Desoxycholate Amphotericin B Research Articles

Leishmanicidal activity of amphotericin B encapsulated in PLGA–DMSA nanoparticles to treat cutaneous leishmaniasis in C57BL/6 mice

The major goal of this work was to design a new nanoparticle drug delivery system for desoxycholate amphotericin B (D-AMB), based on controlled particle size, looking for the most successful release of the active agents in order to achieve the best site-specific action of the drug at the therapeutically optimal rate and dose regimen. For this, AMB nanoencapsulated in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles (Nano-D-AMB) has been developed, and its efficacy was evaluated in the treatment of experimental cutaneous leishmaniasis in C57BL/6 mice, to test if our nano-drug delivery system could favor the reduction of the dose frequency required to achieve the same therapeutic level of free D-AMB, and so, an extended dosing interval. Magnetic citrate-coated maghemite nanoparticles were added to this nanosystem (Nano-D-AMB-MG) aiming to increase controlled release of AMB by magnetohyperthermia. Female mice (N=6/group) were infected intradermally in the right footpad with promastigotes of Leishmania amazonensis in the metacyclic phase, receiving the following intraperitoneal treatments: 1% PBS for 10 consecutive days; D-AMB at 2mg/kg/day for 10days (totalizing 20mg/kg/animal); Nano-D-AMB and Nano-D-AMB-MG at 6mg/kg on the 1st, 4th and 7th days and at 2mg/kg on the 10th day, also totalizing 20mg/kg/animal by treatment end. The Nano-D-AMB-MG group was submitted to an AC magnetic field, allowing the induction of magnetohyperthermia. The evaluations were through paw diameter measurements; parasite number and cell viability were investigated by limiting dilution assay. D-AMB-coated PLGA–DMSA nanoparticles showed the same efficacy as free D-AMB to reduce paw diameter; however, the Nano-D-AMB treatment also promoted a significantly greater reduction in parasite number and cell viability compared with free D-AMB. The nano-drug AMB delivery system appeared more effective than free D-AMB therapy to reduce the dose frequency required to achieve the same therapeutic level. It thus favors a longer interval between doses, as expected with development of a new nano drug delivery system, and may be useful in the treatment of many different pathologies, from cancer to neurodegenerative diseases.

Invasive Aspergillosis: New Insights into Disease, Diagnostic and Treatment

Aspergillus infections are a threat to in patients with hematological malignancies. Known risk factors are profound and long lasting neutropenia, uncontrolled graft versus host disease, continuous administration of steroids and environmental factors such as hospital construction. Numerous efforts have been undertaken for prophylaxis of invasive aspergillosis in high-risk populations. Most of them failed to demonstrate survival advantages. Prophylaxis makes sense, since diagnosis and treatment of invasive aspergillosis remain difficult. The introduction of non-culture based tools for the diagnosis of invasive aspergillosis is an important step forward for early and sensitive diagnosis of invasive aspergillosis. Early treatment is the cornerstone of a successful management of invasive aspergillosis. Substantial improvement came with the introduction of lipid formulations of amphotericin B in the early 1990s. Voriconazole was the first azole that improved the overall survival for patients with invasive aspergillosis. Newer azoles and the echinocandins were introduced for the treatment of invasive aspergillosis in the late 1990s. Voriconazole and liposomal amphotericin B allow a safer and more effective treatment of invasive aspergillosis when compared with amphotericin B-desoxycholate. Combination of antifungal agents has been introduced in clinical trials. Up to now no significant benefit has been obtained with antifungal combination compared to voriconazole alone. Because mortality of invasive aspergillosis remains up to more than 50%, prophylaxis, early diagnosis and early initiation of antifungal therapy are of utmost importance for the reduction of invasive aspergillosis related mortality. Despite all advances in the management of invasive aspergillosis important questions remain unresolved. This article reviews the current state and new insights in the management of invasive aspergillosis and points out clinicians unmet needs.

Dose Range Evaluation of Mycograb C28Y Variant, a Human Recombinant Antibody Fragment to Heat Shock Protein 90, in Combination with Amphotericin B-Desoxycholate for Treatment of Murine Systemic Candidiasis

Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t(1/2)(α) of ~0.198 h and a t(1/2)(β) of ~1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis.

Leitliniengerechte Therapie der invasiven Aspergillose

Invasive fungus infections caused by aspergillus spp. occur most frequently in immunocompromised patients. A high infection-associated death rate of up to and over 50% is attributed even today to these fungi. The disease in humans is caused mainly by Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Other species, for example, Aspergillus terreus or Aspergillus nidulans are quantitatively less prevalent. Evidence based treatment of invasive aspergillosis has become safer and more effective within the last ten years through the introduction of the new azoles and the echinocandines. Voriconazole has become the medication of choice for initial therapy. The efficacy of voriconazole is well documented, to include the treatment of disseminated infections of the central nervous system. Amphotericin B-desoxycholate is associated with definite side-effects in intravenous therapy. On the grounds of its substantial toxicity, the North American Infectious Disease Society's (IDSA) Guidelines of 2008 recommend amphotericin B-desoxycholate for regions with restricted resources only, which could be the case in underdeveloped countries. Liposomal amphotericin B in the daily standard dose of 3 mg/kg offers a rate of response similar to the one with voriconazole in the first-line treatment of invasive aspergillosis. However, a direct comparison with voriconazole on the basis of randomized studies is not available. As a secondary therapeutic treatment, in case of failure or intolerance of the primary treatment, caspofungin, micafungin and posaconazole have recently been under study. Both the echinocandines and posaconazole have proven effective in daily clinical practise. In refractory cases of invasive aspergillosis a combination therapy has been employed clinically. The results of prospective comparative controlled studies on combination therapy versus monotherapy will not be available until after 2010.

Nebulization of four commercially available amphotericin B formulations in persistently granulocytopenic rats with invasive pulmonary aspergillosis: evidence for long-term biological activity

The nebulization of amphotericin B desoxycholate (AMB-DOC), liposomal amphotericin B (L-AMB), amphotericin B lipid complex (ABLC) and amphotericin B colloidal dispersion (ABCD) has been investigated. Particle sizes of generated aerosol droplets were measured. Pulmonary amphotericin B deposition and amphotericin B concentration in blood directly after nebulization and at six weeks after nebulization was measured in healthy rats. The efficacy of nebulized amphotericin B formulations was evaluated in persistently granulocytopenic rats with invasive pulmonary aspergillosis. Treatment was given either after or before fungal inoculation. The endpoint was survival of animals. Aerosol particle sizes, expressed as the values for the mass median diameter were 1.38, 2.43, 0.90 and 2.29 microm for AMB-DOC, L-AMB, ABLC and ABCD, respectively. Amphotericin B concentrations in the lungs directly after nebulization exceeded the minimum inhibitory concentration of Aspergillus fumigatus and amphotericin B was still detected in lungs of rats at six weeks after nebulization. Treatment, started at 16 h after fungal inoculation, resulted in a significantly prolonged survival as compared with sham-treated rats for all four formulations. Prophylactic treatment at one week before fungal inoculation resulted in a significantly prolonged survival for all four formulations. Aerosol treatment given at two weeks before inoculation was effective only for AMB-DOC and L-AMB, whereas treatment given at six weeks resulted in a significantly prolonged survival for L-AMB only. All commercially available amphotericin B preparations could be nebulized efficiently and may be of value in the prophylactic treatment of invasive pulmonary aspergillosis.

Amphotericin B in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles against paracoccidioidomycosis

The present study reports on the preparation and testing of a desoxycholate amphotericin B (D-AMB) sustained delivery system based on poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) polymeric blends (Nano-D-AMB) aimed at reducing the number of AMB administrations required to treat mycosis. BALB/c mice were infected with the yeast Paracoccidioides brasiliensis intravenously to mimic the chronic form of paracoccidioidomycosis. At 30 days post-infection, the animals were treated with Nano-D-AMB [6 mg/kg of encapsulated D-AMB, intraperitoneally (ip), interval of 72 h] or D-AMB (2 mg/kg, ip, interval of 24 h). Drug efficacy was investigated by the fungal burden recovery from tissues. Toxicity was assessed by renal and hepatic biochemical parameters, physical appearance of the animals and haematological investigation. The control groups used were non-infected and the infected mice mock treated with PBS. Nano-D-AMB presented results comparable to free D-AMB, with a marked antifungal efficacy. The Nano-D-AMB-treated group presented lower loss of body weight and absence of stress sign (piloerection and hypotrichosis) observed after D-AMB treatment. No renal [blood urea nitrogen (BUN), creatinine] or hepatic (pyruvic and oxalacetic glutamic transaminases) biochemical abnormalities were found. The micronucleus assay showed no significant differences in both the micronucleus frequency and percentage of polychromatic erythrocytes for Nano-D-AMB, indicating the absence of genotoxicity and cytotoxic effects. The D-AMB-coated PLGA-DMSA nanoparticle showed antifungal efficacy, fewer undesirable effects and a favourable extended dosing interval. Nano-D-AMB comprises an AMB formulation able to lessen the number of drug administrations. Further studies would elucidate whether Nano-D-AMB would be useful to treat systemic fungal infections such as paracoccidioidomycosis, candidiasis, aspergillosis and cryptococcosis.

Meta‐analysis of nebulized amphotericin B to prevent or treat pulmonary aspergillosis in immunosuppressed animals

Pulmonary aspergillosis in immunosuppressed patients is associated with significant mortality and morbidity. We assessed the prophylactic and therapeutic effect of nebulized amphotericin B (AmB) on mortality of immunosuppressed animals with pulmonary aspergillosis in this meta-analysis. Literature search was based on MEDLINE (1966 to January 15, 2007), EMBASE, and Cochrane-Controlled Trials Register (2006 issue 4) databases. Only randomized-controlled studies comparing nebulized AmB with placebo on immunosuppressed animals were included. Two reviewers reviewed and extracted the data independently. Eight studies including 839 immunosuppressed animals were considered. The overall mortality of the immunosuppressed animals with pulmonary aspergillosis treated with nebulized AmB was reduced (69.4% versus 87.7%, odds ratio [OR] 0.12, 95% confidence interval [CI]: 0.08-0.19; P < 0.0001). The effect of nebulized AmB on mortality was more significant when the drug was administered as prophylaxis before the inoculation of Aspergillus fumigatus (OR 0.07, 95% CI: 0.03-0.12, P = 0.0001; I(2) = 0) than as a therapeutic agent (OR 0.20, 95% CI: 0.11-0.36, P < 0.0001; I(2) = 0) (ratio of the 2 ORs = 2.86, 95% CI: 1.03-6.11; P = 0.04). The effectiveness of AmB desoxycholate (OR 0.16, 0.06-0.38, P < 0.0001; I(2) = 0) and lipid-associated (liposomal, or lipid complex or colloidal dispersion) amphotericin (OR 0.11, 95% CI: 0.06-0.20, P < 0.0001; I(2) = 0) was not significantly different (ratio of the 2 ORs = 1.46, 95% CI: 0.49-4.39; P = 0.50). Three studies evaluated the potential side effects of nebulized amphotericin and showed that there was no significant renal toxicity but the amphotericin desoxycholate had some detrimental effects on pulmonary surfactant function. The use of nebulized amphotericin, either as a prophylactic or therapeutic agent, is effective in reducing mortality of immunosuppressed animals with pulmonary aspergillosis. A large randomized controlled trial is needed to confirm whether combining nebulized amphotericin with systemic antifungal agents will reduce mortality in immunosuppressed patients with suspected or proven pulmonary aspergillosis.

Comparison of the effect of amphotericin B desoxycholate and amphotericin B colloidal dispersion on renal functions and renal morphology in rats

Amphotericin B (AmB) desoxycholate remains as one of the most efficacious agents currently available for the treatment of systemic fungal infections; however, amphotericin B colloidal dispersion (ABCD) has been developed because of AmB desoxycholate nephrotoxicity. The goal of our study was to compare the effect of administration of AmB desoxycholate and ABCD on renal functions and renal morphology in rats. Amophotericin B desoxycholate as well as ABCD causes damage to renal tubuli and polyuria. Amophotericin desoxycholate causes considerably more severe damage to tubuli than ABCD, but the morphological damage to renal glomeruli is minimal in both formulas. In tubular cells, AmB desoxycholate causes severe damage to mitochondria, vacuolation of cytoplasm, and increased values of volume density of peroxisomes. None of these formulas causes a decrease in glomerular filtration in rats when animals are properly hydrated.

Efficacy of PLD-118, a novel inhibitor of candida isoleucyl-tRNA synthetase, against experimental oropharyngeal and esophageal candidiasis caused by fluconazole-resistant C. albicans.

PLD-118, formerly BAY 10-8888, is a synthetic antifungal derivative of the naturally occurring beta-amino acid cispentacin. We studied the activity of PLD-118 in escalating dosages against experimental oropharyngeal and esophageal candidiasis (OPEC) caused by fluconazole (FLC)-resistant Candida albicans in immunocompromised rabbits. Infection was established by fluconazole-resistant (MIC > 64 microg/ml) clinical isolates from patients with refractory esophageal candidiasis. Antifungal therapy was administered for 7 days. Study groups consisted of untreated controls; animals receiving PLD-118 at 4, 10, 25, or 50 mg/kg of body weight/day via intravenous (i.v.) twice daily (BID) injections; animals receiving FLC at 2 mg/kg/day via i.v. BID injections; and animals receiving desoxycholate amphotericin B (DAMB) i.v. at 0.5 mg/kg/day. PLD-118- and DAMB-treated animals showed a significant dosage-dependent clearance of C. albicans from the tongue, oropharynx, and esophagus in comparison to untreated controls (P </= 0.05, P </= 0.01, P </= 0.001, respectively), while FLC had no significant activity. PLD-118 demonstrated nonlinear plasma pharmacokinetics across the investigated dosage range, as was evident from a dose-dependent increase in plasma clearance and a dose-dependent decrease in the area under the plasma concentration-time curve. The biochemical safety profile was similar to that of FLC. In summary, PLD-118 demonstrated dosage-dependent antifungal activity and nonlinear plasma pharmacokinetics in treatment of experimental FLC-resistant oropharyngeal and esophageal candidiasis.

Dose-dependent pharmacokinetics of amphotericin B lipid complex in rabbits.

Amphotericin B lipid complex (ABLC) was recently approved by the Food and Drug Administration for treatment of patients with invasive fungal infections who are intolerant of or refractory to conventional amphotericin B therapy. Little is known, however, about the pharmacokinetics of this new antifungal compound. We therefore investigated the pharmacokinetics of ABLC in comparison with those of conventional desoxycholate amphotericin B (DAmB) in rabbits. The pharmacokinetics of DAmB in a rabbit model were similar to those previously reported in humans. The pharmacokinetics of ABLC differed substantially from those of DAmB. Plasma amphotericin B levels following ABLC administration were 10 times lower than those following administration of an equal dosage of DAmB. The levels of ABLC in whole blood were approximately 40 times greater than those in plasma. The ABLC model differed from the DAmB model by (i) a dose- and time-dependent uptake and return between the plasma compartment and apparent cellular components of the blood-sediment compartment and (ii) time-dependent tissue uptake and return to plasma from serially connected compartments. Following infusion of ABLC, there was a nonlinear uptake into the apparent cellular components of the blood-sediment compartment. This uptake was related to the reciprocal of the integral of the total amount of drug infused (i.e., the more drug infused the greater the fractional uptake between 0.5 and 5 mg/kg of body weight for ABLC). The transfer of drug from plasma to the cellular components of the blood-sediment compartment resulted in initial uptake followed by rapid redistribution back to the plasma. The study describes a detailed model of the pharmacokinetics of ABLC and characterizes a potential role of the cellular components of the blood-sediment compartment in the distribution of this new antifungal compound in tissue.

Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice.

Heated (20 min at 70 degrees C) amphotericin B-desoxycholate (hAMB-DOC) was further characterized, as was another formulation obtained after centrifugation (60 min, 3000 x g), hcAMB-DOC. Conventional AMB-DOC consisted of individual micelles (approximately 4 nm in diameter) and threadlike aggregated micelles, as revealed by cryo-transmission electron microscopy. For both hAMB-DOC and hcAMB-DOC, pleiomorphic cobweb structures were observed with a mean particle size of approximately 300 nm as determined by laser diffraction. The potent antifungal activity of AMB-DOC against Candida albicans is not reduced by heating. Effective killing of C. albicans (>99.9% within 6 h) was obtained at 0.1 mg/liter with each of the AMB formulations. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) from C. albicans of > or =50% was observed at 0.8, 0.4, and 0.4 mg/liter, respectively. After heating of AMB-DOC, toxicity was reduced 16-fold as determined by red blood cell (RBC) lysis. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, hemolysis of > or =50% was observed at 6.4, 102.4, and 102.4 mg/liter, respectively. In contrast, AMB-DOC and its derivates showed similar toxicities in terms of cation release from RBC. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) of > or =50% was observed at 1.6, 0.8, and 0.8 mg/liter, respectively. In persistently leukopenic mice with severe invasive candidiasis, higher dosages of both hAMB-DOC and hcAMB-DOC were tolerated than those of conventional AMB-DOC (3 versus 0.8 mg/kg of body weight, respectively), resulting in significantly improved therapeutic efficacy. In conclusion, this new approach of heating AMB-DOC may be of great value for further optimizing the treatment of severe fungal infections.

Biodistribution of liposomal amphotericin B (AmBisome) and amphotericin B-desoxycholate (Fungizone) in uninfected immunocompetent mice and leucopenic mice infected with Candida albicans.

The biodistribution of liposomal amphotericin B (L-AmB; AmBisome) and amphotericin B-desoxycholate were compared after a single injection of drug in uninfected immunocompetent mice and in leucopenic mice 6 h after inoculation with Candida albicans. Amphotericin B-desoxycholate was administered at the maximum tolerated dose (MTD) of 0.3 mg/kg whereas L-AmB was given at either 0.3 mg/kg or the MTD of 7 mg/kg. Amphotericin B (AmB) concentrations in the blood, liver, spleen, lungs and kidneys were determined by HPLC analysis at various intervals during the 48 h after administration. The biodistribution of both preparations of AmB followed similar patterns in both uninfected immunocompetent mice as well as those that were leucopenic and infected with C. albicans. Administration of L-AmB resulted in increased concentrations of drug in the blood, liver, and spleen but decreased concentrations in the kidney and lung. Hepatosplenic uptake of L-AmB was highly dose dependent with 7 mg/kg resulting in a relatively prolonged blood circulation. Blood and tissues retained high AmB concentrations after administration of L-AmB at the MTD. By using radiolabelled L-AmB, it was found that the high AmB concentrations in blood represented liposome-associated AmB and that during circulation in blood slow release of AmB occurred.

Amphotericin B liposomes with prolonged circulation in blood: in vitro antifungal activity, toxicity, and efficacy in systemic candidiasis in leukopenic mice.

Pegylated amphotericin B (AmB) liposomes (PEG-AmB-LIP) were compared with laboratory-prepared nonpegylated AmB liposomes (AmB-LIP), a formulation with a lipid composition the same as that in AmBisome, as well as with industrially prepared AmBisome regarding their in vitro antifungal activities, toxicities, blood residence times, and therapeutic efficacies. Killing of Candida albicans (> 99.9%) during short-term (6-h) incubation was observed at 0.2 mg of AmB per liter for AmB desoxycholate, 0.4 mg of AmB per liter for PEG-AmB-LIP, 0.8 mg of AmB per liter for AmB-LIP, and 12.8 mg of AmB per liter for AmBisome. The maximum tolerated doses of PEG-AmB-LIP, AmB-LIP, and AmBisome were 15, 19, and > 31 mg of AmB per kg of body weight, respectively. In contrast to AmB-LIP, the blood residence time of PEG-AmB-LIP was prolonged and dose independent. In a model of systemic candidiasis in leukopenic mice at a dose of 5 mg of AmB per kg, PEG-AmB-LIP was completely effective and AmB-LIP was partially effective, whereas AmBisome was not effective. AmB-LIP at 11 mg of AmB per kg was partially effective. AmBisome at 29 mg of AmB per kg was completely effective. In conclusion, the therapeutic efficacies of AmB liposomes can be improved by preparing AmB liposomes in which a substantial reduction in toxicity is achieved while antifungal activity is retained. In addition, therapeutic efficacy is favored by a prolonged residence time of AmB liposomes in blood.

Dose-dependent antifungal activity and nephrotoxicity of amphotericin B colloidal dispersion in experimental pulmonary aspergillosis.

We investigated the safety and efficacy of amphotericin B colloidal dispersion (ABCD) for the treatment of invasive pulmonary aspergillosis in persistently granulocytopenic rabbits. Treatment groups included ABCD in dosages of 1, 5, and 10 mg/kg/day intravenously or conventional desoxycholate amphotericin B (DAmB) at 1 mg/kg/day intravenously. Antifungal activity was directly related to increasing dosage of ABCD as determined by the concentration of Aspergillus fumigatus organisms in lungs and the frequency of hemorrhagic pulmonary lesions. At 5 and 10 mg/kg/day, there was a significant reduction in the tissue burden of A. fumigatus as measured by percent culture-positive lobes and CFU per gram of tissue (P < or = 0.001), whereas at 1 mg/kg/day measured by percent culture-positive lobes and CFU per gram of tissue (P < or = 0.001), whereas at 1 mg/kg/day the tissue burden of A. fumigatus was not significantly different from that in untreated controls. Microbiological clearance was significantly greater at 1 mg of DAmB per kg per day than at 1 mg of ABCD per kg per day (P < or = 0.001). There was no difference in microbiological clearance of bronchoalveolar lavage fluid among the treatment groups as measured by CFU per milliliter. As determined by survival, ABCD at 5.0 mg/kg/day was more effective than DAmB at 1.0 mg/kg/day and ABCD at 10 mg/kg/day. ABCD at 10 mg/kg/day was more nephrotoxic than the lower dosages of ABCD and resulted in higher mortality. Impairment of glomerular filtration developed as a direct function increasing the ABCD dosage (r = 0.77; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B in liposomes: a novel therapy for histoplasmosis.

Incorporation of amphotericin B into liposomes significantly altered its toxicity, tissue distribution, and efficacy. Compared with intravenously administered amphotericin B-desoxycholate, liposome-amphotericin B showed a reduced acute toxicity and a maximal tolerable dose 9 times greater than amphotericin B-desoxycholate. Liposome-amphotericin B also produced higher tissue and lower serum concentrations than amphotericin B-desoxycholate, and was significantly more effective in prolonging survival of mice infected with Histoplasma capsulatum.