Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4245—Oxadiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
  • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
  • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
  • C07D271/10—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• the present invention relates to certain substituted oxadiazole nonpeptides. which are useful as inhibitors of serine proteases.
• the serine proteases are a class of enzymes, which includes elastase, chymotrypsin, cathepsin G, trypsin and thrombin. These proteases have in common a catalytic triad consisting of Serine- 195, Histidine-57 and Aspartic acid- 102
• HNE Human neutrophil elastase
• PMNs polymorphonuclear leukocytes
• ⁇ pPI ⁇ i-proteinase inhibitor
• stimulated PMNs produce a burst of active oxygen metabolites, some of which (hypochlorous acid for example) are capable of oxidizing a critical met ionine residue in ⁇ pPI.
• Oxidized ⁇ pPI has been shown to have limited potency as an PINE inhibitor and it has been proposed that alteration of this protease/antiprotease balance permits HNE to perform its degradative functions in localized and controlled environments.
• HNE- mediated processes are implicated in other conditions such as arthritis, periodontal disease, glomerulonephritis, dermatitis, psoriasis, cystic fibrosis, chronic bronchitis, atherosclerosis, Alzheimer's disease, organ transplantation, comeal ulcers, and invasion behavior of malignant tumors.
• X and Y are independently O or N;
• Ri is alkyl
• R 2 and R 3 are independently H or alkyl; or together form a ring consisting of 3-5 carbons optionally substituted with one or more heteroatoms selected from O, S or N wherein N is optionally substituted with H or alkyl; n is O or 1;
• D is a direct bond or valine;
• A is -C(O)- or -OC(O)-; and
• i is alkyl or aryl substituted with one or two N atoms; or a pharmaceutically acceptable salt thereof.
• compounds of the present invention comprise a 1,2,4-oxadiazole (i.e., X is 0; Y is N) or 1 ,3,4 oxadiazole ring (i.e., X is N; Y is O).
• R ⁇ is alkyl, such as tert-butyl.
• R 2 and R 3 are independently alkyl, such as isopropyl, or H.
• R 2 is isopropyl and R 3 is H.
• R 4 is alkyl, such as an w ⁇ -propyl or tert-butyl group.
• R is an aryl group substituted with one or two N atoms, such as a 2-pyridyl or 5-imidazolyl group.
• alkyl means C1-C15, and preferably C
• alkyloxycarbonyl means alkyl-O-C(O)- wherein the meaning of alkyl is defined above.
• alkyloxycarbonyl examples include methyloxycarbonyl, iO-propyloxycarbonoyl and tert-butyloxycarbonyl.
• Pharmaceutically acceptable salts of the compounds described above are within the scope of the invention.
• Figure 1 is a schematic representation of the synthetic scheme for the Boc protected amino alcohol intermediates used in the invention.
• Figure 2 is a schematic representation of the synthetic scheme for the compounds of the invention.
• FIG. 3 shows structural representations of certain embodiments of the invention.
• FIG. 4 shows structural representations of certain embodiments of the invention.
• the compounds of the present invention have been found to be potent inhibitors of the serine protease human neutrophil elastase (HNE). They are reversible inhibitors that presumably form a transition state intermediate with the active site serine residue.
• HNE serine protease human neutrophil elastase
• the compounds are characterized by their low molecular weights, high selectivity with respect to HNE and stability regarding physiological conditions. Therefore, the compounds can be implemented to prevent, alleviate and/or otherwise treat diseases, which are mediated by the degradative effects associated with the presence of HNE. Their usage is of particular importance as they relate to various human treatment in vivo but may also be used as a diagnostic tool in vitro.
• the present invention provides, but is not limited to, specific embodiments set forth in the Examples as well as those set forth below.
• Example 2 N-/_. ⁇ -Propyloxycarbonyl-(l ,2,3,4-tetrahydroisoquinoline)-3-(S)-N- [l-(2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(5)-methylpropyl]amide
• Example 3 N-(Imidazoy l-5-carbonyl)-S-( 1,2,3, 4-tetrahydroisoquinoline)-3 -
• Example 7 N-wo-Propyloxycarbonyl-(2,3-dihydro-lH-indole)-2-(S)-N-[l -(2- [5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide
• Example 8 N-(Imidazoyl-5-carbonyl)-(2,3-dihydro-lH-indole)-2-(S)-N-[l-(2-
• Elastase is a member of the class of enzymes known as serine proteases.
• This enzyme class also includes, for example, chymotrypsin, cathepsin G, trypsin and thrombin.
• These proteases have in common a catalytic triad consisting of Serine- 195, Histidine-57 and Aspartic acid-102 (chymotrypsin numbering system). The precise hydrogen bond network that exists between these amino acid residues allows the Serine- 195 hydroxyl to form a tetrahedral intermediate with the carbonyl of an amide substrate.
• this intermediate results in the release of a free amine and the acylated enzyme.
• this newly formed ester is hydrolyzed to give the native enzyme and the carboxylic acid.
• this carboxyl component helps characterize the specificity for the enzyme.
• the carboxyl component is a peptide
• the alpha-substituent of the amino acid is predominately responsible for the specificity toward the enzyme.
• the amino acid residues in the substrate that undergo the cleavage are defined as P ⁇ ...P n toward the N-terminus and P ⁇ '...P n ' toward the C-terminus. Therefore, the scissile bond is between the Pi and the Pi' residue of the peptide subunits.
• a similar nomenclature is utilized for the amino acid residues of the enzyme that make up the binding pockets accommodating the subunits of the substrate, where the binding pocket for the enzyme is designated by S ⁇ ...S n instead of Pi...P suspend as for the substrate.
• the characteristics for the Pi residue defining serine proteinase specificity is well established.
• the proteinases may be segregated into three subclasses: elastases, chymases and tryptases based on these differences in the Pi residues.
• the elastases prefer small aliphatic moieties such as valine whereas the chymases and tryptases prefer large aromatic hydrophobic and positively charged residues respectively.
• propyl endopeptidase One additional proteinase that does not fall into one of these categories is propyl endopeptidase.
• the Pi residue defining the specificity is a proline.
• This enzyme has been implicated in the progression of memory loss in Alzheimer's patients.
• Inhibitors consisting of ⁇ -keto heterocycles have recently been shown to inhibit propyl endopeptidase (Tsutsumi et al., J. Med. Chem., 37, 3492-3502 ' (1994)).
• ⁇ -keto heterocycles as defined herein allow for an increased binding in P' region of the enzyme.
• the present invention relates to P ⁇ -P n ' modifications, specifically, certain alpha-substituted keto-heterocycles composed of 1,2,4 oxadiazoles and 1,3,4-oxadiazoles.
• the alpha-substituent e.g., small aliphatic groups for elastase.
• HNE human neutrophil elastase
• endotoxin induced acute lung injury model in minipigs ⁇ AARD, 142:782-788 (1990)
• human polymorphonuclear elastase-induced lung hemorrhage model in hamsters (European Patent Publication No. 0769498) may be used;
• the method according to the canine model of reperfusion injury (J. Clin. Invest., 81 : 624-629 ( 1988)) may be used.
• the compounds of the present invention, salts thereof, and their intermediates can be prepared or manufactured as described herein or by various processes known to be present in the chemical art (see e.g., WO 96/16080).
• the compounds of the present invention may be prepared as described in Figures 1 and 2.
• Figure 1 relates to the synthesis of the Boc protected amino alcohol intermediates used in the invention.
• Figure 2 shows the use of the intermediates for the synthesis compounds of the invention.
• the 2-substituted 1,3,4-oxadiazoles (3) may be prepared via formation of methyl esters from the corresponding acids (1) utilizing, for example, thionyl chloride and methanol, followed by treatment with hydrazine in a suitable solvent to yield hydrazonic acids (2).
• esters can be prepared by methods known to one skilled in the art or those methods described in Comprehensive Organic Transformations (R. Larock, VCH Publishers 1989, 966-972). Reaction of (2) with triethyl orthoformate or trimethyl orthoformate and TsOH gives the requisite 2-substituted 1,3,4-oxadiazoles (3).
• Intermediate (3') can be formed utilizing standard conditions (e.g., butyllithium or other known alkyl lithium reagents, at low temperature in a polar aprotic solvent, and further, if desired, reacting with MgBrOEt?) and subsequently added to aldehyde (4) to give alcohol (5).
• standard conditions e.g., butyllithium or other known alkyl lithium reagents, at low temperature in a polar aprotic solvent, and further, if desired, reacting with MgBrOEt?) and subsequently added to aldehyde (4) to give alcohol (5).
• the aldehyde (4) may be prepared via any of three methods as described in Figure 1.
• One method reduces the intermediate that is formed between Boc-Val-OH and iso- propylchloro formate with sodium borohydride to give Boc-Valinol (12).
• the Boc-Valinol is oxidized with S0 3 -Pyin DMSO to give aldehyde (4).
• Another such method takes the Weinreb amide (13) that is prepared from Boc-Val-OH (11) and reduces it to the aldehyde using diisobutylalluminum hydride (DIBAL).
• DIBAL diisobutylalluminum hydride
• one may generate the ester (14) of the amino acid followed by reduction with DIBAL to afford aldehyde (4).
• the compounds described herein may be administered as pure chemicals, it is preferable to present the active ingredient as a pharmaceutical composition.
• the invention thus further provides the use of a pharmaceutical composition comprising one or more compounds together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients.
• the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
• compositions include those suitable for oral or parenteral (including intramuscular, subcutaneous and intravenous) administration.
• the compositions may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combination thereof, and then, if necessary, shaping the product into the desired delivery system.
• compositions suitable for oral administration may be presented as discrete unit dosage forms such as hard or soft gelatin capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or as granules; as a solution, a suspension or as an emulsion.
• the active ingredient may also be presented as a bolus, electuary or paste.
• Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
• the tablets may be coated according to methods well known in the art, e.g., with enteric coatings.
• Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non- aqueous vehicles (which may include edible oils), or preservatives.
• the compounds may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small bolus infusion containers or in multi-dose containers with an added preservative.
• compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• the compounds may be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch.
• Suitable transdermal delivery systems are disclosed, for example, in Fisher et al.- (U.S. Patent No. 4,788,603) or Bawas et al. (U.S. Patent No. 4,931,279, 4,668,504 and 4,713,224).
• Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
• the active ingredient can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122, 4383,529, or 4,051,842.
• compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• unit dosage forms such as lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.
• compositions can be adapted to provide sustained release of the active ingredient employed, e.g., by combination thereof with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
• compositions according to the invention may also contain other adjuvants such as flavorings, coloring, antimicrobial agents, or preservatives.
• the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
• a suitable dose will be in the range of from about 0.5 to about 100 mg kg/day, e.g., from about 1 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg day, most preferably in the range of 15 to 60 mg/kg/day.
• the compound is conveniently administered in unit dosage form, for example, containing 0.5 to 1000 mg, conveniently 5 to 750 mg, and most conveniently, 10 to 500 mg of active ingredient per unit dosage form.
• the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 ⁇ M, more preferably, about 1 to 50 ⁇ M, and most preferably, about 2 to about 30 ⁇ M. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 0.5- 500 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about
• the desired dose may be conveniently presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub- doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations, such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye:
• the final step in the process defined here is an oxidation of a 2° alcohol to a ketone.
• this transformation from an alcohol to ketone was preformed using dimethylsulfoxide and oxalyl chloride followed by base, which is known as the Swem oxidation.
• modifications of the Swem oxidation are known in the art and are acceptable in this present invention.
• alternative electrophilic molecules can be substituted for oxalyl chloride such as dicyclohexylcarbodumide, acetic anhydride, trifluoroacetic anhydride or sulfur trioxide (Mancuso et al., Synthesis 165 (1981)).
• N-chlorosuccinimide N-chlorosuccinimide
• base as described by the inventors in U.S. Pat. No. 5,618,792 or periodinane such as the Dess-Martin reagent.
• periodinane such as the Dess-Martin reagent.
• Still other methods may also be appropriate as described in Oxidation in Organic Chemistry (M. Hudlicky, ACS Monograph 186 (1990)).
• Symbols have the standard meanings as familiar to one skilled in the art, by way of example the following have been used: ml (milliliters), g (grams), TLC (thin layer chromatography), R (the ratio of the distance moved by a compound to the distance that the solvent front moved during the same time on a TLC plate), ⁇ NMR (proton nuclear magnetic resonance), DMSO-d ⁇ (deuterodimethylsulfoxide) and CDC1 3 (deuterochloroform).
• Trimethyl orthoformate was removed (50°C/43 mm Hg) and the residue was distilled at 120°C/23 mm Hg to give 2-tert-Butyl-l,3,4-oxadiazole (131 g) having the following physical data.
• the compound was prepared by oxidizing N-. o-propyloxycarbonyl-(l,2,3,4- tetrahydroisoquinoline)-3-(S)-N-[l-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(5)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swem Oxidation.
• Example 3 N-(Imidazoyl-5-carbonyl)-S-(l,2,3,4-tetrahydroisoquinoline)-3-(S)-N-[l- (2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide
• the compound was prepared by oxidizing N-(imidazoyl-5-carbonyl)-S-( 1 ,2,3 ,4- tetrahydroisoquinoline)-3-(S)-N-[l-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swem Oxidation.
• the compound was prepared by oxidizing N-methyloxycarbonyl-Z-valyl-S- (l,2,3,4-tetrahydroisoquinoline)-3-(S)-N-[l-(2-[5-(tert-butyl)-oxadiazolyl]hydroxy- methyl)-2-(S)-methylpropyl]amide using a procedure known to one skilled in the art, such as the Swem Oxidation.
• the compound was prepared by oxidizing N-tert-butyloxycarbonyl-S-(2,3- dihydro-lH-indole)-2-(S)-N-[l-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swern
• the compound was prepared by oxidizing N-/jo-propyloxycarbonyl-S-(2,3- dihydro-lH-indole)-2-(S)-N-[l-(2-[5-(tert-but ⁇ 'l)-oxadiazolylJhydroxymethyl)-2-(S)- methylpropylj amide using a procedure known to one skilled in the art, such as the Swem Oxidation.
• N- j o-propyloxycarbonyl-(2,3-dihydro-lH-indole)-2-(5)-N-[l- (2-[5-(tert-butyl)-oxadiazolylJhydroxymethyl)-2-(S)-methylpropylJamide was prepared using N- jO-propyloxycarbonyl-(2,3-dihydro-lH-indole)-2-(S)-carboxylic acid and 1 -[2- (5-tert-butyl)-l,3,4-oxadiazolylJ-2-(S)-amino-3-methylbutan-l-ol hydrochloride and a coupling method known to one skilled in the art.
• the compound was prepared by oxidizing N-(Imidazoyl-5-carbonyl)-(2,3- dihydro-lH-indoIe)-2-(S)-N-[l-(2-[5-(tert-butyl)-oxadiazolylJhydroxymethyl)-2-(S)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swern Oxidation.
• the compound was prepared by oxidizing N-(methyloxycarbonyl)-I-valyl-(2,3- dihydro- 1 H-indole)-2-(S)-N-[ 1 -(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swern Oxidation.
• the compound was prepared by oxidizing N-(pyridyl-3-carbonyl)-I-valyl-(2,3- dihydro-lH-indole)-2-(5)-N-[l-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)- methylpropyljamide using a procedure known to one skilled in the art, such as the Swern
• N-[l-(2-[5-(tert-butyl)-oxadiazolylJhydroxymethyl)-2-(S)-methylpropylJamide was prepared using N-(pyridyl-3-carbonyl)-Z-valyl-(2,3-dihydro-lH-indole)-2-(S)-carboxylic acid and l-[2-(5-tert-butyl)-l,3,4-oxadiazolylJ-2-(S)-amino-3-methylbutan-l-ol hydrochloride and a coupling method known to one skilled in the art.
• Example 11 In Vitro Inhibition of Elastase The following protocol was used to determine inhibitory activity of compounds described herein.
• the elastase used in the protocol was derived from human sputum ( ⁇ SE).
• a mother solution of the ⁇ SE enzyme was prepared from commercially available ⁇ SE (875 U/mg protein, SE-563, Elastin Product Co., Inc, Missouri, USA) by diluting with saline to 1,000 U/ml, which was further diluted to 2 U/ml at 0°C prior to use.
• a solution was prepared by mixing 100 ⁇ l 0.2 M ⁇ EPES- ⁇ aO ⁇ buffer (p ⁇ 8.0),
• Optical density (SPECTRA MAX 250, Molecular Devices) at 405 run due to p- nitroaniline generated by the enzyme reaction was measured at 37 * C in order to measure the reaction rate during the period that the production rate of p-nitroaniline remains linear.
• the rate, mO.D./min. was measured for 10 minutes at 30 second intervals immediately after the addition of the enzyme solution.
• IC 50 values were determined by log-logit method and converted to Kj values by Dixson plot method.
• a blood sample (0.9 ml) is collected through the abdominal aorta by a syringe containing 0.1 ml of a 3.8% sodium citrate solution.
• the blood sample is processed as follows: 60 ⁇ l of (final 0.1-1 mg/ml) a suspended solution of opsonized zymosan in Hank's buffer is added to the preincubated whole blood (540 ⁇ l) for 5 minutes at 37 "C, and the resulting mixture is incubated for 30 minutes at the same temperature. The reaction is terminated by immersing the test tube into ice water. The reaction mixture is then centrifuged at 3,000 rpm for 10 minutes at 4 * C. Twenty (20) ⁇ l of each of the resulting supernatant (the Sample) is measured for elastase activity.
• a test sample mixed with l-methyl-2-pyrrolidone instead of the substrate is regarded as Substrate (-).
• a test sample mixed with saline instead of the Sample is regarded as Blank.
• the remaining elastase activity in the Sample is calculated according to the following: optical density of Substrate (+) - (optical density of Substrate (-) + optical density ofBlank) as a total production of p-nitroaniline over 24 hours based on a standard curve for the amount of p-nitroaniline.
• An average activity is calculated based on the test sample of 5-6 animals.
• An agent at 3, 10 or 30 mg/kg is orally given by a forced administration to a 24 hour fasted animal at 60 minutes before the blood sampling.
• Optical density is measured by SPECTRA MAX 250 (Molecular Devices).

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