Related Application

This application is a continuation in part of Application Serial No. 08/410,312 entitled "MERCAPTO DERIVATIVES AS INHIBITORS OF NITRIC OXIDE SYNTHASE" filed on March 24, 1995, which is incorporated herein in its entirety by reference.

Background of the Invention

The present invention relates to the use of mercapto derivatives as inhibitors of cyclooxygenases (COX).

Prostaglandins (PG's) are synthesized from arachidonic acid by a family of enzymes termed cyclooxygenases (COX). One isoform (COX-1 ) is constitutively present in a variety of tissues and releases PG's in low amounts. The continuous release of PG from COX-1 serves physiological purposes. For instance, prostacyclin, a vasodilatory and anti-aggregatory prostaglandin, reduces the adhesion of platelets to the endothelial surface.

The inducible isoform of COX (COX-2) is expressed in

response to immunological stimuli in multiple cell types including

macrophages, vascular smooth muscle cells and epithelial cells, and

produces large amounts of PG's, which can result in tissue injury.

There is substantial evidence that COX-2 plays an

important role in the pathogensis of a variety of inflammatory conditions. In animal models of endotoxic shock, endotoxin causes

induction of COX-2. In addition, it is now thought that excess PG

production may be involved in a number of other inflammatory

conditions, including arthritis and ulcerative colitis.

Various inhibitors of COX have been proposed for

therapeutic use. For example, non-steroidal anti-inflammatory drugs

(acetylsalicylic acid, ibuprofen, etc.) are inhibitors of COX. Examples of United States patents directed to COX inhibition include 5, 1 55, 1 1 0;

5,360,925; 5,399,970; 5,409,944; 5,474,995; and 5,475,021 .

Although the COX inhibitors discussed above have therapeutic use, it

is important to identify additional compounds which inhibit COX. It

also is desirable to identify additional compounds which may have

combined actions, i.e., inhibiting the activity of COX, as well as of

other pro-inflammatory enzymes.

Summary of the Invention

This invention is directed to a pharmacologically

acceptable composition for inhibiting COX in a mammal. The

composition includes a mercapto derivative and a pharmaceutically

acceptable carrier, with the mercapto derivative present in the

composition in an effective amount to inhibit COX in the mammal.

In this regard, the above-identified patent application

relates to the use of the same mercapto compounds in the treatment

of conditions associated with the overproduction of nitric oxide, a

potent cytotoxic free radical. Thus, the mercapto compounds as COX

inhibitors described in the present application have a distinct advantage

over other classes of COX inhibitors, in that they also inhibit another class of inflammatory enzymes (nitric oxide synthases).

The invention also is directed to a method of inhibiting

COX in a mammal, which includes the step of administering to the

mammal a mercapto derivative in a pure form or in a pharmaceutically acceptable carrier.

The mercapto compound or derivative of the composition

and method is defined by a formula selected from the group consisting

of:

N- R,

I

R _t - Y - Z - X - C

I

NH - R,

and

NH- R', N - R ₃

I I

C-X ^' -Z' -S-S-Z-X-C

N- R' ₃ NH - R ₂

or a salt thereof, wherein

R _T is H, alkyl, alkenyl, phenyl, alkylene, alkenylene, or phenylalkylene or a substituted derivative thereof;

When R, is alkylene or alkenylene, R, optionally may be

joined to either of the amidino Ns, to Z or to X of the above formula

containing R to form a 5-, 6- or 7- membered heterocyclic ring, with

the proviso that, when R, is attached to Z, Z is alkylene or alkenylene or a substituted derivative thereof, and, when R, is attached to X, X is either CR ₅ or N;

R ₂ , R ₃ , R ^' ₂ and R ^' ₃ are independently H, lower alkyl, alkenyl, alkylene, alkenylene, amino, phenyl or phenylalkylene, or a

substituted derivative thereof;

When R ₂ or R ' ₂ is alkylene or alkenylene, R ₂ or R ' ₂

optionally may be joined to the imino N residing on the adjacent amidino C to form a 5- or 6- membered heterocyclic ring;

Z and Z ^' are independently alkylene, alkenylene,

cycloalkylene or cycloalkenylene, or a substituted derivative thereof;

When R ₂ , R ₃ , R ' ₂ or R ^' ₃ is alkylene or alkenylene, R ₂ , R ₃ ,

R ^' ₂ or R ' ₃ optionally may be joined to the adjacent Z or Z ^' to form a

5- or 6- membered heterocyclic ring including N, C and not more than

one atom of O or S, with the proviso that said Z or Z' so joined is an

alkylene or alkenylene, said heterocyclic ring optionally being

substituted with a lower alkyl, alkoxy, halo, hydroxy or amino;

X is N, NR ₄ , O, CR ₅ or CR ₄ R ₅ ;

X ' is N, NR ' ₄ , O, CR ' ₅ or CR ^' ₄ R ^' ₅ ;

Y is S;

R ₄ and R ^' ₄ are independently H, alkyl, alkylene, alkenylene, thioalkylene or thioesteralkylene;

R ₅ and R ^' ₅ are independently H, alkyl, alkylene,

alkenylene, thioalkylene, thioesteralkylene, amino or carboxyl; and

When R ₄ or R ^' ₄ is alkylene, alkenylene, thioalkylene, or thioesteralkylene, R ₄ or R ^' ₄ optionally may be joined to R ₂ , R ₃ , R ' ₂ or

R ^' ₃ to form a 5- or 6- membered heterocyclic ring including N, C and

not more than one atom of O or S, with the proviso that said R ₂ , R ₃ , R ^' ₂ or R ^' ₃ so joined is alkylene, alkenylene, amino, phenyl,

phenylalkylene, or a substituted derivative thereof wherein the

substituted derivative is lower alkyl or halo.

Brief Description of the Drawings

Fig. 1 is a graph of the effect of mercaptoethγlguanidine

(MEG) on 6-keto prostagiandin F1 alpha production by (A) non-

stimulated macrophages in the presence of arachidonic acid, and

(B) immunostimulated J774 macrophages (stimulated with LPS and

interferon-gamma for 6h).

Fig. 2 is a graph of the effect of mercaptoethylguanidine

(MEG) on thromboxane B2 production by (A) non-stimulated

macrophages in the presence of arachidonic acid, and

(B) immunostimulated J774 macrophages (stimulated with LPS and

interferon-gamma for 6h).

Fig. 3 is a graph of the effect of mercaptoethylguanidine

(MEG), on 6-keto prostaglandin F1 alpha production by (A) purified COX-1 , and (B) purified COX-2 in a cell-free system (N = 3-6).

Detailed Description of the Invention

This invention is directed to a pharmacologically

acceptable composition for inhibiting COX in a mammal. The

composition includes a mercapto derivative and a pharmaceutically

acceptable carrier, with the mercapto derivative present in the

composition in an effective amount to inhibit COX in the mammal. The

invention also is directed to a method of inhibiting COX in a mammal,

which includes the step of administering to the mammal a mercapto

derivative in pure form or in a pharmaceutically acceptable carrier.

Suitable mercapto derivatives for use in the composition

or method may be made according to the methods of synthesis taught in the following articles which are incorporated herein in their entirety

by reference:

( 1 ) Joseph X. Khym et al., "Ion Exchange Studies of Transguanylation Reactions. I. Rearrangement of S,2- Aminoethylisothiourea to 2-Mercaptoethylguanidine and 2- Am ^' mothiazoVme" , Journal of the American Chemical Society , Vol. 79, pp. 5663-5666, November 5, 1 957;

(2) David G. Doherty, et al., "Synthesis of Aminoalkylisothiuronium Salts and their Conversion to Mercaptoalkylguanidines and Thiazolines", Journal of the American Chemical Society, Vol. 79, pp. 5667-5671 , November 5, 1 957;

(3) Joseph X. Khym, et al., "Ion Exchange Studies of Transguanylation Reactions. II. Rearrangement of 3- Aminopropylisothiourea and N-Substituted Aminoethyl- and Aminopropylisothioureas to Mercaptoalkylguanidines and 2-

Aminothiazolines or Penthiazolines", Journal of the American Chemical Society, Vol.80, pp.3342-3349, July 5, 1958;

(4) David G. Doherty et al. "Synthesis of D- and L-2- Aminobutylisothiourea Dihydrobromide Isomers and their Conversion to Guanidothiols, Disulfides, and Thiazolines", Journal of Organic

Chemistry, Vol.28, pp. 1339-1342, 1963.

(5) Shih-Hsi Chu et al., "Potential Antiradiation Agents. II. Selenium Analogs of 2-Aminoethylisothiouronium Hydrobromide and Related Compounds", Journal of the American Chemical Society, Vol.27, pp.2899-2901, August, 1962.

(6) Tohru Hino et al., "Radiation-protective Agents. I. Studies on N-Alkylated-2-(2-aminoethyl)thiopseudoureas and 1,1- (Dithioethyiene)diguanidines", Chemical & Pharmaceutical Bulletin, Vol. 14, No.11, pp.1193-1201, November, 1966.

Suitable mercapto derivatives also may be made according

to the examples provided at the end of this detailed description of the

invention.

The mercapto derivative of the composition and method

is defined by a formula selected from the group consisting of:

N - R ₃

II

R, - Y - Z - X - C

NH- R ₂

and

NH - R ^' ₂ N- R ₃

I II c- x -Z' - s-s- z-x- c

II I N-R' ₃ NH - R ₂

or a salt thereof, wherein

R, is H, alkyl, alkenyl, phenyl, alkylene, alkenylene, or

phenylalkylene or a substituted derivative thereof;

When R, is alkylene or alkenylene, R, optionally may be joined to either of the amidino Ns, to Z or to X of the above formula

containing , to form a 5-, 6- or 7- membered heterocyclic ring, with

the proviso that, when R, is attached to Z, Z is alkylene or alkenylene

or a substituted derivative thereof, and, when R, is attached to X, X is either CR ₅ or N;

R ₂ , R ₃ , R ' ₂ and R ^' ₃ are independently H, lower alkyl,

alkenyl, alkylene, alkenylene, amino, phenyl or phenylalkylene, or a substituted derivative thereof;

When" R ₂ or R ' ₂ is alkylene or alkenylene, R ₂ or R ' ₂

optionally may be joined to the imino N residing on the adjacent amidino C to form a 5- or 6- membered heterocyclic ring;

Z and Z ' are independently alkylene, alkenylene,

cycloalkylene or cycloalkenylene, or a substituted derivative thereof;

When R ₂ , R ₃ , R ' ₂ or R ' ₃ is alkylene or alkenylene, R ₂ , R ₃ ,

R ' ₂ or R ' ₃ optionally may be joined to the adjacent Z or Z ^' to form a

5- or 6- membered heterocyclic ring including N, C and not more than

one atom of O or S, with the proviso that Z is an alkylene or

alkenylene, said heterocyclic ring optionally being substituted with a

lower alkyl, alkoxy, halo, hydroxy or amino;

X is N, NR ₄ , O, CR ₅ or CR ₄ R ₅ ;

X ' is N, NR ' ₄ , O, CR ' ₅ or CR ' ₄ R ' ₅ ;

Y is S;

R ₄ and R ' ₄ are independently H, alkyl, alkylene,

alkenylene, thioalkylene or thioesteralkylene;

R ₅ and R ^' ₅ are independently H, alkyl, alkylene, alkenylene, thioalkylene, thioesteralkylene, amino or carboxyl; and

When R ₄ or R ^' ₄ is alkylene, alkenylene, thioalkylene, or

thioesteralkylene, R ₄ or R ^' ₄ optionally may be joined to R ₂ , R ₃ , R ' ₂ or

R ' ₃ to form a 5- or 6- membered heterocyclic ring including N, C and not more than one atom of O or S, with the proviso that R ₂ , R ₃ , R ' ₂ or

R ^' ₃ is alkylene, alkenylene, amino, phenyl, phenylalkylene, or a

substituted derivative thereof wherein the substituted derivative is

lower alkyl or halo.

As used herein, the term "salt" refers to any addition salt

derived from any pharmaceutically acceptable organic or inorganic acid.

Examples of suitable acids include hydrochloric, hγdrobromic, sulfuric,

nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic,

succinic, toluene p sulfonic, tartaric, acetic, citric, methanesulfonic,

formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulphonic

acids. Additionally, as used herein, any alkyl or alkylene may be

straight chain, branched or cyclic, and "halo" includes bromine,

chlorine, fluorine and iodine.

As mentioned above, R, is H, alkyl, alkenyl, phenyl,

alkylene, alkenylene or phenylalkylene, or a substituted derivative

thereof. If desired, this R, derivative may be substituted with one or

more alkoxy, halo, hydroxy, amino or nitro groups. Additionally, as

noted above, R ₂ , R ₃ , R' ₂ and R' ₃ are independently H, lower alkyl, alkenyl, alkylene, alkenylene, amino, phenyl or phenylalkylene, or a substituted derivative thereof. If desired, the R ₂ , R ₃ , R' ₂ and R' ₃

derivative may be substituted with a lower alkyl or halo.

If the R ₄ , R ₅ , R' ₄ or R' ₅ substituent is thioalkylene, the thioalkylene preferably has a formula [-(CH ₂ ) _n -SH] where n is

independently 1 to 4. If R ₄ , R ₅ , R' ₄ or R' ₆ is thioesteralkylene, the

thioesteralkylene preferably has the formula HCH ₂ ) _n -S-R ₆ ] where R ₆ is

independently a lower alkyl and n is independently 1 to 4.

The Z and Z' substituents of the mercapto derivative are

independently alkylene, alkenylene, cycloalkylene or cycloalkenylene,

or a substituted derivative thereof. When such a substituted derivative

is employed, the substituent may include an alkoxy, halo, hydroxy,

amino or nitro group.

A preferred subgroup of the mercapto derivative includes

mercapto derivatives where: R is H or lower alkyl; R ₂ is H; R ₃ is H; R' ₂

is H; R' ₃ is H; X is NR ₄ ; X' is NR' ₄ ; R ₄ and R' ₄ are independently H,

methyl or ethyl; and Z and Z' are independently alkylene. A few

nonlimiting examples include mercaptoethylguanidine,

mercaptopropylguanidine, S-methyl-mercaptoethylguanidine, S-methyl-

mercaptopropylguanidine, and guanidinoethyldisulfide. Another

preferred subgroup of mercapto derivatives is formed wherein: R, is H;

R ₂ is H; R ₃ is H; R' ₂ is H; R' ₃ is H; X is NR ₄ ; X' is NR' ₄ ; R ₄ is H; R' ₄ is

H; and Z and Z' are independently a C,^ alkylene. Nonlimiting

examples include mercaptoethylguanidine, mercaptopropylguanidine, and guanidinoethyldisulfide.

The mercapto derivative, in pure form or in a

pharmaceutically acceptable carrier, will find benefit in treating

conditions and disorders where there is an advantage in inhibiting the

cyclooxygenase enzymes. For example, the mercapto derivative may

be used to treat a circulatory shock including its various aspects such as vascular and myocardial dysfunction, metabolic failure including the

inhibition of mitochondrial enzymes and cytochrome P450-mediated

drug metabolism, and multiple organ dysfunction syndrome including

adult respiratory distress syndrome. Circulatory shock may be a result of gram-negative and gram positive sepsis, trauma, hemorrhage, burn

injury, anaphylaxis, cγtokineimmunotherapy, liver failure, kidney failure

or systemic inflammatory response syndrome. Mercapto derivatives

also may be beneficial for patients receiving therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine-inducing agents,

or as an adjuvant to short term immunosuppression in transplant

therapy. In addition, mercapto derivatives may be useful to inhibit PG

synthesis in patients suffering from inflammatory conditions in which

COX-2 activity contributes to the pathophysiology of the condition,

such as arthritis, inflammatory bowel disease, and myocarditis, for

example.

There is also evidence that COX-2 may be involved in the

pathophysiology of autoimmune and/or inflammatory conditions such

as arthritis, rheumatoid arthritis and systemic lupus erythematosus (SLE) and in insulin-dependent diabetes mellitus, and, therefore,

mercapto derivatives may prove helpful in treating these conditions.

Furthermore, it is now clear that there are a number of

additional inflammatory and noninflammatory diseases that are

associated with COX-2 induction. Examples of such physiological

disorders include: inflammatory bowel diseases such as ileitis and

Crohn's disease; inflammatory lung disorders such as asthma and

chronic obstructive airway disease; inflammatory disorders of the eye

including corneal dystrophy, trachoma, onchocerciasis, uveitis,

sympathetic ophthalmitis and endophthalmitis; chronic inflammatory

disorders of the gum including periodontitis; chronic inflammatory

disorders of the joints including arthritis and osteoarthritis, tuberculosis,

leprosy, glomerulonephritis sarcoid, and nephrosis; disorders of the skin

including sclerodermatitis, psoriasis and eczema; inflammatory diseases

of the central nervous system, including chronic demyelinating diseases

such as multiple sclerosis, dementia including AIDS-related

neurodegeneration and Alzheimer'sdisease, encephalomyelitis and viral

or autoimmune encephalitis; autoimmune diseases including immune-

complex vasculitis, systemic lupus and erythematodes; and disease of

the heart including ischemic heart disease and cardiomyopathy.

Additional diseases which may benefit from the use of mercapto

derivatives include adrenal insufficiency; hypercholesterolemia;

atherosclerosis; bone disease associated with increased bone

resorption, e.g., osteoporosis, pre-eclampsia, eclampsia, uremic complications; chronic liver failure, noninflammatory diseases of the

central nervous system (CNS) including stroke and cerebral ischemia; and various forms of cancer.

Pharmaceutical formulations of the mercapto derivative

may include those suitable for oral, rectal, nasal, topical (including

buccai and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by

inhalation or insufflation. The formulations may, where appropriate, be

conveniently presented in discrete dosage units and may be prepared

by any of the methods well known in the art of pharmacy. All such pharmacy methods include the steps of bringing into association the

active compound with liquid carriers or finely divided solid carriers or

both as needed and then, if necessary, shaping the product into the

desired formulation.

Pharmaceutical formulations suitable for oral

administration may conveniently be presented: as discrete units, such

as capsules, cachets or tablets, each containing a predetermined

amount of the active ingredient; as a powder or granules; or as a

solution, a suspension or as an emulsion. The active ingredient may

also be presented as a bolus electuary or paste, and be in a pure form,

i.e., without a carrier. Tablets and capsules for oral administration may

contain conventional excipients such as binding agents, fillers,

lubricants, disintegrant or wetting agents. A tablet may be made by

compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in

a suitable machine the active ingredients in a free-flowing form such as

a powder or granules, optionally mixed with a binder, lubricant, inert

diluent, lubricating, surface active or dispersing agent. Molded tablets

may be made by molding in a suitable machine a mixture of the

powdered compound moistened with an inert liquid diluent. The tablets

may be coated according to methods well known in the art. Oral fluid

preparations may be in the form of, for example, aqueous or oily

suspensions, 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 tablets

may optionally be formulated so as to provide slow or controlled

release of the active ingredient therein.

Formulations for parenteral administration include:

aqueous and non-aqueous sterile injection solutions which may contain

anti-oxidants, buffers, bacteriostats and solutes which render the

formulation isotonic with the blood of the intended recipient; and

aqueous and non-aqueous sterile suspensions which may include

suspending agents and thickening agents. The formulations may be

presented in unit dose or multi-dose containers, for example sealed

ampoules and vials, and may be stored in a freeze-dried (lyophilized)

condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use.

Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be

prepared from sterile powders, granules and tablets of the kind

previously described.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or

polyethylene glycol. Formulations for topical administration in the

mouth, for example buccally or sublingually, include lozenges,

comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in

a base such as gelatin and glycerin or sucrose and acacia. For intra-

nasal administration the compounds of the invention may be used as

a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising

one or more dispersing agents, solubilizing agents or suspending

agents. Liquid sprays are conveniently delivered from pressurized

packs.

For administration by inhalation the compounds according

to the invention are conveniently delivered from an insufflator, nebulizer

pressurized packs or other convenient means of delivering an aerosol

spray. Pressurized packs may comprise a suitable propellant such as

dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the

case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or

insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the

compound and a suitable powder base such as lactose or starch. The

powder composition may be presented in unit dosage form, in for

example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

When desired the above described formulations, adapted

to give sustained release of the active ingredient, may be employed.

The pharmaceutical compositions according to the invention may also

contain other active ingredients such as antimicrobial agents,

immunosuppressants or preservatives.

The compounds of the invention may also be used in

combination with other therapeutic agents, for example, anti-

inflammatory agents, particularly nitric oxide synthase inhibitors,

superoxide or peroxynitrite scavengers, vasodilator prostaglandins

including prostacyclin and prostaglandin E _{1 f} cancer chemotherapeutic

agents including cisplatin, NO donors or NO inhalation therapy, or PAF

- receptor antagonists.

It should be understood that in addition to the ingredients

particularly mentioned above, the formulations of this invention may

include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral

administration may include flavoring agents.

Preferred unit dosage formulations are those containing an

effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient.

For each of the aforementioned conditions, the mercapto

derivative may be administered orally or via injection at a dose of from

0.1 to 250 mg/kg per day. The dose range for adult humans is

generally from 5 mg to 1 7.5 g/day, preferably 5 mg to 10 g/day and

most preferably 100 mg to 3 g/day. Tablets or other forms of

presentation provided in discrete units may conveniently contain an

amount which is effective at such dosage or as a multiple of the same,

for instance, units containing 5 mg to 500 mg, usually around 1 00 mg

to 500 mg.

The pharmaceutical composition preferably is administered

orally or by injection (intravenous or subcutaneous), and the precise

amount administered to a patient will be the responsibility of the

attendant physician. However, the dose employed will depend upon

a number of factors, including the age and sex of the patient, the

precise disorder being treated, and its severity. Also the route of

administration may vary depending upon the condition and its severity.

The following Examples are provided by way of

illustration, and are not intended to limit the scope of the invention.

EXAMPLE 1

This example (Fig. 1 ) illustrates the effect of mercaptoethyguanidine on arachidonic acid or immunostimulation- induced 6-keto prostaglandin F1 alpha formation in J774.2 macrophages. J774 macrophage cell lines were obtained from the

American Type Culture Collection (ATCC) and were grown using standard methods in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, glutamine, penicillin (10,000 U/l) and streptomycin (10,000 U/l). Cells were grown in 96-well plates for measure of the production of prostaglandin metabolites and cell viability. All the experiments were carried out without fetal calf serum in order to avoid interference with radioimmunoassay. Concentration of 6-keto prostaglandin 1 alpha, the stable metabolite product of prostacyclin in the culture medium, was determined by radioimmunoassay. Supernatant or reaction samples were diluted 1 :5 in a buffer containing 0.1 % polyvinylpyrolidine, 0.9%

NaCI, 50 M Tris base, 1.7 mM MgSO ₄ and 0.16 mM CaCI ₂ (pH 7.4) before radioimmunoassay. The stable metabolite of prostacyclin,

6-keto-PGF1 alpha, was determined by radioimmunoassay as described (Wise WC, Cook JA, Haluskha PV. "Arachidonic Acid Metabolism in

Endotoxin Tolerance", Adv. Shock, Vol. 10, pp. 131 -142, 1983) Measurement of cyclooxygenase 1 activity in J774 cells

Cells were plated in 96-well plates at 90-100% confluence

(200 /;M-3mM). Inhibitors were given as a 30-minute pretreatment.

Cells were then stimulated with arachidonic acid (16 μM) in order to activate the constitutive cyclooxygenase (COX-1 ). Cells were then incubated for a further period of 30 minutes and the supernatant was collected for the measurement of arachidonic acid metabolism evaluation by radioimmunoassay.

Measurement of cyclooxygenase 2 activity in J774 cells

Cells were place in 96-well plates at 90-100% confluence

(200 μL final volume) and pretreated for 30 minutes with MEG (1 μM-

3mM). Inhibitors were given as a 30-minute pretreatment. Cells were then stimulated with endotoxin of E.co/H LPS, 10μg/mL) and interferon

Y (IFN, 150 Uml) in order to induce the expression of the inducible cyclooxygenase (COX-2). Cells were then incubated for a further

period of 6 hours and the supernatant was collected for the measurement of arachidonic acid metabolism evaluation by radioimmunoassay.

Mitochondrial respiration, an indicator of cell viability, was

assessed by the mitochondrial-dependent reduction of MTT [3- (4,5 - dimethylthiazol-2-yl) - 2,5 - diphenyltetrazolium bromide] to formazan. Cells in 96-well plates were incubated (37 ^β C) with MTT (0.2 mg/ml for

60 minutes). Culture medium was removed by aspiration and the cells solubilized in dimethylsulf oxide (DMSO) (100 μ\). The extent of reduction of MTT to formazan within cells was quantitated by measurement of OD ₅₅₀ using a microplate reader. The calibration curve for the reduction of MTT to formazan was prepared in DMSO.

Formazan production by cells was expressed as a percentage of the values obtained from untreated cells.

Cells stimulated with arachidonic acid (panel a) or endotoxin (panel b) released 6-keto prostaglandin 1 alpha into the culture medium. This was dose-dependently inhibited by the mercapto derivatives, mercaptoethylguanidine (MEG). The inhibition of PG production was not due to cell killing, as these agents in their effective doses did not decrease cellular viability (not shown). Similar to MEG, other related mercapto derivatives exhibited inhibition of COX-2 activity to a variable degree (Table 1 ).

TABLE 1

Half-maximal inhibitory potencies of selected mercapto derivatives on COX-2 activity in immunostimulated J744 macrophages

Compound EC50 U/M)

MEG 55

S-methyl-MEG 40

N-methyl-MEG 55

MPG 55

GED 75

EXAMPLE 2

This example (Fig. 2) illustrates the effect of mercaptoethylguanidine on arachidonic acid or immunostimulation- induced thromboxane B2 formation in J774.2 macrophages. J774 macrophage cell lines were cultured and treated as described in

example 1 . Concentration of thromboxane B2, the stable metabolite of thromboxane A2 was determined by radioimmunoassay. Supernatant or reaction samples were diluted 1 :5 in a buffer containing 0.1 % polyvinylpyrolidine, 0.9% NaCI, 50mM Tris base, 1.7 mM MgSo ₄

and 0.16 mM CaCI ₂ (pH 7.4) before radioimmunoassay. Thromboxane

B2, the stable metabolite of thromboxane A2, was determined by radioimmunoassay as described (Wise WC, Cook JA, Haiuskha PV, "Arachidonic Acid Metabolism in Endotoxin Tolerance", Adv. Shock, Vol. 10, pp. 131-142, 1983). Cells stimulated with arachidonic acid (A) or endotoxin (B) released thromboxane A2 into the culture medium. This was dose- dependently inhibited by the mercapto derivatives,

mercaptoethylguanidine (MEG). The inhibition of PG production was not due to cell killing, as these agents in their effective doses did not decrease cellular viability (not shown).

EXAMPLE 3

This example (Fig. 3) illustrates the effect of mercaptoethylguanidine on 6-keto prostaglandin F1 alpha formation by purified COX-1 I (A) and COX-2 (B) isoenzymes. In a test tube containing 0.1 M Tris-HCL buffer (pH 8.0, final volume 2 mL) with 1

mM EDTA, o.2 mM phenol and 1 μM hemin, 10 units of COX-1 of COX-2 were allowed to react with 100 μM arachidonic acid for 2 minutes at 37° C in the presence or absence of MEG (1 μM-3mM). The reaction was quenched by addition of 50 μL stannous chloride

solution (100 mg/ml in 1 M HCI). the reaction was allowed to proceed for an additional 10 minutes and it was stopped thereafter by addition of 5 mL of a buffer containing 0.1 % polyvinylpyrolidine, 0.9% NaCI, 50 mM Tris base, 1 .7 mM MgSo ₄ and 0.16 mM CaCI ₂ (pH 7.4). Concentration of 6-keto prostaglandin 1 alpha, the stable metabolite product of prostacyclin in the culture medium, was

determined by radioimmunoassay. Supernatant or reaction samples were diluted 1 :5 in a buffer containing 0.1 % polyvinylpyrolidine, 0.9% NaCI, 50 mM Tris base, 1 .7 mM MgSo ₄ and 0.16 mM CaCI ₂ (pH 7.4) before radioimmunoassay. The stable metabolite of prostacyclin,

6-keto-PGF1 a, was determined by radioimmunoassay as described (Wise WC, Cook JA, Haluskha PV, "Arachidonic Acid Metabolism in Endotoxin Tolerance", Adv. Shock, Vol. 10, pp. 131-142, 1983).

There was a significant production of 6-keto prostaglandin 1 alpha into the reaction mixture. This was dose-dependently inhibited by the mercapto derivatives, mercaptoethylguanidine (MEG).

EXAMPLE 4 This Example illustrates a method for synthesizing mercaptoethylguanidine sulphate. Mercaptoethylamine hydrochtoride (2g) was dissolved in methanol (5 ml) and cooled in a salt/ice bath. A

cold solution of potassium hydroxide (0.99 g) in methanol (10 ml) was added and the mixture stirred. After 1 hour, the solution was filtered

and S-methylisothiourea (2g) was added to 12 ml of the filtrate. The solution was stirred at room temperature ( 18 " C) for 16 hours under

nitrogen. The solution then was filtered and ether was added to precipitate the crude product which was then recrystallized from an ether/ethanol mixture.

The detailed description of the invention presented above is provided by way of illustration, and it is not intended to limit the scope of the invention which is to be determined by the following claims.

What is claimed is: