Pavel Gershkovich

Development and characterization of oral lipid-based amphotericin B formulations with enhanced drug solubility, stability and antifungal activity in rats infected with Aspergillus fumigatus or Candida albicans.

OBJECTIVE To develop an oral formulation of Amphotericin B (AmpB) with: (A) medium chain triglycerides, fatty acids and nonionic surfactants as a self-emulsifying drug delivery system (SEDDS); or (B) glyceryl mono-oleate (Peceol) with poly(ethylene glycol) (PEG)-phospholipids. METHODS SEDDS formulations were prepared by simple mixing at 40 degrees C. Peceol/DSPE-PEG-lipid formulations were prepared by solvent evaporation. Parameters evaluated included: miscibility, solubility and emulsion droplet size after incubation in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) via dynamic light scattering. The stability of AmpB in Peceol/DSPE-PEG was evaluated in SGF and SIF. Phase stability of AmpB in Peceol+/-DSPE-PEG following thermal cycling was evaluated by atomic force microscopy (AFM). Aspergillus fumigatus (2.9-3.45 x 10(7) colony forming units per mL [CFU]) or Candida albicans (3-3.65 x 10(6) CFU per mL) were injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single oral gavage of a Peceol-DSPE/PEG2000-based AmpB (10 mg AmpB/kg and 5 mg AmpB/kg for the Candida albicans study only) twice daily for 2 consecutive days, a single intravenous (i.v.) dose of Abelcet (5mg AmpB/kg), or physiologic saline (non-treated controls; n=9) once daily for 2 consecutive days. Antifungal activity was assessed by organ CFU concentrations and plasma galactomannan levels in the case of A. fumigatus and organ CFU concentrations in the case of Candida albicans. Plasma samples were taken from each animal prior to infection, 48 h after initiation of infection but prior to drug treatment and at the end of the study for plasma creatinine determinations as a measure of renal toxicity. RESULTS Mean diameter of SEDDS after 30 min in 150 mM NaCl at 37 degrees C was 200-400 nm. However, the Peceol/DSPE-PEG, where PEG MW was 350, 550, 750 or 2000, showed a greater solubilization of AmpB (5 mg/mL) compared to SEDDS formulations (100-500 microg/mL). Upon dispersion in SIF, Peceol/DSPE-PEG formulations generated submicron emulsion particle sizes varying slightly with PEG MW. Stability of the AmpB in Peceol/DSPE-PEG formulations in SGF or SIF was >80% after 2 h, and best for formulations containing DSPE-PEG 750 or 2000 compared to 350, 550 or Peceol only. Monoglyceride-Peceol-DSPE/PEG2000-based oral AmpB treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by >80% compared to non-treated controls without significant changes in plasma creatinine levels in the A. fumigatus infected rats. In addition, this formulation significantly decreased kidney fungal CFU concentrations by >75% at the 5 mg/kg dose and by >95% at the 10 mg/kg dose compared to non-treated controls without significant changes in the plasma creatinine levels in the Candida albicans-infected rats. CONCLUSIONS Novel lipid-based AmpB oral formulations were prepared that provide excellent drug solubilization, drug stability in simulated gastric and intestinal fluids and antifungal activity without renal toxicity in rats infected with A. fumigatus and C. albicans.

Highly Effective Oral Amphotericin B Formulation against Murine Visceral Leishmaniasis

Visceral leishmaniasis is a deadly parasitic disease caused by obligate intramacrophage protozoans of the Leishmania genus. The World Health Organization estimates the annual death toll to be 50,000, with 500,000 new cases each year. Without treatment, visceral leishmaniasis is inevitably fatal. For the last 70 years, the first line of defense has been pentavalent antimonials; however, increased resistance has brought amphotericin B to the forefront of treatment options. Unfortunately, the difficult route of drug administration, toxicity issues, and cost prevent amphotericin B from reaching the infected population, and mortality continues to rise. Our reformulation of amphotericin B for oral administration has resulted in a highly efficacious antileishmanial treatment that significantly reduces or eradicates liver parasitemia in a murine model of visceral leishmaniasis. This formulation has overcome amphotericin Bs significant physicochemical barriers to absorption and holds promise for the development of a self-administered oral therapy for the treatment of visceral leishmaniasis.

Pharmacokinetics and biodistribution of amphotericin B in rats following oral administration in a novel lipid-based formulation

OBJECTIVES To assess the pharmacokinetics and biodistribution of amphotericin B (AmB) following oral administration in a novel mono/diglyceride-phospholipid formulation and to compare with intravenous (iv) administrations using commercial formulations. METHODS Rats were allocated into the following treatment groups: oral gavage of AmB dispersed in mono/diglyceride-phospholipid formulation at doses of 4.5 and 10 mg/kg; iv bolus administration of 0.8 mg/kg Fungizone; iv bolus of 5 mg/kg Abelcet and iv bolus of 5 mg/kg AmBisome. Blood was sampled from jugular vein cannula at certain time points. The animals were sacrificed 72 h following administration of AmB and multiple tissues were harvested. The concentration of AmB in plasma and tissues was determined by means of HPLC. The plasma creatinine concentrations were determined using an enzymatic kit. RESULTS The pharmacokinetics and tissue distribution of AmB following iv administrations of the commercial formulations were found to be highly formulation dependent. The terminal half-life and biodistribution of orally administered AmB in a mono/diglyceride-phospholipid formulation resembled those of Fungizone. The larger volume of the co-administered lipid-based formulation in the case of the higher dose of orally administered AmB resulted in flip-flop kinetics and in preferential distribution into the kidneys. No nephrotoxicity was detected for any formulation and route of administration. CONCLUSIONS Oral administration of AmB in a mono/diglyceride-phospholipid formulation to rats resulted in significant intestinal absorption into the systemic circulation with pharmacokinetic and biodistribution properties similar to a micellar iv preparation.

A Novel Tropically Stable Oral Amphotericin B Formulation (iCo-010) Exhibits Efficacy against Visceral Leishmaniasis in a Murine Model

Purpose To develop an oral formulation of amphotericin B (AmB) that is stable at the temperatures of WHO Climatic Zones 3 and 4 (30–43°C) and to evaluate its efficacy in a murine model of visceral leishmaniasis (VL). Methods The stability testing of four novel oral lipid AmB formulations composed of mono- and di-glycerides and pegylated esters (iCo-010 to iCo-013) was performed over 60 d and analyzed by HPLC-UV. In addition, the four formulations were incubated 4 h in fasted-state simulated intestinal fluid. AmB concentration was measured spectrophotometrically and emulsion droplet diameter was assessed by dynamic light scattering. Antileishmanial activity of iCo-010 was evaluated at increasing oral doses (2.5 to 10 mg/kg) in a murine model of VL. Results AmB stability in the lipid formulation (iCo-010) was >75% over 60 days. After 4 h in fasted-state simulated intestinal fluid, AmB concentration was >95%. iCo-010 demonstrated significant efficacy when orally administered to VL-infected mice bid for five days (inhibition of 99%, 98%, and 83% at 10, 5 and 2.5 mg/kg compared to the vehicle control). In addition, the qd dose of 20 mg/kg provided 96% inhibition compared to the vehicle control. Conclusions The oral AmB formulation iCo-010 is stable at the temperatures of WHO Climatic Zones 3 and 4 (30–43°C). iCo-010 showed excellent antileishmanial activity at both 10 mg/kg po bid for 5 days (<99% reduction in parasitic infection) and 20 mg/kg po qd for 5 days (95% inhibition when compared to control).

Translational insight into statin-induced muscle toxicity: from cell culture to clinical studies

Statins are lipid-lowering drugs used widely to prevent and treat cardiovascular and coronary heart diseases. These drugs are among the most commonly prescribed medicines intended for long-term use. In general, statins are well tolerated. However, muscular adverse effects appear to be the most common obstacle that limits their use, resulting in poor patient compliance or even drug discontinuation. In addition, rare but potentially fatal cases of rhabdomyolysis have been reported with the use of these drugs, especially in the presence of certain risk factors. Previous reports have investigated statin-induced myotoxicity in vivo and in vitro using a number of cell lines, muscle tissues, and laboratory animals, in addition to randomized clinical trials, observational studies, and case reports. None of them have compared directly results from laboratory investigations with clinical observations of statin-related muscular adverse effects. To the best of our knowledge this is the first review article that combines laboratory investigation with clinical aspects of statin-induced myotoxicity. By reviewing published literature of in vivo, in vitro, and clinically relevant studies of statin myotoxicity, we aim to translate this important drug-related problem to establish a clear picture of proposed mechanisms that explain the risk factors and describe the diagnostic approaches currently used for evaluating the degree of muscle damage induced by these agents. This review provides baseline novel translational insight that can be used to enhance the safety profile, to minimize the chance of progression of these adverse effects to more severe and potentially fatal rhabdomyolysis, and to improve the overall patient compliance and adherence to long-term statin therapy.

Dissolution methodology for taste masked oral dosage forms

Conventional adult dosage forms are often not suitable for the paediatric and geriatric populations due to either swallowing difficulties or patient repulsion and a requirement for tailored dosing to individual compliance or physiological needs. Alternative formulations are available; however these often require the incorporation of more complex taste masking techniques. One approach to taste masking is to reduce contact between the bitter Active Pharmaceutical Ingredient (API) and oral cavity taste bud regions. This is achieved by hindering release in the oral cavity, or including competitive inhibition of bitter sensation for example by using flavours or sweeteners. There may also be other sensational complications from the API such as residual burning, reflux or metallic taste sensations to deal with. In vitro dissolution testing is employed to elucidate taste masking capability by quantifying release of the drug in simulated oral cavity conditions. Dissolution testing approaches may also be used to potentially predict or quantify the effect of the taste masking technique on the resultant pharmacokinetic profile. The present review investigates the anatomy and physiology of the oral cavity and current approaches to taste masking. In vitro dissolution methodologies adopted in the evaluation of taste masked formulations are discussed for their relative merits and drawbacks. A vast array of methodologies has been employed, with little agreement between approaches, and a lack of biorelevance. Future directions in dissolution methodology such as TNO Intestinal Model (TIM) and the Artificial Stomach and Duodenum model (ASD) are also discussed.

Long-circulating non-toxic blood pool imaging agent based on hyperbranched polyglycerols

PURPOSE Currently, in vivo or in vitro(99m)Tc-radiolabelled red blood cells are the standard blood pool imaging agents. Due to risks associated with handling of blood and the problems with the current (99m)Tc shortage, we were interested in a long-circulating biocompatible synthetic macromolecule that would be simple to prepare and could also be used for PET imaging. METHODS A high molecular weight hyperbranched polyglycerol (HPG) of 500 kDa was derivatized to coordinate radioactive gallium and to establish its labelling efficiency, stability and pharmacokinetics. RESULTS The resulting radiopharmaceutical in kit form was labelled rapidly within a couple of minutes at room temperature, was stable in transferrin and EDTA challenge tests, and was non-toxic in both cell viability and different hemocompatibility assays. A pharmacokinetic biodistribution study showed that the (67)Ga-HPGN was confined to the blood compartment with a biological half life of 50.7h. CONCLUSION (67)Ga-HPGN is thus a simple to prepare blood pool imaging agent for applications where a long biological half-life is essential, i.e., the diagnosis of internal bleeding. Since radiolabelling of the same kit with (68)Ga was also confirmed, we plan to evaluate it shortly as a PET blood pool imaging agent for cardiac applications.

Characterisation of human saliva as a platform for oral dissolution medium development

Human saliva is a biological fluid of great importance in the field of dissolution testing. However, until now, no consensus has been reached on its key characteristics relevant to dissolution testing. As a result, it is difficult to select or develop an in vitro dissolution medium to best represent human saliva. In this study, the pH, buffer capacity, surface tension, viscosity and flow rate of both unstimulated (US) and stimulated (SS) human saliva were investigated in order to provide a platform of reference for future dissolution studies using simulated salivary fluids. Age and gender related differences in a sample size of 30 participants for each parameter were investigated. Significant differences were established between US and SS for all characteristics except surface tension. Therefore, the requirement for using two simulated salivary fluids should be considered when developing an oral dissolution model.

Tropically stable novel oral lipid formulation of amphotericin B (iCo-010): biodistribution and toxicity in a mouse model

BackgroundThe purpose of this study was to evaluate the biodistribution and toxicity of amphotericin B (AmB) following multiple oral administrations of a novel tropically stable lipid-based formulation (iCo-010).MethodsBALB/c mice were allocated into six groups: oral iCo-010 twice daily for 5 days in the dose of 20, 10, 5 and 2.5 mg/kg; vehicle control; and intravenous boluses of Fungizone® 2 mg/kg once daily for 5 days. The animals were sacrificed 12 h following the last administration and blood and tissues were collected.ResultsThe plasma concentrations of AmB were similar to previously reported after administration of iCo-009. Somewhat lower concentrations of AmB were detected in reticulo-endothelial system in the case of iCo-010 when compared with iCo-009. The concentration in kidney was higher with iCo-010 than with iCo-009. The creatinine levels in all oral treatment groups were in a normal range as in the case of iCo-009. Administration of Fungizone® resulted in elevated plasma creatinine levels. Histopathology analysis detected no GI, liver or kidney toxicity following multiple dose oral administration of iCo-010. Fungizone® treatment induced necrotic changes in hepatic and kidney tissues.ConclusionsGiven the tropical stability of iCo-010, near identical activity against visceral leishmaniasis and significant concentrations in target organs this formulation has a potential to become a treatment of choice in tropical developing countries.

Inhibition of intestinal absorption of cholesterol by surface‐modified nanostructured aluminosilicate compounds

The aim of this work was to assess the ability of aqueous suspensions of surface-modified nanostructured aluminosilicate (NSAS) compounds to reduce the intestinal absorption of cholesterol in a rat model. The rats were divided into 10 treatment groups which included several NSAS compounds at various doses, ezetimibe at 10 mg/kg, stigmastanol at 50 mg/kg, and normal saline. All compounds and controls were independently administered by oral gavage and then a mixture of [(3)H]cholesterol and cold cholesterol in 10% Intralipid(R) was immediately administered orally to the animals. Systemic blood was sampled and the concentration of cholesterol in plasma was determined by means of radioactivity. Protonation of NSAS using an ion-exchange column resulted in significant inhibition of cholesterol absorption relative to the control group (31.5% and 38.6% reduction in absorption of cholesterol for 50 and 100 mg/kg doses, respectively). Other surface-ion modifications of NSAS compounds did not show significant effect on intestinal cholesterol absorption. The inhibition of cholesterol absorption by ezetimibe was superior and by stigmastanol was equal to the effect of protonated NSAS in the doses investigated in this study. In conclusion, protonated NSAS material seems to inhibit significantly the intestinal absorption of dietary cholesterol in a rat model.