The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax's limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.

Abstract Introduction: Chimeric degradation activation compound (CDACs) are heterobifunctional molecules causing target protein degradation by simultaneously binding to the target protein as well as an E3-ubiquitin ligase. As an emerging new therapeutic modality, it is critical to understand the contribution of compound-specific parameters (e.g. drug exposure and binding potency to the target protein) and the system-specific parameters (e.g. target protein turnover) of CDACs to its in vivo effect. Method: To identify the key parameters for guiding compound optimization and to find the correlation between BTK degradation and the extent of tumor growth inhibition, a mechanistic PK/PD model was built (Phoenix WinNonlin 8.1) on a dataset generated from a BTK CDAC (Compound A). The PK/PD model was simplified mathematically compared to the full mechanistic models published previously1, since the binding affinity of Compound A to BTK was much higher than the binding affinity of CDAC to E3 ligase. This model can be linked to a tumor growth inhibition (TGI) mathematical model to find the correlation between target degradation and efficacy.Result: In mouse Rec-1 xenograft model, the BTK degradation and TGI was studied. The mice were dosed with Compound A orally at 0.3, 1, or 3 mg/kg, dose-dependent PK and BTK degradation in tumor was observed. After a single dose administration of Compound A, ~50% BTK degradation was observed in tumor at 24 h post dosing and the tumor BTK rebound to baseline at 72 hours post dosing at 3 mg/kg. The tumor BTK degradation was deeper after repeated dosing. A PK/PD model was built based on the data in this PK/PD study. The calculated BTK turnover half-life in Rec-1 model is about 16 h. In the efficacy study, Compound A was dosed orally once a day to the mice at 0.3, 1, or 3 mg/kg. The 3 mg/kg group achieved ~90% tumor growth inhibition in Rec-1 xenograft model. The PK/PD model was then linked with a mathematical model describing TGI2. A threshold level of BTK degradation in tumor to achieve tumor stasis was identified to be 96% for Rec-1 tumor model. At 90% tumor growth inhibition, average of 70% BTK was predicted to be degraded in tumor at steady state. The model was subsequently used to characterize the PK/PD relationship of BGB-16673. The efficacy of BGB-16673 was predicted using the model and it agreed with the observed value.Conclusions: The current model deepened our understanding of the PK/PD relationship of CDACs. The approach can be used to simulate different scenarios, e.g. different BTK turnover, potency or PK exposure. It can also be used in compound selection and optimization to predict repeated dose PD and efficacy from a single dose PK/PD data.1. J Pharmacokinet Pharmacodyn. 2021 Feb;48(1):149-163.2. Cancer Res. 2004 Feb 1;64(3):1094-101. Citation Format: Yue Wu, Shuran Li, Fan Wang, Nan Hu, Longbo Yin, Xiaomin Song, Jiye Zhang, Aiying Xu, Shasha Yang. Characterization of the correlation between BTK degradation and tumor growth inhibition of the BTK target protein degraders using PK/PD modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2110.

Bruton's tyrosine kinase (BTK) plays an essential role in B-cell receptor (BCR)-mediated signaling as well as the downstream signaling pathway for Fc receptors (FcRs). Targeting BTK for B-cell malignancies by interfering with BCR signaling has been clinically validated by some covalent inhibitors, but suboptimal kinase selectivity may lead to some adverse effects, which also makes the clinical development of autoimmune disease therapy more challenging. The structure-activity relationship (SAR) starting from zanubrutinib (BGB-3111) leads to a series of highly selective BTK inhibitors, in which BGB-8035 is located in the ATP binding pocket and has similar hinge binding to ATP but exhibits high selectivity over other kinases (EGFR, Tec, etc.). With an excellent pharmacokinetic profile as well as demonstrated efficacy studies in oncology and autoimmune disease models, BGB-8035 has been declared a preclinical candidate. However, BGB-8035 showed an inferior toxicity profile compared to that of BGB-3111.

Aberrant activation of Bruton's tyrosine kinase (BTK) plays an important role in pathogenesis of B-cell lymphomas, suggesting that inhibition of BTK is useful in the treatment of hematological malignancies. The discovery of a more selective on-target covalent BTK inhibitor is of high value. Herein, we disclose the discovery and preclinical characterization of a potent, selective, and irreversible BTK inhibitor as our clinical candidate by using in vitro potency, selectivity, pharmacokinetics (PK), and in vivo pharmacodynamic for prioritizing compounds. Compound BGB-3111 (31a, Zanubrutinib) demonstrates (i) potent activity against BTK and excellent selectivity over other TEC, EGFR and Src family kinases, (ii) desirable ADME, excellent in vivo pharmacodynamic in mice and efficacy in OCI-LY10 xenograft models.

The discovery and optimization of a novel series of GPR142 agonists are described. These led to the identification of compound 21 (LY3325656), which demonstrated anti-diabetic benefits in pre-clinical studies and ADME/PK properties suitable for human dosing. Compound 21 is the first GPR142 agonist molecule advancing to phase 1 clinic trials for the treatment of Type 2 diabetes.

A pharmacokinetic [PK]-driven screening process was implemented to select new agents for brain tumor chemotherapy from a series of low molecular weight anticancer agents [ON27x] that consisted of 141 compounds. The screening procedures involved a combination of in silico, in vitro and in vivo mouse studies that were cast into a pipeline of tier 1 and tier 2 failures that resulted in a final investigation of 2 analogues in brain tumor-bearing mice. Tier 1 failures included agents with a molecular weight of > 450 Da, a predicted log P (log P) of either <2 or > 3.5, and a cytotoxicity IC50 value of > 2 uM. Next, 18 compounds underwent cassette dosing studies in normal mice that identified compounds with high systemic clearance, and low blood–brain barrier [BBB] penetration. These indices along with a derived parameter, referred to as the brain exposure index, comprised tier 2 failures that led to the administration of 2 compounds [ON27570, ON27740] as single agents [discrete dosing] to mice bearing intracerebral tumors. Comparison of ON27570’s resultant PK parameters to those obtained in the cassette dosing format suggested a drug-drug interaction most likely at the level of BBB transport, and prompted the use of the in vitro MDCK-MDR1 transport model to help assess the nature of the discrepancy. Overall, the approach was able to identify candidate compounds with suitable PK characteristics yet further revisions to the method, such as the use of in vitro metabolism and transport assays, may improve the PK-directed approach to identify efficacious agents for brain tumor chemotherapy.

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

[Objective]To investigate the clinical efficacy for edaravone treatment of patients with acute ischemic stroke.[Methods]60 acute ischemic stroke patients in the department of neurology of our hospital from June 2008 to June 2010 were randomly divided into the treatment group with 30 cases and the control group with 30 patients.The treatment group was taken with edaravone treatment and compared with the control group about neurological score,infarct size and clinical efficacy.[Results]The total effective rate of the treatment group was 90%.The total effective rate in the control group was 66.7%,the difference was statistically significant(P﹤0.05).The difference of the neurological deficit scores in the treatment group and control group were statistically significant(P﹤0.05).The differences of the reducing infarct size in the two groups were statistically significant(P﹤0.05).[Conclusion]The application of edaravone treatment for acute ischemic stroke has better clinical efficacy.

e13584 Background: Neuroblastoma is an aggressive malignancy that accounts for 15% of pediatric cancer deaths. There are limited well characterized neuroblastoma models for use in preclinical trials. We characterized a human neuroblastoma orthotopic xenograft, performed comprehensive preclinical studies, and evaluated the pharmacokinetics of drugs targeting the PI3K pathway. Methods: Tumor tissue (MAST3) was obtained from a 2 year old with stage IV metastatic MYCN amplified neuroblastoma. Using ultrasound guidance, MAST3 cells were injected into the para-adrenal space of nod-scid mice. Tumors were monitored for growth with routine ultrasound and passaged. The initial patient tumor (MAST3) and tumor from each passage were extensively analyzed including: histology, electron microscopy (EM), gene expression profiling, single nucleotide polymorphism (SNP ) microarrays, whole genome sequencing (WGS), and spectral karyotyping (SKY). Using this orthotopic xenograft model, a randomized preclinical trial was conducted to evaluate the response to standard chemotherapeutic agents. Pharmacokinetic studies of oral BEZ-235, BKM-120, OSI-906 and everolimus were conducted. Results: Analysis of the histology, EM, gene expression profiling, SNP microarrays, WGS, and SKY showed minimal variability between the MAST3 tumor and the passaged tumors in the orthotopic xenografts. Notably, MAST3 metastasized to the liver, lung and spleen in vivo. Administering standard chemotherapeutic agents every 3 weeks for a total of 6 courses, we observed a significant response in 40% of treated mice. Further ongoing preclinical trials will evaluate the comparative efficacy of IGFR/PI3K/mTOR molecular targeted therapies. Conclusions: We developed the first well characterized neuroblastoma orthotopic xenograft. Our comprehensive characterization of this xenograft indicates that it retains many of the molecular, cellular and genetic properties of the primary lesion. A preclinical trial using standard chemotherapeutic agents has provided a valuable baseline for comparison with novel therapeutics and will inform our selection of the most promising agents to move into clinical trials.

Abstract Cerebral microdialysis, a technique used to monitor anticancer drug disposition in the central nervous system (CNS), is commonly used to sample extracellular fluid (ECF) for analysis of a single anticancer agent. However, combining anticancer drugs represents a promising strategy to overcome the highly resistant nature of some CNS tumors, such as malignant glioma. To date, the feasibility of simultaneous sampling multiple anti-cancer agents via cerebral microdialysis has not been reported. Given the role of PDGFRα and EGFR in providing multiple inputs for sustaining PI3K signaling, critical for glioma cell survival and proliferation, combining PDGFRα and EGFR inhibitors represents a valid treatment strategy for these tumors. The goals of the current study are to 1) to optimize microdialysis conditions to sample crenolanib and erlotinib as single agents or in combination from brain ECF (CbECF) and tumor ECF (CtECF) and 2) to characterize CNS penetration of crenolanib as a single agent and in combination with erlotinib. In vitro microdialysis experiments to sample crenolanib and erlotinib from aCSF determined that addition of 10% hydroxylpropyl betacyclodextrin increased the recovery of crenolanib and erlotinib by 16.1 and 2.6 fold, respectively. Zero-flow rate recovery method estimated stock concentrations with >85% accuracy. Sensitive LC-MS/MS methods will allow us to detect very low concentrations in the CNS (LLOQ for crenolanib and erlotinib is 0.1 and 0.5ng/mL, respectively). Unbound crenolanib concentrations in brain and tumor ECF were determined in a spontaneous glioma model under steady-state conditions achieved by using a miniosmotic pump loaded with crenolanib which administered a dose of 300 mg/kg/day. Plasma steady-state levels (Cpss) were 516.8±92.9ng/mL, mean ± SD. Crenolanib penetration into tumors [PTumor = CtECF/Cpss] was 3-fold higher than in normal brain [PBrain = CbECF/Cpss] (1.0±0.5 and 0.3±0.02, respectively). In a murine orthotopic xenograft model bearing SJG2 pediatric glioma tumors, crenolanib (300 mg/kg/day via miniosmotic pumps) inhibited PDGFRα phosphorylation (in vitro IC50 =8.1ng/mL) in 75% (3/4) of SJG2 tumors but failed to inhibit AKT phosphorylation or to induce apoptosis. We subsequently treated these animals with an EGFR inhibitor, erlotinib, and tried to determine whether co-administration of erlotinib affected the CNS penetration of crenolanib. Administration of 200 mg/kg/day crenolanib and erlotinib simultaneously via a miniosmotic pump resulted in a crenolanib Cpss of 210.1± 57 ng/mL and an erlotinib Cpss of 634±208 ng/mL. To conclude, optimizing microdialysis technique enhances the ability to simultaneously dialyze crenolanib and erlotinib in vitro. Studies to determine the effect of co-administration of erlotinib on crenolanib CNS penetration are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2742. doi:1538-7445.AM2012-2742

Cerebral microdialysis is used to study anticancer drug penetration in the central nervous system (CNS) and brain tumors in animal models. Genetically engineered murine models (GEMMs) have been recently used to study many aspects of CNS tumors since they represent a more relevant model than orthotopic brain tumor xenograft models. However, it is challenging to implant microdialysis cannula in these animals because T2-weighted magnetic resonance imaging (MRI) does not show the reference point (bregma) traditionally used to obtain stereotactic coordinates. Thus, an alternative reference point that can be visualized on MRI images is needed. In this study, a novel reference point, identified as the intersection between the olfactory bulb/frontal lobe border and the midline between cerebral hemispheres on T2-weighted MRI images, was used to calculate anterior–posterior and medial–lateral coordinates of brain tumors in a GEMM. This point overlies a visible crossover between the rostral rhinal vein and the midline suture on the mouse skull, allowing for the conversion of the MRI coordinates into surgical stereotactic coordinates. Postmortem MRI and histological examination confirmed accurate probe placement. This procedure will facilitate the accurate and precise implantation of microdialysis probes for the study of anticancer drug penetration in brain tumors of GEMMs. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4210–4214, 2011

A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [13C,d3]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid–liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6μm, 50mm×2.1mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate=50/50, v/v, at pH 5.0) delivered at 0.11mL/min. The retention time of both compounds was at 1.60min in a runtime of three min. Detection was achieved by an API-3200 LC–MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25–50ng/mL (R2≥0.990) for the CSF curve and from 1.0 to 3000ng/mL (R2≥0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤8.8% and ≤5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF=0.98, %CV≤4.97, n=6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.

OBJECTIVETo report real-time ophthalmoscopic findings during superselective intraophthalmic artery chemotherapy (SSIOAC) in a nonhuman primate model.METHODSSix adult male Rhesus macaques (Macacca mulatta) were randomly assigned to 1 of 2 treatment cohorts: melphalan (5 mg/30 mL) or carboplatin (30 mg/30 mL). Each animal underwent 3 separate SSIOAC procedures at 3-week intervals. Digital retinal images were obtained during each infusion. Intravenous fluorescein angiography was performed immediately after each procedure.RESULTSAll SSIOAC procedures were successfully completed. Toxicities were equally distributed between drug cohorts. Systemic toxicities included mild bone marrow suppression in all animals and anorexia in 1. One animal had greater than 50% narrowing of the treated ophthalmic artery after its second infusion. All 18 procedures (100%) resulted in pulsatile optic nerve and choroid blanching, retinal artery narrowing, and retinal edema. Of the 18 procedures, retinal artery sheathing was found during 17 (94%), and retinal artery precipitates were seen in 10 (56%); choroidal hypoperfusion was seen by fluorescein angiogram in 18 (100%).CONCLUSIONReal-time ophthalmic investigations are useful and, in our nonhuman primate model, indicate prevalent, acute ocular vascular toxicities during SSIOAC.CLINICAL RELEVANCEReal-time retinal imaging is feasible in a nonhuman primate model of SSIOAC. Application to SSIOAC in children may shed insight into reported vascular toxicities.

Nicotine self-administration causes adaptation in the mesocorticolimbic glutamatergic system, including the up-regulation of ionotropic glutamate receptor subunits. We therefore determined the effects of nicotine self-administration and extinction on NMDA-induced glutamate neurotransmission between the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA). On day 19 of nicotine SA, both regions were microdialyzed for glutamate while mPFC was sequentially perfused with Kreb's Ringer buffer (KRB), 200 mu M NMDA, KRB, 500 mu M NMDA, KRB, and 100 mM KCl. Basal glutamate levels were unaffected, but nicotine self-administration significantly potentiated mPFC glutamate release to 200 mu M NMDA, which was ineffective in controls. Furthermore, in VTA, nicotine self-administration significantly amplified glutamate responses to both mPFC infusions of NMDA. This hyper-responsive glutamate neurotransmission and enhanced glutamate subunit expression were reversed by extinction. Behavioral studies also showed that a microinjection of 2-amino-5-phosphonopentanoic acid (NMDA-R antagonist) into mPFC did not affect nicotine or sucrose self-administration. However, in VTA, NBQX (AMPA-R antagonist) attenuated both nicotine and sucrose self-administration. Collectively, these studies indicate that mesocortical glutamate neurotransmission adapts to chronic nicotine self-administration and VTA AMPA-R may be involved in the maintenance of nicotine self-administration.

PURPOSE:Tetrahydroisoquinolines (THIs) have demonstrated anti-cancer activity in rodent models of glioma, a form of brain cancer refractory to therapeutic intervention. In this study, peripheral and cerebrospinal fluid (CSF) pharmacokinetics in rats were determined to assess the drug developability of the novel THI EDL-155 for the treatment of glioma.METHODS:Serial blood and CSF samples were collected from rats following intravenous bolus administration of EDL-155 (10-20 mg/kg). Samples were analyzed by LC/MS/MS. Pharmacokinetic analyses using compartmental and noncompartmental methods were performed using the computer program WinNonlin. Plasma protein binding was measured using the charcoal adsorption method. The in vivo efficacy of EDL-155 (i.p. 20 mg/kg twice daily for 7 days) was assessed in rats with stereotactically implanted C6 glioma cells into the caudate.RESULTS:EDL-155 plasma concentration data were described by a one-compartment model. EDL-155 demonstrated rapid clearance (342.5+/-49.9 ml/min/kg), high volume of distribution (13.0+/-1.2 l/kg) and a terminal half-life of 23.7+/-1.5 min. Dose-normalized CSF area under the curve (AUC(CSF)) as a percentage of peripheral exposure (AUC(Plasma)) was 1.4%. EDL-155 was highly bound to plasma proteins (>93%). Intracranial tumor volume at 7 days post-implantation was approximately 30% smaller in animals treated with EDL-155 when compared to vehicle control animals (13.2+/-5.3 mm(3) vs. 18.7+/-6.3 mm(3); P=0.04).CONCLUSION:High clearance and extensive protein binding limit the brain availability of EDL-155 following systemic administration. EDL-155 treatment resulted in reduced tumor size despite limited blood brain barrier penetrability, which suggests that analogs with increased metabolic stability and brain penetrability may provide a therapeutic option for primary central nervous system tumors such as glioma. On-going studies are focused on the design, synthesis, and testing of novel analogs based upon these findings.

Nicotine, an addictive substance, is the major psychoactive component in cigarette smoke. Both α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate (NMDA) receptors are essential for the acute stimulative effects of nicotine on the mesocorticolimbic dopamine system, yet little is known about the effects of chronic nicotine treatment on glutamate receptors. Therefore, we used a model of chronic nicotine self-administration (SA), which emulates important aspects of nicotine intake by humans, to determine whether glutamate receptor subunits were affected. After 18 days of saline vs nicotine SA, ionotropic glutamate receptor subunit levels were determined in brain regions within the mesocorticolimbic system by Western blotting. In prefrontal cortex (PFC), the levels of NMDA receptor subunit 2A (NR2A) and NR2B were increased by 67% (p=0.04) and 83% (p=0.027), respectively. In the ventral tegmental area (VTA), glutamate receptor subunit 2/3 (GluR2/3) increased by 34% (p=0.011). Nicotine SA did not affect the expression of these subunits in dorsal striatum and nucleus accumbens. These findings suggest that chronic nicotine SA selectively increased the levels of ionotropic glutamate receptor subunits in a brain region-specific manner.