Interspecies Differences in the Metabolism of a Multiester Prodrug by Carboxylesterases

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA) referred to as C2E5, is being developed as an orally bioavailable radionuclide decorporation agent. The predicted human efficacy obtained in these experimental animals is confounded by interspecies variations of metabolism. Therefore, in the present study, carboxylesterase-mediated metabolism of [14C]-C2E5 was compared in the S9 intestinal and hepatic fractions of human, dog and rat and their respective plasma. Intestinal hydrolysis of C2E5, resulting in the formation of the tetraethyl ester of DTPA (C2E4), was only detected in human and rat. The primary metabolite in human and dog hepatic fractions was C2E4 whereas the predominant species identified in rat hepatic fractions was the triethyl ester (C2E3). Hepatic hydrolysis of C2E5 causes the formation of C2E4 in human, dog and rat and C2E3 in rat only. Minimal C2E5 hydrolysis was observed in human and dog plasma whereas in rat plasma C2E5 converted to C2E3 rapidly, followed by slower further metabolism. Both recombinant CES1 and CES2 play roles in C2E5 metabolism. Together, these data suggest that dogs may be the most appropriate species for predicting human C2E5 metabolism whereas rats might be useful for clarifying the potential toxicity of C2E5 metabolites

Modulation of the severe CD4+ T-cell loss caused by a pathogenic simian–human immunodeficiency virus by replacement of the subtype B vpu with the vpu from a subtype C HIV-1 clinical isolate

AbstractPreviously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIVSCVpu, in which the subtype B vpu gene from the pathogenic SHIVKU-1bMC33 was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIVSCVpu replicated with slightly reduced kinetics when compared with the parental SHIVKU-1bMC33 and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIVKU-1bMC33. To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIVSCVpu and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIVSCVpu caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIVKU-1bMC33. These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model

The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4+ T cell loss caused by the simian–human immunodeficiency virus SHIVKU-lbMC33 in pig-tailed macaques

AbstractThe simian–human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIVS52,56G). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIVS52,56G and virus burdens and circulating CD4+ T cells monitored up to 1 year. Our results indicate that SHIVS52,56G caused rapid loss in the circulating CD4+ T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4+ T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIVKU-1bMC33 and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIVKU-1bMC33, all of which developed severe CD4+ T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIVKU-1bMC33 and suggest that the SHIVKU-1bMC33/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1

Nonaqueous Gel for the Transdermal Delivery of a DTPA Penta-ethyl Ester Prodrug

Diethylenetriamine pentaacetic acid penta-ethyl ester, designated as C2E5, was successfully incorporated into a nonaqueous gel for transdermal delivery. The thermal and rheological properties of a formulation containing 40% C2E5, 20% ethyl cellulose, and 40% Miglyol 840® prepared using the solvent evaporation method demonstrated that the gel had acceptable content uniformity and flow properties. In vitro studies showed that C2E5 was steadily released from the gel at a rate suitable for transdermal delivery. Topical application of the gel at a 200 mg C2E5/kg dose level in rats achieved significantly higher plasma exposures of several active metabolites compared with neat C2E5 oil at the same dose level. The results suggest that transdermal delivery of a chelator prodrug is an effective radionuclide decorporation strategy by delivering chelators to the circulation with a pharmacokinetic profile that is more consistent with the biokinetic profile of transuranic elements in contaminated individuals

Orally administered DTPA di-ethyl ester for decorporation of 241 Am in dogs: Assessment of safety and efficacy in an inhalation-contamination model

Currently two injectable products of diethylenetriaminepentaacetic acid (DTPA) are U.S. Food and Drug Administration (FDA) approved for decorporation of 241Am, however, an oral product is considered more amenable in a mass casualty situation. The diethyl ester of DTPA, named C2E2, is being developed as an oral drug for treatment of internal radionuclide contamination

Molecular Mechanism of Polybrominated Diphenyl Ether Disposition in the Liver

Polybrominated diphenyl ethers (PBDEs) were introduced in the late 1970's as additive flame retardants incorporated into textiles, electronics, plastics and furniture. Although 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE209) is the only congener currently on the market, 2,2`,4,4`-tetrabromodiphenyl ether (BDE47), 2,2`,4,4`,5-pentabromodiphenyl ether (BDE99), and 2,2`,4,4`,5,5`-hexabromodiphenyl ether (BDE153) are the predominant congeners detected in human and wildlife samples. Upon exposure, PBDEs enter the liver where they are biotransformed to potentially toxic metabolites. Although the human liver burden of PBDEs is not clear, the presence of PBDEs in human liver is particularly alarming because it has been demonstrated in rodents that hydroxylated metabolites may play a pivotal role in PBDE-mediated toxicity. The mechanism by which PBDEs enter the liver was not known. However, due to their large molecular weights (MWs ~485 to 1000 Da), they were not likely to enter hepatocytes by simple diffusion. Organic anion transporting polypeptides (OATPs: human; Oatps: rodents) are responsible for hepatic uptake of a variety of amphipathic compounds of MWs larger than 350 Da. Therefore, I tested the hypothesis that OATPs/Oatps expressed in human and mouse hepatocytes are responsible for the uptake of PBDE congeners 47, 99, and 153 by using Chinese hamster ovary (CHO) cell lines expressing OATP1B1, OATP1B3, or OATP2B1 and Human Embryonic Kidney 293 (HEK293) cells transiently expressing Oatp1a1, Oatp1a4, Oatp1b2, or Oatp2b1. Direct uptake studies illustrated that PBDE congeners are substrates of human and mouse hepatic OATPs/Oatps, except for Oatp1a1. Detailed kinetic analysis revealed that OATP1B1, OATP1B3, Oatp1a4, and Oatp1b2 transport BDE47 with the highest affinity followed by BDE99 and BDE153. However, both OATP2B1 and Oatp2b1 transported all three congeners with similar affinities. The importance of hepatic Oatps for the accumulation of BDE47 in liver was confirmed using Oatp1a4- and Oatp1b2-null mice. These results clearly suggest that uptake of PBDEs via these OATPs/Oatps are responsible for liver-specific accumulation of PBDEs. In mouse liver, PBDEs induce drug metabolizing enzymes, namely cytochrome P450s (Cyps). However, the molecular mechanisms underlying this induction was unknown. Cyp2b10 and 3a11 are target genes of the xenobiotic nuclear receptors, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both of which are responsible for mediating induction of Cyp2b10 and Cyp3a11, respectively. I hypothesized that PBDE congeners are CAR and/or PXR activators. Using reporter-gene luciferase assays I showed that BDE47, BDE99 and BDE209 activate human and mouse CAR and PXR in a concentration-dependent manner. Furthermore, induction of Cyp2b10 and Cyp3a11 was markedly suppressed in CAR- and PXR-null mice, respectively, indicating that PBDE congeners activate these receptors in vivo. BDE47 and BDE99, the primary congeners detected in humans in the United States, are capable of inducing Cyp2b and Cyp3a enzymes in rodents. However, it is not clear which Cyp isoform, if any, is preferentially induced upon exposure to BDE47 or BDE99. Induction of mouse hepatic Cyp2b10 and 3a11 by PBDEs showed distinct dose-responses, with Cyp2b10 being induced at lower doses and Cyp3a11 at much higher doses, indicating PBDEs are more likely to induce hepatic enzymes at doses that humans are exposured to. Currently, daily exposure of PBDEs is estimated to be 0.003mg/kg for adults. This study shows that effects of PBDEs are seen in animal models at concentrations within ~10-fold of the high end of the human population. Together, the results from the current dissertational study demonstrate that PBDEs are substrates of OATPs/Oatps and activators of CAR and PXR. This study not only provides a molecular basis for understanding PBDE disposition and toxicity in the liver but also cautions PBDE exposure may result in broader impact on liver physiology and toxicology

Mechanism of Polybrominated Diphenyl Ether Uptake into the Liver: PBDE Congeners Are Substrates of Human Hepatic OATP Transporters

Polybrominated diphenyl ethers (PBDEs) are flame-retardants that upon chronic exposure enter the liver where they are biotransformed to potentially toxic metabolites. The mechanism by which PBDEs enter the liver is not known. However, due to their large molecular weights (MWs ∼485 to 1000 Da), they cannot enter hepatocytes by simple diffusion. Organic anion–transporting polypeptides (OATPs) are responsible for hepatic uptake of a variety of amphipathic compounds of MWs larger than 350 Da. Therefore, in the present study, Chinese hamster ovary cell lines expressing OATP1B1, OATP1B3, and OATP2B1 were used to test the hypothesis that OATPs expressed in human hepatocytes would be responsible for the uptake of PBDE congeners 47, 99, and 153. The results demonstrated that PBDE congeners inhibited OATP1B1- and OATP1B3-mediated uptake of estradiol-17-β-glucuronide as well as OATP2B1-mediated uptake of estrone-3-sulfate in a concentration-dependent manner. Direct uptake studies confirmed that all three PBDE congeners are substrates for the three tested hepatic OATPs. Detailed kinetic analysis revealed that OATP1B1 transported 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) with the highest affinity (Km = 0.31μM) followed by 2,2′,4,4′,5-pentabromodiphenyl ether (BDE99) (Km = 0.91μM) and 2,2′,4,4′,5,5′-hexabromodiphenyl ether (BDE153) (Km = 1.91μM). For OATP1B3, the order was the same (BDE47: Km = 0.41μM; BDE99: Km = 0.70μM; BDE153: Km = 1.66μM), while OATP2B1 transported all three congeners with similar affinities (BDE47: Km = 0.81μM; BDE99: Km = 0.87μM; BDE153: Km = 0.65μM). These results clearly suggest that uptake of PBDEs via these OATPs is a mechanism responsible for liver-specific accumulation of PBDEs