Deepa Patel

Oral Bioavailability Enhancement of Acyclovir by Self-Microemulsifying Drug Delivery Systems (SMEDDS)

Acyclovir is a potent anti-viral agent useful in the treatment of Herpes Simplex Virus (HSV) infections. Acyclovir exerts its antiviral activity by competitive inhibition of viral DNA through selective binding of acyclovir to HSV-thymidine kinase. The main purpose of this work was to develop self-microemulsifying drug delivery system (SMEDDS) for oral bioavailability enhancement of acyclovir. Solubility of acyclovir was determined in various vehicles. SMEDDS is mixture of oils, surfactants, and co-surfactants, which are emulsified in aqueous media under conditions of gentle agitation and digestive motility that would be encountered in the gastro-intestinal (GI) tract. Pseudoternary phase diagrams were constructed to identify the efficient self-emulsifying region dilution study was also performed for optimization of formulation. SMEDDS was evaluated for its percentage transmittance, Assay of SMEDDS, phase separation study, droplet size analysis, zeta potential, electrophoretic mobility, and viscosity. The developed SMEDDS formulation contained acyclovir (50 mg), Tween 60 (60%), glycerol (30%) and sunflower oil (9%) was compared with the pure drug solution by oral administrating to male albino rats. The absorption of acyclovir from SMEDDS form resulted about 3.5 fold increase in bioavailability compared with the pure drug solution. Our studies illustrated the potential use of SMEDDS for the delivery of hydrophobic compounds such as acyclovir by oral route.

Recent advances in liposomal dry powder formulations: preparation and evaluation

Liposomal drug dry powder formulations have shown many promising features for pulmonary drug administration, such as selective localization of drug within the lung, controlled drug release, reduced local and systemic toxicities, propellant-free nature, patient compliance, high dose carrying capacity, stability and patent protection. Critical review of the recent developments will provide a balanced view on benefits of liposomal encapsulation while developing dry powder formulations and will help researchers to update themselves and focus their research in more relevant areas. In liposomal dry powder formulations (LDPF), drug encapsulated liposomes are homogenized, dispersed into the carrier and converted into dry powder form by using freeze drying, spray drying and spray freeze drying. Alternatively, LDPF can also be formulated by supercritical fluid technologies. On inhalation with a suitable inhalation device, drug encapsulated liposomes get rehydrated in the lung and release the drug over a period of time. The prepared LDPF are evaluated in vitro and in vivo for lung deposition behavior and drug disposition in the lung using a suitable inhaler device. The most commonly used liposomes are composed of lung surfactants and synthetic lipids. Delivery of anticancer agents for lung cancer, corticosteroids for asthma, immunosuppressants for avoiding lung transplantation rejection, antifungal drugs for lung fungal infections, antibiotics for local pulmonary infections and cystic fibrosis and opioid analgesics for pain management using liposome technology are a few examples. Many liposomal formulations have reached the stage of clinical trials for the treatment of pulmonary distress, cystic fibrosis, lung fungal infection and lung cancer. These formulations have given very promising results in both in vitro and in vivo studies. However, modifications to new therapies for respiratory diseases and systemic delivery will provide new challenges in conducting well-designed inhalation toxicology studies to support these products, especially for chronic diseases.

Self Micro-Emulsifying Drug Delivery System: Formulation Development and Biopharmaceutical Evaluation of Lipophilic Drugs

Several methods are being employed to improve the oral bioavailability of lipophilic drugs like using inert lipid vehicles such as Oils, Surfactant dispersions, Self Microemulsifying Drug Delivery Systems (SMEDDS) and Liposomes. SMEDDS is formulated with the help of oil or suitable lipid materials, Surfactants and hydrophilic Co-surfactants. Mixture of above ingredients can be filled in a soft capsule and ingested. When the mixture comes in contact with gastrointestinal fluid it forms a stable O/W Microemulsion. A pseudo ternary phase diagram is used for identifying the efficient Self micro-emulsification region. SMEDDS can be formulated to give sustained release by use of inert polymers. When this polymer comes in contact with GI fluids, it forms gelled polymeric matrix, which releases the micro-emulsified active principle in a continuous and prolonged manner. Physical characterization of SMEDDS should be carried out by droplet size analysis and spontaneity of emulsification. The purpose of this review is to provide a brief out line of the formulation of SMEDDS, possible mode for improving bioavailability, fate and evaluation of the same.

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

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.

Gene Delivery Using Viral Vectors

Successful gene delivery at the desired site of action is one of the major challenges for gene therapy over many decades. A successful gene delivery vector should be well equipped, with the ability to deliver an intact gene to the nucleus of targeted cells and express stably without any harmful toxicological and immunogenic responses. The development of viral vectors, such as adenovirus, adeno-associated virus, retrovirus, and herpesvirus, has advanced to clinical trials, with an aim to develop gene-based products for clinical use having adequate safety and biological functions. Viral vectors such as baculovirus, lentivirus, influenza viruses, human papilloma virus, and hepatitis B virus are also being researched for successful gene delivery vectors, as well as to find their application in the treatment of various genetic disorders. Various techniques such as single-photon emission computed tomography, magnetic resonance imaging, and ultrasmall superparamagnetic iron oxide particles are also being examined for their use in qualitative and quantitative information regarding the biodistribution of gene therapy vectors. In this chapter, attention is also given to describing the methods used in designing and constructing the vector-based treatment within safe dosage limits, after about a review of the biology of and required treatments for particular genetic disorders.

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 2268: Intracellular delivery of RGDfk grafted docetaxel encapsulated PLGA nanoparticles for breast cancer therapy

The purpose of the study was to prepare, characterized, assess in vitro antiproliferative activity, and determine DNA content and mode of cell death of RGDfk grafted Docetaxel encapsulated PLGA nanoparticles on breast cancer cells. DC- encapsulated nanoparticles were prepared using solvent evaporation technique. RGD-conjugation was done by carbodiimide coupling method. The nanoparticles were characterized for particle size and zeta potential by zeta sizer (Nano-ZS), entrapment efficiency and in-vitro drug release profile and RGD-conjugation efficiency. To establish antiproliferative effect, the RGD-conjugated nanoparticles of DC(PLGA-DC-RGD), unconjugated nanoparticles (PLGA-DC) and equal amounts of the free drug were studied on BT-20 and 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. The DC encapsulated RGD conjugated and unconjugated PLGA nanoparticles were found to be nanosized. The cytotoxicity indicated by IC 50 values suggests that RGD conjugated nanoparticles at 72hrs are 2.1 and 4 times more cytotoxic than unconjugated nanoparticles and drug solution for BT-20 cell lines. Similarly, for MDA-MB-231 cells, the RGD conjugated nanoparticles are 2 and 4 time more cytotoxic than unconjugated nanoparticles and drug solution. The results shows that the RGD conjugated nanoparticles at the concentration of 5nM showed 51% G2 phase arrest as compared to 30% G2 phase arrest with unconjugated nanoparticles at the same concentration at 24hrs. Two types of mode of cell death were found during the FITC-Annexin V assay. At 24 hrs, the treatment of 5nM of drug solution with cells resulted in 13.6% & 3.4 % necrotic & apoptotic cell fragments respectively. While at similar drug equivalent concentration, the PLGA-DC-RGD and PLGA-DC nanoparticles showed 6.4% & 28.3% and 5.1% & 25.6% necrotic & apoptotic cell fragments respectively. With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. To conclude, RGD conjugation to nanoparticles improved antiproliferative activity when assessed in vitro in breast cancer cell lines compared to free drug and unconjugated nanoparticles. The DNA content analysis depicted the cell cycle arrest in G 2 phase was more even at lower drug equivalent concentration of PLGA-RGD. 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 nanoparticles for breast cancer cell lines. 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. 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 2268.

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.