Sachin Naik

Comparison of active and passive targeting of docetaxel for prostate cancer therapy by HPMA copolymer-RGDfK conjugates.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α(v)β(3) integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α(v)β(3) integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.

Improved transnasal transport and brain uptake of tizanidine HCl‐loaded thiolated chitosan nanoparticles for alleviation of pain

The aim of this study was to prepare and characterize thiolated chitosan (TC) nanoparticles (NPs) of tizanidine HCl (TZ) and to evaluate its transport across monolayer of RPMI 2650 cells (Human nasal septum carcinoma cell line) followed by assessment of their pharmacokinetic and pharmacodynamic attributes, after intranasal (i.n.) administration. Chitosan was thiolated by carbodiimide method and thiolation was confirmed qualitatively and quantitatively. NPs were prepared using ionotropic gelation and evaluated for mucoadhesion using sheep nasal mucosa for drug permeation and cytotoxicity using monolayer of RPMI 2650 cells. Drug biodistribution was evaluated after technetium-99m labeling, visualized using gamma camera, and evaluated pharmacodynamically by measuring antinociceptive activity in mice. High mucoadhesion and permeation of drug were observed for TC NPs with least toxicity to nasal epithelial cells. Brain uptake and antinociceptive effect of the drug were significantly enhanced after thiolation of chitosan. This concludes that TC NPs, after i.n. administration, show significant increase in the mucoadhesion, reduction in cytotoxicity, enhanced permeation across cells monolayer, higher TZ brain uptake, and considerable increase in antinociceptive activity of TZ in mice. These features make TC an interesting polymer for demonstrating appreciable improvement of transnasal permeation of hydrophilic drugs, such as TZ, known to have limited permeation across blood-brain barrier.

In vitro mechanistic study of cell death and in vivo performance evaluation of RGD grafted PEGylated docetaxel liposomes in breast cancer.

Objectives of the investigations were to prepare RGD grafted docetaxel liposomes (RGD-PEG-LP-DC) using supercritical fluid technology and evaluate it in vitro for cytotoxicity, DNA content analysis, mechanism of cell death, and in vivo for pharmacokinetic and biodistribution studies in BALB/c mice. The RGD-PEG-LP-DCs were found to be most cytotoxic in BT-20 and MDA-MB-231 cell lines. The flowcytometry results shows at 48 hours, 96% G2 phase arrest for RGD-PEG-LP-DC at 5 nM drug concentration. The mode of cell death was found to be mainly by necrosis at low drug equivalent concentration (1 nM) and by apoptosis at high drug equivalent concentration (10 nM). With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. Biodistribution demonstrated that site specific drug distribution, t(1/2), and MRT improved significantly for RGD-PEG-LP-DC. From the studies site specific and sustained intracellular drug delivery from RGD-PEG-LP-DCs may provide promising strategy in enhancing embattled against breast cancer treatment. FROM

Microemulsion based intranasal delivery system for treatment of insomnia

The aim of this investigation was to prepare and characterize microemulsions/mucoadhesive microemulsions of Diazepam (D), Lorazepam (L) and Alprazolam (A), evaluate their pharmacodynamic performances by performing comparative sleep induction studies in male albino rats to assess their role in effective management of insomnia patients. Microemulsions of Diazepam (DME), Lorazepam (LME) and Alprazolam (AME) were prepared by titration method and characterized for drug content, globule size distribution and zeta potential, nasal toxicity and sleep induction. DME, LME and AME were transparent and stable with mean globule size and zeta potential in the range of 95.6 nm to 141.7 nm and -2.205 to -0.111 mV respectively. The prepared microemulsions exhibited reversible nasal toxicity. Onset of sleep and duration of sleep were observed in the following order: Lorazepam > Alprazolam>Diazepam. Faster onset of sleep following intranasal administration of microemulsions (<20 min) compared to oral administration (29-33 min) and control group (>45 min) for all three drugs suggested selective nose-to-brain transport of drug(s). Intranasal administration of microemulsion based formulations resulted in even faster onset of sleep (<12 min) with intranasal mucoadhesive microemulsion(s) resulting in fastest onset of sleep (<9 min). Duration of sleep was longest with the intranasal mucoadhesive microemulsions. These results are suggestive of larger extent of distribution of drug(s) to brain after intranasal administration of mucoadhesive microemulsion(s). These results are further corroborated with by loss or rightening reflex and startle reflex at earlier time points (within 10 min and 15 min respectively) with mucoadhesive microemulsions. Thus, the results of this investigation indicated rapid and larger extent of drug transport to the rat brain resulting in rapid induction of sleep followed by prolonged duration of sleep in rats following intranasal administration of mucoadhesive microemulsion(s). However, the role of microemulsion based formulations developed in this investigation in clinical practice can only be established after animal studies in two different animal models followed by extensive clinical trials.

Intranasal delivery of cyclobenzaprine hydrochloride-loaded thiolated chitosan nanoparticles for pain relief

Abstract The purpose of present investigation was to formulate and characterize the cyclobenzaprine HCl (CBZ)-loaded thiolated chitosan nanoparticles and assessment of in-vitro cell viability, trans-mucosal permeability on RPMI2650 cell monolayer, in-vivo pharmacokinetic and pharmacodynamic study of thiolated chitosan nanoparticles on Swiss albino mice after intranasal administration. A significant high permeation of drug was observed from thiolated chitosan nanoparticles with less toxicity on nasal epithelial cells. Brain uptake of the drug after 99mTc labeling was significantly enhanced after thiolation of chitosan. CBZ-loaded thiolated chitosan NPs significantly reverse the N-Methyl-d-Aspartate (NMDA)-induced hyperalgesia by intranasal administration than the CBZ solution. The studies of present investigation revealed that thiolation of chitosan significantly reduce trans-mucosal toxicity with enhanced trans-mucosal permeability via paracellular pathway and brain uptake of a hydrophilic drug (normally impermeable across blood brain barrier) and pain alleviation activity via intranasal route.

Development of Novel Lyophilized Mixed Micelle Amphotericin B Formulation for Treatment of Systemic Fungal Infection

The purpose of the study was to develop a stable, controlled release Amphotericin B (Amph B) lyophilized mixed micelle (MM) formulation using hydrogenated soya phosphatidylcholine (HSPC) and bile salts in monomeric form and evaluate it for therapeutic performance and side effects. The MM formulations of Amph B were prepared using sodium deoxycholate (NDC)/sodium taurocholate (NTC)/sodium cholate (NC), and HSPC. The optimization of bile salt: HSPC ratio in the MM formulation was done using 2(4) factorial designs. MM formulations were lyophilized using sucrose as a cryoprotectant and analyzed for per cent micelle yield, per cent drug loading and per cent entrapment efficiency. Comparative in vitro diffusion studies, hemolytic activity, and minimum inhibitory concentration (MIC) of developed MM formulations and marketed formulation (Fungizone) were evaluated using cellophane membrane, human red blood cells and Candida albicans respectively. In vivo studies of MM formulations were also carried out on Candida albicans infected white albino rats and compared with Fungizone. The optimized molar ratio of bile salt: HSPC was found to be 8:11. Among all MM formulations prepared, NDC/ HSPC formulation found to have maximum per cent drug loading (4.96+/-0.8%), per cent entrapment efficiency (93.2+/-1.5%) and per cent micelle yield (96.4+/-1.4%). The in vitro drug diffusion studies of developed MM formulations show close to zero-order diffusion kinetics. All MM formulations show improved therapeutic index and reduced side effects compared to reference formulation Fungizone. The NDC/HSPC MM formulation was found to have least hemolytic activity, MIC and mortality rate at all dosage levels. Improved antifungal activity and reduced toxicity of Amph B solubilized in MM may be due to higher cellular uptake of the drug by fungal cells of infected tissues from MM formulations. Hence, Amph B MM formulation could be a safe and effective viable alternative in the treatment of systemic fungal infections.

Abstract 3235: Improved in vitro efficacy study of RGD grafted docetaxel encapsulated solid lipid nanoparticles on breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL DC- encapsulated solid lipid nanoparticles (SLN) were prepared using Supercritical fluid technology using CO2 as an anti-solvent. RGD-conjugation was done by carbodiimide coupling method. The SLN were characterized for particle size and zeta potential by zeta sizer (Nano-ZS), entrapment efficiency and in-vitro drug release profile using HPLC assay, and RGD-conjugation efficiency by amino acid analysis method. The possibility of drug-lipid -RGD interaction was ascertained using a differential scanning colorimetry and X-ray diffraction techniques. To establish antiproliferative effect, the RGD-conjugated SLN of DC(RGD-SLN-DC), unconjugated SLN(DC-SLN) and equal amounts of the free drug were studied on MDA-MB-231 cells by CCK-8 assay method. DNA content analysis was done on flowcytometry by propadium iodide staining. The mode of cell death at different time and concentration was determined by FITC-Annexine V assay. Results: The DC encapsulated RGD conjugated and unconjugated SLN were found to be nanosized with optimum drug entrapment. The in-vitro release profiles of unconjugated and conjugated SLN were found to be similar except the fast drug release was observed in case of RGD conjugated pegylated SLN due to the fast hydration process of PEG molecules on the surface of the particles. The cytotoxicity indicated by IC50 values suggests that RGD conjugated SLN at 72hrs are 2.1 and 6.3 times more cytotoxic than unconjugated SLN and drug solution for for MDA-MB-231 cells. The results shows that the RGD conjugated SLN at the concentration of 4nM showed 97% G2 phase arrest as compared to 65% G2 phase arrest with unconjugated SLN at the same concentration at 48hrs. Two types of mode of cell death were found during the FITC-Annexin V assay. At 48 hrs, the treatment of 4nM of drug solution with cells resulted in 23.5% & 2.8 % necrotic & apoptotic cell fragments respectively. While at similar drug equivalent concentration, the RGD-SLN-DC and DC-SLN showed 2.9% & 76.3% and 3.4% & 42.3% necrotic & apoptotic cell fragments respectively. With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. Conclusions: To conclude, RGD conjugation to SLN improved antiproliferative activity when assessed in vitro in breast cancer cells compared to free drug and unconjugated SLN. The DNA content analysis depicted the cell cycle arrest in G2 phase was more even at lower drug equivalent concentration of RGD-SLN-DC. The mode of cell death was found to be mainly by necrosis at low drug equivalent concentration (1nM) and by apoptosis at high drug equivalent concentration (10nM) of RGD conjugated drug encapsulated SLN for breast cancer cells. Hence, it may be concluded that drug nanocarriers once conjugated with RGD can provide prolonged drug release to cytoplasm and can affectively target breast cancer and can probably reduce the limitations associated with breast cancer chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3235. doi:10.1158/1538-7445.AM2011-3235

Abstract 2772: Biodistribution and pharmacokinetics of RGD grafted PLGA nanoparticles after radiolabling with 99mTC on tumor bearing rat model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aim of the studies was to investigate influence of targeting (RGD) moiety on biodistribution of Docetaxel (DC) encapsulated PLGA-NPs in tumor bearing rats. Nanoparticles (Nps) were prepared by emulsification technique. RGD-conjugation was done by carbodiimide method. NPs were characterized for size, zeta potential, drug encapsulation efficiency, RGDfk content and drug interaction with PLGA and RGDfk. Radiolabeling of DC, DC loaded nanoparticles (PLGA-DC-NPs) and DC loaded and RGD grafted nanoparticles (PLGA-DC-RGDfk-NPs) formulations were achieved using stannous chloride as reducing agent (Babbar et al., 1991). The quality control of labeling efficiency was performed by ascending TLC using silica gel coated fiber sheets using acetone as a mobile phase. Stability of the Tc-99m labeled DC and formulations was determined in vitro in rabbit serum, normal saline by ascending TLC technique. Tc-99m labeled DC solution; PLGA-DC-NPs & PLGA-DC-RGDfk-NPs were administered i.v. via the tail vein at a dose of 20mg/kg body weight into the tumor bearing rats weighing about 250-300 gm. The blood samples were collected at 0.5, 1,2,4,6, 12 and 24 hrs post injection into anticoagulant. The different organs were isolated and homogenized at 1, 2, 4 and 24hrs. Radioactivities in the samples were measured using gamma counter. Pharmacokinetic parameters were calculated using Kinetica 4.4. Gamma scintigraphy study was done at 4, 24 and 48hrs. The drug loaded Nps were found to be in nano size with 72.5% drug entrapment efficiency and negative zeta potential. 99m Tc labeling efficiency was found to be high with stability up to 24hr. Calculated plasma AUC(0→24), AUC(0→∞), MRT, and t1/2 of DC solution and NPs were found to be in increasing order of PLGA-DC-RGDfk-NPs>PLGA-DC-NPs > DC solution. Which reveals the long circulation and slow clearance of drug loaded targeted NPs compared to non RGD grafted NPs and drug solution. Gamma scintigraphy studies reveals that the drug solution was rapidly eliminates from the tumor site as well as from the body. The DC-PLGA-RGDfk-NPs accumulate in the tumor very fast and show significantly high radioactivity than the PLGA-DC-NPs and drug solution alone for longer period of time. DC loaded Nps were prepared successfully exhibited in nano size with excellent resdispersibility. Labeling of DC and DC Nps with 99mTc resulted in stable complexes. The improved Pharmacokinetic and biodistribution data suggest the slow clearance of PLGA-DC-RGDfk with compared to PLGA-DC-NPs and drug solution from the body. Gamma schintigraphy studies demonstrated the high accumulation of RGD grafted Nps in the tumor for longer period of time. These findings of the studies are suggestive of drug localization in breast cancer site after grafting of Nps with RGD in animals and more extensive studies are necessary to develop a product suited for clinical use for better outcome then present chemotherapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2772.