
[0118] A suspension of 5 mmol 2,4,5,6-tetraminopyrimidine dibromide
in 50 ml methanol was treated with a solution of 7.5 mmol
.beta.-bromopyruvaldoxime in 10 ml of methanol at reflux
temperature for 2 h. The 2,4-diamino-6-(bromomethyl)pteridine was
collected after neutralization with concentrated NH.sub.3 at room
temperature, washed with methanol, ether and dried at 100.degree.
C. in an oven. .sup.1H NMR (250 MHz, ppm, DMSO-d.sub.6), .delta.
8.84 (s, 1H, C.sub.7--H). The yield was 88%.
Example 7
Active Pharmaceutical Ingredient Containing Aminopterin (4)
[0119] A mixture of 2 (168 mg, 0.500 mmole) and
N-(4-aminobenzoyl)-L-glutamic acid, compound 3 (400 mg, 1.50
mmoles) in DMAC (2 ml) was stirred at 25.degree. C. under N.sub.2
in a stoppered flask protected from light. Solution occurred after
2 hrs. After 18 hrs, the orange solution was mixed with H.sub.2O
(15 ml) with stirring to give a finely divided, yellow precipitate.
The mixture was centrifuged, and the supernatant removed by
decantation. The yellow solid was stirred with four 15-ml portions
of H.sub.2O, each of which was removed by decantation after
centrifugation. The solid was then suspended in EtOH (15-20 ml),
collected by filtration, washed with Et.sub.2O, and dried in vacuo
(25.degree. C., P.sub.2O.sub.5) to give hydrated 4 in 68% yield
(160 mg). Anal. Calcd for C.sub.19H.sub.20N.sub.8O.sub.5.1.75
H.sub.2O: C, 48.36; H, 5.02; N, 23.74. Found: C, 48.72; H, 4.91; N,
23.36. Spectral data: .lamda.max, nm (.epsilon..times.10.sup.-3),
0.1 N HCl, 244 (18.2), 290 (20.5), 335 (11.0); pH 7, 260 (26.7),
283 (25.5), 370 (8.00); 0.1 N NaOH, 260 (26.9), 283 (25.3), 370
(8.00); .sup.1H NMR (DMSO-d.sub.6), .delta.2.02 (m, 2,
CHCH.sub.2CH.sub.2), 2.32 (m, 2, CH.sub.2CO.sub.2H), 4.36 (m, 1,
NHCHCO.sub.2H), 4.52 (s, 2, CH.sub.2N), 6.85 (m, 4, 2 phenylene
protons plus NH.sub.2), 7.72 (m, 2, phenylene), 7.86 (broad s, 2,
NH.sub.2), 8.13 (d, 1, NHCO), 8.72 (s, 1, C.sub.7--H). Examination
by tlc revealed one uv-absorbing spot and no fluorescence at any
point. The product may be used directly as the active
pharmaceutical ingredient, but is occasionally subjected to one or
more re-crystallizations from water or formamide to improve the
aminopterin purity slightly. The active pharmaceutical ingredient
is stored in the presence of desiccant.
Examples 8-13
Analysis of Active Pharmaceutical Ingredient
[0120] Three different 1 mM solutions of a first batch of the
active pharmaceutical ingredient prepared according to Example 59
were prepared by weighing out 2.3 mg, 4.5 mg and 1.8 mg of the
active pharmaceutical ingredient as if it were 100% aminopterin (FW
440.42 g/mole), and dissolving it into 5.223 ml, 10.218 ml, and
4.087 ml of 0.001 N NaOH, respectively. Twenty .mu.l of each 1 mM
solution was subjected to HPLC analysis by injecting each onto a
C18 column (Waters .mu.Bondapak 125 .ANG., 10 .mu.m, 3.9.times.150
mm) using an isocratic mobile phase consisting of 5 mM PicA, 10 mM
NH.sub.4H.sub.2PO.sub.4 and 20% methanol at a pH of 6.8. The flow
rate of the mobile phase was 1 ml/min, and the analysis was
performed at room temperature. Using a Waters 996 PDA UV
spectrophotometer, absorbance data from 210 nm to 400 nm was
captured. The data was analyzed with the Waters Millennium software
by extracting the chromatogram at 282 nm and calculating the peak
area percentages. The data was also analyzed by extracting the
spectra of individual peaks, allowing the identification of
pABAGlu, folic acid, aminopterin and pterin species by their
characteristic spectra.
[0121] The average of the HPLC analyses of the three separate 1 mM
solutions revealed that the first batch of active pharmaceutical
ingredient consisted of approximately 96.27 area % aminopterin and
3.73 area % impurities, wherein the impurities were made up of 0.23
area % folic acid, 2.18 area % N-(4-aminobenzoyl)-L-glutamic acid
(i.e. pABAGlu), and 0.92 area % of a pterin, probably
2,4-Diamino-6-(bromomethyl)pteridine, and 0.4 area % of at least
two other unidentified impurities (Example 8, Table I). A similar
HPLC analysis revealed that a second batch of active pharmaceutical
ingredient consisted of 97.23 area % aminopterin and 2.775 area
impurities, wherein the impurities were made up of 1.82 area %
folic acid, 0.556 area N-(4-aminobenzoyl)-L-glutamic acid (i.e.
pABAGlu), 0.343 area % pterin, probably
2,4-diamino-6-(bromomethyl)pteridine), and 0.056 area % of an
unidentified impurity (Example 9, Table I). By comparison, the
prior art active pharmaceutical ingredients contained aminopterin
and variable amounts of impurities ranging up to 41% (Examples
10-13, Table I).
TABLE-US-00001 TABLE I AMT total Example AMT purity impurities
Composition of total impurities 8 96.27% 3.730% FA (0.23%); pABAGlu
(2.18%); pterins (0.92%); other (0.4%) 9 97.23% 2.775% FA (1.82%);
pABAGlu (0.556%); pterins (0.343%); other (0.056%) 10*.sup.,a
70-80% 20-30% not specified 11*.sup.,b <80% >20% FA (20%);
others unspecified 12*.sup.,c 80% 20% FA (15%); pterins (5%)
13*.sup.,d 59% 41% not specified Percentages are HPLC peak areas.
Abbreviations: FA, folic acid; AMT, aminopterin; and pABAGlu,
N-(4-aminobenzoyl)-L-glutamic acid. *Not part of the subject
invention. .sup.aSeeger, et al., J. Am. Chem. Soc. 71: 1753, 1949.
.sup.bHeinrich et al., J. Am. Chem. Soc. 75: 5425. .sup.cLoo, J.
Med. Chem. 8: 139, 1965. .sup.dSirotnak and Donsbach, Biochem.
Pharmacol. 24: 156, 1975.
Example 14
Pharmaceutical Compositions
[0122] This example illustrates the preparation of pharmaceutical
compositions substantially free of impurities. As used in this
example, "API" means an active pharmaceutical ingredient
substantially free of impurities, wherein the antifolate in the API
is aminopterin, or a pharmaceutically acceptable salt thereof. The
aminopterin purity of the API is used to established how much API
is required in the pharmaceutical composition to obtained a desired
final amount of aminopterin, or a pharmaceutically acceptable salt
thereof, in the pharmaceutical composition.
[0123] Tablets: Surface deposit aminopterin by combining 16.8 grams
API, 791.0 grams microcrystalline cellulose, 553.7 grams lactose
monohydrate, 1.5 g NaOH and 632 grams of sterile water. Mix and dry
overnight. Add 1311 grams of this surface deposited aminopterin to
171 grams of lactose, 3.9 grams colloidal silicon dioxide, 46.2
grams sodium croscarmellose, and 7.7 grams magnesium stearate to
provide a total weight 1540 grams. Compress into approximately
15,000 tablets using a tableting machine, wherein each tablet
weighs approximately 100 mg and contains about 1 mg
aminopterin.
[0124] Gelatin capsules: Prepare by mixing 0.5 grams of API with
0.5 grams magnesium stearate, and 99 grams of lactose. Dispense 100
mg of this mixture into hard-shell gelatin capsules to provide each
capsule with about 0.5 mg of aminopterin.
[0125] Suspension: An aqueous suspension is prepared for oral
administration so that each 5 ml contains about 2 mg aminopterin by
mixing 40 mg of API with 20 grams of sodium carboxymethyl
cellulose, 0.5 grams of sodium benzoate, 100 grams of sorbitol
solution U.S.P., and 2.5 ml of vanillin.
[0126] Liquid for injection or oral administration: Clean glassware
to be used with 2 M NaOH for 3-5 minutes and the rinse thoroughly
with deionized water. Prepare a first saline buffer by adding
2.7017 grams of dibasic sodium phosphate, USP to 5 liters of 0.9%
sodium chloride for injection, USP. Prepare a second saline buffer
by adding 1.39 grams of monobasic sodium phosphate, USP to 1 liter
of 0.9% sodium chloride for injection, USP. While stirring the
first saline buffer solution, slowly add the second saline buffer
solution until the final pH is 7.9-8.1. Record the final volume.
Add sufficient API to provide 0.4 mg/ml aminopterin (i.e. 2 mg
aminopterin per 5 ml) and filter through a 0.2 micron membrane
filter and package under sterile conditions in 10-ml vials each
containing 5 ml. Use within 2 months.
[0127] Injectable: A parenteral formulation is prepared by mixing
0.200 grams of API with 20.0 grams of propylene glycol, 20.0 grams
of polyethylene glycol 400, and 1.0 gram of polysorbate 80. A
sufficient quantity of 0.9% saline solution is then added with
stirring to provide 100 ml of solution which is filtered through a
0.2 micron membrane filter and packaged under sterile conditions in
2-ml vials each containing 1 ml, or a total of about 2 mg/vial
aminopterin. The solution is diluted 10-fold with 0.9% saline
solution prior to administration via an intravenous route.
Example 15
Mean and Standard Deviation of Dose in Pharmaceutical
Compositions
[0128] The average dose and the dose uniformity (i.e. standard
deviation) of aminopterin among 10 randomly selected tablets were
determined for a batch of 15,000 l mg aminopterin tablets prepared
according to Example 14 (Table II). The mean dose and dose
uniformity were obtained by scanning spectrophotometry and a
radioligand binding assay using dihydrofolate reductase (DHFR) as
binder and methotrexate as a standard.
TABLE-US-00002 TABLE II aminopterin dose by radioligand Random
aminopterin dose by binding assay Tablet spectrophotometry (mg)
(mg) 1 0.99 1.03 2 0.98 0.93 3 0.99 1.04 4 0.97 1.04 5 0.94 0.96 6
0.96 1.05 7 0.99 0.99 8 0.94 1.03 9 0.97 1.01 10 1.01 1.03 mean
0.974 1.01 standard 2.271% 3.929% deviation
[0129] For scanning spectrophotometry, each of ten tablets are
dissolved in 11.35 ml of water by rotating in a 15 ml tube for 30
minutes at room temperature to make an approximately 200 .mu.M
solution (i.e. about 1 mg aminopterin with a FW of 440 g/mole in
11.35 ml). The tubes are spun, and a portion of the supernatant is
diluted ten-fold with 0.1 N NaOH to a final concentration of 20
.mu.M. The absorbance of the diluted supernatant is read at 282 nm
and 260 nm on a Perkin-Elmer Lambda 4B scanning spectrophotometer
(1 cm path length) and the concentration of the diluted sample
calculated using the relationships, concentration (in .mu.M) at 282
nm=10.times.(OD.sub.282/0.264) and concentration (in .mu.M) at 260
nm=10.times.(OD.sub.260/0.285). The mg/ml at 282 nm and 260 nm is
then calculated using the relationship, mg/ml=concentration (in
.mu.M).times.0.00044. The total mg of aminopterin in each tablet is
then determined by multiplying the mg/ml determined at 282 nm and
260 nm by 113.5. The mg of aminopterin per tablet is then reported
as the average of the mg of aminopterin in each tablet determined
at 282 nm and 260 nm. For example, using the above procedure, a 1
mg aminopterin tablet yields an OD.sub.282 of 0.5227, and a
calculated concentration (in .mu.M), mg/ml, and total mg of
aminopterin of 19.8 .mu.M, 0.008712 mg/ml, and 0.9888 mg,
respectively. Using the above procedure, the same 1 mg aminopterin
tablet yields an OD.sub.260 of 0.5641, and a calculated
concentration (in .mu.M), mg/ml, and total mg of aminopterin of
19.8 .mu.M, 0.008712 mg/ml, and 0.9888 mg, respectively. The
reported amount of aminopterin in the tablet is the mean of total
aminopterin in the tablet calculated at 282 nm and 260 nm, or
0.9888 mg.
[0130] The radioligand binding assay was performed essentially as
described previously for methotrexate [see Kamen et al., Anal.
Biochem. 70:54, 1976 and Ratliff et al., J. Clin. One. 16:1458,
1998]. Briefly, a standard curve was developed for 0.2 to 1.0
.mu.mol methotrexate binding to partially purified chicken liver
DHFR, wherein binding of a known amount of non-radioactive
methotrexate results in the displacement of an amount of tritiated
methotrexate (i.e. radioactive .sup.3H-methotrexate). Aminopterin
and methotrexate are equivalent in the assay in terms of their
displacement and binding, and the absolute detection limit of the
assay is 0.05 to 0.10 .mu.mol of antifolate. After the standard
curve is established, a portion of the 200 .mu.M supernatant
prepared above for an aminopterin tablet is further diluted 10,000
using two serial 100-fold dilutions with water to provide an
approximately 20 nM solution of aminopterin. The DHFR assay is
performed with 100 .mu.L of the 20 nM solution, as well as several
2-fold serial dilutions, and the result expressed as pmol/ml
aminopterin. The total number of pmol of aminopterin is calculated
using the relationship, pmol/ml.times.10,000.times.11.35. The total
number of mg in the tablet is then calculated using the
relationship, total pmol.times.(10.sup.-6 mmole/.mu.mol).times.0.44
mg/.mu.mmole.
[0131] As determined by scanning spectrophotometry, the average
dose and the dose uniformity (i.e. standard deviation) of
aminopterin among 10 randomly selected tablets from a batch of
15,000 l mg aminopterin tablets was 0.974 mg and 2.271%,
respectively (Table II). As determined by radioligand binding
assay, the average dose and the dose uniformity (i.e. standard
deviation) of aminopterin among the same 10 tablets was 1.01 mg and
3.929%, respectively (Table II).
Example 16
Animal Model of Multiple Sclerosis
[0132] Using the rat EAE animal model of multiple sclerosis, a
series of prophetic studies are performed wherein aminopterin is
given to animals using both active and passive protocols for
inducing EAE (Burton, Agents and Actions (1989) 27 (3/4): 351-355).
In the active protocol, animals are intradermally injected with
myelin basic protein (MBP), Freund's adjuvant and mycobacteria,
whereas in the passive protocol separate donor rats are immunized
actively and then 10-12 days after active immunization, donor
splenocytes are collected and stimulated in vivo with MBP for 3
days, then 1-2.times.10.sup.8 leukocytes injected iv into each
irradiated (600 rads) recipient rat. Aminopterin at doses of
0.0005, 0.001, 0.002, 0.005, 0.008, 0.010, 0.050, 0.1 and 0.5 mg/kg
are administered ip on the day after active or passive
sensitization. One or more of the doses of aminopterin inhibits the
onset of active EAE by greater than 80% and passive EAE by greater
than 80%. The inhibition will persist for up to 14 days after the
single dose, whereas in untreated controls actively stimulated EAE
begins on day 7 and continues for 20 days before beginning to
reverse, and in passively stimulated EAE symptoms begin on day 5
and begin to reverse on day 12. Animals will show a clear
dose-response over the range of aminopterin doses whether
sensitized actively or passively.
Example 17
Human Multiple Sclerosis
[0133] A prophetic randomized, double-blinded, placebo-controlled,
clinical trial of low-dose, weekly, oral aminopterin is performed
in 60 patients with clinically definite chronic progressive
multiple sclerosis (MS) attending a referral-based outpatient MS
clinic. Study patients are 21 to 60 years old with a disease
duration of longer than 1 year. Patients' Expanded Disability
Status Scale scores were 3.0 to 6.5 (ambulatory with moderate
disability). Patients are first stratified by Expanded Disability
Status Scale scores, 3.0 to 5.5 and 6.0 to 6.5, and then were
randomized to receive aminopterin or placebo treatment. Treatment
consists of weekly, oral, low-dose (1.0-2.0 mg) aminopterin or
placebo for 2 years, followed by observation for as long as 1 year.
A composite outcome measurement instrument is used and consists of
(1) Expanded Disability Status Scale (EDSS), (2) ambulation index,
(3) Box and Block Test, and (4) 9-Hole Peg Test. Failure of therapy
is indicated by a designated change that is sustained for more than
2 months in one or more components of this composite measure.
Significantly less progression of impairment is measured by one or
more validated tests of EDSS, ambulation index, Box and Block Test,
9-Hole Peg Test, upper-extremity function or a composite score
thereof in the aminopterin treatment group in the absence of
clinically significant toxicity. The change in T2-weighted total
lesion area (T2W-TLA) is significantly related to sustained change
in performance in one or more of the above validated tests.
Example 18
Canine Atopic Dermatitis Treated with Aminopterin
[0134] The efficacy of aminopterin in the treatment of canine
atopic dermatitis (CAD) was demonstrated in a 4-week open-label
trial followed by an optional one year extension. Five dogs were
treated orally with 0.01 mg/kg/week of aminopterin, except for one
subject whose dose was escalated to 0.015 mg/kg/week at week 4.
Subjects were assessed at baseline and at weeks 2 and 4 of the
trial, and every other week of the 1-year extension phase. Outcome
measures included (1) investigator grading of lesions using the
Canine Atopic Dermatitis Extent and Severity Index (CADESI, a
composite index used in the approval of cyclosporine for CAD), (2)
owner assessment of pruritis using a Pruritis Visual Analog Scale
(PVAS); and overall assessment of efficacy by investigators and
owners categorized as no (<10%), poor (10-25%), fair (25-50%),
good (50-75%), very good (75-90%) and excellent (>90%)
efficacy.
[0135] All 5 dogs completed the original 4 week segment of the
pilot trial, where the median improvements (range) from baseline in
outcome measures were: CADESI score=-59% (-11 to -91%); PVAS
score=-4 (-4 to +2); composite CADESI.times.pruritis score=-60%
(-97 to +24%); overall evaluation of efficacy by clinicians=2
(fair_(1 to 5); and overall evaluation of efficacy by owners=2
(fair) (1 to 4). Importantly, there has been no clinical evidence
of hematologic (CBC) or liver toxicity (LFTs) observed in the dogs
treated for CAD at up to 20 weeks at 0.01 mg/kg/week, a dose
equivalent to 0.7 mg/week in a 70-kg human.
[0136] The reduction in the mean percent CADESI and pruritis PVAS
scores during the pilot and extension phases are graphically
depicted for the subjects remaining enrolled at each week of the
study (FIG. 4). During the extension phase, 3 dogs were withdrawn
at various times for: unrelated medical reasons (1), increase in
pruritis (1) and lack of compliance with drug administration (1).
One dog appeared to be in near or complete remission. One dog had
signs that failed to respond to aminopterin in the first phase,
dosage was increased at week 4 and prednisone was added for 2 weeks
for humane reasons. In total, prednisone was used intermittently in
3 dogs for humane considerations, antihistamines were used in 2
dogs without benefit in 1, and antibiotics were used in 1 dog.
Example 19
Canine Atopic Dermatitis Treated with Methotrexate
[0137] The efficacy of methotrexate in the treatment of CAD was
demonstrated in a 4-week open-label trial. Five dogs were treated
orally with about 0.2 mg/kg/week of methotrexate. Subjects were
assessed at baseline and at weeks 2 and 4. Three dogs did not do
well, one had not improved as of the last assessment and one dog
improved markedly. It was observed that methotrexate took longer to
produce efficacy compared to aminopterin.
Example 20
Adenosine Release by Aminopterin
[0138] Various concentrations of aminopterin were incubated with
ma104 cells for 8 hours, and the release of adenosine measured
(FIG. 5). Aminopterin caused a dose-dependent release of adenosine
with an EC.sub.50 of 0.5 nM. The prior art shows the EC.sub.50 of
methotrexate to be approximately 1 nM (Cronstein '91).
Example 21
DHFR and MTX Binding in Liver
[0139] The animals that were treated in Example 1 were further
analyzed with respect to the free dihydrofolate reductase (DHFR)
available in their livers (i.e., amount of DHFR unbound, or not
inhibited by either AMT or MTX) (FIG. 6). The following procedure
was used. Portions of frozen liver were weighed and homogenized in
three volumes of assay buffer (10 mM KPO.sub.4, 150 mM KCl, 1 mg/ml
charcoal-treated BSA, 0.1 mg/ml NADPH, pH 7.5) on ice. After
centrifugation for 30 min at 16000.times.G at 4.degree. C., tissue
extract was assayed for free DHFR by binding of 3H-MTX. Three
serial 1:2 dilutions were made in assay buffer (total 100
.mu.l/tube), starting with 10 .mu.l extract for control samples
(+190 .mu.l buffer) or 100 .mu.l for antifolate-treated samples.
Approximately 2 pmoles of 3H-MTX (.about.100,000 dpms) in 100 .mu.l
buffer was added to each tube and mixed by addition of another 100
.mu.l buffer and agitation. After 30 mins on ice, unbound 3H-MTX
was removed by addition of 200 .mu.A dextran-coated activated
charcoal (2.5% activated charcoal and 0.5% dextran MW 10,000) and
centrifugation. Radioactivity in the supernatant (=bound 3H-MTX)
was counted after careful removal from the charcoal pellet and
addition of Ultima Gold scintillation fluid. A charcoal "blank"
consisting of buffer, 3H-MTX and charcoal was subtracted from each
value. Results were calculated by averaging the dpms bound/100
.mu.A of the three dilutions, converting to picomoles MTX (specific
activity 49,000 dpms/picomole, determined by competitive
radioligand assay), and multiplying by 30 to give picomoles/gm
tissue.
[0140] The total amount of AMT and MTX in the liver at the
indicated days after the cessation of dosing is plotted together
with the amount of free DHFR as measured using the method described
above (FIG. 6). The drug content data is reproduced from Example 1.
This example shows that at the indicated weekly dosing frequency,
AMT binds (i.e., inhibits) about 95% of the available DHFR in the
liver 1 day (d) after the last of 4 sequential weekly doses; at 8 d
after the last dose about 83% of the available DHFR has been
inhibited; and that at 22 d after this last dose about 70% of the
available DHFR has been inhibited. The amount of free (uninhibited)
DHFR has increased via either loss of bound AMT and/or synthesis of
new DHFR. The amount of AMT remaining in the tissue at 22 d after
the cessation of dosing exhibits a reciprocal decrease to the
increased amount of free DHFR. A substantially similar pattern of
events happens in a separate cohort of mice treated identically
using a 20-fold larger dose of MTX.
[0141] The present disclosure is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a number of aspects of the disclosure and any
embodiments which are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the disclosure
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within appended claims.
[0142] A number of references have been cited, the entire
disclosures of which are incorporated herein by reference.
* * * * *