Martin J. D’Souza

Improved validated assay for the determination of mefloquine and its carboxy metabolite in plasma, serum and whole blood using solid-phase extraction and high-performance liquid chromatography

An improved high-performance liquid chromatography method using a low silanol activity octadecylsilica column and a solid-phase extraction technique is validated for the simultaneous analysis of mefloquine and its carboxy metabolite in whole blood, plasma and serum. An octadecylsilica column with high silanol activity is compared to a column of low activity in terms of pH dependent variability of chromatographic retention times for mefloquine and its carboxy metabolite. The low silanol activity column showed a relatively large mobile phase pH range where retention times for both components are consistent. The solid-phase extraction procedure consists of a simple protein precipitation step followed by sample concentration and extraction using a C18 membrane disk. The inter- and intra-assay variability for a therapeutic concentration of mefloquine (1000 ng/ml) is less than 2% in whole blood, plasma and serum while carboxymefloquine (1000 ng/ml) is 2.3% or less. At concentrations as low as 100 ng/ml the inter-assay variability is 6.2% or less for both analytes. This method shows a robust analytical procedure for the simultaneous analysis of mefloquine and its carboxy metabolite where precise measurements are useful in pharmacokinetic studies and in estimating drug compliance.

Formulation development of albumin based theragnostic nanoparticles as a potential delivery system for tumor targeting.

Background: Generally, chemotherapeutic drugs attack on both normal and tumor cells non-specifically causing life threatening side effects, necessitating targeted drug delivery to tumors. Purpose: The purpose of this study is to formulate albumin-based nanoparticles for tumor targeted drug delivery and noninvasive diagnosis. Methods: Albumin based nanoparticles (NPs) were developed as a potential tumor theragnostic agent by entrapping an anti cancer drug, doxorubicin and a near infrared dye, indocyanine green. Theragnostic nanoparticles were prepared using a well established coacervation/nanoprecipitation method followed by lyophilization. The formulation was optimized by varying process parameters using full factorial design of experiments. Release of dye and drug from NPs and physical state of the drug in NPs was studied using DSC. The NPs were injected into tumor bearing mice intravenously and imaged using a bio-imager. Results: The optimized nanoparticle formulation had a particle size of 125.0 ± 1.8 nm, poly dispersity index of 0.180 ± 0.057 and zeta potential of −32.7 ± 0.9 mV. The release of dye and drug from the nanoparticles was determined to be quasi-fickian diffusion mediated. Differential scanning calorimetry (DSC) studies revealed the stability of drug in the NP. The in-vivo studies showed enhanced accumulation of the dye loaded NPs at the tumor site than the dye solution, thus allowing noninvasive tumor monitoring. Conclusion: These results project the newly proposed and evaluated nanoparticle formulation as a potential tumor targeting and imaging delivery system.

Implementation of mixture design for formulation of albumin containing enteric-coated spray-dried microparticles

Context: Oral delivery of proteins has been a challenging as well as rapidly developing field. Objective: To implement mixture design of experiment to develop enteric-coated microparticles containing bovine serum albumin. Materials and methods: Microparticles were prepared using Buchi Spray Dryer 191. Simplex lattice mixture design computed using JMP software was implemented to compare the gastric protection rendered by Eudragit FS30D, Eudragit L100-55, and Eudragit S100 in microparticulate form. Further, an extreme vertices mixture design was used to incorporate hydroxypropyl methylcellulose (HPMC) Chitosan in the formulation to delay the release. Microparticle recovery yield and protein content in microparticles were evaluated. Results and discussions: The design was statistically significant with Eudragit S100 resulting in protein release of < 5% in acidic buffer. The selected optimal formulation had 70% of Eudragit S, 25% HPMC, and 5% Chitosan. The release profiles of protein from Eudragit S alone and along with HPMC were compared. About 25% decrease in the amount of protein release was observed 6 h post exposure of microparticle to buffer of pH 6.8. The microparticle recovery yield reduced from 77.99% to 71.56% which is due to addition of HPMC into the formulation matrix. Conclusion: Although all three Eudragit polymers can be used for enteric coating, in the microparticulate form Eudragit S resulted in higher gastric protection. Also use of HPMC along with Eudragit S resulted in further sustained release.

Oral delivery of microparticles containing plasmid DNA encoding hepatitis-B surface antigen

The role of albumin-based chitosan microparticles on enhancing immune response of plasmid DNA (pDNA) to hepatitis-B surface antigen (HBsAg) vaccine after oral administration was investigated in mice. The pDNA encoding HBsAg was entrapped in albumin microparticles using a one-step spray drying technique optimized in our laboratory. The encapsulated particles were also characterized in vitro for their shape, size, encapsulation efficiency, content, and stability. Albumin microparticles could protect the DNA from nuclease degradation as confirmed in our agarose gel study. Further immune modulating effect was studied in our formulation by measuring IgG antibodies in serum as well as IgA antibodies in fecal extracts. The mice were immunized with a prime dose of 100 μg of pDNA in microparticle formulations with and without interleukins biweekly until week 7 followed by a booster dose of equivalent strength on week 33 to compare the response with the subcutaneous group. The oral immunization with the pDNA to HBsAg microparticles gave significantly higher titer level of both sIgA and IgG at week 9 and 34, respectively, in oral vaccine with interleukins group when compared with the subcutaneous group. Thus, we observed an augmentation of both humoral and cellular immune responses for prolonged periods after immunization.

Spray-dried doxorubicin-albumin microparticulate systems for treatment of multidrug resistant melanomas

As multidrug resistance continues to be a problem in cancer treatment, controlled release delivery systems, such as microspheres, may aid to give a slower release of anticancer drugs into drug resistant tumor cells. In this study doxorubicin microspheres microencapsulated in an albumin matrix were prepared via the spray-drying method and characterized for particle size, content analysis, and release studies. They were then evaluated in vitro using drug resistant murine melanoma tumor cells for uptake and efflux studies. Spray-drying produced a dispersed powder with a mean particle size of 4.91 ± 1.2 µm, 60% product yield, and encapsulation efficiency of 85% and a ζ potential range of 37 to −40 mV. Intracellular doxorubicin concentrations were higher in drug resistant tumor cells treated with microspheres as opposed to solution, and efflux of doxorubicin from the tumor cell was inhibited. Greater cytotoxic effects were seen in tumor cells treated with doxorubicin microspheres versus solution up to and after 3 days. In vivo pharmacokinetic studies conducted in male Sprague–Dawley rats, revealed a plasma-level time curve indicative of a two-compartment model, and showed prolonged half-life of doxorubicin, greater area under the plasma concentration time curve, and increased plasma concentrations of doxorubicin in rats at 8 and 24 h after administration of doxorubicin microspheres.

Potentiation of pro-inflammatory cytokine suppression and survival by microencapsulated dexamethasone in the treatment of experimental sepsis

Cytokine inhibiting drugs are much more effective when delivered intracellularly to phagocytic cells in the microencapsulated form. Dexamethasone is a powerful inhibitor of TNF-α cytokine through inhibition of NF-κB which is a gene regulator of multiple pro-inflammatory cytokines. We have determined the effect of microencapsulated dexamethasone in pro-inflammatory cytokine release both in in vitro using whole blood model, and in vivo using peritonitis model of septic shock. Microspheres of 1–4 μm mean size were prepared by using albumin polymer matrix in a one-step spray drying method. Microencapsulated form of dexamethasone with concentration of 10−1, 10−2 and 10−3 M was compared to an equivalent concentration of solution form of dexamethasone in the in vitro whole blood model. The results show microencapsulated dexamethasone inhibited tumor necrosis factor-alpha (TNF-α) and interleukin-beta (IL-1β) significantly in comparison with the solution form of dexamethasone. The in vivo peritonitis model also demonstrated significant inhibition of TNF-α and IL-1β cytokines in microencapsulated form in comparison with solution form of dexamethasone. In the in vivo study, the animal survival rate after 5 days was 90%, dexamethasone in solution with gentamicin was 40% and gentamicin alone was 30%. This study demonstrates significantly improved inhibition of TNF-α and IL-1β both in vivo and in vitro when dexamethasone was used in microencapsulated form.

Oral delivery of particulate prostate cancer vaccine: In vitro and in vivo evaluation

Background: Various approaches have been evaluated for generation of efficient immune response against tumor antigens. Our approach exploits usage of particulate delivery to generate immune response against prostate cancer antigens. Purpose: The aim of this study was to evaluate the efficacy of prostate cancer vaccine derived from a murine prostate cancer cell line, TRAMP C2 in murine model via oral route using aleuria aurantia lectin as a targeting ligand for M-cells in the intestinal Peyer’s patches. Methods: The whole cell lysate (WCL) was obtained from TRAMP C2 murine prostate cancer cell line and was formulated into particles using one step spray drying process. For in vivo studies, 4–6 week old C57BL/6 male mice were vaccinated orally biweekly for 10 weeks. Serum samples were analyzed at regular intervals to determine serum IgG levels. The mice were then challenged with live TRAMP C2 cells to determine efficacy of the vaccine. Results: The serum IgG levels of vaccinated animals were higher compared to that of the controls. Moreover, the tumor growth was retarded significantly in the vaccinated mice compared to that of controls (p < 0.001). Conclusions: The above findings suggest that oral particulate WCL vaccine can trigger an immune response against prostate cancer antigens.

Enhanced bioavailability of orally administered antisense oligonucleotide to nuclear factor kappa B mRNA after microencapsulation with albumin

Abstract Antisense molecules that pertain to ribonucleic acid (RNA) and complementary to the messenger RNA (mRNA) are produced by transcription of a given gene. Antisense oligonucleotides have emerged as potential gene-specific therapeutic agents that are currently undergoing evaluation in clinical trials for a variety of diseases. When administered orally, antisense oligionucleotides have poor bioavailability as they are rapidly degraded by the acid in the stomach and by the enzymes in the intestine. Therefore, the enhancement of bioavailability after oral administration is highly desirable. This article shows the enhanced bioavailability of antisense oligonucleotides that targets nuclear factor kappa B (NF-κB) mRNA after encapsulating in an inert, biodegradable albumin polymer matrix that was administered via the oral route into a rat model. The bioavailability of the antisense oligonucleotides to NF-κB in microencapsulated form was compared to the solution form of the drug upon oral administration. The solution form had a low bioavailability of 9%, whereas the bioavailability for the microencapsulated form of the drug increased up to 70%. Moreover, the other pharmacokinetic parameters including half-life (t1/2) and volume of distribution (Vd) increased for the microencapsulated form compared to the solution form of the drug.

The effect of intracellular antioxidant delivery (catalase) on hydrogen peroxide and proinflammatory cytokine synthesis: a new therapeutic horizon

Reactive oxygen species synthesized by endothelial cells may be responsible for cell damage and altered physiologic function. After endotoxin stimulation, free radicals including H2O2 are produced. We have developed a method of intracellular drug delivery using albumin microcapsules. Catalase would be an excellent compound to alter H2O2 production. However, the large molecular size of catalase limits cellular penetration. Endothelial cells have been previously shown to readily phagocytoze albumin microcapsules. Methods: Catalase was added to an albumin solution to form a 10% solution of catalase. Microspheres from 2 to 7 μm in size were formed using a Bucchi spray dryer. Human endothelial cells were incubated with varying concentrations of microencapsulated catalase. The cells were then exposed to Escherichia coli endotoxin to determine if increased intracellular penetration of catalase would inhibit H2O2, nitrate, and cytokine synthesis. Results: There was a 7.2-fold increase in endothelial intracellular catalase after 48 h incubation. H2O2 was inhibited by 72%, nitrate 96%, TNF 90%, IL1 21%, IL6 42%. Conclusions: These results demonstrate that inhibition of H2O2 as a result of increased intracellular delivery of catalase inhibits proinflammatory cytokine synthesis after endotoxin exposure.

The effect of intracellular delivery of catalase and antisense oligonucleotides to NF-κB using albumin microcapsules in the endotoxic shock model

Microencapsulated (MC) catalase has been shown to inhibit H2O2 and tumor necrosis factor (TNF) in vitro after endotoxin stimulation. It is the purpose of this study to determine whether MC catalase improves pro-inflammatory cytokine inhibition and mortality in an endotoxic shock model in vivo. We also examined whether MC catalase and antisense oligonucleotides (ASO) to nuclear factor κB (NF-κB) together improved survival by inhibiting pro-inflammatory cytokines using different mechanisms. Methods: Albumin microcapsules containing catalase and ASO to NF-κB were prepared 2–7 μm in size by using a Büchi spray dryer. Progressively increasing doses of MC catalase, MC ASO to NF-κB, and the combination were given to rats before the administration of Escherichia coli endotoxin. Results demonstrated 60% survival in rats given 15 mg/kg MC catalase, 70% survival with 20 mg/kg MC ASO NF-κB, and 80% survival with the combination. TNF was inhibited by 53% in the MC catalase group 4 h after endotoxin administration, 43% in the ASO NF-κB group, and 78% in the combination group compared to controls. In conclusion, this study demonstrates the effectiveness of MC intracellular delivery of the naturally occurring antioxidant catalase in improving animal survival. The addition of ASO to NF-κB improved both cytokine inhibition and animal survival in endotoxic shock.