A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

A Phase 0/I Pharmacokinetic and Pharmacodynamics and Safety and Tolerability Study of Letrozole in Combination with Standard Therapy in Recurrent High-Grade Gliomas.

PURPOSE: High-grade gliomas (HGG) carry a poor prognosis, with glioblastoma accounting for almost 50% of primary brain malignancies in the elderly. Unfortunately, despite the use of multiple treatment modalities, the prognosis remains poor in this population. Our preclinical studies suggest that the presence of aromatase expression, encoded by CYP19A1, is significantly upregulated in HGGs. Remarkably, we find that letrozole (LTZ), an FDA-approved aromatase inhibitor, has marked activity against HGGs. PATIENTS AND METHODS: We conducted a phase 0/I single-center clinical trial (NCT03122197) to assess the tumoral availability, pharmacokinetics (PK), safety, and tolerability of LTZ in recurrent patients with HGG. Planned dose cohorts included 2.5, 5, 10, 12.5, 15, 17.5, and 20 mg of LTZ administered daily pre- and postsurgery or biopsy. Tumor samples were assayed for LTZ content and relevant biomarkers. The recommended phase 2 dose (R2PD) was determined as the dose that resulted in predicted steady-state tumoral extracellular fluid (ECF; Css,ecf) >2 µmol/L and did not result in ≥33% dose-limiting adverse events (AE) assessed using CTCAE v5.0. RESULTS: Twenty-one patients were enrolled. Common LTZ-related AEs included fatigue, nausea, musculoskeletal, anxiety, and dysphoric mood. No DLTs were observed. The 15 mg dose achieved a Css,ecf of 3.6 ± 0.59 µmol/L. LTZ caused dose-dependent inhibition of estradiol synthesis and modulated DNA damage pathways in tumor tissues as evident using RNA-sequencing analysis. CONCLUSIONS: On the basis of safety, brain tumoral PK, and mechanistic data, 15 mg daily is identified as the RP2D for future trials.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Potentiation of temozolomide activity against glioblastoma cells by aromatase inhibitor letrozole.

PURPOSE: The DNA alkylating agent temozolomide (TMZ), is the first-line therapeutic for the treatment of glioblastoma (GBM). However, its use is confounded by the occurrence of drug resistance and debilitating adverse effects. Previously, we observed that letrozole (LTZ), an aromatase inhibitor, has potent activity against GBM in pre-clinical models. Here, we evaluated the effect of LTZ on TMZ activity against patient-derived GBM cells. METHODS: Employing patient-derived G76 (TMZ-sensitive), BT142 (TMZ-intermediately sensitive) and G43 and G75 (TMZ-resistant) GBM lines we assessed the influence of LTZ and TMZ on cell viability and neurosphere growth. Combination Index (CI) analysis was performed to gain quantitative insights of this interaction. We then assessed DNA damaging effects by conducting flow-cytometric analysis of Ë H2A.X formation and induction of apoptotic signaling pathways (caspase3/7 activity). The effects of adding estradiol on LTZ-induced cytotoxicity and DNA damage were also evaluated. RESULTS: Co-treatment with LTZ at a non-cytotoxic concentration (40 nM) reduced TMZ IC50 by 8, 37, 240 and 640 folds in G76, BT-142, G43 and G75 cells, respectively. The interaction was deemed to be synergistic based on CI analysis. LTZ co-treatment also significantly increased DNA damaging effects of TMZ. Addition of estradiol abrogated these LTZ effects. CONCLUSIONS: LTZ increases DNA damage and synergistically enhances TMZ activity in TMZ sensitive and TMZ-resistant GBM lines. These effects are abrogated by the addition of exogenous estradiol underscoring that the observed effects of LTZ may be mediated by estrogen deprivation. Our study provides a strong rationale for investigating the clinical potential of combining LTZ and TMZ for GBM therapy.

Brain pharmacokinetics and metabolism of the AMP-activated protein kinase selective inhibitor SBI-0206965, an investigational agent for the treatment of glioblastoma.

PURPOSE: Emerging evidence suggests that 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), a key regulator of cellular bioenergetics, is a novel target for the treatment of glioblastoma (GBM), a lethal brain tumor. SBI-0206965, an aminopyrimidine derivative, is a potent AMPK inhibitor being investigated for the treatment of GBM. Here we characterized the systemic and brain pharmacokinetics (PK) and hepatic metabolism of SBI-0206965. METHODS: We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in jugular vein cannulated rats. We assessed systemic PK of SBI-0206965 in rats and C57BL/6 mice following oral administration. Employing human, mouse, and rat liver microsomes we characterized the metabolism of SBI-0206965. RESULTS: SBI-0206965 is quickly absorbed, achieving plasma and brain extracellular fluid (ECF) peak levels within 0.25 - 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma (corrected for protein binding), brain partitioning is ~ 0.6-0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in mouse, rat and human was 325, 76 and 68 mL/min/kg, respectively. SBI-0206965 metabolites included desmethylated products, and the metabolism was strongly inhibited by ketoconazole, a CYP3A inhibitor. CONCLUSION: SBI-0206965 has adequate brain permeability but low relative oral bioavailability which may be due to rapid hepatic metabolism, likely catalyzed by CYP3A enzymes. Our observations will facilitate further development of SBI-0206965, and/or other structurally related molecules, for the treatment of GBM and other brain tumors.

A First-In-Class, Humanized Antibody Targeting Alternatively Spliced Tissue Factor: Preclinical Evaluation in an Orthotopic Model of Pancreatic Ductal Adenocarcinoma.

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

Gender-based differences in brain and plasma pharmacokinetics of letrozole in sprague-dawley rats: Application of physiologically-based pharmacokinetic modeling to gain quantitative insights.

Based on the discovery that the estrogen synthase aromatase (CYP19A1) is abundantly expressed in high- grade gliomas, the aromatase inhibitor, letrozole is being investigated in pre-clinical models as a novel agent against this malignancy. Here, we investigated the systemic and brain pharmacokinetics of letrozole following single and steady state dosing in both male and female Sprague-Dawley rats. Furthermore, we employed physiologically-based pharmacokinetic (PBPK) modeling to gain quantitative insights into the blood-brain barrier penetration of this drug. Letrozole (4 mg/kg) was administered intraperitoneally daily for 5 days (for males) and 11 days (for females) and intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured using HPLC and non-compartmental analysis was conducted employing WinNonlin®. Simcyp animal simulator was used for conducting bottom-up PBPK approach incorporating the specified multi-compartment brain model. Overall, marked gender-specific differences in the systemic and brain pharmacokinetics of letrozole were observed. Letrozole clearance was much slower in female rats resulting in markedly higher plasma and brain drug concentrations. At steady state, the plasma AUC 0-24 was 103.0 and 24.8 µg*h/ml and brain ECF AUC 0-12 was 24.0 and 4.8 µg*h/ml in female and male rats, respectively. The PBPK model simulated brain concentration profiles were in close agreement with the observed profiles. While gender-specific differences in letrozole PK are not observed in the clinical setting, these findings will guide the dose optimization during pre-clinical investigations of this compound. The PBPK model will serve as an important clinical translational tool.

Biotherapy of Brain Tumors with Phosphatidylserine-Targeted Radioiodinated SapC-DOPS Nanovesicles.

Glioblastoma multiforme (GBM), a common type of brain cancer, has a very poor prognosis. In general, viable GBM cells exhibit elevated phosphatidylserine (PS) on their membrane surface compared to healthy cells. We have developed a drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), that selectively targets cancer cells by honing in on this surface PS. To examine whether SapC-DOPS, a stable, blood-brain barrier-penetrable nanovesicle, could be an effective delivery system for precise targeted therapy of radiation, we iodinated several carbocyanine-based fluorescent reporters with either stable iodine (127I) or radioactive isotopes (125I and 131I). While all of the compounds, when incorporated into the SapC-DOPS delivery system, were taken up by human GBM cell lines, we chose the two that best accumulated in the cells (DiI (22,3) and DiD (16,16)). Pharmacokinetics were conducted with 125I-labeled compounds and indicated that DiI (22,3)-SapC-DOPS had a time to peak in the blood of 0.66 h and an elimination half-life of 8.4 h. These values were 4 h and 11.5 h, respectively, for DiD (16,16)-SapC-DOPS. Adult nude mice with GBM cells implanted in their brains were treated with 131I-DID (16,16)-SapC-DOPS. Mice receiving the radionuclide survived nearly 50% longer than the control groups. These data suggest a potential novel, personalized treatment for a devastating brain disease.

Phase 1 dose-finding study of metformin in combination with concurrent cisplatin and radiotherapy in patients with locally advanced head and neck squamous cell cancer.

BACKGROUND: The 5-year overall survival (OS) rate remains at 50% for patients with locally advanced head and neck squamous cell carcinoma (LAHNSCC), thereby underscoring the need for improved treatments. An antidiabetic agent, metformin, was found in retrospective studies to improve survival in patients with HNSCC. Therefore, the authors conducted a phase 1 dose escalation study combining metformin with chemoradiotherapy in patients with LAHNSCC. METHODS: Nondiabetic patients with LAHNSCC were enrolled in the current study to receive escalating doses of metformin and CRT based on the modified toxicity probability interval design. Metformin cohort doses included 2000 mg, 2550 mg, and 3000 mg daily in divided doses in addition to cisplatin (at a dose of 100 mg/m2 on days 1, 22, and 43) and standard radiotherapy (70 grays). Adverse events were categorized as per the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03). RESULTS: Twenty patients were enrolled, 2 of whom withdrew consent. The median age of the patients was 56 years and the majority were male (83%), were white (88%), had p16-positive disease (72%), and were tobacco users (61%). The median length of metformin exposure was 28.5 days. The most common grade ≥3 toxicities were nausea (11%), vomiting (11%), mucositis (6%), acute kidney injury (17%), anemia (6%), and leukopenia (11%). Dose-limiting toxicities included diarrhea and acute kidney injury. After a median follow-up of 19 months, the 2-year overall survival and progression-free survival rates were 90% and 84%, respectively. No hypoglycemia events or lactic acidosis were observed. Cisplatin administration did not appear to affect metformin pharmacokinetics. The maximum tolerated dose for metformin could not be determined given the limited number of patients who tolerated metformin during chemoradiotherapy. CONCLUSIONS: To the authors' knowledge, the current study is the first phase 1 trial combining metformin with chemoradiotherapy. Rates of overall survival and progression-free survival were encouraging in this limited patient population, and warrant further investigation in a phase 2 trial.

Plasma and brain pharmacokinetics of letrozole and drug interaction studies with temozolomide in NOD-scid gamma mice and sprague dawley rats.

PURPOSE: The aromatase inhibitor, letrozole, is being investigated in experimental animal models as a novel treatment for high-grade gliomas (HGGs). To facilitate optimal dosing for such studies, we evaluated the plasma and brain pharmacokinetics (PK) of letrozole in NOD-scid gamma (NSG) mice, which are frequently employed for assessing efficacy against patient-derived tumor cells. Furthermore, we evaluated the potential PK interactions between letrozole and temozolomide (TMZ) in Sprague-Dawley rats. METHODS: NSG mice were administered letrozole (8 mg/kg; i.p) as a single or multiple dose (b.i.d, 10 days). Brain tissue and blood samples were collected over 24 h. Letrozole and TMZ interaction study employed jugular vein-cannulated rats (three groups; TMZ alone, letrozole alone and TMZ + letrozole). Intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured employing HPLC and PK analysis was conducted using Phoenix WinNonlin®. RESULTS: In NSG mice, peak plasma and brain tissue letrozole concentrations (Cmax) were 3-4 and 0.8-0.9 µg/ml, respectively. The elimination half-life was 2.6 h with minimal accumulation following multiple dosing. In the drug interaction study, no PK changes were evident when TMZ and letrozole were given in combination. For instance, peak plasma and brain ECF TMZ levels when given alone were 14.7 ± 1.1 and 4.6 ± 0.6 µg/ml, respectively, and 12.6 ± 2.4 and 3.4 ± 0.8 µg/ml, respectively, when given with letrozole. CONCLUSIONS: These results will guide the optimization of dosing regimen for further development of letrozole for HGG treatment.

Preclinical pharmacokinetic evaluation to facilitate repurposing of tyrosine kinase inhibitors nilotinib and imatinib as antiviral agents.

BACKGROUND: Several tyrosine kinase inhibitors (TKIs) developed as anti-cancer drugs, also have anti-viral activity due to their ability to disrupt productive replication and dissemination in infected cells. Consequently, such drugs are attractive candidates for "repurposing" as anti-viral agents. However, clinical evaluation of therapeutics against infectious agents associated with high mortality, but low or infrequent incidence, is often unfeasible. The United States Food and Drug Administration formulated the "Animal Rule" to facilitate use of validated animal models for conducting anti-viral efficacy studies. METHODS: To enable such efficacy studies of two clinically approved TKIs, nilotinib, and imatinib, we first conducted comprehensive pharmacokinetic (PK) studies in relevant rodent and non-rodent animal models. PK of these agents following intravenous and oral dosing were evaluated in C57BL/6 mice, prairie dogs, guinea pigs and Cynomolgus monkeys. Plasma samples were analyzed using an LC-MS/MS method. Secondarily, we evaluated the utility of allometry-based inter-species scaling derived from previously published data to predict the PK parameters, systemic clearance (CL) and the steady state volume of distribution (Vss) of these two drugs in prairie dogs, an animal model not tested thus far. RESULTS: Marked inter-species variability in PK parameters and resulting oral bioavailability was observed. In general, elimination half-lives of these agents in mice and guinea pigs were much shorter (1-3 h) relative to those in larger species such as prairie dogs and monkeys. The longer nilotinib elimination half-life in prairie dogs (i.v., 6.5 h and oral, 7.5 h), facilitated multiple dosing PK and safety assessment. The allometry-based predicted values of the Vss and CL were within 2.0 and 2.5-fold, respectively, of the observed values. CONCLUSIONS: Our results suggest that prairie dogs and monkeys may be suitable rodent and non-rodent species to perform further efficacy testing of these TKIs against orthopoxvirus infections. The use of rodent models such as C57BL/6 mice and guinea pigs for assessing pre-clinical anti-viral efficacy of these two TKIs may be limited due to short elimination and/or low oral bioavailability. Allometry-based correlations, derived from existing literature data, may provide initial estimates, which may serve as a useful guide for pre-clinical PK studies in untested animal models.

Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids.

The incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active against Cryptococcus neoformansin vitro and had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

Renal Function Descriptors in Neonates: Which Creatinine-Based Formula Best Describes Vancomycin Clearance?

Growth and maturational changes have been identified as significant covariates in describing variability in clearance of renally excreted drugs such as vancomycin. Because of immaturity of clearance mechanisms, quantification of renal function in neonates is of importance. Several serum creatinine (SCr)-based renal function descriptors have been developed in adults and children, but none are selectively derived for neonates. This review summarizes development of the neonatal kidney and discusses assessment of the renal function regarding estimation of glomerular filtration rate using renal function descriptors. Furthermore, identification of the renal function descriptors that best describe the variability of vancomycin clearance was performed in a sample study of a septic neonatal cohort. Population pharmacokinetic models were developed applying a combination of age-weight, renal function descriptors, or SCr alone. In addition to age and weight, SCr or renal function descriptors significantly reduced variability of vancomycin clearance. The population pharmacokinetic models with Léger and modified Schwartz formulas were selected as the optimal final models, although the other renal function descriptors and SCr provided reasonably good fit to the data, suggesting further evaluation of the final models using external data sets and cross validation. The present study supports incorporation of renal function descriptors in the estimation of vancomycin clearance in neonates.

Evaluation of Vancomycin Use in Late-Onset Neonatal Sepsis Using the Area Under the Concentration-Time Curve to the Minimum Inhibitory Concentration ≥400 Target.

AIM: To develop a vancomycin population pharmacokinetic model and assess the probability of attaining a pharmacodynamic target associated with clinical and microbiological success, a ratio of the 24-hour area under the concentration-time curve to the minimum inhibitory concentration (MIC) ≥ 400, in a 5-year clinical cohort of preterm and term neonatal patients with late-onset staphylococcal sepsis. METHODS: Therapeutic drug monitoring data were obtained from septic neonates with ≥1 vancomycin concentration(s) from January 2006 to September 2011. Only neonates with a postnatal age of >72 hours and a positive microbiological culture were included. Population pharmacokinetic model was developed using nonlinear mixed effects modeling (NONMEM 7.2). Eleven demographic characteristics were evaluated as covariates. Probabilities of achieving the pharmacodynamic target were evaluated. RESULTS: A 1-compartment model with first-order elimination was constructed from 528 vancomycin concentrations collected from 152 preterm and term neonates. Body weight, creatinine clearance (CL), and postmenstrual age were identified as significant covariates. Estimated vancomycin CL and volume of distribution for typical neonates were 0.068 ± 0.03 L·h·kg and 0.62 ± 0.13 L/kg, respectively. Coagulase-negative staphylococci (85.5%) and Staphylococcus aureus (14.5%) were the common pathogenic organisms. The distribution of vancomycin MIC breakpoints was composed of approximately 70% MIC breakpoint of ≤2 mcg/mL. Approximately 54% of neonates, with a median serum creatinine concentration of 0.44 mg/dL, achieved the target ratio of 24-hour area under the concentration-time curve to the MIC ≥ 400 with a median daily dose of 30 (interquartile range, 21-42) mg/kg. CONCLUSIONS: Body weight, creatinine CL, and postmenstrual age significantly influenced vancomycin CL. The current vancomycin doses are acceptable at MICs ≤1 mcg/mL because they are likely to achieve the pharmacodynamic target in the majority of neonatal patients, although higher doses may be considered for more resistant staphylococcal infections.

Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

UNLABELLED: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

Safety, tolerability and pharmacokinetics of 2-pyridylacetic acid, a major metabolite of betahistine, in a phase 1 dose escalation study in subjects with ADHD.

Betahistine, a potent histamine H3 receptor antagonist, is being developed for the treatment of attention deficit hyperactivity disorder (ADHD) that manifests with symptoms such as hyperactivity, impulsivity and inattention. This study describes the pharmacokinetics of betahistine in ADHD subjects at doses higher than 50 mg. These assessments were made during a randomized, placebo-controlled, single blind, dose escalation study to determine the safety, tolerability and pharmacokinetics of once daily doses of 50 mg, 100 mg and 200 mg of betahistine in subjects with ADHD. Plasma levels of 2-pyridylacetic acid (2-PAA), a major metabolite of betahistine were quantified using a validated LC-MS/MS method and used for pharmacokinetic analysis and dose proportionality of betahistine. A linear relationship was observed in Cmax and AUC0-4 of 2-PAA with the betahistine dose (R2 0.9989 and 0.9978, respectively) and dose proportionality coefficients (ß) for the power model were 0.8684 (Cmax) and 1.007 (AUC0-4). A population pharmacokinetic model with first-order absorption of betahistine and metabolism to 2-PAA, followed by a first-order elimination of 2-PAA provides estimates of clearance that underscored the linear increase in systemic exposure with dose. There were no serious adverse events reported in the study, betahistine was safe and well tolerated at all the dose levels tested.

Preclinical pharmacological evaluation of letrozole as a novel treatment for gliomas.

We present data that letrozole, an extensively used aromatase inhibitor in the treatment of estrogen receptor-positive breast tumors in postmenopausal women, may be potentially used in the treatment of glioblastomas. First, we measured the in vitro cytotoxicity of letrozole and aromatase (CYP19A1) expression and activity in human LN229, T98G, U373MG, U251MG, and U87MG, and rat C6 glioma cell lines. Estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cells served as controls. Cytotoxicity was determined employing the MTT assay, and aromatase activity using an immunoassay that measures the conversion of testosterone to estrogen. Second, in vivo activity of letrozole was assessed in Sprague-Dawley rats orthotopically implanted with C6 gliomas. The changes in tumor volume with letrozole treatment (4 mg/kg/day) were assessed employing µPET/CT imaging, employing [(18)F]-fluorodeoxyglucose (F18-FDG) as the radiotracer. Brain tissues were collected for histologic evaluations. All glioma cell lines included here expressed CYP19A1 and letrozole exerted considerable cytotoxicity and decrease in aromatase activity against these cells (IC50, 0.1-3.5 µmol/L). Imaging analysis employing F18-FDG µPET/CT demonstrated a marked reduction of active tumor volume (>75%) after 8 days of letrozole treatment. Immunohistochemical analysis revealed marked reduction in aromatase expression in tumoral regions of the brain after letrozole treatment. Thus, employing multifaceted tools, we demonstrate that aromatase may be a novel target for the treatment of gliomas and that letrozole, an FDA-approved drug with an outstanding record of safety may be repurposed for the treatment of such primary brain tumors, which currently have few therapeutic options.

Manipulation of olfactory tight junctions using papaverine to enhance intranasal delivery of gemcitabine to the brain.

CONTEXT: Delivery of drugs from the nasal cavity to the brain is becoming more widely accepted, due to the non-invasive nature of this route and the ability to circumvent the blood brain barrier (BBB). OBJECTIVE: Because of similarities in the proteins comprising the olfactory epithelial tight junction (TJ) proteins and those of the BBB, we sought to determine whether papaverine (PV), which is known to reversibly enhance BBB permeability, could increase the delivery of intranasally administered gemcitabine to the central nervous system in rats. Experimental methods: Included intranasal administration of gemcitabine, fluorescein isothiocyanate-dextran beads and PV, histopathology, immunostaining, RT-PCR, western blot analysis, immunofluorescence localization, spectrofluorometric analysis, in vivo brain microdialysis, HPLC analysis and in vitro gemcitabine recovery. RESULTS AND DISCUSSION: PV transiently decreased the levels and altered immunolocalization of the TJ protein phosphorylated-occludin in the olfactory epithelium, while causing an approximately four-fold increase in gemcitabine concentration reaching the brain. The enhanced delivery was not accompanied by nasal epithelial damage or toxicity to distant organs. CONCLUSIONS: The ability to transiently and safely increase drug delivery from the nose to the brain represents a non-invasive way to improve treatment of patients with brain disorders.

The pharmacokinetics of letrozole in brain and brain tumor in rats with orthotopically implanted C6 glioma, assessed using intracerebral microdialysis.

PURPOSE: Emerging evidence suggests that primary and metastatic brain tumors may be sensitive to hormonal manipulations. However, the pharmacokinetics of compounds against such targets in the brain and, more importantly, in the brain tumor are not well characterized. Here, we investigated the pharmacokinetics of letrozole, a third-generation aromatase inhibitor, in the normal brain and in orthotopically implanted C6 glioma in Sprague-Dawley rats. METHODS: Intracerebral microdialysis was employed to determine the concentrations of unbound letrozole in the brain extracellular fluid (ECF) while simultaneously collecting blood samples (via jugular vein) to assess plasma levels of letrozole. Letrozole was administered intravenously at doses of 4, 6, 8 and 12 mg/kg, and ECF and blood samples were collected over 8 h. For assessing normal versus tumoral brain pharmacokinetics, letrozole (4 or 8 mg/Kg; i.v.) was administered 10 days after implantation of C6 glioma in the brain. Dual-probe intracerebral microdialysis was employed for assessing ECF samples from tumor-free and tumor-bearing regions of the brain. RESULTS: Normal brain ECF and plasma C max and AUC0-8h increased linearly with letrozole doses up to 8 mg/kg dose, but at 12 mg/kg, the pharmacokinetics were nonlinear. The relative brain distribution coefficients, AUCECF/AUCplasma (ub), were 0.3-0.98. The tumoral uptake of letrozole was 1.5- to 2-fold higher relative to tumor-free region. CONCLUSIONS: Thus, letrozole permeability across the blood brain barrier is high, and the exposure to the brain is dose dependent. Furthermore, the brain tumoral letrozole levels are markedly higher than those in the tumor-free regions, which underscore potential selectivity of its activity against tumor cells.