United States Patent
`
`[191
`
`Kerwar et al.
`
`[11] Patent Number:
`
`5,593,671
`
`[45] Date of Patent:
`
`Jan. 14, 1997
`
`|||1ll|||||Il|Illl||1||||||1||||||l||lllll||||||||||||l|||||1|||||I||1|||||
`US0055936'.71A
`
`[54] METHOD OF ATTENUATING LUNG
`CAPILLARY LEAK IN A MAMIMAL
`
`['15]
`
`Inventors: Suresh S. Ker-war, Ossining, N.Y.;
`Michael M. Wick, Brooidine, Mass.
`
`[731 Assignee: American Cyanamid Company,
`'Wayne, NJ.
`
`[21] Appl. No.: 269,702
`
`[22] Filed:
`
`Jul. 1, 1994
`
`[51]
`
`Int. Cl.“
`
`[52] U.S. Cl.
`
`[53] Field of Search
`
`ASIK 33120; A61K 45105;
`A61K 38:20; A61K 31505
`4241852; 514.02; 514121;
`5141258; 530.-'35]; 5441260
`4241852; 51412,
`514.321, ‘.140, 258; 530.3357; 544.1260
`
`[56]
`
`References Cited
`PUBLICATIONS
`
`l. P. Seigel, R. K. Puri, Interleultin-2 'Ibxicity—Journal of
`Clinical Oncology, vol. 9. No. 4 {Apr}. 1991 pp. 694-904.
`John T. Vetto et a1, Reduction of Toxicity of Interleukin—2
`and Lumpholine-Activated Killer Cells in 1-Imans by the
`Administration of Corticosteroids—Joumal of Clinical
`Oncology, vol. 5 No. 3 (Mar.), 1987 pp. 496-503.
`M. Z. Papa et al. Eifect of Corticosteroid on the Antitumor
`Activity of Lympholine-activated Killer Cells and Interleu-
`ltin—2 in Mice—Cancer Research 46, 5618-5623, Nov.
`1986.
`
`D. R. Seeger et a1. Analogs of Peroylutamic Acid. II].
`4-Amino Derivatives—May 1949 4—Amino Derivatives of
`pteroylglutamic Acid pp. 1753-1758.
`M. V. Freeman, The Fluormetric measurement of the
`Absorption, Distribution and Excretiion of Single Doses of
`4-Amino-10-Methyl Pteroylglutarnic Acid (Amethopterin)
`in man—C1inical Pharmacology and Experimental Thera-
`peutics Section, national Cancer Institute pp. 154-162.
`E. S. Henderson et al. The Metabolic Fate of Tritiated
`metholrexate 11. Absorption and Excretion in Man pp.
`1018-1024.
`P.
`'I'. Condit, Studies of the Folic Acid Vitamins. 11. The
`Acute Toxicity of Amethopterin in Man and Studies on the
`Folic Acid Vitamins, III. The Duration of the Efiects of Folic
`Acid Antagonists in Mar1—American Cancer Society 1960
`vol. 13 pp. 221-235.
`P. T. Condit. et al, Renal Toxicity of MeL1:1otrexate-—01riah-
`moma Medical Research Foundation, Jun. 3, 1968 pp.
`126-131.
`K. B. Bisehoif, et al, Preliminary Model for Methotrexate
`Pharmacokinetics—Researcl1 Articles vol. 59 No. 2 Feb.
`
`1970 pp. 149-154.
`K. B. Bisclzolf, et al. Methotrexate PharInacoltinetics—
`Journal of Pharmaceutical Scientx-.s—-pp. 1128-1133.
`W. E. Evans, Mcti-1otrexate—pp. 518-548.
`J. R. Bertino. Clinical use of mcthotrcxate-Willi Emphasis
`on use of High Doses—Caneer Treatment reports, vol. 65,
`Supplement 1, 1981 pp. 131-135.
`J . Jolivet, at al, Medical progress. The Pharmacology and
`Clinical Use of Methol:rexate—The New England Journal of
`Medicine. Nov. 3. 1983, pp. 1094-1104.
`
`A. Wcinstein. Methotrexate: A Perspective on Its Use for
`Rheumatoid Arthritis, Journal of Rheumatology Supplement
`No. 12, 1985—pp. 1-2.
`.1. R. Ward, Historical Perspective on the Use of Method-
`exate for the Treatment of Rheumatoid Arthritis, Journal of
`Rheumatology Supplement No. 12, 1985 pp. 3-6.
`D. E. Trentham. The Imrnunopathogenesis of Rheumatoid
`Arthritis, Journal of Rheumatology Supplement No. 12,
`1985, pp. 7-10.
`D. E. Furst, Clinical Pharmacology of Very Low Dose
`Methotrexate or Use in Rheumatoid Arthritis, Journal of
`Rheumatology Supplement No. 12, pp. 11-14.
`W. E. Evans, M. K. Christensen, Drug Interactions with
`methotrexate-—-Journal of Rheumatology Supplement No.
`12, 1985. IJP~ 15-20.
`R. F. Willkens, Short Term Eificacy of Methotrexate in the
`Treatment of Rheumatoid Arthritis—Journal of Rheumatoi-
`ogy Supplement No. 12, 1985. pp. 21-24.
`J. M. Kremer, Longterm Methotrexate Therapy in Rheuma-
`toid Arthritis: AReview—-Journal of Rlieumatotogy Supple-
`ment No. 12, 1985 pp. '25-28.
`K. G. Tolman, et al, Methotrexate and the Liver—Jouma] of
`Rhcurnatology Supplement No. 12, pp. 29-34.
`M. E. Weinblatt. Toxicity of Low Dose Methotrexate in
`Rheumatoid ArLhritis—Journal of Rheumatology Supple-
`ment No. 12, 1985, pp. 35-39.
`J. R. Ward. Summary. Journal of Rheumatology Supplement
`No. 12. 1985, pp. 40414.
`M. Rosenstein, Extravasation of lntravascular Fluid Medi-
`ated by the Systemic Administration of recombinant Inter-
`Ieukin
`2—T11e
`Journal of
`Immunology,
`vol.
`137,
`1':'35—l'.-'42. Sep. 1, 1966.
`_
`Package Insert, Methotrexate ‘Tablets. Methotrexatc Sodium
`and Methotrexate LPF* Sodium Parenteral, May 1988.
`D. L. Fraiter, et a], Passive Immunizatin Against Tumor
`necrosis Factor partially Abrogates Interleukin 2 Toxicity-
`'I'he Journal of Experimental Medicine vol. 170 Sep. 1989
`pp. 1015-1020.
`M. Fleisher, Antifolate Analogs: mechanism of Action,
`Analytical methodology, and Clinical Efiicacy—T11erapeu—
`tic Drug Monitoring, 15 pp. 521-526 1993.
`K. S. Amman, et al, Effect of Recombinant Human Granu-
`locyte-Macrophage Colony-Stimulating Factor on Chemo-
`therapy—Induced Mye1osuppression—The New England
`Journal of Medicine, vol. 319, No. 10, Sep. 1988. pp.
`593-598.
`S. J. Brandt, et al, Efiect of Recombinant Human Granule-
`cyte—Macrophage Colony-Stiniuiating Factor on Hemato-
`poietic reconstitution After Higl:t—Dose Chemotherapy and
`Autologous Bone Marror Transplantation, The New
`England of medicine, vol. 318, No. 14, Apr. 1988, pp.
`869-896.
`Package Insert, Proleukin Aidesleultiu for Injection, Nov.
`1992.
`
`Primary Exarm'ner—Ponnatl1apura Achutarnurthy
`Attorney, Agent, or Ft'm1—'I'. G. Szatkowski; R. F. Boswell,
`Jr.
`
`[511]
`
`ABSTRACT
`
`The invention provides a method of attenuating lung capil-
`lary leak in a mammal by administering an efiective amount
`of an antifolate, ruethotrexate.
`
`9 Claims, No Drawings
`MEDAC Exhibit 2007
`MEDAC Exhibit 2007
`ANTARES v. MEDAC
`ANTARES v. MEDAC
`IPR2014-01091
`IPR2014-01091
`Page 00001
`Page 00001
`
`

`
`1
`METHOD OF ATTENUATING LUNG
`CAPILLARY LEAK IN A MAMMAL
`
`FIELD OF THE INVENTION
`
`This invention relates to a method of attenuating capillary
`leak in a mammal by administering an eifective amount of
`an antifolatc, mcthotrcxate.
`
`BACKGROUND OF THE INVENTION
`
`5,593,671
`
`2
`
`The invention also provides a composition of matter
`containing an amount of methotrexate etfective to attenuate
`lung capillary leak in a maternal
`in association with a
`pharmaeeutically acceptable carrier.
`DETAILED DESCRIPTION OF THE
`IINIVENTION
`
`In accordance with the present invention, lung capillary
`leak in a mammal is attenuated by administering an effective
`amount of an antifolate methotrexale.
`
`5
`
`10
`
`Lung capillary leak syndrome is a dose limiting toxicity
`brought about by administration of cytokines, lymphokincs,
`growth factors, recombinant proteins and the like to patients
`
`Methotrexaie, is known as N-[4-[[(2,4-diami11o-6-pteridi-
`nyl)methyl]methylaminolhenrioyl]-L-glutarnic acid and has
`the structural formula:
`
`HEN
`
`N
`
`N
`
`m... ‘EH3
`
`/
`
`N
`
`ft?’
`
`N
`
`/ N
`H2N
`
`°°'l‘"
`HOOC -—- (|I—- -CH;CI-LCOOH
`H
`
`and it produces major morbidity. Lung capillary leak is due
`to an increase in vascular permeability and is manifested by
`multl-organ system dysfunction and generalized fluid accu-
`mulation and in particular the accumulation of fluid in the
`lung. Patients with lung capillary leak require respiratory
`and ventiiatory support (see J. P. Siegel and R. K. Puri,
`Intcrleukin—2 toxicity, Journal of Clinical Oncology, Vol. 9,
`pp 694-104, 1991) adding to the expense of medical care.
`The most troublesome effect of lung capillary leak syndrome
`is the extravasation of fluid into the lungs, thereby producing
`interstitial pulmonary edema, often requiring the termination
`of cytokine, lymphokine, growth factor or protein therapy
`and the use of supplemental oxygen and other respiratory
`and ventilator)! support measures.
`Heretofore. corticosteroids have been utilized to attenuate
`lung capillary leak syndrome induced by the lympholcine
`Interleukin-2 (IL-E). However, the corticosteroids adversely
`afiect the antitumor eificacy of the IL.-2(Vetto, J. T.. M. Z.
`Papa, M. T. Loitze J. Clin. Oncol. 51496-503 1987; Papa M.
`Z., J. T. Vetto, S. E. Ettzinghausen, J. J. Mule and S. A.
`Rosenberg. Cartcer Res. 465618-S623, 1986).
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a method of attenuating
`lung capillary leak in a mammal which comprises adminis-
`tering to said mammal an amount of an antifolate, merhot:r-
`cxate, cfieetivc to attenuate lung capillary leak. The effective
`amount of the antifolate, methotrexate. is from about 0.025
`mg}Kg to about 2.0 mg;’Kg. The arltifolate, metllotrexate, is
`administered orally,
`intraperitoneally, subcutaneously or
`intraveneously.
`The method of the present invention is particularly eii'ee—
`tive in attenuating lung capillary leak induced by the lym-
`phokine, Interleukin-2. In utilizing the method of the present
`invention to attenuate lung capillary leak induced by Inter-
`leukin-2, the antifolate methotrexate is administered adjunct
`to the administration of the Interleukin-2. In particular the
`antifolate methotrexate is administered simultaneously with,
`prior to or alter the intraperitoneal administration of IL-2.
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`The following references describe the preparation of meth-
`otrexate (see Seeger et al., J.Am.Chem.Soc. 71, 1'.-'53( 1949);
`the metabolism of methotrexate (see Freeman, J.Pharma-
`col.Exp.Ther. 122, 154(1958J and Henderson ct al., Cancer
`Res. 25. 1008, 10l8{l965)); the toxicity of Inethotrexatc:
`Condit et al., Cancer 13, 222-2-49(1960),
`ibid. 23,
`l26(1969); the pharmacokinetic models of methouexatc:
`Bischolf, et al., J.Pharm.Sci 59, l49(19'?0); eidem, ihid. 60,
`ll28(197l); the metabolism and pharmacokinetics of meth-
`otrcxatc: W. E. Evans, Appl.Pharrnaeoltinct. 1980. 518—548;
`the clinical pharmacology of methotrexate: J. R. Bertino,
`Cancer Chemother. 3, 359—375(l98l); J. Jolivet et al.,
`N.Engl.J.Med. 309, 1094—l104(19S3) and the clinical expe-
`rience of methottexate in rheumatoid arthritis; J.Rheuma.toI.
`12, Suppl, 12, 141-'-K1985).
`Methotrexate inhibits dihydrofolic acid reductase. Folic
`acid must be reduced to tetrahydrofolie acid by this enzyme
`in the process of DNA synthesis, repair and cellular repli-
`cation. Therefore, methotrexatc interferes with cellular
`reproduction. See rnelhotrexate package insert. Lederle
`Laboratories, Pearl River, NJ’. 10965 and references
`therein. Methotrexate has not been reported heretofore to
`attenuate capillary leak.
`The amount of the antifolatc, rnethotrexate. efl'eet.ive to
`attenuate lung capillary leak in a mammal is from about
`0.025 mglKg to about 2.0 mgl'Kg. A preferred range is
`between 0.025 mglltg to 0.5 mgfkg. The antifolate methotr—
`exate is administered orally.
`intraperitoneally. subcutane-
`ously or intraveneously. Oral administration is preferred.
`A distinct advantage of the present invention is that
`attenuation of capillary leak by administering an elfective
`dose of methotrexate will decrease the costs of hospital care.
`Patients need not be treated in intensive care units and
`respiratory and vcntilatory support will not be required.
`Unlike corticosteroids, heretofore utilized to attenuate IL-2
`induced lung capillary leak, methotrexate does not.eornpro-
`misc the antitumor cflicaey of IL-2. Thus, in addition, it may
`be possible to administer higher doses of IL-2 or other
`cytolrine, lympholrine or protein such that the anticancer
`response rates may be increased.
`Interleukin—2 (L2) is one of the first purified recombi-
`nant lymphol-tines to be administered to humans and is the
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:19)(cid:19)(cid:19)(cid:19)(cid:21)
`Page 00002
`
`

`
`5,593,671
`
`3
`most studied lymphokine for the treatment of certain human
`malignancies (renal cell carcinoma and melanoma). A dose
`limiting toxicity associated with IL-2 therapy for cancer is
`capillary leak in the lung.
`The human recombinant interleukin-2 product PROLEU-
`K1N® is a highly purified protein with a molecular weight
`of approximately 15,300 daltons. See package insert Chiron
`Corporation, Emcryville, Calif. and references therein. The
`chemical name is des-alanyl-1, serine-I25 human interleu-
`kin-2. The product is produced by recombinant DNA tech-
`nology using genetically engineered E. colt‘ strain containing
`an analog of the human interleukin-2 gene. In vitro studies
`performed on human cell lines demonstrate the immuno-
`regulatory properties of PROLEUICING9 including:
`a}
`enhancement of lymphocyte mitogcncsis and stimulation of
`long—term growth of human InterleLtkin—2 dependent cell
`lines; b) enhancement of lymphocyte eytotoxicity; c) induc-
`tion of killer cell lymphokine-activated (LAK) and natural
`(NK) activity; and d) induction of interferon-gamma pro-
`duction. When tested in vivo in select murine tumor models
`and in the clinic. PROLEUKlN® produces muitiple immu-
`nological effects in a dose dependent manner. These effects
`include activation of cellular immunity with profound lym-
`phocytosis, eosinophilia, and thromboeytopenia. and the ‘
`production of cytokines including tumor necrosis factor,
`IL-1 and gamma interferon. In vivo experiments in murine
`tumor models have shown inhibition of tumor growth.
`The eflicacy of methotrexate in attenuating capillary leak
`induced by IL-2 is demonstrated by the murine model of
`
`4
`
`Male CSTIBI6 mice (Charles River, Wilmington, Mass.)
`are used. Human IL-2, PROLEUKlN® (specific activity
`l6.4Xl0° IU.-’mg) is purchased from Cetus Oncology Prod-
`ucts, Eineryville, Calif. Mice are treated innapcritoneally
`with 9 doses of IL-2 (500,000 IU.-’dosc) over a four day
`period. Two doses of IL-2 are administered on day 1, three
`closes on day 2 and day 3 and a single dose on day 4. Three
`hours after the last dose of IL-2, mice are injected intrave-
`nously with 1.0 ].lCi of lzslodinated bovine serum albumin.
`One hour after radioactive albumin administration, the mice
`are weighed, sacrificed and an aliquot of blood is collected
`for analysis of radioactivity. The lungs are flushed with two
`ml of phosphate buffered saline (PBS), excised, blotted,
`weighed and analyzed for radioactivity. Radioactivity mea-
`surements are conducted with a gamma counter and the
`results are shown as counts per minute (cpm).
`To ascertain the effect of methotrexate on IL-2 induced
`
`capillary leak. doses of 0.5 naglfig or 0.25 mgllig or 0.125
`mg.-‘Kg of methotrexate are administered orally one hour
`before each dose of IL-2 (hence 9 doses of methotrexate are
`administered over the four day period each one being
`administered one hour before each administration of IL-2).
`Mcthotrcxatc is obtained from Clinical Research Section,
`Lederle Laboratories, Pearl River, N.Y. Results of this
`experiment are described in Table 1.
`
`TABLE 1
`
`Lung
`Wis (mg i
`SE)
`
`Body
`Wts (gm i
`SE)
`
`Group
`
`cpm in lung
`(iSE)
`
`cpm in
`blood ($517.)
`
`19 t 0.2
`
`137 i 3
`
`13.450 11026
`
`‘F122 1 202
`
`19 i 0.2
`I9 1 0.2
`
`175 i 10*
`133 i 51‘
`
`14,205 i 174-4‘
`15.306 1 11411’
`
`6899 i 35'!
`1069 i 254
`
`Phosphate
`Buffered Saline
`(PBS) Control
`IL-2 alone"
`metliolrexate
`(05 marks)
`followed in
`1 hour by
`mzii
`methotreatate
`(0.25 mgfltg)
`followed in
`1 hour by
`Lgvv
`methotrexate
`(0.125 mgfltg)
`followed in
`1 hour by
`Lztt
`
`
`I9 $6.3
`
`I401 31‘
`
`16.1941: 12181
`
`'l'24l}t 252
`
`I9 1 0.2
`
`146 i 51'
`
`18.099 1 2248
`
`6527 i 596
`
`“‘Siguifica.ntly diflczent from control mice that received phosphate buflered saline (PBS). (Student
`
`t test, ‘E <0.t}5).
`‘l‘Sigm cantly diferent from the IL-2 group (student I. test. p <0.05).
`“Each dose of IL-2 = $11,000 ill.
`Faich group contains 10-15 mice. SE = standard error of the mean.
`S5
`
`IL-2 induced capillary leak (M. Rosettstein, S. E. Etting-
`hausen and S. A. Rosenberg, Extravasation of Inn-avascular
`Fluid Mediated by the Systemic Administration of Recom-
`binant Interlcukin—2, Journal of Immunology, Vol. 137. pp
`1735-l’l42, 1986). Methotreitate is administered adjunct to
`the administration of IL-2. Such adjunct administration
`includes administration simultaneously, prior to and after
`administration of IL-2. For adrninistration of methottcxatc
`prior to IL-2 administration a time of from about 1 hour to
`about 5 hours prior to administration of IL-2 may be
`employed. A time of about 1 hour prior to IL-2 administra-
`tion is preferred.
`
`65
`
`Because increase in lung weights and radioactivity accu-
`mulating in the lung are parameters used to demonstrate
`IL-2 induced capillary leak in mice (M. Rosenstein, S. E.
`Ettinghausen and S. A. Rosenberg. Extravasation of Intra-
`vascular Fluid Mediated by the Systemic Administration of
`Recombinant Interleukin-2, Jountal of Immunology, Vol.
`137, pp l735—1'i'42, 1936), the results shown above indicate
`that methotrexate is exceptionally effective in attenuating
`capillary leak induced by IL-2. Lung weights and radioac-
`tivity accumulating in the lung are significantly lower for
`mice pretreated with methotrexate when compared to the
`mice in the IL-2 group. As shown in the above Table l the
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:19)(cid:19)(cid:19)(cid:19)(cid:22)
`Page 00003
`
`

`
`5,593,671
`
`5
`minimal effective dose of methotrexate is 0.25 rnglkg
`administered orally, one hour before IL-2 treatment. Radio-
`activity present in the blood is similar in all groups indicat-
`ing that an equivalent amount of radioactive albumin is
`administered to mice in the various treatment groups.
`Using the same testing protocol as described above, a
`single daily dose of 0.5 rng;"Kg or 1 mgfKg or 2 mg/Kg of
`methoucxate administered one hour before the administra-
`tion of thc flrst daily dose of IL2 is also eifective in the
`attenuation of capillary leak induced by IL-2.
`Male C5'h'B16 mice (Charles River. Wilmington, Mass.)
`are used. Human ILr2, PROLEUKIN-® (specific activity
`l6.4><l0° IUlmg) is purchased from Chiron Corporation.
`Emeryville, Calii‘. Mice are treated intraperitoneally with 9
`doses of IL-2 £500,000 lUl'dosc) over a four day period. Two 15
`doses of IL-2 are administered on day 1. three doses on day
`2 and day 3 and a single dose on day 4. A single daily dose
`of methotrexate was administered orally one hour before the
`first IL.-2 dose. Therefore four doses of methotrexate were
`administered over the four day period. ‘Three hours after the 20
`last dose of IL-2, mice are injected intravenously with 1.0
`uCi of 125Iodinated bovine serum albumin. One hour after
`radioactive albumin administration, the mice are weighed,
`sacrificed and an aliquot of blood is collected for analysis of
`radioactivity. The lungs are flushed with two ml of phos- 25
`phate buffered saline (PBS), excised, blotted, weighed and
`analyzed for radioactivity. Radioactivity measurements are
`conducted with a gamma counter and the results are shown
`as counts per minute {cpm). Results of this experiment are
`shown in Table 2.
`
`6
`rnethotrexate on IL-2 induced capillary leak is not schedule
`dependent. Therefore single or multiple dosing regimens
`known to those slcilled in the art can be utilized to deliver a
`therapeutically effective dose of methotrexate. In addition.
`the timing of methotrexate administration relative to IL-2
`treatment can be adjusted so as to achieve the maximal
`beneficial eifcct of reduction in capillary lcak. A preferred
`regimen is to orally administer 0.025 rng;'Kg to 2.0 mg!Kg
`of methotrexate l to 5 hours before the administration of
`IL-2. Most preferred is to orally administer 0.025 mgncg to
`0.5 rngl’Kg of methotrcxate 1 hour before the administration
`of IL-2. The most particularly preferred regimen is to orally
`administer 0.5 rnglKg of methotrexate 1 hour before admin-
`istration of IL-2.
`Corticosteroids have heretofore been used in this rnurinc
`model to treat capillary leak, however, treatment with this
`agent compromises the antitumor effect of IL-2 (M. Z.
`Pappa, J. '1‘. Vetto, S. E. Ettinghauscn, J. J. Mule and S. A.
`Rosenberg. Eifect of Corticosteroids on the Antitumor
`Activity of Lymphokinc Activated Killer Cells and Interleu-
`l<in—2 in Mice. Cancer Research, Vol. 46, pp 5618-5623,
`1986).
`In contrast to corticosteroids, methotrexate does not inter-
`fere with the eflicacy of IL-2 as shown by the results using
`the following protocol:
`Male C57.-’Bl6 mice are injected intravenously with
`1x10“ cells derived front MCA-205 tumors. Earlier studies
`have demonstrated that these tumor cells derived from
`methyl cholanthrcnc induced carcinoma (cx.. MCA—105 or
`MCA-205) respond to IL-2 therapy (M. Z. Pappa, J. T. Vetto,
`
`body
`weights
`(gm 1 SE)
`
`13.51 0.2
`
`TABLE 2
`
`lung
`wt (mg 1
`SE)
`
`12'.’ i 2
`
`cpm in lungs
`(tSE)
`
`H.901-1 1239
`
`cpmin
`blood ($55)
`8046 1356
`
`19.2 1 0.3
`18.6 i 0.3
`
`16?‘ i 4*
`136 i 81
`
`19,371 1' 1255*
`13.103 1 9621‘
`
`6902 1 343
`7693 i 196
`
`17.8 i 0.3
`
`129 i 3‘l‘
`
`13,623 1- 13331‘
`
`1350x509
`
`333 i 0.2
`
`142 1-61’
`
`12,219 3‘: 11 ill“
`
`8160 i 170
`
`Phosphate
`Bufiered
`Saline (PBS)
`control
`IL-2*‘
`single dose
`of
`mcthotrcsatc
`(2 mgflrg)
`prior to
`daily IL-2
`mzmpy.-no
`single dose
`of
`melholrexate
`
`(1_=rIsfks)
`prior to
`daily LL.-
`meflpymt
`single dose
`of
`methotrexate
`(0-5 meme)
`prior to
`daily 1]..-
`Iherapytt
`
`‘p (0.05 as compared to phosphate bufiered saline (PBS) control. student 1 test.
`11) <0.05 as competed with IL-2 group.
`SE = standard error of the mean.
`n = 10 per group
`"Each dose of IL-2 = 500.000 IU.
`
`Since methotrexate administration regimens may fall into
`two categories (single or multiple doses), the two experi-
`ments described above indicate that the beneficial effect of
`
`S. E. Ettinghausen, J. J. Mule and S. A. Rosenberg. Effect of
`Corticosteroids on the Antiturnor Activity of Lymphokine
`Activated Killer Cells and Interleukin-2 in Mice. Cancer
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:19)(cid:19)(cid:19)(cid:19)(cid:23)
`Page 00004
`
`

`
`5,593,671
`
`7
`Research, Vol. 46, pp 5618-5623, 1986}. After 24 hr,
`MCA—205 treated mice are divided into groups (n=St'group).
`One group of mice is treated with phosphate bulfered saline
`{PBS control). A second group of mice is treated with
`500,000 IU of IL-2 three times daily. Still other groups of
`mice are treated with oral doses of 0.5 mgt'Kg or 0.25 mg.-’Kg
`of methotrcxate three times daily followed in l hour by IL-2.
`A total of 15 doses of IL-2 or IL-2 plus methotrexate are
`adrninistered over a five day period. Methotrexate is admin-
`istered orally one hour before each dose of IL-2. After 5 days
`of therapy, mice are sacrificed, the lungs stained with India
`ink (to reveal tumors) and destained with Fekettes solution.
`The lungs are then scored for the number of tumor nodules
`present. This protocol for detennining the eflicacy of IL-2 is
`adapted from a protocol used by Fralcer et al (D. L. Fraker,
`J. Langstein, J. A. Norton, Passive immunization Against
`Tumor Necrosis Factor Partially Abrogates Inter1eukin~2
`Toxicity, Journal of Experimental Medicine, Vol. 170, pp
`1015-1020, 1989). The results of this experiment are shown
`in Table 3:
`
`TABLE 3
`
`‘NUMBER
`OF MICE
`
`NUMBER OF TUMOR
`NODUIESILUNG
`('.t:SEJ
`
`5
`5
`5
`
`5
`
`5
`
`5
`
`68 :1: 19
`9 i 4'
`6 i 1‘
`
`15 i 6'
`
`34 1 141'
`
`2'? t Bi
`
`GROUP
`
`Control (PBS)
`IL.-2“
`l\«'IeI.hotrcxate (0.5 mgflrg)
`followed in
`1 hour by I.L-2“
`Melhorexatc
`(025 msfks)
`followed in 1
`hour by l]_r2"‘
`Mctbotrexate alone
`(0.5 mgfkg)
`Mclhotrexatc alone
`(0.25 mgfkg)
`
`‘Significantly tiiIIcrt-.nt from control group, p 4.0.03, student I test;
`SE = standard error.
`1'Nct different from control group, student t test.
`“Each dose of IL-2 = 500.000 RI
`
`The above experiment indicates that methotrcxatc does
`not interfere with the eflicacy of l].r2 in this IL-2 responsive
`murine tumor model. Therefore,
`it
`is expected that
`in
`patients receiving IL.~2 therapy, the efiicacy of IL-2 will not
`be compromised and the capillary leak induced by IL-2 will
`be attenuated. In addition, the above results indicate that
`methotrcxatc in the absence of IL~2 does not exhibit anti-
`tumor properties in this animal model.
`The mechanism of action of methotrexate in attenuating
`lung capillary leak in the above experiments has not been
`established. However, methotrexate is known to inhibit the
`enzymatic activity of dihydmfolate neductase (M. Fleisher,
`Antifolate Analogs, Mechanism of Action, Methodology
`and Clinical Eflicacy. Therapeutic Drug Monitoring, Vol.
`15, pp 521-526, 1993]. When methotrexate is administered
`to a mammal,
`it is also converted to methotrexate poly-
`glutamate and polyglutamates of methotrexate are also
`known to inhibit other enzymes for example 5-aminoin:ti-
`dazolc—4-carboxarnide rihonuelcotide transformylase that
`require tetrahydrofolate as a cofactor. It is therefore con-
`templated that other inhibitors of dihydrofolate reductase,
`such as (ex. Trimetrcxate®, Bdatrexate®) or inhibitors of
`5-a.minoimida.zole~4-carboxamide
`ribouucleotide
`trans-
`forrnylasc will also be useful in attenuating lung capillary
`leak.
`
`It is contemplated that capillary leak induced by other
`cytokines, lymphokines, growth factors and other recombi-
`
`nant proteins (K. S. Antmao, J. D. Griffin, A. Elias et al
`Effect of Recombinant Human Granulocyte Macrophase
`Colony Stimulating Factor on Chemotherapy Induced
`Myelosuppression. New England J. Med, Vol. 319:
`593-598, 1988; S. l. Brandt, W. P. Peters, S. K. Atwater et
`al, Effect of Human Granulocyte Macrophage Colony
`Stimulating Factor on Hematopoietic Reconstitution After
`High Dose Chemotherapy and Autologous Bone Marrow
`Transplantalion, New Eng. J. Med, Vol. 313: 869-376,
`1988} may also be attenuated by the administration of an
`antifolate methotrexate.
`Methotrexate may be orally administered, for example,
`with an inert diluent or with an assimilable edible carrier, or
`it may be enclosed in hard or soft shell gelatin capsules, or
`it may be compressed into tablets, or it may be incorporated
`directly with the food of the diet. For oral
`therapeutic
`administration, methotrexate may be incorporated with
`excipieots and used in the form of ingestiblc tablets, buccal
`tablets, trochcs, capsules, elixers, suspension, syrups, wafer,
`and the like. Such compositions and preparations should
`contain at least 0.05% of methotrexate. The percentage of
`the compositions and preparations may, of course. be varied
`and may conveniently be between about 2 to 60% of the
`weight of the unit. The amount of methotrexate in such
`therapeutically useful compositions is such that a suitable
`dosage will be obtained. Preferred compositions or prepa-
`rations according to the present invention are prepared so
`that an oral dosage unit form contains between 0.025 and 2.0
`mg of methotrexate.
`The tablets, troches, pills, capsules and the like may also
`contain the following: a binder, such as gum tragacanth,
`acacia. corn starch or gelatin; excipients such as dicalcium-
`phosphate; a disintegrating agent such as corn starch, alginic
`acid and the like; a lubricant such as magnesium stearate;
`and a sweetening agent such as sucrose, lactose or saccharin
`may be added or a flavoring agent such as peppermint, oil of
`wintergreeu or cherry flavoring. When the dosage unit form
`is a capsule, it may contain, in addition to material of the
`above type, a liquid carrier. Various other material may be
`present as coating or to otherwise modify the physical form
`of the dosage unit. For instance, tablets, pills, or capsules
`may be coated with shellac, sugar or both. A syrup or eiixer
`may contain methotrexate, sucrose as a sweetening agent.
`methyl and propylparabcns as preservative, a dye and fia-
`voring such as cherry or an orange llavor. Of course, any
`material used in preparing any dosage unit form should be
`pharmaceutically pure and substantially non-toxic in the
`amounts employed. In addition, methotrexate may be incor-
`porated into sustained-release preparations and formula-
`tions.
`Methotrexate may also be administered parentcrally or
`intraperitonedly. Solutions of methotrexate can be prepared
`in glycerol,
`liquid polyethylene glycols. and mixtures
`thereof and in oils. Under ordinary conditions of storage and
`use, these preparations contain a preservative to prevent the
`growth of microorganisms.
`The pharmaceutical forms suitable for injectable use
`include sterile aqueous solutions or dispersions and sterile
`powders for the extcmporaneous preparation of sterile
`injectable solutions or dispersions. In all cases, the fon-n
`must be sterile and must be fluid to the extent that easy
`syringability exists. The form must be stable under the
`conditions of manufacture and storage and must be pre-
`servcd against the contamination action of microorganisms
`such as bacteria and fungi. The carrier can be a solvent or
`dispersion medium containing, for example, water, ethyl
`alcohol, polyol (for example, glycerol, propylene glycol, and
`
`5
`
`30
`
`50
`
`65
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:19)(cid:19)(cid:19)(cid:19)(cid:24)
`Page 00005
`
`

`
`5,593,671
`
`9
`
`liquid polyethylene glycol and the like), suitable mixtures
`thereof, and vegetable oils. The proper fluidity can be
`maintained, for example, by the use of a coating such as
`lecithin, by the maintenance of the required particle size in
`the case of dispersion and the use of surfactants. The
`prevention of the action of microorganisms can be brought
`about by various antibacterial and antifungal agents, for
`example, parabens, ehlorobutanol, phenol, sorbic acid,
`tbimerosal and the like. In many cases, it will be preferable
`to include isotonic agents, for example, sugars or sodium
`chloride. Prolonged absorption of the injectable composi-
`tions can be brought about by the use in the compositions of
`agents delaying absorption,
`for
`example,
`aluminum
`monostearate and gelatin.
`Sterile injectable solutions are prepared by incorporating
`methotrexate in the required amount in the appropriate
`solvent with various of the other ingredients enumerated
`above, as required, followed by filtered sterilization. Gen-
`erally, dispersions are prepared by incorporating methotuex-
`ate into a sterile vehicle which contains the basic dispersion
`medium and the required other ingredients from those
`enumerated above. In the case of sterile powder, for the
`preparation of sterile injectable solutions,
`the preferred
`methods of preparation are vacuum drying and the freeze-
`drying technique which yield a powder of nletbotrexate, plus
`any additional desired ingredient from a previously sterile-
`filtered solution thereof.
`As used herein, "pharmaceutieally acceptable carrier“
`includes any and all solvents, dispersion media, coating,
`antibacterial and antifungal agents, isotonic and absorption
`delaying agents and the like. The use of such media and
`agents for pbarmaeeutically active substances is well known
`in the art. Except insofar as any conventional media or agent
`is incompatible with the active ingredient, it's use in the
`therapeutic compositions is contemplated. Supplementary
`active ingredients can also be incorporated into the compo-
`sitions.
`
`It is especially advantageous to formulate parenteral com-
`positions in dosage unit form for case of administration and
`unifomuty of dosage. Dosage unit from as used herein refers
`to physically discrete units suited as unitary dosages for the
`mammalian subjects to be treated; each unit containing a
`predetermined quantity of active material calculated to pro-
`duce the desired therapeutic effect in association with the
`required pharmaceutical carrier. The specification for the
`novel dosage unit forms of the invention are dictated by and
`directly dependent on (a) the unique characteristics of meth-
`otrexate and the particular therapeutic elfect to be achieved
`(attentuation of lung capillary leak syndrome) and (la) the
`limitations inherent in the art of compounding methotrexate
`for the lung capillary leak syndrome in living subjects
`
`20
`
`30
`
`35
`
`45
`
`50
`
`10
`having a diseased condition in which bodily health is
`impaired as herein disclosed in detail.
`Methotrexate is compounded for convenient and eifective
`administration in elfective amounts with a suitable pharma-
`ceutically acceptable carrier in dosage unit form as herein
`before disclosed. A unit dosage form can, for example,
`contain methotrexatc in amounts ranging from about 0.1 to
`400 mg, with from 1 to 20 mg being preferred. Expressed in
`proportions, methotrexate is generally present in from about
`0.1 to about 40 mgfml of carrier. In the case of compositions
`containing supplementary active ingredients, the dosages are
`determined by reference to the usual dose and manner of
`administration of the said ingredients.
`A single intravenous dosage, slow constant infusion, or
`repeated daily dosages can be administered. Daily dosages
`up to about 1 to 10 days are often suflicient. It is also
`possible to dispense one daily dosage or multiple daily doses
`or one dose on alternate or less frequent days. As can be seen
`from the dosage regimens,
`the amount of methotrexate
`administered is to be suflicient to attenuate Interleukin-2
`induced pulmonary capillary leak syndrome.
`We claim:
`1. A method of attenuating lung capillary leak in a
`mammal induced by the administration of interleukin-2
`which comprises administering to said mammal an amount
`of methotrexate effective to attenuate lung capillary leak.
`2. The method of clai