In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450.

The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

Antiinflammatory effects of phosphonomethoxyethyl analogues of adenine in a model of adjuvant arthritis.

The 9-(2-phosphonomethoxyethyl)adenine (PMEA) and its more bioavailable bis(pivaloyloxymethyl) ester, (bis-POM-PMEA), applied s.c. at doses of 5-50 mg/kg, profoundly suppress symptoms of rat adjuvant arthritis, such as paw swelling, sple-nomegaly, fibroadhesive perisplenitis and systemic NO levels. The 9-(2-phosphonomethoxypropyl)adenine, PMPA and bis-POC-PMPA are ineffective. The antiarthritic effect does not depend on the influence of the drugs on macrophage NO production.

The effect of silibinin on experimental cyclosporine nephrotoxicity.

The immunosuppressive drug cyclosporine A (CsA), is metabolized by cytochrome P-450 IIIA. It causes acute reversible as well as chronic largely irreversible nephrotoxic effects. This effect is bases on vasoconstriction of the afferent and efferent glomerular arterioles which leads to a reduction in glomerular plasma flow and glomerular filtration rate. The mechanisms of the vasoconstriction are unclear with a number of different pathways under discussion. Silibinin is the main constituent of silymarin. Silibinin inhibits lipid peroxidation on hepatic microsomes and mitochondria of rats and is also able to reduce the activity of various monooxygenases. Cyclosporin-induced lipid peroxidation and affected cytochrome P-450 may even contribute to cyclosporine nephrotoxicity. We examined the possibility that silibinin had a protective effect as a result of its radical scavenging properties. Silibinin, 5 mg/kg BW i.p., was administered 30 min before cyclosporine application at dose of 30 mg/kg BW daily i.p. The biochemical parameters, total malondialdehyde (MDA) in whole blood and kidney homogenates and specific content of cytochrome P-450 in microsomal liver suspension were estimated. Three groups were studied: controls (con), cyclosporine alone (CsA), and cyclosporine plus silibinin (CsA + Sili). Creatinine was significantly increased after 2 weeks in both cyclosporine treated groups compared to controls (CsA 60.2 +/- 10.6 versus 45.8 +/- 10.4 mumol/L, p < 0.05; and CsA + Sili 72.0 +/- 8.3 versus 45.8 +/- 10.4 mumol/L, p < 0.001) and glomerular filtration rate (GFR) was significantly decreased (p < 0.0001) in the same groups. Total MDA was elevated only in CsA rats (2.26 +/- 0.35 mumol/L, p < 0.05) in comparison with controls (1.60 +/- 0.44 mumol/L, p < 0.05) and with rats treated by CsA + Sili (1.65 +/- 0.27 mumol/L, p < 0.05). The specific content of cytochrome P-450 in microsomal liver suspension was increased in group CsA + Sili (1.179 +/- 0.115 nmol/mg prot) compared to control group (0.775 +/- 0.086 nmol/mg prot., p < 0.05) and also CsA group (0.806 +/- 0.098 nmol/mg prot., p < 0.05). In conclusion, silibinin decreased cyclosporine-induced lipid peroxidation without a protective effect on GFR. These data indicate that this pathway is not be important in cyclosporine-induced nephrotoxicity. Administration of both drugs (CsA + sili) increased the specific content of cytochrome P-450 in liver microsomes. This suggests that the effect of silibinin on cyclosporine biotransformation in the liver is via cytochrome P-450.

Kidney function changes in rats after single-dose administration of borocaptate sodium.

Kidney function changes after single-dose administration of borocaptate sodium (mercaptoundecahydro-closododecaborate, B12H11SH, BSH) were studied in rats. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance, renal plasma flow rate (3H-p-aminohippuric acid clearance) and urine flow rate (UFR) after a slow intravenous injection of BSH (25 mg/kg b.w.) were investigated in rats under pentobarbital anaesthesia. It was found that a slow BSH injection induces a gradual decrease of renal plasma flow and glomerular filtration rate resulting in an almost constant reduction of the filtration fraction. These alterations were accompanied by a temporary increase of urine flow rate. Although a direct effect of BSH on the nephron cannot be excluded, it is suggested that the observed changes in kidney function might at least partly be mediated by disturbances in the function of the cardiovascular system following BSH injection. The role of the dianionic sulfhydryl group present in the borocaptate molecule in inducing these renal functional changes is discussed.

The diuretic effect of borocaptate sodium in rats and in patients with brain tumors.

Kidney function changes after single-dose administration of borocaptate sodium were studied in rats and in patients with brain tumors. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance and urine flow rate (UFR) after a slow intravenous injection of BSH (25 and 50 mg/kg b.w., respectively) were investigated in rats under pentobarbital anesthesia. The effect of BSH has been compared with that of its disulfide (BSSB) which is spontaneously generated by oxidation of BSH during storage. It was found that BSH decreases GFR in relation to dose and, in the same way, causes a temporary increase of UFR. On the other hand, BSSB (50 mg/kg) induced a large reversible decrease of GFR as well as a decrease of urine excretion. Measurements of GFR (inulin clearance), renal plasma flow (PAH clearance) and urine excretion were taken in a group of patients with brain tumors in which boron disposition after an infusion of BSH (25 mg/kg b.w. over 1 h) had been studied. An increase in urine production was the dominant effect (up to 200% of the initial value), with the alterations of GFR and RPF being of minor significance except in one patient with a GFR reduction up to almost 50% the original value. Kidney function changes after BSH or BSSB administration are supposedly related to the high retention of BSH in kidney.

The in vitro effect of sodium mercaptoundecahydrododecaborate (borocaptate) on sulfhydryl groups of different rat tissues.

The effect of borocaptate on structural protein sulfhydryl (SH) groups of different organs and kidney subcellular fractions was studied in vitro. It was shown that the binding capacity of borocaptate is not the same for different tissues and subcellular fractions. The highest binding capacity was in the liver, while kidney and brain values were significantly lower. Therefore, it appears that the same concentration of borocaptate may have different effects in various organs.

Nephrotoxicity of borocaptate after short-term administration in rabbits.

Fifteen Chinchilla rabbits were treated by seven daily i.v. injections of two doses (25 or 50 mg/kg body wt.) of sodium borocaptate (Na2B12H11SH), an agent now widely investigated for potential use in Boron Neutron Capture Therapy (BNCT) of malignant brain tumors. Definite nephrotoxic lesions hyperemic and dilated glomeruli, degenerative necrobiosis with desquamation of tubular epithelium, granular casts in the distal convoluted and collecting tubuli were detected by histopathological examination in the kidneys of all animals without relation to dose of borocaptate. The accumulation of urea in blood and a reduction in red blood cell counts were, however, statistically significant only in rabbits receiving the higher dose (50 mg/kg body wt.) of borocaptate. In the brain the prevalent finding was dilation of perivascular (Virchow-Robin's) spaces. The growth of the animals was retarded and three animals died after injection of the 5th daily dose of borocaptate. With respect to these findings borocaptate sodium can by no means be regarded as an agent which is fully devoid of activity towards healthy non-tumor tissue. Therefore, recent proposals to enhance the effectiveness of BNCT by repeated borocaptate treatment should be considered with caution.

Dose-dependent disposition kinetics and tissue accumulation of boron after intravenous injections of sodium mercaptoundecahydrododecaborate in rabbits.

Kinetics of boron disposition after single intravenous injections of two different doses (25 and 50 mg/kg) of mercaptoundecahydrododecaborate sodium (Na2B12H11SH; BSH) was studied in rabbits. Residual boron concentrations in various organs and tissues (heart, lungs, liver, spleen, kidney, adrenals, and brain) were also determined after seven daily injections of the same doses of BSH. Boron blood and tissue concentrations were measured by atomic emission spectrometry. In the majority of animals, the decline of boron blood concentrations after a single intravenous injection of either dose was biphasic, being consistent with a two-compartment model of boron disposition in the body. Although mean boron blood concentrations were roughly proportional to the BSH dose delivered, the mean total body clearance of boron from the body was 3 times lower (6.5 +/- 1.9 ml min-1 kg-1) after a dose of 50 mg/kg than after the injection of 25 mg/kg (22.4 +/- 7.9 ml min-1 kg-1), the difference between the means being statistically significant (P less than 0.05). Moreover, the mean terminal half-life of boron in blood was prolonged after the injection of 50 mg/kg (14.5 +/- 5.5 h) as compared with that found after the 25-mg/kg dose (3.5 +/- 0.9 h). On the other hand, the different BSH doses did not result in marked differences in the mean values obtained for the volume parameters - the volume of the central compartment (1.3 +/- 0.4 vs 1.3 +/- 0.5 l kg-1) and the volume of distribution at steady state (4.7 +/- 1.3 vs 6.0 +/- 4.0 l kg-1) - both of which were high, indicating extensive binding of the compound not only in the blood but also in tissues. Residual concentrations of boron found after seven daily injections of both doses of BSH were highest in the kidneys, the difference in the mean values being relatively small (33.6 +/- 6.1 vs 39.0 +/- 10.7 micrograms/g tissue). In the majority of other organs (heart, lung, liver, spleen, brain, adrenals), the residual concentrations after a dose of 50 mg/kg were disproportionately higher than those measured after the injection of 25 mg/kg, and the mean values corresponded to the reduced total body clearance rather than to the increased BSH dose. The saturability of BSH binding to blood and tissue proteins is suggested as a possible explanation for the dose dependency of the total clearance of boron from the body and the accumulation of BSH in organs and tissues.

The effect of some immunomodulators administration to rats on palmitic and oleic acids incorporation into the lipids of liver cell organelles.

The incorporation of equimolar doses of [14C]-palmitic and [3H]-oleic acids into the lipid moiety of hepatocytes after muramyl dipeptide (MDP), adamantylamide dipeptide (AdDP) and levamisole (LM) administration were investigated. The utilization of [14C]-palmitic acid for the synthesis of neutral lipids and phospholipids of crude nuclear fraction increased significantly after MDP and AdDP administration and to a lesser extent after LM. While the incorporation of [3H]-oleic acid did not change significantly after MDP and AdDP, LM significantly increased the incorporation of this acid into the phospholipids of nuclear and microsomal fractions. The changes in the incorporation of saturated palmitic and unsaturated oleic acids suggest a possible influence on deacylation-reacylation mechanisms. These changes could alter the physical properties of membrane and affect certain membrane functions, including the activity of membrane bound enzymes and transport mechanisms.

Pharmacokinetic profile of the immunomodulating compound adamantylamide dipeptide (AdDP), a muramyl dipeptide derivative in mice.

A pharmacokinetic profile of 14C-AdDP with uniformly labelled alanine was investigated. It was shown that the distribution phase after an i.v. administration is very short with a half-life of 2.1 min. The half-life of elimination phase after the i.v. administration is about 2.85 hours, that is longer than those of MDP and its derivatives. The total body clearance (30 ml/min/kg) is caused predominantly by metabolism of the compound. All the radioactivity found in urine in a 48 hours interval after a s.c. administration represents only 3.1% of the administered dose. Only a smaller part of the excreted radioactivity is formed by unmetabolised AdDP. The concentration curve after a s.c. administration is characterized by a very fast absorption with a half-life shorter than 1 minute. The distribution and elimination phases are prolonged (20 min, 11 hours respectively) in comparison with an i.v. injection. The decreased absolute bioavailability after a s.c. administration (65%) is probably not biologically significant because of a slower release of the compound from the site of the s.c. administration. A relatively very high radioactivity was found in liver, kidney, thymus, spleen and brain very soon which suggest a very good penetration into tissues. It is an agreement with the high apparent distribution volume of peripheral compartment and higher lipophilicity of AdDP as compared to MDP.

[Kemantane pharmacokinetics in rats]. / Farmakokinetika kemantana u krys.

Pharmacokinetics of novel immunostimulating drug kamantane was studied by using gas-liquid chromatography in experiments on rats. It was found that kemantane biotransformed rapidly after oral administration with the forming of active metabolite. Kemantane and its metabolites are distributed rapidly from the blood to organs. The drug is eliminated from the organism of rats as metabolite.

Muramyl dipeptide and carbon tetrachloride hepatotoxicity in rats: involvement of plasma membrane and calcium homeostasis in protective effect.

The present study was carried out to investigate the effect of single and multiple doses of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) on CCl4-induced hepatotoxicity, and hence some of the mechanisms involved. MDP (8.26 mumol/kg i.v.) was administered to rats according to different protocols followed by a single dose of CCl4 (5.2 mmol/kg i.p.), and either the hepatocytes were subsequently isolated and tested for viability and lipid peroxides formation or the level of serum aminotransferases and lactate dehydrogenase (LD) was measured. The results clearly indicate that MDP pretreatment in a single dose reduced CCl4 hepatotoxicity as judged from viability tests as well as reduction of elevated lipid peroxides induced by CCl4 administration. The level of lactate dehydrogenase was also brought to normal value by single MDP administration. MDP also decreased significantly the CCl4-elevated Ca2+ content of isolated hepatocytes and postmicrosomal supernatant Ca2+. 14C-palmitic acid incorporation was increased significantly for neutral lipids and/or phospholipids in hepatocytes and certain subcellular fractions under MDP treatment in vivo. A different effect was seen after multiple MDP administration which further increased CCl4-induced elevation of aminotransferases. Even the repeated administration of MDP without CCl4 increased the level of the latter enzymes. It may be concluded that a single administration of MDP can protect liver cells from CCl4 injury by a mechanism affecting the plasma membrane.

Possible effects of muramyl dipeptide on liver cell membranes.

Three different approaches were utilized for illustration of membranous effect of muramyl dipeptide (MDP) on liver cells. Toxicologic approach using a hepatoprotective effect against CCL4 as well as from previous results with other toxins. Histochemistry and microfluorometry after chemical injury. Biochemical as assessed by modulation of 14C palmitic acid incorporation under MDP effects in neutral lipids and phospholipids content of cell membranes. By this integrated study we may assume MDP played a role in one or more cell membrane(s). This assumption was supported by reduction of hepatocyte injury as assessed by enzyme analysis in serum or in the cells. Moreover, MDP pretreatment influenced the increase in the incorporation of labeled palmitic acid in neutral lipids and phospholipids. Microscopical image analysis and histochemical results supported the aforementioned findings.

Study of relationship between allogeneic transplantability of tumors and their effects on drug-metabolizing system in rats.

Effects of three different fibrosarcomas on the hepatic mixed-function oxidase system were studied in males of the Lewis inbred strain of rats. No association between graded potentiality of these tumors to grow across histocompatibility barriers and their suppressive effects upon the microsomal drug-metabolizing system was found.

The effect of adjuvant-induced arthritis on rat liver microsomal phospholipid metabolism.

Rats given a single subdermal injection into the left hind paw of 0.5 mg Mycobacterium tuberculosis suspended in 0.1 ml of mineral oil, after several days of latency, developed arthritis accompanied by depression in liver microsomal phosphatidylcholine and phosphatidylethanolamine synthesis at 7, 14 and 21 days. The depression of liver microsomal phosphatidylcholine and phosphatidylethanolamine concentrations of experimental animals was accompanied by decreased incorporation of radioactive palmitate into both phospholipids. Nevertheless, the biosynthesis of phosphatidylcholine via the methylation pathway was unaffected. These observations suggest that adjuvant arthritis affects quantitatively the phospholipid composition of the liver endoplasmic reticulum, which consequently may lead to impairment of the microsomal drug-metabolizing enzyme system.

Biotransformation of drugs in rats treated with a synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP).

Natural immunoadjuvant mixtures like BCG and FCA are known to produce gross alterations of drug-metabolizing systems in the rat. Since it has been shown that the smallest structure of various bacterial peptidoglycans, possessing adjuvant activity, is muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), the possibility has been tested whether this substance is involved in production of the observed metabolic changes. Synthetic compound was applied subcutaneously for the period of 21 days, and the in vitro activity of 7-ethoxycoumarin-O-de-ethylase, aminopyrine-N-demethylase, together with the microsomal content of cytochrome P-450 and b5, and the in vivo acetylation of sulphadimidine, were investigated. No effect of MDP on any of these tests was noted in both the Lewis arthritic strain and the AVN disease-free strain. It is suggested that MDP is metabolically inactive and that the defects in metabolism of drugs, following bacterial-adjuvant treatment, are likely to be due to some additional cell-wall components, other than peptidoglycans. Furthermore, our data support the view of no relationship between the development of metabolic changes and the established arthritic lesions in rats.

Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine.

Following the administration of D-galactosamine the utilization of [2-14C] orotic acid for the synthesis of the cytidine components of the acid-soluble extract and liver RNA cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application of low doses of the drug which do not interfere with the specific activity of the uridine components of the acid-soluble extract or of liver RNA uracil. Simultaneously the administration of [U-14C]cytidine paralleled by its enhanced liver uptake. The total amount of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied by the increased level of microsomal cytochrome P-450.

Alterations in metabolism of cytidine components in rat liver after oral administration of butylated hydroxytoluene (in vivo study).

The administration of the antioxidant, butylated hydroxytoluene (BHT) to rats decreased the utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides in the acid-soluble extract and RNA of the liver. The specific activity of the uridine components was slightly decreased. The depression of the specific activity of the cytidine components depended on the dose of the drug. Simultaneously preformed [U-14C]cytidine in experimental rats was to a higher degree transported to the liver and incorporated into RNA cytosine; its deamination was markedly suppressed. Both phenomena depend on the BHT dose. The concentration of both the uridine and the cytidine components of the acid-soluble extract remained unaffected by the administration of BHT. The utilization of [2-14C]orotic acid for the synthesis of DNA cytosine was depressed after the administration of BHT; by contrast, the specific activity of DNA thymine was higher. The incorporation of [1-14C]palmitic acid into microsomal phospholipids was not substantially influenced over the dose range 25--500 mg BHT/kg. The specific activity of neutral lipids in microsomes increased.