Peritoneal retention of liposomes: Effects of lipid composition, PEG coating and liposome charge.

In the treatment of peritoneal carcinomatosis, systemic chemotherapy is not quite effective due to the poor penetration of cytotoxic agents into the peritoneal cavity, whereas intraperitoneal administration of chemotherapeutic agents is generally accompanied by quick absorption of the free drug from the peritoneum. Local delivery of drugs with controlled-release delivery systems like liposomes could provide sustained, elevated drug levels and reduce local and systemic toxicity. In order to achieve an ameliorated liposomal formulation that results in higher peritoneal levels of the drug and retention, vesicles composed of different phospholipid compositions (distearoyl [DSPC]; dipalmitoyl [DPPC]; or dimiristoylphosphatidylcholine [DMPC]) and various charges (neutral; negative, containing distearoylphosphatidylglycerol [DSPG]; or positive, containing dioleyloxy trimethylammonium propane [DOTAP]) were prepared at two sizes of 100 and 1000nm. The effect of surface hydrophilicity was also investigated by incorporating PEG into the DSPC-containing neutral and charged liposomes. Liposomes were labeled with (99m)Tc and injected into mouse peritoneum. Mice were then sacrificed at eight different time points, and the percentage of injected radiolabel in the peritoneal cavity and the tissue distribution in terms of the percent of the injected dose/gram of tissue (%ID/g) were obtained. The ratio of the peritoneal AUC to the free label ranged from a minimum of 4.95 for DMPC/CHOL (cholesterol) 100nm vesicles to a maximum of 24.99 for DSPC/CHOL/DOTAP 1000nm (DOTAP 1000) vesicles. These last positively charged vesicles had the greatest peritoneal level; moreover, their level remained constant at approximately 25% of the injected dose from 2 to 48h. Among the conventional (i.e., without PEG) 100nm liposomes, the positively charged vesicles again showed the greatest retention. Incorporation of PEG at this size into the lipid structures augmented the peritoneal level, particularly for negatively charged liposomes. The positively charged PEGylated vesicles (DOTAP/PEG 100) had the second-greatest peritoneal level after DOTAP 1000; however, their peritoneal-to-blood AUC ratio was low (3.05). Overall, among the different liposomal formulations, the positively charged conventional liposomes (100 and 1000nm) provided greater peritoneal levels and retention. DOTAP/PEG100 may also be a more efficient formulation because this formulation can provide a high level of anticancer drug into the peritoneal cavity and also can passively target the primary tumor.

Preclinical pharmacokinetics of KBF611, a new antituberculosis agent in mice and rabbits, and comparison with thiacetazone.

Thiacetazone (TAZ), one of the oldest known antituberculosis drugs, causes severe skin reactions in patients co-infected with tuberculosis and human immunodeficiency virus (HIV). KBF611 is a new fluorinated thiacetazone analogue that has shown strong antituberculosis effects. In order to provide valuable information for subsequent preclinical development, pharmacokinetics of KBF611 and its analogue (TAZ) were studied and compared in two animal species (mice and rabbits) following intravenous and oral administration, and pharmacokinetic parameters were characterized. According to the calculated parameters, KBF611 showed a more favourable pharmacokinetics profile than TAZ in terms of half-life (0.89 h compared with 0.57 in mice, p < 0.05, and 2.71 compared with 0.98 in rabbits, p < 0.001) and volume of distribution (1.45 l kg(-1) compared with 0.86 l kg(-1) in mice, p < 0.05, and 1.01 l kg(-1) compared with 0.41 l kg(-1) in rabbits, p < 0.001) for tuberculosis therapy. In rabbits, the oral bioavailability of KBF611 was markedly lower than mice (39% compared with 82%), which may be attributed to a higher presystemic metabolism in rabbit liver. The results of in vivo studies on the metabolism of KBF611, supported by liquid chromatography-mass spectrometry (LC-MS) analysis, showed that the incorporation of a fluorine atom to the TAZ structure made the molecule susceptible to N-deacetylation, a pathway not seen in TAZ metabolism. In summary, KBF611 could be considered a suitable candidate for further preclinical and clinical evaluation.

Effect of liposome size on peritoneal retention and organ distribution after intraperitoneal injection in mice.

Peritoneal carcinomatosis is a serious concern when treating digestive or ovarian tumors. Treatment with systemic chemotherapy suffers from poor penetration of cytotoxic agents into the peritoneal cavity and is not quite effective. Local delivery of drugs, especially as controlled-release delivery systems like liposomes, could provide sustained and higher drug levels and reduce systemic toxicity. In order to investigate the effect of liposome size on peritoneal retention, liposomes composed of distearoylphosphatidylcholine and cholesterol (DSPC/CHOL, molar ratio 2:1) were prepared at four sizes of 100, 400, 1000 and 3000 nm. Subsequently, these liposomes were labeled with (99m)Tc complex of hexamethylpropyleneamineoxime ((99m)Tc-HMPAO) and injected into mouse peritoneum. Then, mice were sacrificed at eight different time points and the percentage of injected radiolabel in the peritoneal cavity and the organ distribution in terms of percentage injected dose/gram tissue (%ID/g) were obtained. Results showed that the free label ((99m)Tc-HMPAO) was cleared very rapidly from the cavity so that after 5 min and 7h only 6.89+/-2.51% and 0.91+/-0.51% of the injected dose was recovered, respectively. However, for the liposomal formulations, this recovery value ranged from 8.47+/-1.62% to 29.99+/-12.06% at 7h. Peritoneal retention of the vesicles was increased with their size, and the highest retention rate was obtained with 1000 nm liposomes with an AUC value 15.51 times that of (99m)Tc-HMPAO. In blood, as expected, 100 nm liposomes showed much higher levels because of their greater stability. Their greater blood concentration also caused increased levels in the heart and kidneys, although their organ to blood AUC ratio was the lowest. Overall, among the different sized neutral liposomes investigated, the 1000 nm vesicles seemed to be the most optimal, achieving a greater peritoneal level and retention.

The effect of PEG coating on in vitro cytotoxicity and in vivo disposition of topotecan loaded liposomes in rats.

Amphoteric drugs encapsulated in PEGylated liposomes may not show superior therapeutic antitumor activity due to increased leakage rate of these drugs in presence of PEG-lipids. In order to investigate the effect of PEG coating on in vitro and in vivo characteristics of topotecan loaded liposomes, an amphoteric anticancer drug, PEGylated and conventional liposomes were prepared by lipid film hydration method. Various properties of the prepared nanoliposomes such as encapsulation efficiency, size, zeta potential, physical stability as well as the chemical stability of lactone form of topotecan, cytotoxicity and topotecan pharmacokinetics were evaluated. In vitro cytotoxic activity was evaluated on murine Lewis lung carcinoma (LLC) and human mammary adenocarcinoma (BT20) cells. Pharmacokinetic was evaluated in Wistar rats after i.v. injection of topotecan, formulated in PBS pH 7.4 or in conventional or in PEGylated liposomes. The conventional liposome (CL) formulation was composed of DSPC/cholesterol/DSPG (molar ratio; 7:7:3), while for PEGylated liposome the composition was DSPC/cholesterol/DSPG/DSPE-PEG(2000) (molar ratio; 7:7:3:1.28). The size of both liposomes was around 100 nm with polydispersity index of about 0.1. In comparison with free drug, liposomal topotecan showed more stability for topotecan lactone form in vitro. Compared to free topotecan, PEGylated and conventional liposomes improved cytotoxic effect of topotecan against the two cancer cell line studied. The results of pharmacokinetic studies in rats showed that both CL and PEGylated liposomal formulations increased the concentration of total topotecan in plasma, however, initial concentration and the values of AUC, MRT and t(1/2 beta) were much higher (P<0.001) for PEGylated liposomal drug than for conventional one or free drug. PEGylated liposome resulted in a 52-fold and 2-fold increases in AUC(0-infinity) compared with that of free topotecan and CL, respectively. These results indicated that PEG modified liposome might be an effective carrier for topotecan.

Encapsulation of 9-nitrocamptothecin, a novel anticancer drug, in biodegradable nanoparticles: factorial design, characterization and release kinetics.

This study was aimed at developing a polymeric drug delivery system for a new and potent antitumor drug, 9-nitrocamptothecin (9-NC), intended for both intravenous administration and improving the therapeutic index of the drug. To achieve these goals, 9-NC loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles were prepared by nanoprecipitation method and characterized. The full factorial experimental design was used to study the influence of four different independent variables on response of nanoparticle drug loading. Analysis of variance (ANOVA) was used to evaluate optimized conditions for the preparation of nanoparticles. The physical characteristics of PLGA nanospheres were evaluated using particle size analyzer, scanning electron microscopy, differential scanning calorimetry and X-ray diffractometry. The results of optimized formulations showed a narrow size distribution with a polydispersity index of 0.01%, an average diameter of 207+/-26 nm, and a drug loading of more than 30%. The in vitro drug release profile showed a sustained 9-NC release up to 160 h indicating the suitability of PLGA nanoparticles in controlled 9-NC release. Thus prepared nanoparticles described here may be of clinical importance in both stabilizing and delivering camptothecins for cancer treatment.

The effect of gender on the pharmacokinetics of verapamil and norverapamil in human.

The effects of gender on the pharmacokinetics of verapamil and its active metabolite, norverapamil, following single oral dose (80 mg, Isoptin) to 12 healthy male (mean age: 25.75+/-2.42 years, mean body weight: 70.59+/-9.94 kg) and 12 healthy female subjects (mean age: 24.08+/-2.84 years, mean body weight: 56.67+/-5.23 kg) were investigated in the present study. Plasma concentrations of verapamil and norverapamil were analysed using a modified high-pressure liquid chromatography method. Pharmacokinetic parameters were calculated by non-compartmental analysis for each subject. For verapamil the half-life (t1/2) and mean residence time (MRT) were significantly shorter in women than men (p<0.01 and p<0.05, respectively). For other pharmacokinetic parameters of verapamil there were no significant differences between males and females. For norverapamil, t1/2, MRT and time to reach to the maximum plasma concentration (Tmax) showed statistically significant differences between the two genders. The AUC(0-24) and AUC(0-infinity) ratios of norverapamil to verapamil were also calculated. The ratios were significantly higher in women compared with men. These observations indicate that the elimination rate of verapamil is faster in women than men which may be attributed to the higher activity of CYP3A4 or lower activity of P-glycoprotein in women compared with men. A contribution of both factors in the appearance of gender differences in verapamil pharmacokinetics is also possible.

Liquid chromatographic quantitation of the lactone and the total of lactone and carboxylate forms of 9-nitrocamptothecin in human plasma.

Simple and sensitive high-performance liquid chromatography (HPLC) assays were developed and validated for the quantitation of the investigational anticancer drug 9-nitrocamptothecin (9-NC) as the lactone form and as the total of the lactone(I) and carboxylate(II) forms in human plasma. For the assay of lactone form (9NC-lac), the analytical method involved a protein precipitation step with adding a mixture of cold acetonitril-chloroform (5:1 (v/v), -20 degrees C) to plasma sample that stabilized the pH-dependent conversion of I to II. After evaporation under gentle stream of nitrogen gas (40 degrees C) the dry extract was dissolved in mobile phase (pH 5.5). For determination of the total of the lactone and carboxylate forms of the drug (9NC-tot), plasma samples were deproteinated with cold acetonitril (-20 degrees C) acidified with perchloric acid (5%), which resulted in the conversion of the carboxylate into the lactone form. After centrifugation the upper solvent was evaporated (nitrogen, 40 degrees C) and the dry extract was dissolved in mobile phase (pH 3.5). All separations were performed on a RP-C(8) column, using a mixture of acetonitril-water as eluent (pH 3.5 for total form and pH 5.5 for lactone form) and UV detection. The presented assay was linear over a concentration range of 25-1500 ng/ml with lower limit of quantitation of 25 ng/ml for both 9NC-tot and 9NC-lac. Within-run and between-run precision was always less than 7.5% in the concentration range of interest. The reported assay method showed good characteristics of linearity, sensitivity, selectivity and precision allowing applying in pharmacokinetic studies.

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma.

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (-20 degrees C) for sample preparation followed by separation on a Novapack C(18) column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at -70 degrees C.

Determination of cyproterone acetate in plasma samples by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the determination of cyproterone acetate in human plasma has been developed. The chromatographic separation was performed on an analytical mbondapak C8 column (125 yen 4.6 mm, i.d) with an isocratic mobile phase consisting of methanol-water (62.38 v/v). Using ultra violet detection at 282 nm, the detection limit for cyproterone acetate in plasma was 10 ng/ml. The calibration curve was linear over the concentration range 50-160. 0 ng/ml. Cyproterone was isolated from plasma by liquid-liquid extraction and the recovery was about 90% for plasma. The inter-day and intra-day assay coefficients of variation were found to be less than 10%.

Time dependent pharmacokinetics of albendazole in human.

The pharmacokinetics of the main metabolites of albendazole (albendazole sulphoxide (ABZ-SO) and albendazole sulphone (ABZ-SO2) were studied in 12 healthy human volunteers in a double blind design on the first and last days of oral administration of 800 mg albendazole daily for 15 days. No significant differences were observed in C(max), T(max) and V(d)/F of ABZ-SO, whereas the AUC, AUMC and T(1/2) of this metabolite were significantly reduced and Cl/F was significantly increased in multiple dosing. There were also no significant differences in the C(max), T(max), V(d)/F and T(1/2) of ABZ-SO2, whereas the AUC and AUMC of this metabolite were significantly reduced and Cl/F was significantly increased in multiple dosing. These observations suggest time dependent pharmacokinetics of albendazole (observed for ABZ-SO and ABZ-SO2), which was explained on the basis of the induction of enzymes involved in the metabolism of ABZ-SO (albendazole sulphoxide) to metabolites other than albendazole sulphone in multiple dosing.

Dose dependent pharmacokinetics of albendazole in human.

Pharmacokinetics of albendazole sulphoxide (ABZ-SO) in three different single oral doses of albendazole (ABZ) (400, 800 and 1200 mg) was studied in 10 healthy human volunteers in a double blind three-way crossover design. The serum levels of albendazole main metabolite, albendazole sulphoxide (ABZ-SO), were analysed by a modified high-pressure liquid chromatography method. (ABZ is not detectable in biological fluids itself.)For ABZ-SO, there was no significant difference in the biological half life, normalized serum peak concentration (C(max-ABZ-SO)/Dose(ABZ)), time to reach peak concentration (T(max)) and mean residence time (MRT), whereas apparent clearance (Cl(p)/F), apparent distribution volume (V(d)/F), normalized area under the serum concentration-time curve (AUC(ABZ-SO)/Dose(ABZ)) and normalized area under the first moment curve (AUMC(ABZ-SO)/Dose(ABZ)) of albendazole main metabolite (ABZ-SO) were statistically different at different doses of the parent drug, resulting in substantially lower serum concentration and thereafter AUC(ABZ-SO)/Dose(ABZ) and AUMC(ABZ-SO)/Dose(ABZ) in higher doses. These observations indicate dose dependent pharmacokinetics of albendazole (observed for ABZ-SO), which were explained on the basis of a change in fraction of dose absorbed (F) as a result of slow and incomplete dissolution of the main drug in the GI tract.

A high performance liquid chromatography method for simultaneous determination of albendazole metabolites in human serum.

A simple assay for albendazole (ABZ) main metabolites-albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO(2)) and albendazole amino sulphone (ABZ-SO(2)-NH(2))-in serum using high performance liquid chromatography was developed. The method involves liquid-liquid extraction of the serum by ethyl acetate, clean up with n-hexane and re-extraction with ethyl acetate, followed by separation on RP-C(8) column with a mixture of methanol: acetonitrile: acetic acid: water (40:1:10:49) as the eluting solvent. ABZ-SO and mebendazole-used as internal standard-were detected by UV (lambda=286 nm), and ABZ-SO(2) and ABZ-SO(2)-NH(2) with fluorescence spectrophotometer at (Excitation=286 nm, Emission=333 nm) and (Excitation=286 nm, Emission=315 nm), respectively. The assay was accurate and reproducible with a detection limit of 10 ng/ml for ABZ-SO, 2 ng/ml for ABZ-SO(2) and 4 ng/ml for ABZ-SO(2)-NH(2). Disregarding ABZ determination, which is not of pharmacokinetic importance as it is not found in human plasma after oral administration, the proposed method is appropriate for further pharmacokinetic and metabolism study of this drug.

Effect of gender in the disposition of albendazole metabolites in humans.

The pharmacokinetics of albendazole in different single oral doses (400 mg, 800 mg & 1200 mg) was studied and compared in healthy male and female human volunteers using a double-blind design. The serum levels of albendazole main metabolites (albendazole sulphoxide and albendazole sulphone) were analysed using a modified high-pressure liquid chromatography method. For both metabolites, there was no significant difference in the biological half-life ( t(1/2)), time to reach peak concentration (t(max)) and mean residence time (MRT) between men and women, whereas apparent oral clearance (Cl(p)/F) and apparent distribution volume (V(d)/F) were less and serum peak concentration (C(max)), area under the serum concentration-time curve (AUC) and area under the first moment curve (AUMC) were more in women than in men. These observations indicate sex dimorphism in pharmacokinetics of albendazole (observed for albendazole sulphoxide and albendazole sulphone) which were explained on the basis of a change in fraction of the main drug turned to metabolite as a result of more extensive first-pass metabolism of the main drug in the liver of adult female subjects.

Dose dependent pharmacokinetics of theophylline: Michaelis-Menten parameters for its major metabolic pathways.

Dose Dependency for pharmacokinetics of theophylline and the formation of its major metabolites, 3-methylxanthine (3-MX); 1-methyluric acid (1-MU); 1,3-dimethyluric acid (DMU), were examined by administering three single oral doses (250, 375, 500 mg) of theophylline to six healthy adult volunteers. The serum and urine concentrations of theophylline and the metabolites in serum and urine were determined by high-performance liquid chromatography. Total clearance of theophylline decreased and its half life increased over the range of doses administered (p<0.01). There was a significant dose related decrease in the fractional recovery of 3-MX and 1-MU (p<0.001) and a dose related increase in fractional excretion of DMU and unchanged theophylline (p<0.01 and p<0.001 respectively). No significant dose related changes were observed in the renal clearance of 3-MX, 1-MU and DMU, indicating linear urinary excretion kinetics of the metabolites. Theophylline metabolic clearance to 3-MX as well as to 1-MU decreased with increasing dose but clearance to DMU remained unnaffected by the size of dose. The individual Michaelis-Menten parameters Km and Vmax were estimated for six subjects receiving three different single doses. The Km values for theophylline metabolism to 3-MX, 1-MU and DMU were 2.4+/-0.6, 5.1+/-1.8+/- and 112.3+/-36.8 mg/L respectively and the Vmax values were 3.5+/-0.7, 7.5+/-2.6 and 112.3+/-36.8 mg/hr respectively. The Km values for the N-demethylation pathways (3MX and 1-MU) were lower corresponding to therapeutic serum concentrations of drug. These results suggest that the elimination kinetics of theophylline is nonlinear in the human in the therapeutic range of serum concenntrations and can be explained by saturable formation kinetics of 3-MX and 1-MU. In contrast to previous studies we didn't find obvious indication for nonlinear formation of DMU at therapeutic concentration range.

Urinary excretion and external radiation dose from patients administered thallium-201 and technetium-99M methoxy isobutyl isonitrile.

In this study, the dose rates at various distances of 5, 10, 50 and 100 cm from 70 patients, who were administered diagnostic amounts of 201Tl-chloride and 99Tcm-MIBI, was measured using an ionisation chamber. The maximum values of external dose rates of 201Tl and 99Tcm-MIBI were 11.2 microSv.h-1 +/- 2.3 and 43.1 microSv.h-1 +/- 11.9 respectively at 5 cm from the patients. Significant exposure from patients after injection of 99Tcm-MIBI was limited on the day of administration. Departure dose rates of 201Tl fell gradually so by 3 days after administration were significant. All excreted urine was also collected from 16 patients for the 24 days after administration. The urinary excretion rate of 201Tl was slow; about 2% of the activity within 24 h after injection. The urinary excretion of 99Tcm-MIBI was rapid, and a large amount of activity was excreted in a few hours after injection; 22% within 20 h after injection.

Determination of amitriptyline in plasma samples by high-performance liquid chromatography.

A simple and selective high-performance liquid chromatographic method for the determination of amitriptyline in human plasma has been developed. For plasma samples, the protein was removed with 1 M NaOH and 0.7 M ZnSO4. aqueous solutions. The chromatographic separation was performed on an analytical mbondapak C18 column (250 3.9 mm, i.d) with an isocratic mobile phase consisting of phosphate buffer-acetonitrile-triethylamine (65:35:0.1 v/v/v) adjusted to pH 5.1. Clomipramine was used as an internal standard. Using ultraviolet detection at 239 nm, the detection limit for amitriptyline in plasma was 5 ng/ml. No interferences were found with tricyclic antidepressant drugs, which allows this method to be used in clinical studies. The calibration curve was linear over the concentration range 5-200 ng/ml. The recovery was complete for plasma. The inter-day and intra-day assay coefficients of variation were found to be less than 10%.

An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine.

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using beta-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 microgram ml-1 for theophylline and 0.5 micrograms ml-1 for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.