Long hydrocarbon chain diols and diacids with central ether or ketone moieties that favorably alter lipid disorders.

Long hydrocarbon chain derivatives with bis-terminal hydroxyl or carboxyl groups and various central moieties (ketone, ether, ester, amide, carbamate, etc.) have been synthesized and evaluated for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes as well as for their effects on lipid, glycemic and body weight variables in female obese Zucker fatty rats following one and two weeks of oral administration. The most active compounds were found to be symmetrical with four to five methylene groups separating the ether or ketone central functionality from the gem dimethyl, cycloalkyl or methyl/aryl substituents. Cycloalkyl substitution alpha to the carboxyl group in keto-acids lowered the in vitro activity to micromolar values. Furthermore, in vivo biological activity was found to be greatest for cyclopropyl-substituted ketone derivatives, particularly the ketodiacid with five methylene groups on each side of the central ketone functionality, which was identified as an HDL elevator and was also found to reduce insulin and glucose.

Metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon.

Preclinical drug safety evaluation studies, typically conducted in two or more animal species, reveal and define dose-dependent toxicities and undesirable effects related to pharmacological mechanism of action. Idiosyncratic toxic responses are often not detected during this phase in development due to their relative rarity in incidence and differences in species sensitivity. This paper reviews and discusses the metabolic idiosyncratic toxicity and species differences observed for the experimental non-benzodiazepine anxiolytic, panadiplon. This compound produced evidence of hepatic toxicity in Phase 1 clinical trial volunteers that was not predicted by rat, dog or monkey preclinical studies. However, subsequent studies in Dutch-belted rabbits revealed a hepatic toxic syndrome consistent with a Reye's Syndrome-like idiosyncratic response. Investigations into the mechanism of toxicity using rabbits and cultured hepatocytes from several species, including human, provided a sketch of the complex pathway required to produce hepatic injury. This pathway includes drug metabolism to a carboxylic acid metabolite (cyclopropane carboxylic acid), inhibition of mitochondrial fatty acid beta-oxidation, and effects on intermediary metabolism including depletion of glycogen and disruption of glucose homeostasis. We also provide evidence suggesting that the carboxylic acid metabolite decreases the availability of liver CoA and carnitine secondary to the formation of unusual acyl derivatives. Hepatic toxicity could be ameliorated by administration of carnitine, and to a lesser extent by pantothenate. These hepatocellular pathway defects, though not directly resulting in cell death, rendered hepatocytes sensitive to secondary stress, which subsequently produced apoptosis and hepatocellular necrosis. Not all rabbits showed evidence of hepatic toxicity, suggesting that individual or species differences in any step along this pathway may account for idiosyncratic responses. These differences may be roughly applied to other metabolic idiosyncratic hepatotoxic responses and include variations in drug metabolism, effects on mitochondrial function, nutritional status, and health or underlying disease.

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes.

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0.5-1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2.5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor beta subunit; she phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK.

Disruption of mitochondrial activities in rabbit and human hepatocytes by a quinoxalinone anxiolytic and its carboxylic acid metabolite.

The quinoxalinone anxiolytic, panadiplon, was dropped from clinical development due to unexpected hepatic toxicity in human volunteers. Subsequent experimental studies in rabbits demonstrated a hepatic toxicity that resembled Reye's syndrome. In the present studies, we examined the effects of panadiplon and a metabolite, cyclopropane carboxylic acid (CPCA) on hepatic mitochondrial activities in vitro and ex vivo. Acute inhibition of beta-oidation of [14C]palmitate was observed in rabbit and human hepatocyte suspensions incubated with 100 microM panadiplon. Panadiplon (30 microM) also reduced mitochondrial uptake of rhodamine 123 (R123) in cultured rabbit and human, but not rat hepatocytes, following 18 h exposure. CPCA also impaired beta-oxidation and R123 uptake in rabbit and human hepatocytes. R123 uptake and beta-oxidation in cells from some donors was not impaired by either agent, and cell death was not observed in any experiment. Hepatocytes isolated from panadiplon-treated rabbits had reduced palmitate beta-oxidation rates and inhibited mitochondrial R123 uptake; R123 uptake remained inhibited until 48-72 h in culture. Rabbit mitochondrial respiration experiments revealed a slightly lower ratio of ATP formed/oxygen consumed in panadiplon-treated animals: direct exposure of normal rabbit liver mitochondria to panadiplon did not have this effect. Hepatocytes isolated from panadiplon-treated rabbits showed reduced respiratory control ratios and lower oxygen consumption compared to controls. Our results indicate that panadiplon induces a mitochondrial dysfunction in the liver, and suggest that this dysfunction may be attributed to the carboxylic acid metabolite.

Potentiation of hypoxic injury in cultured rabbit hepatocytes by the quinoxalinone anxiolytic, panadiplon.

The quinoxalinone anxiolytic, panadiplon, produces hepatic metabolic inhibition (mitochondrial impairment), microvesicular steatosis and centrilobular necrosis in rabbits. Metabolic inhibition occurs in cultured hepatocytes without cytotoxicity, suggesting that hepatic injury is influenced by additional factors. The present experiments were conducted to determine if metabolic inhibition by panadiplon predisposed hepatocytes to hypoxic injury. Injury (cell death) was evaluated by lactate dehydrogenase (LDH) release from cells; ATP and glycogen levels were also evaluated. Under hypoxic conditions, control cultures showed a 6.5-fold increase in LDH release compared to normoxic controls, with a coincident 80% decrease in ATP and 50% decrease in glycogen levels. Under normoxic conditions 10 microgram/ml panadiplon treatment for 48 h reduced ATP and glycogen levels by 40% but did not cause an increase in LDH leakage. Cells treated with panadiplon, then exposed to hypoxia conditions, showed a significant level of injury compared to normoxic control cultures, and a further reduction in ATP. No additional decrease in glycogen ws observed. In an attempt to prevent panadiplon-mediated injury, glycolytic substrates (dihydroxyacetone or pyruvate) were included during normoxic and hypoxic incubations. Both cotreatments reduced the level of LDH leakage produced by panadiplon during hypoxia. Cotreatment did not generally increase ATP or glycogen levels (compared to panadiplon treatment groups) during hypoxia, though individual experiments showed a slight increase in ATP levels. During normoxia both cotreatments with panadiplon resulted in significantly higher glycogen levels than in panadiplon cultures alone. These results suggest that cellular glycogen and subsequently ATP levels are reduced during panadiplon exposure, metabolically predisposing hepatocytes to hypoxic injury.

Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development.

Drugs can fail at any phase during discovery, preclinical or clinical development due to unacceptable levels of toxicity, and liver is commonly the principle target organ. Investigational toxicology methods, using appropriate models and hypotheses, can often resolve problems, identify toxic chemical substituents and salvage therapeutic discovery programs. While in vivo models are used to investigate hepatic drug effects in the context of toxicokinetics and systemic influences, cell culture models provide in vitro systems for investigating specific mechanisms in a precisely controlled environment. Using primary hepatocytes isolated from laboratory animals, we have explored several drug-induced hepatic disorders that surfaced during different phases of drug discovery and development. Additionally, the use of human hepatocytes has allowed us to address concerns for human exposure, examine human relevance of animal data, and provide perspective on problems encountered in clinical trials.

Upregulation of glucose-6-phosphate dehydrogenase in response to hepatocellular oxidative stress: studies with diquat.

The expression of hepatic glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is hypothesized to be modulated by free radicals during oxidative stress. The ability of diquat, a compound known to enhance oxidative stress through generation of reactive oxygen species, to modulate the expression of G6PDH in primary cultures of Fischer-rat hepatocytes was examined. Diquat-treated hepatocytes maintained in a chemically defined medium showed both a time- and concentration-dependent increase in G6PDH enzyme activity. This increase in enzyme activity was accounted for by an increase in both G6PDH mRNA and immunoreactive protein, suggesting control at a pretranslational level. The possibility that diquat increased transcription by transfecting cells with a chimeric gene containing 935 bp of the G6PDH promoter (-878 to +57) linked to the gene for chloramphenicol acetyl-transferase (CAT) was examined. Hepatocytes transiently transfected with this chimera, and subsequently treated with diquat, exhibited an increase in CAT activity. However, hepatocytes transfected with a chimera containing 287 bp of the G6PDH promoter (-230 to +57) exhibited only basal CAT activity in the presence of diquat. These results suggest that regions in the DNA sequences required for diquat-induced expression of G6PDH lie between base pairs -878 and -230 of the G6PDH gene. These findings are suggestive that oxidative stress in hepatocytes increased the expression of G6PDH activity and protein and that the increased expression is controlled at the transcriptional level.

Induction of a hepatic toxic syndrome in the Dutch-belted rabbit by a quinoxalinone anxiolytic.

The non-benzodiazepine anxiolytic, panadiplon, was discontinued from clinical development due to evidence of hepatic toxicity in human volunteers that was not predicted by rat or monkey preclinical development studies. The present study was conducted to examine potential toxicity in the rabbit. Three groups of female rabbits were administered vehicle, 10 mg/kg per day or 20 mg/kg per day of panadiplon by oral gavage for 14 days. Animals in the 20 mg/kg group lost weight, and 6/10 developed a profound lethargy. Hepatic toxicity was observed in treated animals, evidenced by dose- and time-related increases in serum transaminase activities, gross hepatic lesions and multifocal centrilobular necrosis. Hepatic microvesicular steatosis was evident in treated animals; lipid analysis revealed a 123% increase in hepatic triglyceride. A time-dependent increase in serum triglyceride levels was observed in the high-dose group beginning on day 4. Hepatic glycogen was reduced, and histochemical examination revealed the reduction to be heterogeneous across the lobule with some areas showing a complete absence of glycogen. One rabbit in each drug-treated group showed mild hypoglycemia at day 12, and 4/10 rabbits in the high-dose group showed hyperglycemia at days 12-14. We conclude that panadiplon produced a microvesicular steatosis and hepatic toxicity in the rabbit. The observed toxicity resembled a Reye's syndrome-like toxicity produced by a variety of mitochondrial fatty acid oxidation inhibitors.

Cytotoxicity and lamellar body induction potential of a racemic benzamide antiarrhythmic compound and enantiomers in cultured rat hepatocytes.

A wide variety of cationic amphiphilic compounds induce cytoplasmic lamellar bodies, accompanied by phospholipidosis and often coincidental with toxicity. The in vitro cytotoxic and lamellar body-inducing potentials of a racemic cationic amphiphilic benzamide antiarrhythmic compound and its trans-enantiomers were examined. Cultured rat hepatocytes were treated for 24 hr with the racemate and (+) and (-) trans-enantiomers in increasing concentrations to 1.0 mm. All compounds produced significant cytotoxicity at concentrations of 250 mum and greater, as assessed by lactate dehydrogenase release, but the (-) enantiomer was less cytotoxic than the racemate and (+) enantiomer at 500 mum. Electron microscopy revealed significant formation of lamellar bodies in all treated cultures at concentrations of 100 mum and higher for all compounds, with the (-) enantiomer producing significantly fewer lamellar bodies than the (+) enantiomer or racemic mixture. Acid phosphatase cytochemistry indicated that most, but not all, lamellar bodies were lysosomes. Fluorescence distributional studies with a phospholipid analogue, 1-acyl-2-[12-(7-nitro-2-1-,3-benzoxadiazol-4-yl)amino]dodecanoyl phosphatidylcholine, in living cells confirmed drug-induced cytoplasmic vacuoles to be formed from labelled plasma membrane at racemate drug concentrations as low as 10 mum, and suggested that lamellar body formation proceeds by a dynamic process involving the plasma membrane. These data indicate stereo-selective lamellar body-and cytotoxicity-inducing potentials between enantiomers, and that these are dissociable processes.

Potential to induce lamellar bodies and acute cytotoxicity of 6'-alkyl analogues of spectinomycin in primary cultures of rat hepatocytes.

A study was undertaken to investigate the potential of 6'-alkyl analogues of spectinomycin (SPE) to induce cytotoxicity and the formation of lamellar bodies in rat hepatocytes in culture. The cultured hepatocytes were treated with SPE, 6'-propylspectinomycin (trospectomycin sulphate; TRO), 6'-pentylspectinomycin (PES) or 6'-ocytlspectinomycin (OCS) in increasing concentrations up to 2.5 mm in the tissue-culture medium for 24 hr. Assay of lactate dehydrogenase (LDH) release into the medium was used to assess cytotoxicity, and the formation of lamellar bodies was assessed by transmission electron microscopy (TEM). Acid phosphatase cytochemistry was used in conjunction with TEM to determine the relationship of lamellar bodies to the lysosome. Neither SPE nor TRO produced significant cytotoxicity at the concentrations tested. Cytotoxicity was observed with PES at all concentrations >/=0.5 mm, and with OCS at all concentrations >/=0.1 mm. The number of lamellar bodies per cell section was correlated with the length of the 6'-alkyl side chain, and the capacity of the compounds to induce lamellar bodies was ranked in the order OCS > PES > TRO. Lamellar bodies were not observed above control levels in SPE-treated hepatocytes. Lamellar bodies were observed to stain positive for acid phosphatase activity, indicating that they were lysosomal. We conclude that the length of the 6'-alkyl side chain of spectinomycin analogues is correlated with acute cytotoxicity and the induction of lamellar bodies.

Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis).

Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey, Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4 X 10(9) cells with viabilities of 90.8 +/- 5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5'-nucleotide phosphodiesterase remained unchanged. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of alpha-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness, demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.

Hepatic changes produced by 30-day administration of a novel aminocyclitol antibiotic, trospectomycin sulfate, to laboratory animals.

The studies described here were done to characterize the hepatic response to a new aminocyclitol antibiotic, trospectomycin sulfate, administered intravenously (beagle dog) or subcutaneously (Sprague-Dawley rat) at a variety of dose levels, to investigate reversibility of observed changes, and to document any untoward effects of subchronic trospectomycin sulfate administration. Both species showed significant elevations in serum levels of alanine and aspartate transaminases in higher dose groups. In the dog only, a transient neuromuscular blockade was also observed within higher dose groups. No other functional, morphological, or serum chemical changes were observed. Examination of liver by electron microscopy revealed the presence of cytoplasmic lamellar inclusion bodies, concentrated in the bile canalicular region of the hepatocytes. Occurrence of the lamellar bodies and coincident transaminase increases were found to be reversible upon discontinuance of treatment (studied in the dog). Electron microscopy of acid phosphatase cytochemistry in the rat indicated that most, but not all, of the lamellar bodies contained this enzyme. This observation suggests that they may be derived from the lysosome, or once formed become lysosomal.

An ultrathin sectioning alignment tool with application to cell monolayers.

Details are given concerning the construction and use of a simple tool to help align a specimen block face in the vertical axis for subsequent ultrathin sectioning. Further instructions are given for the preparation of cell monolayers for ultrathin sectioning. The advantage is simple, repetitive and quick alignment of the specimen block face for ultrathin sectioning.

The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier.

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 x 10(4) cells/cm2, cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37 degrees C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an "internal standard" procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil-mediated transport of liposomes across the Madin Darby canine kidney epithelial cell monolayer.

Targeted drug delivery to peripheral blood neutrophils (PMNs) should be of therapeutic potential in various disease states. In addition, substances taken up by PMNs in the circulation may be delivered to an extravascular site via the naturally occurring cell infiltration. The present study employs an in vitro chemotaxis model to test whether particulate drug carriers such as liposomes can be transported across a cellular barrier by migrating PMNs. The system contained 10(7) human PMNs/ml, 0.3-micron liposomes at a total lipid concentration of 2.5 mM, and 10% autologous human serum in the apical side of a confluent Madin Darby canine kidney (MDCK) epithelial cell monolayer of 4.71 cm2. The MDCK cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix, and 10(-7) M f-Met-Leu-Phe was added to the basolateral side as a trigger of chemotaxis. The aqueous phase of the reverse-phase evaporation vesicles (REVs) contained lucifer yellow CH (LY) and [14C]sucrose. The lipid bilayer of the REVs was spiked with [3H]dipalmitoylphosphatidylcholine (DPPC). Transmission electron micrographs showed that, in response to the formyl peptide, PMNs adhered to the apical surface of MDCK cells, emigrated across the MDCK cell layer, passed through the 3-micron pores in the polycarbonate membrane, and finally, appeared in the bottom well. Epifluorescence micrographs showed that most, if not all, of the migrated PMNs contained punctate fluorescence derived from LY. Transport data over a 3.5-hr period indicated that those markers that appeared in the basal side were indeed transported by phagocytosis of REVs by PMNs and that intact serum was an essential component in the process.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute hepatocellular effects of erythromycin, gentamicin, and trospectomycin in the perfused rat liver: lack of correlation between lamellar body induction potency and cytotoxicity.

The formation of multilamellar inclusion bodies in cytoplasm is a generalized cellular response to treatment with a variety of chemical agents. The present study was conducted to determine if a correlation exists between acute lamellar body induction potency and cytotoxicity in the perfused rat liver. Livers were perfused for 3 hrs with various concentrations of erythromycin, gentamicin, sulfate, or trospectomycin sulfate, all of which are known to produce lamellar bodies in the rat in vivo. At the end of the experiments, the livers were perfusion fixed for transmission electron microscopy. Based on the bile flow rate, perfusion rate at constant pressure, and cytoplasmic enzyme release, neither gentamicin nor trospectomycin was hepatotoxic at concentrations up to 1.8 mM, whereas erythromycin was toxic at 0.1 mM. Gentamicin caused no ultrastructural changes compared to controls, but trospectomycin caused the dose-dependent formation of lamellar bodies in hepatocytes without other cytoplasmic alterations. Erythromycin caused cellular degeneration accompanied by an increase in the number of secondary lysosomes, but these lacked lamellated inclusions. It is concluded that hepatic lamellar bodies can be induced in acute ex vivo experiments, but that their formation does not appear to be linked with acute cytotoxicity.