Targeting lung macrophages for fungal and parasitic pulmonary infections with innovative amphotericin B dry powder inhalers.

The incidence of fungal pulmonary infections is known to be on the increase, and yet there is an alarming gap in terms of marketed antifungal therapies that are available for pulmonary administration. Amphotericin B (AmB) is a highly efficient broad-spectrum antifungal only marketed as an intravenous formulation. Based on the lack of effective antifungal and antiparasitic pulmonary treatments, the aim of this study was to develop a carbohydrate-based AmB dry powder inhaler (DPI) formulation, prepared by spray drying. Amorphous AmB microparticles were developed by combining 39.7 % AmB with 39.7 % γ-cyclodextrin, 8.1 % mannose and 12.5 % leucine. An increase in the mannose concentration from 8.1 to 29.8 %, led to partial drug crystallisation. Both formulations showed good in vitro lung deposition characteristics (80 % FPF < 5 µm and MMAD < 3 µm) at different air flow rates (60 and 30 L/min) when used with a DPI, but also during nebulisation upon reconstitution in water.

Efficacy of low doses of amphotericin B plus allicin against experimental visceral leishmaniasis.

OBJECTIVES: To evaluate the efficacy of the combination of allicin and amphotericin deoxycholate (AmB) in the chemotherapy of Leishmania infantum infection with the final aim of reducing the dose of AmB in the chemotherapy of visceral leishmaniasis. METHODS: Hamsters were intraperitoneally (ip) infected with L. infantum (10(7) stationary phase promastigotes). On day 45 post-infection animals were treated ip with AmB (1 or 5 mg/kg/day), allicin (5 mg/kg/day) or a combination of AmB (1 mg/kg/day) + allicin (5 mg/kg/day) for 5 days. Animals were clinically and biopathologically monitored and the antibody response (IgG, IgG1, IgG2) was determined. Parasite burdens were estimated by limiting dilution and AmB biodistribution was determined by HPLC in plasma, kidney, spleen and liver. RESULTS: No clinical signs or liver and kidney alterations were observed. AmB (1 mg/kg/day) did not clear the Leishmania infection and no parasites were detected in two animals treated with 5 mg/kg/day allicin. Combination therapy (5 mg/kg allicin + 1 mg/kg AmB) reduced the L. infantum burden by >95%. Antileishmanial activity of the combination was comparable (P < 0.05) to the standard AmB treatment (5 mg/kg). CONCLUSIONS: Allicin alone (5 mg/kg/day for 5 days) significantly reduced the Leishmania burden in spleen and liver of infected hamsters. Co-administration of allicin (5 mg/kg/day for 5 days) and AmB (1 mg/kg/day for 5 days) showed a partial additive effect on the reduction of leishmanial burden in both target organs.

A novel formulation of solubilised amphotericin B designed for ophthalmic use.

Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. However, there are no licensed topical formulations with AmB in most developed countries. Extemporaneous preparations of AmB are frequently prepared from available marketed parenteral formulations. Herein, a solution of AmB with γ-cyclodextrin is described as suitable for topical administration as eye drops. This novel formulation is characterised by its ability to solubilise AmB and to maintain its antifungal activity, physicochemical stability and sterility over 30 days. Antifungal activity against Candida albicans was significantly higher (35%) for the new formulation than that obtained with Fungizone(®) based extemporaneous prepared suspension. Optimal 0.1% AmB-10% cyclodextrin formulation remained sterile and with an acceptable osmolarity, pH and particle size for ophthalmic use over 4 weeks. Complexation with γ-cyclodextrins improved AmB chemical stability compared to the reference eye drops suspension based on Fungizone(®). These results illustrate the feasibility of an ophthalmic AmB formulation easy enough to be licensed or prepared in community and hospital pharmacies.

Effect of PLGA hydrophilia on the drug release and the hypoglucemic activity of different insulin-loaded PLGA microspheres.

The effects of viscosity and hydrophilic characteristics of different PLGA polymers on the microencapsulation of insulin have been studied in vitro and in vivo after subcutaneous administration to hyperglycemic rats. Hydrophilic PLGA polymers produced a higher burst effect than the hydrophobic ones. Moreover, an incomplete insulin release was observed with the hydrophilic PLGA polymers in comparison with the hydrophobic ones. An explanation for that incomplete release can be the development of polymer-insulin interactions associated to the polymer hydrophilic/hydrophobic character, as detected by DSC analysis. Differences in the release rate of microsphere formulations lead to differences in the hypoglycemic action and the weight of animals. Hydrophobic PLGA was able to prolong the hypoglycemic action up to 4 weeks which is at least double than that obtained with hydrophilic PLGA of a similar viscosity. Comparing insulin microspheres with an immediate release formulation, microspheres can increase insulin relative bioavailability up to four times.

Effects of hydroxypropyl-beta-cyclodextrin on the chemical stability and the aqueous solubility of thalidomide enantiomers.

The aim of this work was to study the effect of hydroxypropyl-beta-cyclodextrin on the solubility and stability of thalidomide enantiomers in aqueous solutions for clinical oral administration to be used in HIV-infected children. For this reason racemic thalidomide was added to solutions containing different concentrations of hydroxypropyl-beta-cyclodextrin. True complexes were obtained by using hydroxypropyl-beta-cyclodextrin and the solubility of both thalidomide enantiomers was increased directly depending on the amount of hydroxylpropyl-beta-cyclodextrin in the medium although no enantioselective differences were observed at 37 degrees C. The chemical stability of thalidomide enantiomers is clearly improved by hydroxypropyl-beta-cyclodextrin. No enantioselective degradation of thalidomide was observed in sodium chloride solution (0.9%) samples stored at 6 degrees C for nine days when hydroxypropyl-beta-cyclodextrin was employed as excipient. Therefore a thalidomide solution suitable for oral administration can be prepared by adding hydroxypropyl-beta-cyclodextrin at 10% (w/v).

In vivo distribution and therapeutic efficacy of a novel amphotericin B poly-aggregated formulation.

OBJECTIVES: The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB). METHODS: The therapeutic efficacy and toxicity of amphotericin B formulations was studied in an immunocompetent murine model of systemic candidiasis. A pharmacokinetic study was also performed to measure the plasma, kidney, liver and spleen amphotericin B concentrations after administration of the three formulations to mice. RESULTS: The acute toxicity of P-AMB in mice is lower than that of the conventional amphotericin B reference formulation (D-AMB). The 50% lethal doses were increased at least eight times. Intravenous bolus administration of doses up to 40 mg/kg of body weight of poly-aggregated amphotericin B, either P-AMB or MP-AMB, did not produce acute symptoms of toxicity. Interestingly, in the pharmacokinetic study, significant (P < 0.05) lower plasma and kidney amphotericin B concentrations and higher liver and spleen amphotericin B concentrations were achieved after poly-aggregated amphotericin B formulation (P-AMB and MP-AMB) administration in relation to the reference formulation (D-AMB). At high amphotericin B doses, no significant differences in efficacy (P > 0.05) were observed among the formulations (D-AMB, P-AMB and MP-AMB). CONCLUSIONS: Although the efficacy in the candidiasis treatment was decreased as a consequence of amphotericin B aggregation, it can be compensated by the possibility of increasing the doses with lower nephrotoxicity. Moreover, due to its lower toxicity while maintaining its effectiveness, the poly-aggregated formulations (P-AMB and MP-AMB) have a better therapeutic index than the conventional formulation (D-AMB).

HPLC assay for determination of amphotericin B in biological samples.

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1-10.0 microg/mL was obtained. Intra-day and inter-day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided.

Amphotericin B formulations and drug targeting.

Amphotericin B is a low-soluble polyene antibiotic which is able to self-aggregate. The aggregation state can modify its activity and pharmacokinetical characteristics. In spite of its high toxicity it is still widely employed for the treatment of systemic fungal infections and parasitic disease and different formulations are marketed. Some of these formulations, such as liposomal formulations, can be considered as classical examples of drug targeting. The pharmacokinetics, toxicity and activity are clearly dependent on the type of amphotericin B formulation. New drug delivery systems such as liposomes, nanospheres and microspheres can result in higher concentrations of AMB in the liver and spleen, but lower concentrations in kidney and lungs, so decreasing its toxicity. Moreover, the administration of these drug delivery systems can enhance the drug accessibility to organs and tissues (e.g., bone marrow) otherwise inaccessible to the free drug. During the last few years, new AMB formulations (AmBisome, Abelcet, and Amphotec) with an improved efficacy/toxicity ratio have been marketed. This review compares the different formulations of amphotericin B in terms of pharmacokinetics, toxicity and activity and discusses the possible drug targeting effect of some of these new formulations.

Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation.

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.

Influence of the vehicle on the properties and efficacy of microparticles containing amphotericin B.

New microparticles containing amphotericin B (AMB) have been developed and manufactured by spray drying. To this end albumin, polylactic-co-glycolic acids (PLGA) and poly(sebacic anhydride) have been employed as drug carriers. The selection of the solvent used to disperse the drug and the vehicle before spray drying was critical on production yields and physical properties of the microparticles. Once particle size, morphology and dispersability in some aqueous media were shown to be acceptable for an intravenous administration, in vivo efficacy was evaluated and compared with the reference medicine Fungizone. Microparticles prepared with albumin, albumin heated at a high temperature, some kinds of PLGA or polyanhydride, as well as Fungizone, were tested in an experimental hamster model of infection with Leishmania infantum, by evaluating the evolution of parasitic burdens in spleen, liver and antibody responses. After the injection of three doses corresponding to 2 mg of AMB per kilogram each, diverse reactions were reported depending on the vehicle. The best dispersability, reduction of parasites and antibody response were achieved when the treatment was performed with AMB in albumin microspheres.

Amphotericin B molecular organization as an essential factor to improve activity/toxicity ratio in the treatment of visceral leishmaniasis.

An in vivo study has been performed in order to determine the influence of amphotericin B (AMB) molecular organization on the toxicity and activity of this drug in the treatment of experimental visceral leishmaniasis. Three formulations with similar composition but different drug molecular self-association in aqueous media were prepared. Acute toxicity was evaluated by injecting them in healthy hamsters. Sub-acute toxicity and efficacy were studied administering them to animals previously infected with Leishmania infantum. The preparation with drug molecules completely dissolved into monomers (formulation "C") and produced the highest acute toxicity. The formulation whose AMB molecules were disposed as non-water-soluble multi-aggregates (formulation "B") proved to provide the lowest acute toxicity. This formula also showed an improved activity, mainly in the liver, if compared with the third tested formulation containing AMB molecules disposed as smaller dimerical "water-soluble" aggregates (formulation "A"). As a conclusion, molecular aggregation in biological media should be an important factor to consider when researching or optimizing medicines containing AMB. The liberation of molecules as large dispersed non-water-soluble multi-aggregates seems to improve the narrow therapeutic margin attached to the use of this drug.

Anti-leishmanial activity of a new formulation of amphotericin B.

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post-infection (p.i.) to treat animals previously infected with 10(7) stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4(+) subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non-encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.

Treatment of experimental visceral leishmaniasis with amphotericin B in stable albumin microspheres.

Hydrophilic albumin microspheres are proposed as a new delivery system for amphotericin B (AMB; AMB microspheres). The acute toxicity of AMB microspheres was lower than that of the AMB-deoxycholate (AMB-Doc) reference formulation in hamsters. Lethal doses in healthy and infected animals were improved at least eight times. Intravenous bolus administration of doses of AMB microspheres up to 40 mg/kg of body weight did not produce acute symptoms of toxicity. The efficacy of this new formulation was tested against Leishmania infantum-infected hamsters at doses of 2, 10, 20, and 40 mg/kg. With the 2-mg/kg dose, the activity of AMB, as assessed through the parasite load reductions in the liver and spleen and the evolution of antibody levels, was also improved (P < 0.05) by use of the AMB microsphere system. At the higher doses of 10, 20, and 40 mg/kg, reductions in parasite levels of more than 99% were achieved in the liver and spleen after the administration of AMB microspheres. A pharmacokinetic study was performed to study the serum, liver, and spleen AMB concentrations after administration of AMB microspheres and the reference formulation. Interestingly, a significant accumulation of AMB in the spleen and liver was observed after AMB microsphere administration. Our results suggest that this new formulation is a promising alternative to the conventional AMB-Doc formulation for the treatment of visceral leishmaniasis.

Ex vivo anthelmintic activity of albendazole-sulphoxide enantiomers.

The antiparasitic activity of racemic albendazole-sulphoxide (Ricobendazole = racRBZ) and its (+) and (-) enantiomers was tested in an ex vivo murine model for Trichinella spiralis infection. Larvae were isolated from the muscle of infected mice and exposed to concentrations between 0.01 and 1 microg/ml of the racemic mixture or to each of its enantiomers. The activity of each compound was then assayed by measuring the ability of the treated larvae to infect naive mice (larval viability). At a concentration of 0.5 microg/ml, all 3 compounds were highly effective in reducing the viability of the larvae, achieving reductions of 91.26% (racRBZ), 96.7% (+), and 89.2% (-), when compared with untreated controls. At lower concentrations (0.1 microg/ml), only treatment with (+)RBZ rendered a significant reduction in larval viability in comparison with controls (84.3%; P < 0.01), whereas at 0.01 microg/ml, none of the compounds altered larval viability (P > 0.05).

The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs.

The effect of solubilization by complexation with povidone on the oral bioavailability of three anthelmintic benzimidazole carbamate drugs: mebendazole (MBZ), albendazole (ABZ) and ricobendazole (RBZ), was studied in mice. The following in vitro characteristics of the initial raw materials and the drug-povidone complexes were evaluated: melting point (MP); mean dissolution time (MDT); solubility constants (Cs) in n-octanol, acid (pH 1.2) and neutral (pH 7.4) aqueous media; apparent partition coefficients (P) and capacity factors (k'W) determined by HPLC. The following in vivo parameters were also evaluated: AUC(0-infinity), C(max), T(max) and MRT. The possible relationship between in vitro characteristics and in vivo parameters was explored and it was found that an increase in solubility, especially in acidic medium, leads to an increase in AUC and C(max) and a decrease in T(max). Therefore, dissolution seems to be the absorption limiting step for these drugs. For the in vivo parameters related to the amount of absorbed drug (AUC and C(max)), the best correlation was obtained with the in vitro characteristics related to solubility which are Cs, MP and MDT. On the other hand, there were good linear correlations between T(max) which is an in vivo parameter related to the rate of drug absorption, and the lipophilia/hydrophilia (logP and log k'W) relation-parameters.

Microspheres containing insulin-like growth factor I for treatment of chronic neurodegeneration.

The therapeutic potential of peptide growth factors as insulin-like growth factor I (IGF-I) is currently under intense scrutiny in a wide variety of diseases, including neurodegenerative illnesses. A new poly(lactic-co-glycolide)-based microsphere IGF-I controlled release formulation for subcutaneous (SC) delivery has been developed by a triple emulsion method. The resulting microspheres displayed a mean diameter of 1.5microm, with an encapsulation efficiency of 74.3%. The protein retained integrity after the microencapsulation process as evaluated by circular dichroism and SDS-PAGE. The administration of IGF-I in microspheres caused at least a 30-fold increase in IGF-I mean residence time in rats and mice when compared with the conventional SC solution. Therefore, dosing can be changed from the conventional twice a day to once every 2 weeks. Therapeutic efficacy of this new formulation has been studied in mutant mice with inherited Purkinje cell degeneration (PCD). These mice show a chronic limb discoordination that is resolved after continuous systemic delivery of IGF-I. Normal motor coordination was maintained as long as IGF-I microsphere therapy is continued. Moreover, severely affected PCD mice, with marked ataxia, muscle wasting and shortened life-span showed a significant improvement after continuous IGF-I microsphere therapy as determined by enhanced motor coordination, marked weight gain and extended survival. This new formulation can be considered of great therapeutic promise for some chronic brain diseases.

Effect of gamma-sterilization process on PLGA microspheres loaded with insulin-like growth factor-I (IGF-I).

The influence of gamma-sterilization on the physicochemical properties of a controlled release formulation for the insulin-like growth factor-I (IGF-I) was investigated in this study. Recombinant human insulin-like growth factor-I (rhIGF-I) was efficiently entrapped in poly (D,L-lactide-co-glycolide) (PLGA) microspheres by water-in-oil-in-water (W/O/W) solvent evaporation technique. Microspheres were irradiated at a dose of 25kGy and evaluated by means of scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The stability of the released protein was investigated by circular dichroism (CD) and sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). No difference was noticed in microsphere size and morphology before and after irradiation. Drug loading remains essentially the same after the sterilization process. However, rhIGF-I aggregation was detected by electrophoresis. In addition, subtle changes in DSC pattern were noticed for irradiated microspheres. In vitro drug release from irradiated microspheres was also affected, showing an increased burst effect. From this results it can be concluded that gamma-sterilization process causes changes in the properties of rhIGF-I loaded microspheres.

In vitro release of valerenic and hydroxyvalerenic acids from valerian tablets.

Although most commercial valerian formulations are coated tablets not any comparison study of their drug release profiles has been published so far. The main objective of this work is to establish a drug release test suitable for studying and comparing different valerian tablets. Thus, hydroxyvalerenic and valerenic acid concentrations were assayed by HPLC using a C18 Kromasil (200 x 4.6 mm, 5 microm) column and a mobile phase containing methanol and an orthophosphoric acid solution 0.5% v/v in water at a ratio of 75:25 at a constant flow rate of 1 ml/min. Saturation solubilities for hydroxyvalerenic and valerenic acid at pH 6.8 were 26 +/- 5.1 and 1 +/- 0.6 microg/ml, respectively. Usually for drugs with such low solubility values, their oral absorption and hence bioavailability are limited by their dissolution characteristics. A dissolution test was conducted according to the general method 2 (paddles) of USP 24 using 500 ml buffer medium (pH 6.8) at 50 rpm. Five different formulations were studied and compared: one uncoated tablet formulation and four marketed coated tablets. The uncoated tablet formulation had the fastest release profile, whereas the coated tablets manifested very different release patterns, depending on the type of formulation. Because of these differences in drug release pattern not every tablet formulation may be appropriate for the same clinical indications. Clinical data are required to confirm the correlation between drug release pattern and the therapeutically value of each formulation.

Micromeritic and packing properties of diclofenac pellets and effects of some formulation variables.

The effects of two common diluents (microcrystalline cellulose and calcium phosphate dihydrate), two binding agents (gelatin and methacrylic polymer), and spheronization on the micromeritic (size, shape, density), flow, and packing properties of sodium diclofenac pellets were examined. The shape was assessed as the aspect ratio and was correlated to the flow rate and to the deviation of the tapped porosity from the value of 26%, which corresponds to the ideal rhombohedral packing of spheres. It was found that porosity deviation decreased greatly with spheronization, but it increased with hinder addition. Porosity deviation was proportional to the aspect ratio, while flow rate decreased logarithmically with porosity deviation. Porosity deviation may be a useful index for monitoring the quality of pellets, similar to the aspect ratio, as a successful, simple, and indirect indication of sphericity and of surface roughness as well.

Spray-dried powders as nasal absorption enhancers of cyanocobalamin.

The aim of this work is to describe and characterize a new spray-drying procedure for the production of nasal powders as an alternative to the conventional freeze-drying method. Cyanocobalamin was chosen as the active ingredient and loaded into five different nonsoluble vehicles with high water absorption ability. Then these hydrated particles were suspended in methylene chloride and spray-dried. Particle size, morphology, true, bulk and tapped density, percentage of compressibility, moisture content, water intake, and drug diffusion were studied and significant differences were obtained depending on the nature of the vehicle. The drying method, either the new spray- or the conventional freeze-drying, was less important. Interestingly, an inverse correlation was found between water uptake and drug diffusion. Microcrystalline cellulose, dextran microspheres, and crospovidone were chosen for an in vivo bioavailability study in rabbits. Three other nasal reference formulations and an intravenous solution were also administered. The spray-dried powders showed higher bioavailability than the three nasal reference formulations. The highest absorption enhancement was observed with cellulose microcrystalline powders, which provided a 25% mean absolute bioavailability, followed by crospovidone and dextran microspheres formulations with mean bioavailability values of 14% and 7%, respectively. In conclusion, the new spray-drying method is useful for the production of cyanocobalamin nasal powders.