COMPOSITIONS OF THERAPEUTIC AGENTS SUITABLE FOR ORAL ADMINISTRATION
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates generally to the field of drug delivery and, more specifically, to compositions of therapeutic agents that can administered orally to a subject .
BACKGROUND INFORMATION
The ability to isolate biologically active substances in large amounts has provided accessability to agents that potentially can be useful as therapeutics. In addition, the ability to screen large libraries of synthetic molecules such as peptides has made available new drugs that can be directed to specific targets known to be involved in disease. Unfortunately, while such biologically active substances can show a specific effect when examined using in vi tro assays, the same substances often are not useful when administered to an individual. In particular, potential protein or peptide drugs often are not effective when administered orally due, for example, to enzymatic degradation in the digestive tract or to lack of absorption from the intestinal lumen.
Efforts to modify such biologically active substances have been successful to various degrees. For example, peptides potentially useful as drugs have been modified by incorporating (D) -amino acids in place of one or more corresponding naturally occurring (L) -amino acids in a peptide. Such modified peptides often can be resistant to enzymatic degradation due to steroselectivity of digestive enzymes. However, such
modifications that alter the stereochemistry of a peptide also can result in the peptide not interacting with its biological target and, therefore, losing its efficacy.
In order to avoid problems associated with chemical modification of a potential biologically active substance, such substances have been formulated into compositions that physically protect the agent from degradation or improve the absorption of the agent from the gut into the circulation. Although such compositions have found use in making various biologically active substances orally available, more effective compositions continually are being sought. Thus, a need exists for compositions that permit oral administration of a biologically active substance that otherwise lacks efficacy when administered orally. The present invention satisfies this need and provides related advantages as well .
SUMMARY OF THE INVENTION
The present invention provides compositions of biologically active substances that are suitable for oral administration. For example, the invention provides compositions comprising a microemulsion and a cytokine regulatory agent having the structure
X,-X5- (D)Phe-Arg- (D)Trp-X3 or X«-X5- (D) Phe-Arg- (D)Trp-X3, where Xl t X2, X3, X and X5 are amino acids or amino acid analogs, or modified forms of such structures.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 compares the effect of orally administered CRA-1 with indomethacin on arachidonic acid induced ear swelling and demonstrates the dose dependent effect obtained with orally administered CRA-1.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions useful for formulating a biologically active substance such that the substance can be administered as an oral medicament. In particular, a composition of the invention comprises a microemulsion and an adjuvant. The invention is exemplified by the preparation of a composition comprising a microemulsion containing salicylic acid as an adjuvant, the composition further comprising the cytokine regulatory agent (CRA) , Ac-Nle- Gln-His- (D)Phe-Arg- (D) Trp-Gly-NH2 ("CRA-1"). CRA's are known in the art and described, for example, in U.S. Patent No. 5,420,109; issued May 30, 1995, which is incorporated herein by reference (CRA's previously were known as "cytokine restraining agents") .
As used herein, the term "microemulsion" has its commonly understood meaning of a liquid dispersion of water and oil made homogenous, transparent and stable by addition of a surfactant and a cosurfactant (see Gennaro, "Remington's Pharmaceutical Sciences" (Mack Publishing
Co. 1990, which is incorporated herein by reference) . In general, a microemulsion contains oil globules dispersed in the aqueous phase or water globules dispersed in the oil phase. The size of the globules generally ranges from about 10 nm to about 100 nm.
Microemulsions have been formed, for example, by dispersing an anionic surfactant such as sodium lauryl sulfate in benzene, then adding a small amount of water followed by gradual addition of a cosurfactant such as pentanol . Microemulsions are used, for example, in cosmetics, foods, dry cleaning agents, and waxes and polishes. A microemulsion useful in the invention is exemplified herein by a Formulation of "LABRAFAC LIPOPHILE L 1349" (medium chain triglycerides) as the
oil, "PLUROL OLEIQUE CC 497" (polyglyceryl oleate FCC) as the surfactant, and "LABRASOL" (satureated polyglycolyzed C5-C10 glycerides) as the cosurfactant (Gattefosse; estwood NJ; see Example I) .
As used herein, the term "adjuvant" means an agent that enhances the bioavailability of a biologically active substance. For example, an adjuvant can result in increased absorbability of the biologically active substance from the gastrointestinal tract into the circulation or can prevent the nonspecific binding of the substance so as to increase the effective concentration of the substance in a subject. An adjuvant can act, for example, by complexing with a biologically active substance, thereby enhancing the solubility of the substance. Thus, caffeine has been used an adjuvant in combination with benzocaine to enhance the dissolution of benzocaine. Similarly, hydroquinone has been used as an adjuvant in combination with digoxin to enhance dissolution of digoxin.
As disclosed herein, salicylic acid (SIGMA
Chemical Co.; St. Louis MO) is another adjuvant, which is useful in the present invention (see Example I) . Although the mechanism by which salicylic acid acts as an adjuvant, it does not appear to effect absorption of the examined biologically active substance (see Example II) .
As used herein, the term "biologically active substance" means a chemical or biological molecule that is useful as a therapeutic agent. Thus, a biologically active substance can be, for example, an organic molecule or can be a peptide, polypeptide or protein. The usefulness of the claimed composition is demonstrated by the oral administration of a CRA, which is a modified peptide that is not therapeutically effective when administered orally in a free form.
In particular, the effectiveness of the claimed composition for permitting oral delivery of a biologically active substance was demonstrated by showing that a CRA having the structure Ac-Nle-Gln-His- (D) Phe- Arg- (D)Trp-Gly-NH2 ("CRA-1"), when formulated in a composition of the invention, effectively regulates lipopolysaccharide (LPS) induced interleukin-10 ("IL-10") and tumor necrosis factor-α ("TNF ") levels and effectively reduces arachidonic acid induced dermal swelling in an experimental animal model (see Example II and Figure 1) . Remarkably, the therapeutic effect of orally administered CRA-1 formulated in a composition of the invention occurred in the absence of a significant increase in plasma CRA-1 levels.
The efficacy of a composition of the invention in providing a vehicle that allows oral administration of a biologically active substance was demonstrated using a cytokine regulatory agent (CRA) . In general, a CRA has the structure:
x1 - X2 -His - (D)Phe - Arg - (D)Trp - X3, where
and
where Y1 and Y2 are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; Rx is H, COCH3, C2H5, CH2Ph, COPh, COO-t-butyl, COOCH2Ph, CH2CO- (polyethylene glycol) or A; R2 is H or COCH3; R3 is a linear or branched alkyl group having 1 to 6 carbon atoms or a cyclic alkyl group having 3 to 6 carbon atoms; R4 is (CH2) m- C0NH2 , (CH2) m- CONHRj or (CH2)m-C0NHA; R5 is OH, 0R3, NH2, SH, NHCH3, NHCH2Ph or A; and R6 is H or R3;
and where "Ph" is C€H5, "m" is 1, 2 or 3 , ""nn II is 0, 1, 2 or 3, and "A" is a carbohydrate having the general formula:
(U.S. Patent No. 5,420,109; supra , 1995]
In addition, a CRA can have the structure;
X4 - X5 - (D)Phe - Arg - (D)Trp - X3, where
H , COCH3 or absent ;
X5 is His , H or COCH3 ; and
where Y1 and Y2 are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; Rx is H, COCH3, C2H5, CH2Ph, COPh, COO-t-butyl, COOCH2Ph, CH2CO- (polyethylene glycol) or A; R2 is H or COCH3; R4 is (CH2) ra-CONH2, (CH2) m-CONHRi or (CH2)πι-CONHA; R5 is OH, OR3, , NH2 , SH, NHCH3, NHCH2Ph or A; and R6 is H or R3;
and where "Ph" is C6H5, "m" is 1, 2 or 3 , "n" is 0, 1, 2 or 3, and "A" is a carbohydrate having the general formula
(see U.S. Patent No. 5,420,109, supra, 1995, which also discloses methods for making a CRA) .
In general, a CRA is a peptide or a peptide- like structure such as a peptidomimetic or a peptoid (see Ecker and Crooke, Biotechnology 13:351-360 (1995), and Blondelle et al . , Trends Anal. Chem. 14:83-92 (1995), and the references cited therein, each of which is incorporated herein by reference) . Amino acids are indicated herein by their commonly known three letter code, where " (D) " designates an amino acid having the "D" configuration, as compared to the naturally occurring (L) -amino acids; "Nle" is the three letter code for norleucine. Where no specific configuration is indicated, one skilled in the art would understand the amino acid to be an (L) -amino acid. In the CRA structures shown above, "Ph" indicates a "phenyl" group (C6H5) . CRA peptides are written in the conventional manner, such that the amino-terminus (N-terminus) is shown to the left and the carboxy-terminus (C-terminus) is shown to the right.
One skilled in the art would know that the choice of amino acids or amino acid analogs incorporated into the peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the CRA. Selective modification of a reactive group in a peptide also can impart desirable characteristics to a CRA. For example, the N-terminus can be modified by acetylation or the C-terminus can be modified by amidation. Methods for modifying the N-terminus or C-terminus of a peptide are well known in the art (see, for example, in U.S. Patent No. 5,420,109, supra, 1995).
The choice of modifications made to the reactive groups present on the peptide is determined by a desirable characteristic required in the CRA. CRA-1, which has the structure Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH2, is an
example of a CRA that is modified both by acetylation at the N-terminus and by amidation at the C-terminus.
A cyclic peptide also can be an effective CRA. A cyclic peptide can be obtained by inducing the formation of a covalent bond between, for example, the amino group at the N-terminus of the peptide and the carboxyl group at the C-terminus. For example, the peptide, cyclo (His- (D) he-Arg- (D) Trp) , can be produced by inducing the formation of a covalent bond between His and (D)Trp. Alternatively, a cyclic peptide can be obtained by forming a covalent bond between a terminal reactive group and a reactive amino acid side chain or between two reactive amino acid side chains such as the sulfhydryl reactive groups present in cysteine residues. One skilled in the art would know that the choice of a particular cyclic peptide is determined by the reactive groups present on the peptide as well as the desired characteristic of the peptide. Cyclization of a CRA peptide can provide the CRA with increased stability in vivo.
In addition to the examples provided above, other representative cytokine regulatory agents include:
1) Ac-Nle - Gin - His - (D) Phe - Arg - (D)Trp -Gly-OH;
2) Ac-Nle - Gin - His - (D)Phe - Arg - (D)Trp -Gly-OC2H5; 3) Ac-Nle - Gin - His - (D)Phe - Arg - (D)Trp -Gly-NH-NH2 ;
4) Ac-Nle - Asn - His - (D)Phe - Arg - (D)Trp -Gly-NH2;
5) Ac-Nle - Asn - His - (D) Phe - Arg - (D)Trp -Gly-OH;
6) Ac-Nle - Gin - His - (D)Phe - Arg - (D)Trp - Gly-
NHCH2CH2Ph; 7) Ac-Nle - Gin - His - (D)Phe - Arg - (D)Trp - Gly- NHCH2Ph;
8)
9) Ac-Gin - His - (D) Phe - Arg - (D)Trp - Gly-NH
2; 10) Ac-Nle - Gin - His - (D) Phe - Arg - (D)Trp-NH
2;
11) His- (D) Phe-Arg- (D)Trp-NH
2;
12) Ac-His - (D)Phe - Arg - (D)Trp-OH;
13) Ac-His - (D)Phe - Arg - (D)Trp - Gly-NH2; and
14) Ac-His- (D) Phe-Arg- (D)Trp- (CH2NHAc) -Gly-NH2, where " - (CH2NHAc) - " indicates a modified peptide bond between (D) Trp and Gly.
Peptide cytokine regulatory agents as described above are characterized, in part, by a core structure (D) Phe-Arg- (D) Trp, where the amino acids are indicated by their commonly known three letter code and where " (D) " designates an amino acid having the "D" configuration, as opposed to the naturally occurring L-amino acids. Where no specific configuration is indicated, one skilled in the art would understand the amino acid to be an (L) -amino acid. In the peptides exemplified above, "Nle" is the three letter code for norleucine and "Ph" indicates a "phenyl" group (C6H5) .
Cytokine regulatory agents are synthesized using a modification of the solid phase peptide synthesis method of Merrifield (J. Am. Chem. Soc.. 85:2149 (1964), which is incorporated herein by reference; see U.S. Patent No. 5,420,109, supra , 1995) or can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M. , Principles of Peptide Synthesis 2nd revised ed. (Springer-Verlag, 1988 and 1993) , which is incorporated herein by reference) . Peptides prepared by the method of Merrifield can be synthesized using an automated peptide synthesizer such as the Applied Biosystems 431A-01 Peptide Synthesizer (Mountain View, CA) or using the manual peptide synthesis technique described by Houghten, Proc . Natl . Acacj. Sci .. USA 82:5131 (1985), which is incorporated herein by reference .
CRA-1 was synthesized using amino acids or amino acid analogs, the active groups of which were protected as required using, for example, a t-butyldicarbonate (t-BOC) group or a fluorenylmethoxy carbonyl (FMOC) group. Amino acids and amino acid analogs can be purchased commercially (Sigma Chemical Co.; Advanced Chemtec) or synthesized using methods known in the art. Peptides synthesized using the solid phase method can be attached to resins including 4-methylbenzhydrylamine (MBHA) , 4- (oxymethyl) -phenyl acetamido methyl and 4- (hydroxymethyl) phenoxymethyl- copoly (styrene-1% divinylbenzene) (Wang resin), all of which are commercially available, or to p-nitro benzophenone oxime polymer (oxime resin) , which can be synthesized as described by De Grado and Kaiser, J. Org. Che . 47:3258 (1982), which is incorporated herein by reference (see Example I) .
One skilled in the art would know that the choice of amino acids or amino acid analogs incorporated into the peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the cytokine regulatory peptide. The skilled artisan further would recognize that selective modification of a peptide such as a CRA can impart desirable characteristics such as increased solubility on a CRA.
With regard to selective modification of the reactive groups in a peptide, the peptides can be manipulated while still attached to the resin to obtain N-terminal modified compounds such as an acetylated peptide or can be removed from the resin using hydrogen fluoride or an equivalent cleaving reagent and then modified. Compounds synthesized containing the C-terminal carboxy group (Wang resin) can be modified after cleavage from the resin or, in some cases, prior to solution phase synthesis. Methods for modifying the
N-terminus or C-terminus of a peptide are well known in the art and include, for example, methods for acetylation of the N-terminus or methods for amidation of the C-terminus. Similarly, methods for modifying side chains of the amino acids or amino acid analogs are well known to those skilled in the art of peptide synthesis. The choice of modifications made to the reactive groups present on the peptide will be determined by the characteristics that the skilled artisan requires in the peptide.
A newly synthesized peptide can be purified using a method such as reverse phase high performance liquid chromatography (RP-HPLC; see U.S. Patent No. 5,420,109, supra, 1995) or other methods of separation based on the size or charge of the peptide. Furthermore, the purified peptide can be characterized using these and other well known methods such as amino acid analysis and mass spectrometry.
A composition of the invention, which comprises a microemulsion and an adjuvant, that contains a biologically active substance such as a CRA also can contain an additional material such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
In addition, a composition of the invention can contain a physiologically acceptable compound that acts, for example, to stabilize the biologically active substance or increase the absorption of the substance. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or
glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the particular physico-chemical characteristics of the specific biologically active substance .
The concentration of a biologically active substance required for a therapeutic effect will depend, for example, on the particular substance and on the disease to be treated. For example, CRA's are known to be useful for treating various conditions associated with altered cytokine activity (see U.S. Patent No. 5,420,109, supra, 1995). Thus, CRA's are useful for treating, for example, inflammatory reactions and patho-immunogenic diseases such as rheumatoid arthritis.
In order to effectively treat a condition characterized, in part, by altered cytokine activity, a CRA must be administered in an effective dose, which is about 0.01 to 200 mg/kg body weight per administration. The total treatment dose can be administered to a subject as a single dose or can be administered as a series of multiple doses over a period of time. One skilled in the art would know that the amount of a CRA required to obtain an effective dose in a subject depends on many factors including the specific CRA being administered and the age and general health of the subject. In view of these factors, the skilled artisan would recognize that the amount of a biologically active substance required to obtain an effective dose for treating a particular condition can be determined by monitoring the treated patient's clinical course using routine methods such as radiologic, immunologic and, where indicated, histopathologic methods.
The efficacy of using a composition of the invention for orally administering a biologically active substance was confirmed by demonstrating that oral administration of CRA-1 can regulate cytokine activity in mice exposed to bacterial lipopolysaccharide (LPS) and can decrease the amount of dermal swelling in mice treated with arachidonic acid. These animal models are recognized as useful models for bacterial sepsis and inflammation, respectively (see Inflammation 17:723-741 (1993); Am. J. Pathol. 143:1121-1130 (1993), each which is incorporated herein by reference) .
The following examples are intended to illustrate but not limit the invention.
EXAMPLE I
PREPARATION OF A COMPOSITION COMPRISING
A MICROEMULSION AND AN ADJUVANT
This example describes methods for preparing a composition of the invention, which permits oral administration of a CRA that otherwise is not therapeutically effective when administered orally.
A cytokine regulatory agent having the amino acid sequence Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH2 (CRA-1) was prepared as described in U.S. Patent No. 5,420,109 (supra , 1995). A stock solution of 60 mg/ml salicylic acid in water was prepared.
Twenty grams of a mixture of 8.429 g "LABRASOL, " 3.571 g "PLUROL OLEIQUE CC 497" and 8.000 g "LABRAFAC LIPOPHILE" (Gattefosse) was prepared and vortexed to a homogeneous, clear solution ("premicroemulsion") . A 6 mg/ml stock salicylic acid/microemulsion was prepared by transferring 5 g
premicroemulsion into a clean glass vial, adding 500 μl 60 mg/ml salicylic acid and vortexing until the solution was clear.
A formulation of 27 mg/ml CRA-1 in 2 mg/ml salicylic acid/microemulsion was prepared by transferring 70.0 mg CRA-1 into a clean glass vial, adding 300 μl water and vortexing and sonicating until the CRA-1 was dissolved, then transferring 190 μl of the CRA-1 solution to a clean vial containing 0.996 g premicroemulsion, vortexing the mixture until clear, adding 0.496 g 6 mg/ml salicylic acid/microemulsion and vortexing until a clear, single phase microemulsion was obtained.
A formulation of 10 mg/ml CRA-1 in 2 mg/ml salicylic acid/microemulsion was prepared by transferring 39.1 mg CRA-1 into a clean glass vial, adding 300 μl water and vortexing and sonicating until the CRA-1 was dissolved, then transferring 250 μl of the CRA-1 solution to a clean vial containing 1.992 g premicroemulsion, vortexing the mixture until clear, adding 1.008 g 6 mg/ml salicylic acid/microemulsion and vortexing until a clear, single phase microemulsion was obtained.
A "placebo" vehicle containing 2 mg/ml salicylic acid was prepared by adding 1.337 g premicroemulsion, 200 μl water and 0.675 g 6 mg/ml salicylic acid together and vortexing until the solution was clear.
EXAMPLE II
ORAL ADMINISTRATION OF CRA-1 FORMULATED TN A MICROEMULSION AND ADJUVANT IS THERAPEUTICALLY EFFECTIVE
This example compares the plasma levels of CRA-1 following intravenous injection or oral administration and demonstrates that CRA-1 formulated in a composition of the invention is therapeutically effective when administered orally.
A. Plasma concentration of CRA-1
Plasma concentrations of CRA-1 were examined following intravenous injection of free CRA-1 or oral administration of various concentrations of CRA-1 formulated in a composition of the invention (4 mice/ group) . Plasma CRA-1 concentrations were determined by a scintillation proximity assay based radioimmunoassay using a Packard Tri-Carb 1099 TR scintillation counter (Packard Instruments; Downers Grove IL) .
Mice injected intravenously with 13.5 mg/kg CRA-l (approximately 270 μg/mouse) had a level of about 65,000 ng CRA-l/ml blood plasma 5 min after injection. This level decreased to about 8000 ng/ml one hr after injection and essentially was a zero by 4 hr.
In comparison, mice that received 13.5 mg/kg CRA-l, which was formulated in a microemulsion and salicylic acid, by oral administration attained a maximum level of only about 600 ng/ml 10 min after administration. This level decreased to about 100 ng/ml after 1.5 hr and essentially was at zero after about 2.5 hr. In a second group of mice that received oral administration of 26.9 mg/kg CRA-l, a maximum plasma level of about 1750 ng CRA-l/ml plasma was reached by
10 min. This level decreased to about 250 ng/ml after 1.5 hr and essentially was at zero after 1.5 hr.
These results indicate that oral administration of a biologically active substance such as CRA-l in a composition comprising a microemulsion and an adjuvant such as salicylic acid results in about a 100 fold lower plasma level of the substance as compared to intravenous administration of the substance.
B. Effect of orally administered CRA-l on TNF and IL-10 levels following treatment with LPS
The effectiveness of CRA-l administered orally or for decreasing tumor necrosis factor (TNF) levels and for increasing IL-10 levels in lipopolysaccharide (LPS; endotoxin) treated mice was examined.
Balb/c female mice weighing approximately 20 g were placed into six groups of eight mice each, except as indicated, as follows: two control groups, one of which was injected intraperitoneally (ip) with phosphate buffered saline (PBS) and other of which received placebo (2 mg/ml salicylic acid in microemulsion) orally; one group treated by ip injection of 300 μg CRA-l; and four groups, each of which received orally administered 0.25 mg, 0.5 mg, 1.0 mg or 2.7 mg CRA-l formulated in microemulsion/salicylic acid (see Example I) . One minute after administration of CRA-l, 100 μg LPS in 0.9% saline was administered by ip injection into the mice. In addition, a seventh group of five mice was orally administered 1.0 mg CRA-l, then LPS injection was delayed for 15 min.
Blood samples were collected from the orbital sinus of the mice 90 min after LPS was administered. The plasma was separated by centrifugation at 3000 x g for 5
min, then diluted with four volumes of lx phosphate buffer saline (pH 7.4) containing 1% bovine serum albumin. 100 μl samples of serum were assayed by ELISA using commercially available kits for TNFα (Genzyme; Cambridge MA) or for IL-10 (R & D Systems; Minneapolis MN) .
The mean (+/- SEM) TNFα and IL-10 levels were determined for each group of mice and the percent of control (PBS) for TNF and IL-10 levels were calculated. TNFα and IL-10 levels in mice injected with placebo microemulsion were essentially identical with the control (PBS) mice. Mice injected ip with 300 μg CRA-l had TNFα levels that were about 25% of control mice and IL-10 levels that were about 325% of control. These results indicate that orally administered CRA-l can regulate cytokine levels in LPS treated mice.
£_, Effect of Orally Administered CRA-l on Arachidonic
Acid- Induced Dermal Swelling in Mice
The effect of orally administered CRA-l on arachidonic acid-induced dermal swelling in mice was examined .
Experiments were performed using female Balb/c mice weighing approximately 20 g. Saline (control; 10 mice) , indomethacin (50 mg/kg; 5 mice) or 25 (10 mice) , 50 (10 mice) or 135 (9 mice) mg/kg CRA-l in microemulsion and salicylic acid were administered orally to the mice. Thirty min later, a 10 μl pipet was used to apply 10 μl arachidonic acid (AA) solution (100 mg/ml ethanol; Calbiochem-Novabiochem; San Diego CA) to the inner and outer surfaces of the right ear of each mouse.
Ear thickness was measured with a hand-held spring loaded caliper immediately before and 60 min after
AA application. Increase in ear thickness was calculated by subtracting the ear thickness prior to AA administration from the thickness 60 min after AA administration. 50 mg/kg indomethacin reduced dermal swelling to about 40% of the swelling in control mice
(Figure 1) . In comparison, orally administered CRA-l at 25, 50 or 135 mg/kg reduced swelling to about 82%, 60% or 24% of the swelling in control mice (Figure 1) . These results indicate that orally administered CRA-l reduces arachidonic acid induced ear swelling in a dose dependent manner, despite the observation that oral administration of CRA-l did not produce a correspondingly high plasma concentration of the agent.
Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.