ABBV-319: A CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies.

Glucocorticoids are key components of the current standard-of-care regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for treatment of B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. CD19 displays restricted expression in normal B-cells and is up-regulated in B-cell malignancies. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payloads. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity when multiple MOAs are enabled. ABBV-319 also showed durable anti-tumor activity across multiple B-cell lymphoma PDX models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma post R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in Phase I clinical trial (NCT05512390).

Long-Acting Tumor-Activated Prodrug of a TGFßR Inhibitor.

Inhibition of TGFß signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFß receptor (TGFßR) inhibitors in cancer therapy. To restrict the activity of TGFßR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFßR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

In Vivo Efficacy of Tesevatinib in EGFR-Amplified Patient-Derived Xenograft Glioblastoma Models May Be Limited by Tissue Binding and Compensatory Signaling.

Tesevatinib is a potent oral brain penetrant EGFR inhibitor currently being evaluated for glioblastoma therapy. Tesevatinib distribution was assessed in wild-type (WT) and Mdr1a/b(-/-)Bcrp(-/-) triple knockout (TKO) FVB mice after dosing orally or via osmotic minipump; drug-tissue binding was assessed by rapid equilibrium dialysis. Two hours after tesevatinib dosing, brain concentrations in WT and TKO mice were 0.72 and 10.03 µg/g, respectively. Brain-to-plasma ratios (Kp) were 0.53 and 5.73, respectively. With intraperitoneal infusion, brain concentrations were 1.46 and 30.6 µg/g (Kp 1.16 and 25.10), respectively. The brain-to-plasma unbound drug concentration ratios were substantially lower (WT mice, 0.03-0.08; TKO mice, 0.40-1.75). Unbound drug concentrations in brains of WT mice were 0.78 to 1.59 ng/g. In vitro cytotoxicity and EGFR pathway signaling were evaluated using EGFR-amplified patient-derived glioblastoma xenograft models (GBM12, GBM6). In vivo pharmacodynamics and efficacy were assessed using athymic nude mice bearing either intracranial or flank tumors treated by oral gavage. Tesevatinib potently reduced cell viability [IC50 GBM12 = 11 nmol/L (5.5 ng/mL), GBM6 = 102 nmol/L] and suppressed EGFR signaling in vitro However, tesevatinib efficacy compared with vehicle in intracranial (GBM12, median survival: 23 vs. 18 days, P = 0.003) and flank models (GBM12, median time to outcome: 41 vs. 33 days, P = 0.007; GBM6, 44 vs. 33 days, P = 0.007) was modest and associated with partial inhibition of EGFR signaling. Overall, tesevatinib efficacy in EGFR-amplified PDX GBM models is robust in vitro but relatively modest in vivo, despite a high brain-to-plasma ratio. This discrepancy may be explained by drug-tissue binding and compensatory signaling.

Discovery of Orally Active Isofuranones as Potent, Selective Inhibitors of Hematopoetic Progenitor Kinase 1.

While the discovery of immune checkpoint inhibitors has led to robust, durable responses in a range of cancers, many patients do not respond to currently available therapeutics. Therefore, an urgent need exists to identify alternative mechanisms to augment the immune-mediated clearance of tumors. Hematopoetic progenitor kinase 1 (HPK1) is a serine-threonine kinase that acts as a negative regulator of T-cell receptor (TCR) signaling, to dampen the immune response. Herein we describe the structure-based discovery of isofuranones as inhibitors of HPK1. Optimization of the chemotype led to improvements in potency, selectivity, plasma protein binding, and metabolic stability, culminating in the identification of compound 24. Oral administration of 24, in combination with an anti-PD1 antibody, demonstrated robust enhancement of anti-PD1 efficacy in a syngeneic tumor model of colorectal cancer.

Enhanced antitumor immunity by a novel small molecule HPK1 inhibitor.

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy. METHODS: Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo. RESULTS: CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease. CONCLUSION: CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance.

Pharmacodynamics-based approach for efficacious human dose projection of BMS-986260, a small molecule transforming growth factor beta receptor 1 inhibitor.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine that has a wide array of biological effects. For decades, tumor biology implicated TGF-ß as an attractive therapeutic target due to its immunosuppressive effects. Toward this end, multiple pharmaceutical companies developed a number of drug modalities that specifically target the TGF-ß pathway. BMS-986260 is a small molecule, selective TGF-ßR1 kinase inhibitor that was under preclinical development for oncology. In vivo studies across mouse, rat, dog, and monkey and cryopreserved hepatocytes predicted human pharmacokinetics (PK) and distribution of BMS-986260. Efficacy studies of BMS-986260 were undertaken in the MC38 murine colon cancer model, and target engagement, as measured by phosphorylation of SMAD2/3, was assessed in whole blood to predict the clinical efficacious dose. The human clearance is predicted to be low, 4.25 ml/min/kg. BMS-986260 provided a durable and robust antitumor response at 3.75 mg/kg daily and 1.88 mg/kg twice-daily dosing regimens. Phosphorylation of SMAD2/3 was 3.5-fold less potent in human monocytes than other preclinical species. Taken together, the projected clinical efficacious dose was 600 mg QD or 210 mg BID for 3 days followed by a 4-day drug holiday. Mechanism-based cardiovascular findings in the rat ultimately led to the termination of BMS-986260. This study describes the preclinical PK characterization and pharmacodynamics-based efficacious dose projection of a novel small molecule TGF-ßR1 inhibitor.

Immunoaffinity microflow liquid chromatography/tandem mass spectrometry for the quantitation of PD1 and PD-L1 in human tumor tissues.

RATIONALE: High tumor expression of programmed cell death protein (PD1) and programmed death-ligand 1 (PD-L1) is thought to be associated with positive clinical outcomes after treatment with anti-PD1 or anti-PD-L1 agents. Several sensitive methods based on immunohistochemistry, ligand binding assay (LBA), and liquid chromatography/mass spectrometry involving the measurement of PD1 and PD-L1 expression have been reported. Here, we expand on the characterization of different tumor types using a highly specific, sensitive, and robust immunoaffinity liquid chromatography/tandem mass spectrometry (IA-LC/MS/MS)-based method for the simultaneous quantitation of PD1 and PD-L1 in tumor tissues. METHODS: Human tumor tissue samples were homogenized using a Precellys Evolution homogenizer. The samples were incubated with anti-PD1 and anti-PD-L1 capture polyclonal antibodies, which were bound to magnetic beads. Following enrichment, samples were digested with trypsin. A Waters iKEY HSS T3 1.8 um (150 µm × 100 mm) column with a gradient flow rate of 3 µL/min was used for chromatographic separation, and a Waters TQ-S triple quadrupole mass spectrometer was used for detection. Selected reaction monitoring (SRM) transitions with unit resolution for precursor/product ion masses were optimized for PD1 and PD-L1 surrogate peptides. RESULTS: The surrogate peptides LAAFPEDR for PD1 and FTVTVPK for PD-L1 yielded the most intense SRM transitions at m/z 459.7 > 516.2 and m/z 396.2 > 543.3, respectively, and thus were selected for the quantitation of PD1 and PD-L1. The lower limit of quantitation for PD1 and PD-L1 was 0.062 ng/mL with an assay range up to 10 ng/mL. Using this method, human PD1 and PD-L1 were detected and quantified from four different types of tumor tissues. The data show that PD1 expression level was highly correlated with that of PD-L1 in all tumor tissues analyzed here. CONCLUSIONS: A highly specific and sensitive immunoaffinity microflow LC/MS/MS method for the simultaneous quantification of PD1 and PD-L1 in tumor tissues was developed and implemented. This method combines the advantage of immuno-capture for analyte enrichment with the high specificity of detection of multiple surrogate peptides by LC/MS/MS. The quantification of PD1 and PD-L1 co-expression in tumor could help evaluate their role in assessing tumor type selection and patient stratification.

Enhancing Brain Retention of a KIF11 Inhibitor Significantly Improves its Efficacy in a Mouse Model of Glioblastoma.

Glioblastoma, the most lethal primary brain cancer, is extremely proliferative and invasive. Tumor cells at tumor/brain-interface often exist behind a functionally intact blood-brain barrier (BBB), and so are shielded from exposure to therapeutic drug concentrations. An ideal glioblastoma treatment needs to engage targets that drive proliferation as well as invasion, with brain penetrant therapies. One such target is the mitotic kinesin KIF11, which can be inhibited with ispinesib, a potent molecularly-targeted drug. Although, achieving durable brain exposures of ispinesib is critical for adequate tumor cell engagement during mitosis, when tumor cells are vulnerable, for efficacy. Our results demonstrate that the delivery of ispinesib is restricted by P-gp and Bcrp efflux at BBB. Thereby, ispinesib distribution is heterogeneous with concentrations substantially lower in invasive tumor rim (intact BBB) compared to glioblastoma core (disrupted BBB). We further find that elacridar-a P-gp and Bcrp inhibitor-improves brain accumulation of ispinesib, resulting in remarkably reduced tumor growth and extended survival in a rodent model of glioblastoma. Such observations show the benefits and feasibility of pairing a potentially ideal treatment with a compound that improves its brain accumulation, and supports use of this strategy in clinical exploration of cell cycle-targeting therapies in brain cancers.

Discovery of BMS-986260, a Potent, Selective, and Orally Bioavailable TGFßR1 Inhibitor as an Immuno-oncology Agent.

Novel imidazole-based TGFßR1 inhibitors were identified and optimized for potency, selectivity, and pharmacokinetic and physicochemical characteristics. Herein, we report the discovery, optimization, and evaluation of a potent, selective, and orally bioavailable TGFßR1 inhibitor, 10 (BMS-986260). This compound demonstrated functional activity in multiple TGFß-dependent cellular assays, excellent kinome selectivity, favorable pharmacokinetic properties, and curative in vivo efficacy in combination with anti-PD-1 antibody in murine colorectal cancer (CRC) models. Since daily dosing of TGFßR1 inhibitors is known to cause class-based cardiovascular (CV) toxicities in preclinical species, a dosing holiday schedule in the anti-PD-1 combination efficacy studies was explored. An intermittent dosing regimen of 3 days on and 4 days off allowed mitigation of CV toxicities in one month dog and rat toxicology studies and also provided similar efficacy as once daily dosing.

Brain Distributional Kinetics of a Novel MDM2 Inhibitor SAR405838: Implications for Use in Brain Tumor Therapy.

Achieving an effective drug concentration in the brain is as important as targeting the right pathway when developing targeted agents for brain tumors. SAR405838 is a novel molecularly targeted agent that is in clinical trials for various solid tumors. Its application for tumors in the brain has not yet been examined, even though the target, the MDM2-p53 interaction, is attractive for tumors that could occur in the brain, including glioblastoma and brain metastases. In vitro and in vivo studies indicate that SAR405838 is a substrate of P-glycoprotein (P-gp). P-gp mediated active efflux at the blood-brain barrier plays a dominant role in limiting SAR405838 brain distribution. Even though the absence of P-gp significantly increases the drug exposure in the brain, the systemic exposure, including absorption and clearance processes, were unaffected by P-gp deletion. Model-based parameters of SAR405838 distribution across the blood-brain barrier indicate the CLout of the brain was approximately 40-fold greater than the CLin The free fraction of SAR405838 in plasma and brain were found to be low, and subsequent Kpuu values were less than unity, even in P-gp/Bcrp knockout mice. These results indicate additional efflux transporters other than P-gp and Bcrp may be limiting distribution of SAR405838 to the brain. Concomitant administration of elacridar significantly increased brain exposure, also without affecting the systemic exposure. This study characterized the brain distributional kinetics of SAR405838, a novel MDM2 inhibitor, to evaluate its potential in the treatment of primary and metastatic brain tumors. SIGNIFICANCE STATEMENT: This paper examined the brain distributional kinetics of a novel MDM2-p53 targeted agent, SAR405838, to see its possible application for brain tumors by using in vitro, in vivo, and in silico approaches. SAR405838 is found to be a substrate of P-glycoprotein (P-gp), which limits its distribution to the brain. Based on the findings in the paper, manipulation of the function of P-gp can significantly increase the brain exposure of SAR405838, which may give an insight on its potential benefit as a treatment for primary and metastatic brain cancer.

Brain Distribution and Active Efflux of Three panRAF Inhibitors: Considerations in the Treatment of Melanoma Brain Metastases.

Targeted inhibition of RAF and MEK by molecularly targeted agents has been employed as a strategy to block aberrant mitogen-activated protein kinase (MAPK) signaling in melanoma. While the use of BRAF and MEK inhibitors, either as a single agent or in combination, improved efficacy in BRAF-mutant melanoma, initial responses are often followed by relapse due to acquired resistance. Moreover, some BRAF inhibitors are associated with paradoxical activation of the MAPK pathway, causing the development of secondary malignancies. The use of panRAF inhibitors, i.e., those that target all isoforms of RAF, may overcome paradoxical activation and resistance. The purpose of this study was to perform a quantitative assessment and evaluation of the influence of efflux mechanisms at the blood-brain barrier (BBB), in particular, Abcb1/P-glycoprotein (P-gp) and Abcg2/breast cancer resistance protein (Bcrp), on the brain distribution of three panRAF inhibitors: CCT196969 [1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea], LY3009120 1-(3,3-Dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea, and MLN2480 [4-pyrimidinecarboxamide, 6-amino-5-chloro-N-[(1R)-1-[5-[[[5-chloro-4-(trifluoromethyl)-2-pyridinyl]amino]carbonyl]-2-thiazolyl]ethyl]-]. In vitro studies using transfected Madin-Darby canine kidney II cells indicate that only LY3009120 and MLN2480 are substrates of Bcrp, and none of the three inhibitors are substrates of P-gp. The three panRAF inhibitors show high nonspecific binding in brain and plasma. In vivo studies in mice show that the brain distribution of CCT196969, LY3009120, and MLN2480 is limited, and is enhanced in transgenic mice lacking P-gp and Bcrp. While MLN2480 has a higher brain distribution, LY3009120 exhibits superior in vitro efficacy in patient-derived melanoma cell lines. The delivery of a drug to the site of action residing behind a functionally intact BBB, along with drug potency against the target, collectively play a critical role in determining in vivo efficacy outcomes.

Discovery of 4-Azaindole Inhibitors of TGFßRI as Immuno-oncology Agents.

The multifunctional cytokine TGFß plays a central role in regulating antitumor immunity. It has been postulated that inhibition of TGFß signaling in concert with checkpoint blockade will provide improved and durable immune response against tumors. Herein, we describe a novel series of 4-azaindole TGFß receptor kinase inhibitors with excellent selectivity for TGFß receptor 1 kinase. The combination of compound 3f and an antimouse-PD-1 antibody demonstrated significantly improved antitumor efficacy compared to either treatment alone in a murine tumor model.

Efficacy of the MDM2 Inhibitor SAR405838 in Glioblastoma Is Limited by Poor Distribution Across the Blood-Brain Barrier.

Controversy exists surrounding whether heterogeneous disruption of the blood-brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893-901. ©2018 AACR.

Barriers to Effective Drug Treatment for Brain Metastases: A Multifactorial Problem in the Delivery of Precision Medicine.

The treatment of metastatic lesions in the brain represents a serious unmet medical need in the field of neuro-oncology. Even though many effective compounds have demonstrated success in treating peripheral (non-CNS) tumors with targeted agents, one aspect of this lack of success in the brain may be related to poor delivery of otherwise effective compounds. Many factors can influence the brain delivery of these agents, but one key barrier is a heterogeneously "leaky" BBB that expresses efflux transporters that limit the BBB permeability for many targeted agents. Future success in therapeutics for brain metastases must take into account the adequate delivery of "active, free drug" to the target, and may include combinations of targeted drugs that are appropriate to address each individual patient's tumor type. This review discusses some issues that are pertinent to precision medicine for brain metastases, using specific examples of tumor types that have a high incidence of brain metastases.

Pharmacokinetic Assessment of Cooperative Efflux of the Multitargeted Kinase Inhibitor Ponatinib Across the Blood-Brain Barrier.

A compartmental blood-brain barrier (BBB) model describing drug transport across the BBB was implemented to evaluate the influence of efflux transporters on the rate and extent of the multikinase inhibitor ponatinib penetration across the BBB. In vivo pharmacokinetic studies in wild-type and transporter knockout mice showed that two major BBB efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), cooperate to modulate the brain exposure of ponatinib. The total and unbound (free) brain-to-plasma ratios were approximately 15-fold higher in the triple knockout mice lacking both P-gp and Bcrp [Mdr1a/b(-/-)Bcrp1(-/-)] compared with the wild-type mice. The triple knockout mice had a greater than an additive increase in the brain exposure of ponatinib when compared with single knockout mice [Bcrp1(-/-) or Mdr1a/b(-/-)], suggesting functional compensation of transporter-mediated drug efflux. Based on the BBB model characterizing the observed brain and plasma concentration-time profiles, the brain exit rate constant and clearance out of the brain were approximately 15-fold higher in the wild-type compared with Mdr1a/b(-/-)Bcrp1(-/-) mice, resulting in a significant increase in the mean transit time (the average time spent by ponatinib in the brain in a single passage) in the absence of efflux transporters (P-gp and Bcrp). This study characterized transporter-mediated drug efflux from the brain, a process that reduces the duration and extent of ponatinib exposure in the brain and has critical implications for the use of targeted drug delivery for brain tumors.

Restricted Delivery of Talazoparib Across the Blood-Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma.

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2-M arrest in vitroIn vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp-/- versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b-/- mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735-46. ©2017 AACR.

Heterogeneous Binding and Central Nervous System Distribution of the Multitargeted Kinase Inhibitor Ponatinib Restrict Orthotopic Efficacy in a Patient-Derived Xenograft Model of Glioblastoma.

This study investigated how differences in drug distribution and free fraction at different tumor and tissue sites influence the efficacy of the multikinase inhibitor ponatinib in a patient-derived xenograft model of glioblastoma (GBM). Efficacy studies in GBM6 flank (heterotopic) and intracranial (orthotopic) models showed that ponatinib is effective in the flank but not in the intracranial model, despite a relatively high brain-to-plasma ratio. In vitro binding studies indicated that flank tumor had a higher free (unbound) drug fraction than normal brain. The total and free drug concentrations, along with the tissue-to-plasma ratio (Kp) and its unbound derivative (Kp,uu), were consistently higher in the flank tumor than the normal brain at 1 and 6 hours after a single dose in GBM6 flank xenografts. In the orthotopic xenografts, the intracranial tumor core displayed higher Kp and Kp,uu values compared with the brain-around-tumor (BAT). The free fractions and the total drug concentrations, hence free drug concentrations, were consistently higher in the core than in the BAT at 1 and 6 hours postdose. The delivery disadvantages in the brain and BAT were further evidenced by the low total drug concentrations in these areas that did not consistently exceed the in vitro cytotoxic concentration (IC50). Taken together, the regional differences in free drug exposure across the intracranial tumor may be responsible for compromising efficacy of ponatinib in orthotopic GBM6.

ABCG2 and ABCB1 Limit the Efficacy of Dasatinib in a PDGF-B-Driven Brainstem Glioma Model.

Dasatinib is a multikinase inhibitor in clinical trials for glioma, and thus far has failed to demonstrate significant efficacy. We investigated whether the ABC efflux transporters ABCG2 and ABCB1 expressed in the blood-brain barrier (BBB), are limiting the efficacy of dasatinib in the treatment of glioma using genetic and pharmacologic approaches. We utilized a genetic brainstem glioma mouse model driven by platelet-derived growth factor-B and p53 loss using abcg2/abcb1 wild-type (ABC WT) or abcg2/abcb1 knockout mice (ABC KO). First, we observed that brainstem glioma tumor latency is significantly prolonged in ABC KO versus ABC WT mice (median survival of 47 vs. 34 days). Dasatinib treatment nearly doubles the survival of brainstem glioma-bearing ABC KO mice (44 vs. 80 days). Elacridar, an ABCG2 and ABCB1 inhibitor, significantly increases the efficacy of dasatinib in brainstem glioma-bearing ABC WT mice (42 vs. 59 days). Pharmacokinetic analysis demonstrates that dasatinib delivery into the normal brain, but not into the tumor core, is significantly increased in ABC KO mice compared with ABC WT mice. Surprisingly, elacridar did not significantly increase dasatinib delivery into the normal brain or the tumor core of ABC WT mice. Next, we demonstrate that the tight junctions of the BBB of this model are compromised as assessed by tissue permeability to Texas Red dextran. Finally, elacridar increases the cytotoxicity of dasatinib independent of ABCG2 and ABCB1 expression in vitro In conclusion, elacridar improves the efficacy of dasatinib in a brainstem glioma model without significantly increasing its delivery to the tumor core. Mol Cancer Ther; 15(5); 819-29. ©2016 AACR.

Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases.

Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood-brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood-brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show that P-gp and Bcrp are involved in limiting the brain distribution of GSK2126458 and provide a rationale for the lack of efficacy of GSK2126458 in the orthotopic PDX model.

Strategies to improve delivery of anticancer drugs across the blood-brain barrier to treat glioblastoma.

Glioblastoma (GBM) is a lethal and aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. Molecularly targeted agents hold great promise in treating these genetically heterogeneous tumors, yet have produced disappointing results. One reason for the clinical failure of these novel therapies can be the inability of the drugs to achieve effective concentrations in the invasive regions beyond the bulk tumor. In this review, we describe the influence of the blood-brain barrier on the distribution of anticancer drugs to both the tumor core and infiltrative regions of GBM. We further describe potential strategies to overcome these drug delivery limitations. Understanding the key factors that limit drug delivery into brain tumors will guide future development of approaches for enhanced delivery of effective drugs to GBM.