PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING INFLAMMATION
Background of the Invention
Technical Field
This invention relates to methods for treating inflammatory conditions employing certain H-fluorenyl-methoxycarbonyl-amino acids. The invention further relates to pharmaceutical composition suitable for use in such methods.
Background Information
The treatment of inflammatory conditions, such as atopic dermatitis, contact dermatitis, psoriasis, rheumatoid arthritis, glomerulonephritis, osteoarthritis, lupus erythematosis, scleroderma, asthma and irritable bowel disease has, in the past, involved the use of agents such as aspirin-like nonsteroidal anti-inflammatory agents, glucocorticoids, methotrexate and cyclophosphamide. Unfortunately these agents generally produce unwanted side effects. Specifically, the nonsteroidal anti-inflammatory drugs often cause gastrointestinal and renal side effects. Glucocorticoids suppress the immune system, thus producing opportunistic infection and endocrinopathy. Methotrexate has been associated with patient death, and cyclophosphamide has carcinogenic liability. Thus, new agents for treating inflammatory conditions that are free of these adverse side effects are needed.
Objects of the Invention
It is a general object of the present invention to provide a method of treating a subject suffering from an inflammatory condition while avoiding the adverse side effects associated with art-recognized anti-inflammatory agents.' It is a further object of the invention to provide a pharmaceutical composition suitable for use in such a method.
Further objects and advantages of the invention will be clear to one skilled in the art from a reading of the description that follows.
Summary of the Invention
The present invention relates to a method of treating a subject having an inflammatory condition, such as atopic or contact dermatitis, psoriasis, rhematoid arthritis, glomerulonephritis, osteoarthritis, lupus erythematosis, scleroderoma, asthma or irritable bowel disease. The invention also relates to a pharmaceutical composition suitable for use in such a method comprising as an active ingredient a Jl-fluorenyl-methoxycarbonyl- amino acid.
In one embodiment, the present invention relates to a method of treating an inflammatory condition comprising administering to an animal in need of such treatment at least one N-fluorenyl- methoxycarbonyl-amino acid of formula I:
Formula
R, and R2 are hydrogen, halogen or nitro;
R3 and R4 are hydrogen, alkyl or aryl containing up to eight carbons; and Rs is a lipophilic aromatic or aliphatic amino acid residue, or a pharmaceutically acceptable salt thereof, in an amount sufficient to reduce or eliminate the inflammatory condition.
In another embodiment, the present invention relates to a pharmaceutical composition suitable for use in the above-described method comprising, as an active ingredient, an anti- inflammatory amount of at least one ϋ-[ (9H-fluoren- 9-ylmethoxy)carbonyl]-amino acid, or salt thereof, as defined above, together with a pharmaceutically acceptable carrier or diluent.
Detailed Description of the Invention
The N-[ (9H-fluoren-9-ylmethoxy)carbony1]- amino acids which serve as active ingredients in the pharmaceutical compositions used in the method of treating an inflammatory condition of the present invention, are represented by the following formula:
Formula I
wherein: R, and R, are hydrogen, halogen or nitro;
' R-, and R< are hydrogen, alkyl or aryl containing up to eight carbons; and
R, is an lipophilic aromatic or aliphatic amino acid residue. (See United States Patents
3,835,175 and 3,906,031.) In a preferred embodiment, R,. R2, R3 and R4 are hydrogen and Rj is a L-leucine residue, i.e., CH(COOH)CH2CH(CH3)-».
The above compounds of Formula I are prepared by synthetic methods known in the art.
Details of such procedures have been described by
L.A. Carpino and G.Y. Han (U.S. Patents 3,835,175 and 3,906,031). Typically a 9H-fluorene is utilized as the starting material. This is converted to a corresponding 9H-fluoren-9-ylmethanol, for example, by condensation of a 9H-fluorene with methyl formate in the presence of sodium ethoxide, followed by reduction of the intermediate
9H-fluorene-9-carboxaldehyde. Alternatively, 9H-fluorene can be condensed directly with formaldehyde in the presence of a strong base such as sodium hydride or sodium amide to give the 9-methanol derivative. Compounds in which the alpha carbon atom is substituted may be prepared by reaction between the selected 9H-fluorene and an aldehyde other than formaldehyde or a ketone, such as acetone or acetophenone, in the presence of a strong base.
Introduction of substituents in the benzo fused rings of the 9H-fluorene can be achieved by known procedures as, for example, by direct halogenation or nitration. 9H-Fluoren-9-ylmethanols are converted to
9H-fluoren-9-ylmethanol halofor ates, carbonates, thiocarbonates, i idylcarbonates or other formate derivatives bearing a grouping ("leaving group") that is readily displaced by a nucleophilic nitrogen of an alpha amino acid. The resulting carbonyl derivatives of an activated 9H-fluoren-9-ylmethanol are condensed with an alpha aminocarboxylic acid to
form a 9H-fluoren-9-ylmethoxycarbonyl derivative of the general Formula I. If the "leaving group" is halogen, especially chlorine, reaction may be effected in a polar organic solvent such as dioxane, tetrahydrofuran, dimethylformamide or pyridine under alkaline conditions (preferably mild) at a low temperature, for example from 0°C to 25°C during a period of from about 2 to 3 hours. A preferred solvent is a mixture of dioxane and water. Normally, the N-[ (9H-fluoren-9-ylmethoxy)carbonyl]- a ino acid precipitates from solution and may be purified, for example, by recrystallization. Utilization of other "leaving groups" may require somewhat elevated temperatures, for example, 25°C to 50°C and longer reaction times, for example, 8 to 12 hours.
The N-[9H-fluoren-9-ylmethoxy)carbonyl]- amino acids of the present invention are generally utilized as the free acid or in the form of a pharmaceutically acceptable salt with various inorganic or organic bases. 'Typical salts include the alkali metal or alkaline earth salts, although it will be appreciated that other nontoxic salts can also be used. Advantageously, compounds suitable for use in this invention are administered as sodium, potassium, ammonium, choline or ethylenediamine salts. Sodium salts are preferred. As will be understood by those skilled in the art, the compounds of this invention can be present as D or L optical isomers or, in some cases, as diastereoiso ers as well as racemates and diastereoiso eric mixtures. Unless otherwise specified, the compounds of Formula I suitable for use in the methods and pharmaceutical compositions of the present invention include all isomers of such compounds, whether separated or mixtures thereof.
The activity of a compound of Formula I as an anti-inflammatory agent can be demonstrated in animals, such as mice, for example, by measuring the ability of the compound to inhibit edema caused by a variety of inflammatory agents that are generally accepted as producing irritation by differing mechanisms. Such inflammatory agents typically include tetradecanoylphorbolacetate, arachidonic acid, xylene, capsaicin, oxazolone, carrageenan and the like. The reverse passive Arthus test offers another measure of the compound's utility in preventing an inflammatory response (Chang et al, Eur. J. Pharm. 69:155-164 (1981)). Test compounds are typically administered intraperitoneally or topically. For intraperitoneal administration, the test compound can be given in dimethyl sulfoxide or in 0.5% methylcellulose 30 minutes prior to administration of the irritant. For topical administration, the test compound can be dissolved in, for example, acetone, ethanol or dimethyl sulfoxide and applied about 15 minutes prior to application of the irritant. Results can be expressed as the percent decrease in swelling in the compound-treated animals as compared to control animals that receive only the irritant.
The pharmaceutical compositions of the present invention comprise, as an active ingredient, at least one N-[ (9H-fluoren-9-ylmethoxy)carbonyl]- amino acid of Formula I (see above) , together with a pharmaceutically acceptable carrier. The active ingredient is present in the composition in an amount sufficient to produce an anti-inflammatory effect. The composition of the invention can be formulated so as to be suitable, for example, for oral", nasal, parenteral, topical, transdermal or rectal administration.
Preferably, the pharmaceutical composition of the invention includes the active ingredient of Formula I in a quantity selected from 25 mg to 500 mg, advantageously, from about 50 mg to 250 mg, per dosage unit, depending on the route of administration. Appropriate concentrations and dosage unit sizes can be readily determined by one skilled in the art.
The pharmaceutical carriers used in the compositions of the invention may be, for example, in solid or liquid form. Exemplary of solid carriers are lactose, magnesium stearate, terra alba, sucrose, talc, stearic acid, gelatin, agar, pectin or acacia. The amount of solid carrier present in the composition will vary greatly but preferably will be from about 25 mg to 1 g. Exemplary of liquid carriers are syrup, peanut oil, olive oil, sesame oil, propylene glycol, polyethylene glycol (mol. wt. 200-400) and water. The carrier or diluent may include a time delay material well known to the art' such as, for example, glyceryl monostearate or glyceryl distearate alone or with a wax.
As indicated above, the pharmaceutical composition of the invention can be present in dosage unit form. For example, the composition can take the form of a tablet (preferrably enteric coated) , capsule (preferrably enteric coated) , powder, troche, lozenge, inhalant, syrup, emulsion, gel, ointment, cream, lotion tranεder al patch, suppository, sterile injectable liquid as well as a liquid suspension or solution. The pharmaceutical compositions of the present invention are prepared by conventional techniques such as by mixing, granulating and compressing or dissolving the ingredients as may be appropriate for the desired preparation.
8
The method of treating an inflammatory condition according to this invention comprises administering to a subject in need of such treatment an amount of at least one H~ [ (9H~ luoren-9- ylmethoxy)carbonyl]-amino acid of Formula I (see above) sufficient to produce an anti-inflammatory effect. The compounds of Formula I can be administered orally, nasally, topically, transdermally, parenterally or anally, as may be required to effect the desired anti-inflammatory effect.
The active ingredient of Formula I (see above) will normally be administered in a daily dosage regimen selected from about 100 mg to 1 g, most preferably from about 200 mg to about 500 mg. Advantageously, equal doses will be administered, preferably, between one time per day to one time per week. The frequency of administration and the amount of active ingredient to be administered to effect treatment of a particular inflammatory condition can readily be determined by one skilled in the art. For inflammatory conditions of the lungs, an aerosol dispensing system wherein the active medicament is incorporated with Freon® (fluorohydrocarbon) or other inert propellant in an aerosol container is of particular applicability. Such an aerosol system will deliver a etered dose of about 100 meg to about 650 meg, administered once or twice at a time as needed. The following non-limiting Examples, which are illustrative of the compounds suitable for use in the methods and compositions of the present invention, demonstrate the activity of these compounds as well as processes for their preparation.
EXAMPLE 1
H-[ (9H-Fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine
9H-Fluoren-9-ylmethylchloroformate (51.4 g, 0.143 mole) and ϋ-hydroxysuccinimide (29.0 g, 0.252 mole) were dissolved in 350 ml of dry, distilled dioxane. The mixture was cooled in ice and 27.9 ml of triethyla ine was added slowly, so as to maintain the temperature of the mixture below
10°C. After four hours, the mixture was filtered to remove triethylammonium chloride. The solid was well washed with dioxane and the combined filtrates concentrated under reduced pressure. The product, 9-fluorenylmethyl-succinimidyl carbonate, 74 g, was crystallized by addition of petroleum ether and cooling to 4°C.
Phenylalanine (27.25 g, 0.165 mole) was dissolved in a solution of sodium carbonate (31.8 g, 0.3 mole) in 320 ml of water. This mixture was added to a solution of 9-fluorenylmethylsuccinimidyl carbonate (50.8 g, 0.15 mole) dissolved in a minimum amount of dioxane (approximately 90 ml being required) . The mixture was stirred vigorously (mechanical stirring) at room temperature for 22 hours and then diluted with water. The reaction mixture was extracted twice with ethyl ether and then acidified to pH 2 with concentrated hydrochloric acid in the presence of 750 ml of ethyl acetate. The organic layer was separated, washed twice with 1 N hydrochloric acid, twice with water, once with brine, dried over anhydrous magnesium εulfate, and concentrated under reduced pressure. The product was crystallized by addition of hexane to the boiling ethyl acetate solution, to give 18 g of ϋ~[ (9H-fluoren-9-ylmethyloκy)carbonyl]-L- phenylalanine, p 179-181°C.
EXAMPLE 2
N-[(9g-Fluoren-9-ylmethoxy)carbonyl]-L-leucine
To a solution of (1.31 g, 10 mole) of
L-leucine in 27 ml of water was added (2.5 g, 23 mmole) of sodium carbonate and the mixture was cooled in ice. To this was added a solution of (2.58 g, 7.2 mmole) of 9-fluorenylmethyl chloroformate in 20 ml of dioxane. The mixture was stirred at room temperature for 2.5 hours and diluted with 500 ml of water. The reaction mixture was extracted twice with ethyl ether. The aqueous layer was made acidic to Congo Red indicator paper with concentrated hydrochloric acid and the precipitate collected by filtration. The solid was recrystallized from ethyl acetate to give 0.9 g of H~[(9H-fluoren-9-ylmethyloxy)carbonyl) ]-L-leucine, mp 151-155°C.
EXAMPLE 3
Inhibition of Ear Edema Caused by Tetradecanoylphorbolacetate.
CF-1 Mice, 25-30 g body weight, six animals per group, were used. Test compounds (compounds of Formula I where Rt, R2, R, and R4 are hydrogen and R, is as indicated in Table 1) were administered intraperitoneally (100 g/kg) or topically as follows. For intraperitoneal administration, the test compound was dissolved in dimethyl sulfoxide or 0.5% methylcellulose and 100 μl was injected 30 minutes prior to irritant (100 mg/kg, i.p.). For topical administration, the test compound was dissolved in either acetone, ethanol or dimethyl sulfoxide and 5 μl (100 μg) applied to the
11 upper surface (1 cm7) of the ear and an additional 5 μl (100 μg) applied to the lower surface (1 cm7) of the ear fifteen minutes prior to application of the irritant. A solution of the irritant, tetradecanoylphorbolacetate, 200 μg/ml, was added to the surface of the ear, 5 μl added to the upper surface and 5 μl to the lower surface. After three hours, the thickness of the ear was measured to 0.01 mm by a micrometer with loose drag positioned at the lateral-most edge of the mid-point of the pinna. Data were calculated as the inhibition by the test compound of increased ear thickness compared to control animals receiving only the irritant. The results are reported in Table 1.
Table 1
Inhibition of Ear Edema Caused by Tetradecanoylphorbolacetate
% Inhibition, %Inhibition
Amino Acid intraperitoneal topical
L-Glycine 38
L-Alanine "26 —
L-Proline 58
L-Isoleucine 48 —
L-Valine 46
L-Homophenylalanine 3 —
L-Asparagine 9 —
L-Lysine 14
L-Tryptophan 5
D-Phenylalanine 35
L-Phenylalanine 78
D-Leucine 54 21
L-Leucine 54 41
L-Isoleucine 48 39
L-Methionine 19
Piroxica (Feldene') 40
(Reference Standard)
Dexamethasone 50
(Reference Standard, 10 mg/kg)
12 EXAMPLE 4
Inhibition of Ear Edema Caused by Arachidonic Acid
CF-1 Mice, 25-30 g body weight, six animals per group, were used. Test compounds (compounds of Formula I where R,, R2, R3 and R4 are hydrogen and j is as indicated in Table 2) were administered intraperitoneally (100 mg/kg) as follows. For intraperitoneal administration, test compound was dissolved in DMSO or 0.5% methylcellulose and 100 μl was injected 30 minutes prior to i.p. administration of 100 mg/kg of arachidonic acid. A solution of the irritant, arachidonic acid, 100 mg/ml in ethanol, was added to the surface of the ear, 5 μl added to the upper surface and 5 μl to the lower surface. After sixty minutes, the thickness of the ear was measured to 0.01 mm by a micrometer with loose drag positioned at the lateral-most edge of the mid-point of the pinna. Data were calculated as the percent inhibition by the test compound of increased ear thickness compared to control animals receiving only the irritant. The results are reported in Table 2.
13
Table 2 Inhibition of Ear Edema Caused by Arachidonic Acid
Amino Acid % Inhibition, % Inhibition, intraperitoneal tooical
L-Glycine 75 —
L-Alanine 62 _______
L-Proline 45 —
L-Isoleucine 0 —
L-Valine 0 —
L-Homophenylalanine 24 —
D-Phenylalanine 70 —
L-Phenylalanine 70 —
D-Leucine 9 21
L-Leucine 62 41
L-Isoleucine 0 39
Piroxicam (Feldene®) 86 —
EXAMPLE 5
Inhibiton of Ear Edema Caused by Xylene
CF-1 Mice, 25-30 g body weight, six animals per group, were used. Test compounds (compounds of Formula I where R,, R2, RΛ and R4 are hydrogen and R, is as indicated in Table 3) were administered intraperitoneally (100 mg/kg) or topically as follows. For intraperitoneal administration, the test compound was dissolved in DMSO or 0.5% methylcellulose and 100 μl was injected 30 minutes prior to irritant. For topical administration, test compound was dissolved in either acetone, ethanol or dimethyl sulfoxide and 5 μl (10 μg) applied to the upper surface (1 cm3) of the ear and an additional 5 μl (10 μg) applied to the lower surface (1 cm') of the ear fifteen minutes
prior to application of the irritant. The irritant, xylene, was added to the surface of the ear, 20 μl added to the upper surface and 20 μl to the lower surface. After two hours, the thickness of the ear was measured to 0.01 mm by a micrometer with loose drag positioned at the lateral-most edge of the mid¬ point of the pinna. Data were calculated as the inhibition by the test compound of increased ear thickness compared to that of control animals receiving only the irritant. The results are reported in Table 3.
Table 3 Inhibition of Ear Edema Caused by Xylene
% Inhibition, %Inhibition,
Amino Acid intraperitoneal topical
L-Glycine 12 22
L-Alanine 39 27
L-Proline 43 —
L-Valine 27 19
L-Homophenylalanine 57 27
D-Phenylalanine 45 —
L-Phenylalanine 49 50
D-Leucine 35 0
L-Leucine 47 66
L-Isoleucine 40 39
Piroxicam 74 —
D-Pro2-D-Trp7''-Substance P 44 —
(Reference Standard, 100 μg/ear)
EXAMPLE 6
Inhibition of Ear Edema Caused by Capsaicin
CF-1 Mice, 25-30 g body weight, six animals per group, were used. Test compounds (compounds of Formula I where R,, R,, R3 and R4 are hydrogen and R, is as indicated in Table 4) were administered intraperitoneally (100 mg/kg) as follows. The test compound was dissolved in DMSO or 0.5% methylcellulose and 100 μl was injected 30 minutes prior to irritant. The irritant, capsaicin, 25 mg/ml, was added to the ear, 5 μl added to the upper surface and 5 μl to the lower surface. After thirty minutes, the thickness of the ear was measured to 0.01 mm by a micrometer with loose drag positioned at the lateral-most edge of the mid-point of the pinna. Data were calculated as the inhibition by the test compound of increased ear thickness compared to control animals receiving only the irritant. The results are reported in Table 4.
Table 4
Inhibition of Ear Edema Caused by Capsaicin
% Inhibition, Amino Acid intraperitoneal
L-Proline 65 L-Valine 45
L-Phenylalanine
D-Leucine 66
L-Leucine 74
L-Isoleucine 44 D-Pro2-D-Trp;7'-Substance P 34
(Reference Standard, 100 μg/ear)
1 6 EXAMPLE 7
Inhibition of Ear Edema Caused by Oxazolone
CF-1 Mice, 25-30 g body weight, six animals per group, were used. The mice were sensitized to the irritant two weeks prior to the test by dribbling 100 μl of a 3% solution of oxazolone in acetone onto the abdominal skin of the animal. Test compounds (compounds of Formula I where Rιr R2, R3 and R4 are hydrogen and Rj is as indicated in Table 5) were administered intraperitoneally as follows. The test compound was dissolved in DMSO or 0.5% methylcellulose and 100 μl (100 mg/kg) was injected 30 minutes prior to irritant. The irritant, 3% oxazolone in acetone, was added to the surface of the ear, 5 μl added to the upper surface and 5 μl to the lower surface. After twenty four hours, the thickness of the ear was measured to 0.01 mm by a -micrometer with loose drag positioned at the lateral-most edge of the mid¬ point of the pinna. Data were calculated as the inhibition by the test compound of increased ear thickness compared to control animals receiving only the irritant. The results are reported in Table 5.
1 7 Table 5
Inhibition of Ear Edema Caused by Oxazolone
% Inhibition,
Amino Acid intraperitoneal
L-Glycine 45
L-Alanine 46 L-Proline 51
L-Valine 42
L-Homophenylalanine 58
D-Phenylalanine 52
L-Phenylalanine 62 D-Leucine 25
L-Leucine 58
L-Isoleucine 48
Dexamethasone 48 (at 10 mg/kg) Reference Standard
EXAMPLE 8
Reverse Passive Artus Reaction
Male CD rats weighing between 200 and 250 g were used. Test compounds (compounds of Formula I where R,, R2, R3 and R4 are hydrogen and R, is as indicated in Table 6) were dissolved in dimethyl sulfoxide and 200 μl of this solution (100 mg/kg) were injected intraperitoneally one hour before administration of the antigen. The animals were anesthetized inhalationally with isoflurane and then were injected through the penile vein with 1 ml of a solution of 2.5 mg of Evan's blue dye and 5.0 mg of human serum albumin in 1 ml of saline. This treatment was followed immediately by intracutaneous
18 injections of 0.03 ml of anti-human albumin diluted to contain 3.65 mg of antibody at 3 sites along the midline back. Anesthesia was terminated and after three hours the animals were sacrificed. The skin was removed and the blue stained areas cut out. The skin patches were soaked overnight in stoppered tubes containing 1 ml of 1 N potassium hydroxide at 37°C. Then 9 ml of a mixture of five parts of 0.6 N phosphoric acid and thirteen parts of acetone was added to the tubes. The tube contents were agitated and centrifuged, and the absorbance of the supernatant liquid was measured at 620 nm. The data were calculated as inhibition of blueing by test compound compared to control animals receiving only antigen and antibody. The results are reported in Table 6.
Table 6
Reverse Passive Artus Reaction
% Inhibition,
Amino-Acid intraperitoneal
L-Glycine 0
L-Alanine 18
L-Proline 41
L-Valine 30
L-Homophenylalanine 76
L-Phenylalanine 47
D-Leucine 42
L-Leucine 48
L-Isoleucine 31
Colchicine 64
(Reference Standard 1 mg/kg)
19
For purposes of completing this disclosure, all references cited hereinabove are hereby incorporated by reference.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art on reading this disclosure will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.