The Potentially Significant Role of CYP3A-Mediated Oxidative Metabolism of Dabigatran Etexilate and Its Intermediate Metabolites in Drug-Drug Interaction Assessments Using Microdose Dabigatran Etexilate.

Dabigatran etexilate (DABE), a double ester prodrug of dabigatran, is a probe substrate of intestinal P-glycoprotein (P-gp) commonly used in clinical drug-drug interaction (DDI) studies. When compared with its therapeutic dose at 150 mg, microdose DABE (375 µg) showed approximately 2-fold higher in DDI magnitudes with CYP3A/P-gp inhibitors. In this study, we conducted several in vitro metabolism studies to demonstrate that DABE, at a theoretical gut concentration after microdosing, significantly underwent NADPH-dependent oxidation (~40%-50%) in parallel to carboxylesterase-mediated hydrolysis in human intestinal microsomes. Furthermore, NADPH-dependent metabolism of its intermediate monoester, BIBR0951, was also observed in both human intestinal and liver microsomes, accounting for 100% and 50% of total metabolism, respectively. Metabolite profiling using high resolution mass spectrometry confirmed the presence of several novel oxidative metabolites of DABE and of BIBR0951 in the NADPH-fortified incubations. CYP3A was identified as the major enzyme catalyzing the oxidation of both compounds. The metabolism of DABE and BIBR0951 was well described by Michaelis-Menten kinetics, with Km ranging 1-3 µM, significantly below the expected concentrations following the therapeutic dose of DABE. Overall, the present results suggested that CYP3A played a significant role in the presystemic metabolism of DABE and BIBR0951 following microdose DABE administration, thus attributing partly to the apparent overestimation in the DDI magnitude observed with the CYP3A/P-gp inhibitors. Therefore, DABE at the microdose, unlike the therapeutic dose, would likely be a less predictive tool and should be considered as a clinical dual substrate for P-gp and CYP3A when assessing potential P-gp-mediated impacts by dual CYP3A/P-gp inhibitors. SIGNIFICANT STATEMENT: This is the first study demonstrating a potentially significant role of cytochrome P450-mediated metabolism of the prodrug DABE following a microdose but not a therapeutic dose. This additional pathway, coupled with its susceptibility to P-glycoprotein (P-gp), may make DABE a clinical dual substrate for both P-gp and CYP3A at a microdose. The study also highlights the need for better characterization of the pharmacokinetics and metabolism of a clinical drug-drug interaction probe substrate over the intended study dose range for proper result interpretations.

In vitro and in vivo characterisation of the metabolism and disposition of suvorexant in humans.

1. Suvorexant (MK-4305, Belsomra®) is a first-in-class dual orexin receptor antagonist approved in the USA and Japan for the treatment of insomnia. The current studies describe suvorexant's absorption, disposition and potential for CYP-mediated drug interactions in humans. 2. Following single oral administration of [(14)C]suvorexant to healthy human subjects, 90% of the radioactivity was recovered (66% in faeces, 23% in urine), primarily as oxidative metabolites. 3. In plasma, suvorexant and M9 were predominant, accounting for 30 and 37% of the total radioactivity, respectively. Metabolite M17 became more prominent (approaching 10%) following multiple daily doses of unlabelled suvorexant. M9 and M17 are not expected to contribute to the pharmacological activity of suvorexant due to reduced orexin receptor binding affinity and limited brain penetration. 4. CYP3A was determined to be the predominant enzyme mediating suvorexant oxidation. In vitro, suvorexant demonstrated reversible inhibition of CYP3A4 and 2C19 (IC50 ∼ 4-5 µM), and weak time-dependent inhibition of CYP3A4 (KI = 12 µM, kinact = 0.14 min(-1)). Suvorexant was also a weak inducer of CYP3A4, 1A2 and 2B6. Given the low plasma concentrations at clinical doses, suvorexant was not anticipated to cause significant drug interactions via inhibition and/or induction of major CYPs in vivo.

Discovery of piperidine ethers as selective orexin receptor antagonists (SORAs) inspired by filorexant.

Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.

Discovery of diazepane amide DORAs and 2-SORAs enabled by exploration of isosteric quinazoline replacements.

Dual orexin receptor antagonists (DORAs), or orexin 1 (OX1) and orexin 2 (OX2) receptor antagonists, have demonstrated clinical utility for the treatment of insomnia. Medicinal chemistry efforts focused on the reduction of bioactivation potential of diazepane amide 1 through the modification of the Western heterocycle resulted in the discovery of suvorexant, a DORA recently approved by the FDA for the treatment of insomnia. A second strategy towards reducing bioactivation risk is presented herein through the exploration of monocyclic quinazoline isosteres, namely substituted pyrimidines. These studies afforded potent DORAs with significantly reduced bioactivation risk and efficacy in rodent sleep models. Surprisingly, side products from the chemistry used to produce these DORAs yielded isomeric pyrimidine-containing diazepane amides possessing selective OX2R antagonist (2-SORA) profiles. Additional exploration of these isomeric pyrimidines uncovered potent 2-SORA diazepane amides with sleep efficacy in mouse EEG studies.

Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin.

AIMS: Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion-transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. METHODS: The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. RESULTS: Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration-time curve were 5.7- and 7.6-fold for pitavastatin and 4.4- and 3.3-fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. CONCLUSIONS: The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose.

siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Discovery of dual orexin receptor antagonists with rat sleep efficacy enabled by expansion of the acetonitrile-assisted/diphosgene-mediated 2,4-dichloropyrimidine synthesis.

Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX1R and OX2R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.

Discovery of MK-3697: a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia.

Orexin receptor antagonists have demonstrated clinical utility for the treatment of insomnia. The majority of clinical efforts to date have focused on the development of dual orexin receptor antagonists (DORAs), small molecules that antagonize both the orexin 1 and orexin 2 receptors. Our group has recently disclosed medicinal chemistry efforts to identify highly potent, orally bioavailable selective orexin 2 receptor antagonists (2-SORAs) that possess acceptable profiles for clinical development. Herein we report additional SAR studies within the 'triaryl' amide 2-SORA series focused on improvements in compound stability in acidic media and time-dependent inhibition of CYP3A4. These studies resulted in the discovery of 2,5-disubstituted isonicotinamide 2-SORAs such as compound 24 that demonstrated improved stability and TDI profiles as well as excellent sleep efficacy across species.

Preclinical characterization of a novel potent core protein assembly modulator for the treatment of chronic hepatitis B viral infection.

The hepatitis B virus (HBV) capsid or core protein is a promising drug target currently being investigated for potential curative therapies for chronic HBV infection. In this study, we performed extensive in vitro and in vivo characterization of a novel and potent HBV core protein assembly modulator (CpAM), CU15, for both anti-HBV activity and druggability properties. CU15 potently inhibited HBV DNA replication in in vitro HBV-infected HepG2.2.15 cells (EC50 of 8.6 nM), with a low serum shift. It was also effective in inhibiting HBV DNA and cccDNA formation in de novo HBV-infected primary human hepatocytes. Furthermore, CU15 was active across several HBV genotypes and across clinically relevant core protein variants. After oral administration to an in vivo HBV mouse model, CU15 significantly reduced plasma HBV DNA and RNA levels, at plasma exposure consistent with the estimated in vitro potency. In vitro, CU15 exhibited excellent passive permeability and relatively high metabolic stability in liver preparations across species (human > dog> rat). In vitro human liver microsomal studies suggest that the compound's major metabolic pathway is CYP3A-mediated oxidation. Consistent with the in vitro findings, CU15 is a compound with a low-to-moderate clearance and high oral bioavailability in rats and dogs. Based on the apparent in vitro-in vivo correlation observed, CU15 has the potential to exhibit low clearance and high oral bioavailability in humans. In addition, CU15 also showed low drug-drug interaction liability with an acceptable in vitro safety profile (IC50 > 10 µM).

Assessing the relative contribution of CYP3A-and P-gp-mediated pathways to the overall disposition and drug-drug interaction of dabigatran etexilate using a comprehensive mechanistic physiological-based pharmacokinetic model.

Dabigatran etexilate (DABE) is a clinical probe substrate for studying drug-drug interaction (DDI) through an intestinal P-glycoprotein (P-gp). A recent in vitro study, however, has suggested a potentially significant involvement of CYP3A-mediated oxidative metabolism of DABE and its intermediate monoester BIBR0951 in DDI following microdose administration of DABE. In this study, the relative significance of CYP3A- and P-gp-mediated pathways to the overall disposition of DABE has been explored using mechanistic physiologically based pharmacokinetic (PBPK) modeling approach. The developed PBPK model linked DABE with its 2 intermediate (BIBR0951 and BIBR1087) and active (dabigatran, DAB) metabolites, and with all relevant drug-specific properties known to date included. The model was successfully qualified against several datasets of DABE single/multiple dose pharmacokinetics and DDIs with CYP3A/P-gp inhibitors. Simulations using the qualified model supported that the intestinal CYP3A-mediated oxidation of BIBR0951, and not the gut P-gp-mediated efflux of DABE, was a key contributing factor to an observed difference in the DDI magnitude following the micro-versus therapeutic doses of DABE with clarithromycin. Both the saturable CYP3A-mediated metabolism of BIBR0951 and the solubility-limited DABE absorption contributed to the relatively modest nonlinearity in DAB exposure observed with increasing doses of DABE. Furthermore, the results suggested a limited role of the gut P-gp, but an appreciable, albeit small, contribution of gut CYP3A in mediating the DDIs following the therapeutic dose of DABE with dual CYP3A/P-gp inhibitors. Thus, a possibility exists for a varying extent of CYP3A involvement when using DABE as a clinical probe in the DDI assessment, across DABE dose levels.

In vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters.

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.

The impact of glycosylation on the pharmacokinetics of a TNFR2:Fc fusion protein expressed in Glycoengineered Pichia Pastoris.

PURPOSE: P. pastoris has previously been genetically engineered to generate strains that are capable of producing mammalian-like glycoforms. Our objective was to investigate the correlation between sialic acid content and pharmacokinetic properties of recombinant TNFR2:Fc fusion proteins generated in glycoengineered P. pastoris strains. METHODS: TNFR2:Fc fusion proteins were generated with varying degrees of sialic acid content. The pharmacokinetic properties of these proteins were assessed by intravenous and subcutaneous routes of administration in rats. The binding of these variants to FcRn were also evaluated for possible correlations between in vitro binding and in vivo PK. RESULTS: The pharmacokinetic profiles of recombinant TNFR2:Fc produced in P. pastoris demonstrated a direct positive correlation between the extent of glycoprotein sialylation and in vivo pharmacokinetic properties. Furthermore, recombinant TNFR2:Fc produced in glycoengineered Pichia, with a similar sialic acid content to CHO-produced etanercept, demonstrated similar in vivo pharmacokinetic properties to the commercial material. In vitro surface plasmon resonance FcRn binding at pH6.0 showed an inverse relationship between sialic acid content and receptor binding affinity, with the higher affinity binders having poorer in vivo PK profiles. CONCLUSIONS: Sialic acid content is a critical attribute for modulating the pharmacokinetics of recombinant TNFR2:Fc produced in glycoengineered P. pastoris.

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs).

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX1R/OX2R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.

Binding characteristics of pyrrole-scaffold hepatitis B virus capsid inhibitors and identification of novel potent compounds.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) are currently being evaluated in clinical trials as potential curative therapies for HBV. This study used in silico computational modeling to provide insights into the binding characteristics between the HBV core protein and two pyrrole-scaffold inhibitors, JNJ-6379 and GLP-26, both in the CAM-Normal (CAM-N) series. Molecular dynamics simulations showed that the pyrrole inhibitors displayed similar general binding-interaction patterns to NVR 3-778, another CAM-N, with hydrophobic interactions serving as the major driving force. However, consistent with their higher potency, the pyrrole inhibitors exhibited stronger nonpolar interactions with key residues in a solvent-accessible region as compared to NVR 3-778. This feature was facilitated by distinct hydrogen bond interactions of the pyrrole scaffold inhibitors with the residue 140 in chain B of the HBV core protein (L140B). Based on these findings, novel CAM-N compounds were designed to mimic the interaction with L140B residue while maximizing nonpolar interactions in the solvent-accessible region. Several 1H-pyrrole-2-carbonyl substituted pyrrolidine-based compounds with various hydrophobic side chains were synthesized and evaluated. Through analyses of the structure-activity and structure-druggability relations of a series of compounds, CU15 emerged as the most promising lead CAM-N compound, exhibiting sub-nanomolar potency and good pharmacokinetic profiles. Overall, the study demonstrated a practical approach to leverage computational methods for understanding key target binding features for rationale-based design, and for guiding the identification of novel compounds.

Lymphatic transport and catabolism of therapeutic proteins after subcutaneous administration to rats and dogs.

The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ∼70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph. In both LDC and intact rats, the total radioactivity recoveries in excreta after subcutaneous administration were high (70-80%), indicating that catabolism, not poor absorption, was the main cause for the observed low bioavailability (30-40%). Moreover, catabolism of PEGylated EPO was found with both rat subcutaneous tissue homogenate and lymph node cell suspensions, and a significant amount of dose-related breakdown fragments was found in the lymph of LDC rats. In addition, the bioavailability of PEGylated EPOs was shown to be 2- to 4-fold lower in "fat rats," indicating that physiologic features pertinent to lymphatic transport can have a profound impact on subcutaneous absorption. Limited studies in dogs also suggested similar subcutaneous absorption mechanisms. Collectively, our results suggest that the lymphatic absorption mechanism for macromolecules is probably conserved among commonly used preclinical species, e.g., rats and dogs, and that mechanistic understanding of the subcutaneous absorption mechanism and associated determinants should be helpful in biologic drug discovery and development.

Discovery of the selective androgen receptor modulator MK-0773 using a rational development strategy based on differential transcriptional requirements for androgenic anabolism versus reproductive physiology.

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

Monoclonal antibodies with identical Fc sequences can bind to FcRn differentially with pharmacokinetic consequences.

The neonatal Fc receptor (FcRn) is a key determinant of IgG homeostasis. It binds to the Fc domain of IgG in a strictly pH-dependent manner and protects IgG from lysosomal degradation. The impact of FcRn salvage pathway on IgG monoclonal antibody (mAb) pharmacokinetics (PK) has been well established. In this report, a set of mAbs with wild-type human Fc sequences but different Fab domains were used to examine the potential impact of Fab domain on in vitro FcRn binding and in vivo PK. We were surprised to find that mAbs with the same wild-type human Fc sequences but different Fab domains were shown to bind FcRn with considerable differences in both the binding at acidic pH and the dissociation at neutral pH, suggesting that the Fab domain may also have an impact on FcRn interaction. For these mAbs, no relationship between the FcRn binding affinity at acidic pH and in vivo PK was found. Instead, an apparent correlation between the in vitro FcRn dissociation at neutral pH and the in vivo PK in human FcRn mice, nonhuman primates and humans was observed. Our results suggested that the Fab domain of mAbs can affect their interaction with FcRn and thus their pharmacokinetic properties and that in vitro FcRn binding/dissociation assays can be a useful screening tool for pharmacokinetic assessment of mAbs with wild-type Fc sequences.

Theoretical analysis of interplay of therapeutic protein drug and circulating soluble target: temporal profiles of 'free' and 'total' drug and target.

PURPOSE: To systemically investigate, for a therapeutic protein with a circulating soluble target, how the interplay of target dynamics and drug pharmacokinetics defines the 'total' and 'free' drug and target temporal profiles. METHOD: By extending the established rapid-binding target-mediated drug disposition (TMDD) pharmacokinetic model to circulating soluble targets, the temporal profiles of 'total' and 'free' drug and target were simulated with varying binding affinity (K(D)), target baseline (R(ss)), target turnover, and drug dose level. Two sets of published experimental data were compared with the simulated results. RESULTS: Binding to a circulating soluble target could lead to a divergence of the 'free' drug from the 'total' drug. Simulations show this divergent magnitude determined by K(D) and R(ss), with the temporal profile being defined by target turnover and drug dose level. As divergence proceeds, starting at the distribution phase, 'free' drug would decline faster but eventually parallel 'total' drug at the terminal phase, giving rise to a steeper distribution phase and comparable terminal half-life, relative to the 'total' form. The model also allows for estimation of the dynamic change of 'total' and 'free' target in response to the treatment of a therapeutic protein drug, facilitating dose level and regimen design to achieve desired 'free' target suppression. Model predictions compared favorably with two sets of published experimental data. CONCLUSIONS: Theoretical analyses identified key variables governing the different temporal profiles of 'total' and 'free' drug and target. The rapid-binding TMDD model reasonably captured the features of the interplay of drug pharmacokinetics and target dynamics for two reported cases.

Pyridyl amides as potent inhibitors of T-type calcium channels.

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.

Pharmacokinetics of IgG1 monoclonal antibodies produced in humanized Pichia pastoris with specific glycoforms: a comparative study with CHO produced materials.

A glycoengineered Pichia pastoris host was used to produce an IgG1 with either afucosylated N-glycosylation (afucosylated biantennary complex) or without N-glycosylation (N297A) while a wild type P. pastoris host was used to produce an IgG1 containing fungal-type N- and O-linked glycosylation. The PK properties of these antibodies were compared to a commercial IgG1 produced in CHO cells following intravenous administration in wild type C57B6, FcγR-/- or hFcRn transgenic mice. MAbs produced in glycoengineered yeast exhibited similar PK properties in wild type mice or FcγR-/- mice with respect to clearance (CL), volume of distribution at steady-state (Vss) and half-life (t(1/2)) to that produced in mammalian (CHO) cells, while the mAb produced in wild type yeast exhibited ∼2-3-fold faster CL, which might be due to the high mannose content interacting with mannose receptors. Furthermore, in vitro binding affinity to human FcRn or mouse FcRn was similar between the reference mAb and mAbs produced in humanized yeast, and the glycovariants produced in humanized yeast exhibited similar PK patterns in human FcRn transgenic mice and in wild type mice. These results suggest the potential application of P. pastoris as a production platform for clinically viable mAbs.