The extent of DNA damage and cellular proliferation induced in rat kidneys by single doses of the diabetogenic alkylating agent streptozotocin (STZ) and the time course of repair of that damage were ...evaluated using an in vivo alkaline elution assay for DNA strand breaks and a bromodeoxyuridine (BrdUrd) labeling assay for cell replication. Male Sprague-Dawley rats were given iv injections of 0.25 to 60 mg/kg STZ and kidneys were harvested 3 hr later for alkaline elution. A dose of 2.5 mg/kg STZ was the lowest dose to induce detectable DNA strand breaks and extensive damage was produced by the commonly used diabetogenic dose of 60 mg/kg. To characterize the repair of the drug-induced DNA damage, kidneys were harvested from a 60 mg/kg group of animals 3 hr to 27 days after dosing. BrdUrd-labeled kidney sections were also evaluated to assess any cellular proliferative response associated with STZ administration. Significant DNA damage was detected up to 14 days after dosing with return to near background levels by 20 days. Similarly, treatment with 60 mg/kg STZ was associated with increases in BrdUrd labeling indices 4 and 9 days after treatment with resolution by 27 days. These results indicate that the cellular and molecular repair responses to a single diabetogenic dose of STZ are prolonged, requiring up to 3 weeks to complete. Thus, to avoid potential additive or synergistic effects on STZ-induced nephrotoxicity and/or genotoxicity, a delay in the start of experimental therapies in this model (other than insulin) should be considered.
Recent clinical evidence indicates a potential for skeletal muscle toxicity after therapy with HMG-CoA reductase inhibitors (HMGRIs) in man. Although the incidence of drug-induced skeletal muscle ...toxicity is very low (0.1-0.2%) with monotherapy, it may increase following concomitant drug therapy with the immunosuppressant, cyclosporine A (CsA), and possibly with certain other hypolipidemic agents. In the Sprague-Dawley rat, very high, pharmacologically comparable dosages (150-1200 mg/kg/day) of structurally similar HMGRIs (lovastatin, simvastatin, pravastatin and L-647, 318) produced dose-related increases in the incidence and severity of skeletal muscle degeneration. Physical signs included inappetence, decreased activity, loss of body weight, localized alopecia and mortality. To evaluate the interaction between HMGRIs and CsA, a rat model of CsA-induced cholestasis was developed. In this 2-week model, the skeletal muscle toxicity of the HMGRIs was clearly potentiated by CsA (10 mg/kg/day). Doses of HMGRIs which did not produce skeletal muscle toxicity when given alone caused between 75 and 100% incidence of myopathy (very slight to marked skeletal muscle degeneration) when CsA was coadministered. Typical light microscopic changes included myofiber necrosis with interstitial edema and inflammatory infiltration in areas of acute injury. Histochemical characterization of the muscle lesion indicated that type 2B fibers (primarily glycolytic white fibers) were most sensitive to this toxicity but that, with prolonged administration, all fiber types were ultimately affected. Results of pharmacokinetic studies in rats treated with various HMGRIs +/- CsA indicated that coadministration of CsA alters the disposition of these compounds, resulting in increased systemic exposure (e.g., increased area under the plasma drug concentration vs. time curve-AUC) and consequent (up to 13-fold) increases in skeletal muscle drug levels. Evaluation of the potential interaction between the HMGRI, lovastatin and CsA at the level of hepatic microsomal metabolism indicated that CsA did not inhibit the metabolism of lovastatin in isolated microsomes from female rats. In light of the above findings, it appears that HMGRI-induced myopathy is a class effect in the rat, which is potentiated by CsA as the result of altered clearance and resultant increased tissue exposure. Cholestasis associated with CsA and HMGRIs may form the basis for decreased elimination and the resultant elevated systemic exposure. Furthermore, this toxicity is muscle fiber-selective and may be associated with impaired skeletal muscle energy metabolism.
Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, ...recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.
L-694,492 (DUP 532), an angiotensin II (AII) receptor antagonist, was given orally at 125 mg/kh/day to rats and monkeys for up to 6 mo to assess the effects of the compound on juxtaglomerular (JG) ...cells. In rats, mild JG cell hypertrophy/hyperplasia occurred and was associated with a 12-fold increase in the bromodeoxyuridine-labeling index of JG cells and a 10-fold increase in renal renin content. Ultrastructurally, intermediate cells with characteristics of both smooth muscle cells and granulated renin-producing cells as well as hypertrophied renin-synthesizing cells were seen in the afferent arterioles. In monkeys, marked hypertrophy and hyperplasia were seen with an 80% increase in JG cell numbers, mitotic activity, and a greatly increased renin content compared to controls. Three mo after drug withdrawal, an increased number of cells remained, which showed features of smooth muscle cells with essentially no renin. These results show that AII receptor antagonism stimulates increased renal renin production by hypertrophy of existing granulated cells, metaplasia of smooth muscle cells to renin-synthesizing cells, and cell proliferation. When treatment was discontinued, the renin-producing cells redeveloped the features of smooth muscle cells, but, as we have shown with enalapril (augioteusin-converting enzyme inhibitor), the increase in their number persists for at least 3 mo.
Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed ...as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as α-naphtylisothiocyanate and ethinyl estradiol.
L-694,492 (DUP 532), an angiotensin II (AII) receptor antagonist, was given orally at 125 mg/kg/day to rats and monkeys for up to 6 mo to assess the effects of the compound on juxtaglomerular (JG) ...cells. In rats, mild JG cell hypertrophy/hyperplasia occurred and was associated with a 12-fold increase in the bromodeoxyuridine-labeling index of JG cells and a 10-fold increase in renal renin content. Ultrastructurally, intermediate cells with characteristics of both smooth muscle cells and granulated renin-producing cells as well as hypertrophied renin-synthesizing cells were seen in the afferent arterioles. In monkeys, marked hypertrophy and hyperplasia were seen with an 80% increase in JG cell numbers, mitotic activity, and a greatly increased renin content compared to controls. Three mo after drug withdrawal, an increased number of cells remained, which showed features of smooth muscle cells with essentially no renin. These results show that AII receptor antagonism stimulates increased renal renin production by hypertrophy of existing granulated cells, metaplasia of smooth muscle cells to renin-synthesizing cells, and cell proliferation. When treatment was discontinued, the renin-producing cells redeveloped the features of smooth muscle cells, but, as we have shown with enalapril (angiotensin-converting enzyme inhibitor), the increase in their number persists for at least 3 mo.
Juxtaglomerular (JG) cell hypertrophy and hyperplasia were investigated in rhesus monkeys given angiotensin II (AII) AT1 receptor antagonists L-158,338 and DUP 753 (MK-0954, losartan).
In 2 initial ...studies, L-158,338 was given orally at 10, 30, and 90 mg/kg/day for 3 or 14 weeks. To investigate the observed JG hypertrophy and hyperplasia, in a third 5-week experiment L-158,338 was given alone at 90 mg/kg/day, or with physiologic saline supplementation at 25 ml/kg/day, or coadministered with the angiotensin converting enzyme inhibitor enalapril at 10 mg/kg/day. Physiologic saline was given to attempt to suppress renin release through volume expansion and/or sodium retention. Enalapril was given to lower plasma AII levels and observe whether JG cell hypertrophy and hyperplasia were increased or decreased. For comparison, DUP 753 was given at 90 and 300 mg/kg/day. Plasma renin activity and AII concentration were measured in this study.
Dose- and time-dependent increases in JG cell hypertrophy and hyperplasia were seen in the 2 initial experiment. In the third experiment, plasma renin activity and AII concentration were increased 3-fold and 6-fold over pretest values by L-158,338 at 90 mg/kg/day for 5 weeks. Saline supplementation had no effect on these parameters but diminished the group mean severity grade for JG hypertrophy and hyperplasia from 1.5 to 1.0. Enalapril coadministration had no effect on plasma renin activity, whereas it blunted the plasma AII increase caused by L-158,338 and increased the group mean grade to 2.5. DUP 753 at 300 mg/kg/day produced similar increases in plasma renin activity and AII concentration but only resulted in grade 1 JG cell hypertrophy and hyperplasia.
L-158,338-induced JG cell hypertrophy and hyperplasia is an exaggerated pharmacologic response that can be modulated by saline supplementation and angiotensin converting enzyme inhibition. These results suggest that decreased renal perfusion or altered sodium homeostasis and plasma AII concentration are important variables that contribute to AT1 receptor blockade to induce JG cell hypertrophy and hyperplasia.
Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III ...(AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.
The experience with the submission of a nonclinical (pharmacology and toxicology) computer-assisted New Drug Application (CANDA) is reviewed. This system consisted of a stand-alone personal computer ...running several commercial programs in Microsoft Windows to access both text and data. WordPerfect was used as the word processor that contained all the documents and data tables (in read-only format) that were submitted in hard copy, and Andyne GQL was used as a tool to query the data in an Oracle relational database. Microsoft Excel was provided as a spreadsheet for graphics and analysis of data. Documents appeared virtually identical to those in the hard copy NDA submission. Searching the text was facilitated by the use of buttons on the screen, which allowed the NDA to be searched for a particular term. Data could be located either in WordPerfect documents, or in an Oracle database (using Andyne GQL) by querying the data. The data queries could be performed ad hoc, in which the reviewer selected all the parameters for a search, or with predefined query buttons, which retrieved data for principal treatment-related changes. This type of system also could serve as a useful model for both inhouse nonclinical review and the submission of INDs and IND amendments.
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