Preclinical Assessment Addressing Intravenous Administration of a [68Ga]Ga-PSMA-617 Microemulsion: Acute In Vivo Toxicity, Tolerability, PET Imaging, and Biodistribution.

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [68Ga]Ga-PSMA-617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [68Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results.

Functionalization of PLGA Nanoparticles with 1,3-ß-glucan Enhances the Intracellular Pharmacokinetics of Rifampicin in Macrophages.

PURPOSE: Mycobacterium tuberculosis which causes tuberculosis, is primarily resident within macrophages. 1,3-ß-glucan has been proposed as a ligand to target drug loaded nanoparticles (NPs) to macrophages. In this study we characterized the intracellular pharmacokinetics of the anti-tubercular drug rifampicin delivered by 1,3-ß-glucan functionalized PLGA NPs (Glu-PLGA). We hypothesized that Glu-PLGA NPs would be taken up at a faster rate than PLGA NPs, and consequently deliver higher amounts of rifampicin into the macrophages. METHODS: Carbodiimide chemistry was employed to conjugate 1,3-ß-glucan and rhodamine to PLGA. Rifampicin loaded PLGA and Glu-PLGA NPs as well as rhodamine functionalized PLGA and Glu-PLGA NPs were synthesized using an emulsion solvent evaporation technique. Intracellular pharmacokinetics of rifampicin and NPs were evaluated in THP-1 derived macrophages. A pharmacokinetic model was developed to describe uptake, and modelling was performed using ADAPT 5 software. RESULTS: The NPs increased the rate of uptake of rifampicin by a factor of 17 and 62 in case of PLGA and Glu-PLGA, respectively. Expulsion of NPs from the macrophages was also observed, which was 3 fold greater for Glu-PLGA NPs than for PLGA NPs. However, the ratio of uptake to expulsion was similar for both NPs. After 24 h, the amount of rifampicin delivered by the PLGA and Glu-PLGA NPs was similar. The NPs resulted in at least a 10-fold increase in the uptake of rifampicin. CONCLUSIONS: Functionalization of PLGA NPs with 1,3-ß-glucan resulted in faster uptake of rifampicin into macrophages. These NPs may be useful to achieve rapid intracellular eradication of Mycobacterium tuberculosis.

Bioaccumulation and Subchronic Toxicity of 14 nm Gold Nanoparticles in Rats.

Colloidal suspensions of 14 nm gold nanoparticles (AuNPs) were repeatedly administered intravenously at three dose levels (0.9, 9 and 90 µg) to male Sprague Dawley rats weekly for 7 weeks, followed by a 14-day washout period. After sacrificing, the amount of gold was quantified in the liver, lungs, spleen, skeleton and carcass using neutron activation analysis (NAA). During the study, pre- and post (24 h) administration blood samples were collected from both the test and control groups, the latter which received an equal injection volume of normal saline. General health indicators were monitored together with markers of kidney and liver damage for acute and subchronic toxicity assessment. Histopathological assessments were done on the heart, kidneys, liver, lungs and spleen to assess any morphological changes as a result of the exposure to AuNPs. The mass measurements of all the groups showed a steady increase with no signs of overt toxicity. The liver had the highest amount of gold (µg) per gram of tissue after 56 days followed by the spleen, lungs, skeleton and carcass. Markers of kidney and liver damage showed similar trends between the pre and post samples within each group and across groups. The histopathological examination also showed no hepatotoxicity and nephrotoxicity. There was accumulation of Au in tissues after repeated dosing, albeit with no observable overt toxicity, kidney or liver damage.

Nanomedicine: Past, present and future - A global perspective.

Nanomedicine is an emerging and rapidly evolving field and includes the use of nanoparticles for diagnosis and therapy of a variety of diseases, as well as in regenerative medicine. In this mini-review, leaders in the field from around the globe provide a personal perspective on the development of nanomedicine. The focus lies on the translation from research to development and the innovation supply chain, as well as the current status of nanomedicine in industry. The role of academic professional societies and the importance of government funding are discussed. Nanomedicine to combat infectious diseases of poverty is highlighted along with other pertinent examples of recent breakthroughs in nanomedicine. Taken together, this review provides a unique and global perspective on the emerging field of nanomedicine.

Curdlan-Conjugated PLGA Nanoparticles Possess Macrophage Stimulant Activity and Drug Delivery Capabilities.

PURPOSE: There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells. We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system. METHODS: Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using (1)H and (13)C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTT assay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug. RESULTS: Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca(2+) dependent uptake of nanoparticles by the macrophages. CONCLUSIONS: PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy.

Nanoparticle formulation of a poorly soluble cannabinoid receptor 1 antagonist improves absorption by rat and human intestine.

The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.

Rift Valley Fever vaccine strategies: Enhanced stability of RVF Clone 13.

Rift Valley Fever virus (RVFV) causes the zoonotic RVF disease, which results in substantial economic losses in livestock industries. Regular vaccination of livestock against RVF is necessary to generate long-term immunity and avoid the loss of livestock. The live attenuated vaccine based on Clone 13 virus strain has been used to reduce the negative impact of RVF disease. The vaccine strain is heat labile and requires stringent conditions for storage and handling. This research evaluated lactose and sucrose-based stabilizers coupled with lyophilisation to enhance stability of the RVF Clone 13 vaccine strain. The glass transition temperature (Tg) of the sucrose-RVF vaccine was 97.0 °C with average residual moisture of below 2 %. The lactose formulation was characterised with Tg of 83.5 °C and residual moisture of above 2 %. The RVF Clone 13 sucrose-based formulation maintained higher antigen titres during lyophilisation compared to the lactose-formulated vaccine. Cellular-mediated and humoral immunity was evaluated and compared for the two newly formulated vaccines. Pheroid® technology was also investigated as a potential adjuvant and its ability to further enhance the immunogenicity conferred by the RVF Clone 13 vaccine formulations in Merino sheep. No adverse reactions were observed following injection of the vaccine formulations in mice, guinea pigs and Merino sheep. Comparable protective humoral immune responses against RVF were obtained for all animals vaccinated with the lactose and sucrose-based stabilisers with and without the Pheroid® adjuvant. No proliferation of CD8+ and CD4+ T-cells as well as expression of IFN-γ was observed for all animals group vaccinated with Pheroid® only. Specific CD8+ IFN-γ+T-cells were expressed at higher levels compared to the CD4+ IFN-γ+T-cells in the RVF Clone 13 vaccines, suggesting that cellular immunity against RVF is through the Class I antigen presentation pathway.

Phytochemical Profile, Safety and Efficacy of a Herbal Mixture Used for Contraception by Traditional Health Practitioners in Ngaka Modiri Molema District Municipality, South Africa.

The use of medicinal plants for contraception remains a common practice among South African ethnic groups. The present study assessed the phytochemical profile, cytotoxicity, acute oral toxicity and efficacy of a herbal mixture used for contraception by the Batswana of South Africa. An aqueous extract was prepared from equal quantities (in terms of weight) of Bulbine frutescens (roots), Helichrysum caespititium (leaves) and Teucrium trifidum (leaves) based on a recipe used by traditional health practitioners. The phytochemical profiles of the freeze-dried herbal mixture were analyzed using gas chromatography-mass spectrometry (GC-MS). In addition, cytotoxicity was determined using an MTT assay on Vero cells and in vivo contraceptive efficacy was evaluated using seven Sprague Dawley rats per control and treatment groups. The control group received distilled water while test groups received 5, 50 and 300 mg/kg of the herbal mixture, which was administered orally once a day for three consecutive days. Subsequently, female rats were paired 1:1 with males for 3 days. Their weights were measured weekly and incidence of pregnancy was recorded. The GC-MS chromatogram revealed the presence of 12 identified and 9 unidentified compounds. In terms of safety, the herbal mixture had an IC50 value of 755.2 µg/mL and 2000 mg/kg, which was the highest tested dose that caused no mortality or morbidity in the rats. A contraceptive efficacy of 14.5% was exerted with 50 mg/kg herbal mixture extract while other doses had no effects given that all the rats were pregnant. Based on a chi-square test (p < 0.05), there was no correlation between the tested herbal mixture doses and contraception, nor on the weight of the rats. Overall, the herbal mixture extract was found to be safe but had limited contraceptive efficacy at the tested doses. In future studies, exploring increased dose range, solvent extract types and hormonal analysis will be pertinent.

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13.

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 µM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 µM and 0.011 µM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.

Development and Evaluation of a Reconstitutable Dry Suspension Containing Isoniazid for Flexible Pediatric Dosing.

Tuberculosis (TB) is a major cause of childhood death. Despite the startling statistics, it is neglected globally as evidenced by treatment and clinical care schemes, mostly extrapolated from studies in adults. The objective of this study was to formulate and evaluate a reconstitutable dry suspension (RDS) containing isoniazid, a first-line anti-tubercular agent used in the treatment and prevention of TB infection in both children and adults. The RDS formulation was prepared by direct dispersion emulsification of an aqueous-lipid particulate interphase coupled with lyophilization and dry milling. The RDS appeared as a cream-white free-flowing powder with a semi-crystalline and microparticulate nature. Isoniazid release was characterized with an initial burst up to 5 minutes followed by a cumulative release of 67.88% ± 1.88% (pH 1.2), 60.18% ± 3.33% (pH 6.8), and 49.36% ± 2.83% (pH 7.4) over 2 hours. An extended release at pH 7.4 and 100% drug liberation was achieved within 300 minutes. The generated release profile best fitted the zero order kinetics (R2 = 0.976). RDS was re-dispersible and remained stable in the dried and reconstituted states over 4 months and 11 days, respectively, under common storage conditions.

Development and validation of an LC-MS/MS method for the quantification of goserelin in a Pheroid® formulation, in simulated intestinal fluid.

The purpose of this reported study was to develop and validate an LC-MS/MS method for the quantification of goserelin in a Pheroid® formulation simulated intestinal fluid. Biopharmaceuticals are formulated in drug delivery systems to improve their gastrointestinal stability. Goserelin, a peptide drug was formulated in Pheroid® delivery system and its gastrointestinal stability assessed using simulated intestinal fluid, which required an assay to determine the varying amounts of goserelin remaining after a specific time. Several extraction methods and solvents investigated to extract goserelin from complex matrix led to either poor recovery, peak shape or high background interference. A rapid gradient reversed-phase method coupled to tandem mass spectrometry detection was optimized for the separation and quantification of the extracted peptide. A simple, reproducible and good recovery extraction procedure for goserelin quantification was achieved through simultaneous acetonitrile protein precipitation and water-saturated n-butanol liquid-liquid extraction with water dilution. The method was found to be rapid, specific, precise and accurate, and successfully applied to determine goserelin remaining content in a simulated intestinal fluid, with potential use in other lipid-based formulation evaluated in simulated intestinal fluids.

Spray-Dried, Nanoencapsulated, Multi-Drug Anti-Tuberculosis Therapy Aimed at Once Weekly Administration for the Duration of Treatment.

Aiming to improve the treatment outcomes of current daily tuberculosis (TB) chemotherapy over several months, we investigated whether nanoencapsulation of existing drugs would allow decreasing the treatment frequency to weekly, thereby ultimately improving patient compliance. Nanoencapsulation of three first-line anti-TB drugs was achieved by a unique, scalable spray-drying technology forming free-flowing powders in the nanometer range with encapsulation efficiencies of 82, 75, and 62% respectively for rifampicin, pyrazinamide, and isoniazid. In a pre-clinical study on TB infected mice, we demonstrate that the encapsulated drugs, administered once weekly for nine weeks, showed comparable efficacy to daily treatment with free drugs over the same experimental period. Both treatment approaches had equivalent outcomes for resolution of inflammation associated with the infection of lungs and spleens. These results demonstrate how scalable technology could be used to manufacture nanoencapsulated drugs. The formulations may be used to reduce the oral dose frequency from daily to once weekly in order to treat uncomplicated TB.

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories.

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro.

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC(50) values of 1.5 microM, 3.5 microM, 4 microM, 4.3 microM and 4.8 microM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K(i) of 0.24 microM and 0.14 microM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K(i) = 0.4 microM) whilst GG1 produced mixed type of inhibition. The K(i) and K(i)' for GSH for Tral-1 and GG1 were 0.2 microM and 0.1 microM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.

The inhibition of human glutathione S-transferases activity by plant polyphenolic compounds ellagic acid and curcumin.

Glutathione S-transferases (GSTs) are multifunctional detoxification proteins that protect the cell from electrophilic compounds. Overexpression of GSTs in cancer results in resistance to chemotherapeutic agents and inhibition of the over expressed GST has been suggested as an approach to combat GST-induced resistance. The inhibition of human recombinant GSTs by natural plant products was investigated in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) as a substrate, ellagic acid and curcumin were shown to inhibit GSTs A1-1, A2-2, M1-1, M2-2 and P1-1 with IC(50) values ranging from 0.04 to 5 microM whilst genistein, kaempferol and quercetin inhibited GSTs M1-1 and M2-2 only. The predominant mode of inhibition with respect to the G and H-sites were mixed inhibition and uncompetitive to a lesser extent. The K(i) (K(i)(')) values for ellagic acid and curcumin with respect to GSH and CDNB were in the range 0.04-6 microM showing the inhibitory potency of these polyphenolic compounds. Ellagic acid and curcumin also showed time- and concentration-dependent inactivation of GSTs M1-1, M2-2 and P1-1 with curcumin being a more potent inactivator than ellagic acid. These results facilitate the understanding of the interaction of human GSTs with plant polyphenolic compounds with regards to their role as chemomodulators in cases of GST-overexpression in malignancies.

The potential inhibitory effect of antiparasitic drugs and natural products on P-glycoprotein mediated efflux.

The potential inhibitory effect on P-glycoprotein (Pgp) by antiparasitic drugs and natural compounds was investigated. Compounds were screened for Pgp interaction based on inhibition of Pgp mediated [3H]-taxol transport in Caco-2 cells. Bidirectional transport of selected inhibitors was further evaluated to identify potential Pgp substrates using the Caco-2 cells. Of 21 antiparasitics tested, 14 were found to inhibit Pgp mediated [3H]-taxol with K(iapp) values in the range 4-2000 microM. The antimalarial quinine was the most potent inhibitor with a K(iapp) of 4 microM. Of the 12 natural compounds tested, 3 inhibited [3H]-taxol transport with K(iapp) values in the range 50-400 microM. Quinine, amodiaquine, chloroquine, flavone, genistein, praziquantel, quercetin and thiabendazole were further investigated in bidirectional transport assays to determine whether they were substrates for Pgp. Transport of quinine in the secretory direction exceeded that in the absorptive direction and was saturable, suggesting quinine being a Pgp substrate. The rest of the compounds inhibiting Pgp showed no evidence of being Pgp substrates. In conclusion, we have demonstrated that a substantial number of antiparasitic and natural compounds, in a range of concentrations, are capable of inhibiting Pgp mediated [3H]-taxol efflux in Caco-2 cells, without being substrates and this may have implications for drug interactions with Pgp.

Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin.

Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 µM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters k inact and K I were found to be 0.020 ± 0.001 min(-1) and 7.5 ± 2.1 µM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs.