ITMI981079A1 - BILIARY ACIDS AS INDUCERS OF THE CYTOCHROME P450-EMPLOYEE SYSTEM IN PARTICULAR TO ANTI-COLESTATIC ACTIVITY - Google Patents

Description

DESCRIPTION

The present invention relates to bile acids as inducers of the cytochrome P450-dependent system, particularly useful for the treatment of pathologies induced by cholestasis.

In particular, the present invention relates to the use of bile acids and similar compounds to eliminate and prevent hepatic damage induced by lipophilic and toxic bile acids and by other toxins in general.

It is known that bile acids initially found use in litholytic therapy due to their ability to desaturate cholesterol bile, while subsequently they were used in the treatment of chronic liver diseases.

Among them, chenodeoxycholic acid (CDCA) was the first to find use in the clinic. However, the administration of this acid, since it induces a transient and reversible hypertransaminasemia in 30% of subjects treated for litholytic purposes, has suggested its potential hepatic toxicity.

These drawbacks have been overcome with the advent of the 7 beta epimer of CDCA, ursodeoxycholic acid (UDCA), whose use in litholytic therapy has proven to be practically free of side effects in the liver. This fact has made it possible to extend the use of UDCA for lytic purposes even in chronic liver disease patients.

It dates back to 1985 that the administration of UDCA in six patients with chronic active hepatitis and suffering from gallstones induced a consistent reduction in the serum levels of transaminases. It has therefore been postulated that retention and increased hepatic perfusion with endogenous, detergent and therefore potentially hepatotoxic bile acids, such as CDCA and deoxycholic acid, may play an important role in the pathogenesis of chronic cholestatic liver diseases.

On the contrary, it has been thought that the administration of exogenous UDCA, determining the enrichment of the overall pool with a hydrophilic and less detergent bile acid at the expense of the endogenous bile acids, can determine a protective action on the hepatocyte.

In light of these considerations, several studies have been carried out aimed at highlighting an improvement in biohumoral indices in chronic liver disease patients treated with UDCA.

Recently, the scientific community has therefore proposed a series of hypotheses on the mechanisms of action by which UDCA determines an improvement in the indices of necrosis and cholestasis. Among these the most accredited lead us to think:

- a stabilizing effect on cell membranes, with a reduction in their permeability,

- an antagonistic effect against the increased synthesis of alkaline phosphatase resulting from the retention of endogenous bile acids in the course of cholestasis,

- an action favoring the excretion of hepatic enzymes in the bile, due to a decreased return of the enzymes from the bile into the sinusoidal blood as a function of a protective effect exerted by the UDCA on the canalicular tight junctions or to a real choleretic effect associated with an increased secretion of bicarbonates.

Experimental studies have also shown that, in rats with choledochus ligament, there is a reduction in the levels of cytochrome ≤> 450 microsomal and that in vitro the bile acids produce a general reduction of the catalytic activity of cytochrome P450 and other microsomal liver enzymes. .

At present, it is therefore known that bile acids are inhibitors of these activities which indicate liver toxicity in cases of chronic liver disease with cholestasis, where high intrahepatic concentrations of endogenous bile acids occur. This fact occurs in other intestinal pathologies in which, due to an alteration in the intestinal microflora, there is a production of more lipophilic and hepatotoxic secondary bile acids.

A general object of the present invention is to provide new uses in the therapeutic field for selected bile acids.

One of the main purposes of the present invention is to provide new drugs or medicaments with anticolestatic activity the administration of which involves limited systemic side effects.

A further object of the present invention is to provide drugs for the treatment of both type and hepatic diseases

type induced by exogenous toxic substances that are easily available on the market at competitive costs.

Not least object is to provide medicaments which are capable of preventing the damage induced by lipophilic and toxic bile acids and other toxins which can be used both in the treatment of human beings and animals.

In the light of these purposes and of others still which will become more evident in the following, the use of bile acids of formula (I) is provided in accordance with the present invention:

in which

R ^ is H, RgCOO wherein it is an alkyl with from 1 to 6 C atoms, RgCOO <"> in which Rg is alkyl with from 1 to 6 C atoms, and their pharmaceutically acceptable salts, for the production of a medicament to induce the cytochrome P4S0-P-glycoprotein system. In particular, said bile acids of formula (I) and their pharmaceutically acceptable salts are useful for the production of a medicament for the prevention or treatment of pathologies induced or associated with cholestasis.

Among the bile acids of formula (I) those in which it is selected from H, -CHjCOO, salts with alkali metals are preferred.

Within the scope of the present invention, the term pathologies induced or associated with cholestasis means diseases both in the medical and veterinary field, including primary biliary cirrhosis, chronic cholestatic liver disease, infantile liver disease associated, or not, with cystic fibrosis, liver disease induced by toxins of various kinds, including drugs, environmental toxicants, endogenous substances. Intestinal diseases are also included in the pathologies associated with cholestasis.

In prophylactic or therapeutic use as molecules / agents with anticolestatic activity, the bile acids of formula (I) of the invention are administered in a quantity preferably between 5 and 20 mg / kg body, through a single or multiple daily administration.

The specific dosages will vary according to the needs of the subject to be treated, the severity of the disease in progress and the activity of the compound used. Determining the optimal dosage to administer in each particular situation is within the physician's choice.

In the production of a medicament comprising a bile acid of formula <(> I <)> according to the invention, pharmaceutically acceptable carriers are used, both in liquid and solid form.

Solid preparations include tablets, capsules, veterinary-type boluses, cachets and powders.

Pharmaceutically acceptable carriers include one or more excipients, diluents, dispersing and solubilizing agents, lubricants, disaggregating binders and substances for sustained release of the active ingredient.

In the production of tablets, the bile acid of formula (I) is in admixture with one or more vehicles, in suitable proportions and in a compacted and shaped form.

By way of example, the medicaments and pharmaceutical formulations produced in accordance with the invention contain from 5 to 30% by weight of at least one bile acid of formula (I).

Suitable vehicles in the preparation of anti-cholestatic medicaments include magnesium carbonate, magnesium stearate, talc, lactose, corn starch, sucrose, pectin, dextrin, methylcellulose, sodium carboxymethylcellulose, cocoa butter, low melting waxes and the like.

Among the pharmaceutical forms which can be produced by using the bile acids of formula (I) are included the ready-release forms and the delayed or controlled release forms.

The liquid form preparations include solutions suitable for parenteral and oral administration.

The forms for parenteral use are advantageously in the form of sterile aqueous solutions and can include further solvents such as ethanol, propylene glycol.

The administration of the medicaments of the invention is advantageously carried out in humans before meals (30-60 minutes before) to allow the induction of the P450 dependent mechanism to take place and to predispose the liver to remove the bile acid bolus in a short time. endogenous which as a result of the meal are removed from the gallbladder, released in the intestine, absorbed and, through the portal vein, reach the liver.

Under these conditions, induction of the P450-P-glycoprotein system facilitates the transport and excretion of potentially toxic bile acids that accumulate in the liver.

The administration of bile acids of formula (I) induces the activity of cytochrome P450 which is associated with the induction of the P-glycoprotein, a special ATP-dependent pump, of a protein nature present in numerous tissues with the function of eliminating substances from the cells cytotoxic. This induction of the cytochrome P450-glycoprotein-P system is closely associated with the ability of these molecules to prevent or minimize the damage induced by the subsequent administration of lipophilic and toxic bile acids, such as, for example, chenodeoxycholic and deoxycholic acid. both in the free and conjugated forms with gliein and taurine which are rapidly excreted by the liver with a maximum transport value increased by at least 10 times, through the induction of the removal pump (P-glycoprotein).

According to a particularly preferred embodiment of the present invention, said bile acids are selected from the group reported in the following scheme I, among these, ursodeoxycholic acid and / or tauroiodesoxycholic acid being particularly preferred.

Scheme I

It has been verified that the bile acids of formula (I) have a lipophilicity and detergency lower than that of endogenous bile acids with lower log P values and higher critical micellar concentrations, as shown in the following Table 1.

Table 1: chemical-physical properties of the bile acids studied.

It has been verified that the bile acids of formula (I) according to the invention induce, in both mice and rats, the cytochrome P450-dependent monooxygenase system, with particular reference to the isoforms P450 3A which is specifically associated with the induction of glycoprotein P.

According to another aspect, the present invention relates to the use of a pharmacological active principle capable of inducing the cytochrome P450 / P-glycoprotein system for the preparation of a medicament for the treatment of liver and / or intestinal diseases.

Said active ingredients are advantageously selected from glucocorticoid derivatives such as dexamethasone, betamethasone, 9alpha-fluorohydrocortisone, alpha-methylprednisolone, triamcinolone acetonide, fluocinolone acetonide, triamcinolone, llbeta-hydroxyprogesterone, prednisolone, corticone, hydrogen peroxide; derivatives of pregnenolone 16alpha-carbonitrile such as salfamethyl-16,17-epoxy-pregnenolone, 16 alpha methylpregnolone, dehydropregnolone; estrogens such as beta-estradiol, progesterone; testosterone derivatives such as alpha methyltestosterone, corticosteroids such as aldosterone spironolactone, barbiturates, macrolides, imidazoles, antibiotics such as rifampicin, reserpine, phenobarbital, verapamil, midazolam, repamycin, clotrimazole, FK506, isosaphrole, amiodolycin, dedomycin, dedomycin, dedomycetil phenytoin and their mixtures.

The following examples are provided for illustrative purposes only of the present invention and are not to be construed as limiting the scope of the present invention, as defined by the attached claims.

EXAMPLE 1

Male Spague Dawley rats weighing approx. 300 +/- 20 g intravenously with a toxic bile acid (TCDCA) at a dose of 8 μηιοΐ x min <"1> x Kg <" 1> for one hour. An equimolar dose of THDCA, endowed with anticolestatic activity, was then co-infused IV. Controls consist of rats treated only with saline solution and with only toxic or therapeutic bile acid as reported in Table 1. The animal model envisages rats with external biliary fistula to collect bile for a further two hours from the start of the experiment and for the analysis of the multiple enzymatic activities of P45Q in the liver removed at the end of the experiment. Testosterone, due to its ability to be metabolized in a stereo and regio selective manner by the different isoforms of cytochrome P450, has been used as a multi-substrate marker of the different isoforms.

In the following Table 2, the values related to the

modulation of the monooxygenase system in the rat at time tQ (baseline),

t1 (1 hour of infusion) and t2 (2 hours of infusion). From data analysis

reported in Table 2, where the expression of testosterone is reported

hydroxylase in liver microsomes of animals treated with TCDCA, it is inferred

the powerful inactivating effect induced by the acid already after an hour of infu¬

sion (the percentage of decline reaches as much as 97% for the activity

testosterone 16alpha and 2alpha-hydroxylase) indicating the high toxicity of

this bile acid.

Conversely, THDCA was able to determine a potent e

significant inductive effect on the P450 system with particular

reference to cytochrome P4503A (testosterone Sbeta and 2beta-hydroxylase).

TABLE 2 - TESTOSTERONE HYDROXYLASE IN LIVER MICROSOMES 01 RATS

TREATED WITH TCDCA

EXAMPLE 2

The inductive effect (Table 3) after one hour reached increases of the order of 28.29% (16alpha-hydroxytestosterone) and 48.42% (2alphahydroxytestosterone) and was further strengthened with a 2-hour infusion reaching the limit increase of 93.38% (2alphahydroxytestosterone). The testosterone 6beta-hydroxylase activity, associated with the P450 3A isoform, is the most induced, not in percentage terms, as it reaches 29.33%, but rather in terms of "catalytic power <1>". in nmol x mg <"1> x min <" 1>, the induced enzyme is able to metabolize about 14000 pmol more in the unit of time per milligram of protein, a value much higher than that obtained with the induction of the other activities expressed in pmol x mg <"1> x min <" 1> reported in the following Table 3.

TABLE 3 - TESTOSTERONE HYDROXYLASE IN LIVER MICROSOMES OF RATS

TREATED WITH TCOCA-THOCA

When THDCA was co-infused with toxic bile acid

{TCDCA), surprisingly, the drastic precedent e is not recorded

sudden effect inactivating the enzymes of the metabolism of xenobiotics

but, on the contrary, THDCA is capable of carrying out a protective effect

on the liver by the aforementioned hepatotoxic effect.

As evidence of this fact, all the activities associated with the molecule

of testosterone were found, albeit to a lesser extent than in trat¬

tamento with only THDCA, markedly induced by the co-treatment, as evi¬

referenced in Table 4.

TABLE 4 - TESTOSTERONE HYDROXYLASE IN LIVER MICROSOMES OF RATS

TREATED WITH THDCA

EXAMPLE 3

Aminopyrine was used as the preferential substrate for the P450 3A isoforms. The biochemical-enzymological picture confirmed: a drastic decrease in activity due to the treatment with TCDCA, strong inductive effect on cytochrome P450 caused by the infusion with THDCA and protective effect (intermediate induction) after co-infusion of the two molecules, as highlighted in Table 5 below.

TABLE 5 - AMINOPYRINE N-DEMETHYLASE IN HEPATICIC MICROSOMES IN RATS

TREATED WITH ACIDIBILIARIES

EXAMPLE 4

A parallel study was performed on the mouse Swiss Albino ODI strain, male, 30 / -3 g.

The animals were treated intraperitoneally (i.p.) with THDCA, UDCA, or TDCA, at a dose of 12.2 or 24.4 mg / kg and the monooxygenase system studied on hepatic subcellular fractions (microsomes at 1050000 x g) obtained 24 h after the treatment.

The results highlighted the modulatory capacity of the hepatic monooxygenase particulate system by THDCA and UDCA; on the contrary, the TDCA does not induce significant changes.

In Table 6 below, the expression of metabolizing enzymes using different substrates as probes for the different P450 isoforms is reported. In particular, aminopyrine was used (for the P450 3A1 / 2 isoforms, as previously reported for the rat), para-nitrophenol (isoform P4502E1), ethoxycumarin (mixed), pentoxiresoruphine (isoform P4S02B1), methoxyresoruphine (isoforms P4501A2) and 1 <1> ethoxyresoruphine (isoforms P4501A1). Finally, total P450 content and associated (NADPH-dependent) reductase were measured.

TABLE 6 - EFFECTS OF THDCA ON THE MONOOXYGENASE SYSTEM IN

MICROSOM1 HEPATIC WALLS

The data relating to THDCA demonstrate an inductive effect on the total P450 as well as the reductase activity associated with it. This induction is reflected in the monooxygenase activities correlated with these components, as evidenced by the significant increase in the hydroxylation of p-nitrophenol, dealkylation of pentoxiresorufine, demethylation of methoxyresorufine, but, above all, by the demethylation of aminopyrine (P450 3A). On the contrary, TDCA treatment does not give rise to substantial modulations, some of these being statistically significant but biologically not relevant as shown in the following Table 7.

Similarly to THDCA, UDCA was able to markedly induce a mixed function oxidase system as shown by the significant increase in all the parameters considered, with particular reference to the aminopyrine N-demethylase activity, probe for the P450 3A isoforms associated with induction of P-glycoprotein (Table 8).

In order to confirm the effects of the bile acids of formula ø of the invention, on the monooxygenase system, the samples were tested using testosterone as a multibiomarker. Table 9 shows the results obtained with treatment with THDCA: from the analysis of the results, a powerful inductive effect on the different activities carried out on testosterone is clearly observed, which include cytochrome P450-3A / PgP dependent activity (testosterone 6 beta- hydroxylase). While TDCA, also studied with testosterone, was unable to modulate the related oxidase activities (Table 10), UDCA produced a significant increase in all testosterone metabolites, including 3A-dependent isoforms (6beta). -linked) Table 11.

TABLE 10 - TESTOSTERONE HYDROXYLASE IN LIVER MICROSOML OF MICE

TREATED WITH TDCA

TABLE 11 - TESTOSTERONE HYDROXYLASE IN LIVER MICROSOML OF MICE

TREATED WITH UDCA

EXAMPLE 6

Male rats were pre-infused (IV) for 30 minutes with a bile acid of formula (I) according to the invention, having inductive properties (THDCA) and subsequently infused with a bile acid having toxic properties (TCDCA) at equimolar doses ( 8 μτηοΐ x min <-1> x Kg <"1>, in the presence of, or, in the absence of vinblastine, a known inhibitor of the take-away pump (glycoprotein P), administered (IV) at a dose of 2 / absolute ichthyols in a single bolus.

The results, reported in Table 12, indicate a generalized induction of the P4SO-dependent machinery in rats subjected to protection (i.e. retentive THDCA and TCDCA) with respect to the control. Conversely, a significant reduction (approximately 30%) of testosterone 6beta and 2beta-hydroxylase activity (associated with the P450-glycoprotein-P complex) is observed in the inhibitor-receiving group. It is believed that despite the presence of a high induction of P450, the presence of vinblastine, inhibiting the. ATP-dependent removal pump, it maintains, inside the cell, a quantity of TCDCA sufficient to inactivate, at least partially, the monooxygenase system such as to significantly reduce the inductive effect recorded.

TABLE 12 - LOROXYLASE TESTOSTERONE IN HEPATIC MICROSOMIES Ol RAT

TREATED WITH TCDCA-THDCA IN THE PRESENCE OR ABSENCE OF VIN8LAST1NA

Claims (11)

CLAIMS 1. Use of bile acids of formula (I): in which R4 is H, RgCOO <*> where Rg is an alkyl with 1 to 6 C atoms, RgCOO where Rg is alkyl with 1 to 6 C atoms, and their pharmaceutically acceptable salts, for the production of a medicament for inducing the cytochrome P450-P-glycoprotein system. Use according to claim 1, wherein R ^ is selected from: Use according to claim 1 or 2, characterized in that said bile acid is selected from the group consisting of ursodeoxycholic acid, iodesoxycholic acid, ursocolic acid, iocolic acid, cholic acid, glicoursodeoxycholic acid, tauroursodeoxycholic acid, glycoiodeoxycholic acid, tauroiodesoxycholic acid, alpha-glycomuricolate acid, alpha tauromulicolate acid, beta glycomuriculate acid, betatauromuriculate acid, glycoursocolic acid, tauroursocolic acid, gl coiocolic acid, tauroiocolic acid, glycolic acid, taurocholic acid. 4. Use of bile acids of formula (I); in which is H, alpha / beta OH; R2 is H or alpha OH; R3 is H or beta / alpha OH; R4 is H, R ^ COO <’> in which R3⁄4 is an alkyl with from 1 to 6 atoms of C, RgCOO in which Rg is alkyl with from 1 to 6 atoms of C, and their pharmaceutically acceptable salts, for the production of a medicament for the treatment or prevention of pathologies induced or associated with the treatment of cholestasis with toxic bile acids. Use according to claim 4, wherein R ^ is selected from: <+> Use according to claim 4 or 5, characterized in that said bile acid is selected from the group consisting of ursodeoxycholic acid, iodesoxycholic acid, ursocolic acid, iocolic acid, cholic acid, glicoursodeoxycholic acid, tauroursodeoxycholic acid, glycoiodeoxycholic acid, tauroiodesoxycholic acid, alpha-glycolic acid, alpha tauromulicolate acid, beta glycomuricolate acid, betatauromuricolate acid, glycoursocolic acid, tauroursocolic acid, glycoiocolic acid, tauroiocolic acid, glycolic acid, taurocholic acid. Use according to any one of claims 4-6, characterized in that said pathologies induced or associated with cholestasis are hepatopathies or intestinal diseases. Use according to any one of claims 4-7, characterized in that said pathologies induced or associated with cholestasis are primary biliary cirrhosis, chronic cholestatic liver disease, infantile liver disease associated with cystic fibrosis. 9. Use of a pharmacological active principle capable of inducing cytochrome P450 for the production of a medicament for the treatment of liver and / or intestinal diseases. 10. Use according to claim 9, wherein said active principle is selected from glucocorticoid derivatives such as dexamethasone, betamethasone, 9alpha-fluorohydrocortisone, alpha-methylprednisolone, triamcinolone acetonide, fluocinolone acetonide, triamcinolone, llbeta-hydroxyprogesterone, prednisolone, cortisolone, hydrocortisolone; pregnenolone 16-alpha-carbonitrile derivatives such as 16-alpha-methyl-16,17-epoxy-pregnenolone, 16 alpha-methylpregnolone, 16-alpha-dehydropregnolone; oestrogens such as beta-estradiol, progesterone, testosterone derivatives such as alpha methyltestosterone, corticosteroids such as aldosterone spironolactone, barbiturates, macrolides, imidazoles, antibiotics such as rifampicin, reserpine, phenobarbital, verapamil, midazolam, repamycin, clotrimazole, trimazole , erythromycin, dexamethasone, nifedipine, phenytoin and their mixtures. Use according to claim 9 or 10, characterized in that said hepatopathies consist of primary biliary cirrhosis, chronic cholestatic liver diseases, infantile liver diseases associated with cystic fibrosis.