Discovery of GNE-502 as an orally bioavailable and potent degrader for estrogen receptor positive breast cancer.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

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.

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in Vivo Activity.

IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.

Development of Potent and Selective Pyrazolopyrimidine IRAK4 Inhibitors.

A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high-throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by cocrystal structures with the protein and small-molecule crystal structures, yielded a series of dihydrobenzofurans. This semisaturated bicycle provided superior druglike properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed target-based inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.

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.

Solubilization of a Poorly Soluble B-Raf (Rapidly Accelerated Fibrosarcoma) Inhibitor: From Theory to Application.

The oral bioavailability of a drug candidate is influenced by its permeability, metabolism, and physicochemical properties. Among the physicochemical properties, solubility and dissolution rate often are the most critical factors affecting the oral bioavailability of a compound. The increasing challenge for the pharmaceutical industry is to achieve reasonable oral bioavailability of poorly water-soluble drug candidates. G-F is a potent and selective B-Raf (rapidly accelerated fibrosarcoma) inhibitor with poor water solubility and moderate permeability, which resulted in an absorption-limited exposure in preclinical safety studies. The intrinsic solubility of G-F is 8 µg/mL (i.e., 0.0188 nM). In this study, pH adjustment combined with cosolvency, micellization, or complexation was applied as a technique to enhance the solubility of G-F. pH 9.5 and 4 buffers were selected to combine with the solubilization agents based on G-F's acidic pKa of 7.47. The solubilization power of each solubilization agent was determined based on the experimental data. The solubility G-F can be increased up to 4000-fold in a selected combination. The advantage of combination over individual solubilization agent was demonstrated. In this study, the understanding of the solubilization power of each solubilization agent played an important role in the formulation development of this development candidate.

Individual serum bile acid profiling in rats aids in human risk assessment of drug-induced liver injury due to BSEP inhibition.

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP).

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

From a novel HTS hit to potent, selective, and orally bioavailable KDM5 inhibitors.

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C-C bond between the pyrrolidine and pyridine. Replacing this with a C-N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (LogD) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5mg/kg resulted in unbound Cmax ∼2-fold of its cell potency (PC9 H3K4Me3 0.96µM), meeting our criteria for an in vivo tool compound from a new scaffold.

Compatibility study of a parenteral microdose polyethylene glycol formulation in medical devices and identification of degradation impurity by 2D-LC/MS.

Polyethylene glycol (PEG) based formulation and polyvinylchloride (PVC) tubing are frequently used for drug delivery and administration. The compatibility of a parenteral drug microdose formulation in intravenous infusion (IV) devices was studied to support the clinical determination of absolute bioavailability by the microdosing method. The investigational microdose formulation containing PEG was found prone to significant loss of potency within hours of storage in the PVC IV tubing due to degradation. Degradation occurred only when both PEG and PVC tubing were present. The degradation product could not be detected by LC/MS due to the significant interference from the high concentration of PEG (4%) matrix and the extremely low level of drug (0.6ppm). To obtain structural information of the degradation impurity and understand the cause of the degradation, a simple heart-cutting 2D-LC/MS approach was utilized to effectively separate the impurity from the complex PEG oligomers and overcome the matrix interference, enabling mass spectrometric analysis of the impurity. An oxidation- dominated mechanism was proposed in which the combination of PEG auto-oxidation and dehydrochlorination of the PVC tubing yielded an oxidative environment that enhanced radical propagation and accelerated degradation of the investigational parent drug.

Nanosuspension delivery of paclitaxel to xenograft mice can alter drug disposition and anti-tumor activity.

Paclitaxel is a common chemotherapeutic agent that is effective against various cancers. The poor aqueous solubility of paclitaxel necessitates a large percentage of Cremophor EL:ethanol (USP) in its commercial formulation which leads to hypersensitivity reactions in patients. We evaluate the use of a crystalline nanosuspension versus the USP formulation to deliver paclitaxel to tumor-bearing xenograft mice. Anti-tumor efficacy was assessed following intravenous administration of three 20 mg/kg doses of paclitaxel. Paclitaxel pharmacokinetics and tissue distribution were evaluated, and differences were observed between the two formulations. Plasma clearance and tissue to plasma ratio of mice that were dosed with the nanosuspension are approximately 33- and 11-fold higher compared to those of mice that were given the USP formulation. Despite a higher tumor to plasma ratio for the nanosuspension treatment group, absolute paclitaxel tumor exposure was higher for the USP group. Accordingly, a higher anti-tumor effect was observed in the xenograft mice that were dosed with the USP formulation (90% versus 42% tumor growth inhibition). This reduction in activity of nanoparticle formulation appeared to result from a slower than anticipated dissolution in vivo. This study illustrates a need for careful consideration of both dose and systemic solubility prior utilizing nanosuspension as a mode of intravenous delivery.

Systemic in vitro and in vivo evaluation for determining the feasibility of making an amorphous solid dispersion of a B-Raf (rapidly accelerated fibrosarcoma) inhibitor.

It is well acknowledged that oral bioavailability of a drug candidate is often influenced by factors such as the permeability, physico-chemical properties, and metabolism of the drug. Among the physico-chemical properties, solubility and dissolution rate are considered the most critical factors affecting the oral bioavailability of a compound G-F is a potent and selective B-Raf inhibitor with poor solubility and adsorption is limited by solubility at high doses. In order to overcome this issue using a spray-dried amorphous dispersion (SDD) formulation was evaluated. A combination of theoretical solubility prediction and in vitro dissolution, were used to predict the in vivo exposure of G-F. The predicted value was found to have good agreement with the in vivo exposure from dosing the crystalline and amorphous form of G-F. In general, this combined approach demonstrated that the amorphous form of G-F offers an advantage over the crystalline form of G-F in terms of solubility; in vitro dissolution and in vivo absorption were predictable and consistent with the literature. This systemic approach provides a great value for compound development.

In vitro and in vivo evaluation of amorphous solid dispersions generated by different bench-scale processes, using griseofulvin as a model compound.

Drug polymer-based amorphous solid dispersions (ASD) are widely used in the pharmaceutical industry to improve bioavailability for poorly water-soluble compounds. Spray-drying is the most common process involved in the manufacturing of ASD material. However, spray-drying involves a high investment of material quantity and time. Lower investment manufacturing processes such as fast evaporation and freeze-drying (lyophilization) have been developed to manufacture ASD at the bench level. The general belief is that the overall performance of ASD material is thermodynamically driven and should be independent of the manufacturing process. However, no formal comparison has been made to assess the in vivo performance of material generated by different processes. This study compares the in vitro and in vivo properties of ASD material generated by fast evaporation, lyophilization, and spray-drying methods using griseofulvin as a model compound and hydroxypropyl methylcellulose acetate succinate as the polymer matrix. Our data suggest that despite minor differences in the formulation release properties and stability of the ASD materials, the overall exposure is comparable between the three manufacturing processes under the conditions examined. These results suggest that fast evaporation and lyophilization may be suitable to generate ASD material for oral evaluation. However, caution should be exercised since the general applicability of the present findings will need to be further evaluated.

Evaluation of drug load and polymer by using a 96-well plate vacuum dry system for amorphous solid dispersion drug delivery.

It is well recognized that poor dissolution rate and solubility of drug candidates are key limiting factors for oral bioavailability. While numerous technologies have been developed to enhance solubility of the drug candidates, poor water solubility continuously remains a challenge for drug delivery. Among those technologies, amorphous solid dispersions (SD) have been successfully employed to enhance both dissolution rate and solubility of poorly water-soluble drugs. This research reports a high-throughput screening technology developed by utilizing a 96-well plate system to identify optimal drug load and polymer using a solvent casting approach. A minimal amount of drug was required to evaluate optimal drug load in three different polymers with respect to solubility improvement and solid-state stability of the amorphous drug-polymer system. Validation of this method was demonstrated with three marketed drugs as well as with one internal compound. Scale up of the internal compound SD by spray drying further confirmed the validity of this method, and its quality was comparable to a larger scale process. Here, we demonstrate that our system is highly efficient, cost-effective, and robust to evaluate the feasibility of spray drying technology to produce amorphous solid dispersions.

Potent and selective aminopyrimidine-based B-Raf inhibitors with favorable physicochemical and pharmacokinetic properties.

Recent clinical data provided proof-of-concept for selective B-Raf inhibitors in treatment of B-Raf(V600E) mutant melanoma. Pyrazolopyridine-type B-Raf inhibitors previously described by the authors are potent and selective but exhibit low solubility requiring the use of amorphous dispersion-based formulation for achieving efficacious drug exposures. Through structure-based design, we discovered a new class of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system, amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties, emerged as lead compounds with strong efficacy in a B-Raf(V600E) mutant mouse xenograft model. Synthesis, SAR, lead selection, and evaluation of key compounds in animal studies will be described.

Pyrazolopyridine inhibitors of B-Raf(V600E). Part 3: an increase in aqueous solubility via the disruption of crystal packing.

A single crystal was obtained of a lead B-Raf(V600E) inhibitor with low aqueous solubility. The X-ray crystal structure revealed hydrogen-bonded head-to-tail dimers formed by the pyrazolopyridine and sulfonamide groups of a pair of molecules. This observation suggested a medicinal chemistry strategy to disrupt crystal packing and reduce the high crystal lattice energy of alternative inhibitors. Both a bulkier group at the interface of the dimer and an out-of-plane substituent were required to decrease the compound's melting point and increase aqueous solubility. These substituents were selected based on previously developed structure-activity relationships so as to concurrently maintain good enzymatic and cellular activity against B-Raf(V600E).

Novel nanoparticles formulation for cassette dosing via intravenous injection in rats for high throughput pharmacokinetic screening and potential applications.

In recent years, one of the biggest challenges for pharmaceutical industry is to increase the speed of finding new medicines while at the same time controlling the ever rising cost of drug discovery and development. In order to increase the speed at which drug candidates are identified, high throughput assays (HTS) have been developed and have been widely implemented in the pharmaceutical industry. Cassette (or N-in-1) dosing for pharmacokinetic (PK) evaluation is the process of generating in vivo PK data in a higher throughput manner by dosing multiple compounds to individual animals. However, due to generally poor solubility of compounds being tested, high percentages of organic solvents are often used in the formulation vehicle in order to solubilize compounds for cassette studies. Utilization of high organic content in formulation vehicles can result in unwanted side effects and animal tolerability issues. The current study evaluates the suitability of using nanoparticles in an aqueous suspension for cassette IV dosing. Nanoparticles of 10 poorly soluble marketed drugs covering a wide range of clearances were prepared using an electrospray device and evaluated. PXRD, TGA and particle size data were obtained in order to ensure the quality for in vivo evaluation. Phosphate buffered saline (PBS) was used as the vehicle in IV cassette study using nanoparticles and pharmacokinetic estimates from this study were comparable to those from a traditional high organic formulation approach. The use of nanoparticles in an aqueous suspension formulation was demonstrated to be appropriate for cassette dosing.

Preclinical assessment of novel BRAF inhibitors: integrating pharmacokinetic-pharmacodynamic modelling in the drug discovery process.

The objective of these studies were to determine the preclinical disposition of the two BRAF inhibitors, G-F and G-C, followed by pharmacokinetic (PK)-pharmacodynamic (PD) modelling to characterize the concentration-efficacy relationship of these compounds in the Colo205 mouse xenograft model. With G-F, the relationship of pERK inhibition to concentration was also characterized. Compounds G-F and G-C were administered to mice, rats and dogs and the pharmacokinetics of G-F and G-C was determined. In addition, using indirect response models the concentration-efficacy relationship was described. The clearance of G-F was low; 0.625 and 4.65 mL/min/kg in rat and dog respectively. Similarly, the clearance of G-C was low in rat and dog, 0.490 and 4.43 mL/min/kg, respectively. Both compounds displayed low volumes of distribution (0.140-0.267 L/kg), resulting in moderate half-lives across species (~2.5 to 4 h). Bioavailability was formulation dependent and decreased with increasing dose. Using the indirect response models, the KC(50) (50% K(max); maximal response) value for tumor growth inhibition for G-F and G-C were 84.5 and 19.2 µM, respectively. The IC(50) for pERK inhibition in Colo205 tumors by G-F was estimated to be 29.2 µM. High exposures of G-F and G-C were required for efficacy. Despite good PK properties of low CL and moderate half-life, limitations in obtaining exposures adequate for safety testing in rat and dog resulted in development challenges.

Investigation of utilization of nanosuspension formulation to enhance exposure of 1,3-dicyclohexylurea in rats: Preparation for PK/PD study via subcutaneous route of nanosuspension drug delivery.

1,3-Dicyclohexylurea (DCU), a potent soluble epoxide hydrolase (sEH) inhibitor has been reported to lower systemic blood pressure in spontaneously hypertensive rats. One limitation of continual administration of DCU for in vivo studies is the compound's poor oral bioavailability. This phenomenon is mainly attributed to its poor dissolution rate and low aqueous solubility. Previously, wet-milled DCU nanosuspension has been reported to enhance the bioavailability of DCU. However, the prosperities and limitations of wet-milled nanosuspension have not been fully evaluated. Furthermore, the oral pharmacokinetics of DCU in rodent are such that the use of DCU to understand PK/PD relationships of sEH inhibitors in preclinical efficacy model is less than ideal. In this study, the limitation of orally delivered DCU nanosuspension was assessed by a surface area sensitive absorption model and pharmacokinetic modeling. It was found that dosing DCU nanosuspension did not provide the desired plasma profile needed for PK/PD investigation. Based on the model and in vivo data, a subcutaneous route of delivery of nanosuspension of DCU was evaluated and demonstrated to be appropriate for future PK/PD studies.