Method of attenuating lung capillary leak in a mammal

The invention provides a method of attenuating lung capillary leak in a mammal by administering an effective amount of an antifolate, methotrexate.

FIELD OF THE INVENTION 
This invention relates to a method of attenuating capillary leak in a 
mammal by administering an effective amount of an antifolate, 
methotrexate. 
BACKGROUND OF THE INVENTION 
Lung capillary leak syndrome is a dose limiting toxicity brought about by 
administration of cytokines, lymphokines, growth factors, recombinant 
proteins and the like to patients and it produces major morbidity. Lung 
capillary leak is due to an increase in vascular permeability and is 
manifested by multi-organ system dysfunction and generalized fluid 
accumulation and in particular the accumulation of fluid in the lung. 
Patients with lung capillary leak require respiratory and ventilatory 
support (see J. P. Siegel and R. K. Puri, Interleukin-2 toxicity, Journal 
of Clinical Oncology, Vol. 9, pp 694-704, 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 ventilatory support measures. 
Heretofore, corticosteroids have been utilized to attenuate lung capillary 
leak syndrome induced by the lymphokine Interleukin-2 (IL-2). However, the 
corticosteroids adversely affect the antitumor efficacy of the IL-2(Vetto, 
J. T., M. Z. Papa, M. T. Loitze J. Clin. Oncol. 5:496-503 1987; Papa M. 
Z., J. T. Vetto, S. E. Ettinghausen, J. J. Mule and S. A. Rosenberg. 
Cancer Res. 46:5618-5623, 1986). 
SUMMARY OF THE INVENTION 
The present invention provides a method of attenuating lung capillary leak 
in a mammal which comprises administering to said mammal an amount of an 
antifolate, methotrexate, effective 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 antifolate, methotrexate, is administered 
orally, intraperitoneally, subcutaneously or intraveneously. 
The method of the present invention is particularly effective in 
attenuating lung capillary leak induced by the lymphokine, Interleukin-2. 
In utilizing the method of the present invention to attenuate lung 
capillary leak induced by Interleukin-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 after the intraperitoneal administration of IL-2. 
The invention also provides a composition of matter containing an amount of 
methotrexate effective to attenuate lung capillary leak in a mammal in 
association with a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, lung capillary leak in a mammal 
is attenuated by administering an effective amount of an antifolate 
methotrexate. 
Methotrexate, is known as 
N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic 
acid and has the structural formula: 
##STR1## 
The following references describe the preparation of methotrexate (see 
Seeger et al., J.Am.Chem.Soc. 71, 1753(1949); the metabolism of 
methotrexate (see Freeman, J.Pharmacol.Exp.Ther. 122, 154(1958) and 
Henderson et al., Cancer Res. 25, 1008, 1018(1965)); the toxicity of 
methotrexate: Condit et al., Cancer 13, 222-249(1960), ibid. 23, 
126(1969); the pharmacokinetic models of methotrexate: Bischoff, et al., 
J.Pharm.Sci 59, 149(1970); eidem, ibid. 60, 1128(1971); the metabolism and 
pharmacokinetics of methotrexate: W. E. Evans, Appl.Pharmacokinet. 1980, 
518-548; the clinical pharmacology of methotrexate: J. R. Bertino, Cancer 
Chemother. 3, 359-375(1981); J. Jolivet et al., N.Engl.J.Med. 309, 
1094-1104(1983) and the clinical experience of methotrexate in rheumatoid 
arthritis; J.Rheumatol. 12, Suppl, 12, 1-44(1985). 
Methotrexate inhibits dihydrofolic acid reductase. Folic acid must be 
reduced to tetrahydrofolic acid by this enzyme in the process of DNA 
synthesis, repair and cellular replication. Therefore, methotrexate 
interferes with cellular reproduction. See methotrexate package insert, 
Lederle Laboratories, Pearl River, N.Y. 10965 and references therein. 
Methotrexate has not been reported heretofore to attenuate capillary leak. 
The amount of the antifolate, methotrexate, effective to attenuate lung 
capillary leak in a mammal is from about 0.025 mg/Kg to about 2.0 mg/Kg. A 
preferred range is between 0.025 mg/kg to 0.5 mg/kg. The antifolate 
methotrexate is administered orally, intraperitoneally, subcutaneously or 
intraveneously. Oral administration is preferred. 
A distinct advantage of the present invention is that attenuation of 
capillary leak by administering an effective dose of methotrexate will 
decrease the costs of hospital care. Patients need not be treated in 
intensive care units and respiratory and ventilatory support will not be 
required. Unlike corticosteroids, heretofore utilized to attenuate IL-2 
induced lung capillary leak, methotrexate does not compromise the 
antitumor efficacy of IL-2. Thus, in addition, it may be possible to 
administer higher doses of IL-2 or other cytokine, lymphokine or protein 
such that the anticancer response rates may be increased. 
Interleukin-2 (IL-2) is one of the first purified recombinant lymphokines 
to be administered to humans and is the 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 PROLEUKIN.RTM. is a highly 
purified protein with a molecular weight of approximately 15,300 daltons. 
See package insert Chiron Corporation, Emeryville, Calif. and references 
therein. The chemical name is des-alanyl-1, serine-125 human 
interleukin-2. The product is produced by recombinant DNA technology using 
genetically engineered E. coli strain containing an analog of the human 
interleukin-2 gene. In vitro studies performed on human cell lines 
demonstrate the immunoregulatory properties of PROLEUKIN.RTM. including: 
a) enhancement of lymphocyte mitogenesis and stimulation of long-term 
growth of human Interleukin-2 dependent cell lines; b) enhancement of 
lymphocyte cytotoxicity; c) induction of killer cell lymphokine-activated 
(LAK) and natural (NK) activity; and d) induction of interferon-gamma 
production. When tested in vivo in select murine tumor models and in the 
clinic, PROLEUKIN.RTM. produces multiple immunological effects in a dose 
dependent manner. These effects include activation of cellular immunity 
with profound lymphocytosis, eosinophilia, and thrombocytopenia, 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 efficacy of methotrexate in attenuating capillary leak induced by IL-2 
is demonstrated by the murine model of IL-2 induced capillary leak (M. 
Rosenstein, S. E. Ettinghausen and S. A. Rosenberg, Extravasation of 
Intravascular Fluid Mediated by the Systemic Administration of Recombinant 
Interleukin-2, Journal of Immunology, Vol. 137, pp 1735-1742, 1986). 
Methotrexate is administered adjunct to the administration of IL-2. Such 
adjunct administration includes administration simultaneously, prior to 
and after administration of IL-2. For administration of methotrexate 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 administration is preferred. 
Male C57/B16 mice (Charles River, Wilmington, Mass.) are used. Human IL-2, 
PROLEUKIN.RTM. (specific activity 16.4.times.10.sup.6 IU/mg) is purchased 
from Cetus Oncology Products, Emeryville, Calif. Mice are treated 
intraperitoneally with 9 doses of IL-2 (500,000 IU/dose) over a four day 
period. Two doses of IL-2 are administered on day 1, three doses 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 intravenously with 1.0 .mu.Ci of .sup.125 
Iodinated 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 measurements 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 mg/Kg or 0.25 mg/Kg 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). Methotrexate is 
obtained from Clinical Research Section, Lederle Laboratories, Pearl 
River, N.Y. Results of this experiment are described in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Body Lung 
Wts (gm .+-. 
Wts (mg .+-. 
cpm in lung 
cpm in 
Group SE) SE) (.+-.SE) blood (.+-.SE) 
__________________________________________________________________________ 
Phosphate 
19 .+-. 0.2 
137 .+-. 3 
13,450 .+-. 1026 
7122 .+-. 202 
Buffered Saline 
(PBS) Control 
IL-2 alone** 
19 .+-. 0.2 
175 .+-. 10* 
24,205 .+-. 2744* 
6899 .+-. 357 
methotrexate 
19 .+-. 0.2 
138 .+-. 5.dagger. 
15,806 .+-. 1141.dagger. 
7069 .+-. 254 
(0.5 mg/kg) 
followed in 
1 hour by 
IL-2** 
methotrexate 
19 .+-. 0.3 
140 .+-. 3.dagger. 
16,194 .+-. 1218.dagger. 
7240 .+-. 252 
(0.25 mg/kg) 
followed in 
1 hour by 
IL-2** 
methotrexate 
19 .+-. 0.2 
146 .+-. 5.dagger. 
18,099 .+-. 2248 
6527 .+-. 596 
(0.125 mg/kg) 
followed in 
1 hour by 
IL-2** 
__________________________________________________________________________ 
*Significantly different from control mice that received phosphate 
buffered saline (PBS). (Student t test, p &lt;0.05). 
.dagger.Significantly different from the IL2 group (student t test, p 
&lt;0.05). 
**Each dose of IL2 = 500,000 IU. 
Each group contains 10-15 mice. SE = standard error of the mean. 
Because increase in lung weights and radioactivity accumulating 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 
Intravascular Fluid Mediated by the Systemic Administration of Recombinant 
Interleukin-2, Journal of Immunology, Vol. 137, pp 1735-1742, 1986), the 
results shown above indicate that methotrexate is exceptionally effective 
in attenuating capillary leak induced by IL-2. Lung weights and 
radioactivity 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 1 the minimal effective dose of methotrexate 
is 0.25 mg/kg administered orally, one hour before IL-2 treatment. 
Radioactivity present in the blood is similar in all groups indicating 
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 mg/Kg or 1 mg/Kg or 2 mg/Kg of methotrexate administered one hour 
before the administration of the first daily dose of IL-2 is also 
effective in the attenuation of capillary leak induced by IL-2. 
Male C57/B16 mice (Charles River, Wilmington, Mass.) are used. Human IL-2, 
PROLEUKIN.RTM. (specific activity 16.4.times.10.sup.6 IU/mg) is purchased 
from Chiron Corporation, Emeryville, Calif. Mice are treated 
intraperitoneally with 9 doses of IL-2 (500,000 IU/dose) over a four day 
period. Two 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 last dose of IL-2, mice are injected intravenously 
with 1.0 .mu.Ci of .sup.125 Iodinated 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 
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. 
TABLE 2 
__________________________________________________________________________ 
body lung 
weights 
wt (mg .+-. 
cpm in lungs 
cpm in 
(gm .+-. SE) 
SE) (.+-.SE) blood (.+-.SE) 
__________________________________________________________________________ 
Phosphate 
18.5 .+-. 0.2 
127 .+-. 2 
11,904 .+-. 1239 
8046 .+-. 356 
Buffered 
Saline (PBS) 
control 
IL-2** 19.2 .+-. 0.3 
167 .+-. 4* 
19,371 .+-. 1755* 
6902 .+-. 348 
single dose 
18.6 .+-. 0.3 
136 .+-. 8.dagger. 
13,103 .+-. 962.dagger. 
7698 .+-. 196 
of 
methotrexate 
(2 mg/kg) 
prior to 
daily IL-2 
therapy** 
single dose 
17.8 .+-. 0.3 
129 .+-. 3.dagger. 
13,623 .+-. 1333.dagger. 
7850 .+-. 509 
of 
methotrexate 
(1 mg/kg) 
prior to 
daily IL- 
therapy** 
single dose 
18.3 .+-. 0.2 
142 .+-. 6.dagger. 
12,219 .+-. 1111.dagger. 
8160 .+-. 170 
of 
methotrexate 
(0.5 mg/kg) 
prior to 
daily IL- 
therapy** 
__________________________________________________________________________ 
*p &lt;0.05 as compared to phosphate buffered saline (PBS) control, student 
test. 
.dagger.p &lt;0.05 as compared with IL2 group. 
SE = standard error of the mean. 
n = 10 per group 
**Each dose of IL2 = 500,000 IU. 
Since methotrexate administration regimens may fall into two categories 
(single or multiple doses), the two experiments described above indicate 
that the beneficial effect of methotrexate on IL-2 induced capillary leak 
is not schedule dependent. Therefore single or multiple dosing regimens 
known to those skilled 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 effect of reduction in capillary 
leak. A preferred regimen is to orally administer 0.025 mg/Kg to 2.0 mg/Kg 
of methotrexate 1 to 5 hours before the administration of IL-2. Most 
preferred is to orally administer 0.025 mg/Kg to 0.5 mg/Kg of methotrexate 
1 hour before the administration of IL-2. The most particularly preferred 
regimen is to orally administer 0.5 mg/Kg of methotrexate 1 hour before 
administration of IL-2. 
Corticosteroids have heretofore been used in this murine model to treat 
capillary leak, however, treatment with this agent compromises the 
antitumor effect of IL-2 (M. Z. Pappa, J. T. Vetto, S. E. Ettinghausen, J. 
J. Mule and S. A. Rosenberg. Effect of Corticosteroids on the Antitumor 
Activity of Lymphokine Activated Killer Cells and Interleukin-2 in Mice. 
Cancer Research, Vol. 46, pp 5618-5623, 1986). 
In contrast to corticosteroids, methotrexate does not interfere with the 
efficacy of IL-2 as shown by the results using the following protocol: 
Male C57/B16 mice are injected intravenously with 1.times.10.sup.4 cells 
derived from MCA-205 tumors. Earlier studies have demonstrated that these 
tumor cells derived from methyl cholanthrene induced carcinoma (ex., 
MCA-105 or MCA-205) respond to IL-2 therapy (M. Z. Pappa, J. T. Vetto, S. 
E. Ettinghausen, J. J. Mule and S. A. Rosenberg. Effect of Corticosteroids 
on the Antitumor Activity of Lymphokine Activated Killer Cells and 
Interleukin-2 in Mice. Cancer Research, Vol. 46, pp 5618-5623, 1986). 
After 24 hr, MCA-205 treated mice are divided into groups (n=5/group). One 
group of mice is treated with phosphate buffered 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 mg/Kg or 
0.25 mg/Kg of methotrexate three times daily followed in 1 hour by IL-2. A 
total of 15 doses of IL-2 or IL-2 plus methotrexate are administered over 
a five day period. Methotrexate is administered 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 determining the efficacy of IL-2 is adapted 
from a protocol used by Fraker et al (D. L. Fraker, J. Langstein, J. A. 
Norton, Passive Immunization Against Tumor Necrosis Factor Partially 
Abrogates Interleukin-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 TUMOR 
NUMBER NODULES/LUNG 
GROUP OF MICE (.+-.SE) 
______________________________________ 
Control (PBS) 5 68 .+-. 19 
IL-2** 5 9 .+-. 4* 
Methotrexate (0.5 mg/kg) 
5 6 .+-. 1* 
followed in 
1 hour by IL-2** 
Methorexate 5 15 .+-. 6* 
(0.25 mg/kg) 
followed in 1 
hour by IL-2** 
Methotrexate alone 
5 34 .+-. 14.dagger. 
(0.5 mg/kg) 
Methotrexate alone 
5 27 .+-. 9.dagger. 
(0.25 mg/kg) 
______________________________________ 
*Significantly different from control group, p &lt;0.05, student t test; 
SE = standard error. 
.dagger.Not different from control group, student t test. 
**Each dose of IL2 = 500,000 IU 
The above experiment indicates that methotrexate does not interfere with 
the efficacy of IL-2 in this IL-2 responsive murine tumor model. 
Therefore, it is expected that in patients receiving IL-2 therapy, the 
efficacy 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 
methotrexate in the absence of IL-2 does not exhibit antitumor 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 dihydrofolate reductase (M. 
Fleisher, Antifolate Analogs, Mechanism of Action, Methodology and 
Clinical Efficacy. Therapeutic Drug Monitoring, Vol. 15, pp 521-526, 
1993). When methotrexate is administered to a mammal, it is also converted 
to methotrexate polyglutamate and polyglutamates of methotrexate are also 
known to inhibit other enzymes for example 5-aminoimidazole-4-carboxamide 
ribonucleotide transformylase that require tetrahydrofolate as a cofactor. 
It is therefore contemplated that other inhibitors of dihydrofolate 
reductase, such as (ex. Trimetrexate.RTM., Edatrexate.RTM.) or inhibitors 
of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase will also 
be useful in attenuating lung capillary leak. 
It is contemplated that capillary leak induced by other cytokines, 
lymphokines, growth factors and other recombinant proteins (K. S. Antman, 
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. J. 
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 Transplantation, New 
Eng. J. Med., Vol. 318: 869-876, 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 
excipients and used in the form of ingestible tablets, buccal tablets, 
troches, 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 preparations 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 dicalciumphosphate; 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 
wintergreen 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 elixer 
may contain methotrexate, sucrose as a sweetening agent, methyl and 
propylparabens as preservative, a dye and flavoring such as cherry or an 
orange flavor. 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 incorporated into 
sustained-release preparations and formulations. 
Methotrexate may also be administered parenterally or intraperitoneally. 
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 
extemporaneous preparation of sterile injectable solutions or dispersions. 
In all cases, the form 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 preserved 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 
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, chlorobutanol, 
phenol, sorbic acid, thimerosal 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 compositions 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. Generally, dispersions are prepared by incorporating 
methotrexate 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 methotrexate, plus any 
additional desired ingredient from a previously sterile-filtered solution 
thereof. 
As used herein, "pharmaceutically 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 pharmaceutically 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 compositions. 
It is especially advantageous to formulate parenteral compositions in 
dosage unit form for ease of administration and uniformity 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 
produce 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 methotrexate and the particular therapeutic effect to 
be achieved (attentuation of lung capillary leak syndrome) and (b) the 
limitations inherent in the art of compounding methotrexate for the lung 
capillary leak syndrome in living subjects having a diseased condition in 
which bodily health is impaired as herein disclosed in detail. 
Methotrexate is compounded for convenient and effective administration in 
effective amounts with a suitable pharmaceutically acceptable carrier in 
dosage unit form as herein before disclosed. A unit dosage form can, for 
example, contain methotrexate 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 mg/ml 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 sufficient. 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 sufficient to attenuate Interleukin-2 induced 
pulmonary capillary leak syndrome.