Enhanced FGFR signalling predisposes pancreatic cancer to the effect of a potent FGFR inhibitor in preclinical models.

BACKGROUND: Fibroblast growth factor receptor (FGFR) signalling has been implicated in pancreas carcinogenesis. We investigated the effect of FGFR inhibition in pancreatic cancer in complementary cancer models derived from cell lines and patient-derived primary tumour explants. METHODS: The effects of FGFR signalling inhibition in pancreatic cancer were evaluated using anti-FRS2 shRNA and dovitinib. Pancreatic cancers with varying sensitivity to dovitinib were evaluated to determine potential predictive biomarkers of efficacy. Primary pancreatic explants with opposite extreme of biomarker expression were selected from 13 tumours for in vivo dovitinib treatment. RESULTS: Treatment with anti-FRS2 shRNA induced significant in vitro cell kill in pancreatic cancer cells. Dovitinib treatment achieved similar effects and was mediated by Akt/Mcl-1 signalling in sensitive cells. Dovitinib efficacy correlated with FRS2 phosphorylation status, FGFR2 mRNA level and FGFR2 IIIb expression but not phosphorylation status of VEGFR2 and PDGFRß. Using FGFR2 mRNA level, a proof-of-concept study using primary pancreatic cancer explants correctly identified the tumours' sensitivity to dovitinib. CONCLUSION: Inhibiting FGFR signalling using shRNA and dovitinib achieved significant anti-cancer cancer effects in pancreatic cancer. The effect was more pronounced in FGFR2 IIIb overexpressing pancreatic cancer that may be dependent on aberrant stimulation by stromal-derived FGF ligands.

Paclitaxel pharmacodynamics: application of a mechanism-based neutropenia model.

Antineoplastic agents exert adverse effects that impact both dose and scheduling of drug administration. Our objective was to develop a quantitative relationship between paclitaxel (taxol) exposure and pharmacodynamic endpoints, such as neutropenia or body weight loss. Paclitaxel in liposomes or Cremophor EL was administered to rats at doses of 20 or 40 mg/kg. Body weight and absolute neutrophil count were determined daily. The decrease in body weight was greater for paclitaxel in Cremophor EL than for liposomal paclitaxel, but hematological toxicity was similar. The hematological data was fit using a pharmacodynamic model to investigate the temporal delay between drug exposure and neutropenia. From the model, the lifespan of neutrophils (T(N)), of surviving precursor cells in bone marrow (T(P)), and a killing rate constant (K) were determined. The values of T(N), T(P), and K for liposomal paclitaxel were 95 h, 82 h, and 0.735 (microM h)(-1), respectively, and for paclitaxel in Cremophor EL, 86 h, 78 h, and 0.475 (microM h)(-1), respectively. Simulations of various doses indicated a dependency of the neutropenia time course on paclitaxel exposure. The entire time course of changes in neutrophil count is more informative than a single measurement if myelosuppression is prolonged and at a level associated with increased incidence of clinical adverse effects.

Influence of cationic lipids on the stability and membrane properties of paclitaxel-containing liposomes.

Paclitaxel (taxol) is a poorly soluble anticancer agent that is in widespread clinical use. Liposomes provide a less toxic vehicle for solubilizing the drug and increasing the therapeutic index of paclitaxel in model tumor systems. The role of liposome membrane composition in the stability of paclitaxel-containing formulations is understood partially for neutral and anionic liposomes, but poorly for other compositions. We investigated the effect of dialkyl cationic lipids on the stability and physical properties of paclitaxel-containing liposomes, using circular dichroism (CD), fluorescence spectroscopy, and differential interference contrast microscopy (DIC). DOTAP (1,2-dioleoyl-3-trimethylammonium propane), a cationic lipid used frequently for gene delivery, was combined at various ratios with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or distearoylphosphatidylcholine (DSPC). In the absence of DOTAP, the stability of liposomes containing > or =3 mol% paclitaxel was observed to follow the following rank order: DPPC >DSPC > DMPC. Increasing concentrations of DOTAP increased the physical stability of all compositions, and maximal stabilization was achieved at 30-50 mol% DOTAP, depending on the paclitaxel concentration and the acyl chain length of the phosphatidylcholine. The relationship between stability and mole fraction of DOTAP was complex for some compositions. DOTAP exerted a major fluidizing effect on DMPC, DPPC, and DSPC membranes, and the addition of paclitaxel at 3-8 mol% did not increase fluidity further. Studies of membrane phase domain behavior using the probe Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) indicated that both paclitaxel and DOTAP were miscible with the phosphatidylcholine phase. The physical events leading to destabilization of formulations are hypothesized to arise from concentration-dependent paclitaxel self-association rather than immiscibility of the membrane lipids. Given the increased incorporation and stability of paclitaxel in DOTAP-containing membranes and the potential for enhanced interaction with cells, cationic liposomes may provide a therapeutic advantage over previously described liposome formulations.

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties.

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.

Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.

PURPOSE: The advent of recombinant DNA technology has made possible the pharmaceutical use of a wide range of proteins and peptides. However, the complex structure of proteins renders them susceptible to physical instabilities such as denaturation, aggregation and precipitation. We tested the hypothesis that partial unfolding and exposure of hydrophobic domains leads to physical instability, and investigated approaches to stabilize protein formulations. METHODS: KP6 beta, an 81 amino acid killer toxin from Ustilago maydis, was used as a model protean. Circular dichroism and fluorescence spectroscopy were used to study the temperature dependent folding/ unfolding characteristics of KP6 beta. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent probe that partitions into hydrophobic domains, was used to detect exposure of hydrophobic domains. RESULTS: As the temperature was elevated, near-UV CD indicated progressive loss of KP6 beta tertiary structure, while far-UV CD indicated retention of secondary structure. Increasing exposure of hydrophobic domains was observed, as indicated by the penetration of ANS. At elevated temperatures (60 degrees C), KP6 beta3 conserved most secondary structural features. However, tertiary structure was disordered, suggesting the existence of a partially folded, structured intermediate state. Liposomes bound to partially unfolded structures and prevented the formation of aggregates. CONCLUSIONS: Partial unfolding resulted in increased exposure of hydrophobic domains and aggregation of KP6 beta, but with preservation of secondary structure. Liposomes interacted with the structured intermediate state, stabilizing the protein against aggregation. These results suggest a general formulation strategy for proteins, in which partially unfolded structures are stabilized by formulation excipients that act as molecular chaperones to avoid physical instability.

Biopharmaceutics of boronated radiosensitizers: liposomal formulation of MnBOPP (manganese chelate of 2,4-(alpha, beta-dihydroxyethyl) deuterioporphyrin IX) and comparative toxicity in mice.

Binary treatment modalities such as photodynamic therapy (PDT) and neutron capture therapy (NCT) combine low-toxicity electromagnetic irradiation with an appropriate radiation sensitizer to enhance selectivity for tumor targets. The porphyrin derivative tetrakiscarborane carboxylate ester of 2,4-(alpha, beta-dihydroxyethyl) deuterioporphyrin IX (BOPP) shows tumor-selective uptake and is active in both treatment modalities. BOPP also chelates paramagnetic ions such as Mn(2+), and therefore its tissue accumulation and selectivity can be detected noninvasively by using magnetic resonance imaging. However, local and systemic toxicity appears elevated for the Mn(2+) chelate (MnBOPP), but is poorly characterized. Here we have developed a liposomal formulation of MnBOPP and compared its toxicity with that of MnBOPP administered to mice in saline. The optimal liposome composition and maximal capacity to accommodate MnBOPP were investigated by differential scanning calorimetry and by encapsulation efficiency. MnBOPP was encapsulated quantitatively at up to 12 mol % (drug:lipid) in liposomes of varying composition, and remained incorporated during extended dialysis. Phase separation of drug- and lipid-rich domains was observed above 12% drug. MnBOPP in buffered saline was lethal to animals at 90 micromol/kg, and caused severe necrosis at the injection site at dose levels of 60 micromol/kg or greater. In contrast, MnBOPP formulated in liposomes was well tolerated at the highest tested dose of 135 micromol/kg, with the elimination of local toxicity.

Functional magnetic resonance (fMR) imaging of a rat brain tumor model: implications for evaluation of tumor microvasculature and therapeutic response.

Functional MR (fMR) imaging techniques based on blood oxygenation level dependent (BOLD) effects were developed and applied to a rat brain tumor model to evaluate the potential utility of the method for characterizing tumor growth and regression following treatment. Rats bearing 9L brain tumors in situ were imaged during inhalation of room air and after administration of 100% oxygen + acetazolamide (ACZ) injected 15 mg/kg intravenously. Pixel-to-pixel fMR maps of normalized signal intensity change from baseline values were calculated from T2 weighted spin echo (SE) images acquired pre- and post- oxygen + ACZ administration. Resultant fMR maps were then compared to gross histological sections obtained from corresponding anatomical regions. Regions containing viable tumor with increased cellular density and localized foci of necrotic tumor cells consistent with hypoxia were visualized in the fMR images as regions with decreased signal intensities, indicating diminished oxyhemoglobin concentration and blood flow as compared to normal brain. Histological regions having peritumor edema, caused by increased permeability of tumor vasculature, were visualized in the fMR images as areas with markedly increased signal intensities. These results suggest that fMR imaging techniques could be further developed for use as a non-invasive tool to assess changes in tumor oxygenation/hemodynamics, and to evaluate the pharmacologic effect of anti-neoplastic drugs.

Candidacidal activity of salivary histatins. Identification of a histatin 5-binding protein on Candida albicans.

Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane. 125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (KD = 1 microM), numbering 8.6 x 10(5) sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 to C. albicans resulted in the appearance of a specific 73-kDa 125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.

Cellular retention of liposome-delivered anionic compounds modulated by a probenecid-sensitive anion transporter.

PURPOSE: Drug carriers such as liposomes may enhance the intracellular delivery of therapeutic agents for infectious or neoplastic diseases. However, the mechanisms affecting cellular retention of liposome contents are understood poorly. We tested the hypothesis that retention of anionic compounds may be modulated by a nonspecific probenecid-sensitive anion transport mechanism, and that liposome composition may determine the impact of such transporters on drug retention by cells. METHODS: The fluorescent anionic dye hydroxy-pyrene-[1,3,6]-trisulfonate (HPTS) was transferred to the cytoplasm of cultured CV-1 or J774 cells by direct needle-microinjection or by ATP-induced permeabilization of the-plasma membrane, respectively, to investigate whether the cells have anion transport mechanisms capable of extruding HPTS from the cytoplasm. Cellular retention of dye was monitored in the presence and absence of the anion transport inhibitors probenecid or sulfinpyrazone. Liposomes containing HPTS were co-labeled with tetramethylrhodamine-labeled phosphatidylethanolamine (Rho-PE) as a marker of liposome membrane fate, and uptake was investigated using J774 cells. RESULTS: Needle-injected HPTS underwent both sequestration in early endocytic vesicles and rapid extrusion into the extracellular medium. Probenecid or sulfinpyrazone reduced the extrusion of HPTS. Thus HPTS is a substrate for a probenecid-sensitive anion transporter in J774 and CV1 cells. After delivery via fluid liposomes composed of phosphatidylglycerol:phosphatidylcholine:cholesterol (3:7:5 mole ratio) and co-labeled with Rho-PE, cell-associated HPTS declined more rapidly than did Rho-PE. Exposure of cells to 5 mM probenecid doubled the quantity of HPTS retained by cells, without changing the retention of the Rho-PE membrane marker. In contrast, the effect of probenecid was negligible when gel-phase liposomes of distearoylphosphatidylglycerol:cholesterol (10:5 mole ratio) were used. CONCLUSIONS: Probenecid-sensitive nonspecific anion transporters can mediate the extrusion of model anions delivered via liposomes. However, liposome composition modulates the amount of material subject to extrusion from cells, possibly by altering the endocytic compartment in which liposomes release their contents.

Liposome-mediated therapy of intracranial brain tumors in a rat model.

PURPOSE: Malignant brain tumors represent a serious therapeutic challenge, and survival often is low. We investigated the delivery of doxorubicin (DXR) to rat brain tumors in situ via liposomes, to test the hypothesis that intact liposomes undergo deposition in intracranial tumor through a compromised blood-tumor vasculature. Both therapeutic effect and intra-tumor drug carrier distribution were evaluated to identify variables in carrier-mediated delivery having impact on therapy. METHODS: The rat 9L gliosarcoma tumor was implanted orthotopically in Fischer 344 rats in the caudate-putamen region. The tumor-bearing rats were treated with DXR, either free or encapsulated in long-circulating, sterically-stabilized liposomes. Anti-tumor efficacy was assessed by survival time. In parallel, liposomes labeled with a fluorescent phospholipid analog were injected into tumor-bearing rats. At predetermined intervals, the brains were perfused with fixative, sectioned, and imaged with laser scanning confocal microscope (LSCM) to investigate the integrity of the tumor vascular bed and the intratumor deposition of liposomes. RESULTS: Free DXR given in 3 weekly iv injections was ineffective in increasing the life span of tumor-bearing rats at cumulative doses < or = 17 mg/kg, and at the highest dose (17 mg/kg) decreased survival slightly, compared to saline-treated controls. In contrast, DXR encapsulated in long-circulating liposomes mediated significant increases in life span at 17 mg/kg. Rats showed a 29% percent increase in median survival, respectively, compared to saline-control animals. The delay of treatment after tumor implantation was a major determinant of therapeutic effect. Fluorescent liposomes were deposited preferentially in tumor rather than normal brain, and were distributed non-uniformly, in close proximity to tumor blood vessels. CONCLUSIONS: Liposomes can be used to enhance delivery of drugs to brain tumors and increase therapeutic effect. The therapeutic effect may arise from release of drug from liposomes extravasated in discrete regions of the tumor vasculature and the extravascular space.

Salicylic acid induces changes in the physical properties of model and native kidney membranes.

Salicylic acid (SA) can inhibit the facilitated transport of inorganic sulfate in the kidney, placenta, and erythrocytes. One mechanism of this inhibition could involve the interaction of SA with membranes, resulting in altered function of transporter protein(s) due to changes in membrane fluidity. Such membrane effects could result in altered membrane transport and consequently in changes in the pharmacokinetics and the therapeutic activity of both xenobiotics and endogenous substrates. We investigated the effect of SA on the fluidity of brush border membrane (BBM) and basolateral membrane (BLM) isolated from rat kidney and also on the physical properties (such as phase transition temperature and fluidity) of model membranes by fluorescence polarization and differential scanning calorimetry (DSC) techniques. SA decreased the lipid order parameter (S) of BBM and BLM membranes in a concentration-dependent manner, indicating that the addition of SA makes the membrane more fluid. The fluidizing effect of SA was more pronounced than that of benzyl alcohol. Studies were carried out with protein-free model membranes composed of dipalmitoylphosphatidylcholine (DPPC) to investigate the effects of SA on the bilayer membrane lipids. SA decreased the fluorescence polarization of DPH (1,6-diphenyl 1,3,5-hexatriene) incorporated in DPPC vesicles. DSC studies demonstrated that SA broadened the phase transition temperature of DPPC vesicles and suggested that SA is located in the C1-C8 region of the acyl chain. In protein-free model membranes, SA exerted fluidizing effects through its incorporation into the cooperative hydrophobic region of the bilayer. The perturbation of membrane physical properties induced by SA and its hydrophobic localization in the membrane bilayer may be important in the SA-induced alteration of sulfate membrane transport.

Paclitaxel-liposomes for intracavitary therapy of intraperitoneal P388 leukemia.

Paclitaxel, a recently approved antineoplastic agent, is cleared slowly from the peritoneal cavity after i.p. injection, and therefore appears to be promising for intracavitary therapy of malignancies confined to the peritoneal cavity. However the dose-limiting toxicity of Taxol, the clinical formulation of paclitaxel, was severe abdominal pain, likely caused by the excipients (Cremophor EL and ethanol) that are required to overcome low drug solubility. We tested the hypothesis that a liposome-based formulation could modulate paclitaxel toxicity independent of antitumor activity. The dose-dependence of toxicity and antitumor effect of paclitaxel liposomes was evaluated after i.p. administration against i.p. P388 leukemia. Liposomal paclitaxel showed antitumor activity similar to that of free paclitaxel (as Taxol), but was better tolerated by both healthy and tumor-bearing mice.

Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.

Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.

Antitumor efficacy of taxane liposomes on a human ovarian tumor xenograft in nude athymic mice.

Taxanes such as paclitaxel (Taxol) and docetaxel (Taxotere) are promising agents for use against ovarian cancer and other malignancies. Recently, SB-T-1011, a semisynthetic taxane, has been prepared from 14-hydroxy-10-deacetylbaccatin III. SB-T-1011 shows similar or greater in vitro cytostatic activity than paclitaxel, depending on the tumor cell line. The administration of taxanes is problematic due to their low solubility in most pharmaceutically acceptable solvents; formulations used clinically contain Cremophor/ethanol (diluent 12) or polysorbate 80/ethanol, excipients which may cause serious adverse effects. To eliminate these vehicles, we have prepared paclitaxel liposome formulations. The objective of the present work was to evaluate the antitumor activity of paclitaxel and two semisynthetic analogs in Cremophor-based and liposomal formulations. Antitumor activity was evaluated against A121a, a taxane-sensitive human ovarian tumor, growing as subcutaneous xenografts in athymic nude mice. Free and liposomal formulations of each taxane showed similar antitumor effect. The antitumor activity of paclitaxel and SB-T-1011 was similar, and docetaxel was more potent than either paclitaxel or SB-T-1011. Overall, taxane liposomes were better tolerated and more easily administered iv than taxane formulated in Cremophor/ethanol.

Pharmaceutical and physical properties of paclitaxel (Taxol) complexes with cyclodextrins.

Paclitaxel (as Taxol) is under clinical investigation for treatment of a variety of cancers. Because of its low aqueous solubility, paclitaxel is administered in polyethoxylated castor oil (Cremophor EL) and ethanol, a vehicle associated with severe hypersensitivity reactions. Cyclodextrins (CyDs) are molecular complexing agents that can increase the solubility and stability of some poorly soluble drugs and were investigated here as a means to obviate the requirement for Cremophor. A variety of beta- and gamma-cyclodextrins were tested; (hydroxypropyl)-(HP beta CyD), (hydroxyethyl)-(HE beta CyD), and dimethyl-(DM beta CyD) beta CyD increased paclitaxel solubility 2 x 10(3)-fold or more and did not alter the cytostatic properties of paclitaxel in vitro. The quantity of drug solubilized increased with the CyD concentration, but precipitation upon dilution occurred with some CyDs or stoichiometries. Thermal and spectroscopic (fluorescence, IR, NMR, and circular dichroism) analyses provided evidence of complex formation that was stable in the solid state but weak in solution, suggesting an explanation for the observed precipitation upon dilution. DM beta CyD solutions of < or = 3.7 mol % (mole of drug:mole of CyD) showed no precipitation upon dilution, nor did HP beta CyD solutions of < or = 0.14 mol %. Maximum tolerated dose (MTD) experiments showed uncomplexed DM beta CyD to be toxic in mice at doses of 2 g CyD/kg body weight, the quantity required to administer paclitaxel at 10 mg/kg. HP beta CyD allowed paclitaxel administration at higher doses and had an MTD of 25 mg drug/kg. The CyDs tested are marginal in feasibility for paclitaxel administration, and their use in taxane formulation will require a reduction of the dose-limiting toxicity of the CyD itself.

Formulation and efficacy of liposome-encapsulated antibiotics for therapy of intracellular Mycobacterium avium infection.

Mycobacterium avium is an intracellular pathogen that can invade and multiply within macrophages of the reticuloendothelial system. Current therapy is not highly effective. Particulate drug carriers that are targeted to the reticuloendothelial system may provide a means to deliver antibiotics more efficiently to M. avium-infected cells. We investigated the formulation of the antibiotics ciprofloxacin and azithromycin in liposomes and tested their antibacterial activities in vitro against M. avium residing within J774, a murine macrophage-like cell line. A conventional passive-entrapment method yielded an encapsulation efficiency of 9% for ciprofloxacin and because of aggregation mediated by the cationic drug, was useful only with liposomes containing < or = 50 mol% negatively charged phospholipid. In contrast, ciprofloxacin was encapsulated with > 90% efficiency, regardless of the content of negatively charged lipids, by a remote-loading technique that utilized both pH and potential gradients to drive drug into preformed liposomes. Both the cellular accumulation and the antimycobacterial activity of ciprofloxacin increased in proportion to the liposome negative charge; the maximal enhancement of potency was 43-fold in liposomes of distearoylphosphatidylglycerol-cholesterol (DSPG-Chol) (10:5). Azithromycin liposomes were prepared as a freeze-dried preparation to avoid chemical instability during storage, and drug could be incorporated at 33 mol% (with respect to phospholipid). Azithromycin also showed enhanced antimycobacterial effect in liposomes, and the potency increased in parallel to the moles percent of negatively charged lipids; azithromycin in DSPG-Chol (10:5) liposomes inhibited intracellular M. avium growth 41-fold more effectively than did free azithromycin. Thus, ciprofloxacin or azithromycin encapsulated in stable liposomes having substantial negative surface charge is superior to nonencapsulated drug in inhibition of M.avium growth within cultured macrophages and may provide more effective therapy of M.avium infections.

Physicochemical, pharmacokinetic and pharmacodynamic evaluation of liposomal tacrolimus (FK 506) in rats.

PURPOSE: Tacrolimus (FK 506) is a new potent immunosuppressant. Because of poor water solubility, the conventional intravenous dosage forms of FK 506 (C-FK 506) contain surfactants such as HCO-60 which may cause adverse effects. We sought a liposomal formulation of FK 506 (L-FK 506) containing endogenous phospholipids to target drug to the spleen, a major organ controlling the immune system. METHODS: L-FK 506, consisting of 0.1 micron diameter vesicles of phosphatidylcholine and phosphatidylglycerol (molar ratio 9:1) and 7.5 mole% drug, was evaluated for in vitro stability. The intravenous disposition profile, spleen distribution, and immunosuppression of L-FK 506 was compared with that of C-FK 506 in the rat after single doses of 0.3 mg/kg. RESULTS: The L-FK 506 showed good in vitro stability. L-FK 506 exhibited an increased volume of distribution at steady-state (Vss) (from 3.41 to 14.71 L/kg) and increased mean residence time (MRT) (from 2.83 to 16.07 hr). FK 506 concentrations in spleen were increased by 40% at 10 hr after administration of the liposomal formulation. The pharmacodynamics of L-FK 506, evaluated by the extent of inhibition of splenocyte proliferation, was comparable to that of C-FK 506. CONCLUSIONS: Liposomal FK 506 may be an improved dosage form for parenteral use.

Solvent- and concentration-dependent molecular interactions of taxol (Paclitaxel).

Taxol (paclitaxel) is a promising anticancer agent that has been approved for the treatment of ovarian cancer and is under investigation for the therapy of other tumors. Paclitaxel is poorly soluble in water, and information on its physical behavior in hydrophilic and hydrophobic environments is limited. Circular dichroism (CD) and nuclear magnetic resonance spectroscopy were used to investigate the effect of solvent and drug concentration on the solution conformation of paclitaxel. CD is sensitive to paclitaxel's environment, owing to the presence of effective chromophores in the vicinity of several chiral centers. It was found that (i) the conformation of the paclitaxel side chain depends on the polarity of the solvent and (ii) paclitaxel has a tendency to undergo concentration-dependent aggregation in solvents such as chloroform. To account for the observations, a model is proposed in which paclitaxel molecules are held together in stacks by intermolecular hydrogen bonds involving all four exchangeable protons. Intermolecular interactions and self-association of paclitaxel may have impact not only on the physical stability of the drug in existing formulations or investigational vehicles but also on the effect of paclitaxel in the stabilization of cellular microtubules.

Taxol-lipid interactions: taxol-dependent effects on the physical properties of model membranes.

Taxol (paclitaxel) is a diterpenoid anticancer agent undergoing intensive human clinical evaluation. The poor aqueous solubility of taxol necessitates administration in excipients causing a variety of adverse effects, including anaphylactoid hypersensitivity reactions. Recently, taxol has been formulated in better-tolerated drug carriers such as liposomes. We investigated the conformation of taxol and the interaction of taxol with dipalmitoylphosphatidylcholine (DPPC) liposomes using fluorescence, circular dichroism, differential scanning calorimetry, fluorescence polarization, and X-ray diffraction. The conformation of taxol in DPPC membranes was similar to that observed in nonpolar solvents such as chloroform. Taxol was found to partition into the bilayer, perturbing the hydrocarbon chain conformation. The taxol C13 side chain was found to be fluorescent, and it displays an environment-sensitive shift in emission spectrum; taxol fluorescence was used to confirm the insertion of the drug into the bilayer. Taxol induces a broadening of the DPPC phase transition, and the location of the drug in the bilayer depends on drug concentration. Incorporation of taxol affects other physical properties of the bilayer such as the lipid order parameter, and this fluidizing effect was also observed upon incorporation of taxol in biological membranes isolated from basolateral plasma membranes of rat liver. These studies demonstrate that taxol incorporated into liposomes penetrates into the acyl chain domain of the bilayer and alters the physical properties of both artificial and biological membranes.

Novel taxol formulations: preparation and characterization of taxol-containing liposomes.

Taxol is a promising anticancer agent under investigation for therapy of ovarian, breast, colon, and head and neck cancer. One problem associated with the administration of taxol is its low solubility in most pharmaceutically-acceptable solvents; the formulation used clinically contains Cremophor EL (polyethoxylated castor oil) and ethanol as excipients, which cause serious adverse effects. To eliminate this vehicle and possibly improve the antitumor efficacy of taxol, we have formulated taxol in liposomes of various compositions. Liposome formulations containing taxol and phospholipid in the molar ratio 1:33 were prepared from phosphatidylglycerol (PG) and phosphatidylcholine (PC) (1:9 molar ratio), and were physically and chemically stable for more than 2 months at 4 degrees C, or for 1 month at 20 degrees C. A method of producing taxol-liposomes by lyophilization has been developed, by which large batches can be prepared reproducibly in a 'pharmaceutically rational' manner. Taxol-liposomes retained the growth-inhibitory activity of the free drug in vitro against a variety of tumor cell lines. In mice, taxol-liposomes were well-tolerated when given in bolus doses by both iv and ip routes. The Maximum Tolerated Dose (MTD) was > 200 mg/kg; it exceeded that of free taxol, which had a MTD of 30 mg/kg by iv or 50 mg/kg by ip administration. Free taxol administered in the Cremophor vehicle was toxic at doses > 30 mg/kg, as was the equivalent volume of vehicle without drug.(ABSTRACT TRUNCATED AT 250 WORDS)