Development and validation of LC/MS/MS method for Triciribine and its monophosphate metabolite in plasma and RBC: Application to mice pharmacokinetic studies.

Triciribine (TCN) is a tricyclic nucleoside analog of adenosine and an inhibitor of Akt kinase. Triciribine 5'-monophosphate (TCNP) is a water-soluble analog of Triciribine and has progressed to Phase I and II clinical trials in oncology. TCNP is also an endogenous anabolite of TCN similar to other nucleoside phosphates. Clinical development of TCNP has been hampered by high pharmacokinetic variability due to complex interplay of TCN-TCNP conversion and reconversion in plasma, erythrocytes (RBC) and peripheral organs. TCN has been demonstrated to be an efficacious agent in mice models of acute lung injury at low doses (0.5 mg/kg/day) although its pharmacokinetic-pharmacodynamic (PK/PD) relationship remained unclear. We have developed and validated a sensitive, specific and robust LC/MS/MS assay for quantitation of TCN and TCNP in plasma and RBC. Using a simple protein precipitation method, quantitation of these analytes was accomplished with recoveries exceeding 85% and with a run time of 4 min. This assay was used to determine the pharmacokinetic parameters of TCN and TCNP in mice after single dose intravenous administration at 1, 3 and 10 mg/kg. TCNP accumulates in RBC, has low clearance and a half-life of 18 to 23 h. Unlike other nucleoside phosphates, TCNP was found to be relatively stable in mice plasma serving as a secondary depot. TCN levels were low and with high clearance relative to hepatic blood flow. A combination of sustained levels of TCNP in RBC and plasma serves as a depot for TCN to elicit robust therapeutic activity in acute lung injury mice models.

Orally available stilbene derivatives as potent HDAC inhibitors with antiproliferative activities and antitumor effects in human tumor xenografts.

Herein we report the synthesis and activity of a novel class of HDAC inhibitors based on 2, 3-diphenyl acrylic acid derivatives. The compounds in this series have shown to be potent HDAC inhibitors possessing significant antiproliferative activity. Further compounds in this series were subjected to metabolic stability in human liver microsomes (HLM), mouse liver microsomes (MLM), and exhibits promising stability in both. These efforts culminated with the identification of a developmental candidate (5a), which displayed desirable PK/PD relationships, significant efficacy in the xenograft models and attractive ADME profiles.

An improved method of transcutaneous cisterna magna puncture for cerebrospinal fluid sampling in rats.

A simple, reproducible and chronic technique of cerebrospinal fluid (CSF) collection in rats was developed by direct cisterna magna (CM) puncture utilizing stereotaxic apparatus. CSF collection apparatus was constructed using 1 mL syringe, silicone tubing, 21G disposable needle and water. Animal was placed on an elevated platform over stereotaxic apparatus base and puncture site was identified with the aid of stereotaxic co-ordinates. The volume of CSF collected varied from 100 to 180 µL with mean CSF volume of 150 µL. Neurological deficits were recorded according to the modified Bederson's scoring system 24h post CSF collection and differential cell count in CSF samples was performed. Animals continued to be normal with regular feed intake and gained body weight (∼24%) even after repeated sampling for four weeks and showed no severe neurological deficits (mean Bederson score<1 for four weeks). Neuropharmacokinetic data for Phenytoin sodium, MS 275 and Valproic acid (VPA) demonstrated CSF uptake with CSF(AUC)/plasma(AUC) ratio (K(p,CSF)) of 0.09, 0.01 and 0.33, respectively. This model exemplifies the 3R's of animal use and has been successfully implemented at Orchid Chemicals and Pharmaceuticals Limited for lead optimization of CNS penetrating HDAC inhibitors.

Therapeutic targeting of STAT3 (signal transducers and activators of transcription 3) pathway inhibits experimental autoimmune uveitis.

Mice with targeted deletion of STAT3 in CD4(+) T-cells do not develop experimental autoimmune uveitis (EAU) or experimental autoimmune encephalomyelitis (EAE), in part, because they cannot generate pathogenic Th17 cells. In this study, we have used ORLL-NIH001, a small synthetic compound that inhibits transcriptional activity of STAT3, to ameliorate EAU, an animal model of human posterior uveitis. We show that by attenuating inflammatory properties of uveitogenic lymphocytes, ORLL-NIH001 inhibited the recruitment of inflammatory cells into the retina during EAU and prevented the massive destruction of the neuroretina caused by pro-inflammatory cytokines produced by the autoreactive lymphocytes. Decrease in disease severity observed in ORLL-NIH001-treated mice, correlated with the down-regulation of α4ß1 and α4ß7 integrin activation and marked reduction of CCR6 and CXCR3 expression, providing a mechanism by which ORLL-NIH001 mitigated EAU. Furthermore, we show that ORLL-NIH001 inhibited the expansion of human Th17 cells, underscoring its potential as a drug for the treatment of human uveitis. Two synthetic molecules that target the Th17 lineage transcription factors, RORγt and RORα, have recently been suggested as potential drugs for inhibiting Th17 development and treating CNS inflammatory diseases. However, inhibiting STAT3 pathways completely blocks Th17 development, as well as, prevents trafficking of inflammatory cells into CNS tissues, making STAT3 a more attractive therapeutic target. Thus, use of ORLL-NIH001 to target the STAT3 transcription factor, thereby antagonizing Th17 expansion and expression of proteins that mediate T cell chemotaxis, provides an attractive new therapeutic approach for treatment of posterior uveitis and other CNS autoimmune diseases mediated by Th17 cells.

Assessment of the renal toxicity of novel anti-inflammatory compounds using cynomolgus monkey and human kidney cells.

PF1, an anti-inflammatory drug candidate, was nephrotoxic in cynomolgus monkeys in a manner that was qualitatively comparable to that observed with the two previous exploratory drug candidates (PF2and PF3). Based on the severity of nephrotoxicity, PF1 ranked between the other two compounds, withPF2 inducing mortality at all doses and PF3 eliciting only mild nephrotoxicity. To further characterize nephrotoxicity in monkeys and enable direct comparisons with humans, primary cultures of proximal tubular (PT) cells from monkey and human kidneys were used as in vitro tools, using lactate dehydrogenase release as the biomarker of cytotoxicity. In both human and monkey PT cells, PF2was by far the most cytotoxic compound of the three drugs. PF1 exhibited modest cytotoxicity at the highest concentration tested in human PT cells but none in monkey kidney cells whereas PF3 exhibited the reverse pattern.Because these drugs are organic anions, mechanistic studies using human organic anion transporters 1 and 3 (hOAT1 andhOAT3) transfected cell lines were pursued to evaluate the potential of these compounds to interact with these transporters. All three drugs exhibited high affinity for hOAT3 (PF1 exhibited the lowest IC50 of 6M) but only weakly interacted with hOAT1 (with no interaction found for PF2). PF2 was a strong hOAT3 (not hOAT1) substrate, whereas PF1 and PF3 were substrates for both hOAT1 and hOAT3.Upon pretreatment of monkeys with the OAT substrate probenecid, PF3 systemic exposure (AUC) and half-life (t1/2) increased approximately 2-fold whereas clearance (CL) and volume of distribution (Vdss) decreased, as compared to naïve monkeys. This indicated that PF3 competed with probenecid for hOAT1 and/or hOAT3mediated elimination of PF3. Thus, hOAT1 and/or hOAT3 may be responsible for the uptake of this series of drugs in renal PT cells, which may directly or indirectly lead to the observed nephrotoxicity in vivo.

A novel method for assessing inhibition of ibuprofen chiral inversion and its application in drug discovery.

An inhibition assay to assess the potential for chiral inversion of compounds was developed using R(-)-ibuprofen as the probe substrate. Inhibition of the chiral inversion of R(-)-ibuprofen by structurally similar compounds in cyropreserved rat hepatocytes was studied using chiral HPLC and LC/MS methods for the chromatographic separation and detection of enantiomers. Concept validation of this assay was performed with three commercially available compounds and four Pfizer compounds. The results of these studies demonstrated that compounds that are structurally similar to ibuprofen inhibited the formation of S(+)-ibuprofen, suggesting that they may undergo similar enzymatic chiral inversion pathways or compete for the same enzyme active sites. Additionally, an application of this assay in early drug discovery for a specific class of compounds was demonstrated. Thirty-three in-house compounds were screened for their chiral inversion potential utilizing this assay to investigate the structure activity relationship (SAR) for this class of compounds.

The road map to oral bioavailability: an industrial perspective.

Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnology industries. The number of potential drug candidates requiring in vivo evaluation has significantly increased with the advent of combinatorial chemistry. In addition, drug discovery programmes are increasingly forced into more lipophilic and lower solubility chemical space. To aid in the use of in vitro and in silico tools as well as reduce the number of in vivo studies required, a team-based discussion tool is proposed that provides a 'road map' to guide the selection of profiling assays that should be considered when optimising oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metabolism. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal - acceptable oral bioavailability using minimal resources in an acceptable time frame.

Evaluation of an integrated in vitro-in silico PBPK (physiologically based pharmacokinetic) model to provide estimates of human bioavailability.

PK express module is a physiologically based model of first pass metabolism, which integrates in vitro data with an in silico physiologically based pharmacokinetic (PBPK) model to predict human bioavailability (F(H)). There are three required inputs: FDp (Fraction dose absorbed, final parameter from iDEA absorption module), protein binding (fu) and disappearance kinetics in human hepatocytes. Caco-2 permeability, aqueous solubility (at multiple pH's), estimated dose and chemical structure are inputs required for the estimation of FDp (Norris et al., 2000; Stoner et al., 2004) and were determined for all compounds in our laboratory or obtained from literature. Protein binding data was collected from literature references and/or Pfizer database. Human hepatocyte data was generated in-house using an automated human hepatocyte method (using Tecan Genesis Workstation) as described previously (). Sixteen compounds (commercial and Pfizer compounds) were chosen to evaluate the PK express model and the bioavailability predicted from the module was compared with known clinical endpoints. For majority of the 16 compounds (approximately 80%), the PK express model F(H) values were comparable to the known human bioavailability (F(H)) (within 23.7 units of the known human (true) F, except for PF 3, PF 4, PF 6). In conclusion, the PK express model integrates a number of key readily available discovery parameters and provides estimates of human performance by integrating in silico and experimental variables built on a physiological based pharmacokinetic model. Information from this model in conjunction with other ADME data (e.g., P450 inhibition) will enable progression of most promising compounds for further in vivo PK and/or efficacy studies.

Validation of a semi-automated human hepatocyte assay for the determination and prediction of intrinsic clearance in discovery.

An automated high throughput human hepatocyte assay has been established with a 96-well format using a Tecan Genesistrade mark Workstation. Validation of this assay was performed with nine commercially available compounds and an additional 10 Pfizer compounds with varying hepatic extraction ratios (E(H)) ranging from 0.02 to approximately 1. The incubation conditions in the automated assay are readily and precisely controlled and cell viability of over 80% was achieved in the automated assay further confirming its utility for absorption, distribution, metabolism, and excretion (toxicity) (ADME (T)) screening. The results of the nine commercial compounds correlate with both manually executed (R(2)=0.97) and literature reported experimental results (R(2)=0.93). Overall, measured E(H)s were within two-fold of the literature values for approximately 90% of the 19 compounds tested. Additionally, good inter- and intra-day reproducibility was observed for all the 19 compounds. In conclusion, an automated and robust assay suitable for simultaneously testing up to 48 compounds with multiple time points has been validated. Throughput of 192 compounds per run can be achieved using 384-well plates to meet increasing needs in drug discovery. Currently, this automated assay is used to support early discovery profiling towards lead optimization of various discovery targets/programs.

Implementation of an ADME enabling selection and visualization tool for drug discovery.

The pharmaceutical industry has large investments in compound library enrichment, high throughput biological screening, and biopharmaceutical (ADME) screening. As the number of compounds submitted for in vitro ADME screens increases, data analysis, interpretation, and reporting will become rate limiting in providing ADME-structure-activity relationship information to guide the synthetic strategy for chemical series. To meet these challenges, a software tool was developed and implemented that enables scientists to explore in vitro and in silico ADME and chemistry data in a multidimensional framework. The present work integrates physicochemical and ADME data, encompassing results for Caco-2 permeability, human liver microsomal half-life, rat liver microsomal half-life, kinetic solubility, measured log P, rule of 5 descriptors (molecular weight, hydrogen bond acceptors, hydrogen bond donors, calculated log P), polar surface area, chemical stability, and CYP450 3A4 inhibition. To facilitate interpretation of this data, a semicustomized software solution using Spotfire was designed that allows for multidimensional data analysis and visualization. The solution also enables simultaneous viewing and export of chemical structures with the corresponding ADME properties, enabling a more facile analysis of ADME-structure-activity relationship. In vitro and in silico ADME data were generated for 358 compounds from a series of human immunodeficiency virus protease inhibitors, resulting in a data set of 5370 experimental values which were subsequently analyzed and visualized using the customized Spotfire application. Implementation of this analysis and visualization tool has accelerated the selection of molecules for further development based on optimum ADME characteristics, and provided medicinal chemistry with specific, data driven structural recommendations for improvements in the ADME profile.

Integrated oral bioavailability projection using in vitro screening data as a selection tool in drug discovery.

The objective of the analysis described herein is to examine the in vitro/in vivo relationship of estimated bioavailability values and also the applicability of the estimated in vitro bioavailability to lead candidate selection in drug discovery. To this end, in vitro ADME data from screening assays as well as in vivo rat pharmacokinetic (PK) data were compiled for 140 compounds across therapeutic areas from the Pfizer library in Ann Arbor. The compounds span a broad range of structural types, including neutral, basic, and acidic compounds. Solubility and Caco-2 permeability data from in vitro ADME screening were used to calculate the fraction dose absorbed (FDp) using the physiologically based IDEA model. In vitro metabolic stability (t(1/2)) from human and rat liver microsomal incubations was converted to an in vitro intrinsic clearance value (CL(int)'), which was then scaled up to reflect in vivo clearance (CL) and hepatic extraction as described by Obach et al. [J. Pharmcol. Exp. Ther. 283 (1997) 46]. Subsequently, the in vitro/in vivo relationship between the measured bioavailability (F(obs)) in rats and the estimated bioavailability (F(est)) from FDp and predicted CL values was examined. The observed data suggest that compounds with low estimated in vitro bioavailability (F(est)<15%) are more likely to have low in vivo bioavailability (F(obs)<30%). Therefore, the present study indicates that in vitro estimation of bioavailability is an efficient tool to eliminate compounds having low bioavailability prior to in vivo characterization and therefore can be used to reduce attrition due to poor ADME properties in drug development.

Improved intestinal transport of PD 158473, an N-methyl-D-aspartate (NMDA) antagonist, by involvement of multiple transporters.

The objective of this research was to determine the characteristics of intestinal transport of PD 0158473 using in vitro Caco-2 cells as well as in situ single-pass rat intestinal perfusion models. Because apical (AP)-to-basolateral (BL) transport was greater (1.8- fold) than BL-to-AP transport in the Caco-2 cell model, identification of carrier system(s) involved was further investigated. Cellular uptake of PD 0158473 was concentration and/or pH dependent and significantly inhibited by substrates of dipeptide and monocarboxylic acid transporters. Although this compound is an analog of L-Phe and previous studies demonstrated a high affinity of this compound to large neutral amino acid transporter, the involvement of amino acid carriers did not appear to be significant in the Caco-2 cell model. Subsequently, in situ single-pass intestinal perfusion studies in rats demonstrated that intestinal absorption of PD 0158473 was not concentration dependent at a concentration range tested but significantly inhibited by various dipeptides as well as substrates of dipeptide transporters. The difference in the concentration-dependent transport of PD 0158473 between Caco-2 cells and the rat perfusion model could be explained by the difference in the affinity (apparent K(m)) of PD 0158473 between Caco-2 cells (107 microM) and rat tissue (>1 mM). The present study suggested that multiple transporters are involved in the transcellular transport of this amino-acid analogue compound, of which peptide transporters could play a major role.