Stealth monensin immunoliposomes as potentiator of immunotoxins in vitro.

Stealth monensin liposomes (SML) were prepared using dipalmitoyl phosphatidylcholine, cholesterol, distearoyl glycerophosphoethanolamine coupled to polyethylene glycol, stearylamine, and N-succinimidyl pyridodithiopropionate linked to stearyl amine, in the molar ratio of 10:5:1.4:1.4:1.5. SML was conjugated to the anti-MY9 antibody by a disulfide linkage to form stealth monensin immunoliposomes (SMIL) by an already established procedure. The encapsulation concentrations of monensin in SML and SMIL were 10(-7) and 4.9x10(-8) M, respectively. More than 20% of monensin remained in circulation after 24 h in BALB/c mice. The ability of SML and SMIL to potentiate the effect of anti-MY9 immunotoxin (anti-MY9-IT) was tested against human leukemia HL-60 sensitive and resistant tumor cells in vitro. SML and SMIL potentiated the activity of anti-MY9-IT by 10-20 times against HL-60 sensitive tumor cell lines. However, greater potentiation of anti-MY9-IT was observed in combination with SML and SMIL against HL-60 resistant tumor cells, found to be 200 and 500 times, respectively. The potentiation of anti-MY9-IT by SMIL was more than two-fold compared with SML against both HL-60 sensitive and resistant tumor cells. Transmission electron microscopy studies conducted with HL-60 resistant cells incubated with anti-MY9-IT and monensin liposomes showed significant dilation of the golgi, which was reversible after re-incubation in fresh medium. Our studies show that SML and SMIL can be successfully used to potentiate the activity of ricin based anti-MY9-IT in vitro, and further in vivo studies will demonstrate the usefulness of this approach.

Stealth monensin liposomes as a potentiator of adriamycin in cancer treatment.

Small unilamellar stealth monensin liposomes (SMLs) were prepared from multilamellar liposomes (MLVs). The MLVs were prepared by using dipalmitoyl phosphatidylcholine (DPPC), cholesterol, distearoyl glycerophosphoethanolamine coupled to poly(ethylene glycol) (DSPE-PEG) and stearylamine in the molar ratio of 10:5:1.4:1.4 (32.8 mM total lipid). The encapsulation efficiencies of monensin in MLVs and small unilamellar vesicles (SUVs) was 6x10++(-6) and 10(-7) M, respectively. The stability of SMLs was studied at 4 degrees C. The amount of leakage of monensin from SMLs was less than 20% after four weeks of storage. The in vitro release of monensin from SMLs in human serum was determined, and t1/2 was found to be 10 h. Pharmacokinetic studies on SMLs were carried out in BALB/c mice. More than 20% of SMLs remained in blood circulation after 24 h. SMLs increased the uptake of adriamycin (AM) in HL-60-resistant cells by more than two fold, compared to monensin in solution. SMLs potentiated the effect of AM against both sensitive and resistant HL-60 cells (six- and tenfold potentiation, respectively) and human LOVO tumor cells (four- and 200-fold potentiation, respectively). However, the highest potentiation was observed against resistant human breast tumor MCF7 cells, and was found to be 2400 times in comparison to AM alone. Transmission electron microscopic (TEM) studies carried out with HL-60-resistant tumor cells incubated with SMLs showed that SMLs caused dilation of the golgi of tumor cells within 10 min. The dilation of golgi was reversible after reincubation of the cells in fresh medium. SMLs showed considerable potential as a potentiator in combination with AM in overcoming drug resistance.

Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro.

Monensin is a carboxylic ionophore which can potentiate the activity of ricin based immunotoxins (IT) in vitro and in vivo against a variety of human tumours. Monensin was encapsulated into nanoparticles (NP) by using biodegradable poly(DL-lactide-co-glycolide) (PLGA, 50:50). The NP were prepared by modified emulsification-solvent evaporation method. High shear homogenization followed by simultaneous stirring and bath sonication were used for preparing NP. The size of NP was determined by photon correlation spectroscopy using a BI 90 particle sizer (Brookhaven Instruments). The average size of NP could be decreased from 567 nm to 163 nm by increasing the concentration of polyvinyl alcohol from 10% to 100% of PLGA. The NP were spherical in shape as observed by Atomic Force Microscopy. The concentration of monensin in the NP was analyzed by HPLC and the entrapment efficiency was found to be more than 12%. The zeta potential of NP was -25.8 (+/- 1.3) mv, which did not change significantly after resuspension of the freeze dried sample. The NP were tested against HL-60 and HT-29 human tumour cell lines in vitro. Monensin NP potentiated the activity of IT by 40 to 50 times against these cell lines. There was however, no difference between the NP and liposomes for their potentiating affect of IT against the two tumour cell lines.

Development of liposomal amphotericin B formulation.

A considerable effort has been spent in the past three decades to investigate various aspects of liposomes as novel drug delivery systems. In 1990, the first amphotericin B (AmB) liposomal preparation (L-AmB) under the brand name AmBisome was introduced into the market by Vestar. The successful marketing of the product moved liposomes out of the stage of experimental obscurity to the realistic stage of clinical utility. The launch of AmBisome sparked off the introduction of other lipid-based AmB products marketed by Liposome Technology (Amphocil) and The Liposome Co. (Abelcet). The drive behind the development of a modified formulation of AmB was to improve the therapeutic index of this drug with respect to its major drawback associated with both acute and chronic toxic effects. In a 30-year-long experience with AmB, several reports were recorded in the literature of acute adverse effects, such as fever, rigors, vomiting, cardiotoxicity and hypotension occurring during infusion; while long-term therapy was reported to be associated with hypokalemia, renal dysfunction and hematological abnormalities. Another serious problem encountered with the drug had been the poor response obtained in immunocompromised patients like those with AIDS, neutropenia and cancer patients on chemotherapy. The encapsulation of amphotericin B in liposomal vesicles was hence targeted not only to obtain an improvement in the therapeutic index but also to see if it was useful in eradicating deep-seated fungal infections in immunocompromised patients. The liposomal AmB was found to have a better therapeutic index and lower toxicity than the commercial AmB preparations. The LD50 of AmBisome in mouse was 175 mg/kg compared with 3.7 mg/kg for Fungizone, the commercial preparation of AmB. Additionally, L-AmB has prolonged circulation time, and extravasates into the site of infection and delivers the drug directly to the site, with no nephrotoxicity and neurotoxicity as experienced with AmB. This review traces the course of development of L-AmB and discusses the rationale behind the development of its liposomal preparation. The results in in vitro, in vivo and clinical studies, mechanism of action, biodistribution, and formulation considerations of L-AmB are described. The clinical experience with the marketed preparation is reviewed.

A modified HPLC method for monensin analysis in liposomes and nanocapsules and its comparison with spectrophotometric and radioactive methods.

Monensin is a carboxylic ionophore which can potentiate the immunotoxin activity against human tumors in vitro and in vivo. Currently monensin is being encapsulated in liposomes and nanocapsules in our laboratory. The reported methods for monensin analysis by spectrophotometric and HPLC lack the required sensitivity. We have developed a sensitive HPLC method for analysis on monensin. Separation was achieved on a Beckman C18 reverse phase column with methanol-acetonitrile-methylene chloride-water-acetic acid (45:20:25:9.5: 0.5) as the mobile phase. The eluent was reacted with vanillin reagent in the post column reactor at 70 degrees C. The reagent reacted with monensin and formed a pink color, which was detected at 520 nm. The retention time of monensin was found to be 6 min. By using this method it was possible to quantify monensin down to 100 ng ml-1, with a signal to noise ration of > 17:1. Linearity was observed within the range of 10 to 100 ng (r2 > 0.99). Inter-day standard deviations for monensin samples of 20, 50 and 80 ng were 0.675, 0.543 and 0.736 respectively. Alternative methods of analysis include using radioactive [3H]monensin in liposomes which can be quantified by scintillation counter. The results from the HPLC, spectrophotometric and radioactive method were compared and were found to be within acceptable limits. The HPLC method is being utilized in our laboratory for quantitative analysis of monensin in liposomes and nanocapsules.

Effect of ultraviolet light on the release of theophylline from ethylcellulose-based sustained-release microcapsules.

Sustained-release theophylline microcapsules were prepared using the emulsion solvent-evaporation technique. Three viscosity grades of ethylcellulose and varying concentrations of theophylline were used for the formulation. The microcapsules were exposed to short-wave ultraviolet (UV) light for 7 days. The surface and the cross-section of microcapsules were analyzed by scanning electron microscopy (SEM). The release of drug was determined by using the USP dissolution apparatus. Differential scanning calorimetry (DSC) was used to study the thermal properties of theophylline and the polymer. SEM revealed numerous pores on the surface of microcapsules. The size of the pores increased on exposure to light. Theophylline release from microcapsules was found to follow first-order kinetics both before and after UV light exposure. The drug release rate from microcapsules exposed to short-wave UV light increased by about 80%-450% compared with the unexposed samples. The release of theophylline from the microcapsules was more faster with higher concentration of ethylcellulose polymer. Both SEM and DSC showed uniform dispersion of drug with the polymer within the microcapsules. The melting point of the polymers changed, but it remained unchanged for theophylline on exposure to UV light. The UV light caused photodegradation or depolymerization of the polymer and thus caused a faster release of drug from the sustained-release microcapsules. The finding underscores the need for setting standards and requirements for light-stability testing for sustained-release formulations, where there may be substantial change in the release pattern of drug on exposure to light.

Photodegradation of furosemide solutions.

Photodegradation of furosemide was studied under the influence of fluorescent and UV lights. Photodecomposition of furosemide solutions appeared to follow first-order kinetics. The compound was found to be most stable at an optimum pH of 7. Long-wave UV light caused the fastest rate of degradation of furosemide. Glutathione, thiourea, EDTA, sodium thiosulfate, sodium metabisulfite, glycine, sodium benzoate and uric acid were found to be ineffective as photoprotective agents. Increase in ionic strength did not cause any change in the degradation rate constant. Amber glass vials and transparent glass vials covered with aluminum foil offered complete protection against fluorescent light. A vehicle consisting of a mixture of 50% (v/v) propylene glycol in phosphate buffer offered slight photoprotection.

Size-dependent release of carboxyfluorescein from cetylmannoside-modified liposomes in human plasma.

The interaction of liposomes with human plasma was investigated using 6(5)-carboxyfluorescein (CF) as an aqueous phase marker of cetylmannoside-modified multilamellar vesicles (Man-MLVs) of various sizes. The release of CF decreased with increasing liposome concentration. The time courses of the CF release from Man-MLVs were monitored continuously and were analysed kinetically. The curves were characterized by two phases, the first-order release process and the maximum release, which represent the rate and the extent of CF release, respectively. The increase of liposome size increased the rate of release by 42% and the extent of release by 121%, respectively. These effects of liposome size on the release processes were suggested to result from the size-dependent affinities of liposomes to the human complement system. The assay system of liposomally bound fragments of complement component 3 (C3), such as C3b and/or iC3b, was developed by applying a sandwich enzyme-linked immunospecific assay. The percentage of C3 fragments to total proteins bound to liposomes increased with the size of liposomes and there was a good correlation between the extent of CF release and the percentage of C3 fragments bound. These results indicated that Man-MLVs released entrapped CF via activating the human complement system and the affinity of Man-MLV to complement increased with the size of Man-MLVs in human plasma. These in vitro results suggest the role of complement as an opsonin in the disposition of Man-MLVs in humans.

Amount of polyvinyl alcohol (pva) polymer adsorbed on the oil globules in liquid paraffinemulsions prepared by pva.

A modified method for estimating polyvinyl alcohol (PVA) in solution has been developed based on the principle of formation of PVA-iodine complex which has a characteristic absorption maxima. The amount of PVA adsorbed on the oil globules in liquid paraffin oil-water emulsions prepared by the polymer was determined. This was found to be dependent on the concentration of the polymer in the continuous phase of the emulsion until it reached a saturation point. Beyond that concentration, the amount of adsorption remained steady.

Application of the WATR technique for water suppression in 1H NMR spectroscopy in determination of the kinetics of hydrolysis of neostigmine bromide in aqueous solution.

Both ammonium chloride and guanidinium chloride were used to secure water suppression in 1H NMR spectra using the 'Water Attenuation by T2 Relaxation' (WATR) technique. The effect of phosphate buffer in the suppression was investigated over a range of pH values at 80 MHz. The spin-spin relaxation time of water protons at 80 MHz was found to reach a minimum at pH 7.3 in the presence of 0.1 M phosphate buffer and 1 M guanidinium chloride; these conditions were therefore chosen for subsequent use of the WATR technique in a study of the kinetics of hydrolysis of neostigmine bromide. The method was found to be very convenient for studies of the hydrolysis of this representative amide.

Viscosity and stability stijdies of hydroxypropyl methylcellulose polymer solutions.

Viscosity studies of three grades of hydroxypropyl methylcellulose polymer solutions have been done in detail. The solutions showed pseudoplastic behaviour and their viscosity increased with increase in concentration of the polymer, but rise in temperature significantly lowered the viscosity. The effect of aging on solutions' viscosity at 25 degrees C was measured.

1H NMR as an analytical tool for the investigation of hydrolysis rates: a method for the rapid evaluation of the shelf-life of aqueous solutions of drugs with ester groups.

The rate of hydrolysis of esters of primary and secondary alcohols can be determined quickly and easily by 1H NMR in aqueous solution, provided that the water signal is suppressed by the WATR (Water Attenuation by T2 Relaxation) method. To evaluate this approach, Arrhenius plots have been constructed for hydrolysis of acetylcholine, carbachol and atropine, and the effect of pH on the hydrolysis of procaine has been determined over a limited range. The results agree well with literature values for rate constants.

In vitro testing of the leaf extracts of Lawsonia alba for antimicrobial properties.

Leaf extracts (chloroform, ethanol and aqueous extracts) of Lawsonia elba were tested in vitro for their antimicrobial activity against seventeen strains of pathogenic bacteria and twelve fungi including seven pathogenic strains. Each of the extract showed good activity against most of the strains tested. The sensitivity of the bacteria and fungi to the extracts was compared to that of ampicillin, tetracycline, co-trimoxazole and griseofulvin sensidiscs.

Antibacterial activity of clove extracts against phagogenic strains including clinically resistant isolates of Shigella and Vibrio cholerae.

Clove extracts (petroleum ether, chloroform and ethanol extracts) were tested in vitro for their antibacterial activity against forty isolates of pathogenic bacteria including clinically resistant (resistant to ampicillin and nalidixic acid) strains of twenty-five Shigella and four Vibrio cholerae. All of the isolates except Pseudomonas aeruginosa showed promising sensitivity to the extracts.