Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 µM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Comparative Study of Dermal Pharmacokinetics Between Topical Drugs Using Open Flow Microperfusion in a Pig Model.

PURPOSE: Accurate methods to determine dermal pharmacokinetics are important to increase the rate of clinical success in topical drug development. We investigated in an in vivo pig model whether the unbound drug concentration in the interstitial fluid as determined by dermal open flow microperfusion (dOFM) is a more reliable measure of dermal exposure compared to dermal biopsies for seven prescription or investigational drugs. In addition, we verified standard dOFM measurement using a recirculation approach and compared dosing frequencies (QD versus BID) and dose strengths (high versus low drug concentrations). METHODS: Domestic pigs were topically administered seven different drugs twice daily in two studies. On day 7, drug exposures in the dermis were assessed in two ways: (1) dOFM provided the total and unbound drug concentrations in dermal interstitial fluid, and (2) clean punch biopsies after heat separation provided the total concentrations in the upper and lower dermis. RESULTS: dOFM showed sufficient intra-study precision to distinguish interstitial fluid concentrations between different drugs, dose frequencies and dose strengths, and had good reproducibility between studies. Biopsy concentrations showed much higher and more variable values. Standard dOFM measurements were consistent with values obtained with the recirculation approach. CONCLUSIONS: dOFM pig model is a robust and reproducible method to directly determine topical drug concentration in dermal interstitial fluid. Dermal biopsies were a less reliable measure of dermal exposure due to possible contributions from drug bound to tissue and drug associated with skin appendages.

ADME and DMPK considerations for the discovery and development of antibody drug conjugates (ADCs).

 The therapeutic concept of antibody drug conjugates (ADCs) is to selectively target tumour cells with small molecule cytotoxic drugs to maximise cell kill benefit and minimise healthy tissue toxicity.An ADC generally consists of an antibody that targets a protein on the surface of tumour cells chemically linked to a warhead small molecule cytotoxic drug.To deliver the warhead to the tumour cell, the antibody must bind to the target protein and in general be internalised into the cell. Following internalisation, the cytotoxic agent can be released in the endosomal or lysosomal compartment (via different mechanisms). Diffusion or transport out of the endosome or lysosome allows the cytotoxic drug to express its cell-killing pharmacology. Alternatively, some ADCs (e.g. EDB-ADCs) rely on extracellular cleavage releasing membrane permeable warheads.One potentially important aspect of the ADC mechanism is the 'bystander effect' whereby the cytotoxic drug released in the targeted cell can diffuse out of that cell and into other (non-target expressing) tumour cells to exert its cytotoxic effect. This is important as solid tumours tend to be heterogeneous and not all cells in a tumour will express the targeted protein.The combination of large and small molecule aspects in an ADC poses significant challenges to the disposition scientist in describing the ADME properties of the entire molecule.This article will review the ADC landscape and the ADME properties of successful ADCs, with the aim of outlining best practice and providing a perspective of how the field can further facilitate the discovery and development of these important therapeutic modalities.

Human Recombinant Alkaline Phosphatase (Ilofotase Alfa) Protects Against Kidney Ischemia-Reperfusion Injury in Mice and Rats Through Adenosine Receptors.

Adenosine triphosphate (ATP) released from injured or dying cells is a potent pro-inflammatory "danger" signal. Alkaline phosphatase (AP), an endogenous enzyme that de-phosphorylates extracellular ATP, likely plays an anti-inflammatory role in immune responses. We hypothesized that ilofotase alfa, a human recombinant AP, protects kidneys from ischemia-reperfusion injury (IRI), a model of acute kidney injury (AKI), by metabolizing extracellular ATP to adenosine, which is known to activate adenosine receptors. Ilofotase alfa (iv) with or without ZM241,385 (sc), a selective adenosine A2A receptor (A2AR) antagonist, was administered 1 h before bilateral IRI in WT, A2AR KO (Adora2a-/- ) or CD73-/- mice. In additional studies recombinant alkaline phosphatase was given after IRI. In an AKI-on-chronic kidney disease (CKD) ischemic rat model, ilofotase alfa was given after the three instances of IRI and rats were followed for 56 days. Ilofotase alfa in a dose dependent manner decreased IRI in WT mice, an effect prevented by ZM241,385 and partially prevented in Adora2a-/- mice. Enzymatically inactive ilofotase alfa was not protective. Ilofotase alfa rescued CD73-/- mice, which lack a 5'-ectonucleotidase that dephosphorylates AMP to adenosine; ZM241,385 inhibited that protection. In both rats and mice ilofotase alfa ameliorated IRI when administered after injury, thus providing relevance for therapeutic dosing of ilofotase alfa following established AKI. In an AKI-on-CKD ischemic rat model, ilofotase alfa given after the third instance of IRI reduced injury. These results suggest that ilofotase alfa promotes production of adenosine from liberated ATP in injured kidney tissue, thereby amplifying endogenous mechanisms that can reverse tissue injury, in part through A2AR-and non-A2AR-dependent signaling pathways.

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.

Dose Predictions for Drug Design.

The efficacious dose of a drug is perhaps the most holistic metric reflecting its therapeutic potential. Dose is predicted at many stages in drug discovery and development. Prior to the 1990s, dose prediction was limited to the drug "working" at a reasonable dose and dose regimen in an animal model. Through the early 2000s, dose predictions were generated at candidate nomination and then refined during clinical development. Currently, dose predictions can be made early in drug discovery to enable drug design. Dose predictions at this stage can identify critical drug properties for a viable dose regimen and provide clinically relevant context to lead optimization. In this paper, we give an overview of the opportunities and challenges associated with dose prediction for drug design. A number of general considerations, approaches, and case examples are discussed.

Clearance in Drug Design.

Due to its implications for both dose level and frequency, clearance rate is one of the most important pharmacokinetic parameters to consider in the design of drug candidates. Clearance can be classified into three general categories, namely, metabolic transformation, renal excretion, and hepatobiliary excretion. Within each category, there are a host of biochemical and physiological mechanisms that ultimately determine the clearance rate. Physiochemical properties are often indicative of the rate-determining mechanism, with lipophilic molecules tending toward metabolism and hydrophilic, polar molecules tending toward passive or active excretion. Optimization of clearance requires recognition of the major clearance mechanisms and use of the most relevant in vitro and in vivo tools to develop structure-clearance relationships. The reliability of methods to detect and predict human clearance varies across mechanisms. While methods for metabolic and passive renal clearance have proven reasonably robust, there is a clear need for better tools to support the optimization of transporter-mediated clearance.

Discovery of a Novel Small-Molecule Modulator of C-X-C Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis.

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic ß 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

Relevance of Half-Life in Drug Design.

Drug half-life has important implications for dosing regimen and peak-to-trough ratio at the steady state. A half-life of 12-48 h is generally ideal for once daily dosing of oral drugs. If the half-life is too short, it may require more frequent dosing in order to maintain desired exposures and avoid unnecessarily high peak concentrations. This may pose challenges to achieving optimal efficacy, safety, and patient compliance. If the half-life is too long, the time over which accumulation and subsequent elimination occur may be prolonged. This may pose problems with managing adverse effects and the design of efficient clinical trials. Half-life is a key parameter for optimization in research and development. Structural modification to affect clearance, and to a lesser extent volume of distribution, is the preferred means of modulating half-life. An effective approach to half-life optimization requires an understanding of the many pitfalls associated with its estimation and interpretation.

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide-Peptoid Hybrid CXCR7 Modulators.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide-peptoid hybrids with high CXCR7 binding affinities (Ki < 100 nM) and measurable passive permeability (Papp > 5 × 10-6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

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 cross-species translational pharmacokinetic-pharmacodynamic evaluation of core body temperature reduction by the TRPM8 blocker PF-05105679.

PF-05105679 is a moderately potent TRPM8 blocker which has been evaluated for the treatment of cold pain sensitivity. The TRPM8 channel is responsible for the sensation of cold environmental temperatures and has been implicated in regulation of core body temperature. Consequently, blockade of TRPM8 has been suggested to result in lowering of core body temperature. As part of the progression to human studies, the effect of PF-05105679 on core body temperature has been investigated in animals. Safety pharmacology studies showed that PF-05105679 reduced core body temperature in a manner that was inversely related to body weight of the species tested (greater exposure to PF-05105679 was required to lower temperature by 1°C in higher species). Based on an allometric (body weight) relationship, it was hypothesized that PF-05105679 would not lower core body temperature in humans at exposures that could exhibit pharmacological effects on cold pain sensation. On administration to humans, PF-05105679 was indeed effective at reversing the cold pain sensation associated with the cold pressor test in the absence of effects on core body temperature.

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.

Clinical Micro-Dose Studies to Explore the Human Pharmacokinetics of Four Selective Inhibitors of Human Nav1.7 Voltage-Dependent Sodium Channels.

BACKGROUND: The emergence of genetic data linking Nav1.7 sodium channel over- and under- expression to human pain signalling has led to an interest in the treatment of chronic pain through inhibition of Nav1.7 channels. OBJECTIVE: We describe the pharmacokinetic (PK) results of a clinical microdose study performed with four potent and selective Nav1.7 inhibitors and the subsequent modelling resulting in the selection of a single compound to explore Nav1.7 pharmacology at higher doses. METHODS: A clinical microdose study to investigate the intravenous and oral PK of four compounds (PF-05089771, PF-05150122, PF-05186462 and PF-05241328) was performed in healthy volunteers. PK parameters were derived via noncompartmental analysis. A physiologically-based PK (PBPK) model was used to predict exposure and multiples of Nav1.7 50 % inhibitory concentration (IC50) for each compound at higher doses. RESULTS: Plasma clearance, volume of distribution and bioavailability ranged from 45 to 392 mL/min/kg, 13 to 36 L/kg and 38 to 110 %, respectively. The PBPK model for PF-05089771 predicted a 1 g oral dose would be required to achieve exposures of approximately 12× Nav1.7 IC50 at maximum concentration (C max), and approximately 3× IC50 after 12 h (minimum concentration [C min] for a twice-daily regimen). Lower multiples of Nav1.7 IC50 were predicted with the same oral doses of PF-05150122, PF-05186462, and PF-05241328. In a subsequent single ascending oral dose clinical study, the predictions for PF-05089771 compared well with observed data. CONCLUSION: Based on the human PK data obtained from the microdose study and subsequent modelling, PF-05089771 provided the best opportunity to explore Nav1.7 blockade for the treatment of acute or chronic pain conditions.

Discovery of a Selective TRPM8 Antagonist with Clinical Efficacy in Cold-Related Pain.

The transient receptor potential (TRP) family of ion channels comprises nonselective cation channels that respond to a wide range of chemical and thermal stimuli. TRPM8, a member of the melastatin subfamily, is activated by cold temperatures (<28 °C), and antagonists of this channel have the potential to treat cold induced allodynia and hyperalgesia. However, TRPM8 has also been implicated in mammalian thermoregulation and antagonists have the potential to induce hypothermia in patients. We report herein the identification and optimization of a series of TRPM8 antagonists that ultimately led to the discovery of PF-05105679. The clinical finding with this compound will be discussed, including both efficacy and its ability to affect thermoregulation processes in humans.