Patent Publication Number: US-5627206-A

Title: Tricyclic inhibitor of matrix metalloproteinases

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to a novel tricyclic compound useful as a pharmaceutical agent to pharmaceutical compositions which include this compound and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. The novel compound of the present invention is an inhibitor of matrix metalloproteinases, e.g., gelatinase A (72 kDa gelatinase). More particularly, the novel compound of the present invention is useful in the treatment of multiple sclerosis and other autoimmune or inflammatory disorders dependent on the tissue invasion of leukocytes or other activated migrating cells. 
     Gelatinase A and stromelysin-1 are members of the matrix metalloproteinase (MMP) family (Woessner J.F., FASEB J. 1991;5:2145-2154). Other members include fibroblast collagenase, neutrophil collagenase, gelatinase B (92 kDa gelatinase), stromelysin-2, stromelysin-3, matrilysin, collagenase 3 (Freije J.M., Diez-Itza I., Balbin M., Sanchez L.M., Blasco R., Tolivia J., and Lopez-Otin C. J. Biol. Chem., 1994;269:16766-16773), and the newly discovered membrane-associated matrix metalloproteinases (Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., and Seiki M., Nature, 1994;370:61-65). 
     Matrix metalloproteinases share high sequence homology and the catalytic domains of each of the MMPs can be identified by sequence alignment. The gene for the catalytic domain of stromelysin-1, SCD, was constructed by removing the propeptide and C-terminal domain (Ye Q.-Z., Johnson L.L, Hupe D.J., and Baragi V., &#34;Purification and Characterization of the Human Stromelysin Catalytic Domain Expressed in Escherichia coli&#34;, Biochemistry, 1992;31:11231-11235). The gelatinase A catalytic domain, GCD, was similarly constructed with the additional removal of the fibronectin-like insert which interrupts the catalytic domain (Ye Q.-Z., Johnson L.L, Yu A.E., and Hupe D., &#34;Reconstructed 19 kDa Catalytic Domain of Gelatinase A is an Active Proteinase&#34;, Biochemistry, 1995;34:4702-4708). Both truncated proteins cleave synthetic peptide substrates and the natural substrates proteoglycan and gelatin in a manner similar to the full-length enzymes and can be used to identify matrix metalloproteinase inhibitors. 
     The catalytic zinc in matrix metalloproteinases is the focal point for inhibitor design. The modification of substrates by introducing chelating groups has generated potent inhibitors such as peptide hydroxymates, thio-containing peptides, and N-carboxyalkyl peptides. Peptide hydroxymates and the natural endogenous inhibitors of MMPs (TIMPs) have been used successfully to treat animal models of cancer and inflammation. However, except for amino acid derivatives with weak potency (Ye Q.-Z., Johnson L.L., Nordan I., Hupe D., and Hupe L., J. Med. Chem. 1994;37(1):206-209), few non-peptide inhibitors have been described or shown to have in vivo activity. 
     The ability of the matrix metalloproteinases to degrade various components of connective tissue makes them potential targets for controlling pathological processes. Gijbels, et al., (J. Clin, Invest. 1994;94:2177-2182) recently described a peptide hydroxymate, GM6001, that suppressed the development or reversed the clinical expression of experimental allergic encephalomyelitis (EAE) in a dose dependent manner, suggesting the use of MMP inhibitors in the treatment of autoimmune inflammatory disorders such as multiple sclerosis. 
     Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system (CNS) characterized by inflammation, disruption of the bloodbrain barrier, selective destruction of the myelin sheaths with glial scar formation and loss of neuronal cell conductivity leading to neurological deficits. The underlying cause is unknown, but it has been established as a T-cell mediated autoimmune disease (Lawrence Steinman, &#34;Autoimmune Disease&#34;, Scientific American, Sep. 1993;269(3):106-114). While there are no spontaneous animal models for the disease, experimental allergic encephalomyelitis (EAE) has been used successfully to study many aspects of MS pathogenesis, and the work of Paterson and others has clearly and convincingly demonstrated the validity of this model as the only accepted preclinical test for efficacy of agents in MS (Paterson P., &#34;Going to the Rats and Dogs to Study the Patient,&#34; Cell-Immunol., 1983;82(1):55-74). Bornstein&#39;s work using mammalian organotypic cultures showed that the CNS tissue responded with identical patterns of demyelination, swollen myelin sheaths, and eventual &#34;sclerosis&#34; when exposed to serum from EAE-affected animals and MS patients (Bornstein M.B., Miller A.I., Slagle S., Arnon R., Sela M., and Teitelbaum D., &#34;Clinical Trials of Copolymer I in Multiple Sclerosis,&#34; in Annals of the New York Academy of Sciences, eds Labe Scheinberg and Cedric S. Raine, 1984;36:366-372). Analysis of the receptors on T-cells isolated from brain lesions of MS patients reveal that they are reactive to a peptide fragment of myelin basic protein analogous to the antigen used to precipitate the EAE model (Lawrence Steinman and Paul Conlon, &#34;Designing rational therapies for multiple sclerosis,&#34; Bio/technology, Feb. 1995:118-120). 
     Several successful therapeutic strategies for treating MS target the T-cell response modeled in EAE. Beta-interferon (betaseron), acts in part by downregulating the expression of histocompatibility locus antigen (HLA) DR2. EAE studies have been used as the primary experimental basis to develop many of the current treatments for MS. Copolymer-1 and oral administration of myelin basic protein act by inducing immune tolerance to the myelin basic protein (MBP) antigen. The EAE model was used to develop a therapy in which peptides derived from the T-cell receptor V region recognizing an MBP fragment are used to immunize patients with relapsing-remitting MS. Monoclonal antibodies (Mab) against the CD4 receptor prevented the clinical and histological manifestations of EAE (Steinman L., Lindsey J.W., Alters S., and Hodgkinson S., &#34;From treatment of experimental allergic encephalomyelitis to clinical trials in multiple sclerosis,&#34; Immunol-Ser., 1993;59:253-60). Clinical trials of CD4 Mabs are being conducted by several companies. 
     In MS and EAE, the blood-brain barrier has been shown to be defective both with respect to exclusion of blood-borne substances from the CNS and infiltration of lymphocytes (Lam D.K.C., &#34;The central nervous system barrier in acute experimental allergic encephalomyelitis,&#34; in The Blood Brain Barrier in Health and Disease, edited by Suckling A.J., Rumsby M.G., and Bradbury M.W.B., 1986:158-164, Ellis Horwood, Ltd., Chichester, UK). Using gadolinium-DTPA enhanced magnetic resonance imaging (MRI), Miller, et al. (Miller D.H., Rudge P., Johnson G., Kendall B.E., MacManus D.G., Moseley I.F., Barnes D., and McDonald W.I., &#34;Serial gadolinium enhanced magnetic resonance imaging in multiple sclerosis, &#34; Brain, 988;111:927-939) showed in a serial study of MS patients that in recognizable new lesions or in new parts of existing lesions, blood-brain barrier impairment was always present. It appears that bloodbrain barrier disruption is the beginning of an irreversible cascade of events leading to demyelination (Barkhof F., Hommes O.R., Scheltens P., and Valk J., &#34;Quantitative MRI changes in gadolinium-DPTA enhancement after high-dose intravenous methylprednisolone in multiple sclerosis,&#34; Neurology, 1991;14:1219-1222) and is necessary for the development of the disease (Moor A.C.E., De Vries H.E., De Boer A.G., and Breimer D.D., Biochemical Pharmacology, 1994;47:1717-1724). Previously, it has been shown that neutralizing antibodies to cell adhesion molecules prevent lymphocyte infiltration in the EAE, and that this inhibits the inflammatory response initiating the encephalomyelitis (Yednock T.A., Cannon C., Fritz L.C., Sanchez-Madrid F., Steinman L., and Karin N., &#34;Prevention of experimental autoimmune encephalomyelitis by antibodies against α4 β1 integrin,&#34; Nature, Mar. 5, 1992;356(6364):63-66). It has therefore been predicted that agents that prevent such infiltration may be among the most inviting prospects for therapy in MS. 
     The mechanism through which the blood-brain barrier is disrupted during the pathogenesis of MS and other inflammatory diseases of the central nervous system is under intense study. Rosenberg, et al. showed that activated gelatinase A injected intracerebrally attacks extracellular matrix and opens the blood-brain barrier. Treatment with TIMP-2 reduced the proteolysis and protected the blood-brain barrier. (Rosenberg G.A., Kornfeld M., Estrada E., Kelley R.O., Liotta L., and Stetler-Stevenson W.G., &#34;TIMP-2 reduces proteolytic opening of the blood-brain barrier by type IV collagenase&#34;, Brain Research, 1992;576:203-207). A recent study by Madri has elucidated the role of gelatinase A in the extravasation of T-cells from the blood stream during inflammation (Ramanic A.M., and Madri J.A., &#34;The Induction of 72-kD Gelatinase in T Cells upon Adhesion to Endothelial Cells is VCAM-1 Dependent&#34;, J. Cell Biology, 1994;125:1165-1178). This transmigration past the endothelial cell layer is coordinated with the induction of gelatinase A and is mediated by binding to the vascular cell adhesion molecule-1 (VCAM-1). Once the barrier is compromised, edema and inflammation are produced in the CNS. Leukocytic migration across the blood-brain barrier is known to be associated with the inflammatory response in EAE. Inhibition of the metalloproteinase gelatinase A would block the degradation of extracellular matrix by activated T-cells that is necessary for CNS penetration. 
     These studies provide the basis for the expectation that an effective, bioavailable inhibitor of gelatinase A would have value in the treatment of diseases involving disruption of extracellular matrix resulting in inflammation due to lymphocytic infiltration. 
     We have identified a tricyclic compound that is an inhibitor of matrix metalloproteinases, particularly gelatinase A, and is additionally active in an allergic encephalomyelitis model and thus useful as an agent for the treatment of multiple sclerosis or other autoimmune or inflammatory diseases dependent upon tissue invasion by leukocytes. 
     SUMMARY OF THE INVENTION 
     Accordingly, the present invention is a method of using the compound of Formula I ##STR1## and corresponding isomers thereof; or a pharmaceutically acceptable salt thereof; as an agent for the treatment of multiple sclerosis. 
     A further embodiment of the present invention is the use of the compound of Formula I for the treatment of other inflammatory disorders dependent upon tissue invasion by leukocytes. 
     A still further embodiment of the present invention is a pharmaceutical composition for administering an effective amount of the compound of Formula I in unit dosage form in the treatment methods mentioned above. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The compound of Formula I is capable of further forming pharmaceutically acceptable base addition salts. All of these forms are within the scope of the present invention. 
     Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N&#39;-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge S.M., et al., &#34;pharmaceutical Salts,&#34; J. of Pharma Sci., 1977;66:1. 
     The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. 
     The compound of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. 
     The present invention includes all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. 
     The compound of Formula I is a valuable inhibitor of gelatinase A. It has been shown previously that inhibitors of matrix metalloproteinases have efficacy in models of disease states like arthritis and metastasis that depend on modification of the extracellular matrix. We demonstrate here that a potent and specific inhibitor of gelatinase A also has activity in the rat experimental allergic encephalomyelitis model which is predictive for human multiple sclerosis and has previously been used as a basis for predicting the efficacy of other therapeutic agents for MS, including anti-CD4 monoclonal antibody, copolymer I, betaseron, cyclosporin, and MBP oral antigen. 
     In vitro experiments were carried out which demonstrate the efficacy of the compound of Formula I as a potent and specific inhibitor of gelatinase A while showing lesser or no inhibition of other related matrix metalloproteinases. Experiments were carried out with both the full-length enzymes and the catalytic domains. Table I shows the activity of Example 2 versus GCD (recombinant gelatinase A catalytic domain); gelatinase A (recombinant full-length enzyme); SCD (stromelysin-1 catalytic domain); stromelysin-1 (full-length native enzyme); gelatinase B (recombinant full-length enzyme); and collagenase (full-length native enzyme). IC 50  values were determined using a thiopeptolide substrate, Ac-Pro-Leu-Gly-thioester-Leu-Leu-Gly-OEt (Ye Q.-Z., Johnson L.L., Hupe D.J., and Baragi V., &#34;Purification and Characterization of the Human Stromelysin Catalytic Domain Expressed in Escherichia coli&#34;, Biochemistry, 1992;31:11231-11235). Example 2 inhibits the conversion of the substrate by gelatinase A with an IC 50  value of 1.31 μM. The same compound also inhibits stromelysin-1 with an IC 50  value of 7.64 μM. IC 50  values for gelatinase B, and collagenase, were &gt;100 μM. 
     
                       TABLE I                                                     
______________________________________                                    
Enzyme       Example 2 IC.sub.50 (μM)                                  
______________________________________                                    
GCD          0.040                                                        
Gelatinase A 1.31                                                         
SCD          7.12                                                         
Stromelysin  7.64                                                         
Gelatinase B &gt;100                                                         
Collagenase  &gt;100                                                         
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     The activity of Example 2 in suppressing the inflammatory disease associated with EAE was tested using the Lewis rat acute model. Female Lewis rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and were used at 8 to 10 weeks of age. Active EAE was induced by injection of 0.05 mL emulsion of myelin basic protein (MBP) with complete Freund&#39;s adjuvant (CFA), containing 25 μg guinea pig MBP and 100 μg Mycobacterium butyricum (Difco Laboratories, Detroit, MI) into one hind footpad. Immunized rats were observed daily for clinical signs of EAE and scored as follows: 0, no symptoms; 1, loss of tail tonicity; 2, paresis; 3, hind limb paralysis, often accompanied by incontinence; and 4, death. Formalin-fixed brain slice sections were stained with hematoxylin-eosin and evaluated microscopically for perivascular and parenchymal infiltration. Compound was administered by gavage as a solution in sterile saline in a volume of 500 μL. The dose of 50 mg/kg typically required 7.5 mg of compound per rat. Several different protocols were used to define the efficacy and timing requirements for potency. Two separate protocols which demonstrate efficacy are described below. 
     Protocol 1: Compound was administered daily at 50 mg/kg beginning on Day 0 and continuing through Day 14, with MBP in CFA administered on Day 0 also. As shown in Table II, this treatment substantially reduced the average scores for the treated group. The percentage of animals responding is also very high with 100% of treated animals showing either a total inhibition of symptoms (80%) or reduction of symptoms (20%). N=5 for all groups. 
     
                                           TABLE II                                
__________________________________________________________________________
                 Clinical                                                 
                 Grade          Effect on                                 
         Daily Oral                                                       
                 at Peak  Day of                                          
                                EAE Rats                                  
         Dosing Begun                                                     
                 of       Peak  (≧Paresis/                         
Treatment                                                                 
         at Day 0                                                         
                 Disease                                                  
                      SD  Expression                                      
                                Total)                                    
__________________________________________________________________________
CFA      Phosphate-                                                       
                 0    0   --    0/5                                       
         Buffered                                                         
         Saline (PBS)                                                     
CFA +    50 mg/kg                                                         
                 0    0   --    0/5                                       
Example 2                                                                 
CFA + MBP                                                                 
         PBS     3.0  0.0 13 &amp; 14                                         
                                5/5                                       
CFA + MBP +                                                               
         50 mg/kg                                                         
                 1.0  1.22                                                
                          12    1/5                                       
Example 2                                                                 
__________________________________________________________________________
 
    
     Protocol 2: To determine whether less frequent dosing or dosing at lower levels was still effective at suppressing EAE, compound was administered at either 50 mg/kg or at 10 mg/kg on Day 0 and on alternate days through Day 14. As shown in Table III, this protocol demonstrated a similar effect on average clinical scores at the peak of disease expression for the treatment group. The decrease in disease severity was highly significant (p ≦0.01) for Day 15. Controls run in parallel included CFA only and MBP in CFA. N=4 for treatment groups. N=8 for controls. 
     
                                           TABLE III                               
__________________________________________________________________________
                 Clinical                                                 
                 Grade          Effect on                                 
                 at Peak  Day of                                          
                                EAE Rats                                  
                 of       Peak  (≧Paresis/                         
Treatment                                                                 
         Dosage  Disease                                                  
                      SD  Expression                                      
                                Total)                                    
__________________________________________________________________________
CFA      PBS     0    0   --    0/4                                       
CFA + MBP                                                                 
         PBS     2.0  0.7 15    5/7                                       
CFA + MBP +                                                               
         10 mg/kg                                                         
                 0.6  0.7 14    0/4                                       
Example 2                                                                 
CFA + MBP +                                                               
         50 mg/kg                                                         
                 0.8  0.6 15    0/4                                       
Example 2                                                                 
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     Histologically, the Lewis rat EAE model typically shows perivascular and parenchymal inflammation due to an autoimmune myelin-specific T-cell response. Examination of histology specimens of the brain slices from animals subjected to Protocol 1 demonstrated that leukocyte migration across the blood-brain barrier had been completely prevented by the 50 mg/kg daily dosing schedule. There was no cuff formation and little CNS inflammation as a result. 
     The activity of Example 2 as a general inhibitor of inflammation was tested using the Mycobacterium footpad edema assay (MFE) in Wistar rats. Compound was administered daily by gavage at 2 mg/kg, 10 mg/kg, or 50 mg/kg. 
     Male outbred Wistar rats (110-125 g, Charles River Labs, Portage, Mich.) were used in this study. Rats were housed for a minimum of 1 week before use. Food and water were supplied ad libitum. Foot pad edema was induced following the method described by Martel R.R. and Klicius J., &#34;Comparisons in rats of the antiinflammatory and gastric irritant effects of etodolac with several clinically effective antiinflammatory drugs&#34;, Agents and Actions, 1982;12:295. Briefly, male Wistar rats were injected subcutaneously into the right hind footpad with 0.1 mL of 5 mg/mL suspension of killed and dried Mycobacterium butyricum (Difco, Detroit, Mich.) in liquid paraffin. Compound was suspended in 0.5% hydroxypropylmethylcellulose (HPMC) containing 0.2% Tween-80 and administered orally 1 hour before injection. Subsequent doses of the compound were given 24 and 48 hours after the Mycobacterium. Control animals were given vehicle alone. Swelling was assessed on the third day by subtracting the initial volume (determined immediately following the Mycobacterium injection) from the final volume of the treated paw. Paw volume was determined by mercury plethysmography. The percent inhibition of edema achieved in each compound-treated group was determined by comparison with swelling in the vehicle-treated group and the ID 50  values were determined by regression analysis. Statistical significance between experimental groups was evaluated using a Student&#39;s t-test. Data is shown in Table IV. All the values for inhibition of the inflammatory response in this model were statistically significant. 
     
                       TABLE IV                                                    
______________________________________                                    
Dose (mg/kg)  % Inhibition of Swelling                                    
______________________________________                                    
 2            23                                                          
10            27                                                          
50            40                                                          
______________________________________                                    
 
    
     Plasma concentrations of Example 2 peaked at 1 hour post-treatment and declined mono-exponentially through the subsequent 4 hours. Cmax and AUC(0-4) values increase proportionately with dose. Compound plasma concentrations determined from Cmax correspond to 4.62 μM (2-mg/kg dose), 38.8 μM (10-mg/kg dose), and 163 μM (50-mg/kg dose). The bioavailability of this compound is therefore well in excess of the effective range of gelatinase A inhibition determined in the in vitro assays. 
     The compound of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms. Thus, the compound of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compound of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compound of the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either the compound of Formula I or a corresponding pharmaceutically acceptable salt of the compound of Formula I. 
     For preparing pharmaceutical compositions from the compound of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. 
     In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. 
     In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. 
     The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term &#34;preparation&#34; is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration. 
     For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify. 
     Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. 
     Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. 
     Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. 
     Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. 
     The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, cachet, or lozenge itself, or it can be the tablet appropriate number of any of these in packaged form. 
     The quantity of active component in a unit dose preparation may be varied or adjusted from 1 mg to 1000 mg, preferably 10 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents. 
     In therapeutic use as an agent for the treatment of multiple sclerosis or other autoimmune or inflammatory disorders dependent upon tissue invasion by leukocytes, the compound utilized in the pharmaceutical method of this invention is administered at the initial dosage of about 1 mg to about 100 mg per kilogram daily. A daily dose range of about 25 mg to about 75 mg per kilogram is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstance is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. 
    
    
     The following nonlimiting examples illustrate the inventor&#39;s preferred methods for preparing the compound of the invention. 
     EXAMPLE 1 
     γ-Oxo-2-dibenzofuranbutanoic acid 
     A 5-L, three-necked, round-bottom flask is equipped with a mechanical stirrer, thermocouple thermometer, and a powder funnel with a nitrogen inlet. This flask is charged with 1.5 L of dichloromethane, flushed with nitrogen and aluminum chloride (196 g, 1.44 mol) is added portionwise. The resulting slurry is cooled in a dry ice bath while a dry powdered mixture of dibenzofuran (100 g, 0.595 mol) and succinic anhydride (71.5 g, 0.714 mol) is added portionwise. Addition is at a rate sufficient to keep the reaction temperature less than -30° C. and is completed in 15 minutes. The resulting mixture is stirred at this temperature for 2 hours then slowly treated with aqueous HCl (375 mL of conc. HCl in 1 L of solution). During addition of the HCl solution, the temperature is controlled to a maximum of 3° C. and addition is completed in 45 minutes. The dichloromethane is then removed in vacuo, the aqueous slurry is filtered, and the solid is air dried to give 175 g of white solid. This solid is extracted into 4 L of tetrahydrofuran (THF), treated with Darco, and filtered. The filtrate was evaporated leaving a cream-colored solid which was recrystallized from 95% ethanol to give 180 g of crude product. This solid is recrystallized from toluene (10 L in 4 portions), and the solids are washed with hexanes then dried in vacuo to give 119.5 g (mp 186-188° C., 75% yield) of the title compound. 
     EXAMPLE 2 
     4-Dibenzofuran-2-yl-4-hydroximino-butyric acid 
     A solution of γ-oxo-2-dibenzofuranbutanoic acid (Example 1) (75.5 g) and sodium acetate trihydrate (114.9 g) in methanol (2.5 L) is treated with a solution of hydroxylamine hydrochloride (38.9 g) in water (150 mL of solution). The solution is heated to reflux for 3.5 hours then concentrated, cooled, and filtered. This solid was washed with water then dried in vacuo to give the crude title compound (71.32 g, 89.6% of theory). This solid was recrystallized from ethyl acetate, washed with hexane, and dried in vacuo to give 53.04 g (68% yield) of the title compound; mp 167-168° C. (d).