Mechanistic Determinants of Daprodustat Drug-Drug Interactions and Pharmacokinetics in Hepatic Dysfunction and Chronic Kidney Disease: Significance of OATP1B-CYP2C8 Interplay.

Daprodustat is the first oral hypoxia-inducible factor prolyl hydroxylase inhibitor approved recently for the treatment of anemia caused by chronic kidney disease (CKD) in adults receiving dialysis. We evaluated the role of organic anion transporting polypeptide (OATP)1B-mediated hepatic uptake transport in the pharmacokinetics (PKs) of daprodustat using in vitro and in vivo studies, and physiologically-based PK (PBPK) modeling of its drug-drug interactions (DDIs) with inhibitor drugs. In vitro, daprodustat showed specific transport by OATP1B1/1B3 in the transfected cell systems and primary human and monkey hepatocytes. A single-dose oral rifampin (OATP1B inhibitor) reduced daprodustat intravenous clearance by a notable 9.9 ± 1.2-fold (P < 0.05) in cynomolgus monkeys. Correspondingly, volume of distribution at steady-state was also reduced by 5.0 ± 1.1-fold, whereas the half-life change was minimal (1.5-fold), corroborating daprodustat hepatic uptake inhibition by rifampin. A PBPK model accounting for OATP1B-CYP2C8 interplay was developed, which well described daprodustat PK and DDIs with gemfibrozil (CYP2C8 and OATP1B inhibitor) and trimethoprim (weak CYP2C8 inhibitor) within 25% error of the observed data in healthy subjects. About 18-fold increase in daprodustat area under the curve (AUC) following gemfibrozil treatment was found to be associated with strong CYP2C8 inhibition and moderate OATP1B inhibition. Moreover, PK modulation in hepatic dysfunction and subjects with CKD, in comparison to healthy control, was well-captured by the model. CYP2C8 and/or OATP1B inhibitor drugs (e.g., gemfibrozil, clopidogrel, rifampin, and cyclosporine) were predicted to perpetrate moderate-to-strong DDIs in healthy subjects, as well as, in target CKD population. Daprodustat can be used as a sensitive CYP2C8 index substrate in the absence of OATP1B modulation.

Low molecular weight acids and OATP1B mediated hepatic clearance: In vitro and in vivo evaluation using novel hypoxia-inducible factor prolyl hydroxylase inhibitors (Dustats).

Organic anion transporting polypeptide (OATP1B) plays a key role in the hepatic clearance of a majority of high molecular weight (MW) acids and zwitterions. Here, we evaluated the role of OATP1B-mediated uptake in the clearance of novel hypoxia-inducible factor prolyl hydroxylase inhibitors ("Dustats"), which are typically low MW (300-400 daltons) aliphatic carboxylic acids. Five acid dustats, namely daprodustat, desidustat, enarodustat, roxadustat and vadadustat, showed specific transport by OATP1B1/1B3 in transporter-transfected HEK293 cells. Neutral compound, molidustat, was not a substrate to OATP1B1/1B3. None of the dustats showed transport by other hepatic uptake transporters, including NTCP, OAT2 and OAT7. In the primary human hepatocytes, uptake of all acids was significantly reduced by rifampin (OATP1B inhibitor); with an estimated fraction transported by OATP1B (ft ,OATP1B) of up to >80% (daprodustat). Molidustat uptake was minimally inhibited by rifampin; and low permeability acids (desidustat and enarodustat) also showed biliary efflux in sandwich culture human hepatocytes. In vivo, intravenous pharmacokinetics of all 5 acids was significantly altered by a single-dose rifampin (30 mg/kg) in Cynomolgus monkey. Hepatic clearance (non-renal) was about 4-fold (vadadustat) to >11-fod (daprodustat and roxadustat) higher in control group compared to rifampin-treated subjects. In vivo ft ,OATP1B was estimated to be ~70-90%. In the case of molidustat, rifampin had a minimal effect on overall clearance. Rifampin also considerably reduced volume of distribution of daprodustat and roxadustat. Overall, OATP1B significantly contribute to the hepatic clearance and pharmacokinetics of several dustats, which are low MW carboxylic acids. OATP1B activity should therefore by evaluated in this property space. Significance Statement Our in vitro and in vivo results suggest that OATP1B-mediated hepatic uptake play a significant role in the pharmacokinetics of low MW acidic dustats, which are being developed or approved for the treatment of anemia in chronic kidney disease. Significant active uptake mechanisms are not apparent for the neutral compound, molidustat. Characterization of uptake mechanisms is therefore important in predicting human pharmacokinetics and evaluating drug-drug interactions for low MW acids.

Utilization of Rosuvastatin and Endogenous Biomarkers in Evaluating the Impact of Ritlecitinib on BCRP, OATP1B1, and OAT3 Transporter Activity.

PURPOSE: Ritlecitinib, an inhibitor of Janus kinase 3 and tyrosine kinase expressed in hepatocellular carcinoma family kinases, is in development for inflammatory diseases. This study assessed the impact of ritlecitinib on drug transporters using a probe drug and endogenous biomarkers. METHODS: In vitro transporter-mediated substrate uptake and inhibition by ritlecitinib and its major metabolite were evaluated. Subsequently, a clinical drug interaction study was conducted in 12 healthy adult participants to assess the effect of ritlecitinib on pharmacokinetics of rosuvastatin, a substrate of breast cancer resistance protein (BCRP), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporter 3 (OAT3). Plasma concentrations of coproporphyrin I (CP-I) and pyridoxic acid (PDA) were assessed as endogenous biomarkers for OATP1B1 and OAT1/3 function, respectively. RESULTS: In vitro studies suggested that ritlecitinib can potentially inhibit BCRP, OATP1B1 and OAT1/3 based on regulatory cutoffs. In the subsequent clinical study, coadministration of ritlecitinib decreased rosuvastatin plasma exposure area under the curve from time 0 to infinity (AUCinf) by ~ 13% and maximum concentration (Cmax) by ~ 27% relative to rosuvastatin administered alone. Renal clearance was comparable in the absence and presence of ritlecitinib coadministration. PK parameters of AUCinf and Cmax for CP-I and PDA were also similar regardless of ritlecitinib coadministration. CONCLUSION: Ritlecitinib does not inhibit BCRP, OATP1B1, and OAT3 and is unlikely to cause a clinically relevant interaction through these transporters. Furthermore, our findings add to the body of evidence supporting the utility of CP-I and PDA as endogenous biomarkers for assessment of OATP1B1 and OAT1/3 transporter activity.

Characterization of Bile Acid Sulfate Conjugates as Substrates of Human Organic Anion Transporting Polypeptides.

Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Abrocitinib, a Selective Janus Kinase Inhibitor, in Humans.

Abrocitinib is an oral once-daily Janus kinase 1 selective inhibitor being developed for the treatment of moderate-to-severe atopic dermatitis. This study examined the disposition of abrocitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite (M) profiles. The results indicated abrocitinib had a systemic clearance of 64.2 L/h, a steady-state volume of distribution of 100 L, extent of absorption >90%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 60%. The half-life of both abrocitinib and total radioactivity was similar, with no indication of metabolite accumulation. Abrocitinib was the main circulating drug species in plasma (∼26%), with 3 major monohydroxylated metabolites (M1, M2, and M4) at >10%. Oxidative metabolism was the primary route of elimination for abrocitinib, with the greatest disposition of radioactivity shown in the urine (∼85%). In vitro phenotyping indicated abrocitinib cytochrome P450 fraction of metabolism assignments of 0.53 for CYP2C19, 0.30 for CYP2C9, 0.11 for CYP3A4, and ∼0.06 for CYP2B6. The principal systemic metabolites M1, M2, and M4 were primarily cleared renally. Abrocitinib, M1, and M2 showed pharmacology with similar Janus kinase 1 selectivity, whereas M4 was inactive. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of abrocitinib, a Janus kinase inhibitor for atopic dermatitis, in humans, as well as characterization of clearance pathways and pharmacokinetics of abrocitinib and its metabolites.

Pharmacokinetics, mass balance, metabolism, and excretion of the liver-targeted acetyl-CoA carboxylase inhibitor PF-05221304 (clesacostat) in humans.

The disposition of the hepatoselective ACC inhibitor PF-05221304 (Clesacostat) was studied after a single 50-mg oral dose of [14C]-PF-05221304 to healthy human subjects.Mass balance was achieved with 89.9% of the administered dose recovered in urine and faeces, over the 11-day study period. The total administered radioactivity excreted in faeces and urine was 81.7 and 8.2%, respectively. Unchanged PF-05221304 accounted for 35.6% of the radioactive dose in faeces, suggesting ∼64% of the administered dose was absorbed.PF-05221304 was principally metabolised via oxidative and reductive pathways involving: (a) N-dealkylation, (b) isopropyl group monohydroxylation to yield enantiomeric metabolites (M2a and M2b), (c) hydroxylation on the 3-azaspiro[5.5]undecan-8-one moiety to metabolites M5 and 519c, and (d) carbonyl group reduction to enantiomeric alcohol metabolites M3, and M4. Secondary metabolites (521a, 521b, and 533), derived from a combination of oxidation and reduction of the primary metabolites accounted for ∼14.8% of the dose. In plasma, unchanged PF-05221304 represented 96.1% circulating radioactivity. Metabolites M1, M2b, and M2a represented 1.94, 1.76, and 0.18% of circulating radioactivity, respectively.Overall, these data suggest that PF-05221304 is well absorbed in humans and eliminated largely via phase I metabolism.

Effect of a Ketohexokinase Inhibitor (PF-06835919) on In Vivo OATP1B Activity: Integrative Risk Assessment Using Endogenous Biomarker and a Probe Drug.

PF-06835919 is a first-in-class ketohexokinase inhibitor (KHKi), recently under development for the treatment of metabolic and fatty liver diseases, which inhibited organic anion transporting polypeptide (OATP)1B1 in vitro and presented drug-drug interaction (DDI) risk. This study aims to investigate the dose-dependent effect of KHKi on OATP1B in vivo activity. We performed an open-label study comparing pharmacokinetics of atorvastatin (OATP1B probe) dosed alone (20 mg single dose) and coadministered with two dose strengths of KHKi (50 and 280 mg once daily) in 12 healthy participants. Additionally, changes in exposure of coproporphyrin-I (CP-I), an endogenous biomarker for OATP1B, were assessed in the atorvastatin study (1.12-fold and 1.49-fold increase in area under the plasma concentration-time profile (AUC) with once-daily 50 and 280 mg, respectively), and a separate single oral dose study of KHKi alone (100-600 mg, n = 6 healthy participants; up to a 1.80-fold increase in AUC). Geometric mean ratios (90% confidence interval) of atorvastatin AUC following 50 and 280 mg KHKi were 1.14 (1.00-1.30) and 1.54 (1.37-1.74), respectively. Physiologically-based pharmacokinetic modeling of CP-I plasma exposure following a single dose of KHKi predicted in vivo OATP1B inhibition from about 13% to 70% over the 100 to 600 mg dose range, while using the in vitro inhibition potency (1.9 µM). Model-based analysis correctly predicted "no-effect" (AUC ratio < 1.25) at the low dose range and "weak" effect (AUC ratio < 2) on atorvastatin pharmacokinetics at the high dose range of KHKi. This study exemplified the utility of biomarker-informed model-based approach in discerning even small effects on OATP1B activity in vivo, and to project DDI risk at the clinically relevant doses.

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

The association of antidepressant monotherapy and weight change in a Middle Eastern psychiatric population.

Genetic and lifestyle factors influence weight changes secondary to antidepressants. However, the association between antidepressants and weight changes is seldom studied in Middle Eastern populations who have a unique genetic, lifestyle and dietary profile. This is a chart-based retrospective longitudinal study that describes the 6-month evolution of weight in outpatient Lebanese adults initiated on serotonin reuptake inhibitor (SRI) antidepressant monotherapy, with the exclusion of confounders with metabolic implications. A linear mixed model was used to estimate the association of weight with time. Demographic factors were tested for interaction with the model. The sample consisted of 200 antidepressant trials. After adjusting for baseline weight, age, and sex, weight did not change significantly from week 1 to week 4 (P = 0.530) but significantly increased by 0.15 kg/week from week 4 to week 24 (P < 0.05). Sex, baseline weight, alcohol use, tobacco use, and presence of metabolic condition(s) did not affect weight change (P > 0.05). The same changes were observed across all SRIs and diagnoses. Our results show that SRI monotherapy is associated with weight gain throughout the treatment course in a Middle Eastern population. We recommend judicious prescription of SRIs and clinical vigilance over weight throughout the course of treatment in these populations.

Dose-Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs.

To address the most appropriate endogenous biomarker for drug-drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate-3-glucuronide, glycochenodeoxycholate-3-sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose-dependent change in area under the plasma concentration-time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0-24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR-1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B-mediated drug-drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

Attitudes, beliefs, and knowledge of substance use amongst youth in the Eastern Mediterranean region: A systematic review.

BACKGROUND: Substance use has a tremendous impact on the burden of disease. This is particularly true in the Eastern Mediterranean region (EMR), where many countries serve as suppliers of drugs. As risk perception and frequency of use are inversely correlated, targeting perception during adolescence becomes essential for prevention. In this study, we systematically reviewed the literature on attitudes, beliefs, and knowledge of substance use amongst youth in the EMR. METHODS: We reviewed quantitative articles addressing attitudes, beliefs, and knowledge of youth aged between 13 and 25 years towards substance use in the EMR. We searched MEDLINE, PubMed, Cochrane, PsycInfo, and PsycArticles then applied a duplicate independent method for study selection and screening. Two reviewers completed data abstraction and a narrative summary of findings. RESULTS: Our search generated 12,810 articles. Five cross-sectional studies were eligible (two analytic and three descriptive). The analytic studies described a significant correlation between intention to use and both attitudes and subjective norms. The descriptive studies portrayed a negative attitude towards use with a low threshold for considering it as serious. Beliefs pertaining to reasons for use included stress and sleeping, whereas thoughts on treatment were restricted to traditional methods based on personal resilience and religious support. Knowledge about substance use symptoms, withdrawal, and treatment was low. CONCLUSION: Our review ascertains the role of sociocultural moral prohibition and awareness of mental health as major influencers in shaping the perception of substance use. Further research is needed to elaborate culturally-tailored survey tools.

A fully automated and validated human plasma LC-MS/MS assay for endogenous OATP biomarkers coproporphyrin-I and coproporphyrin-III.

AIM: A validated LC-MS/MS assay for the quantitation of coproporphyrin-I and -III (CP-I, CP-III) in human plasma has been developed to understand the utility of both as possible endogenous biomarkers for organic anion-transporting polypeptides (OATP)-mediated drug-drug interactions (DDIs). MATERIALS AND METHODS:  Human plasma extracts were analyzed for CP-I and CP-III using a Sciex API 6500+ mass spectrometer. Results: The assay was utilized for plasma samples from a clinical DDI study involving a new chemical entity that presented as an OATP inhibitor in vitro. A formal DDI study, with a probe drug (atorvastatin), was also included as part of the clinical study. CONCLUSION: Changes in CP-I area under the plasma concentration versus time curve (AUC0-48 h) were observed, which were similar to the AUC ratio obtained with atorvastatin. These results support the idea that plasma CP-I may have utility in Phase I by supporting the rapid assessment of OATP inhibition risk.

A sensitive method for the quantitation of the peptide-based glucagon-like peptide-1 receptor agonist liraglutide in plasma using microfluidics chromatography tandem MS.

AIM: An LC-MS/MS assay for the quantitation of liraglutide, a peptide-based injectable glucagon-like peptide-1 receptor agonist, has been developed as a convenient alternative to the enzyme-linked immunosorbent assay, and used to characterize liraglutide pharmacokinetics in cynomolgus monkeys. RESULTS: Assay calibration curves exhibited a linear dynamic range of 10-5000 ng/ml and correlation coefficient ≥0.98. Following a 30 µg/kg intravenous dose, liraglutide demonstrated low plasma clearance and distribution volume, which led to a terminal half-life of 6.59 h in monkeys. CONCLUSION: The dynamic range of our LC-MS/MS assay provides sufficient coverage of the average efficacious liraglutide concentrations in human plasma, and can be used for pharmacokinetics/pharmacodynamics studies in animals and potentially in humans.

Introducing Specialist Integrated Mental Health Care in Lebanon: The Psychosis Recovery Outreach Program.

The recognition of mental health needs in developing countries is growing in parallel with increased public awareness of and reduced stigma toward mental illness. With resources still limited in these countries, creating economically viable health care models is essential. These models are often adapted from already established and tested systems in industrialized nations. The Psychosis Recovery Outreach Program (PROP) at the American University of Beirut is an initiative that exemplifies the global transfer of knowledge, experience, and skills. Now in its second year, PROP provides specialist care to individuals with severe mental disorders in Lebanon by integrating psychiatry, psychology, nursing, and social work in a challenging social and cultural context.

Liver-Targeted Small-Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis.

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.

A highly selective and sensitive LC-MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting peptide biomarkers.

AIM: Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug-drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials & methods: Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer. Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.14) were used as precursor ions to improve the assay sensitivity and selectivity over the singly charged precursor ions (m/z 655.28). Levels of CP-I and CP-III measured ranged 0.45-1.1 and 0.050-0.50 ng/ml in plasma and 5-35 and 1-35 ng/ml in urine, respectively. CONCLUSION: The described highly selective and sensitive CP-I/CP-III LC-HRMS assay offers options for earlier characterization and clinical safety projections for OATP1B1/3-mediated drug-drug interactions along with pharmacokinetic analyses of a new chemical entity as part of first-in-human clinical studies.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

In Vitro-In Vivo Extrapolation of Intestinal Availability for Carboxylesterase Substrates Using Portal Vein-Cannulated Monkey.

Prediction of intestinal availability (FaFg) of carboxylesterase (CES) substrates is of critical importance in designing oral prodrugs with optimal properties, projecting human pharmacokinetics and dose, and estimating drug-drug interaction potentials. A set of ester prodrugs were evaluated using in vitro permeability (parallel artificial membrane permeability assay and Madin-Darby canine kidney cell line-low efflux) and intestinal stability (intestine S9) assays, as well as in vivo portal vein-cannulated cynomolgus monkey. In vitro-in vivo extrapolation (IVIVE) of FaFg was developed with a number of modeling approaches, including a full physiologically based pharmacokinetic (PBPK) model as well as a simplified competitive-rate analytical solution. Both methods converged as in the PBPK simulations enterocyte blood flow behaved as a sink, a key assumption in the competitive-rate analysis. For this specific compound set, the straightforward analytical solution therefore can be used to generate in vivo predictions. Strong IVIVE of FaFg was observed for cynomolgus monkey with R2 of 0.71-0.93. The results suggested in vitro assays can be used to predict in vivo FaFg for CES substrates with high confidence.

The role of mode of delivery on elastic fiber architecture and vaginal vault elasticity: a rodent model study.

We report on an experimental study of the role of mode of delivery and pregnancy on the architecture of vaginal elastic fibers and vaginal vault elasticity in female Sprague-Dawley rats. In primiparous rats submitted to spontaneous or Cesarean delivery and virgin rats submitted to simulated delivery, the tortuosity of elastic fibers (defined as the ratio of length to end-to-end distance) was observed to decrease when measured from two days to two weeks postpartum. In addition, the measured tortuosity of elastic fibers in multiparous rats was greater than that of virgin rats. The tortuosity of elastic fibers of all rats measured at two days postpartum was found to be similar to that of multiparous rats. At two weeks postpartum the measured tortuosity of vaginal elastic fibers was indistinguishable from virgin rats, regardless of the delivery method. Borrowing from the field of polymer physics, a model is suggested that connects elastic fiber tortuosity to the resulting tension under an applied stress; fibers having high tortuosity are expected to provide less structural support than more linear, low tortuosity fibers. To probe the macroscopic effects in elasticity due to architectural changes observed in elastic fibers, we have measured the stiffness of the vaginal vault in each cohort using a pressure-infusion system. The vaginal vault stiffness of all primiparous rats measured two weeks postpartum was greater than that measured two days postpartum. In addition, the vaginal vault of virgin rats was stiffer than that of multiparous rats. These observations confirmed that vaginal vault elastic fibers undergo significant remodeling due to pregnancy and parturition, and that the complex remodeling may be a significant contributor to tissue elasticity. Remarkably, regardless of the mode of delivery or simulated tissue trauma, elastic fiber tortuosity is observed to decrease from two days to two weeks postpartum indicating the onset of repair and recovery of tissue stiffness.