Predicting In Vivo Efficacy from In Vitro Data: Quantitative Systems Pharmacology Modeling for an Epigenetic Modifier Drug in Cancer.

Reliably predicting in vivo efficacy from in vitro data would facilitate drug development by reducing animal usage and guiding drug dosing in human clinical trials. However, such prediction remains challenging. Here, we built a quantitative pharmacokinetic/pharmacodynamic (PK/PD) mathematical model capable of predicting in vivo efficacy in animal xenograft models of tumor growth while trained almost exclusively on in vitro cell culture data sets. We studied a chemical inhibitor of LSD1 (ORY-1001), a lysine-specific histone demethylase enzyme with epigenetic function, and drug-induced regulation of target engagement, biomarker levels, and tumor cell growth across multiple doses administered in a pulsed and continuous fashion. A PK model of unbound plasma drug concentration was linked to the in vitro PD model, which enabled the prediction of in vivo tumor growth dynamics across a range of drug doses and regimens. Remarkably, only a change in a single parameter-the one controlling intrinsic cell/tumor growth in the absence of drug-was needed to scale the PD model from the in vitro to in vivo setting. These findings create a framework for using in vitro data to predict in vivo drug efficacy with clear benefits to reducing animal usage while enabling the collection of dense time course and dose response data in a highly controlled in vitro environment.

Physiologically Based Absorption Modeling to Explore the Impact of Food and Gastric pH Changes on the Pharmacokinetics of Alectinib.

Alectinib, a lipophilic, basic, anaplastic lymphoma kinase (ALK) inhibitor with very low aqueous solubility, has received Food and Drug Administration-accelerated approval for the treatment of patients with ALK+ non-small-cell lung cancer. This paper describes the application of physiologically based absorption modeling during clinical development to predict and understand the impact of food and gastric pH changes on alectinib absorption. The GastroPlus™ software was used to develop an absorption model integrating in vitro and in silico data on drug substance properties. Oral pharmacokinetics was simulated by linking the absorption model to a disposition model fit to pharmacokinetic data obtained after an intravenous infusion. Simulations were compared to clinical data from a food effect study and a drug-drug interaction study with esomeprazole, a gastric acid-reducing agent. Prospective predictions of a positive food effect and negligible impact of gastric pH elevation were confirmed with clinical data, although the exact magnitude of the food effect could not be predicted with confidence. After optimization of the absorption model with clinical food effect data, a refined model was further applied to derive recommendations on the timing of dose administration with respect to a meal. The application of biopharmaceutical absorption modeling is an area with great potential to further streamline late stage drug development and with impact on regulatory questions.

Investigating the effect of autoinduction in cynomolgus monkeys of a novel anticancer MDM2 antagonist, idasanutlin, and relevance to humans.

1. Idasanutlin (RG7388) is a potent p53-MDM2 antagonist currently in clinical development for treatment of cancer. The purpose of the present studies was to investigate the cause of marked decrease in plasma exposure after repeated oral administration of RG7388 in monkeys and whether the autoinduction observed in monkeys is relevant to humans. 2. In monkey liver and intestinal microsomes collected after repeated oral administration of RG7388 to monkeys, significantly increased activities of homologue CYP3A8 were observed (ex vivo). Investigation using a physiologically based pharmacokinetic (PBPK) model suggested that the loss of exposure was primarily due to induction of metabolism in the gut of monkeys. 3. Studies in monkey and human primary hepatocytes showed that CYP3A induction by RG7388 only occurred in monkey hepatocytes but not in human hepatocytes, which suggests the observed CYP3A induction is monkey specific. 4. The human PK data obtained from the first cohorts confirmed the lack of relevant induction as predicted by the human hepatocytes and the PBPK modelling based on no induction in humans.

Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines.

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galalpha(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.

The discovery of potent, selective, and orally bioavailable PDE9 inhibitors as potential hypoglycemic agents.

Starting from a non-selective pyrazolo-pyrimidone lead, the sequential use of parallel medicinal chemistry and directed synthesis led to the discovery of potent, highly selective, and orally bioavailable PDE9 inhibitors. The availability of these tools allowed for a thorough evaluation of the therapeutic potential of PDE9 inhibition.

SAP regulates T cell-mediated help for humoral immunity by a mechanism distinct from cytokine regulation.

X-linked lymphoproliferative disease is caused by mutations affecting SH2D1A/SAP, an adaptor that recruits Fyn to signal lymphocyte activation molecule (SLAM)-related receptors. After infection, SLAM-associated protein (SAP)-/- mice show increased T cell activation and impaired humoral responses. Although SAP-/- mice can respond to T-independent immunization, we find impaired primary and secondary T-dependent responses, with defective B cell proliferation, germinal center formation, and antibody production. Nonetheless, transfer of wild-type but not SAP-deficient CD4 cells rescued humoral responses in reconstituted recombination activating gene 2-/- and SAP-/- mice. To investigate these T cell defects, we examined CD4 cell function in vitro and in vivo. Although SAP-deficient CD4 cells have impaired T cell receptor-mediated T helper (Th)2 cytokine production in vitro, we demonstrate that the humoral defects can be uncoupled from cytokine expression defects in vivo. Instead, SAP-deficient T cells exhibit decreased and delayed inducible costimulator (ICOS) induction and heightened CD40L expression. Notably, in contrast to Th2 cytokine defects, humoral responses, ICOS expression, and CD40L down-regulation were rescued by retroviral reconstitution with SAP-R78A, a SAP mutant that impairs Fyn binding. We further demonstrate a role for SLAM/SAP signaling in the regulation of early surface CD40L expression. Thus, SAP affects expression of key molecules required for T-B cell collaboration by mechanisms that are distinct from its role in cytokine regulation.

Establishment of correlation between in vitro enzyme binding potency and in vivo pharmacological activity: application to liver glycogen phosphorylase a inhibitors.

In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic Kd for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro Kd determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.

Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source.

The major drawback of liquid chromatography/mass spectrometry (LC/MS) for the analysis of mixtures is the non-quantitative nature of these studies. The ionization efficiency of the various components in the mixture (e.g., a compound and its metabolites) can vary greatly and, therefore, relative intensities of signals cannot be related to relative abundance. A chip-based nanoelectrospray ionization source was used to compare the ionization efficiencies of compounds with different physical-chemical characteristics. The data indicate that the ionization efficiencies vary much less with the chip-based device than by LC/MS. This was ascribed to the generation of a much higher electric field around the nozzles, which supplies a large excess of protons to the small droplets and reduces/eliminates the differences in the ionization efficiency for the analytes.

Identification of a novel glutathione adduct of diclofenac, 4'-hydroxy-2'-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes.

Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4'-OH-3'-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structure-characteristic" fragment for GS adduct, on an automated chip-based nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH+), in addition to the known adduct peak with m/z 617 (MH+). This new adduct was further confirmed to be 4'-OH-2'-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.

SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1.

XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor.

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.

Cysteine residues required for the attachment of the light chain in human IgA2.

In humans, there are two subclasses of IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1), IgA2m(2) and IgA2(n). In IgA1, Cys(133) in C(H)1 forms the disulfide bond to the L chain. Our previous studies indicated that in IgA2 lacking Cys(133), a disulfide bond forms between the alpha-chain and the L chain when Cys(220) is followed by Arg(221), but not when Cys(220) is followed by Pro(221), suggesting that the Cys in C(H)1 might be involved in disulfide bonding to the L chain. However, here we show that covalent assembly of the H and L chains in IgA2(n) requires hinge-proximal Cys(241) and Cys(242) in C(H)2 and not Cys(196) or Cys(220) in C(H)1. Using pulse-chase experiments, we have demonstrated that wild-type IgA2(n) with Arg(221) and Cys(241) and Cys(242) assembles through a disulfide-bonded HL intermediate. In contrast, the major intermediate for IgA2 m(1) with Pro(221) assembly was H(2) even though both Cys(241) and Cys(242) were present. Only a small fraction of IgA2 m(1) assembles through disulfide-bonded HL. Overall, our studies indicate that for IgA2 covalent assembly of the H and L chains requires the hinge-proximal cysteines in C(H)2 and that the structure of C(H)1 influences the efficiency with which this covalent bond forms.