In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative,N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

The effects of amphotericin B on lipid metabolism in cultured human skin fibroblasts

Summary Amphotericin B inhibits hydroxymethylglutarylcoenzyme A (HMGCoA) reductase activity and incorporation of [³H]acetate into sterols and fatty acids of human skin fibroblasts. Delivery of cholesterol to cells via the low density lipoprotein receptor pathway is not altered. The effects of amphotericin B on cell lipid metabolism are partially reversed by both potassium and insulin. Portions of this work were presented at the Southern Regional Meeting of the American Federation for Clinical Research, January 1983.     

The in vitro immunomodulatory effects of sulfasalazine on human polymorphonuclear leukocytes, mononuclear cells, and cultured glomerular mesangial cells

Sulfasalazine (SSA) was investigated for its effects on phagocytic activity of normal human polymorphonuclear neutrophils (PMN), proliferation of mononuclear cells (MNC) and cultured glomerular mesangial cells. At concentrations from 25 to 100 microM, it inhibited phagocytic activity of PMN and the 3H-thymidine incorporation of phytohemagglutinin (PHA)-stimulated human MNC in a dose-dependent manner. At comparable concentrations, sulfapyridine and 5-aminosalicylic acid, two of its major metabolites, did not show similar effects. SSA exhibited an inhibitory effect on both mouse and rat mesangial cells but at rather higher concentrations (0.5 mM). Excretion of interleukin (IL)-8 by lipopolysaccharide (LPS)-stimulated PMN was also markedly deterred in a dose-dependent manner but excretion of IL-8 by LPS-stimulated MNC was not interfered by SSA. Production of tumor necrosis factor (TNF)-alpha and IL-1beta by mouse mesangial cells was not blocked by SSA but production of IL-4 by these cells was inhibited by it (>0.1 mM). Inhibition of MNC was not due directly to cytotoxic effect of SSA on these cells as shown by fluorescein diacetate stain. Collectively, SSA inhibits phagocytosis and IL-8 excretion by PMN as well as mitogen-stimulated MNC reaction. On the other hand, at high concentrations, it inhibits glomerular mesangial cells and their IL-4 excretion but not TNF-alpha and IL-1beta excretion. These results can account for minimal nephrotoxic characteristic of SSA and suggest that it may be helpful in the treatment of immune-mediated glomerulonephritis.     

MFAME, N-methyl-N-D-fructosyl amphotericin B methyl ester, a new amphotericin B derivative of low toxicity: relationship between self-association and effects on red blood cells

In aqueous solutions N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME), a novel amphotericin B derivative with low animal toxicity, similar to its parent antibiotic, exists in three forms: monomeric, soluble and insoluble aggregates in equilibrium [1]. The aim of our work was to examine the influence of medium composition on the MFAME self-association and the relationship between MFAME self-association and its toxicity towards red blood cells. The toxicity of MFAME in aggregated state towards red blood cells was tested by measuring the induction of potassium leakage and extent of haemolysis. The proportions of antibiotic species present in various aqueous media were determined by analysis of the UV-Vis spectra as a function of the antibiotic concentration. Numeric decomposition of the spectra allowed identification of four spectral species present in MFAME solutions: monomeric and three aggregated forms. Our results indicate that these aggregates, named type I, type II and type III, are different in terms of spectral properties, as well as effectiveness towards red blood cells. Soluble aggregate types I and III are the active forms of MFAME towards erythrocytes. The medium composition seems to be the main factor determining which type of antibiotic aggregate prevails in solution.     

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.     

The effects of the antibiotics gramicidina,amphotericin B,and nystatin on the electrical properties of frog skeletal muscle

The antibiotics gramicidin A, amphotericin B, and nystatin drastically decrease the membrane resistance of frog skeletal muscle fibers without changing the total capacitance. The resting potential of muscle fibers treated with these antibiotics is essentially normal if the Ringer solution does not contain Na⁺.     

Lipids and cell fusion in vitro: effect of amphotericin B.

Fusion rate of Cl-1D cells mediated by Sendai virus was decreased by preincubating the cells or the virus in the presence of lysolecithin. However, a significant increase occurred when the cells were preincubated in the presence of amphotericin B. This increase was suppressed by exogenous cholesterol.     

Immunopotentiating effects of amphotericin B. II. Enhanced in vitro proliferative responses of murine lymphocytes.

Enhanced in vitro proliferative responses to DNBSO3 were seen in lymph node cells and spleen cells after in vivo sensitization of mice with DNFB plus AmB compared with mice primed with DNFB alone. The T cell proliferation in the nylon column nonadherent fraction for both groups was highly similar, and the enhanced lymph node cell proliferation with AmB was demonstrated to be in the nylon adherent population consisting of both T and B cells. These and earlier studies of immunopotentiation by AmB are consistent with a mechanism that depends on selective interaction of the polyene with a subset of T cells and a resultant impairment of the normally induced suppressor regulation that limits the magnitude and duration of immune responses.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

地塞米松影响念珠菌对抗真菌药物敏感性的实验研究

目的 探讨地塞米松在体外试验中是否影响念珠菌对抗真菌药物的敏感性,以了解糖皮质激素与抗真菌药物直接作用于念珠菌时是否存在相互作用。方法 用微量液体培养基稀释法分别测定26株白念珠菌与地塞米松（0．2mg／ml）共同孵育前、孵育24～48h及7d时氟康唑、伊曲康唑、两性霉素B的最低抑菌浓度（MIC）值,并作对照。结果 白念珠菌与地塞米松孵育24～48h后、孵育后第7d氟康唑和伊曲康唑的MIC值升高,分别与孵育前的MIC值存在统计学差异,但孵育24～48h后的MIC与孵育后第7d的MIC无统计学差异;白念珠菌与地塞米松共同孵育24～48h后两性霉素B的MIC值也较孵育前升高,但第7d的MIC值与孵育前无差异。结论 地塞米松可增加三种抗真菌药物对于白念珠菌的MIC,但三种抗真菌药物间存在差异,表明地塞米松对于氟康唑和伊曲康唑体外抗白念珠菌的活性有拮抗作用,但没有时间依赖性,地塞米松对于两性霉素B的影响较氟康唑和伊曲康唑小,且影响时间较短。     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

两性霉素B联合氟胞嘧啶与伏立康唑治疗艾滋病合并隐球菌脑膜炎的临床回顾性研究

目的观察两性霉素B联合氟胞嘧啶与伏立康唑治疗艾滋病合并隐球菌性脑膜炎的疗效和安全性,探讨艾滋病合并隐球菌性脑膜炎的新型治疗策略。方法采用回顾性病例对照研究,20例艾滋病合并隐球菌脑膜炎分别接受三联治疗（两性霉素B＋氟胞嘧啶＋伏立康唑,n=10）和传统的两联治疗（两性霉素B＋氟胞嘧啶,n=10）,比较两种治疗方案在降低脑脊液中隐球菌计数的幅度以及临床症状缓解、病死率等方面的差异以及不良反应发生情况。结果治疗2周后,三联治疗患者脑脊液中隐球菌计数下降率明显高于两联治疗患者（下降率分别为0.743和0.408,P=0.009）,三联治疗患者头痛明显减轻或消失的时间短于两联治疗者（分别为12 d和20 d,P=0.009）,两组在2周、4周、8周及12周时的病死率均无统计学差异。两种治疗方案的不良反应发生率相当。结论两性霉素B联合氟胞嘧啶与伏立康唑能有效降低脑脊液中隐球菌计数,可作为艾滋病合并隐球菌脑膜炎诱导期的治疗选择。     

The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss

Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.     

Effect of mycolase and amphotericin B on Candida albicans and Candida pseudotropicalis in vitro and in vivo

A mixture of enzymes (mycolase) capable of lysing yeast cell walls was prepared from culture filtrates of Physarum polycephalum. The enzymes present in mycolase included chitinase, beta-1,3-glucanases and exo-glycosidases. The pH optima of these enzymes were in the range 3.5-5.0 and they had low activities at pH 7.0. Mycolase produced spheroplasts from Candida pseudotropicalis and, unlike commercial enzyme preparations such as L1, chitinase, beta, 1,3-glucanase and beta-glucosidase, had some candicidal activity in vitro against C. pseudotropicalis and C. albicans. Mycolase potentiated the antifungal activity of amphotericin B against C. pseudotropicalis grown in shake flask culture but did not potentiate the antifungal activity of the antibiotic against similar cultures of C. albicans; indeed antagonism between mycolase and amphotericin B was sometimes observed with the latter yeast. Mycolase caused an approximately two-fold increase in the total and viable counts of cultures of C. albicans inoculated with stationary phase cells. These increases, which were observed within about 30 min, were attributed to mycolase inducing the premature release of viable buds from 'lag' phase cells. Mycolase also increased the rate at which C. albicans formed germ tubes when the yeast was cultured in a medium containing serum. Mycolase alone or in combination with amphotericin B did not appreciably enhance phagocytosis or intracellular killing of the yeasts by unstimulated mouse peritoneal macrophages. Studies on mice infected systemically with C. albicans showed that mycolase only slightly enhanced amphotericin B therapy.     

两性霉素B和伏立康唑对临床真菌的体外抗菌活性分析

目的比较两性霉素B和伏立康唑对临床真菌的体外抗菌活性。方法用两性霉素B和伏立康唑的E-test条对分离自临床标本的116株真菌进行体外药敏试验,其中热带念珠菌15株,光滑念珠菌14株,近平滑念珠菌11株,克柔念珠菌6株,新生隐球菌8株,阿萨希毛孢子菌9株,烟曲霉29株,黄曲霉15株,黑曲霉1株,镰刀菌属7株,根霉属1株,以ATCC22019光滑念珠菌为质控菌株。结果两性霉素B对阿萨希毛孢子菌、镰刀菌、黄曲霉的MIC90均为64μg/ml,对其余受试菌株的MIC90均≤1μg/ml,伏立康唑对镰刀菌的MIC90为64μg/ml,对大部分受试菌株的MIC90均≤2μg/ml。结论除对某些真菌可能无效外,两性霉素B和伏立康唑可能适用于治疗大多数的真菌感染。