Pharmacokinetic/Toxicokinetic Considerations

Toxicokinetics (TK) is generation of kinetic data for systemic exposure and toxicity assessment of the drug. These studies help us to estimate the observed toxicity to that dose. TK evaluation is very important in drug development phase in both regulatory and scientific perspective. There are several guidelines to conduct TK study in animals recommended by regulatory bodies (OECD). TK evaluation is useful in selection of dose, dosing form, alternative dosing route, evaluation of toxicological mechanism, and also used for the setting safe dose level in clinical phases. This TK studies also used to reduces the animal number (replacement, reduction and refinement). On the other hand, TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection. Email:marketing@medicilon.com.cn Web:www.medicilon.com

Triptolide's molecular mechanism of action has remained elusive, but triptolide was reported to covalently bind to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and to inhibit its DNA-dependent ATPase activity, leading to inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the many of the known biological activities of triptolide. For example, triptolide binding to XPB lead to the down regulation of a number of growth and survival promoters including NF kappa B (NF-κΒ) and the anti-apoptotic factors Mcl-1 and XIAP. (Titov, et a!., Nat. Chem. Biol. (2011) 7(3): 182-8). Subsequently, the triptolide derivative MRxl02 was also found to have these effects, i.e., reduced mRNA levels, reduced NF-κΒ and reduced Mcl-1 and XIAP. At low nanomolar concentrations, MRxl02 also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. In vivo, MRxl02 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Thus, MRxl02 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation. (Carter, et al, (2012) Leukemia 26:443-50). Furthermore, triptolide and triptolide derivatives can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.

Another consequence of XPB binding is the inhibition of nucleotide excision repair. This activity in blocking DNA repair should enhance the activities of those drugs that have DNA as their target, including cisplatin and topoisomerase 1 inhibitors for solid tumors; both have been shown to act in a synergistic fashion with triptolide. The potential synergy between MRxl02 and two drugs used in AML, cytarabine and idarubicin was investigated using MV4-11 cells in vitro and synergy was demonstrated between MRxl02 and both of these drugs used in AML.

One concern regarding triptolide and triptolide derivatives is their epoxide structure, viewed as potentially toxic; however, proteosome inhibitor anti-cancer drug, carfilzomib (Kyprolis) is a tetrapeptide epoxyketone containing an epoxide, and was recently FDA approved. Furthermore, triptolide, even though it is a triepoxide, was shown by Titov, et al., (supra) to be exquisitely selective, and not promiscuous, in its binding characteristics. Nonetheless, triptolide's reported safety issues in a number of animal studies as well as clinically, have resulted in an "image problem" and potential safety challenges; accordingly, triptolide has not been deemed appropriate for clinical use and has not been commercially developed.

Triptolide prodrugs are generally believed to be safer than triptolide. In an initial rodent toxicology study, PG796(MRxl02) demonstrated no gross or histopathologic toxic effects at intravenous doses up to 1.5 mg/kg/day for seven days. Triptolide prodrugs as an emulsion formulation are believed to have a toxicokinetic profile characterized by a flat AUC with a minimized Cmax. [In conjunction, it was postulated that a sustained inhibition of RNA polymerase is needed for optimum efficacy which in turn requires a pharmcokinetic profile of constant exposure to drug]. Figure 1 shows a side-by-side comparative toxicology study of PG796(MRxl02) and triptolide in which both drugs were administered intravenously to rodents using the novel emulsion formulation disclosed herein demonstrated that PG796(MRxl02) was at least 20 times less toxic than triptolide based on both gross and histopathologic criteria. The no effect dose ("NOAEL") of PG796(MRxl02) again exceeded 1.5 mg/kg/day intravenously for seven days in rodents confirming the initial results. It is interesting to ask why a prodrug of triptolide would be safer than the natural product itself; while not wishing to be bound by theory, perhaps the answer lies in the pharmacokinetic profile of triptolide administered either directly or released from its carrier, PG796(MRxl02). When triptolide is provided alone (see line connecting circles in Fig. 1) it had a very high Cmax as well as a rapid decline such that by two hours post-dose none remained in circulation. However, when the prodrug PG796(MRxl02) was administered, the triptolide Cmax was approximately one-tenth that noted when triptolide was administered directly (see line connecting triangles in Fig. 1) and the triptolide blood levels remained relatively constant and demonstrate a longer AUC ("area under the curve") as seen at the two-hour time point. It also remained above the therapeutic levels (shown as a thick line without symbols). The difference in the Cmax/ AUC profile of PG796(MRxl02) vs. triptolide is believed to be due to the physiochemical properties of the lipid prodrug/emulsion formulation combination. In general, triptolide prodrugs having a cLogP greater than 0.5 are more lipid-soluble than water soluble and are expected to take longer to convert to the drug form; such characteristics may yield a flatter conversion profile and less of a drug-release Cmax spike.

PG490-88 given intravenously, entered clinical trials and showed promising activity in patients with AML. (Xia Zhi Lin and Zhen You Lan, Haematologica, 93 : 14 (2008)). However, as a prodrug, it was incompletely and erratically converted to the active entity, triptolide, and, as such, may provide areason it produced toxicity. However, PG490-88 did have an optimized AUC, relatively flat over time with no intense Cmax. The finding that PG796(MRxl02) was rapidly and completely converted to triptolide using human serum (as well as seen in vivo in rats and dogs) while PG490-88Na was incompletely converted to triptolide in human serum argues that the conversion of PG796(MRxl02) is not dependent on variations in species enzymatic (esterase) activities but is dependent on the physiochemical properties of the lipid prodrug/emulsion formulation.

Lipid emulsions have been studied as drug delivery systems for some time. (See Hippalgaonkar, et al., (2010) AAPS Pharm. Sci. Tech. 11(4): 1526-1540; Stevens, et al., (2003) Business Briefing: Pharmatech 2003, p. 1-4). Solid lipid nanoparticle (SLN) delivery systems may have advantages over conventional formulations of bioactive plant extracts, such as enhancing solubility and bioavailability, offering protection from toxicity, and enhancing pharmacological activity. A tripterygium glycoside (TG) solid lipid nanoparticle (TG-SLN) delivery system was reported to have a protective effect against TG-induced male reproductive toxicity. Triptolide (TP) was used as a model drug in a comparative study of the toxicokinetic study and tissue distribution of TP-SLN and free TP in rats. A fast and sensitive HPLC-APCI- MS MS method was developed for the determination of triptolide in rat plasma. Fourteen rats were divided randomly into two groups of 7 rats each for toxicokinetic analysis, with one group receiving free TP (450μg/kg) and the other receiving the TP-SLN formulation (450μg/kg). Blood was obtained before dosing and 0.083, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 3 and 4h after drug administration. Thirty-six rats were divided randomly into six equal groups for a tissue-distribution study. Half of the rats received intragastric administration of TP (45(^g/kg) and the other half received TP-SLN (450pg/kg). At 15, 45, and 90min after dosing, samples of blood, liver, kidney, spleen, lung, and testicular tissue were taken. TP concentration in the samples was determined by LC-APCI-MS-MS. The toxicokinetic results for the nanoformulation showed a significant increase the area under the curve (AUC) (P<0.05), significantly longer T(max) and mean retention times (MRTs) (0-t) (P<0.05), significantly decreased C(max) (PO.05). The nanoformulation promoted absorption with a slow release character, indicating that toxicokinetic changes may be the most important mechanism for the enhanced efficacy of nanoformulations. Tissue-distribution results suggest a tendency for TP concentrations in the lung and spleen to increase, while TP concentrations in plasma, liver, kidney, and testes tended to decrease in the TP-SLN group. At multiple time points, testicular tissue TP concentrations were lower in the TP-SLN group than in free TP group. This provides an important clue for the decreased reproductive toxicity observed with TP-SLN. Overall, an orally administered lipid nanoparticle formulation of triptolide promoted absorption with a slow release character. (Xue, et al., (2012) Eur. J. Pharm. Sci., 47(4):713-7). The toxicokinetic results for the nanoformulation showed a significant increase in AUC, and a decreased Cmax. These results indicate that toxicokinetic change are a consideration for enhanced safety.