INVESTIGATING APOPTOSIS PATHWAY IN CHRONIC LYMPHOCYTIC LEUKEMIA: STROMAL INFLUENCE AND THERAPEUTIC ACTIVATION

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy. High levels of Bcl-2 and IAP family proteins are responsible for apoptotic-resistance and accumulation of mature CLL lymphocytes in bone-marrow, lymph nodes and peripheral blood. Besides pro-survival proteins, supporting stromal cells as well as soluble factors in the microenvironment of bone-marrow and lymph nodes provide survival advantage to CLL leukemic cells. Though the stromal – leukemia cell interactions has been studied extensively, in-depth-knowledge on the regulation of apoptotic pathway proteins in the context of microenvironment is still limited. To address this, the first part of our study focused on comprehensive analysis of 93 gene transcripts from seven families that are key regulators of apoptosis such as Bcl-2, IAPs, NF-kB, Caspase, TNF receptor superfamily (TNFRSF), Death Domain (DED) and Caspase Activation and Recruitment Domain (CARD) families using “real-time PCR apoptosis array-card”. While shorter incubations with supporting stromal cells induced significant changes in 22/93 transcripts including Bcl-2 (6/22), TNFRSF (5/22), DED (5/22), Caspase (2/22) family members, longer incubations induced significant changes in 8/93 transcripts from NF-kB (4/8) and Bcl-2 (2/8) family members. At the protein level, decrease or stable expressions in pro-apoptotic proteins, but a significant increase in anti-apoptotic proteins was observed. The second portion of this study involved therapeutic manipulation of executioner caspases in restoring apoptotic pathway that is frequently blocked by upstream anti-apoptotic Bcl-2 family members. A novel therapeutic approach that can potentially bypass the functional pro-survival proteins and directly activate terminal executioner procaspases to induce apoptosis was investigated. Executioner procaspase activities are blocked by zinc and zinc removal releases this inhibition. CLL primary cells express high levels of executioner procaspases-7 and -3 compared to normal lymphocytes providing a basis for therapeutic selectivity. Our studies with L14R8, a second generation PAC-1 analog [Procaspase activating compound-1 (PAC-1; Vanquish Oncology)] revealed that L14R8-induced cell death was specific to CLL cells and was independent of prognostic markers and microenvironmental factors. Mechanistically, L14R8 acted through direct activation of executioner procaspases -3 and -7 by removing labile Zinc ions. In summary, our investigations demonstrate cytotoxic actions of L14R8 and elucidate utility of this novel approach for combating CLL

A Possible Positive Feedback Modulation of Acetylcholine Release through the Stimulation of Alpha-7 Nicotinic Acetylcholine Receptors on Bipolar Neurons in Pig Retina

Glaucoma is associated with excitotoxicity in which increased glutamate release leads to apoptotic death of retinal ganglion cells (RGCs). Acetylcholine (ACh) has shown neuroprotection of RGCs through the stimulation of RGCs’ nicotinic acetylcholine receptors (nAChRs). The cholinergic amacrine cells are the only cells in retina which synthesize and release ACh. They get excitatory inputs from bipolar cells. The presence of α7nicotinic acetylcholine receptors (α7nAChRs) on the cholinergic amacrines, bipolar cells and RGCs is documented. Recently, stimulation of presynaptic nAChRs of cholinergic cells was shown to enhance ACh release in the rat superior cervical ganglion. Therefore, we hypothesized that α7nAChRs stimulation by tropisetron and PNU282987 (α7nAChR agonists) could induce ACh release through either direct stimulation of cholinergic amacrine α7nAChRs or indirect stimulation of bipolar α7nAChRs. For ACh release studies, pig eyes were dissected and cholinergic amacrine cells were labeled with 40μCi of 3H-choline in which the retina was flashed with light (3Hz) for 30 minutes to maximize 3H-choline uptake. Then, the eyecup was transferred to a perfusion chamber, washed for 20 minutes. 1 minute output fractions were collected into vials and prepared for liquid scintillation counting. To assess the viability of the preparation, light and kainate were applied. Light (2-3 fold increase), kainate (3-4 fold; 10-100μM), α7nAChR agonists (2-4 fold; 0.01-100nM) evoked ACh release greater than the baseline in the absence of DNQX (a glutamate receptor antagonist). In the presence of DNQX, which blocked bipolar input to cholinergic cells, α7nAChRs stimulation did not increase ACh release from baseline. Hence, the possibility of indirect input of bipolar α7nAChRs for ACh release was supported. Our results indicate that ACh release through α7nAChRs stimulation is possible and specifically the bipolar α7nAChRs release of ACh via an indirect positive feedback mechanism. During excitotoxicity, ACh released by amacrine cells, might feedback on bipolar nAChRs to increase ACh release. The neuroprotective effect of tropisetron on α7nAChRs on isolated RGCs is documented. Our study suggests that tropisetron might also protect RGCs through increased ACh release by possible indirect modulation. This study indicates the possibility of dual therapeutic targets of α7nAChRs in the retina for neuroprotection against RGC excitotoxicity

Controlled Release of Dipyridamole from Floating Matrices Prepared Using Glyceryl Behenate

Peer reviewe

Influence of Drug to Lipid Ratio and Release Modifier on Dipyridamole Release from Floating Lipid Granules

Peer reviewe

Self correcting monolithic floating matrix tablets of dipyridamole : Influence of formulation variables

The present investigation describes the influence of content of polyethylene oxide and ratio of lactose to starch 1500 on dipyridamole release from self correcting floating matrix tablets using 32 full factorial design. Tablets were evaluated for in vitro floating ability and drug release study using USP 24 type II apparatus using 0.1 N HCI at 100 rpm and temperature of 37±0.50. Multiple regression analysis and two way analysis of variance followed by Tukey test were performed for dependent variables. All formulations floated within 2 min regardless of factors studied and had total floating time of more than 12 h. It was observed that both the factors had significant influence on all dependent variable studied ( P 0.05) except the ratio of lactose to starch 1500 did not significantly contribute for Q1 ( P > 0.05). As content of polymer increased the release rate declined with increase in value of diffusion exponent giving anomalous drug release to zero order drug release ( P 0.05). It was observed that above a certain threshold level of polymer content further increase did not contribute significantly for percentage drug release. Lactose gave higher drug release with release mechanism towards zero order compared to starch 1500 which gave slower release with release mechanism towards diffusion based. Although both the factors significantly contribute for percentage drug release at different time point, the content of polymer dominated. It was observed that polymer content was a dominant controlling factor for drug release kinetics and it could be controlled by employing various blends of fillers.Peer reviewe

Statistical evaluation of influence of viscosity and content of polymer on dipyridamole release from floating matrix tablets: a technical note

The present investigation described the influence of viscosity and content of HPMC on dipyridamole release using 32 full factorial design. All formulations had desired floating lag time (<2 minutes) regardless of viscosity and content of polymeric matrices. Results of multiple regression analysis indicate that both factors significantly affect the diffusion exponent (n), release rate constant (k), and percentage drug release at 1 hour, 4 hours, 6 hours, and 12 hour, (P<.05). Mechanism of drug release was found to be anomalous type to case-II transport depending upon the viscosity and content of polymer. It was found that content of HPMC had a dominant role in the initial phase of drug release, while in the later phase viscosity of HPMC PredominatedPeer reviewe

Statistical evaluation of influence of viscosity of polymer and types of filler on dipyridamole release from floating matrix tablets

Present investigation describes statistically the influence of viscosity of hydroxypropyl methylcellulose and types of filler on dipyridamole release from floating matrix tablets using 3 2 full factorial design. Tablets were prepared using direct compression technique. Tablets were evaluated for in vitro floating ability and drug release study using USP 24 types II paddle apparatus using 0.1 N HCl (pH 1.2) at rotation of 100 rpm and temperature of 37±0.5°. Multiple regression analysis was performed for dependent variables studied and to evaluate contribution of factors with their levels two way ANOVA was performed followed by Tukey test. Polynomial equations and response surface plots were generated for all dependent variables. It was observed that both the factors had significant influence on all dependent variable studied (p<0.05). It was observed that as viscosity of polymer increases the release rate constant was decreased. Release rate obtained was highest when microcrystalline cellulose was employed as filler followed by dicalcium phosphate and lactose. Mechanism of drug release was anomalous types and depends upon viscosity of polymer and types of filler used. Microcrystalline cellulose gave release mechanism nearer to diffusion mechanism while dicalcium phosphate and lactose gave anomalous release. It was observed that both the factors had significant contribution on all dependent variables studied. A major controlling factor for kinetics of drug release was viscosity of polymer and it can be modified by incorporation of different types of filler. In initial phase of drug release viscosity of polymer and types of filler both governs the drug release while in later phase only the viscosity of polymer predominatesPeer reviewe

Development and validation of a HPTLC method for the estimation of cefuroxime axetil

A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the determination of cefuroxime axetil in dosage form. The stationary phase used was precoated silica gel 60F 254 . The mobile phase used was a mixture of chloroform:methanol:toluene (4:2:2 v/v). The detection of spot was carried out at 290 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 300 to 800 ng/spot for cefuroxime axetil. The limit of detection and the limit of quantification for the cefuroxime axetil were found to be 50 ng/spot and 100 ng/spot. The proposed method can be successfully used to determine the drug content of marketed formulation.Peer reviewe

Image_3_Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.pdf

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.</p