Plasma Pharmacokinetics of N-Acetylgalactosamine-Conjugated Small-Interfering Ribonucleic Acids (GalNAc-Conjugated siRNAs).

Small-interfering ribonucleic acids (siRNAs) with N-acetylgalactosamine (GalNAc) conjugation for improved liver uptake represent an emerging class of drugs that modulate liver-expressed therapeutic targets. The pharmacokinetics of GalNAc-siRNAs are characterized by a rapid distribution from plasma to tissue (hours) and a long terminal plasma half-life, analyzed in the form of the antisense strand, driven by redistribution from tissue (weeks). Understanding how clinical pharmacokinetics relate to the dose and type of siRNA chemical stabilizing method used is critical, e.g., to design studies, to investigate safety windows, and to predict the pharmacokinetics of new preclinical assets. To this end, we collected and analyzed pharmacokinetic data from the literature regarding nine GalNAc-siRNAs. Based on this analysis, we showed that the clinical plasma pharmacokinetics of GalNAc-siRNAs are approximately dose proportional and similar between chemical stabilizing methods. This holds for both the area under the concentration-time curve (AUC) and the maximum plasma concentration (Cmax). Corresponding rat and monkey pharmacokinetic data for a subset of the nine GalNAc-siRNAs show dose-proportional Cmax, supra-dose-proportional AUC, and similar pharmacokinetics between chemical stabilizing methods​. Together, the animal and human pharmacokinetic data indicate that plasma clearance divided by bioavailability follows allometric principles and scales between species with an exponent of 0.75. Finally, the clinical plasma concentration-time profiles can be empirically described by standard one-compartment kinetics with first-order absorption up to 24 h after subcutaneous dosing, and by three-compartment kinetics with first-order absorption in general. To describe the system more mechanistically, we report a corrected and unambiguously defined version of a previously published physiologically based pharmacokinetic model.

OSWG Recommended Approaches to the Nonclinical Pharmacokinetic (ADME) Characterization of Therapeutic Oligonucleotides.

This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.

Hepatic patatin-like phospholipase domain-containing 3 levels are increased in I148M risk allele carriers and correlate with NAFLD in humans.

In nonalcoholic fatty liver disease (NAFLD) the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well-characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2-4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.

Drug metabolism and pharmacokinetic strategies for oligonucleotide- and mRNA-based drug development.

Oligonucleotide and modified mRNA therapeutics have great potential to treat diseases that are currently challenging to cure and are expanding into global and chronic disease areas such as cancer and various cardiovascular diseases. Advanced drug delivery systems or ligand-drug conjugates are utilized to achieve 'the right dose to the right target' to benefit efficacy and safety in patients. Chemistry and ADME characteristics distinguish these therapeutics from small molecules. Understanding the scalability and translatability between species and compound properties is crucial for robust nonclinical PKPD predictions to support clinical study design. Although the field has been developing for three decades, there is still room for innovation but also a need for nonclinical regulatory guidance to address these new modalities.

Effects of a novel potent melanin-concentrating hormone receptor 1 antagonist, AZD1979, on body weight homeostasis in mice and dogs.

BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non-human primates and dogs express two MCH receptors (MCH1 and MCH2 ). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood. EXPERIMENTAL APPROACH: A novel recently identified potent MCH1 receptor antagonist, AZD1979, was studied in wild type and Mchr1 knockout (KO) mice and by using pair-feeding and indirect calorimetry in diet-induced obese (DIO) mice. The effect of AZD1979 on body weight was also studied in beagle dogs. KEY RESULTS: AZD1979 bound to MCH1 receptors in the CNS and dose-dependently reduced body weight in DIO mice leading to improved homeostasis model assessment-index of insulin sensitivity. AZD1979 did not affect food intake or body weight in Mchr1 KO mice demonstrating specificity for the MCH1 receptor mechanism. In DIO mice, initial AZD1979-mediated body weight loss was driven by decreased food intake, but an additional component of preserved energy expenditure was apparent in pair-feeding and indirect calorimetry studies. AZD1979 also dose-dependently reduced body weight in dogs. CONCLUSION AND IMPLICATIONS: AZD1979 is a novel potent MCH1 receptor antagonist that affects both food intake and energy expenditure. That AZD1979 also lowers body weight in a species expressing both MCH receptors holds promise for the use of MCH1 receptor antagonists for the treatment of human obesity.

Discovery of (3-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone (AZD1979), a Melanin Concentrating Hormone Receptor 1 (MCHr1) Antagonist with Favorable Physicochemical Properties.

A novel series of melanin concentrating hormone receptor 1 (MCHr1) antagonists were the starting point for a drug discovery program that culminated in the discovery of 103 (AZD1979). The lead optimization program was conducted with a focus on reducing lipophilicity and understanding the physicochemical properties governing CNS exposure and undesired off-target pharmacology such as hERG interactions. An integrated approach was taken where the key assay was ex vivo receptor occupancy in mice. The candidate compound 103 displayed appropriate lipophilicity for a CNS indication and showed excellent permeability with no efflux. Preclinical GLP toxicology and safety pharmacology studies were without major findings and 103 was taken into clinical trials.

Identification of positron emission tomography (PET) tracer candidates by prediction of the target-bound fraction in the brain.

BACKGROUND: Development of tracers for imaging with positron emission tomography (PET) is often a time-consuming process associated with considerable attrition. In an effort to simplify this process, we herein propose a mechanistically integrated approach for the selection of tracer candidates based on in vitro measurements of ligand affinity (Kd), non-specific binding in brain tissue (Vu,brain), and target protein expression (Bmax). METHODS: A dataset of 35 functional and 12 non-functional central nervous system (CNS) PET tracers was compiled. Data was identified in literature for Kd and Bmax, whereas a brain slice methodology was used to determine values for Vu,brain. A mathematical prediction model for the target-bound fraction of tracer in the brain (ftb) was derived and evaluated with respect to how well it predicts tracer functionality compared to traditional PET tracer candidate selection criteria. RESULTS: The methodology correctly classified 31/35 functioning and 12/12 non-functioning tracers. This predictivity was superior to traditional classification criteria or combinations thereof. CONCLUSIONS: The presented CNS PET tracer identification approach is rapid and accurate and is expected to facilitate the development of novel PET tracers for the molecular imaging community.

A modeling approach for compounds affecting body composition.

Body composition and body mass are pivotal clinical endpoints in studies of welfare diseases. We present a combined effort of established and new mathematical models based on rigorous monitoring of energy intake (EI) and body mass in mice. Specifically, we parameterize a mechanistic turnover model based on the law of energy conservation coupled to a drug mechanism model. Key model variables are fat-free mass (FFM) and fat mass (FM), governed by EI and energy expenditure (EE). An empirical Forbes curve relating FFM to FM was derived experimentally for female C57BL/6 mice. The Forbes curve differs from a previously reported curve for male C57BL/6 mice, and we thoroughly analyse how the choice of Forbes curve impacts model predictions. The drug mechanism function acts on EI or EE, or both. Drug mechanism parameters (two to three parameters) and system parameters (up to six free parameters) could be estimated with good precision (coefficients of variation typically <20 % and not greater than 40 % in our analyses). Model simulations were done to predict the EE and FM change at different drug provocations in mice. In addition, we simulated body mass and FM changes at different drug provocations using a similar model for man. Surprisingly, model simulations indicate that an increase in EI (e.g. 10 %) was more efficient than an equal lowering of EI. Also, the relative change in body mass and FM is greater in man than in mouse at the same relative change in either EI or EE. We acknowledge that this assumes the same drug mechanism impact across the two species. A set of recommendations regarding the Forbes curve, vehicle control groups, dual action on EI and loss, and translational aspects are discussed. This quantitative approach significantly improves data interpretation, disease system understanding, safety assessment and translation across species.

Discovery of agonists of cannabinoid receptor 1 with restricted central nervous system penetration aimed for treatment of gastroesophageal reflux disease.

Agonists of the cannabinoid receptor 1 (CB1) have been suggested as possible treatments for a range of medical disorders including gastroesophageal reflux disease (GERD). While centrally acting cannabinoid agonists are known to produce psychotropic effects, it has been suggested that the CB1 receptors in the periphery could play a significant role in reducing reflux. A moderately potent and highly lipophilic series of 2-aminobenzamides was identified through focused screening of GPCR libraries. Development of this series focused on improving potency and efficacy at the CB1 receptor, reducing lipophilicity and limiting the central nervous system (CNS) exposure while maintaining good oral absorption. Improvement of the series led to compounds having excellent potency at the CB1 receptor and high levels of agonism, good physical and pharmacokinetic properties, and low penetration into the CNS. A range of compounds demonstrated a dose-dependent inhibition of transient lower esophageal sphincter relaxations in a dog model.

In silico prediction of unbound brain-to-plasma concentration ratio using machine learning algorithms.

Distribution over the blood-brain barrier (BBB) is an important parameter to consider for compounds that will be synthesized in a drug discovery project. Drugs that aim at targets in the central nervous system (CNS) must pass the BBB. In contrast, drugs that act peripherally are often optimised to minimize the risk of CNS side effects by restricting their potential to reach the brain. Historically, most prediction methods have focused on the total compound distribution between the blood plasma and the brain. However, recently it has been proposed that the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) is more relevant. In the current study, quantitative K(p,uu,brain) prediction models have been built on a set of 173 in-house compounds by using various machine learning algorithms. The best model was shown to be reasonably predictive for the test set of 73 compounds (R(2)=0.58). When used for qualitative prediction the model shows an accuracy of 0.85 (Kappa=0.68). An additional external test set containing 111 marketed CNS active drugs was also classified with the model and 89% of these drugs were correctly predicted as having high brain exposure.

Structure-brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids.

New experimental methodologies were applied to measure the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) and the unbound CSF-to-plasma concentration ratio (K(p,uu,CSF)) in rats for 43 structurally diverse drugs. The relationship between chemical structure and K(p,uu,brain) was dominated by hydrogen bonding. Contrary to popular understanding based on the total brain-to-plasma concentration ratio (logBB), lipophilicity was not a determinant of unbound brain exposure. Although changing the number of hydrogen bond acceptors is a useful design strategy for optimizing K(p,uu,brain), future improvement of in silico prediction models is dependent on the accommodation of active drug transport. The structure-brain exposure relationships found in the rat also hold for humans, since the rank order of the drugs was similar for human and rat K(p,uu,CSF). This cross-species comparison was supported by K(p,uu,CSF) being within 3-fold of K(p,uu,brain) in the rat for 33 of 39 drugs. It was, however, also observed that K(p,uu,CSF) overpredicts K(p,uu,brain) for highly effluxed drugs, indicating lower efflux capacity of the blood-cerebrospinal fluid barrier compared to the blood-brain barrier.

(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action.

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABA(B) receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABA(B) receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABA(B) receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABA(B) receptor antagonist and absent in GABA(B)-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABA(B) receptors and may offer a potential new approach to treatment of GERD.

Development of a high-throughput brain slice method for studying drug distribution in the central nervous system.

New, more efficient methods of estimating unbound drug concentrations in the central nervous system (CNS) combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (V(u,brain)). Although the brain slice method is the most reliable in vitro method for measuring V(u,brain), it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4 to 5 h of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programs. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit of buffer volume. The use of cassette experiments for investigating V(u,brain) in a linear drug concentration range increased the throughput of the method. The V(u,brain) for the model compounds ranged from 4 to 3000 ml . g brain(-1), and the sources of variability are discussed. The optimized setup of the brain slice method allows precise, robust estimation of V(u,brain) for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.

In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids.

Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unhelpful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (V(u,brain)). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with V(u,brain) microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the V(u,brain) of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.