Formulation and characterization of polymeric films containing combinations of antiretrovirals (ARVs) for HIV prevention.

PURPOSE: To develop polymeric films containing dual combinations of anti-HIV drug candidate tenofovir, maraviroc and dapivirine for vaginal application as topical microbicides. METHODS: A solvent casting method was used to manufacture the films. Solid phase solubility was used to identify potential polymers for use in the film formulation. Physical and chemical properties (such as water content, puncture strength and in vitro release) and product stability were determined. The bioactivity of the film products against HIV was assessed using the TZM-bl assay and a cervical explant model. RESULTS: Polymers identified from the solid phase solubility study maintained tenofovir and maraviroc in an amorphous state and prevented drug crystallization. Three combination film products were developed using cellulose polymers and polyvinyl alcohol. The residual water content in all films was <10% (w/w). All films delivered the active agents with release of >50% of film drug content within 30 min. Stability testing confirmed that the combination film products were stable for 12 months at ambient temperature and 6 months under stressed conditions. Antiviral activity was confirmed in TZM-bl and cervical explant models. CONCLUSIONS: Polymeric films can be used as a stable dosage form for the delivery of antiretroviral combinations as microbicides.

Tenofovir vaginal film as a potential MPT product against HIV-1 and HSV-2 acquisition: formulation development and preclinical assessment in non-human primates.

Tenofovir (TFV) is an adenosine nucleotide analog with activity against HIV and HSV-2. Secondary analyses of clinical trials evaluating TFV gel as pre-exposure prophylaxis (PrEP) for HIV have shown that gel formulations of TFV provide significant protection against both HIV and HSV-2 acquisition in women who had evidence of use. An alternate quick-dissolving polymeric thin film, to deliver TFV (20 and 40 mg) has been developed as a potential multipurpose technology (MPT) platform. Film formulation was developed based on excipient compatibility, stability, and ability to incorporate TFV doses. Placebo, low dose (20 mg), and high dose (40 mg) films were utilized in these studies. The developed film platform efficiently incorporated the high dose of TFV (40 mg/film), released more than 50% of drug in 15 min with no in vitro toxicity. Pharmacological activity was confirmed in an ex vivo HIV-1 challenge study, which showed a reduction in HIV-1 infection with TFV films. Films were stable at both doses for at least 2 years. These films were found to be safe in macaques with repeated exposure for 2 weeks as evidenced by minimal perturbation to tissues, microbiome, neutrophil influx, and pH. Macaque sized TFV film (11.2 mg) evaluated in a pigtail macaque model showed higher vaginal tissue concentrations of TFV and active TFV diphosphate compared to a 15 mg TFV loaded gel. These studies confirm that TFV films are stable, safe and efficiently deliver the drug in cervicovaginal compartments supporting their further clinical development.

Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro.

Cells of the mononuclear phagocytic system, in particular monocytes/macrophages (Mo/Mac) serve as a reservoir for human immunodeficiency virus (HIV) and are believed to be responsible for its dissemination throughout the body and especially into the brain. Treatment of HIV infection, therefore, must reach these cells in addition to the lymphocytes. The purpose of the present study is to develop poly(propyleneimine) (PPI) dendrimer-based nanocontainers for targeting of efavirenz (EFV) to Mo/Mac. Fifth generation PPI dendrimer, t-Boc-glycine conjugated PPI dendrimer (TPPI) and mannose conjugated dendrimers were synthesized and characterized. While the haemolytic activity and cytotoxicity of PPI dendrimer was found to be very high, the toxicity of t-Boc-glycine conjugated dendrimer and mannose conjugated dendrimers were found to be negligible. The entrapment efficiency of mannose conjugated dendrimer was found to be 47.4%, followed by that of PPI dendrimer (32.15%) and t-Boc-glycine conjugated dendrimer (23.1%). The in vitro drug release profile shows that while PPI dendrimer releases the drug by 24 h, the dendrimer-based nanocontainers prolong the release rate up to 144 h (83 +/- 0.4% in case of t-Boc-glycine conjugated dendrimer and 91 +/- 0.3% in mannose conjugated dendrimer). The cellular uptake of EFV was found to be both concentration and time dependent. Significant increase in cellular uptake of EFV by Mo/Mac cells were observed in case of mannose conjugated dendrimer which is 12 times higher than that of free drug and 5.5 times higher than that of t-Boc-glycine conjugated dendrimer. While mannose conjugated dendrimer was taken up by the lectin receptors of the cells, phagocytosis of t-Boc-glycine conjugated dendrimer might be responsible for its enhanced uptake. Results suggest that the proposed carriers hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.

Polypropylene imine dendrimer mediated solubility enhancement: effect of pH and functional groups of hydrophobes.

Dendrimers today are known for their three dimensional, monodispersed, highly branched, macromolecular nano-scopic architecture with number of reactive end groups. Dendrimers have been reported to act as solubilizing agents to host both hydrophilic and hydrophobic drugs. The present study was performed to investigate the effect of pH on poly(propylene) imine dendrimers (5.0G) mediated solubility enhancement of hydrophobes differing in functional groups (pKa). Weakly basic, (famotidine, -NH2 functional group; pKa 7.1), weakly acidic (indomethacin, -COOH functional group; pKa 4.5) and amphoteric (amphotericin B, -COOH and -NH2 functional groups; pKa 5.7 and 10.0) hydrophobes were selected for the study. The experiment was carried out at pH 4.0, 7.4 and 10.0. The solubility of all the drugs was enhanced at pH 7.4 and 10.0 but not at pH 4.0. The drug-dendrimer complexes followed 1:1 stoichiometry (AL type of curve) and were characterized for stability of complex, complexation efficiency and thermodynamic properties. Thermodynamic properties were utilized to elucidate the mechanism behind dendrimer mediated solubility enhancement. The data suggested that hydrophobic and electrostatic interactions were responsible for solubility enhancement. Conclusively, PPI dendrimers were found useful in solubility enhancement of not only acidic and basic but also amphoteric drugs, their solubilization ability was clearly regulated by pH and chemical nature of drug.

PEGylated dendritic architecture for development of a prolonged drug delivery system for an antitubercular drug.

The present study was aimed at developing and exploring the use of PEGylated poly (propylene imine) dendritic architecture for the delivery of an anti-tuberculosis drug, rifampicin. For this study, PEGylated poly(propylene imine) dendritic architecture was synthesized and loaded with rifampicin. Various physicochemical and physiological parameters UV, IR, NMR, TEM, DSC, drug entrapment, drug release and hemolytic toxicity of both PEGylated and non-PEGylated systems were determined and compared. The PEGylation of the systems was found to have increased their drug-loading capacity, reduced their drug release rate and hemolytic toxicity. The systems were found suitable for prolonged delivery of rifampicin.

Investigations on biodistribution of technetium-99m-labeled carbohydrate-coated poly(propylene imine) dendrimers.

The purpose of this work was to study the biodistribution pattern of the fifth generation of poly(propylene imine) dendrimer (PPI-5.0G)-based carbohydrate (mannose and lactose)-coated glycodendrimers in mice so as to explore the potential of these systems as drug carriers. Plain dendrimers were synthesized and coated with carbohydrates following the reported procedures. The formulations were labeled with radioactive technetium (sodium pertechnetate; 99mTcO4-) and characterized for labeling efficiency as well as in vitro and in vivo stability of the labeled complexes. The blood clearance study was performed in female New Zealand rabbits. The periodic in vivo biodistribution profile of the formulations was investigated in female Balb/c mice. The dendrimeric formulations were labeled with 95% labeling efficiency. The labeled complexes were found to be stable in vitro (97% to 98% stability) and in vivo (89% to 94% stability). All the formulations were cleared rapidly from circulation; clearance of mannose-coated poly (propylene imine) dendrimer (M-PPI) and lactose-coated poly(propylene imine) dendrimer (L-PPI) was faster than PPI-5.0G. All the formulations accumulated in liver to a significant extent, but only those with terminal carbohydrate moieties were retained for a longer period. Significant accumulation of PPI-5.0G and M-PPI was observed in kidneys as against very less activity in the case of L-PPI. Rapid clearance of the dendrimers was in accordance with the earlier reports. Higher and prolonged retention of M-PPI and L-PPI in liver was attributed to lectin-carbohydrate interactions. Lesser accumulation of L-PPI in kidneys was suggestive of its lesser excretion. This observation can be explained on the basis of the molecular weight of L-PPI, which was greater than the threshold of glomerular excretion. In general, it was observed that the carbohydrate-coated dendrimers were distributed in liver to a significant extent. This information could serve as a useful platform in designing carbohydrate-coated dendrimers for selective delivery of bioactive agents to liver.

Ethinylestradiol-loaded ultraflexible liposomes: pharmacokinetics and pharmacodynamics.

This study aimed to develop ultraflexible liposomes as an alternative to the oral route, which would enhance the bioavailability and reduce the toxicity of ethinylestradiol. Ultraflexible liposomes of ethinylestradiol using an optimized concentration of surfactants were prepared and characterized in vitro. The effect of surfactant type under non-occlusive conditions on transdermal permeability was assessed. A histopathological study was performed to assess the action of ethinylestradiol on the uterus and ovaries. The pharmacokinetics of free ethinylestradiol (following single oral administration and one day of application to the skin), ultraflexible liposomal ethinylestradiol and non-flexible liposomal ethinylestradiol were studied in female Sprague-Dawley rats. Insignificant differences in size between the ultraflexible liposomal formulations containing optimized concentrations of different surfactants were observed. Ultraflexible liposomes can penetrate through pores much smaller than their own diameter. The transdermal permeability of lipophilic surfactant was greater than that of hydrophilic surfactant. The release of ethinylestradiol from the proposed formulation through rat skin was found to be constant. The histopathological study showed that the ultraflexible liposomal transdermal drug delivery system for ethinylestradiol provided effective contraception by follicular cell lysis, depletion of zona granulosa and ova, and by increasing the uterine mucosal and endometrial proliferation. Encapsulation of ethinylestradiol in ultraflexible liposomes modified the pharmacodynamics and pharmacokinetics of the contraceptive agent, resulting in a marked improvement in bioavailability and optimized therapy.

Investigations on the toxicological profile of functionalized fifth-generation poly (propylene imine) dendrimer.

Dendrimers have generated tremendous interest in the field of drug delivery. Despite indications of their utility as drug carriers, the inherent cytotoxicity associated with polycationic dendrimers acts as a limiting factor to their clinical applications. Many functionalization strategies have been adopted to mask peripheral amines in order to overcome this limitation. The object of the present investigation was to evaluate the effect of functionalization on the toxicological profile of fifth-generation poly(propylene imine) dendrimer (PPI-5.0G). Four forms of functionalized dendrimers, including protected glycine and phenylalanine, and mannose and lactose functionalized poly(propylene imine) (PPI) dendrimer, were synthesized as prospective drug carriers. These dendrimeric systems were evaluated for haemolytic toxicity, cytotoxicity, immunogenicity and haematological parameters. PPI-5.0G demonstrated a positive charge-based time- and concentration-dependent toxicity profile. Functionalization greatly improved the toxicity profile of the parent dendrimer. Hence it is proposed that these functionalized forms of PPI dendrimer have great potential as bio-compatible drug vehicles.

Stavudine-loaded mannosylated liposomes: in-vitro anti-HIV-I activity, tissue distribution and pharmacokinetics.

Cells of the mononuclear phagocyte system (MPS) are important hosts for human immunodeficiency virus (HIV). Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of these cells of the MPS. Stavudine-loaded mannosylated liposomal formulations were developed for targeting to HIV-infected cells. The mannose-binding protein concanavalin A was employed as model system for the determination of in-vitro ligand-binding capacity. Antiretroviral activity was determined using MT-2 cell line. Haematological changes, tissue distribution and pharmacokinetic studies of free, liposomal and mannosylated liposomal drug were performed following a bolus intravenous injection in Sprague-Dawley rats. The entrapment efficiency of mannosylated liposomes was found to be 47.2 +/- 1.57%. Protein-carbohydrate interaction has been utilized for the effective delivery of mannosylated formulations. Cellular drug uptake was maximal when mannosylated liposomes were used. MT2 cells treated continuously with uncoated liposomal formulation had p24 levels 8-12 times lower than the level of free drug solution. Further, the mannosylated liposomes have shown p24 levels that were 14-20 and 1.4-2.3 times lower than the level of free drug and uncoated liposomal formulation treatment, respectively. Similar results were observed when infected MT2 cells were treated overnight. Stavudine, either given plain or incorporated in liposomes, led to development of anaemia and leucocytopenia while mannosylated liposomes overcame these drawbacks. These systems maintained a significant level of stavudine in the liver, spleen and lungs up to 12 h and had greater systemic clearance as compared with free drug or the uncoated liposomal formulation. Mannosylated liposomes have shown potential for the site-specific and ligand-directed delivery systems with desired therapeutics and better pharmacological activity.

A review of in vitro-in vivo investigations on dendrimers: the novel nanoscopic drug carriers.

Dendrimers have emerged as one of the most interesting themes for researchers as a result of their unique architecture and macromolecular characteristics. Several groups are involved in exploring their potential as versatile carriers in drug delivery. The use of dendrimers in drug delivery has been reviewed extensively. The increasing relevance of the potential of dendrimers in drug delivery emphasizes the need to explore the routes by which they can be administered. The present review focuses on dendrimer-mediated drug delivery based on various routes of administration, a topic that has received little attention in the available literature. With this focus in mind, we present a comprehensive exploration of the recent advances in the investigational aspects of these nanoscopic polymeric devices. Also included are some in vitro studies that present data suggestive of their possible application in different routes of administration.

Transdermal delivery of cyclodextrin-solubilized curcumin.

The purpose of the present investigation was to explore the effect of cyclodextrin (CD) as permeation enhancer through rat skin in the form of a valuable and stable transdermal drug delivery system by exploiting its favorable properties. Phase-solubility studies demonstrated that the CD:drug ratio 1:2 was employed in complexation. Solid-state characterizations of complexes was performed by differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared, nuclear magnetic resonance spectrophotometry analysis, and scanning electron micrograph. The HP-ß-CD by virtue of its greater stability than the pure curcumin (CMN), allowed greater transdermal flux of CMN indicative of enhanced permeation via CMN-2-hydroxy propyl ß cyclodextrins (HP-ß-CD). Permeability studies of drug, complex, and with various penetration enhancers (PEs), were performed through rat skin, highlighted a favorable effect of CDs on drug permeation rate, due to its solubilizing action; in contrast with unpredictably poor skin permeation of pure drug. The complexes were found to cause relatively less irritation as compared to the pure drug and drug with PEs in skin irritation studies. The anti-inflammatory activity using paw odema model showed that the formulations of CMNCur-HP-ß-CD complex exhibited significant (p < 0.001) decrease in paw edema volume than its pure CMN gel demonstrating enhanced biological activity.

Formulation and delivery of microbicides.

The development of pre-exposure prophylactics or microbicide products for the reduction or elimination of the sexual transmission of HIV has numerous challenges or barriers to success. Historically traditional dosage forms such as gels have been developed in the field but more recently controlled release dosage forms such as vaginal rings and novel dosage forms such as polymeric thin films have been studied. Studies have begun to incorporate scientific strategies into the formulation design of microbicide products in order to develop safer and more effective products. In addition advanced drug delivery strategies to overcome barriers to delivery and specific drug targeting methods are being employed. In the present review, a comprehensive discussion of formulation efforts and novel delivery strategies in the field of microbicide product development is presented.

Cyclodextrin-mediated entrapment of curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA in liposomes for treatment of xenograft lung tumor in rats.

We recently reported a novel curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA as a potent anti-proliferative agent, and showed that it induces autophagic cell death in lung cancer cells. We are now reporting a drug-in-CD-in-liposome approach to formulate CLEFMA liposomes that could be labeled with Tc-99m radionuclide for non-invasive imaging of their biodistribution. CLEFMA encapsulation was enabled by hydroxypropyl-ß-cyclodextrin. In vitro studies showed that CLEFMA possessed more potent anti-proliferative activity in lung adenocarcinoma H441 cells than naturally occurring curcumin. At the same time, it had no effect on the proliferative capacity of normal lung fibroblasts. CLEFMA liposomes retained the antiproliferative potency of free CLEFMA, while maintaining its non-toxic nature in normal lung fibroblasts. In nude rats bearing xenograft H441 tumors, the tumor volume significantly reduced after i.v. treatment with CLEFMA liposomes (p<0.05); the tumor inhibition was determined to be 94%. The anti-tumor activity of CLEFMA liposomes was confirmed by the observation that F-18-fluorodeoxyglucose uptake in tumors of treated rats was reduced as compared to those of control rats. Tc-99m-labeled CLEFMA liposomes accumulated in liver (33.7%); spleen showed the largest accumulation on per gram tissue basis (6.2%/g). Upon histopathological examination of liver, lung and kidney, we found no apparent toxicity from multiple CLEFMA liposome administrations. The results demonstrate the utility of liposomes to serve as a carrier for CLEFMA. This study is the first to demonstrate the efficacy of novel curcuminoid CLEFMA in a preclinical model.

Synthesis and in vivo evaluation of p-18F-Fluorohippurate as a new radiopharmaceutical for assessment of renal function by PET.

UNLABELLED: The molecular structure of p-18F-fluorohippurate (18F-PFH) is similar to that of p-aminohippurate, a gold standard for the measurement of effective renal plasma flow. The objective of this study was to investigate 18F-PFH as a new PET renal agent. METHODS: 18F-PFH was synthesized by reacting N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) with glycine at 90°C (pH 8) for 20 min. In vitro stability was determined by incubating 18F-PFH in fresh human plasma at 37°C for 60 min. In vivo stability was determined by high-performance liquid chromatography analysis of urine collected from a normal rat at 40 min after injection of 18F-PFH. The plasma protein binding and erythrocyte uptake were determined using plasma collected from a normal rat at 5 min after injection of 18F-PFH. The plasma clearance of 18F-PFH was determined using a single-injection clearance method in normal and probenecid-treated rats. Biodistribution studies were conducted in normal rats at 10 min and 1 h after injection of 18F-PFH. Dynamic PET/CT studies were conducted in normal rats injected with 18F-PFH. RESULTS: In normal rats, the plasma clearance of 18F-PFH was 4.11±1.09 mL/min/100 g, which reduced by approximately 50% (P=0.03) to 2.01±0.08 mL/min/100 g in probenecid-treated rats. About 45.3% of 18F-PFH was found to associate with plasma proteins in vivo in normal rats. Biodistribution studies of 18F-PFH in normal rats showed 72.1±6.4 percentage injected dose and 88.6±6.2 percentage injected dose, respectively, in urine at 10 min and 1 h after injection. The uptake in other organs was negligible. High-performance liquid chromatography analysis of urine collected from a rat at 40 min after injection of 18F-PFH indicated that it was excreted intact, with no metabolic products. Dynamic PET revealed a rapid clearance of 18F-PFH through the renal-urinary pathway. The PET-derived renograms revealed a time to peak activity of 3.0±1.0 min. CONCLUSION: These combined results warrant further investigation of 18F-PFH as a radiopharmaceutical for the assessment of renal function by PET.

Improved formulation of liposome-encapsulated hemoglobin with an anionic non-phospholipid.

We are developing liposome-encapsulated hemoglobin (LEH) as an artificial oxygen carrier for resuscitation in indications, such as acute blood loss and surgery. Earlier attempts to formulate a viable LEH met with constraints of scale up and limited hemoglobin content. In this work, we report an LEH formulation containing novel anionic non-phospholipid (CHHDA) that enhances the encapsulation efficiency of hemoglobin inside the liposome bilayer. CHHDA was synthesized from inexpensive ingredients in high yields. The formulation was evaluated in vitro to investigate the cytotoxic effects on RAW 264.7 macrophages and HUVEC endothelial cells in culture by LDH, MTT and hexosaminidase assays. Under optimal conditions of manufacturing, the presence of 28 mol% of CHHDA enhanced the hemoglobin content to over 4 g/dl. The LEH containing CHHDA shows some cytotoxicity in HUVEC and RAW cells in vitro, especially by LDH assay. MTT assay was negative for cytotoxicity in both cells lines. By hexosaminidase assay, the proliferation of RAW cells, but not HUVEC cells, was inhibited. When CHHDA-LEH was incubated with isolated human platelets in vitro, no platelet activation was observed. The LEH formulation with novel anionic lipid and high hemoglobin content reported in this article is an improvement from the past preparations.

Dendrimers: novel polymeric nanoarchitectures for solubility enhancement.

Poor solubility and hydrophobicity of drugs/bioactives limit their possible applications in drug delivery and formulation development. Apart from conventional methods of solubility enhancement, there are some novel methods which can be used in solubilization. Dendrimers represent a novel type of polymeric material that has generated much interest in many diverse areas due to their unique structure and properties. Dendrimer-mediated solubility enhancement mainly depends on factors such as generation size, dendrimer concentration, pH, core, temperature, and terminal functionality. Added advantage in solubilization can be achieved considering these factors. Available literature suggests that ionic interaction, hydrogen bonding, and hydrophobic interactions are the possible mechanisms by which a dendrimer exerts its solubilizing property. This review presents various mechanisms and reports relating to solubility enhancement using dendrimers. Also, micellar behavior and future possibilities in relation to solubilization via dendrimers are included.