Pharmacokinetics, distribution and elimination of a synthetic octapeptide analogue of somatostatin in the rat

The in vivo fate of a long acting somatostatin analogue [des-AA1,2,3,4,13,14,D-Trp8,Gaba12]-somatostatin has been studied in the rat using biochemical and autoradiographic techniques. The analogue has a longer plasma half-life than somatostatin. This is due to its greater metabolic stability which renders it resistant to enzymic attack in the tissues. The primary route of elimination is by biliary excretion following clearance from the circulation by the liver. Evidence of enterohepatic circulation was found, but only to a very limited extent. When administered s.c., high plasma concentrations of intact peptide persist for a period of hours due to slow release from a stable depot at the injection site.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

Effect of natural and synthetic glucocorticoids on rat hepatic microsomal drug metabolism

Endogenous and synthetic glucocorticoids of varied biological potency have been used for the treatment of intact male rats in an attempt to determine the interactions of corticosteroids with hepatic mixed-function oxidation. The more potent synthetic steroids increased several components of the microsomal electron transport chain, and the metabolism of biphenyl, aniline, benzo[α]pyrene and ethylmorphine. In contrast, the natural glucocorticoids and their less potent synthetic analogues decreased or had no effect on the activity of these parameters. None of the steroids used affected the spectral interaction kinetics or the apparent K_mvalues of the enzymes responsible for the metabolism of type I and type II substrates. The effects of glucocorticoids on hepatic drug metabolism therefore appear to be different from those of phenobarbital or 3-methylcholanthrene, but show some similarities with those resulting from pregnenolone-16α-carbonitrile or spironolactone pretreatment.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

Age dependent changes in androgen metabolism in the rat prostate

Oxidation and reduction of androstenedione, testosterone, dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (3 alpha- and 3 beta-A'diol) were measured in homogenates from the ventral prostate (VP), dorsal prostate (DP), lateral prostate (LP), the coagulating gland (CG) and seminal vesicles (SV) in intact rats of different ages from young mature (3-6 months) to senescent rats (20-30 months). Some very old intact rats (30-32 months) were treated with testosterone in order to rule out the effect of this hormone on androgen metabolism. The enzymatic activities for young mature rats were significantly altered by increasing age, both with regard to differences between the various organs as well as differences in cofactor requirement. With increasing age, the specific activity of most enzymes gradually decreased. With testosterone as substrate, 5 alpha-reductase activity was significantly reduced in the old rats in all tissues studied and was undetectable in the oldest animals in the VP and the SV. On the other hand, 5 alpha-reductase could not be recorded in any tissue in any tissue in old rats when androstenedione was the substrate. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) in the VP was the only enzyme which did not decrease in activity by increasing age. In the other lobes this enzyme activity decreased similar to 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR) and the 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) activity. Administration of testosterone to old rats increased the specific activity of most of the enzymes studied.     

Altered regulation of hepatic heme metabolism in cadmium exposed chick embryo

An investigation on the process of heme metabolism with special emphasis on ALA synthetase, heme synthetase and heme oxygenase was studied in cadmium exposed chick embryo to enlighten the mechanism of cadmium embryotoxicity. Cadmium chloride injection (2.5-10 mumole/kg) to chick embryo increases the activity of ALA synthetase by 5-7 folds, however, it inhibits the activity of heme synthetase significantly. The activity of heme oxygenase is further shown to be enhanced by cadmium chloride treatment. These changes are accompanied by a marked reduction in hepatic heme content. The induction of ALA synthetase and heme oxygenase was dependent on the initial concentration of exogenous cadmium. Pretreatment with actinomycin D completely blocks the cadmium mediated induction of both ALA synthetase and heme oxygenase. Time course studies on the stimulation of these two enzymes show that cadmium enhances the activity of heme oxygenase to its maximum level after 24 h. of injection, whereas ALA synthetase activity reaches its highest value only by 48 h. and both the enzymes remain elevated at least upto 96 h. This observation can be correlated with the hepatic heme level at different time intervals after cadmium exposure. These observations suggest the presence of regulatory process for heme metabolism which is susceptible to alteration of 'regulatory heme pool' caused by cadmium.     

Effect of acetaminophen on heme metabolism in rat liver

BACKGROUND AND AIMS: Acetaminophen (APAP) or paracetamol is a hepatotoxic drug through mechanisms involving oxidative stress. To know whether mammalian cells possess inducible pathways for antioxidant defense, we have to study the relationship between heme metabolism and oxidative stress. METHODS: fasted female Wistar rats received a single injection of APAP (3.3 mmol kg(-1) body weight) and then were killed at different times. Heme oxygenase-1 (HO), delta-aminolevulinic acid (ALA) synthase, ALA dehydratase, and porphobilinogenase activities, lipid peroxidation, GSH, catalase and glutathione peroxidase, were measured in liver homogenates. The antioxidant properties of bilirubin and S-adenosyl-L-methionine were also evaluated. RESULTS: APAP increased lipid peroxidation (115% +/- 6; S.E.M., n=12 over control values) 1 h after treatment. GSH reached a minimum at 3 h (38% +/- 5) increasing thereafter. At the same time antioxidant enzymes reached minimum values (catalase, 5. 6 +/- 0.4 pmol mg(-1) protein, glutathione peroxidase, 0.101 +/- 0.006 U mg(-1) protein). HO induction was observed 6 h after treatment reaching a maximum value of 2.56 +/- 0.12 U mg(-1) protein 15 after injection. ALA synthase (ALA-S) induction occurred after enhancement of HO, reaching a maximum at 18 h (three-fold the control). ALA dehydratase activity was first inhibited (31 +/- 3%) showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of bilirubin (5 micromol kg(-1) body weight) or S-adenosyl L-methionine (46 micromol kg(-1) body weight) 2 h before APAP treatment entirely prevented the increase in malondialdehyde (MDA) content, the decrease in GSH levels as well as HO and ALA-S induction. CONCLUSION: This study shows that oxidative stress produced by APAP leads to increase in ALA-S and HO activities, indicating that toxic doses of APAP affect both heme biosynthesis and degradation.     

Influence of a rachitogenic regime on hepatic metabolism in the rat

We have previously reported that rats fed on the Steenbock and Black's rickets-inducing diet (deficient in vitamin D and with an altered Ca/P ratio) show metabolic modifications in kidney and intestinal mucosa. We have therefore decided to investigate if also in liver, seat of vitamin D hydroxylation, changes in the metabolic pattern occur. An increase of mitochondrial NAD+-dependent isocitrate dehydrogenase and a decrease of citrate and ATP content was demonstrated in liver of rachitic rats, together with changes in ATP-citrate lyase and glucose-6-phosphate dehydrogenase activity. The inhibitory effect of ATP on liver mitochondria NAD+-dependent isocitrate dehydrogenase was also studied.     

Alterations in hepatic heme metabolism in fish exposed to sublethal cadmium levels

A study on hepatic heme metabolism with special emphasis to ALA synthetase, ALA dehydratase and heme oxygenase was carried out in cadmium exposed freshwater fish Channa punctatus to enlighten the mechanism of cadmium induced toxicity. Cadmium exposure (0.5-5.0 mg/1) for 7 days increased the hepatic level of ALA, along with the depletion in heme content, which are characteristic to chemical porphyria. The resultant enhancement in the activities of ALA synthetase and heme oxygenase were further shown to be dose dependent. ALA dehydratase activity on the other hand was enhanced only at higher exposure. Time course studies on the enzyme activities and heme content showed that ALA synthetase started to increase after 24 hrs., reached maximum at 7 days and came back nearly to normal level after 30 days of exposure. Simultaneously maximum depletion in heme level occurred on 7 days of exposure, tending to return to normal on 30 day. In addition, attempt has been made to correlate alterations in heme metabolism due to cadmium with the histopathological manifestations in liver.     

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.     

Antibodies against synthetic peptides recognize the human and rat androgen receptor

Antibodies against two synthetic peptides (aa 299-311 and aa 544-559) selected in different immunogenic domains of the human AR, were induced in rabbits. Antiserum reactivity against the native receptor was investigated by gel permeation chromatography and sucrose density gradient centrifugation using [3H]mibolerone-labeled rat prostate cytosol and [3H]5 alpha-dihydrotestosterone-labeled T-47D cytosol as a source of AR. The absence of cross-reactivity of the antisera with estrogen, progesterone and glucocorticoid receptor was confirmed by density gradient centrifugation of rat uterus cytosol labeled with [3H]E2 or [3H]ORG 2058 and rat liver cytosol labeled with [3H]dexamethasone. After partial proteolytic breakdown of rat prostate AR by endogenous proteases the steroid-labeled receptor was recognized only by the second peptide (aa 544-559) antibody. This proteolytic breakdown could be prevented to a large degree by addition of a high concentration of soybean trypsin inhibitor. The specific AR antibodies provide new tools for the functional analysis of AR, since they interact selectively with specific domains of the receptor.     

Effects of synthetic androgen fluoxymesterone on triglyceride secretion rates in the rat.

Fluoxymesterone, a C-17 methylated derivative of testosterone employed in the treatment of renal-failure patients with refractory anemia, was administered to Sprague-Dawley rats and the effects on plasma triglyceride (TG) and immunoreactive insulin (IRI) levels and the rate of hepatic triglyceride secretion (TGSR) into plasma determined (Triton). Animals treated with fluoxymesterone demonstrated significantly lower TG (less than 0.05) and no alteration in TGSR. These findings are consistent with other observations which suggest that the triglyceride-lowering effect of androgens results from an accelerated rate of removal of triglyceride-rich lipoproteins from plasma.     

Cyclic oscillations in rat hepatic heme oxygenase and delta-aminolevulinic acid synthetase following intravenous heme administration.

Heme administration in vivo results in the suppression of synthesis of rat hepatic δ-aminolevulinic acid (ALA) synthetase and induction of rat hepatic heme oxygenase. Intravenous heme administration in vivo results in the appearance of cyclic progressively damped oscillations of both hepatic ALA synthetase activity and hepatic heme oxygenase activity. Heme oxygenase induction precedes in time the induction of ALA synthetase. ALA synthetase oscillations are observed in hepatic cell cytosol and mitochondrial fractions as well as in the total homogenate. Cycloheximide pretreatment abolishes both the ALA synthetase and heme oxygenase oscillations, while actinomycin D pretreatment has only a minimal effect on the induction of heme oxygenase. These results suggest that hepatic heme metabolism is closely regulated by rapid changes in the capacity to synthesize and catabolize heme, and the cyclic oscillations following intravenous heme may be a manifestation of the feedback regulation processes involved. This regulatory capacity is dependent on protein synthesis, and the primary site of regulation may be at the translational level on the endoplasmic reticulum.     

Evidence against a regulatory role for heme oxygenase in hepatic heme synthesis

The role of a microsomal enzyme system, heme oxygenase, in the regulation of intracellular heme concentration and of the heme biosynthetic pathway was investigated. It was determined that alterations in heme oxygenase activity were not consistent with the observed alterations of heme biosynthesis produced by the administration of various drugs. It is concluded that heme oxygenase does not play a role in the regulation of heme biosynthesis under these circumstances.     

Antipyrine metabolism in the rat by three hepatic monooxygenases

The relative contribution of 3 oxidative reactions of antipyrine to its metabolism in vivo were assessed by comparing male and female rats, and by studying the effects of phenobarbital (PB) and 3-methylcholanthrene (MC). After [N¹⁴CH₃] antipyrine, 11–15% of the dose was excreted as ¹⁴CO₂ in both sexes as a consequence of N-demethylation. PB pretreatment had no effect but MC doubled ¹⁴CO₂ excretion. The other metabolites, 4-hydroxyantipyrine (4OHA) and 3-hydroxymethylantipyrine (3HMA), in urine were determined by thin-layer chromatography. Of the total activity in the urine, 4OHA represented 30% in male and 40% in female rats; 3HMA represented 35% in male and 20% in female rats. The ratio of 4OHA to 3HMA in both sexes increased after PB and MC, the effect being more pronounced with the latter. The results show that the 3 major oxidative pathways of antipyrine metabolism are mediated by different enzymes, almost certainly different forms of cytochrome P450.     

Effects of repeated administration with CP-55,940, a cannabinoid CB1 receptor agonist on the metabolism of the hepatic heme

Drugs metabolised by cytochrome P450 (CYP) such as analgesics may induce acute attacks in patients with hepatic porphyrias. In recent years, preclinical and clinical studies have suggested that cannabinoid pharmaceutical preparations may be potentially useful in the treatment of pain. The purpose of the study was to examine the effects of CP-55,940, a cannabinoid CB1 receptor agonist, on the hepatic heme metabolism in mice. To this end, hepatic activities of aminolevulinic acid synthase (ALAS), heme oxygenase (HO) and CYP levels were determined in mice treated with CP-55,940 (0.5 mg/kg/day; i.p.; 5 or 24 days). Results showed that treatment with CP-55,940 decreased CYP concentrations by 80% and increased HO activity by 158%. However, ALAS activity also decreased by 37%, suggesting that regulatory free heme pool was not modified. Our findings indicate that CP-55,940 and its metabolites do not behave as porphyrinogenic drugs and may potentially be safe for treating pain in patients with acute porphyrias.     

Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal

The treatment of male rats with Hg2+ resulted in significant alterations in heme and hemoprotein metabolism in the adrenal gland which, in turn, were reflected in abnormal steroidogenic activities and steroid output. Twenty-four hours after the administration of 30 mumol of HgCl2/kg (sc) the mitochondrial heme and cytochrome P-450 concentrations increased by approximately 50%. Also, Hg2+ treatment stimulated a porphyrinogenic response which included an 11-fold increase in the activity of delta-aminolevulinate synthetase. The increase in mitochondrial cytochrome P-450 content was reflected in elevated steroid 11 beta-hydroxylase and cholesterol side-chain cleavage activities. In contrast, Hg2+ treatment resulted in decreased concentrations of microsomal cytochrome P-450 (-75%) and heme (-45%). Similarly, the reduction in the microsomal cytochrome P-450 content was accompanied by reduced steroid 21 alpha-hydroxylase and benzo[alpha]pyrene hydroxylase activities. The mechanisms responsible for the loss of the microsomal cytochrome P-450 content appeared to involve a selective impairment of formation of the holocytochrome as well as an enhanced rate of heme degradation. This suggestion is made on the basis of findings that (a) the decrease in the microsomal cytochrome P-450 content was accompanied by a sevenfold increase in the activity of adrenal heme oxygenase, (b) no decrease in apocytochrome P-450 could be detected in sodium dodecyl sulfate-gel electrophoresis of the solubilized microsomal fractions stained for heme, and (c) the concentration of adrenal microsomal cytochrome b5 was significantly increased in the Hg2+-treated animals. It is suggested that Hg2+ directly caused a defect in adrenal steroid biosynthesis by inhibiting the activity of 21 alpha-hydroxylase. The apparent physiological consequences of this effect included lowered plasma levels of corticosterone and elevated concentrations of progesterone and dehydroepiandrosterone. This abnormal plasma steroid profile is indicative of a 21 alpha-hydroxylase impairment.