Current Approaches for Predicting Human PK for Small Molecule Development Candidates: Findings from the IQ Human PK Prediction Working Group Survey.

Accurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry. The survey revealed a heterogeneity in approaches being used across the industry (e.g., the use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development. The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets.

Application of a high-resolution in vitro human MDR1-MDCK assay and in vivo studies in preclinical species to improve prediction of CNS drug penetration.

P-glycoprotein (P-gp, MDR1) is expressed at the blood-brain barrier (BBB) and restricts penetration of its substrates into the central nervous system (CNS). In vitro MDR1 assays are frequently used to predict the in vivo relevance of MDR1-mediated efflux at the BBB. It has been well established that drug candidates with high MDR1 efflux ratios (ERs) display poor CNS penetration. Following a comparison of MDR1 transporter function between the MDR1-MDCKI cell line from National Institutes of Health (NIH) and our internal MDR1-MDCKII cell line, the former was found to provide better predictions of in vivo brain penetration than our in-house MDR1-MDCKII cell line. In particular, the NIH MDR1 assay has an improved sensitivity to differentiate the compounds with ERs of <3 in our internal cell line and is able to reduce the risk of false negatives. A better correlation between NIH MDR1 ERs and brain penetration in rat and non-human primate (NHP) was demonstrated. Additionally, a comparison of brain penetration time course of MDR1 substrates and an MDR1 non-substrate in NHP demonstrated that MDR1 interaction can delay the time to equilibrium of drug concentration in the brain with plasma. It is recommended to select highly permeable compounds without MDR1 interaction for rapid brain penetration to produce the maximal pharmacological effect in the CNS with a quicker onset.

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Development of a small molecule that corrects misfolding and increases secretion of Z α1 -antitrypsin.

Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency.

The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation.

BACKGROUND: Antenatal corticosteroids (ACS) are the standard of care for maturing the fetal lung and improving outcomes for preterm infants. Antenatal corticosteroid dosing remains nonoptimized, and there is little understanding of how different treatment-to-delivery intervals may affect treatment efficacy. The durability of a lung maturational response is important because the majority of women treated with antenatal corticosteroids do not deliver within the widely accepted 1- to 7-day window of treatment efficacy. OBJECTIVE: We used a sheep model to test the duration of fetal exposures for efficacy at delivery intervals from 1 to 10 days. MATERIALS AND METHODS: For infusion studies, ewes with single fetuses were randomized to receive an intravenous bolus and maintenance infusion of betamethasone phosphate to target 1-4 ng/mL fetal plasma betamethasone for 36 hours, with delivery at 2, 4 ,or 7 days posttreatment or sterile saline solution as control. Animals receiving the clinical treatment were randomised to receive either a single injection of 0.25 mg/kg with a 1:1 mixture of betamethasone phosphate + betamethasone acetate with delivery at either 1 or 7 days posttreatment, or 2 treatments of 0.25 mg/kg betamethasone phosphate + betamethasone acetate spaced at 24 hours (giving ∼48 hours of fetal steroid exposure) with delivery at 2, 5, 7, or 10 days posttreatment. Negative control animals were treated with saline solution. All lambs were delivered at 121 ± 3 days gestational age and ventilated for 30 minutes to assess lung function. RESULTS: Preterm lambs delivered at 1 or 2 days post-antenatal corticosteroid treatment had significant improvements in lung maturation for both intravenous and single-dose intramuscular treatments. After 2 days, the efficacy of 36-hour betamethasone phosphate infusions was lost. The single dose of 1:1 betamethasone phosphate + betamethasone acetate also was ineffective at 7 days. In contrast, animals treated with 2 doses had significant improvements in lung maturation at 2, 5, and 7 days, with treatment efficacy reduced by 10 days. CONCLUSION: In preterm lambs, the durability of antenatal corticosteroids treatment depends on the duration of fetal exposure and is independent of the intravenous or intramuscular maternal route of administration. For acute 24- to 48-hour posttreatment deliveries, a 24-hour fetal antenatal corticosteroids exposure was sufficient for lung maturation. A fetal exposure duration of at least 48 hours was necessary to maintain long-term treatment durability. A single-dose ACS treatment should be sufficient for women delivering within <48 hours of antenatal corticosteroids treatment.

The efficacy of antenatal steroid therapy is dependent on the duration of low-concentration fetal exposure: evidence from a sheep model of pregnancy.

BACKGROUND: Antenatal corticosteroids are among the most important and widely used interventions to improve outcomes for preterm infants. Antenatal corticosteroid dosing regimens remain unoptimized and without maternal weight-adjusted dosing. We, and others, have hypothesized that, once a low concentration of maternofetal steroid exposure is achieved and maintained, the duration of the steroid exposure determines treatment efficacy. Using a sheep model of pregnancy, we tested the relationship among steroid dose, duration of exposure, and treatment efficacy. OBJECTIVE: The study was conducted to investigate the relative importance of duration and magnitude of fetal corticosteroid exposure to mature the preterm fetal ovine lung. STUDY DESIGN: Ewes with single fetuses at 120 days gestation received an intravenous bolus (loading dose) followed by a maintenance infusion of betamethasone phosphate to target 12-hour fetal plasma betamethasone concentrations of (1) 20 ng/mL, (2) 10 ng/mL, or (3) 2 ng/mL. In a subsequent experiment, fetal plasma betamethasone concentrations were targeted at 2 ng/mL for 26 hours. Negative control animals received sterile saline solution. Positive control animals received 2 intramuscular injections of 0.25 mg/kg Celestone Chronodose (betamethasone phosphate + betamethasone acetate) spaced at 24 hours. Preterm lambs were delivered surgically and ventilated 48 hours after treatment commenced. Maternal and fetal plasma betamethasone concentrations were confirmed by mass spectrometry in a parallel study of chronically catheterized, corticosteroid-treated ewes and fetuses. RESULTS: The loading and maintenance doses were achieved and maintained the desired fetal plasma betamethasone concentrations of approximately 20, 10, and 2 ng/mL for 12 hours. Compared with the 12-hour infusion-treated animals, lambs from the positive control (2 intramuscular doses of 0.25 mg/kg Celestone Chronodose) group had the greatest functional lung maturation (compliance, gas exchange, arterial pH) and molecular evidence of maturation (glucocorticoid receptor signaling activation), despite having maximum fetal plasma betamethasone concentrations 2.5 times lower than animals in the 20 ng/mL betamethasone infusion group. Lambs from the 12-hour 2-ng/mL betamethasone infusion group had little functional lung maturation. In contrast, lambs from the 26-hour 2-ng/mL betamethasone infusion group had functional lung maturation equivalent to lambs from the positive control group. CONCLUSION: In preterm lambs that were exposed to antenatal corticosteroids, high maternofetal plasma betamethasone concentrations did not correlate with improved lung maturation. The largest and most consistent improvements in lung maturation were in animals that were exposed to either the clinical course of Celestone Chronodose or a low-dose betamethasone phosphate infusion to achieve a fetal plasma betamethasone concentration of approximately 2 ng/mL for 26 hours. The duration of low-concentration maternofetal steroid exposure, not total dose or peak drug exposure, is a key determinant for antenatal corticosteroids efficacy. These findings underscore the need to develop an optimized steroid dosing regimen that may improve both the efficacy and safety of antenatal corticosteroids therapy.

The Differential Absorption of a Series of P-Glycoprotein Substrates in Isolated Perfused Lungs from Mdr1a/1b Genetic Knockout Mice can be Attributed to Distinct Physico-Chemical Properties: an Insight into Predicting Transporter-Mediated, Pulmonary Specific Disposition.

PURPOSE: To examine if pulmonary P-glycoprotein (P-gp) is functional in an intact lung; impeding the pulmonary absorption and increasing lung retention of P-gp substrates administered into the airways. Using calculated physico-chemical properties alone build a predictive Quantitative Structure-Activity Relationship (QSAR) model distinguishing whether a substrate's pulmonary absorption would be limited by P-gp or not. METHODS: A panel of 18 P-gp substrates were administered into the airways of an isolated perfused mouse lung (IPML) model derived from Mdr1a/Mdr1b knockout mice. Parallel intestinal absorption studies were performed. Substrate physico-chemical profiling was undertaken. Using multivariate analysis a QSAR model was established. RESULTS: A subset of P-gp substrates (10/18) displayed pulmonary kinetics influenced by lung P-gp. These substrates possessed distinct physico-chemical properties to those P-gp substrates unaffected by P-gp (8/18). Differential outcomes were not related to different intrinsic P-gp transporter kinetics. In the lung, in contrast to intestine, a higher degree of non-polar character is required of a P-gp substrate before the net effects of efflux become evident. The QSAR predictive model was applied to 129 substrates including eight marketed inhaled drugs, all these inhaled drugs were predicted to display P-gp dependent pulmonary disposition. CONCLUSIONS: Lung P-gp can affect the pulmonary kinetics of a subset of P-gp substrates. Physico-chemical relationships determining the significance of P-gp to absorption in the lung are different to those operative in the intestine. Our QSAR framework may assist profiling of inhaled drug discovery candidates that are also P-gp substrates. The potential for P-gp mediated pulmonary disposition exists in the clinic.

Development of a Novel Quantitative Structure-Activity Relationship Model to Accurately Predict Pulmonary Absorption and Replace Routine Use of the Isolated Perfused Respiring Rat Lung Model.

PURPOSE: We developed and tested a novel Quantitative Structure-Activity Relationship (QSAR) model to better understand the physicochemical drivers of pulmonary absorption, and to facilitate compound design through improved prediction of absorption. The model was tested using a large array of both existing and newly designed compounds. METHODS: Pulmonary absorption data was generated using the isolated perfused respiring rat lung (IPRLu) model for 82 drug discovery compounds and 17 marketed drugs. This dataset was used to build a novel QSAR model based on calculated physicochemical properties. A further 9 compounds were used to test the model's predictive capability. RESULTS: The QSAR model performed well on the 9 compounds in the "Test set" with a predicted versus observed correlation of R(2) = 0.85, and >65% of compounds correctly categorised. Calculated descriptors associated with permeability and hydrophobicity positively correlated with pulmonary absorption, whereas those associated with charge, ionisation and size negatively correlated. CONCLUSIONS: The novel QSAR model described here can replace routine generation of IPRLu model data for ranking and classifying compounds prior to synthesis. It will also provide scientists working in the field of inhaled drug discovery with a deeper understanding of the physicochemical drivers of pulmonary absorption based on a relevant respiratory compound dataset.

ADME SARfari: comparative genomics of drug metabolizing systems.

MOTIVATION: ADME SARfari is a freely available web resource that enables comparative analyses of drug-disposition genes. It does so by integrating a number of publicly available data sources, which have subsequently been used to build data mining services, predictive tools and visualizations for drug metabolism researchers. The data include the interactions of small molecules with ADME (absorption, distribution, metabolism and excretion) proteins responsible for the metabolism and transport of molecules; available pharmacokinetic (PK) data; protein sequences of ADME-related molecular targets for pre-clinical model species and human; alignments of the orthologues including information on known SNPs (Single Nucleotide Polymorphism) and information on the tissue distribution of these proteins. In addition, in silico models have been developed, which enable users to predict which ADME relevant protein targets a novel compound is likely to interact with.

Predictive approaches to increase absorption of compounds during lead optimisation.

INTRODUCTION: Complex physicochemical and biological processes influence the oral absorption of a drug molecule. Consideration of these processes is an important activity during the optimisation of potential candidate molecules. AREAS COVERED: The authors review the applications of physicochemical and structural requirements for intestinal absorption. Furthermore, they provide examples of how to aid the lead optimisation process through improvement of solubility and permeability. EXPERT OPINION: The physicochemical requirements for absorption are solubility and permeability. Both are influenced by lipophilicity, but in the opposite way. The size of the molecule also affects both solubility and permeability. Several models can be used to estimate oral absorption from chemical structure or from measured physicochemical properties. Thus, logD-cMR model, the 'golden triangle' model, Abraham solvation equations and absorption potential can be used as tools in the lead optimisation process. Measured values of solubility and permeability greatly improve the estimation of in vivo oral absorption of compounds. However, it is important to appreciate that predictions of oral absorption may be confounded by the involvement of active transporters in the gut which may either increase (e.g., active uptake) or decrease (e.g., efflux) the absorption of drug molecules. To evaluate the first-pass metabolism, in vitro clearance measurements using liver microsomes can be used in physiologically based models for the estimation of bioavailability. The general tools discussed in this review are based on the physicochemical property assessment of compound libraries and they help design compounds that occupy desirable property space with increased likelihood of good oral absorption.

Selectivity in the impact of P-glycoprotein upon pulmonary absorption of airway-dosed substrates: a study in ex vivo lung models using chemical inhibition and genetic knockout.

P-glycoprotein (P-gp) mediated efflux is recognised to alter the absorption and disposition of a diverse range of substrates. Despite evidence showing the presence of P-gp within the lung, relatively little is known about the transporter's effect upon the absorption and distribution of drugs delivered via the pulmonary route. Here, we present data from an intact isolated rat lung model, alongside two isolated mouse lung models using either chemical or genetic inhibition of P-gp. Data from all three models show inhibition of P-gp increases the extent of absorption of a subset of P-gp substrates (e.g. rhodamine 123 and loperamide) whose physico-chemical properties are distinct from those whose pulmonary absorption remained unaffected (e.g. digoxin and saquinavir). This is the first study showing direct evidence of P-gp mediated efflux within an intact lung, a finding that should warrant consideration as part of respiratory drug discovery and development as well as in the understanding of pulmonary pharmacokinetic (PK)-pharmacodynamic (PD) relationships.

An evaluation of ondansetron binding interactions with human cytochrome P450 enzymes CYP3A4 and CYP2D6.

The results of an evaluation study of ondansetron binding to human cytochromes P450 CYP3A4 and CYP2D6 is reported. The methodology includes NMR spectroscopic measurements of substrate to heme iron distances together with molecular modelling of the enzyme-substrate interactions. It is shown that there is a generally good agreement between the experimental and calculated binding affinities for ondansetron towards CYP2D6 and CYP3A4 enzymes, based on interactive docking studies. Moreover, the modelled binding orientations for ondansetron in CYP2D6 and CYP3A4 are largely consistent with the NMR data and with the known routes for P450-mediated metabolism of this compound.

Studies on a series of potent, orally bioavailable, 5-HT(1) receptor ligands--part II.

A series of 5-(piperidinylethyloxy)quinoline 5-HT(1) receptor ligands have been studied by elaboration of the series of dual 5-HT(1)-SSRIs reported previously. These new compounds display a different in vitro pharmacological profile with potent affinity across the 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors and selectivity against the serotonin transporter. Furthermore, they have improved pharmacokinetic profiles and CNS penetration.

Discovery of potent, orally bioavailable, selective 5-HT1A/B/D receptor antagonists.

5-HT1 receptor antagonists have been discovered with good selectivity over the 5-HT transporter. This is the first report of highly potent, selective ligands for the 5-HT1A/B/D receptors with low intrinsic activity, which represent a useful set of molecules for further understanding the roles of the 5-HT1 receptor subtypes and providing new approaches for the treatment of depression.

Studies towards the identification of a new generation of atypical antipsychotic agents.

A rational structure-activity relationship study around compound (1) is reported. The lead optimisation programme led to the identification of sulfonamide (25), a molecule combining dopamine D2/D3 receptor antagonism with serotonin 5-HT2A, 5-HT2C, 5-HT6 receptor antagonism for an effective treatment of schizophrenia. Compound (25) was shown to possess the required in vivo activity with no EPS liability.

SB-656104-A, a novel selective 5-HT7 receptor antagonist, modulates REM sleep in rats.

1 (6-((R)-2-[2-[4-(4-Chloro-phenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulphonyl)-1H-indole hydrochloride) (SB-656104-A), a novel 5-hydroxytryptamine (5-HT(7)) receptor antagonist, potently inhibited [(3)H]-SB-269970 binding to the human cloned 5-HT(7(a)) (pK(i) 8.7+/-0.1) and 5-HT(7(b)) (pK(i) 8.5+/-0.2) receptor variants and the rat native receptor (pK(i) 8.8+/-0.2). The compound displayed at least 30-fold selectivity for the human 5-HT(7(a)) receptor versus other human cloned 5-HT receptors apart from the 5-HT(1D) receptor ( approximately 10-fold selective). 2 SB-656104-A antagonised competitively the 5-carboxamidotryptamine (5-CT)-induced accumulation of cyclic AMP in h5-HT(7(a))/HEK293 cells with a pA(2) of 8.5. 3 Following a constant rate iv infusion to steady state in rats, SB-656104 had a blood clearance (CL(b)) of 58+/-6 ml min(-1) kg(-1) and was CNS penetrant with a steady-state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg(-1)), the compound displayed a t(1/2) of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 micro M, respectively. 4 SB-656104-A produced a significant reversal of the 5-CT-induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5-HT(7) receptor interaction in vivo (ED(50) 2 mg kg(-1)). 5 SB-656104-A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg(-1) i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg(-1) i.p. with no significant effect on the latency to, or amount of, non-REM sleep. SB-269970-A produced qualitatively similar effects in the same study. 6 In summary, SB-656104-A is a novel 5-HT(7) receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5-HT(7) receptors in the modulation of REM sleep.