Ocular Pharmacokinetics of Intravitreally Injected Protein Therapeutics: Comparison among Standard-of-Care Formats.

The current standard of care for antivascular endothelial growth factor (VEGF) treatment requires frequent intravitreal (IVT) injections of protein therapeutics, as a result of limited retention within the eye. A thorough understanding of the determinants of ocular pharmacokinetics (PK) and its translation across species is an essential prerequisite for developing more durable treatments. In this work, we studied the ocular PK in macaques of the protein formats that comprise today's anti-VEGF standard of care. Cynomolgus monkeys received a single IVT injection of a single-chain variable fragment (scFv, brolucizumab), antigen-binding fragment (Fab, ranibizumab), fragment crystallizable-fusion protein (Fc-fusion, aflibercept), or immunoglobulin G monoclonal antibody (IgG, VA2 CrossMAb). Drug concentrations were determined in aqueous humor samples collected up to 42 days postinjection using immunoassay methods. The ocular half-life (t1/2) was 2.28, 2.62, 3.13, and 3.26 days for scFv, Fab, Fc-fusion, and IgG, respectively. A correlation with human t1/2 values from the literature confirmed the translational significance of the cynomolgus monkey as an animal model for ocular research. The relation between ocular t1/2 and molecular size was also investigated. Size was inferred from the molecular weight (MW) or determined experimentally by dynamic light scattering. The MW and hydrodynamic radius were found to be good predictors for the ocular t1/2 of globular proteins. The analysis showed that molecular size is a determinant of ocular disposition and may be used in lieu of dedicated PK studies in animals.

Understanding the Half-Life Extension of Intravitreally Administered Antibodies Binding to Ocular Albumin.

The burden associated with frequent injections of current intravitreal (IVT) therapeutics may be reduced by long-acting delivery strategies. Binding to serum albumin has been shown to extend the ocular half-life in rabbits, however, the underlying molecular mechanisms and translational relevance remain unclear. The aim of this work was to characterize the in vitro and in vivo formation of complexes between human serum albumin (HSA) and an antigen-binding fragment of a rabbit antibody linked to an anti-HSA nanobody (FabA). The ocular and systemic pharmacokinetics of 3H-labeled FabA (0.05 mg/eye IVT) co-formulated with HSA (1 and 15 nmol/eye) were assessed in Dutch belted rabbits. Next, FabA was incubated in vitreous samples from cynomolgus monkeys and human donors (healthy and diseased) supplemented with species-specific serum albumin. Finally, the FabA-albumin complexes formed in vitro and in vivo were analyzed by radio-size exclusion chromatography. A 3-fold increase in FabA vitreal exposure and half-life was observed in rabbits co-administered with 15 nmol HSA compared to 1 nmol and a control arm. The different pharmacokinetic behavior was explained with the formation of higher molecular weight FabA-albumin complexes. The analysis of vitreous samples revealed the existence of predominantly 1:1 complexes at endogenous or low concentrations of supplemented albumin. A shift towards 1:2 complexes was observed with increasing albumin concentrations. Overall, these results suggest that endogenous vitreal albumin concentrations are insufficient for half-life extension and warrant supplementation in the dosing formulation.

Ocular Half-Life of Intravitreal Biologics in Humans and Other Species: Meta-Analysis and Model-Based Prediction.

Therapeutic antibodies administered intravitreally are the current standard of care to treat retinal diseases. The ocular half-life (t1/2) is a key determinant of the duration of target suppression. To support the development of novel, longer-acting drugs, a reliable determination of t1/2 is needed together with an improved understanding of the factors that influence it. A model-based meta-analysis was conducted in humans and nonclinical species (rat, rabbit, monkey, and pig) to determine consensus values for the ocular t1/2 of IgG antibodies and Fab fragments. Results from multiple literature and in-house pharmacokinetic studies are presented within a mechanistic framework that assumes diffusion-controlled drug elimination from the vitreous. Our analysis shows, both theoretically and experimentally, that the ocular t1/2 increases in direct proportion to the product of the hydrodynamic radius of the macromolecule (3.0 nm for Fab and 5.0 nm for IgG) and the square of the radius of the vitreous globe, which varies approximately 24-fold from the rat to the human. Interspecies differences in the proportionality factors are observed and discussed in mechanistic terms. In addition, mathematical formulae are presented that allow prediction of the ocular t1/2 for molecules of interest. The utility of these formulae is successfully demonstrated in case studies of aflibercept, brolucizumab, and PEGylated Fabs, where the predicted ocular t1/2 values are found to be in reasonable agreement with the experimental data available for these molecules.

Establishment of an In Vitro-In Vivo Correlation for Melanin Binding and the Extension of the Ocular Half-Life of Small-Molecule Drugs.

A large variety of drugs bind effectively to melanin, and this binding influences their ocular pharmacokinetic and distribution profiles. We aimed to establish a correlation between in vitro melanin binding and in vivo ocular pharmacokinetics (PK). The extent of melanin binding in vitro was determined for a set of model drugs; binding kinetics and binding isotherms were generated and fitted to a mechanistic model to derive the drug-melanin binding parameters (Bmax, KD, kon, and koff). In addition, in vitro ADME properties such as cellular permeability, P-glycoprotein-mediated efflux, plasma protein binding, and octanol partition coefficients were determined. Moreover, cellular uptake was measured in the nonpigmented ARPE-19 cells and in lightly pigmented human epidermal melanocytes. Finally, in vivo ocular PK studies were performed in albino and pigmented rats using intravenous injections. Substantial drug enrichment accompanied by a very long residence time was observed in pigmented ocular tissues, which could be linked to the melanin binding determined in vitro and to the intracellular drug uptake into the pigmented cells. The resulting ocular PK profile is shown to be a consequence of the interplay of melanin binding with concurrent processes such as systemic clearance, plasma protein binding, cellular permeation, P-glycoprotein efflux, pH partitioning, and tissue binding. Understanding this interplay at a mechanistic level could help in the rational design and development of new small-molecule drug candidates with the desired PK/pharmacodynamic profile to target the back of the eye.

Influence of Melanin Characteristics on Drug Binding Properties.

Melanins are biopolymers encompassing a high degree of chemical heterogeneity. Binding of small-molecule drugs to ocular melanin significantly affects the ocular pharmacokinetics, and could serve as a strategy for prolonged drug retention in the eye. The influence of the structural and physical characteristics of melanins originating from different sources on their drug binding properties has not yet been methodically investigated. We performed physical characterization of Sepia officinalis, synthetic and porcine melanin. The particle size distribution was analyzed by laser diffractometry. A dynamic vapor sorption method, requiring small amounts of the material, was developed to analyze the differences in the specific surface area of the melanins. The extent of melanin binding at equilibrium was determined for a set of 34 small-molecule drugs and compared across different melanin types. Despite systematic shifts in the extent of binding within a twofold range, binding data were highly correlated across the melanins. These moderate differences in binding could not be directly explained by the substantial differences in particle size and were more in line with the relatively similar specific surface area of these different melanin materials. Overall, these results suggest that the specific surface area reflects the actual accessibility of a small molecule in the melanin structure and could serve as a surrogate to explain the binding differences observed for the respective melanin materials.

Understanding Molecular Drivers of Melanin Binding To Support Rational Design of Small Molecule Ophthalmic Drugs.

Binding of drugs to ocular melanin is a prominent biological phenomenon that affects the local pharmacokinetics and pharmacodynamics in the eye. In this work, we report on the development of in vitro and in silico tools for an early assessment and prediction of melanin binding properties of small molecules. A robust high-throughput assay has been established to study the binding of large sets of compounds to melanin. The extremely randomized trees approach was used to develop an in silico model able to predict the extent of melanin binding from the molecular properties of the compounds. After the last iteration of the model, strong melanin binders could prospectively be identified with 91% accuracy. On the basis of in vitro data generated for approximately 3400 chemically diverse drug-like small molecules, pronounced correlations were observed between the extent of melanin binding and the basicity, lipophilicity, and aromaticity of the compounds.

Kinetics of lipid bilayer permeation of a series of ionisable drugs and their correlation with human transporter-independent intestinal permeability.

For low molecular weight drugs, lipid bilayer permeation is considered the major route for in vivo cell barrier passage. We recently introduced a fluorescence assay with liposomes to determine permeation kinetics of ionisable compounds across the lipid bilayer by monitoring drug-induced pH changes inside the liposomes. Here, we determined the permeability coefficients (PFLipP, FLipP for "Fluorescence Liposomal Permeability") across 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers of 35 ionisable drugs at pH6.0 and compared them to available in vivo human jejunal permeability (Peff) data. PFLipP values were furthermore compared with published Caco-2 cell permeability coefficients (PCaco-2), permeability coefficients determined with the parallel artificial membrane permeability assay (PAMPA) and with log D (pH6.0). The log PFLipP, corrected for predicted para-cellular diffusion, and log PCaco-2 correlated best with log Peff, with similar adjusted R2 (0.75 and 0.74, n=12). Our results suggest that transporter-independent intestinal drug absorption is predictable from liposomal permeability.

Extension of the dissolution-precipitation model for kinetic elucidation of solvent-mediated polymorphic transformations.

Thorough understanding and control of the different crystal forms of a drug product is key for fine chemistry and materials science; it ultimately determines the product's physicochemical properties and performance. In this work, we extend the application of a mechanistic dissolution-precipitation model to solvent-mediated solid form transformations. To address the relevance of the model, various kinetic solvent-mediated polymorphic transition studies were retrieved from the literature. Our model succeeds in accurately describing the experimental data, shedding light on the molecular steps driving the polymorphic conversion. Given its simplicity and mechanistic character, the model can be viewed as a useful tool to monitor, predict and optimize the solvent-mediated transformations of solid forms.

Incretin-like effects of small molecule trace amine-associated receptor 1 agonists.

OBJECTIVE: Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. METHODS: We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. RESULTS: TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. CONCLUSIONS: We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

Development of a Unified Dissolution and Precipitation Model and Its Use for the Prediction of Oral Drug Absorption.

Drug absorption is a complex process involving dissolution and precipitation, along with other kinetic processes. The purpose of this work was to (1) establish an in vitro methodology to study dissolution and precipitation in early stages of drug development where low compound consumption and high throughput are necessary, (2) develop a mathematical model for a mechanistic explanation of generated in vitro dissolution and precipitation data, and (3) extrapolate in vitro data to in vivo situations using physiologically based models to predict oral drug absorption. Small-scale pH-shift studies were performed in biorelevant media to monitor the precipitation of a set of poorly soluble weak bases. After developing a dissolution-precipitation model from this data, it was integrated into a simplified, physiologically based absorption model to predict clinical pharmacokinetic profiles. The model helped explain the consequences of supersaturation behavior of compounds. The predicted human pharmacokinetic profiles closely aligned with the observed clinical data. In summary, we describe a novel approach combining experimental dissolution/precipitation methodology with a mechanistic model for the prediction of human drug absorption kinetics. The approach unifies the dissolution and precipitation theories and enables accurate predictions of in vivo oral absorption by means of physiologically based modeling.

Estimation of Drug Binding to Brain Tissue: Methodology and in Vivo Application of a Distribution Assay in Brain Polar Lipids.

The unbound drug concentration-effect relationship in brain is a key aspect in CNS drug discovery and development. In this work, we describe an in vitro high-throughput distribution assay between an aqueous buffer and a microemulsion of porcine brain polar lipids (BPL). The derived distribution coefficient LogDBPL was applied to the prediction of unbound drug concentrations in brain (Cu,b) and nonspecific binding to brain tissue. The in vivo relevance of the new assay was assessed for a large set of proprietary drug candidates and CNS drugs by (1) comparing observed compound concentrations in rat CSF with Cu,b calculated using the LogDBPL assay in combination with total drug brain concentrations, (2) comparing Cu,b derived from LogDBPL and total drug brain concentrations to Cu,b estimated using in vitro P-glycoprotein efflux ratio data and unbound drug plasma levels, and (3) comparing tissue nonspecific binding data from human brain autoradiography studies for 17 PET tracer candidates to distribution in BPL. In summary, the LogDBPL assay provides a predicted drug fraction unbound in brain tissue that is nearly identical to brain homogenate equilibrium dialysis with an estimation of in vivo Cu,b that is superior to LogD in octanol. LogDBPL complements the approach for predicting Cu,b based on in vitro P-glycoprotein efflux ratio and in vivo unbound plasma concentration and stands as a fast and cost-effective tool for nonspecific brain binding optimization of PET ligand candidates.

Discovery of highly selective brain-penetrant vasopressin 1a antagonists for the potential treatment of autism via a chemogenomic and scaffold hopping approach.

From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.

3-Amido-3-aryl-piperidines: A Novel Class of Potent, Selective, and Orally Active GlyT1 Inhibitors.

3-Amido-3-aryl-piperidines were discovered as a novel structural class of GlyT1 inhibitors. The structure-activity relationship, which was developed, led to the identification of highly potent compounds exhibiting excellent selectivity against the GlyT2 isoform, drug-like properties, and in vivo activity after oral administration.

Discovery and optimisation of 1-hydroxyimino-3,3-diphenylpropanes, a new class of orally active GPBAR1 (TGR5) agonists.

A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screening campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which displayed a robust secretion of PYY after oral administration in a degree that can be correlated with the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glucose excursion in an oral glucose tolerance test in DIO mice.

PK/PD assessment in CNS drug discovery: Prediction of CSF concentration in rodents for P-glycoprotein substrates and application to in vivo potency estimation.

The unbound drug concentration in brain parenchyma is considered to be the relevant driver for interaction with central nervous system (CNS) biological targets. Drug levels in cerebrospinal fluid (C_CSF) are frequently used surrogates for the unbound concentrations in brain. For drugs actively transported across the blood-brain barrier (BBB), C_CSF differs from unbound plasma concentration (Cu_p) to an extent that is commonly unknown. In this study, the relationship between CSF-to-unbound plasma drug partitioning in rats and the mouse Pgp (Mdr1a) efflux ratio (ER) obtained from in vitro transcellular studies has been investigated for a set of 61 CNS compounds exhibiting substantial diversity in chemical structure and physico-chemical properties. In order to understand the in vitro-in vivo extrapolation of Pgp efflux, a mechanistic model was derived relating in vivo CNS distribution kinetics to in vitro active transport. The model was applied to predict C_CSF from Cu_p and ER data for 19 proprietary Roche CNS drug candidates. The calculated CSF concentrations were correlated with CNS pharmacodynamic responses observed in rodent models. The correlation between in vitro and in vivo potency for different pharmacological endpoints indicated that the predicted C_CSF is a valuable surrogate of the concentration at the target site. Overall, C_CSF proved superior description of PK/PD data than unbound plasma or total brain concentration for Mdr1a substrates. Predicted C_CSF can be used as a default approach to understand the PK/PD relationships in CNS efficacy models and can support the extrapolation of efficacious brain exposure for new drug candidates from rodent to man.

2-Phenoxy-nicotinamides are potent agonists at the bile acid receptor GPBAR1 (TGR5).

Potency with potential: 2-Phenoxy-nicotinamides were identified as potent agonists at the GPBAR1 receptor, a target in the treatment of obesity, type 2 diabetes and metabolic syndrome. Extensive structure-activity relationship studies supported by homology modeling and docking resulted in the identification of optimized GPBAR1 agonists, potent against both human and mouse receptors, endowed with favorable physicochemical properties and good metabolic stability.

Orally active aminopyridines as inhibitors of tetrameric fructose-1,6-bisphosphatase.

A novel sulfonylureido pyridine series exemplified by compound 19 yielded potent inhibitors of FBPase showing significant glucose reduction and modest glycogen lowering in the acute db/db mouse model for Type-2 diabetes. Our inhibitors occupy the allosteric binding site and also extend into the dyad interface region of tetrameric FBPase.

Benzoxazole piperidines as selective and potent somatostatin receptor subtype 5 antagonists.

SAR studies of a recently described SST5R selective benzoxazole piperidine lead series are described with particular focus on the substitution pattern on the benzyl and benzoxazole side-chains. Introduction of a second meta substituent at the benzyl unit significantly lowers residual hH1 activity and insertion of substituents onto the benzoxazole periphery entirely removes remaining h5-HT2B activity. Compounds with single digit nM activity, functional antagonism and favorable physicochemical properties endowed with a good pharmacokinetic profile in rats are described which should become valuable tools for exploring the pharmacological role of the SST5 receptor in vivo.

Thiazolidinedione bioactivation: a comparison of the bioactivation potentials of troglitazone, rosiglitazone, and pioglitazone using stable isotope-labeled analogues and liquid chromatography tandem mass spectrometry.

Troglitazone, a thiazolidinedione (TZD) type insulin sensitizer for the treatment of diabetes, was withdrawn from the U.S. market after several fatal cases of hepatotoxicity. Although the mechanism(s) of these idiosyncratic adverse reactions are not completely understood, circumstantial evidence suggests at least a partial contribution of reactive metabolite formation. Despite isolated case reports of hepatotoxicity, the other TZD derivatives pioglitazone and rosiglitazone are comparatively safe. Herein, we report on the bioactivation potential of these drugs and their TZD ring isotope-labeled 2-(15)N-3,4,5-(13)C(3) analogues in rat and human liver microsomes supplemented with glutathione (GSH). Screening for GSH adducts as surrogate markers for reactive intermediate formation was performed by liquid chromatography tandem mass spectrometry. Chemical characterization of the GSH conjugates was conducted by acquisition of their respective product ion spectra and the comparison between unlabeled and stable isotope-labeled TZD derivatives. The data suggest that all drugs undergo bioactivation processes via a common metabolic activation on the TZD ring, yielding disulfide type GSH conjugates as evidenced by the loss of labeled positions in the TZD moiety. Additional bioactivation processes leading to GSH adducts not involving TZD ring scission were evident for troglitazone. In human liver microsomes at low substrate concentrations, only troglitazone yielded a predominant GSH adduct not involving TZD ring scission. This property may contribute, together with other factors such as the relatively high dose administered as well as its potential to induce hepatic cholestasis and oxidative stress, to the hepatotoxicity of this drug.

Glutathione conjugates of 4-hydroxy-2(E)-nonenal as biomarkers of hepatic oxidative stress-induced lipid peroxidation in rats.

Here we present a simple, specific, and sensitive liquid chromatography/mass spectrometry method to measure 4-hydroxy-2(E)-nonenal-glutathione (HNE-GSH), the major stable hepatic metabolite of HNE after GSH conjugation, as a marker of oxidative stress in rat liver and hepatocytes. Commonly employed methods for the measurement of lipid peroxidation-derived free aldehydes or modified proteins suffer from the artificial formation of HNE or HNE adducts to cellular molecules during sample preparation and derivatization, resulting in an overestimation of background levels. Basal levels of HNE-GSH in liver tissue from untreated rats were detected in amounts of 20 pmol/g liver. Rats exposed to a single dose of iron nitrilotriacetate (Fe(III)NTA; 15 mg Fe/kg bw, ip), a model compound for the induction of oxidative stress, revealed a fivefold increase in the hepatic HNE-GSH levels compared to controls 5 h after dosing. Moreover, a significant increase in HNE-mercapturic acid (HNE-MA) and its reduced metabolite DHN-MA was evident at 5 or 24 h after treatment, which was also reflected in increased plasma concentrations of these secondary HNE-GSH metabolites. In agreement with the in vivo data, a time-dependent increase in the levels of HNE-GSH from <1 to 123 +/- 16 pmol/10(6) cells over 5 h was detected in rat hepatocytes treated with Fe(III)NTA (150 microM). An increase in cellular HNE-GSH from <1.0 to 7.2 +/- 0.3 pmol/10(6) cells could be observed in rat hepatocytes treated with allyl alcohol (500 microM, 3 h), known for generation of HNE in hepatocytes. These data suggest that the direct measurement of the stable GSH conjugation product of cellular HNE in rat primary hepatocytes or its secondary metabolites may represent a reliable biomarker of oxidative stress-induced lipid peroxidation in rat liver in vivo.