Preparation by spray drying of amphotericin B-phospholipid composite particles and their anticellular activity

As a potent drug carrier for systemic fungal infections, amphotericin B(AmB)-phospholipid composite particles (APCPs) were prepared by the spray drying method. AmB and egg phosphatidylcholine, co-dissolved in methanol (0.0425-0.45 mg AmB/ml, 0.17-1.8 mg lipid/ml), was nebulized at 2 ml/min. The aerosol produced was carried by air at 1000 ml/min to the inner tubes of a serially connected distilling column system, of which the outer tubes were supplied with circulating water of 95°C. The particles, by scanning electron microphotography,are spherical and submicronsized. Upon hydration of the particles in phosphate-buffered saline for 30 min at room temperature, liposome-like bilayer vesicles were formed along with AmB-phospholipid complexes, evidenced by the transmission electron microphotographs and the positive peak around 330 nm of the circular dichroism spectrum, respectively. The hemolytic abilities of the APCPs were lower than those of free drug, without loss of the antifungal activity. The suppressed hemolysis could be ascribed to the liposomes and to the complexes that are reconstituted by hydration of APCPs. The dry composite particles could circumvent the inherent instability of liposomal formulations.     

Interactions of liposome-incorporated amphotericin B with kidney epithelial cell cultures

The polyene antibiotic amphotericin B (AmB) is profoundly cytotoxic to both fungal cells and mammalian cells. We have previously shown that the incorporation of AmB into phospholipid vesicles can markedly reduce the toxicity of the drug for mammalian cells (erythrocytes) without changing its antifungal potency [Mol. Pharmacol. 31:1-11 (1987)]. Because the primary site of in vivo toxicity of AmB is the kidney, here we investigate the effects of free AmB and liposomal AmB (L-AmB) on LLCPK1 cells, a porcine kidney cell line with many characteristics typical of proximal tubule cells. Acute exposure (2 hr) to free AmB inhibits protein synthesis and causes cell detachment and protein loss in LLCPK1 cells, with an IC50 of about 30 micrograms/ml. By contrast, certain formulations of L-AmB have little effect on protein synthesis/protein loss at concentrations of up to 2 mg/ml. The action of liposomes in protecting against acute AmB toxicity extends to effects on sugar transport and on cellular morphology in differentiated cultured kidney cells. Thus, the IC50 for inhibition of sodium-stimulated glucose transport by free AmB is 1.5 micrograms/ml whereas concentrations of L-AmB up to 1 mg/ml do not inhibit this process. However, chronic exposure of cells to L-AmB results in profound toxic effects as manifested by changes in cellular transport functions and cell morphology. Our results suggest that extended periods of proximity between cells and liposomes permit the transfer to toxic amounts of AmB. This may be of importance to the therapeutic use of AmB, for which protracted courses of drug administration are common.     

Development and anti-Candida evaluation of the vaginal delivery system of amphotericin B nanosuspension-loaded thermogel

Vulvovaginal candidiasis (VVC) is a typical kind of vaginal mucosal infection. Herein, we developed a novel vaginal delivery system of amphotericin B (AmB) nanosuspension-loaded thermogel (AmB NPs/thermogel) utilising pharmaceutical technique of high-pressure homogenisation and Poloxamer P407/P188 hydrogel. The stabiliser and hydrogel materials of the formulation were tested to maintain proper sol–gel transition as well as the relative stability of the particle size of AmB nanosuspension in the thermogel. The particle size of AmB nanosuspensions in the hydrogel was ～247?nm. Transmission electron microscopy images confirmed the round-shape morphology of AmB nanoparticles in AmB NPs/thermogel, while that of irregular morphology of merely AmB nanosuspensions without stabiliser and hydrogel materials. AmB could be sustained release for ～12?h in vitro. In vivo drug content in the vaginal tissue was also evaluated with 87, 47, 33 and 6.7% drug remaining after 1, 3, 6 and 12?h, respectively. The in vivo anti-Candida test was conducted on candidiasis-infected mice model. In the same drug dose of 2.5?mg/kg, AmB NPs/thermogel showed better anti-Candida efficiency compared with commercial AmB effervescent tablet. This delivery system might show some insights for the vaginal formulation development of other hydrophobic antifungal drugs.     

Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability

The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures. AmB used at concentrations of 0.6 to 1.25 microg ml(-1) led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5 - 5.0 microg ml(-1)) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells. Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity.     

Reduced renal toxicity of nanoparticular amphotericin B micelles prepared with partially benzylated poly-L-aspartic acid

A partially benzylated poly-L-aspartic acid (PBPA) was synthesized and investigated as a potential renal protective agent for the toxicity of amphotericin B (AmB) when the drug was administered as nanoparticular micelle. Nanoparticular AmB micelles were prepared by slow dialysis of the drug against distilled water at the elevated pH of 11.5 and subsequent sonication with the polymer solution. The resulting nanoparticular AmB micelles with PBPA showed an average diameter of about 20 nm and demonstrated significantly less damage to the tubular cells of the rat's kidney in terms of transmission electron microscopic study. There was little or no damage on the brush border of tubular cells 7 d after single intravenous dose of AmB 5 mg/kg in the rats when the drug was administered as nanoparticuar micelles with PBPA. The reduced renal toxicity appears to be due to the alteration of self-aggregation status of AmB. Molar absorptivity of AmB at 412 nm (a marker for the prevalence of non-aggregated AmB) was significantly higher in the nanoparticular AmB micelles with PBPA than those in AmB micelles without PBPA as well as in Fungizone. This result indicates an association of the reduced nephrotoxicity and the prevalence of non-aggregated AmB. Mechanisms for the reduced renal toxicity may also include the formation of electrostatic complex between anionic groups of PBPA and hydroxyl groups of AmB.     

In vitro activities of amphotericin B and AmBisome against Aspergillus isolates recovered from Italian patients treated for haematological malignancies

Although there is evidence that liposomal amphotericin B (AmBisome) is non-inferior to amphotericin B (AmB) in terms of in vivo efficacy, in vitro data regarding the activity of AmBisome against clinical isolates of Aspergillus are rare. In this study, the susceptibilities to AmB and AmBisome of 103 Aspergillus complex isolates (48 Aspergillus flavus, 33 Aspergillus fumigatus, 13 Aspergillus terreus and 9 Aspergillus niger) recovered from haematological patients with invasive infection were compared. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute (CLSI), whilst AmB susceptibility was also determined by Etest. Using a susceptible/resistant MIC cut-off of 1mg/L, all A. fumigatus and A. niger complexes isolates were susceptible to both AmB and AmBisome. In contrast, 38.5% and 30.8% of the A. terreus complex isolates were resistant to AmB and AmBisome, respectively, with good agreement between BMD and Etest methods. With respect to A. flavus complex isolates, 43.7% and 16.7% were resistant by the BMD method to AmBisome and AmB, respectively. For isolates with discrepant results, AmB MICs obtained by Etest were higher than those obtained for AmB by the BMD method and they were closer to those obtained for AmBisome by BMD. Aspergillus flavus AmB MICs ranged from 0.5 mg/L to 2 mg/L by the BMD method and from 1 mg/L to >16 mg/L by the Etest method, and AmBisome MICs ranged from 0.06 mg/L to >16 mg/L by the BMD method. Etest appears to be superior to the CLSI BMD method using AmB in detecting AmB resistance of Aspergillus spp., although the CLSI BMD method might be a suitable procedure if AmBisome is used as the test drug.     

Preparation and evaluation of zinc-pectin-chitosan composite particles for drug delivery to the colon: role of chitosan in modifying in vitro and in vivo drug release

Zinc-pectin-chitosan composite microparticles were designed and developed as colon-specific carrier. Resveratrol was used as model drug due to its potential activity on colon diseases. Formulations were produced by varying different formulation parameters (cross-linking pH, chitosan concentration, cross-linking time, molecular weight of chitosan, and drug concentration). Single-step formulation technique was compared with multi-step technique. Effect of these parameters was investigated on shape, size, weight, weight loss (WL), moisture content (MC), encapsulation efficiency (EE), drug loading (L), and drug release pattern of the microparticles. The formulation conditions were optimized from the drug release study. In vivo pharmacokinetics of the zinc-pectinate particles was compared with the zinc-pectin-chitosan composite particles in rats. Formulations were spherical with 920.48-1107.56 μm size, 21.19-24.27 mg weight of 50 particles, 89.83-94.34% WL, 8.31-13.25% MC, 96.95-98.85% EE, and 17.82-48.31% L. Formulation parameters showed significant influence on drug release pattern from the formulations. Formulation prepared at pH 1.5, 1% chitosan, 120 min cross-linking time, and pectin:drug at 3:1 ratio demonstrated colon-specific drug release. Microparticles were stable at 4 °C and room temperature. Pharmacokinetic study indicated in vivo colon-specific drug release from the zinc-pectin-chitosan composite particles only.     

Comparative Tissue Distribution and Elimination of Amphotericin B Colloidal Dispersion (Amphocil®) and Fungizone® After Repeated Dosing in Rats

The pharmacokinetic profiles of amphotericin B (AmB) after administration of Amphocil^®, an AmB/cholesteryl sulfate colloidal dispersion (ABCD) and the micellar AmB/deoxycholate (Fungizone^®) were compared after repeated dosing in rats. After administration of ABCD and Fungizone at an equal AmB dose (1 mg/kg), AmB concentrations in plasma and most tissues were lower for the ABCD dose, especially in the kidneys where reduced drug concentration correlated with reduced nephrotoxicity. In contrast, AmB concentrations in the liver were substantially higher when ABCD was administered; however, without an accompanying increase in hepato-toxicity. Daily administration of ABCD for 14 days did not lead to AmB accumulation in plasma; while a slight accumulation was observed after multiple administration of Fungizone. AmB was eliminated more slowly from the plasma and various tissues and urinary and fecal recoveries of AmB were reduced after ABCD administration. These results suggest that ABCD may be stored in tissues in a form that is less toxic and is eliminated from the systemic circulation by a different mechanism than the free and protein-bound AmB in plasma. AmB accumulation in the spleen was observed when higher doses of ABCD (5 mg/kg) were administered, which could be due to saturation of hepatic uptake of AmB. Comparison of spleen concentrations of AmB between ABCD and Fungizone^® at 5 mg/kg AmB doses was not possible because of Fungizone's toxicity in rats. In all other organs, AmB concentrations reached or approached a steady state within two weeks of dosing with ABCD. Urinary and fecal clearances of AmB were not different between ABCD and Fungizone administration. In summary, the distribution and elimination characteristics of AmB in rats were substantially altered when it was administered as ABCD in comparison to Fungizone. Nephrotoxicity of AmB in rats was reduced after administration of ABCD apparently because of the altered tissue distribution pattern. Thus, ABCD (Amphocil^®) may be a clinically beneficial formulation of AmB in patients with systemic fungal infections.     

Preparation, characterization and pharmacokinetics of liposomes-encapsulated cyclodextrins inclusion complexes for hydrophobic drugs

Liposomes-encapsulated indomethacin/cyclodextrins (IMC/ CD) inclusion complexes were prepared. The characteristics and pharmacokinetics of the combined system were investigated. The high drug entrapment values of 2.38 +/- 0.16 microg/mg and 2.48 +/- 0.12 microg/mg for liposomes-encapsulated IMC/ beta-CD and IMC/HP-beta-CD inclusion complexes were achieved, as only 1.60 +/- 0.09 microg/mg for conventional liposomes. Encapsulating IMC/CD inclusion complexes into liposomes resulted in a slow release of drug. Following intravenous administration, both liposomes-encapsulated inclusion complexes showed significantly improved AUC(0 - infinity) compared with that of conventional liposomes (p < 0.05). After intramuscular administration, C(max) has been increased to 5.21 +/- 1.14 microg x ml(-1) and 6.02 +/- 1.22 microg x ml(- 1) for liposomes-encapsulated IMC/ beta -CD and IMC/HP-beta -CD inclusion complexes, respectively, whereas only 2.43 +/- 0.69 microg x ml(- 1) for liposomes-encapsulated free drug (p < 0.01).     

Development and characterization of amphotericin B bearing emulsomes for passive and active macrophage targeting

The antifungal and antileishmanial agent amphotericin B (AmB) has been complexed with lipids to develop a less toxic formulation of AmB. Because lipid particles are phagocytized by the reticuloendothelial system, lipid associated AmB should be concentrated in infected macrophages of liver and spleen and be very effective against visceral leishmaniasis (VL) and systemic fungal infections. Therefore, AmB was formulated in trilaurin based nanosize lipid particles (emulsomes) stabilized by soya phosphatidylcholine (PC) as a new intravenous drug delivery system for macrophage targeting. Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range. Formulations were optimized for AmB to lipid ratio, sonication time and PC to trilaurin ratio. Emulsomes were modified by coating them with macrophage-specific ligand (O-palmitoyl mannan, OPM). The surface modified emulsomes and their plain counterparts were characterised for size, shape, lamellarity and entrapment efficiency. Fluorescence microscopy study showed significant localization of plain and coated emulsomes inside the liver and spleen cells of golden hamsters. In vivo organ distribution studies in albino rats demonstrated that extent of accumulation of emulsome entrapped AmB in macrophage rich organs, particularly liver, spleen and lungs was significantly high when compared against the free drug (AmB-deoxycholate or AmB-Doc). The rate and extent of accumulation were found to increase further on ligand anchoring. Further, a significantly higher (P < 0.05) drug concentration in the liver was estimated over a period of 24 h for OPM coated emulsomes than for plain emulsomes. We concluded that OPM coated emulsomes could fuse with the macrophages of liver and spleen due to ligand-receptor interaction and could target the bioactives inside them. The proposed plain and OPM coated emulsome based systems showed excellent potential for passive and active intramacrophage targeting, respectively and the approach could be a successful alternative to the currently available drug regimens of VL and systemic fungal infections.     

Development and characterization of amphotericin B bearing emulsomes for passive and active macrophage targeting

The antifungal and antileishmanial agent amphotericin B (AmB) has been complexed with lipids to develop a less toxic formulation of AmB. Because lipid particles are phagocytized by the reticuloendothelial system, lipid associated AmB should be concentrated in infected macrophages of liver and spleen and be very effective against visceral leishmaniasis (VL) and systemic fungal infections. Therefore, AmB was formulated in trilaurin based nanosize lipid particles (emulsomes) stabilized by soya phosphatidylcholine (PC) as a new intravenous drug delivery system for macrophage targeting. Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range. Formulations were optimized for AmB to lipid ratio, sonication time and PC to trilaurin ratio. Emulsomes were modified by coating them with macrophage-specific ligand (O-palmitoyl mannan, OPM). The surface modified emulsomes and their plain counterparts were characterised for size, shape, lamellarity and entrapment efficiency. Fluorescence microscopy study showed significant localization of plain and coated emulsomes inside the liver and spleen cells of golden hamsters. In vivo organ distribution studies in albino rats demonstrated that extent of accumulation of emulsome entrapped AmB in macrophage rich organs, particularly liver, spleen and lungs was significantly high when compared against the free drug (AmB-deoxycholate or AmB-Doc). The rate and extent of accumulation were found to increase further on ligand anchoring. Further, a significantly higher (P < 0.05) drug concentration in the liver was estimated over a period of 24 h for OPM coated emulsomes than for plain emulsomes. We concluded that OPM coated emulsomes could fuse with the macrophages of liver and spleen due to ligand–receptor interaction and could target the bioactives inside them. The proposed plain and OPM coated emulsome based systems showed excellent potential for passive and active intramacrophage targeting, respectively and the approach could be a successful alternative to the currently available drug regimens of VL and systemic fungal infections.     

Preparation of budesonide/gamma-cyclodextrin complexes in supercritical fluids with a novel SEDS method

The aim was to investigate if solid drug/cyclodextrin complexes could be produced in a single-step process with a solution enhanced dispersion by supercritical fluids (SEDS) method. Budesonide and gamma-cyclodextrin (CD) solutions (50% or 99.5% ethanol) were pumped from the same (conventional method) or separate (modified method) containers together with supercritical carbon dioxide through a coaxial nozzle into a particle formation chamber. The pressure was maintained at 100, 150 or 200 bar with a temperature of 40, 60 or 80 degrees C. SEDS-processed powders were characterised with HPLC, DSC and XRPD for budesonide content, complexation and crystallinity. The budesonide dissolution rate was determined in 1% gamma-CD aqueous solution. Solid, white budesonide/gamma-CD complex particles were formed using the conventional and modified SEDS processes. The complexation efficiency was dependent on the processing conditions. For example, with the conventional method (100 bar, 60 degrees C) the yield of the powder was 65+/-12% with 0.14+/-0.02 mg budesonide/mg powder, corresponding to 1:2 drug:CD molar ratio. The dissolution rate of this complexed budesonide (93+/-2% after 15 min) was markedly higher compared to unprocessed micronised budesonide (41+/-10%) and SEDS-processed budesonide without CD (61+/-3%). As a conclusion, SEDS is a novel method to produce solid drug/CD complexes in a single-step process.     

Chitosan‐coated antifungal formulations for nebulisation

Objectives The aim of this study was to produce and characterise amphotericin B (AmB) containing chitosan‐coated liposomes, and to determine their delivery from an air‐jet nebuliser. Methods Soya phosphatidylcholine : AmB (100 : 1) multilamellar vesicles were generated by dispersing ethanol‐based proliposomes with 0.9% sodium chloride or different concentrations of chitosan chloride. These liposomes were compared with vesicles produced by the film hydration method and micelles. AmB loading, particle size, zeta potential and antifungal activity were determined for formulations, which were delivered into a two‐stage impinger using a jet nebuliser. Key findings AmB incorporation was highest for liposomes produced from proliposomes and was greatest (approximately 80% loading) in chitosan‐coated formulations. Following nebulisation, approximately 60% of the AmB was deposited in the lower stage of the two‐stage impinger for liposomal formulations, for which the mean liposome size was reduced. Although AmB loading in deoxycholate micellar formulations was high (99%), a smaller dose of AmB was delivered to the lower stage of the two‐stage impinger compared to chitosan‐coated liposomes generated from proliposomes. Chitosan‐coated and uncoated liposomes loaded with AmB had antifungal activities against Candida albicans and C. tropicalis similar to AmB deoxycholate micelles, with a minimum inhibitory concentration of 0.5 µg/ml. Conclusions This study has demonstrated that chitosan‐coated liposomes, prepared by an ethanol‐based proliposome method, are a promising carrier system for the delivery of AmB using an air‐jet nebuliser, having a high drug‐loading that is likely to be effectively delivered to the peripheral airways for the treatment of pulmonary fungal infections.     

Gelatin coated hybrid lipid nanoparticles for oral delivery of amphotericin B

Amphotericin B (AmB) loaded polymer lipid hybrid nanoparticles (AmB-PLNs) comprised of lecithin (anionic lipid) and gelatin (Type A, cationic below its isoelectric point 7.0-9.0) were prepared by a two-step desolvation method to improve the oral bioavailability of AmB. The optimized AmB-PLNs were found to have particle size 253 ± 8 nm, polydispersity index (PDI) 0.274 ± 0.008, and entrapment efficiency 50.61 ± 2.20% at 6% w/w of initial theoretical drug loading. Scanning electron microscopy (SEM) revealed spherical shaped nanoparticles whereas confocal laser scanning electron microscopy (CLSM) and fluorescent resonance energy transfer (FRET) analysis confirmed the orientation of the lecithin (located in the core) and gelatin (exterior coat) within the system. The developed formulation exhibited a sustained drug release profile with a release pattern best fitted to Higuchi kinetics. Experiments on Caco-2 cell lines revealed a 5.89-fold increase in the intestinal permeability of AmB-PLNs whereas in vivo pharmacokinetic studies exhibited a 4.69-fold increase in the oral bioavailability upon incorporation of AmB into PLNs as compared to that of free drug. The developed formulation showed significantly lesser hemolytic toxicity as compared to the free drug, Fungizone (micellar solution of AmB) and Fungisome (liposomal formulation of AmB). Furthermore, blood urea nitrogen (BUN) and plasma creatinine levels, indicative of nephrotoxicity, were also found to be significantly lesser for developed PLN formulation as compared to free drug and Fungizone while comparable to that of Fungisome. The histopathology of the kidney tissues further confirmed the absence of any changes in the morphology of the renal tubules.     

Amphotericin B lipid nanoemulsion aerosols for targeting peripheral respiratory airways via nebulization

Amphotericin B (AmB) lipid nanoemulsions were prepared and characterized and their suitability for pulmonary delivery via nebulization was evaluated. AmB nanoemulsions were prepared by sonicating and vortexing the drug with two commercially available lipid nanoemulsions: the Intralipid(?) or Clinoleic(?). Loading the nanoemulsions with the drug slightly increased the size of the lipid droplets and did not affect the zeta potential of the nanoemulsions. The loading efficiency of AmB was found to be 87.46±2.21% in the Intralipid(?) nanoemulsions and 80.7±0.70% in the Clinoleic(?) formulation. This respectively corresponded to 21.86mg and 20.19mg of AmB being successfully loaded in the nanoemulsions. On aerosolization using a Pari Sprint jet nebulizer, both nanoemulsions produced very high drug output which was approximately 90% for both formulations. Using the two-stage impinger, the Clinoleic(?) emulsion had higher fine particle fraction (FPF) than the Intralipid(?), since the Clinoleic(?) displayed higher deposition of AmB in the lower impinger stage (exceeding 80%), compared to 57% for the Intralipid(?). Overall, the ease of preparation of the AmB lipid nanoemulsions, along with their in vitro nebulization performance suggest that lipid nanoemulsions could be successful nanocarriers for delivery of AmB to the peripheral respiratory airways.     

Dose-dependent kinetics of cilastatin in laboratory animals

Cilastatin, a potent inhibitor of renal dehydropeptidase I, was specifically designed to inhibit renal metabolism of the antibiotic imipenem in order to achieve therapeutically relevant imipenem concentrations in the urinary tract. In this study the elimination kinetics of cilastatin in rats at doses of 5, 10, 20, 50, 100, and 200 mg/kg iv were demonstrated to be dose dependent, with total plasma clearance and non-renal clearance falling from 20.2 +/- 3.1 ml/min/kg and 17.7 +/- 3.3 ml/min/kg (mean +/- S.D.) at the 5 mg/kg dose to 11.4 +/- 1.2 ml/min/kg and 5.30 +/- 1.2 ml/min/kg, respectively, at the 200 mg/kg dose, whereas the volume of distribution of the drug remained unchanged. Since cilastatin is mainly eliminated by renal excretion as well as by N-acetylation, the non-renal clearance may reasonably reflect the N-acetylation process. Thus, the dose-dependent kinetics of cilastatin might be explained, at least partly, by the saturation of the N-acetylation of the drug. The dose-related decrease in the fraction (fm) of cilastatin converted to its N-acetylated metabolite provided further evidence for the saturable N-acetylation. The fm values decreased from 0.915 at the 10 mg/kg dose to 0.626 at the 100 mg/kg dose. Although both the total plasma clearance and non-renal clearance decreased with increasing dose, the dose had an opposite effect on the renal clearance of cilastatin. The renal clearance of cilastatin increased from 2.50 +/- 0.40 ml/min/kg at the lowest dose to 6.10 +/- 0.50 ml/min/kg at the highest dose as the dose increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Etoposide encapsulated in positively charged liposomes: pharmacokinetic studies in mice and formulation stability studies.

Etoposide is an antineoplastic agent which acts by forming a ternary complex with topoisomerase II and DNA, causing DNA breaks and cell death. In recent studies we have demonstrated that encapsulation in liposomes increases the antitumour efficacy and reduces the adverse effects associated with etoposide. The present study was thus conducted to evaluate whether encapsulation in cationic liposomes altered the pharmacokinetics of etoposide and to study the effect of cholesterol incorporation on the stability of the liposomes. Etoposide-encapsulated unilammellar liposomes were synthesized by thin film hydration followed by extrusion. The drug was administered to Swiss albino mice at a dose of 10 mg kg(-1). The concentration of the drug in plasma was analysed at different time points till 360 min after injection, using a h.p.l.c. method. The terbium chloride-dipicolinic acid interaction method was applied to study the stability of the formulation in mouse serum and also following storage at 0( composite function)C over a period of time. The effect of the free and liposomal drug on myelosuppression was evaluated at 10 mg m(-2)and 40 mg m(-2)dose levels by quantifying blood cell counts on day 15 and day 21 following a 5 day course of therapy. Encapsulation in cationic liposomes increased the area under the concentration vs time curve to 42.98 microghml(-1)from 24.18 microghml(-1)in the case of the free drug. Half-life (beta) was 58. 62 and 186 min in the case of free and liposomal etoposide, respectively. In the stability studies, incorporation of cholesterol progressively stabilized the formulation in serum. The use of sucrose at increasing concentrations as a cryoprotectant also increased the shelf stability of the formulation at 0( composite function)C. Toxicity studies using a dose of pure drug revealed that though myelosuppression was evident in both liposomal- and free drug-treated groups on day 15 it was reversed by day 21 following initiation of therapy. The present findings suggest that liposomes could serve as an alternative mode of delivery for etoposide.     

Mixed micellar nanoparticle of amphotericin b and poly styrene-block-poly ethylene oxide reduces nephrotoxicity but retains antifungal activity

Mixed micellar nanoparticle consisting of amphotericin B (AmB) and poly styrene-block-poly ethylene oxide (PS-block-PEO) was prepared by high pressure homogenizer. Nephrotoxicity of the nanoparticle was investigated along with antifungal activity and self-aggregation status of the drug in the nanoparticle. Nephrotoxicity was markedly reduced when AmB was intravenously administered to rats as mixed micellar nanoparticle with PS-block-PEO in terms of transmission electron microscopy of tubular cells and creatinine clearance. Antifungal activity of AmB was not altered when the drug was in the form of mixed micellar nanoparticle compared to both conventional formulation and AmB micelle treated by same procedure without PS-block-PEO. Self-aggregation status of AmB molecules revealed monomeric in the mixed micellar nanoparticle with PS-block-PEO up to the therapeutic level of the drug (1-3 mM). The reduced nephrotoxicity of AmB in mixed micellar nanoparticle may be associated with the existence of the drug as monomeric form in the nanoparticle. Based on our result, formulation of AmB as mixed micellar nanoparticle with PS-block-PEO may be a promising alternative for the treatment of fungal diseases in patients who are at risk of renal dysfunction.     

Disposition of acamprosate in the rat: influence of probenecid

The purpose of the present study was to investigate the disposition of acamprosate (calcium bis acetyl-homotaurine) in the rat. Initially, we studied the linearity of acamprosate disposition and the fraction of acamprosate excreted unchanged in the urine of the animals. Rats received 9.3, 36.6 or 73.3 mg/kg of the drug as an intravenous bolus. The statistical analysis of the pharmacokinetic parameters did not reveal any significant difference, indicating that acamprosate disposition was linear within the range of the doses assayed. On average, 95% of the administered dose was excreted unchanged in the urine of the animals in the 0-6 h post-administration period indicating that renal excretion is the main elimination route for this drug. Acamprosate was also administered by the intravenous route at three different constant infusion rates (2.65, 132.5 and 530 microg/min) in order to quantify total (Cl(t)) and renal (Cl(r)) plasma clearances at steady-state conditions. The mean Cl(r) values were, respectively, 4.60+/-0.42, 4.28+/-0.52 and 4.08+/-0.67 ml/min, practically equivalent to the Cl(t) values (4.78+/-0.38, 4.51+/-0.36 and 4.21+/-0.56 ml/min), confirming that the drug is mainly eliminated via renal excretion. Moreover, Cl(r) values were clearly higher than the glomerular filtration rate (2.61+/-0.26 ml/min), suggesting the existence of a highly efficient tubular secretion mechanism in the renal excretion of the drug. To confirm this hypothesis, two groups of rats were intravenously treated with probenecid (33.3 or 66.6 mg/kg) prior to acamprosate administration (9.3 mg/kg). Probenecid provoked a statistically significant dose-dependent reduction in the total clearance of acamprosate (from 5.8+/-0.7 ml/min in the control group to 2.6+/-0.1 ml/min in the animals treated with 66 mg/kg of probenecid) demonstrating the existence of a tubular secretion process on the renal excretion of acamprosate in the rat.