Side effects of brequinar and brequinar analogues, in combination with cyclosporine, in the rat.

Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.

SDZ PRI 053, an orally bioavailable human immunodeficiency virus type 1 proteinase inhibitor containing the 2-aminobenzylstatine moiety.

A series of inhibitors of human immunodeficiency virus type 1 (HIV-1) proteinase containing the 2-aralkyl-amino-substituted statine moiety as a novel transition-state analog was synthesized, with the aim to obtain compounds which combine anti-HIV potency with oral bioavailability. The reduced-size 2-aminobenzylstatine derivative SDZ PRI 053, which contains 2-(S)-amino-3-(R)-hydroxyindane in place of an amino acid amide, is a potent and orally bioavailable inhibitor of HIV-1 replication. The antiviral activity of SDZ PRI 053 was demonstrated in various cell lines, in primary lymphocytes, and in primary monocytes, against laboratory strains as well as clinical HIV-1 isolates (50% effective dose = 0.028 to 0.15 microM). Cell proliferation was impaired only at 100- to 300-fold-higher concentrations. The mechanism of antiviral action of the proteinase inhibitor SDZ PRI 0.53 was demonstrated to be inhibition of gag precursor protein processing. The finding that the inhibitory potency of SDZ PRI 053 in chronic virus infection, determined by p24 release, was considerably lower than that in de novo infection may be explained by the fact that the virus particles produced in the presence of SDZ PRI 053 are about 50-fold less infectious than those from untreated cultures. Upon intravenous administration, half-lives in blood of 100 and 32 min in mice and rats, respectively, were measured. Oral bioavailability of SDZ PRI 053 in rodents was 20 to 60%, depending on the dose. In mice, rats, and dogs, the inhibitor levels after oral administration remained far above the concentrations needed to efficiently block HIV replication in vitro for a prolonged period. This compound is thus a promising candidate for clinical use in HIV disease.

Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog.

(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50% inhibitory concentration = 0.011 to 0.057 micrograms/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.

Pharmacologic interactions between the resistance-modifying cyclosporine SDZ PSC 833 and etoposide (VP 16-213) enhance in vivo cytostatic activity and toxicity.

Cyclosporin A reverses multidrug resistance (MDR) and increases the in vivo cytostatic activity and toxicity of the anticancer agent etoposide (VP 16-213). SDZ PSC 833 (PSC 833), a non-immunosuppressive, non-toxic cyclosporin and very active modifier of P-gp 170-mediated MDR, elicits similar effects when administered with adriamycin. The underlying mechanisms, however, are not yet understood. The present pharmacological interaction study with PSC 833 and VP 16-213 was carried out to reveal the nature of this enhancement of cytostatic activity and toxicity. Rats pre-treated with either PSC-833 or solvent received a single dose of VP 16-213. Plasma levels of VP 16-213 were measured by high-performance liquid chromatography (HPLC). The resulting increase in cytostatic activity and toxicity of VP 16-213 mediated by PSC 833 was paralleled by marked changes in the pharmacokinetic parameters of VP 16-213 in vivo. Bioavailability and blood levels of VP 16-213 were significantly increased 30 min after administration if PSC 833 had been given before. The disappearance rate of VP 16-213 from the intravascular compartment was considerably slowed down by PSC 833. In drug-sensitive xenografts of human colon carcinoma, the PSC-833-induced pharmacologic changes in vivo could be counteracted by dose reduction of VP 16-213 while a full therapeutic potential was maintained. Doses of VP 16-213, 1.5 to 2 times smaller, combined with PSC 833, were as effective in terms of tumor-growth inhibition as the maximum tolerated dose of VP 16-213 alone. Thus, pharmacologic interactions between PSC 833 or other resistance modifiers and VP 16-213 and other cytostatic agents require careful attention if they are to be used in humans to overcome MDR.

Effect of enzyme induction on Sandimmun (cyclosporin A) biotransformation and hepatotoxicity in cultured rat hepatocytes and in vivo.

This study was designed to examine the relationship between the extent of Sandimmun (cyclosporin A, SIM) metabolism and SIM-induced hepatotoxicity both in vivo and in primary cultures of rat hepatocytes. Firstly, SIM (50 mg/kg p.o.) was administered daily to male Wistar rats for 10 days with or without co-administration of Aroclor 1254. SIM-induced hepatotoxicity appeared after 4 days of treatment and was enhanced after 10 administrations of SIM. Total plasma proteins were decreased and hyperbilirubinemia as well as increased levels of plasma bile salts were prominent. Aroclor 1254 stimulated total hepatic cytochrome P-450 3.7-fold, and markedly increased the rate of SIM metabolism and plasma elimination as determined by both HPLC and RIA techniques. However, this induction did not change the degree of SIM-induced hepatotoxicity. Secondly, short-term cultures of hepatocytes obtained from normal rats and from rats pretreated with either Aroclor 1254 or dexamethasone, a specific inducer of the cytochrome P-450 III gene family responsible for the formation of the primary SIM metabolites M1, M17 and M21, were incubated with various concentrations of SIM for up to 17 hr. At 1 microM SIM, both inducers greatly increased the rate of SIM metabolism in vitro, producing, however, different metabolite patterns. In the hepatocyte cultures, SIM inhibited the incorporation of amino acids into proteins. In addition, a small fraction of [3H]-labeled SIM was covalently bound to hepatocellular macromolecules. Although the fraction of covalently bound SIM was markedly increased in cells from dexamethasone-treated rats, the degree of inhibition of hepatocellular protein synthesis was not changed in cells from induced rats. In contrast to SIM-induced nephrotoxicity, these results suggest that increased rates of SIM biotransformation by inducers of drug metabolism are not associated with an attenuation of hepatotoxicity both in vivo and in vitro.

In vitro toxicity assessment of cyclosporin A and its analogs in a primary rat hepatocyte culture model.

The cytotoxicity of cyclosporin A (CsA), a widely used immunosuppressant drug, was evaluated in primary cultures of rat hepatocytes. Furthermore, the concentration-dependent (10(-7) to 10(-5) M) cytotoxic effects of the cyclosporin analogs, CsG, CsH, CsF, and of a major metabolite of CsA, CsA/M17, were assessed in an attempt to classify the different cyclosporin analogs according to their in vitro hepatotoxic potential. All compounds invariably inhibited the net release of taurocholate (de novo synthesized from cholate added to the extracellular medium). This sensitive functional marker did not discriminate between the structural analogs. In addition, all compounds inhibited, to various extents, the biosynthesis and secretion of proteins without affecting the uptake rate of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. These functional changes occurred in the absence of overt irreversible cell damage (no leakage of lactic dehydrogenase up to 10(-5) M cyclosporin during 17 hr of incubation). The relative toxic potential of the drug congeners (CsG greater than CsA greater than CsH = CsF = CsA/M17) correlated well with the degree of their accumulation in the hepatocytes during exposure to equimolar drug concentrations.

Relative cytotoxicity of psychotropic drugs in cultured rat hepatocytes.

The relative cytotoxic effects of ten psychotropic drugs were assessed in rat hepatocyte monolayer cultures. Clear concentration-related toxicity was seen in the narrow range of 10(-5) M to 5 X 10(-5) M. The four cytotoxicity endpoints chosen were: release of the cytosolic enzyme, lactate dehydrogenase, and impairment of biosynthesis and secretion of proteins, bile acids and glycerolipids. LDH leakage and inhibition of protein secretion into the culture medium proved to be the parameters which allowed the best differentiation between the test compounds. The inhibition of glycerolipid secretion was the most sensitive test in relation to concentration and time of exposure. Based on the effects of these endpoints, the following ranking of relative in vitro toxicity, using equimolar drug concentrations, could be established: clomipramine greater than imipramine = thioridazine greater than chlorpromazine greater than amitriptyline = fluperlapine greater than haloperidol greater than promazine greater than clozapine much greater than sulpiride. This ranking order of in vitro cytotoxicity correlated well with the potential of the drugs to impair liver function in man. Only clozapine had to be classified as a false negative. There was, however, no correlation between the cytotoxicity and the intracellular accumulation of the test drugs. Furthermore, the comparison of the data obtained with psychotropics with the data from five other amphiphilic cationic drugs was consistent with the widely accepted concept of a direct toxic interaction of the drugs with cytomembranes. This nonspecific toxicity of the membrane-active drugs was further corroborated by a positive correlation between their potential to induce LDH leakage in hepatocytes and their ability to induce hemolysis in red cells. In conclusion, the results obtained in our study strongly suggest that it is possible to assess the relative cytotoxicity of psychotropic drugs in rat hepatocyte cultures. It is proposed that this in vitro system provides a useful tool to evaluate new drugs at an early stage of their development, and to identify the most promising candidates within a class of structurally related compounds. In addition, it allows information to be obtained on possible mechanisms of cytotoxicity.

Cyclosporine A nephrotoxicity studied by the combined application of kidney cell lines, hepatocytes, and endothelial-platelet cocultures.

Established renal epithelial cell lines of human, pig, and dog origin (293, LLC-PK1, MDCK) were examined in terms of nephrotoxicity and ability to biotransform cyclosporine A (CsA). All three cell lines exhibited a comparable concentration dependent cytotoxicity to CsA treatment. Alterations in cell function included a decreased transport of lysine, an inhibition of growth, and an activation of lysosomal and mitochondrial activity as indicated by the increased uptake of neutral red (NR) and increased reduction of the tetrazolium dye MTT at 1-6 microM CsA. Increased leakage of lactic dehydrogenase and activities of gamma-glutamyl transpeptidase (GGT) and N-acetyl-beta-D-hexosaminidase were observed at 48 h and 12 microM CsA. A discrimination between CsA and the less nephrotoxic cyclosporine-(CsH) was shown for DNA synthesis and NR uptake. The contribution of extrarenal parameters on kidney cell function was studied by the addition of medium from hepatocytes exposed to CsA to the kidney cell lines. A more potent inhibition of DNA synthesis and enhanced reduction of MTT resulted than by addition of equimolar CsA directly to the kidney cells. These data indicate that hepatocyte constituents present in the medium due to CsA treatment affect kidney cell function; additionally, the presence of CsA metabolites may contribute to the CsA-induced nephrotoxicity. The vascular nephrotoxicity induced by CsA, an increased deposition of platelets in the renal arterioles, was mimicked by cocultures of endothelial cell monolayers and platelets. CsA increased the aggregability and adherence of platelets to the endothelial cell monolayers, whereas CsH had no effect.

Psychotropic drugs as inhibitors of glycerolipid biosynthesis and secretion in primary rat hepatocyte cultures.

Glycerolipid synthesis and secretion in rat hepatocyte monolayer cultures have been studied as possible endpoints in cytotoxicity assays. Ten psychotropic drugs of different structural classes were incubated with the hepatocytes at concentrations ranging from 10(-6)m to 5 x 10(-5)m and their effects on the incorporation of [(3)H]glycerol into glycerolipids and on the subsequent lipid secretion into the medium were measured. After 2-hr exposures to eight of the ten drugs, glycerolipid secretion was inhibited as a function of the drug concentration. The integrity of the plasma membrane, however, was still preserved, as no leakage of the cytosolic enzyme, lactate dehydrogenase, occurred. The incorporation of [(3)H]glycerol into the total cell-associated glycerolipid fraction was much less affected by the drugs than was the medium-associated fraction. In this model, the tricyclic antidepressants, imipramine, clomipramine and amitriptyline caused a marked inhibition of in vitro glycerolipid secretion. Chlorpromazine, thioridazine, fluperlapine, haloperidol and promazine were of intermediate potency, and clozapine and sulpiride had no inhibitory effect on glycerolipid secretion. A significant inhibition of glycerolipid secretion was also seen after exposure to low concentrations (10(-5)m) of the non-psychotropic drugs, amiodarone, perhexiline and chloroquine, indicating that the cationic amphiphilic nature of all of the drugs studied accounted for the toxic effect. The results suggest that the assessment of the inhibitory effect on glycerolipid secretion is a sensitive endpoint for cytotoxicity which, in combination with other in vitro tests (e.g. determination of protein and bile acid biosynthesis and secretion, and enzyme leakage), allows a ranking of the relative toxicity of psychotropic drugs.

Pharmacokinetics of cyclosporine in toxicological studies.

Pharmacokinetic studies performed in toxicological studies indicate that CsA is well absorbed in rats and dogs with absolute bioavailability in the range of 10% to 30% when administered by gavage (olive oil). Exceptions are the guinea pig, the rabbit and marmoset with low bioavailability (less than 5%). CsA is also absorbed in rats when given mixed in the feed. Vehicles may have marked effects on the absolute bioavailability. The correlation between areas under the plasma concentration time curves (AUC) and dose levels is linear in most species up to a dose of 50 mg/kg/d. CsA steady state plasma levels are generally 2 to 3 times higher than after single administration. Measures to reduce CsA nephrotoxicity must consider possible changes of oral bioavailability and immunosuppressive activity as shown for dmPGE2. Drug interactions may be predicted in animal experiments.

PGE2 reduces nephrotoxicity and immunosuppression of cyclosporine in rats.

Acute cyclosporine nephrotoxicity is characterized by the dose-dependent reduction of glomerular filtration rate both in patients and experimental animals. Administration of a vasodilator agent such as PGE2 might prevent glomerular vasoconstriction and hence reduce cyclosporine nephrotoxicity. The results of the present investigation in spontaneously hypertensive rats indicate that a synthetic PGE2 prevents cyclosporine nephrotoxicity. Pharmacokinetic studies revealed that CSA plasma peak levels and area under the plasma concentration-time curve were significantly decreased in PGE2 treated rats, indicating reduced bioavailability of oral CSA. Furthermore, the immunosuppressive effect of CSA was completely abolished by concomitant PGE2 administration. In summary, the preventive effect of a synthetic PGE2 analogue on CSA nephrotoxicity in rats is most likely due to a reduced enteral absorption of CSA, resulting in insufficient immunosuppression.

Effects of cyproheptadine on the rat yolk sac membrane and embryonic development in vitro.

Electron-microscopic examinations of rat embryonic yolk sacs treated in vitro with 1.5 X 10(-5) M cyproheptadine showed proliferation of the lysosomal structures; other organelles remained unaffected, and also overall yolk-sac growth and vascularization were comparable to non-treated samples. Radioactive measurements with 125I-labelled albumin showed that yolk sacs and embryos of the cyproheptadine-treated group incorporated less radioactivity than the controls. Embryos inside the yolk sacs, treated either for 24 or 48 h, were severely retarded in growth and differentiation (approximately 50% of the controls). It is suggested that the specific action of cyproheptadine on yolk-sac lysosomal structures, combined with reduced macromolecular transport, is the cause of inhibited embryonic development.