Martin J. D'Souza

Increased carbamazepine plasma concentrations after fluoxetine coadministration

The interaction between fluoxetine and carbamazepine was investigated in six normal, healthy male volunteers (aged 23 to 40 years). Subjects were given carbamazepine, 400 mg every morning, for 3 weeks. Venous carbamazepine blood samples were obtained at baseline and 1, 2, 4, 6, 8, 10, 12, and 24 hours after the morning dose. Fluoxetine, 20 mg every morning, was then coadministered with carbamazepine for 7 days. Venous carbamazepine blood samples were again obtained as described. Carbamazepine and carbamazepine‐10,11‐epoxide (CBZE) were assayed by HPLC. Addition of fluoxetine resulted in a significant increase in the area under the concentration‐time curve of carbamazepine (105.93 ± 18.05 μg/ml · hr versus 134.97 ± 12.15 μg/ml · hr; t = 3.284; df = 5;p = 0.022) and CBZE (11.6 ± 1.93 μg/ml · hr versus 15.2 ± 2.4 μg/ml · hr; t = 2.805; df = 5; p = 0.038). Both oral and intrinsic clearance of carbamazepine was decreased significantly on fluoxetine addition (3.87 ± 0.68 L/hr versus 2.98 ± 0.26 L/hr; t = 3.025; df =5; p = 0.029 and 17.90 ± 4.9 L/hr versus 11.92 ± 1.4 L/hr; t = 3.037; df = 5;p = 0.029, respectively). No significant changes were determined for fraction of absorbed dose, volume of distribution, absorption rate constant, and elimination rate constant. These findings suggest that fluoxetine can inhibit the metabolism of carbamazepine. Careful monitoring of patients is recommended when these two drugs are coadministered.

Mechanisms of T cell‐induced glomerular injury in anti‐glomeruler basement membrane (GBM) glomerulonephritis in rats

The effector mechanisms of T cell‐dependent acute glomerular injury were studied in autologous phase anti‐GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti‐rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4+ and CD8+ T cells. T cell depletion, using an anti‐CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell‐dependent. Inhibition of T helper cell function using an anti‐CD4 MoAb prevented proteinuria and glomerular macrophage and CD4+ T cell influx, but not accumulation of CD8+ T cells. Depletion of CD8+ T cells also prevented proteinuria and the influx of macrophages and CD8+ T cells, but not accumulation of CD4+ T cells. Macrophage depletion, using micro‐encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4+ or CD8+ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell‐dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti‐GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell‐mediated injury (cellular cytotoxicity) is not involved.

Evaluation of PLGA Microspheres as Delivery System for Antitumor Agent-Camptothecin

Abstract Camptothecin (CPT) and its analogues are a new class of anticancer agents that have been identified over the past several years. Camptothecin exists in two forms depending on the pH: An active lactone form at pH below 5 and an inactive carboxylate form at basic or physiological neutral pH. Poly(lactide-co-glycolide) (PLGA) microspheres have been considered good delivery vehicles for CPT because of acidic microenvironment formed through PLGA degradation. The objective of this study is to investigate antitumor activity of CPT after it is encapsulated in PLGA microspheres. In this study, PLGA microspheres containing various CPT loadings were prepared and characterized. Cytotoxicity of these microspheres to B16 melanoma cells was then evaluated, and uptake of microspheres by B16 cells was also studied. Analysis of drug stability revealed that CPT is released from the microspheres in its active lactone form over the entire release duration. It was also found that there was no interaction between CPT and PLGA matrix within microspheres through Differential Scanning Calorimetry (DSC) and Fourien Transform Infrared Spectroscopy (FT-IR) and hign performance liquid chromatography (HPLC) studies. Cytotoxicity assay showed that CPT encapsulated in PLGA microspheres still retained its antitumor potency. Uptake study revealed quick uptake of the microspheres by B16 cells, which was desirable. It was concluded that PLGA microspheres were suitable delivery vehicles to stabilize and deliver CPT for the treatment of cancer.

Development of sulforaphane-encapsulated microspheres for cancer epigenetic therapy

Even though conventional chemotherapeutic management of cancer has reduced morbidity and mortality to a great extent, virtually all chemotherapeutic agents cause damage to healthy cells, necessitating exploration of novel anticancer agents that exert their effects through an alternate mode of action. Objectives of our research were twofold. First, we explored the promising potential of histone deacetylase inhibitor sulforaphane for epigenetic therapy for cancer as this therapeutic approach aims to reverse aberrant epigenetic modifications that affect gene expression. In vitro cell culture studies performed using B16 and S91 melanoma cells showed that sulforaphane inhibited growth and proliferation of cancer cells by downregulating deacetylation enzymes. The second part of our research investigated polymeric drug delivery systems to increase therapeutic efficacy and to minimize potential side effects of R,S-sulforaphane. Albumin microspheres encapsulating sulforaphane were developed by spray drying. Microspheres were characterized for their morphology, size and zeta potential. Cell culture studies using melanoma cells and in vivo studies in melanoma tumor-bearing C57BL/6 mice demonstrated that albumin based polymeric delivery system was efficacious and has the potential to enhance the therapeutic effect and anticancer activity of sulforaphane.

Macrophage Depletion by Albumin Microencapsulated Clodronate: Attenuation of Cytokine Release in Macrophage-Dependent Glomerulonephritis

A macrophage plays an important role in mediating the inflammatory response. Cytokines released by activated macrophages contribute to inflammation in glomerulonephritis (GN). Clodronate, a biphosphonate, causes macrophage depletion when administered in an encapsulated form in liposomes. We used albumin as the polymer matrix to microencapsulate clodronate to the microspheres (MS) in the 1-micron size range. The purpose of this study was to (a) determine macrophage depletion by clodronate MS, (b) determine the effect of clodronate MS on endotoxin-induced cytokine release in vitro, and (c) assess the effect of clodronate MS on macrophage infiltration in experimental antiglomerular basement membrane nephritis. Macrophage depletion by clodronate MS was assessed by staining for the EDI marker. The results indicate greater than 95% depletion of macrophages from the spleen, liver, kidney, and blood. In the whole blood model, clodronate MS attenuated endotoxin-induced tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) release, and the attenuation by the microencapsulated form of clodronate was also more effective than the free (solution) form of clodronate. Macrophage infiltration into the glomerulus in experimental GN was also blocked very effectively by pretreatment with clodronate MS. In conclusion, macrophage depletion by clodronate MS may be beneficial in reducing cytokine release and renal damage in GN.

Evaluation of albumin microspheres as oral delivery system for Mycobacterium tuberculosis vaccines

Mucosal immunization has been suggested to be the best option for preventing Mycobacterium tuberculosis infection. The purpose of this study was to develop albumin microspheres containing Mycobacterium tuberculosis antigens and to determine if oral administration of the microspheres can induce antigen-specific mucosal and systemic immune responses. Albumin microspheres containing Mycobacterium tuberculosis dead cells and cell lysate were prepared. The physico-chemical characteristics of the formulations were determined and the microspheres were administered to animal models to evaluate the induction of immune responses to the antigens. The results showed that the particle sizes, zeta potential and dissolution pattern of the microspheres were ideal for oral delivery of vaccines. In vivo studies showed high production of antigen-specific antibody production in serum, nasal, salivary and faecal samples. From the results of the study, it can be concluded that oral administration of Mycobacterium tuberculosis microspheres was successful in inducing antigen-specific systemic and mucosal immune responses.

Oral delivery of gastro-resistant microencapsulated typhoid vaccine

Oral vaccination has long been regarded as the best alternative to conventional parenteral vaccination considering practical, economical, and immunological aspects. The purpose of this study was to develop albumin–chitosan mixed matrix microsphere-filled coated capsule formulations of Typhoid Vi® antigen and to determine whether it can induce antigen-specific mucosal and systemic immune responses on oral administration. Formulations with Typhoid Vi® antigen were prepared and filled into hard gelatin capsules (size # 9) and enteric coated. Formulations were characterized and administered to Sprague–Dawley rats to evaluate the induction of immune response to the antigen. The results indicated that the particle size, zeta potential, swelling, and disintegration rates were optimal for the oral delivery of microencapsulated vaccines. In vivo studies displayed multifold increase of antigen-specific IgG and IgA levels 8 weeks after oral immunization. No statistically significant difference in the antigen-specific IgG and IgA levels were found between oral and parenteral injection groups 8 weeks after vaccination. On the basis of the results of the study, it can be concluded that the oral administration of Typhoid Vi antigen microspheres was successful in inducing antigen-specific systemic and mucosal immune response.

Prevention of Lethality and Suppression of Proinflammatory Cytokines in Experimental Septic Shock by Microencapsulated CNI-1493

CNI-1493, a newly developed, water-soluble tetravalent guanylhydrazone, is a powerful inhibitor of tumor necrosis factor (TNF) and interleukin-1 (IL-1) synthesis. Microencapsulation of drugs into microcapsules that target macrophages has improved the effectiveness of both TNF and IL-1 neutralizing antibodies in experimental models of septic shock. It is the purpose of this study to determine if microencapsulation of CNI-1493 will improve cytokine inhibition. CNI-1493 was microencapsulated using albumin into 1 microm spheres. Comparable amounts of CNI-1493 in solution and in microencapsulated form were added to 1 ml aliquots of whole blood along with 100 ng of endotoxin. TNF and IL-1 were measured by ELISA. Microencapsulated CNI-1493 was also given to rats with endotoxic shock (15 mg/kg Escherichia coli endotoxin) and rats with peritonitis induced by peritoneally injecting 10(10) CFU E. coli. Equivalent amounts of encapsulated and solution CN I-493 were given intravenously. Endotoxin 15 mg/kg was also given to rats 6 and 24 h after a dose of encapsulated CNI-1493 to determine the duration of action of encapsulated drug. The microencapsulated CNI-1493 produced significantly greater inhibition of TNF and IL-1 at all doses in the whole blood model. There was significantly improved survival and cytokine inhibition in the endotoxic shock model as well as the peritonitis model in rats treated with microencapsulated CNI-1493. There was also 83% survival in rats given endotoxin 24 h after a dose of encapsulated CNI-1493. From these data, we conclude that CNI-1493 is a potent inhibitor of cytokine production and is greatly potentiated by microencapsulation, which transports the drug to macrophages.

Microparticle transport in the human intestinal M cell model

The purpose of this study was to construct a human M cell model to evaluate the transport of polystyrene and albumin microparticles via this model. The integrity of the M cell model was evaluated by determining the transepithelial electrical resistance (TEER), and the alkaline phosphate activity was determined as an indicator of the presence of M-cell-like cells in the coculture. Both Fluoresbrite® microparticles and albumin microparticles were transported by both Caco-2 monoculture and M cell models. The albumin microparticles showed a significantly greater transport via the M-cell-like cells compared to Caco-2 cells monoculture (p < 0.05). The coculture showed comparable TEER and reduction of 15–36% of alkaline phosphatase (AP) activity compared to the Caco-2 cell monoculture. This human M cell model was successfully constructed using 3 and 8 μm transwell inserts and it will serve as a useful tool in formulations screening and optimization of microparticle for oral vaccine delivery.

Formulation and testing of vancomycin loaded albumin microspheres prepared by spray-drying

Microparticles are widely employed as carriers of biologically active compounds with many possible applications. For targeted drug delivery and sustained release purposes, biopolymers (i.e. polysaccharides and proteins) have been proposed. In this study, microsphere formulations of vancomycin were prepared by the spray-drying method. Bovine serum albumin (BSA) was used as a polymer matrix and was cross-linked with glutaraldehyde after microsphere preparation. The product yield obtained from the spray-drying method was ∼75%. The mean particle size was 5 ± 1.6 µm, with the majority of particles between 4 and 8 µm. The extent of cross-linking affected the release of vancomycin from microspheres. Moreover, both rate and extent of vancomycin release from microspheres decreased with increasing glutaraldehyde concentration. Encapsulation of vancomycin did not alter the bioactivity of the drug and it was more effective in killing Staphylococcus aureus than the solution form.