FIELD AMD BACKGROUND OF THE INVENTION

The present invention relates to the use of certain non-peptidic compounds having terminal guanidino and carboxyl functional groups, for the preparation of pharmaceutical compositions for prevention and treatment of hepatic fibrosis and cirrhosis.

Chronic hepatitis and liver cirrhosis affect 3-5% of the population worldwide. Despite its frequent occurrence and high morbidity and mortality, the current medical management of liver cirrhosis is still inadequate. In spite of the introduction of new drugs for the treatment of chronic liver diseases, such as interferon for viral hepatitis B and C, ursodeoxycholic acid for chronic cholestatic disorders and the immunosuppressants for immune- mediated liver diseases, no current therapy prevents the progression of hepatic fibrosis and cirrhosis. In order to develop and select drugs for the effective treatment of chronic liver disorders that progress from hepatic inflammation to fibrosis and cirrhosis, pathogenic mechanisms responsible for hepatic fibrosis must be elucidated.

Fibrosis is initiated by a chronic insult (e.g. infectious, immunologic, toxic, etc.) that induces prolonged inflammatory reaction. In the liver, such an insult disrupts the endothelial cell layer in the space of Disse and exposes

the extracellular matrix (ECM) adjacent to the basement membrane. This basement membrane consists of a type IV collagen core to which are attached proteoglycans and glycoproteins, such as fibronectin (FN) and laminin (LM) . Shortly after the injury in the basement membrane, recruitment of lymphocytes and macrophages to the site of injury occurs. The accumulation of mononuclear cells in the injured area depends on their ability to traffic across the blood vessel wall and their adhesion to the basement membrane in the space of Disse. Both these processes are mediated by integrins, a family of cell surface adhesion molecules-

The cell matrix component (target epitope) to which several integrin receptors bind is the GD (Arg-Gly-Asp) sequence - a cell adhesion motif shared by several matrix- associated glycoproteins, such as FN, VN { itronectin) and fibrinogen. This sequence is recognized by several integrin receptors that mediate RGD-dependent matrix adhesion of cells following cell activation. The interaction of FN with these integrins occurs through multiple sites, including the major RGD-containing epitope. Therefore, when present in solution, peptides containing the RGD sequence prevent cell adhesion by competing with RGD-containing matrix proteins for binding to their respective integrin receptors. During inflammatory processes, immune cells penetrate the walls of blood vessels, its underlying basement membrane and the ECM, while homing to target organs and tissues. Cell adhesion to glycoproteins of the ECM is a prerequisite for their subsequent homing to in lammatory sites . Once at the site of injury, immune cell-FN interactions dictate the subsequent activation, proliferation and secretion of cytokines, such as tumor necrosis factor α (TNFo) and

fibroblast growth factor (FGF) , by the immune cells. These soluble mediators probably link the inflammatory and reparative phases of liver cirrhosis, mainly by the activation of Ito cells, that adhere to the basement membrane, proliferate and deposit collagen and other matrix components in the extracellular space, to complete the fibrotic process. The total collagen content of the cirrhotic liver may increase up to 10-fold above the normal value of about 5 mg/g wet liver weight. Therefore, the management of hepatic fibrosis should involve, in addition to the suppression of the causative agent that induces chronic hepatic inflammation, a specific action on the ECM of the liver.

Several studies using a combination of methods have shown that Ito (fat storing) cells are the major source of matrix components in both normal and fibrotic livers (review in Clement et al., 1992). In chronically injured liver, Ito cells acquire a yofibroblast- i e phenotype. During the reparative phase of hepatic fibrosis, these activated cells proliferate in areas of cell necrosis and inflammation, and play a major role in liver fibrogenesis via increased deposition of a wide spectrum of ECM constituents and through secretion of several growth factors and cytokines. Early in this so-called reparative phase, Ito cells, in response to TNFα, migrate to the site of tissue injury, there these cells adhere to the basement membrane, proliferate, and deposit matrix components in the extracellular space to complete the fibrotic process. The key event in this phase is the adhesion of localized fibroblast to ECM components, particularly FN, and this is a RGD-dependent process.

Recently the usage of RGD-containing peptide ligands in preventing mononuclear and fibroblast cell migration in vivo

has been indicated (D'Souza et al., 1991) . Since RGD- containing ligands are rapidly degraded by proteolytic enzymes, these ligands were used at high concentrations, which rendered them highly immunogenic. International PCT Application WO 93/09795 corresponding to Israel Patent Application No. 100130 of the same applicant of the present application, describes non-peptidic compounds having no sequence of natural α-amino acids and comprising a guanidino and a carboxyl terminal functional groups spaced by a sequence of 11 atoms, at least 5 of which are carbon atoms, and to salts thereof, which are capable of inhibiting cell adhesion. These compounds are mimetics of the RGD sequence and exhibit a high affinity for RGD- dependent integrins. Said RGD surrogates were said to be useful for the treatment of several disorders, such as thrombosis, metastasis, autoimmune diseases and other immune responses such as allergy, graft- versus-host and host- versus-graft reactions, and inhibition of scar-tissue formation. Some of these RGD surrogates were shown indeed to specifically inhibit melanoma cell adhesion to immobilized VN and FN and formation of metastatic melanoma cell colonies in urine lungs in experimental and spontaneous models of metastases. The same RGD surrogates were shown to specifically inhibit integrin-mediated platelet aggregation with IC _JO 'S in the submillimolar range and to interfere with RGD-dependent adhesion of CD4* T-lymphocytes and metastatic tumor cells to immobilized EN and VΝ in vitro (Greenspoon et al., 1993) . In vivo, an RGD surrogate effectively inhibited the elicitation of a delayed-type hypersensitivity (DTH) reaction mediated by CD4* T-cells (Greenspoon et al., 1993) .

SDMMARY OF THE INVENTION

It has now been found in accordance with the present invention that some of the RGD surrogates described before in International Application No. WO 93/09795 are able to inhibit the development of liver fibrosis and cirrhotic lesions in murine models.

The present invention thus relates to pharmaceutical compositions for the prevention and/or treatment of liver fibrosis and cirrhosis comprising as active ingredient a compound of formula I:

H ₂ N-C(-NH) -NH-CHj-A-CH _j -COjH and pharmaceutically acceptable salts thereof, wherein A is a chain of 9 atoms selected from the group consisting of:

(i) -{CH ₂ ) _n -CO-NH-(CH ₂ ) ₇ _ _n ; (ii) -(CH ₂ ) _n -NH-CO-(CH ₂ ) ₇ _ _n ;

(iii) - (CHj) _x -CO-NH- (CH ₂ ) _n -CO-NH- (CH ₂ )„;

(iv) - (CH ₂ ) _x -NH-CO- (CH ₂ ) _n -NH-CO- (CH ₂ ) _Λ ;

(v) -(CH ₂ ) _n -CO-N_5 C ^c O-UH- (CH ₂ ) _m ;

wherein each of x, n and m is at least 1; in chains (i) and (ii) n is at most 6; in chains (iii) and (iv) the sum of x+n+m is 5; and the sum of n+m is 4 in chain (v) and 3 in chain (vi) .

In one embodiment, the pharmaceutical composition comprises as active ingredient the compound 6-aza-7-oxo-12- guanidinododecanoic acid, a compound of formula I(i) above wherein n is 4, herein designated compound SF-6,5. In another embodiment, the composition comprises as active

ingredient the compound NS-11, a compound of formula I(vii) above.

The invention further relates to the use of compounds of formula I for the manufacture of pharmaceutical compositions for the prevention and/or treatment of hepatic fibrosis and cirrhosis.

The invention still further relates to a method for the prevention or treatment of a patient afflicted with hepatic fibrosis or cirrhosis which comprises administering to said patient an effective amount of a compound of formula I herein.

BRIEF DESCRIPTIONS OF THE FIGURES

Fig. 1 shows the mean integrated optical density (OD) values (calculated by computerized imaging morphometry as a method for the quantitation of hepatic fibrosis in the histologic slides) of thioacetamide (TAA) -treated rats without and with treatment with the RGD analog SF-6,5 for 3 and 5 months. Fig. 2 shows the mean spleen weight in rats that received TAA and TAA+RGD analog SF-6,5 (for 3 months) treatment.

DETAILED DESCRIPTION OF THE INVENTION The compounds of formula I used in the composition of the present invention may be prepared by suitable chemical synthesis, for example by the methods described in above- mentioned WO 93/09795, herein incorporated in its entirety by reference. The pharmaceutical compositions according to the invention will comprise as active ingredient a compound of formula I and a pharmaceutically acceptable carrier. The

compositions may be in the form of tablet, capsule, solution or suspension containing from about 0.7 to 70 mg per unit of dosage of an active compound of the formula I or mixtures thereof. The compounds may be compounded in conventional manner with a physiologically acceptable vehicle or carrier, excipiβnt,binder, preservative, stabilizer, etc.

The compounds of the invention can be administered to patients by any suitable route including oral and parenteral routes, e.g., intravenous, subcutaneous or intramuscular injection. An effective but essentially non-toxic quantity of the compound will be employed in the treatment. Effective amounts may be within the range of 0.01 to 1 mg/kg, preferably 0.5 mg/kg body weight, on a regimen in single or several daily doses. To examine the possibility that the nonpeptidic analogues of RGD of formula I may inhibit the development of liver damage and fibrosis in rats, a thioacetamide (TAA)- induced liver cirrhosis animal model system was used (Muller et al., 1988) . In this model, prominent liver fibrosis and regenerative nodules are developed after 3 months of administration of TAA, associated by portal hypertension and a hyperdynamic circulation characteristic to patients with liver cirrhosis.

The following examples are intended to illustrate, by way of example, the principles of the invention, without limiting it thereto.

EXAMPLES

Example 1: Preparation of 6-a«a-7-o_«o-12-g-αan±d±no- dodβeanoic aαi (compound. SF-6,5)

Compound SF-6,5 was prepared by coupling of methyl-5- aminovaleric acid with N- (butyloxycarbonyl) -6-aminohexanoic

acid as previously described (Hershkoviz et al.,1994) .

Briefly, compound SF-6,5 was prepared. The reaction was carried out using 1, 3-dicyclohexylcarbodiimide and 1- carboxamidine nitrate. Compound SF-6,5 was characterized and its purity (over 97%) was determined by HPLC chromatography.

The structure deduced from spectroscopy was consistent with the assigned structure. SF-6,5 dissolved in PBS was administered daily (100-500μg/rat) for 5 days a week by the

I.P. route during the various treatment periods.

Example 2: Induction of liver eirrhoais

Hepatic cirrhosis was induced in rats by long term (3 months) oral administration of thioacetamide (TAA, Sigma, Israel; 0.03% in tap water) . Female Wistar rats (at least 50 per group) weighing 200-250 gm were administered TAA. The rats were kept in the Animal Breeding Center of the Wolfson Medical Center, Holon, Israel, and were fed Purina rodent chow ad libitum and maintained under a constant light cycle. Characteristic lesions in rat liver occurred, which correspond to cirrhosis-like patterns of micronodular cirrhosis. These lesions persist for 2 months after termination of TAA treatment. The administration of TAA for a shorter time period, such as 6 weeks, was not followed by the development of cirrhosis.

Example 3: Treatment of TAA-induced oirrhotie rats with αompound SF-6,5.

(i) Experiment design

The study design consisted of 6 treatment groups of 6 rats each: 1. TAA for 12 weeks with daily injections of NaCl 0.9% (cirrhotic controls for the RGD-treated groups); 2. TAA +SF-6,5 (100 μg/rat/day for 12 weeks); 3. TAA -t-SF-6,5 (500

μg/rat/day for 12 weeks); 4. TAA for 6 weeks; 5. Tap water without TAA for 12 weeks (normal controls); 6. TAA + SF-6,5

(500 μg/rat/day both for 12 weeks) and then SF-6,5 (500 μg/rat/day) for additional 8 weeks. Body weight of the rats in the different treatment groups was recorded monthly and spleen weight was measured upon completion of treatment, after the rats were sacrificed.

(ii) Liver Histopathology

Upon completion of the various treatment periods, rats were sacrificed and idsections of the left lobe of the liver were taken immediately and processed for light microscopy. The specimens were fixed in 5% neutral formol solution. 5 μm paraffin sections were stained with Hematoxyllin & Eosin, and Masson Trichrome, to enable more accurate assessment of the degree of hepatic fibrosis. The tissue slices were scanned, the pathological changes were scored semi-quantitatively (0-3) and the degree of inflammation and fibrosis expressed as the mean of 10 different fields in each slide, and were classified according to Muller et al. (1988) : no change - 0, slight changes - 1, stronger changes - 2, intense changes - 3. The following pathological alterations were scored in the portal tracts: fibrosis, enlargement and inflammatory infiltration, breaking up to the hepatocellular limiting plates, and in the intraacinar mesenchyma: diffuse or spotty inflammation, nodule formation, spotty fibrosis and the presence of active or passive septa, transformation of liver structure with formation of pseudoacini. (iii) Quantitative Analysis of the Mioroaeopiα Slldea Quantitative analysis of the microscopic slides was performed by a computerized video-imaging system (Biological Detection System, Pittsburgh, PA) . To standardize the

optical density (OD) determinations of the microscopic slides (stained by H&E) we proceeded as follows: for each microscopic field an OD value of clear areas, designated as the background level, was defined. For each microscopic field containing tissue the area and the OD were determined.

The integrated OD value (IOD) was calculated by multiplying the area by the OD and subtracting the multiplication of the same area by the background OD. Since fibrotic tissue was characterized by a higher OD value, it enabled the specific determination of its area based on an OD value density slices. The IOD of the fibrotic area was determined in a similar way (as described above) . The data for the fibrotic tissue was obtained by multiplying the OD value by the sum of the area occupied by the fibrotic tissue divided by the IOD value of liver tissue in each microscopic field and expressed as a percentage. Data were analyzed by ANOVA followed by Bartlett's test for homogeneity of variance, and Turkey-Kramer multiple comparisons were applied as the post hoc test. (iv) Induction of Liver Cirrhosis

Administration of 0.03% TAA in drinking water for 3 months resulted in cirrhosis-like structural pattern characterized by mixed-sized fibrotic nodules in treated rats associated by minimal inflammatory response. The administration of TAA for a shorter time period (i.e., 6 weeks) was followed by the development of peri-portal inflammation and slight fibrosis but no cirrhotic nodules or fibrotic septa were observed in this treatment group.

Liver histopathology in rats that received TAA and the RGD analog SF-6,5 for 3 months in a lower dose of 100 μg/rat/day showed cirrhotic lesions similar to the group that was treated by TAA alone for 3 months. In this lower

dose, the RGD analog SF-6,5 was not effective in the prevention of liver cirrhosis (Table 1) . In contrast, in the group that received TAA and 500μg/rat/day SF-6,5 for 3 months, the liver histology showed prominent portal and peri-portal inflammation with mild bridging fibrosis, but cirrhotic nodules or passive fibrotic septa were absent

(pathologic score of 1-2 compared to 3 in the TAA group) . In the TAA-treated group that was given the RGD analog SF-6,5 in a dose of 500 μg/rat/day for 5 months, the liver histology looked near normal except for minimal enlargement and inflammation in the portal space (score 0-1; Table 1) .

As shown previously (Muller et al., 1988), and also consistent with our own observation in the present study, in this model of TAA intoxication the cirrhotic lesions persist for two months after TAA withdrawal. Therefore, these latter results suggest that the administration of the RGD mimetic SF-6,5 for another two months after TAA withdrawal further improves liver histology, and this implies that the RGD analog may have a therapeutic rather than merely a prophylactic effect.

(v) Ef ect on body weight

Control rats treated only by NaCl 0.9% for 3 months gained weight as expected, from a baseline of 238 ± 28 gm to 317 ± 22 in 12 weeks. In contrast, the rats that received only TAA for 3 months showed a slight decrease in body weight from a baseline of 243 ± 28 to 228 ± 12 gm (p-NS compared to pretreatment) in the end of the treatment period. In contrast, rats treated with the RGD analogue SF- 6,5 for 12 weeks, in addition to TAA, increased in body weight from a baseline of 238 ± 18 to 295 ± 28 gm after 3 months (p<0.01 compared to TAA alone - Table 2). (vi) Quantitative Analysis of the Microscopic Slides

The mean integrated OD values of the TAA-treated group

(calculated by computerized imaging morphometry as a method for the quantitation of hepatic fibrosis in the histologic slides) were significantly higher (46.6 ± 1.9) compared to 29.9 ± 3.5 (p<0.001) and 8.9 ± 1.6 (p<0.001) in the TAA +

SF-6,5 (500 μg) and the control (NaCl-treated) groups, respectively. The IOD in the group treated with RGD analog

SF-6,5 for 5 months was 19.9 ± 0.9 (P<0.001 compared to control, Fig. 1) . These results confirm by a computerized morphometric method the semiquantitative histopathologic scoring. (vii) Effects of the RGD analog SF-6,5 on spleen weight

Spleen weights were recorded in all rats upon the end of the treatment periods and used as an indirect measure for the development of portal hypertension. Previous studies have shown that in TAA-induced liver cirrhosis in rats, characteristic hemodyna ic changes developed after 3 months of TAA administration. These included a significant increase in spleen weight, in addition to portal hypertension and an hyperdynamic circulation (Hori et al., 1993). The mean spleen weight in control rats injected daily with NaCl 0.9% was 522 ± 64 mg, compared to 938 ± 82 in TAA-treated cirrhotic rats (p<0.01, Fig. 2). The mean spleen weights in rats that received TAA and SF-6,5 for 3 months was significantly lower 672 ± 59; p<0.01 compared to TAA alone) . These results provide additional evidence that the development of portal hypertension in TAA-treated rats liver cirrhosis was inhibited by the simultaneous administration of the RGD analog SF-6,5.

TABLE 2:. -.ff-f-._- nf ROD analog. SP-6,5 on rat body weight las!

NaCl 0.9% TAA TAA+SF-6,5 500 μg (control) (3 months) (3 months)

Pretreatment 238 ± 28 243 ± 28 238 ± 18 1 month 271 ± 23 236 ± 16 238 ± 10

2 months 294 ± 38 228 ± 12 257 ± 24

3 months 317 ± 22 228 ± 12 295 ± 28*

Mean ± SD, * p<0.01 compared to TAA

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