Nimita Dave

Solubilization of flurbiprofen into aptamer-modified PEG–PLA micelles for targeted delivery to brain-derived endothelial cells in vitro

Abstract Novel aptamer-functionalized polyethylene glycol–polylactic acid (PEG–PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer’s disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.

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 [18F]-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. Mol Cancer Ther; 14(4); 857–64. ©2015 AACR.

Abstract 4241: Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In this study, we investigated the potential role of aromatase (CYP19) as a target for the treatment of CNS malignancies, as well as brain disposition and anti-tumor efficacy of letrozole, an aromatase inhibitor, in Sprague Dawley rats. Cytotoxicity and aromatase activity of letrozole against human glioma cell lines were measured using MTT assay and Enzyme Immunoassay respectively. For brain and brain tumor PK of letrozole, rats with and without orthotopic implantation of C6 glioma received letrozole (4 - 12 mg/kg; i.v. andor oral). Dual probe intracerebral microdialysis was performed to determine the unbound extracellular fluid (ECF) letrozole concentrations. Serial ECF and blood samples were simultaneously collected over 8 hrs. µPET/CT imaging was performed using 18F FDG to evaluate changes in active tumor volumes pre- and post-treatment of letrozole. Brain tissues were collected at the end of the experiment for histological evaluations. All glioma cell lines included in this study expressed CYP19 and letrozole exerted marked cytotoxicity against these cells ( IC50s 0.1 - 3.5 μM). The relative brain distribution coefficients, measured as the ratio of the observed AUC in ECF and AUC of unbound letrozole in plasma (AUCecf/AUCp,ub) ranged from 0.31 - 0.98. Furthermore, the tumoral ECF levels of letrozole was 1.5 - 2 fold higher relative to tumor-free region of the brain, resulting in tumoral ECF Cmax values that were 10 and 35-fold higher than the observed IC50 value of 0.1 µM against C6 gliomas cells in culture. µPET/CT imaging showed a marked reduction of active tumor volume (75-90%) after 8-10 days of letrozole treatment (N=7). Oral administration of letrozole showed similar brain disposition and efficacy of letrozole. Immunohistochemical analysis of aromatase demonstrated selectivelymarkedly higher aromatase expression in tumoral regions of the brain as well asand a clear considerable reduction in aromatase expression in letrozole the treated rats relative to the treatment group as compared to the control group. Thus, employing multifaceted and cutting edge in vitro and in vivo methods, we conclude: a) aromatase is abundantly expressed in glioma cell lines examined, b) letrozole exerts marked cytotoxicity in these cells presumably due to aromatase inhibition, c) letrozole has selectively higher accumulation in tumoral region of the brain and d) In vivo µPET/CT studies show marked efficacy of letrozole on C6 glioma in a preclinical rat model. Note: This abstract was not presented at the meeting. Citation Format: Nimita Dave, Pankaj B. Desai, Gary A. Gudelsky, Kathleen LaSance, Lionel M.L. Chow, Xiaoyang Qi. Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4241. doi:10.1158/1538-7445.AM2014-4241

Abstract 3360: The pharmacokinetics of letrozole in brain and brain tumor in rats (orthotopically implanted C6 glioma) assessed using microdialysis.

The presence of blood-brain or blood-tumor barrier is well known to restrict access of drugs to CNS malignancies. For most drugs, there is limited quantitative information pertaining to the time course of drug absorption, distribution and elimination and the resulting drug exposure. In this study, we employed intracerebral microdialysis to access the extracellular fluid (ECF) to assess the pharmacokinetics of letrozole in brain and primary glioma in rats. Intracerebral microdialysis (perfusion rate; 2 μl/min) was performed in female rats to determine the unbound letrozole ECF concentrations. Serial ECF samples were collected over a 30 min period up to 8 hrs following drug administration. Blood samples were simultaneously collected through the jugular vein cannulation. Letrozole concentrations in extracted plasma and dialysate samples were analyzed employing HPLC. In vivo relative recovery was estimated to be 9.5%. Normal brain versus plasma pharmacokinetic study employed four doses (4, 6, 8 & 12 mg/kg; i.v.). For tumoral pharmacokinetic studies, C6 glioma cells were orthotopically implanted in the right striatum while the left hemisphere served as control. Ten days after tumor implantation, dual probe microdialysis was performed under the aforementioned experimental conditions following letrozole administration at 4 and 8 mg/kg doses. Pharmacokinetic analyses were carried out using WinNonlin 6.2 (Pharsight Inc., CA). Normal brain ECF and plasma peak concentrations (Cmax) and the Area Under the Curve (AUC0-8hr) increased linearly with increase in doses of letrozole administration up to 8mg/kg dose. However, at higher dose of 12mg/kg brain and plasma AUC0-8hr increased non-linearly. The relative brain distribution coefficient, calculated as (AUCbrain ecf/AUCplasma), was 0.122 at the lowest dose of 4mg/kg letrozole, whereas, 0.3 for the other three doses. The distribution of letrozole into the brain was rapid with the time to attain the peak levels (Tmax) ranging between 1.3-1.5 hr for the four doses. Furthermore, the tumoral uptake of letrozole was 1.5 - 2 folds higher relative to tumor-free region of the brain for the two doses of letrozole, 4 and 8 mg/kg. Histological evaluation confirmed the presence and restriction of glioma in the right hemisphere, and the passage of the microdialysis probe in the tumor mass. Our extensive in vivo pharmacokinetic study represents the first quantitative assessment of letrozole exposure to the brain and brain tumor tissues. These studies clearly demonstrate a rapid and high partitioning of letrozole to the brain with higher accumulation in the tumoral region of the brain. Further pharmacokinetic and pharmacodynamics modeling and simulations of the observed unbound ECF concentrations are in progress to assess clinical implications of our findings. Citation Format: Nimita Dave, Xiaoyang Qi, Gary G. Gudelsky, Pankaj B. Desai. The pharmacokinetics of letrozole in brain and brain tumor in rats (orthotopically implanted C6 glioma) assessed using microdialysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2013-3360