Ronald A. Hill

SATURATION TOXICOKINETICS OF THIOACETAMIDE: ROLE IN INITIATION OF LIVER INJURY

Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S,S-dioxide (TASO2), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not dose-proportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA → TASO → TASO2 may follow zero-order kinetics. A 12-fold dose range of TA (50, 300, and 600 mg/kg i.p.) was injected into male Sprague-Dawley rats. TA and TASO were quantified in plasma, liver, and urine by high-performance liquid chromatography. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of 14C-TA-derived radiolabel to liver macromolecules showed a lessthan-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with 14C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared with the lowest dose (50 mg TA/kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 Supersomes, over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated Km values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second (TASO to TASO2) steps of TA bioactivation.

Enhanced Brain Amyloid-β Clearance by Rifampicin and Caffeine as a Possible Protective Mechanism Against Alzheimer’s Disease

Rifampicin and caffeine are widely used drugs with reported protective effect against Alzheimers disease (AD). However, the mechanism underlying this effect is incompletely understood. In this study, we have hypothesized that enhanced amyloid-β (Aβ) clearance from the brain across the blood-brain barrier (BBB) of wild-type mice treated with rifampicin or caffeine is caused by both drugs potential to upregulate low-density lipoprotein receptor related protein-1 (LRP1) and/or P-glycoprotein (P-gp) at the BBB. Expression studies of LRP1 and P-gp in brain endothelial cells and isolated mice brain microvessels following treatment with rifampicin or caffeine demonstrated both drugs as P-gp inducers, and only rifampicin as an LRP1 inducer. Also, brain efflux index (BEI%) studies conducted on C57BL/6 mice treated with either drug to study alterations in Aβ clearance demonstrated the BEI% of Aβ in rifampicin (82.4 ± 4.3%) and caffeine (80.4 ± 4.8%) treated mice were significantly higher than those of control mice (62.4 ± 6.1%, p < 0.01). LRP1 and P-gp inhibition studies confirmed the importance of both proteins to the clearance of Aβ, and that enhanced clearance following drugs treatment was caused by LRP1 and/or P-gp upregulation at the mouse BBB. Furthermore, our results provided evidence for the presence of a yet to be identified transporter/receptor that plays significant role in Aβ clearance and is upregulated by caffeine and rifampicin. In conclusion, our results demonstrated the upregulation of LRP1 and P-gp at the BBB by rifampicin and caffeine enhanced brain Aβ clearance, and this effect could explain, at least in part, the protective effect of rifampicin and caffeine against AD.

Ceramide Glycosylation Catalyzed by Glucosylceramide Synthase and Cancer Drug Resistance

Glucosylceramide synthase (GCS), converting ceramide to glucosylceramide, catalyzes the first reaction of ceramide glycosylation in sphingolipid metabolism. This glycosylation by GCS is a critical step regulating the modulation of cellular activities by controlling ceramide and glycosphingolipids (GSLs). An increase of ceramide in response to stresses, such as chemotherapy, drives cells to proliferation arrest and apoptosis or autophagy; however, ceramide glycosylation promptly eliminates ceramide and consequently, these induced processes, thus protecting cancer cells. Further, persistently enhanced ceramide glycosylation can increase GSLs, participating in selecting cancer cells to drug resistance. GCS is overexpressed in diverse drug-resistant cancer cells and in tumors of breast, colon, and leukemia that display poor response to chemotherapy. As ceramide glycosylation by GCS is a rate-limiting step in GSL synthesis, inhibition of GCS sensitizes cancer cells to anticancer drugs and eradicates cancer stem cells. Mechanistic studies indicate that uncoupling ceramide glycosylation can modulate gene expression, decreasing MDR1 through the cSrc/β-catenin pathway and restoring p53 expression via RNA splicing. These studies not only expand our knowledge in understanding how ceramide glycosylation affects cancer cells but also provide novel therapeutic approaches for targeting refractory tumors.

Discovery of Novel GSK-3β Inhibitors with Potent in Vitro and in Vivo Activities and Excellent Brain Permeability Using Combined Ligand- and Structure-Based Virtual Screening

Dysregulation of glycogen synthase kinase (GSK-3β) is implicated in the pathophysiology of many diseases, including type-2 diabetes, stroke, Alzheimers, and others. A multistage virtual screening strategy designed so as to overcome known caveats arising from the considerable flexibility of GSK-3β yielded, from among compounds in our in-house database and two commercial databases, new GSK-3β inhibitors with novel scaffold structures. The two most potent and selective validated hits, a 2-anilino-5-phenyl-1,3,4-oxadiazole (24) and a phenylmethylene hydantoin (28), both exhibited nanomolar affinity and selectivity over CDK2 and were potent enough for direct in vivo validation. Both were able to cause significant increases in liver glycogen accumulation in dose-dependent fashion. One also exhibited excellent blood-brain barrier permeability, the other adequate for a lead compound. Analogues of the oxadiazole 24 were synthesized to experimentally corroborate or rule out ligand-bound structures arising from docking studies. SAR results supported one docking study among a number of alternatives.

Final Report of the Safety Assessment of Kojic Acid as Used in Cosmetics

Kojic acid functions as an antioxidant in cosmetic products. Kojic acid was not a toxicant in acute, chronic, reproductive, and genotoxicity studies. While some animal data suggested tumor promotion and weak carcinogenicity, kojic acid is slowly absorbed into the circulation from human skin and likely would not reach the threshold at which these effects were seen. The available human sensitization data supported the safety of kojic acid at a use concentration of 2% in leave-on cosmetics. Kojic acid depigmented black guinea pig skin at a concentration of 4%, but this effect was not seen at 1%. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that the 2 end points of concern, dermal sensitization and skin lightening, would not be seen at use concentrations below 1%; therefore, this ingredient is safe for use in cosmetic products up to that level.

Permeability of Chemical Delivery Systems Across Rabbit Corneal (SIRC) Cell Line and Isolated Corneas: A Comparative Study

The purpose of this study was to investigate the corneal permeability of phenylephrone chemical delivery systems (CDS) across isolated cornea and to evaluate the utility of the SIRC cell line (epithelial cells originating from rabbit cornea) as an in vitro model for predicting the ocular permeability. The effect of benzalkonium chloride (BAC) on the drug permeability through SIRC cell layers was also studied. The transport of phenylephrone CDS across the isolated cornea of the albino rabbit was measured at various pH values using a two-chamber glass diffusion cell, and the results were compared with the reported permeability values across SIRC cells of rabbit origin. Corneal membranes showed lower flux values for compounds, especially for hydrophilic compounds, than the SIRC cell line. A significant correlation was observed between the permeability coefficients through corneal membranes and SIRC cells. When the pH of the transport medium was increased, the permeability coefficients increased and lag times decreased in both in vitro models. Furthermore, both in vitro models showed significant correlation between permeability coefficients and lipophilicities of the drugs. The three esters, having higher lipophilic characteristics, showed higher permeability than phenylephrine HCl. The phenylacetyl ester of phenylephrone showed a three-fold increase in penetration across SIRC cell layers in the presence of 0.01% BAC. These results suggest that the use of SIRC cell layers can reasonably predict the permeability of ophthalmic drugs across corneal membranes.

Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

Final Report of the Amended Safety Assessment of Sodium Laureth Sulfate and Related Salts of Sulfated Ethoxylated Alcohols

Sodium laureth sulfate is a member of a group of salts of sulfated ethoxylated alcohols, the safety of which was evaluated by the Cosmetic Ingredient Review (CIR) Expert Panel for use in cosmetics. Sodium and ammonium laureth sulfate have not evoked adverse responses in any toxicological testing. Sodium laureth sulfate was demonstrated to be a dermal and ocular irritant but not a sensitizer. The Expert Panel recognized that there are data gaps regarding use and concentration of these ingredients. However, the overall information available on the types of products in which these ingredients are used and at what concentrations indicates a pattern of use. The potential to produce irritation exists with these salts of sulfated ethoxylated alcohols, but in practice they are not regularly seen to be irritating because of the formulations in which they are used. These ingredients should be used only when they can be formulated to be nonirritating.

Hydroxyl-substituted sulfonylureas as potent inhibitors of specific [3H]glyburide binding to rat brain synaptosomes.

We are seeking to discover potent CNS-active sulfonylureas with structural features that allow for the formation of several types of prodrugs. We report herein the syntheses of compounds comprising an initial series of hydroxyl-substituted analogues of the potent ATP-sensitive potassium channel blockers glyburide (glibenclamide) and gliquidone. Somewhat unexpectedly, several of the compounds were found to be comparably potent to glyburide as inhibitors of specific [(3)H]glyburide binding in rat brain preparations.

Final Amended Safety Assessment of Hydroquinone as Used in Cosmetics

Hydroquinone is an aromatic compound that functions in cosmetics as an antioxidant, fragrance, reducing agent, or polymerization inhibitor. Hydroquinone is also used as a skin bleaching agent. Safety and toxicity information indicate that hydroquinone is dermally absorbed in humans from both aqueous and alcoholic formulations and is excreted mainly as the glucuronide or sulfate conjugates. Hydroquinone is associated with altered immune function in vitro and in vivo in animals and an increased incidence of renal tubule cell tumors and leukemia in F344 rats, but the relevance to humans is uncertain. Quantitatively, however, the use of hydroquinone in cosmetics is unlikely to result in renal neoplasia through this mode of action. Thus, hydroquinone is safe at concentrations of ≤1% in hair dyes and is safe for use in nail adhesives. Hydroquinone should not be used in other leave-on cosmetics.