Analysis of the Bile Acid Composition in a Fibroblast Growth Factor 19-Expressing Liver-Humanized Mouse Model and Its Use for CYP3A4-Mediated Drug-Drug Interaction Studies.

Numerous biomedical applications have been described for liver-humanized mouse models, such as in drug metabolism or drug-drug interaction (DDI) studies. However, the strong enlargement of the bile acid (BA) pool due to lack of recognition of murine intestine-derived fibroblast growth factor-15 by human hepatocytes and a resulting upregulation in the rate-controlling enzyme for BA synthesis, cytochrome P450 (CYP) 7A1, may pose a challenge in interpreting the results obtained from such mice. To address this challenge, the human fibroblast growth factor-19 (FGF19) gene was inserted into the Fah-/- , Rag2-/- , Il2rg-/- NOD (FRGN) mouse model, allowing repopulation with human hepatocytes capable of responding to FGF19. While a decrease in CYP7A1 expression in human hepatocytes from humanized FRGN19 mice (huFRGN19) and a concomitant reduction in BA production was previously shown, a detailed analysis of the BA pool in these animals has not been elucidated. Furthermore, there are sparse data on the use of this model to assess potential clinical DDI. In the present work, the change in BA composition in huFRGN19 compared with huFRGN control animals was systematically evaluated, and the ability of the model to recapitulate a clinically described CYP3A4-mediated DDI was assessed. In addition to a massive reduction in the total amount of BA, FGF19 expression in huFRGN19 mice resulted in significant changes in the profile of various primary, secondary, and sulfated BAs in serum and feces. Moreover, as observed clinically, administration of the pregnane X receptor agonist rifampicin reduced the oral exposure of the CYP3A4 substrate triazolam. SIGNIFICANCE STATEMENT: Transgenic expression of FGF19 normalizes the unphysiologically high level of bile acids in a chimeric liver-humanized mouse model and leads to massive changes in bile acid composition. These adaptations could overcome one of the potential impediments in the use of these mouse models for drug-drug interaction studies.

Application of empirical scalars to enable early prediction of human hepatic clearance using IVIVE in drug discovery: an evaluation of 173 drugs.

The utilization of in vitro data to predict drug pharmacokinetics (PK) in vivo has been a consistent practice in early drug discovery for decades. However, its success is hampered by mispredictions attributed to uncharacterized biological phenomena/experimental artifacts. Predicted drug clearance (CL) from experimental data (i.e. hepatocyte intrinsic clearance: CLint, fraction unbound in plasma: fu,p) is often systematically underpredicted using the well-stirred model (WSM). The objective of this study was to evaluate using empirical scalars in the WSM to correct for CL mispredictions. Drugs (N=28) were used to generate numerical scalars on CLint (α), and fu,p (ß) to minimize the error (AAFE) for CL predictions. These scalars were validated using an additional dataset (N=28 drugs) and applied to a non-redundant AstraZeneca (AZ) dataset available in the literature (N=117 drugs) for a total of 173 compounds. CL predictions using the WSM were improved for most compounds using an α value of 3.66 (~64%<2-fold) compared to no scaling (~46%<2-fold). Similarly, using a ß value of 0.55 or combination of α and ß scalars (values of 1.74 and 0.66, respectively) resulted in a similar improvement in predictions (~64%<2-fold and ~65%<2-fold, respectively). For highly bound compounds (fu,p{less than or equal to}0.01), AAFE was substantially reduced across all scaling methods. Using the ß scalar alone or a combination of α and ß appeared optimal; and produce larger magnitude corrections for highly-bound compounds. Some drugs are still disproportionally mispredicted, however the improvements in prediction error and simplicity of applying these scalars suggests its utility for early-stage CL predictions. Significance Statement In early drug discovery, prediction of human clearance using in vitro experimental data plays an essential role in triaging compounds prior to in vivo studies. These predictions have been systematically underestimated. Here we introduce empirical scalars calibrated on the extent of plasma protein binding that appear to improve clearance prediction across multiple datasets. This approach can be used in early phases of drug discovery prior to the availability of pre-clinical data for early quantitative predictions of human clearance.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Investigating the Utility of Humanized Pregnane X Receptor-Constitutive Androstane Receptor-CYP3A4/7 Mouse Model to Assess CYP3A-Mediated Induction.

Clinical induction liability is assessed with human hepatocytes. However, underpredictions in the magnitude of clinical induction have been reported. Unfortunately, in vivo studies in animals do not provide additional insight because of species differences in drug metabolizing enzymes and their regulatory pathways. To circumvent this limitation, transgenic animals expressing human orthologs were developed. The aim of this work was to investigate the utility of mouse models expressing human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 (Tg-Composite) in evaluating clinical induction. Rifampin, efavirenz, and pioglitazone, which were employed to represent strong, moderate, and weak inducers, were administered at multiple doses to Tg-Composite animals. In vivo CYP3A activity was monitored by measuring changes in the exposure of the CYP3A probe substrate triazolam. After the in vivo studies, microsomes were prepared from their livers to measure changes of in vitro CYP3A4 activity. In both in vivo and in vitro, distinction of clinic induction was recapitulated as rifampin yielded the greatest inductive effect followed by efavirenz and pioglitazone. Interestingly, with rifampin, in vivo CYP3A activity was approximately 4-fold higher than in vitro activity. Conversely, there was no difference between in vivo and in vitro CYP3A activity with efavirenz. These findings are consistent with the report that, although rifampin exhibits differential inductive effects between the intestines and liver, efavirenz does not. These data highlight the promise of transgenic models, such as Tg-Composite, to complement human hepatocytes to enhance the translatability of clinical induction as well as become a powerful tool to further study mechanisms of drug disposition. SIGNIFICANCE STATEMENT: Underprediction of the magnitude of clinical induction when using human hepatocytes has been reported, and transgenic models may improve clinical translatability. The work presented here showcases the human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 model, which was able to recapitulate the magnitude of clinical induction and to differentiate tissue-dependent induction observed with rifampin but not with efavirenz. These results not only foreshadow the potential application of such transgenic models in assessing clinical induction but also in further investigation of the mechanism of drug disposition.

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Evaluation of a Competitive Equilibrium Dialysis Approach for Assessing the Impact of Protein Binding on Clearance Predictions.

Fraction unbound (fu) is an important consideration when characterizing the ADME properties of drug candidates. For highly bound compounds, there can be low confidence in quantifying fu introducing uncertainty in certain parameter estimations. Specifically, predictions of clearance (CL) rely on accurate fu values measured in plasma (fu,p) and microsomes (fu,mic) to scale in vitro intrinsic CL to in vivo CL. However, determining the ratio of fu,p/fu,mic may circumvent the need to measure discrete binding values. The purpose of this study was to evaluate a plasma-to-microsome competitive equilibrium dialysis (cED) method to determine fu,p/fu,mic ratio (fuR) for nine physiochemically-distinct compounds, and to investigate the impact of altering microsomal concentrations on fuR. The values of fuR were comparable to ratios calculated from discretely measured fu,p and fu,mic values. Furthermore, increasing microsomal concentrations increased fuR for basic and neutral compounds. When using fuR values, there was a good in vitro-in vivo correlation (IVIVC) (≤3-fold observed in vivo CL). These results suggest that the cED method used to determine fuR may be an appropriate, alternative IVIVC approach. Application of cED may extend beyond IVIVC of CL to evaluate other parameters such as species differences in protein binding and free tissue to plasma ratios.

Orally Bioavailable Small-Molecule CD73 Inhibitor (OP-5244) Reverses Immunosuppression through Blockade of Adenosine Production.

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,ß-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

Investigating the Impact of Albumin on the Liver Uptake of Pitavastatin and Warfarin in Nagase Analbuminemic Rats.

Albumin has been suggested to enhance the hepatic uptake of organic anion-transporting polypeptide (Oatp) substrates in various in vitro as well as liver perfusion models. However, it is not known whether the interplay between albumin and Oatp substrates is an experimental artifact or if this interaction occurs in vivo. The objective of this work was to investigate the hepatic uptake of warfarin and pitavastatin, which are both extensively bound to albumin but only pitavastatin being an Oatp substrate. Experiments were conducted in Nagase analbuminemic rats (NAR) which exhibit reduced albumin levels compared with F344 (wild type, WT). The fraction unbound (f u) was 140- and 10-fold greater in NAR plasma for warfarin and pitavastatin, respectively, whereas no meaningful differences were observed with tissue binding. In vitro, pitavastatin uptake into hepatocytes reconstituted in WT plasma was 17- and 3-fold greater than when reconstituted in buffer or NAR plasma, respectively. In vivo, the free tissue-to-free plasma ratios (K p,u,u) from brain and liver in intact WT and NAR were not significantly different for warfarin. Contrarily, liver K p,u,u of pitavastatin was 6-fold higher in WT animals, which corresponded to a 2.3-fold reduction in free plasma and 2.6-fold increase in free liver exposure. These results suggest that the enhanced hepatic uptake by albumin is not necessarily an experimental artifact but is also a relevant phenomenon in vivo. This work raises the possibility that other plasma proteins may also effect the function of additional drug transporters, and that modulating plasma protein binding may exhibit meaningful clinical relevance in the disposition of drugs. SIGNIFICANCE STATEMENT: The interplay between albumin and Oatp substrates has been reported in hepatocytes and in liver perfusion studies, but the in vivo relevance of this interaction has yet to be elucidated. Using NAR and its corresponding WT animal, this study demonstrates that albumin may indeed enhance the hepatic uptake of pitavastatin in intact animals. In vivo demonstration of this interplay not only provides further justification for continued investigation into this particular mechanism but also raises the possibility that other plasma proteins may affect additional drug transporters and that modulating plasma protein binding may exhibit meaningful clinical relevance in the disposition of drugs.

Comprehensive Evaluation of Bile Acid Homeostasis in Human Hepatocyte Co-Culture in the Presence of Troglitazone, Pioglitazone, and Acetylsalicylic Acid.

Interruption of bile acid (BA) homeostasis has been hypothesized for a variety of liver diseases and for drug-induced liver injury (DILI). Consequently, BA is gaining increasing prominence as a potential biomarker. The objective of this work was to evaluate the effect of troglitazone (TZN, associated with severe DILI), pioglitazone (PZN, rarely associated with DILI), and acetylsalicylic acid (ASA, or aspirin, not associated with DILI) on the in vitro BA homeostasis in hepatocytes co-cultured with nonparenchymal cells by monitoring the disposition of 36 BAs. The cells were supplemented with 2.5 µM d4-cholic acid, d4-chenodeoxycholic acid, d4-lithocholic acid, d4-deoxycholic acid, d4-ursodeoxycholic acid, and hyodeoxycholic acid. Concentration-time profiles of BAs were used to determine the area under the curve from the supernatant, lysate, or bile compartments, in the presence or absence of TZN, PZN, or ASA. When applicable, IC50 describing depletion of individual BAs was calculated, or accumulation greater than 200% of dimethyl sulfoxide control was noted. Thiazolidinediones significantly altered the concentration of glycine and sulfate conjugates; however, more BAs were impacted by TZN than with PZN. For commonly shared BAs, TZN exhibited 3- to 13-fold stronger inhibition than PZN. In contrast, no changes were observed with ASA. Modulation of BA disposition by thiazolidinediones and ASA was appropriately differentiated. Particularly for thiazolidinediones, TZN was more potent in interrupting BA homeostasis, and, when also considering its higher dose, may explain differences in their clinical instances of DILI. This is one of the first works which comprehensively evaluated the disposition of primary and secondary BAs along with their metabolites in an in vitro system. Differing degrees of BA homeostasis modulation was observed with various perpetrators associated with varying clinical instances of DILI. These data indicate that in vitro systems such as hepatocyte co-cultures may be a promising tool to gain a detailed insight into how drugs affect BA handling to further probe into the mechanism of DILI related to BA homeostasis.

Correction to: Safety, Tolerability, and Pharmacokinetics of GDC-0276, a Novel NaV1.7 Inhibitor, in a First-in-Human, Single- and Multiple-Dose Study in Healthy Volunteers.

The original version of this article unfortunately contained a mistake. A few entries were incorrect in Table 2.

Discovery of a C-8 hydroxychromene as a potent degrader of estrogen receptor alpha with improved rat oral exposure over GDC-0927.

Phenolic groups are responsible for the high clearance and low oral bioavailability of the estrogen receptor alpha (ERα) clinical candidate GDC-0927. An exhaustive search for a backup molecule with improved pharmacokinetic (PK) properties identified several metabolically stable analogs, although in general at the expense of the desired potency and degradation efficiency. C-8 hydroxychromene 30 is the first example of a phenol-containing chromene that not only maintained excellent potency but also exhibited 10-fold higher oral exposure in rats. The improved in vivo clearance in rat was hypothesized to be the result of C-8 hydroxy group being sterically protected from glucuronide conjugation. The excellent potency underscores the possibility of replacing the presumed indispensable phenolic group at C-6 or C-7 of the chromene core. Co-crystal structures were obtained to highlight the change in key interactions and rationalize the retained potency.

Safety, Tolerability, and Pharmacokinetics of GDC-0276, a Novel NaV1.7 Inhibitor, in a First-in-Human, Single- and Multiple-Dose Study in Healthy Volunteers.

BACKGROUND AND OBJECTIVE: Current pain therapies often do not provide adequate pain relief and have dose-limiting adverse effects. Genetic evidence indicates that NaV1.7 sodium channels are required for pain transduction and therefore represent an important therapeutic target. GDC-0276 is a novel NaV1.7 inhibitor developed for the treatment of pain. This first-in-human trial evaluated the safety, tolerability, and pharmacokinetics of orally administered GDC-0276 in healthy subjects. METHODS: This phase I, randomized, double-blind, placebo-controlled study assessed GDC-0276 as powder-in-capsule (PIC) or cyclodextrin solution (CD) single doses (SDs) of 2-270 mg (seven cohorts) and 45-540 mg (five cohorts), respectively. Multiple (MD) PIC doses were administered as total daily doses of 15-540 mg divided into two or three doses/day, up to 10 or 14 days. Safety was assessed by monitoring adverse events (AEs), vital signs, physical examinations, electrocardiograms, and laboratory tests for up to 15 days after the last day of dosing. GDC-0276 plasma pharmacokinetics were also determined. RESULTS: Three stages included 183 randomized subjects. GDC-0276 plasma exposure increased with dose level for all stages. Exposure was higher in the SD-CD cohorts compared with the equivalent SD-PIC dose levels. SDs were adequately tolerated up to 270 mg (SD-PIC) and 360 mg (SD-CD). Hypotension limited tolerability in the 540-mg SD-CD cohort. Multiple PIC doses were tolerated up to 270 mg twice daily, however liver transaminase elevations were frequently observed. No deaths or serious AEs occurred. CONCLUSION: GDC-0276 exhibited a safety and pharmacokinetic profile that supports its future investigation as a potential therapeutic for pain.

Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma.

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.

Unremarkable impact of Oatp inhibition on the liver concentration of fluvastatin, lovastatin and pitavastatin in wild-type and Oatp1a/1b knockout mouse.

1. Oatp inhibitors have been shown to significantly increase the plasma exposure of statins. However, understanding alterations of liver concentration is also important. While modeling has simulated liver concentration changes, availability of experimental data is limited, especially when concerning drug-drug interactions (DDI). The objective of this work was to determine blood and liver concentrations of fluvastatin, lovastatin and pitavastatin, when blocking uptake transporters. 2. In wild-type mouse, rifampin pre-treatment decreased the unbound liver-to-plasma ratio (Kp,uu) of fluvastatin by 4.2-fold to 2.2, lovastatin by 4.9-fold to 0.81 and pitavastatin by 10-fold to 0.21. Changes in Kp,uu were driven by increases in systemic exposures as liver concentrations were not greatly altered. 3. In Oatp1a/1b knockout mouse (KO), rifampin exerted no additional effect on fluvastatin and lovastatin. Contrarily, rifampin further decreased pitavastatin Kp,uu by 3.4-fold, suggesting that the KO is inadequate to completely block liver uptake of pitavastatin as there are additional rifampin-sensitive uptake mechanism(s) not captured in the KO model. 4. This work provides experimental data showing that the plasma compartment is more sensitive to Oatp modulation than the liver compartment, even for rifampin-mediated DDI. Consistent with previous simulations, inhibiting or targeting Oatps may change Kp,uu, but exhibit only a minimal effect on absolute liver concentrations.

Unequal Absorption of Radiolabeled and Nonradiolabeled Drug from the Oral Dose Leads to Incorrect Estimates of Drug Absorption and Circulating Metabolites in a Mass Balance Study.

BACKGROUND: Mass balance studies conducted using radiolabeled material (14C or 3H) definitively characterize the Absorption, Metabolism, and Excretion (AME) of a drug. A critical aspect of these studies is that the radiotracer maintains its proportion to total drug from its administration to its complete elimination from the body. In the study of GDC-0276 in beagle dogs, we observed that the 14C radiotracer proportion (specific activity) varied through the study. METHOD: High resolution-accurate mass spectrometric measurements of 12C and 14C isotopes of GDC- 0276 and its metabolites in plasma and excreta samples were used to determine the apparent specific activities, which were higher than the specific activity of the dosing formulation. Drug concentrations were adjusted to the observed specific activities to correct the readouts for GDC-0276 AME and PK. RESULTS: The enrichment of 14C, which resulted in higher specific activities, was consistent with faster and more extensive absorption of the radiotracer from the dosing formulation. This resulted in overestimating the dose absorbed, the extent of elimination in urine and bile, and the exposures to circulating metabolites. These biases were corrected by the specific activities determined for study samples by mass spectrometry. CONCLUSION: Assuming that the radiotracer was proportional to total drug throughout a radiolabeled study was not valid in a 14C study in beagle dogs. This presumably resulted from unequal absorption of the radiotracer and nonradiolabeled test articles from the oral dose due to inequivalent solid forms. We were able to provide a more accurate description of the AME of GDC-0276 in dogs by characterizing the differential absorption of the radiotracer.

Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors.

Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.

Selective NaV1.7 Antagonists with Long Residence Time Show Improved Efficacy against Inflammatory and Neuropathic Pain.

Selective block of NaV1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several NaV1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.