Effects of parasitic infection on the pharmacokinetics and disposition of pentamidine analogs

Diamidine analogs of pentamidine are under evaluation as new and safer alternatives for treatment of first- and second-stage human African trypanosomiasis (HAT). Bis-O-methylamidoxime prodrugs of diamidines depend on hepatic biotransformation for generation of the pharmacologically active diamidines. The goal of this dissertation project was to investigate whether trypanosomal infection attenuates biotransformation of bis-O-methylamidoxime prodrugs, with the consequent potential to alter systemic exposure to prodrug and/or active diamidines. Biotransformation of the prodrug DB868 to the active diamidine, DB829, under investigation for second-stage HAT, mirrored biotransformation of the prodrug for first-stage HAT, pafuramidine, highlighting the central role of biotransformation for bis-O-methylamidoxime prodrug activation. In addition to the O-demethylation and N-dehydroxylation reactions preceding DB829 generation, a previously unrecognized N-demethoxylation reaction was observed in human and rat liver microsomes. Formation of DB829 in cultured primary hepatocytes from both species was rapid; however, basolateral export and/or intracellular sequestration limited appearance of DB829 diamidine in culture medium. In a rat model of first-stage trypanosomiasis, developed and characterized during this investigation, infection altered significantly the pharmacokinetics of both the prodrug, pafuramidine, and active diamidine, furamidine. Compared to uninfected animals, systemic exposure (AUC) of both prodrug and active diamidine was increased significantly in infected animals, by 1.3- and 3-fold, respectively. The increase in pafuramidine AUC during infection was dependent on dose and route of administration, and conformed to expected behavior for a blood-flow limited compound according to the well-stirred model of hepatic clearance. The increase in furamidine AUC during infection was explained, in part, by decreased biliary excretion of furamidine. Simulations of decreased enzyme capacity using different doses and routes of administration provided a framework for considering the impact of infection on pharmacokinetics. This dissertation project showed that trypanosomal infection is capable of altering the pharmacokinetics of bis-O-methylamidoxime prodrugs and corresponding diamidines. Knowledge gained from this work provides a basis for making predictions of pharmacokinetic outcomes during infection and inflammation

Refining Liver Safety Risk Assessment: Application of Mechanistic Modeling and Serum Biomarkers to Cimaglermin Alfa (GGF2) Clinical Trials

Cimaglermin alfa (GGF2) is a recombinant human protein growth factor in development for heart failure. Phase I trials were suspended when two cimaglermin alfa‐treated subjects experienced concomitant elevations in serum aminotransferases and total bilirubin, meeting current US Food and Drug Administration criteria for a serious liver safety signal (i.e., “Hy's Law”). We assayed mechanistic biomarkers in archived clinical trial serum samples which confirmed the hepatic origin of the aminotransferase elevations in these two subjects and identified apoptosis as the major mode of hepatocyte death. Using a mathematical model of drug‐induced liver injury (DILIsym) and a simulated population, we estimated that the maximum hepatocyte loss in these two subjects was <13%, which would not result in liver dysfunction sufficient to significantly increase serum bilirubin levels. We conclude that the two subjects should not be considered Hy's Law cases and that mechanistic biomarkers and modeling can aid in refining liver safety risk assessment in clinical trials

Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport

Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [3H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Clbiliary) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Clbiliary by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n=2–4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI

Differential Effects of Pravastatin and Simvastatin on the Growth of Tumor Cells from Different Organ Sites

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors, commonly known as statins, may possess cancer preventive and therapeutic properties. Statins are effective suppressors of cholesterol synthesis with a well-established risk-benefit ratio in cardiovascular disease prevention. Mechanistically, targeting HMGCR activity primarily influences cholesterol biosynthesis and prenylation of signaling proteins. Pravastatin is a hydrophilic statin that is selectively taken up by a sodium-independent organic anion transporter protein-1B1 (OATP1B1) exclusively expressed in liver. Simvastatin is a hydrophobic statin that enters cells by other mechanisms. Poorly-differentiated and well-differentiated cancer cell lines were selected from various tissues and examined for their response to these two statins. Simvastatin inhibited the growth of most tumor cell lines more effectively than pravastatin in a dose dependent manner. Poorly-differentiated cancer cells were generally more responsive to simvastatin than well-differentiated cancer cells, and the levels of HMGCR expression did not consistently correlate with response to statin treatment. Pravastatin had a significant effect on normal hepatocytes due to facilitated uptake and a lesser effect on prostate PC3 and colon Caco-2 cancer cells since the OATP1B1 mRNA and protein were only found in the normal liver and hepatocytes. The inhibition of cell growth was accompanied by distinct alterations in mitochondrial networks and dramatic changes in cellular morphology related to cofilin regulation and loss of p-caveolin. Both statins, hydrophilic pravastatin and hypdrophobic simvastatin caused redistribution of OATP1B1 and HMGCR to perinuclear sites. In conclusion, the specific chemical properties of different classes of statins dictate mechanistic properties which may be relevant when evaluating biological responses to statins

Thoracic Lymph Duct Catheterization with a Venous Shunt in the Nonhuman Primate

Background: Biologic therapeutics constitute up to 30% of all drugs approved from 2010 to 2018 and represent a continuous growing market. In contrast to small molecules, biologic therapeutics (>1 kDa MW) are administered parenterally or intravenously due to poor bioavailability when administered orally. The absorption and disposition of biologics that are administered subcutaneously may be absorbed via lymphatic or blood capillaries. Methods: To understand the absorption and distribution of biotherapeutics via the lymphatic system a surgical model was developed in the cynomolgus macaque (Macaca fascicularis) to allow for frequent and chronic collection of lymph fluid. Additionally, the model allowed for the recirculation of the lymph fluid into the blood stream providing true physiologic redistribution of the biologic drug from the bloodstream back into the lymph. Results: To our knowledge, models of lymphatic duct catheterization with recirculation in the NHP have not been reported. The model consisted of two surgically implanted catheters, one in the thoracic lymph duct and one in the azygous vein. These two catheters were then exteriorized and connected to each other to allow for recirculation of lymph back into the venous blood stream. The exteriorized catheters were protected within the pocket of a jacket. Thirty-one surgical procedures were performed with an overall success rate of 70%. Unsuccessful attempts were related to anatomical differences where the lymphatic duct was either not identifiable (n = 3) or too small to catheterize (n = 6). The patency rate was 90% instrumented animals for at least 24 h, up to 168 h. Conclusion: We present the surgical technique, complications, and refinements which resulted in a reliable and reproducible model in the nonhuman primate for the chronic collection and recirculation of lymphatic fluid