Efficacy of liposomal amphotericin B in treatment of systemic murine fusariosis

We have compared the activities of liposomal amphotericin B (LAMB) at 3, 5, 10, and 20 mg/kg/day and amphotericin B deoxycholate (AMB) at 1.5 and 2.5 mg/kg/day in a murine systemic infection by Fusarium verticillioides. Survival was improved by all treatments except AMB at 1.5 mg/kg/day. The tissue burden in liver was reduced by LAMB at all dosages and by AMB at 2.5 mg/kg/day. The two highest dosages of LAMB showed significant reductions in the spleen.     

Comparison of a New Triazole Antifungal Agent, Schering 56592, with Itraconazole and Amphotericin B for Treatment of Histoplasmosis in Immunocompetent Mice

A murine model of intratracheally induced histoplasmosis was used to evaluate a new triazole antifungal agent, Schering (SCH) 56592, for treatment of histoplasmosis. MICs were determined for SCH 56592, amphotericin B, and itraconazole by testing yeast-phase isolates from 20 patients by a macrobroth dilution method. The MICs at which 90% of the isolates are inhibited were for 0.019 μg/ml for SCH 56592, 0.5 μg/ml for amphotericin B, and ≤0.019 μg/ml for itraconazole. Survival studies were done on groups of 10 B6C3F₁ mice with a lethal inoculum of 10⁵. All mice receiving 5, 1, or 0.25 mg of SCH 56592 per kg of body weight per day, 2.5 mg of amphotericin B per kg every other day (qod), or 75 mg of itraconazole per kg per day survived to day 29. Only 44% of mice receiving 5 mg of itraconazole/kg/day survived to day 29. Fungal burden studies done in similar groups of mice with a sublethal inoculum of 10⁴ showed a reduction in CFUs and Histoplasma antigen levels in lung and spleen tissue in animals treated with 2 mg of amphotericin B/kg qod, 1 mg of SCH 56592/kg/day, and 75 mg of itraconazole/kg/day, but not in those treated with lower doses of the study drugs (0.2 mg of amphotericin B/kg qod, 0.1 mg of SCH 56592/kg/day, or 10 mg of itraconazole/kg/day). Serum drug concentrations were measured 3 and 24 h after the last dose in mice (groups of five to seven mice), each treated for 7 days with SCH 56592 (10 and 1 mg/kg/day) and itraconazole (75 and 10 mg/kg/day). Mean levels measured by bioassay were as follows: SCH 56592, 10 mg/kg/day (2.15 μg/ml at 3 h and 0.35 μg/ml at 24 h); SCH 56592, 1 mg/kg/day (0.54 μg/ml at 3 h and none detected at 24 h); itraconazole, 75 mg/kg/day (22.53 μg/ml at 3 h and none detected at 24 h); itraconazole, 10 mg/kg/day (1.33 μg/ml at 3 h and none detected at 24 h). Confirmatory results were obtained by high-pressure liquid chromatography assay. These studies show SCH 56592 to be a promising candidate for studies of treatment of histoplasmosis in humans.     

Pharmacodynamics of Amphotericin B Deoxycholate,Amphotericin B Lipid Complex,and Liposomal Amphotericin B against Aspergillus fumigatus

Amphotericin B is a first-line agent for the treatment of invasive aspergillosis. However, relatively little is known about the pharmacodynamics of amphotericin B for invasive pulmonary aspergillosis. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of amphotericin B deoxycholate (DAMB), amphotericin B lipid complex (ABLC), and liposomal amphotericin B (LAMB) by using a neutropenic-rabbit model of invasive pulmonary aspergillosis. The study endpoints were lung weight, infarct score, and levels of circulating galactomannan and (1→3)-β-d-glucan. Mathematical models were used to describe PK-PD relationships. The experimental findings were bridged to humans by Monte Carlo simulation. Each amphotericin B formulation induced a dose-dependent decline in study endpoints. Near-maximal antifungal activity was evident with DAMB at 1 mg/kg/day and ABLC and LAMB at 5 mg/kg/day. The bridging study suggested that the “average” patient receiving LAMB at 3 mg/kg/day was predicted to have complete suppression of galactomannan and (1→3)-β-d-glucan levels, but 20 to 30% of the patients still had a galactomannan index of >1 and (1→3)-β-d-glucan levels of >60 pg/ml. All formulations of amphotericin B induce a dose-dependent reduction in markers of lung injury and circulating fungus-related biomarkers. A clinical dosage of liposomal amphotericin B of 3 mg/kg/day is predicted to cause complete suppression of galactomannan and (1→3)-β-d-glucan levels in the majority of patients.     

Urinary excretion of cyclic nucleotides and principal electrolytes in healthy humans of different ages

Urinary excretion of cyclic AMP, cyclic GMP, inorganic phosphorus, calcium, sodium, potassium and creatinine was measured in 138 healthy male and 104 healthy female humans from 2 to 68 years old. The range of cyclic nucleotide excretion was as follows: cyclic AMP (μmol/day), 1.01–10.89; cyclic GMP (μmol/day), 0.13–2.00; cyclic AMP (Mmol/g creatinine), 1.52–8.93; cyclic GMP (μmol/g creatinine), 0.11–1.87. The 242 volunteers were grouped into seven classes according to age: A, 2–9 years old; B, 10–19; C, 20–29; D, 30–39; E, 40–49; F, 50–59 and G, 60–68. Average excretion (μmol/day) of cyclic AMP in class A (2.62 ± 0.29 for males and 2.30 ± 0.18 for females) was significantly smaller than that in other classes (4.59 ± 0.12 for males and 3.90 ± 0.13 for females) (p < 0.01). Such a significant difference was not observed in cyclic GMP excretion. In terms of μmol/g creatinine, however, average excretion of both cyclic AMP and cyclic GMP in class A was greater than that in other classes.

The amounts of urinary cyclic AMP and cyclic GMP (μmol/day) were correlated with age in the subjects from 2 to 16 years. A reverse correlation between the amounts of both nucleotides (μmol/g creatinine) and age was found in the young subjects. No correlation between the excretion of either urinary cyclic nucleotide and age was found in adults.

A significantly positive correlation between cyclic AMP (μmol/day) and inorganic phosphorus (g/day) was found (r = 0.50 for males and 0.56 for females) (p < 0.01). This correlation suggests that urinary cyclic AMP might reflect the activity of parathyroid hormone in normal humans. There was no significant correlation between cyclic GMP and electrolytes tested. The above results are considered to provide basic data for clinical evaluation of relevant disorders.     

Efficacy of Liposomal Amphotericin B Combined with Gamma Interferon or Granulocyte-Macrophage Colony-Stimulating Factor for Treatment of Systemic Zygomycosis in Mice

Granulocyte-macrophage colony-stimulating factor enhanced the efficacy of liposomal amphotericin B (LAMB) in a murine model of disseminated infection by Rhizopus oryzae, significantly prolonging survival and reducing tissue burden. The use of gamma interferon (IFN-γ) alone was ineffective, and IFN-γ combined with LAMB did not improve the results obtained with LAMB alone.Zygomycosis is a frequently lethal invasive infection (2). The standard therapy for zygomycosis has not yet been resolved (2). Historically, conventional amphotericin B (AMB) was the drug of choice for invasive zygomycosis, but its use is limited by its potential toxicity. The lipid formulation of AMB (LAMB) allows higher doses to be administered on account of its low toxicity and represents first-line therapy (17). In murine zygomycosis, LAMB has showed efficacy and has been even better than AMB deoxycholate (11, 12). The efficacy of posaconazole is controversial, as some clinical data show good results (9, 20) but some authors have demonstrated that this drug is poorly active against Rhizopus oryzae, the most common species causing zygomycosis (13, 16). Since the mortality rate is often high in disseminated zygomycosis, despite aggressive therapy, new strategies for the treatment of this infection are urgently needed (5).Cytokines are critical components of the functional host defenses promoting activation and recruitment of granulocyte and mononuclear phagocyte effector cells (18). Over the last decade, the usefulness of these compounds as adjunctive agents in antifungal therapy in the treatment of severe fungal infections has been evaluated (2, 10). In particular, gamma interferon (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have showed efficacy as adjunctive agents in the treatment of experimental cryptococcosis and histoplasmosis in mice (3, 4) and also in several clinical cases of zygomycosis (1, 5, 8, 14). Moreover, these cytokines have induced an increase in in vitro polymorphonuclear leukocyte-induced hyphal damage to R. oryzae (6).In this study, we have evaluated the effects of IFN-γ and GM-CSF, alone and combined with LAMB, in a murine model of infection by R. oryzae.Two clinical isolates of Rhizopus oryzae, FMR 6485 and FMR 8542, were used in this study. The isolates were cultured on potato dextrose agar at 35°C. The AMB MICs were identical for both strains (0.5 μg/ml) (16).Male OF1 mice were used in this study. All animal care procedures were supervised and approved by the Universitat Rovira i Virgili Animal Welfare Committee. Animals were immunodepressed 1 day prior to infection by administering a single dose of 200 mg of cyclophosphamide per kg of body weight intraperitoneally plus a single dose of 150 mg of 5-fluorouracil per kg intravenously (i.v.). Mice were challenged with 0.2 × 10⁵ CFU/animal. Ten mice were used for survival studies and 10 for tissue burden studies, with the latter group being identified before the study started. All mice received ceftazidime (5 mg/day subcutaneously [s.c.]) from day 1 to day 7 after infection.LAMB was administered i.v. at doses of 5 or 10 mg/kg of body weight/dose once daily. Human recombinant IFN-γ (GenScript Corporation) was administered i.v. at a dose of 10⁵ U once daily (3); human recombinant GM-CSF (GenScript Corporation) was administered s.c. at a dose of 5 μg/kg/day once daily (19). The different groups were treated as follows: LAMB at 5 or 10 mg/kg of body weight i.v., IFN-γ at 10⁵ U i.v., GM-CSF at 5 μg/kg/day s.c., LAMB at 10 mg/kg i.v. plus IFN-γ at 10⁵ U i.v., and LAMB at 10 mg/kg i.v. plus GM-CSF at 5 μg/kg/day s.c.All treatments began 24 h after challenge, and the therapies lasted for 7 days (16). Survival of mice was evaluated daily for 30 days. For tissue burden studies, mice were sacrificed on day 4 postinfection. Kidneys and brains were removed aseptically and were homogenized in 1 ml of sterile saline; care was taken to minimize tissue trauma. Serial 10-fold dilutions of the homogenates were plated on potato dextrose agar and incubated for 18 to 24 h at 35°C. Mean survival times were estimated by the Kaplan-Meier method and compared among groups by using the log rank test. Colony counts in tissue burden studies were analyzed by the Kruskal-Wallis test. When the results of this test were significant, we used the Mann-Whitney U test to compare treatment pairs. The Bonferroni correction method was used to avoid an increase in type I errors due to multiple comparisons. When P was <0.05, the observed differences were declared to be statistically significant.For both strains, the two doses of LAMB and the combination of LAMB with IFN-γ or GM-CSF significantly prolonged survival with respect to the rates for the control and the groups treated only with IFN-γ or GM-CSF (Fig. ​(Fig.1).1). LAMB combined with GM-CSF was able to prolong survival with respect to the rate for the group treated with LAMB at 10 mg/kg for strain FMR 8542. The combination of LAMB with IFN-γ showed efficacy similar to that of monotherapy with LAMB at 10 mg/kg, with no differences between them. Neither of the two cytokines prolonged survival.Open in a separate windowFIG. 1.Cumulative mortality of mice infected with Rhizopus oryzae FMR 6485 (A) or R. oryzae FMR 8542 (B) and treated with LAMB, GM-CSF, and IFN-γ. ^a, P values of <0.05 for comparison with the control, IFN-γ (100,000 U), and GM-CSF (5 μg/kg); ^b, P values of <0.05 for comparison with LAMB (5 mg/kg); ^c, P values of <0.05 for comparison with LAMB (10 mg/kg).All the treatments, with the exception of IFN-γ, significantly reduced the fungal load in brain and kidney in comparison to that for the control group for both strains. In addition, GM-CSF and IFN-γ plus LAMB failed to reduce tissue burden in kidney for strain FMR 6485 (Fig. ​(Fig.2).2). The combination of LAMB at 10 mg/kg with GM-CSF was the most effective treatment, reducing the fungal load in brain and kidney tissues, although only in brain tissues for strain FMR 8542.Open in a separate windowFIG. 2.Effects of antifungal treatment on colony counts of Rhizopus oryzae strains FMR 6485 (A) and FMR 8542 (B) in brain and kidney tissues of mice. ^a, P values of <0.002 for comparison with the control; ^b, P values of <0.002 for comparison with LAMB (5 mg/kg); ^c, P values of <0.002 for comparison with LAMB (10 mg/kg); ^d, P values of <0.002 for comparison with IFN-γ (100,000 U); ^e, P values of <0.002 for comparison with GM-CSF (5 μg/kg); ^f, P values of <0.002 for comparison with LAMB (10 mg/kg) plus IFN-γ. CFU/g of tissue are expressed as log₁₀ scale. Horizontal lines indicate mean values.AMB showed efficacy similar to that in a previous study (16), which proved the reproducibility of this murine model.LAMB in general shows efficacy in zygomycosis treatment, but in many cases, the patients die. In a recent review of 120 cases of zygomycosis in patients with hematological malignances, LAMB was associated with a 67% survival rate, compared to a 39% survival rate for AMB deoxycholate (7). In severe zygomycosis, correction of metabolic disturbances and reversal of immunosuppression are as essential as the other therapeutic measures for successful management (2). For this reason, we have assessed the efficacy of cytokines as adjuvant agents for returning the host immune response and tried to restore the host''s defenses. In our model, only the combination of LAMB with GM-CSF was significantly better than the treatment with LAMB alone. These results agree with those observed in a few clinical cases of zygomycosis in which GM-CSF was administered successfully as an adjunctive therapy with AMB or its lipid formulation (5, 8, 14).These studies suggest a potential use for GM-CSF as an immunomodulator for improving the benefits of therapy with LAMB against zygomycosis.     

Granulocyte Colony-Stimulating Factor Enhances Endotoxin-Induced Decrease in Biliary Excretion of the Antibiotic Cefoperazone in Rats

We have recently reported that endotoxin (lipopolysaccharide [LPS]) derived from Klebsiella pneumoniae dramatically decreased the biliary excretion of the β-lactam antibiotic cefoperazone (CPZ), which is primarily excreted into the bile via the anion transport system, in rats. The present study was designed to investigate the effect of human recombinant granulocyte colony-stimulating factor (G-CSF), which is reported to be beneficial in experimental models of inflammation, on the pharmacokinetics and biliary excretion of CPZ in rats. CPZ (20 mg/kg of body weight) was administered intravenously 2 h after the intravenous injection of LPS (250 μg/kg). G-CSF was injected subcutaneously at 12 μg/kg for 3 days and was administered intravenously at a final dose of 50 μg/kg 1 h before LPS injection. Peripheral blood cell numbers were also measured. LPS dramatically decreased the systemic and biliary clearances of CPZ and the bile flow rate. Pretreatment with G-CSF enhanced these decreases induced by LPS. The total leukocyte numbers were increased in rats pretreated with G-CSF compared to the numbers in the controls, while the total leukocyte numbers were decreased (about 3,000 cells/μl) by treatment with LPS. Pretreatment with G-CSF produces a deleterious effect against the LPS-induced decrease in biliary secretion of CPZ, and leukocytes play an important role in that mechanism.     

Antifungal activity of amphotericin B cochleates against Candida albicans infection in a mouse model

Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against Candida albicans are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 microg of AmB/ml, and DAMB was highly hemolytic at 10 microg of AmB/ml. CAMB protect ICR mice infected with C. albicans when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.     

Depletion of myocardial adenosine triphosphate during prolonged untreated ventricular fibrillation: effect on defibrillation success

We studied left ventricular endomyocardial adenosine triphospate levels in 13 large mongrel dogs before and during ventricular fibrillation induced cardiac arrest to assess whether myocardial adenosine triphosphate content could predict successful cardiopulmonary resuscitation. Endomyocardial biopsies were performed during sinus rhythm (control), after 15 min of ventricular fibrillation or 10 min of ventricular fibrillation and 5 min of open chest cardiopulmonary resuscitation, after 20 min of ventricular fibrillation and 10 min of open chest cardiopulmonary resuscitation and after 40 min ventricular fibrillation and 15–20 min open chest cardiopulmonary resuscitation. Myocardial adenosine triphosphate was measured utilizing a bioluminescence method adapted for use with endomyocardial biopsies and normalized to protein content. Left ventricular endomyocardial adenosine triphosphate content fell significantly over time from a control level of 8.88 ±0.9 pg/mg protein to 5.73 ± 0.5 pg/mg protein at 15 min of cardiac arrest, to 3.4 ± 0.4 μg/mg protein after 30 min of cardiac arrest and to 1.98 ± 0.3 μg/mg protein after 60 min of cardiac arrest (P < 0.001). Adenosine triphosphate levels were significantly different between animals that received 10 min of ventricular fibrillation and successful open chest cardiopulmonary resuscitation and those that received 40 min of ventricular fibrillation and unsuccessful open chest cardiopulmonary resuscitation (4.35 ± 0.48 vs. 2.11 ± 0.43 μg/mg protein; P < 0.025). Endomyocardial adenosine triphosphate levels falling below 3.5 μg/mg protein were associated with only 2/6 animals being successfully resuscitated, while 6/7 successfully resuscitated animals had adenosine triphosphate levels 3.5 μg/ mg protein (Positive Predictive Value = 0.75, Negative Predictive Value = 0.80). Myocardial adenosine triphosphate content diminishes significantly during prolonged ventricular fibrillation and once levels fall below 3.5 μg/mg protein, successful resuscitation is rare.     

Adjunctive efficacy of granulocyte colony-stimulating factor on treatment of Pseudomonas aeruginosa pneumonia in neutropenic and non-neutropenic hosts

OBJECTIVES: Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation of neutrophils and enhances their phagocytic and microcidal activity. Increasing resistance to existing antibacterials and the dearth of new alternatives have complicated the treatment of Gram-negative infections. The aim of this study was to evaluate the efficacy of G-CSF in the treatment of Pseudomonas aeruginosa pneumonia when administered in combination with ceftazidime in both neutropenic and non-neutropenic hosts. METHODS: A group of mice were rendered neutropenic with cyclophosphamide. Pneumonia was induced by intratracheal instillation of approximately 5 x 10(7) cfu/mL and approximately 5 x 10(9) cfu/mL (LD(100)) of the organism to neutropenic and non-neutropenic mice, respectively. Two hours after inoculation, the mice received normal saline and 5% dextrose, G-CSF (300 micro g/kg per day x 3 days), ceftazidime (2000 mg/kg x 2 doses) or a combination of G-CSF and ceftazidime. Survival was monitored at different time points for 5 days. RESULTS: Treatment with G-CSF showed a dose-dependent increase in survival from 50 to 300 micro g/kg. In neutropenic mice, survival was markedly better in the G-CSF + ceftazidime group compared with controls (P = 0.0001), G-CSF (P = 0.0002) or ceftazidime (P = 0.0172). In non-neutropenic mice, survival in the G-CSF + ceftazidime group (20%) was significantly higher than in the control and G-CSF groups (P = 0.0001) but not significantly higher than ceftazidime alone (9%) (P > 0.05). CONCLUSIONS: G-CSF administered in combination with antibiotic after onset of severe P. aeruginosa pneumonia may improve therapeutic outcome and this suggests a new treatment option in the management of pneumonia especially in neutropenic patients.     

Uniform Susceptibility of Various Strains of Coccidioides immitis to Amphotericin B

The susceptibility of 12 strains of Coccidioides immitis to amphotericin B (amB) was studied in vitro and in vivo. The minimal inhibitory concentration (MIC) of the mycelial phase of these strains was 0.078 to 0.16 μg/ml after 3 days of incubation, but by 15 days all strains were inhibited by 2.5 μg/ml. Mice infected intraperitoneally with these strains were sucessfully treated with 0.5 mg of amB per kg per day. These strains included several studied by others and which reportedly varied widely in susceptibility (MIC from 0.24 to 24.01 μg/ml) to amB. Four of these strains representing this putative broad range of susceptibility were used to infect mice intranasally. Regardless of infecting strain, mice were sucessfully treated with 0.38, 0.75, and 1.5 mg/kg, but 0.19 mg/kg was only partially effective. Thus, in vivo as well as in vitro there was a uniform response of C. immitis strains to amB.     

Liposomal amphotericin B and granulocyte colony-stimulating factor therapy in a murine model of invasive infection by Scedosporium prolificans

We established a reproducible lethal disseminated infection by the opportunistic fungus Scedosporium prolificans in an immunosuppressed murine model. We compared the effectiveness of the combined administration of liposomal amphotericin B (LAMB) and granulocyte colony-stimulating factor (G-CSF) with that of either agent alone and with that of amphotericin B deoxycholate (AMB). LAMB + G-CSF and LAMB treatments improved survival significantly with respect to the untreated control. The mean survival times of these three groups were 13.2, 9.1 and 7.9 days, respectively. Culture results in terms of colony counts for samples of deep organs were lower in mice treated with the combined therapy, although differences were not significant. Combined LAMB + G-CSF therapy could be a promising approach for the treatment of disseminated infections of S. prolificans, although further studies are required to determine the most appropriate doses.     

A protocol for use of medetomidine anesthesia in rats for extended studies using task-induced BOLD contrast and resting-state functional connectivity

The alpha-2-adrenoreceptor agonist, medetomidine, which exhibits dose-dependent sedative effects and is gaining acceptance in small-animal functional magnetic resonance imaging (fMRI), has been studied. Rats were examined on the bench using the classic tail-pinch method with three infusion sequences: 100 μg/kg/h, 300 μg/kg/h, or 100 μg/kg/h followed by 300 μg/kg/h. Stepping the infusion rate from 100 to 300 μg/kg/h after 2.5 h resulted in a prolonged period of approximately level sedation that cannot be achieved by a constant infusion of either 100 or 300 μg/kg/h. By stepping the infusion dosage, experiments as long as 6 h are possible. Functional MRI experiments were carried out on rats using a frequency dependent electrical stimulation protocol—namely, forepaw stimulation at 3, 5, 7, and 10 Hz. Each rat was studied for a four-hour period, divided into two equal portions. During the first portion, rats were started at a 100 μg/kg/h constant infusion. During the second portion, four secondary levels of infusion were used: 100, 150, 200, and 300 μg/kg/h. The fMRI response to stimulation frequency was used as an indirect measure of modulation of neuronal activity through pharmacological manipulation. The frequency response to stimulus was attenuated at the lower secondary infusion dosages 100 or 150 μg/kg/h but not at the higher secondary infusion dosages 200 or 300 μg/kg/h. Parallel experiments with the animal at rest were carried out using both electroencephalogram (EEG) and functional connectivity MRI (fcMRI) methods with consistent results. In the secondary infusion period using 300 μg/kg/h, resting-state functional connectivity is enhanced.     

Effect of recombinant murine granulocyte colony-stimulating factor with or without fluoroquinolone therapy on mixed-infection abscesses in mice

The aim of the study was to determine if immunomodulation of host defense with recombinant murine granulocyte colony-stimulating factor (G-CSF) improves the efficacy of trovafloxacin or moxifloxacin in abscesses containing Bacillus fragilis ATCC 23745 and different Escherichia coli strains varying in virulence. Treatment of mice inoculated with 10(7) CFU B. fragilis and 10(5) CFU low-virulence E. coli with either trovafloxacin (150 mg/kg/day every 24 hours, days 3 to 7) or moxifloxacin (96 mg/kg/day every 12 hours, days 3 to 7), significantly reduced the number of B. fragilis to 6.9 +/- 0.35 and 5.8 +/- 0.10 and that of E. coli to 4.9 +/- 0.09 and 4.2 +/- 0.07 log CFU/abscess for trovafloxacin and moxifloxacin, respectively, compared to controls (B. fragilis 8.7 and E. coli 7.4 log CFU/abscess) on day 8. Also, moxifloxacin was more potent than trovafloxacin. Addition of G-CSF prophylaxis (1 mug once on day -1) or therapy (1 mug/day on days 3 to 7) to fluoroquinolone treatment did not improve the efficacy of fluoroquinolone therapy alone. The effect of moxifloxacin with or without G-CSF prophylaxis on abscesses with a virulent hemolytic E. coli strain was also studied. In moxifloxacin-treated mice, 75% survived infection compared to 10% of controls. Combining moxifloxacin with G-CSF prophylaxis significantly decreased survival (30%) compared to moxifloxacin alone. In addition, G-CSF prophylaxis resulted in a threefold (E. coli) to 100-fold (B. fragilis) increased outgrowth in the abscesses of surviving mice. In conclusion, the addition of G-CSF to a fluoroquinolone is not advisable since, depending on the virulence of the E. coli strains, this might detrimentally influence the outcome of therapy.     

Determination of the ED95 of intranasal sufentanil combined with intranasal dexmedetomidine for moderate sedation during endoscopic ultrasonography

BACKGROUNDSedation during endoscopic ultrasonography (EUS) poses many challenges and moderate-to-deep sedation are often required. The conventional method to preform moderate-to-deep sedation is generally intravenous benzodiazepine alone or in combination with opioids. However, this combination has some limitations. Intranasal medication delivery may be an alternative to this sedation regimen.AIMTo determine, by continual reassessment method (CRM), the minimal effective dose of intranasal sufentanil (SUF) when combined with intranasal dexmedetomidine (DEX) for moderate sedation of EUS in at least 95% of patients (ED₉₅).METHODSThirty patients aged 18-65 and scheduled for EUS were recruited in this study. Subjects received intranasal DEX and SUF for sedation. The dose of DEX (1 μg/kg) was fixed, while the dose of SUF was assigned sequentially to the subjects using CRM to determine ED₉₅. The sedation status was assessed by modified observer’s assessment of alertness/sedation (MOAA/S) score. The adverse events and the satisfaction scores of patients and endoscopists were recorded.RESULTSThe ED₉₅ was intranasal 0.3 μg/kg SUF when combined with intranasal 1 μg/kg DEX, with an estimated probability of successful moderate sedation for EUS of 94.9% (95% confidence interval: 88.1%-98.9%). When combined with intranasal 1 μg/kg DEX, probabilities of successful moderate sedation at each dose level of intranasal SUF were as follows: 0 μg/kg SUF, 52.8%; 0.1 μg/kg SUF, 75.4%; 0.2 μg/kg SUF, 89.9%; 0.3 μg/kg SUF, 94.9%; 0.4 μg/kg SUF, 98.0%; 0.5 μg/kg SUF, 99.0%.CONCLUSIONThe ED₉₅ needed for moderate sedation for EUS is intranasal 0.3 μg/kg SUF when combined with intranasal 1 μg/kg DEX, based on CRM.     

Population pharmacokinetics of liposomal amphotericin B and caspofungin in allogeneic hematopoietic stem cell recipients

Liposomal amphotericin B (LAMB) and caspofungin (CAS) are important antifungal agents in allogeneic hematopoietic stem cell transplant (aHSCT) recipients. Little is known, however, about the pharmacokinetics (PK) of both agents and their combination in this population. The PK of LAMB and CAS and the potential for PK interactions between both agents were investigated within a risk-stratified, randomized phase II clinical trial in 53 adult aHSCT recipients with granulocytopenia and refractory fever. Patients received either LAMB (n = 17; 3 mg/kg once a day [QD]), CAS (n = 19; 50 mg QD; day 1, 70 mg), or the combination of both (CAS-LAMB; n = 17) for a median duration of 10 to 13 days (range, 4 to 28 days) until defervescence and granulocyte recovery. PK sampling was performed on days 1 and 4. Drug concentrations in plasma (LAMB, 405 samples; CAS, 458 samples) were quantified by high-pressure liquid chromatography and were analyzed using population pharmacokinetic modeling. CAS concentration data best fitted a two-compartment model with a proportional error model and interindividual variability (IIV) for clearance (CL) and central volume of distribution (V(1)) (CL, 0.462 liter/h ± 25%; V(1), 8.33 liters ± 29%; intercompartmental clearance [Q], 1.25 liters/h; peripheral volume of distribution [V(2)], 3.59 liters). Concentration data for LAMB best fitted a two-compartment model with a proportional error model and IIV for all parameters (CL, 1.22 liters/h ± 64%; V(1), 19.2 liters ± 38%; Q, 2.18 liters/h ± 47%; V(2), 52.8 liters ± 84%). Internal model validation showed predictability and robustness of both models. None of the covariates tested (LAMB or CAS comedication, gender, body weight, age, body surface area, serum bilirubin, and creatinine clearance) further improved the models. In summary, the disposition of LAMB and CAS was best described by two-compartment models. Drug exposures in aHSCT patients were comparable to those in other populations, and no PK interactions were observed between the two compounds.     

Systemic physostigmine increases the antinociceptive effect of spinal morphine

In this study we evaluated the antinociceptive effect of concurrent intrathecal (i.t.) and subcutaneous (s.c.) administration of morphine and physostigmine, respectively. The experiments were performed on male Wistar rats. Intrathecal administration of morphine was performed through a catheter implanted in the subarachnoid space. The ‘tail-immersion' test was used to measure animals' responses to evoked nociceptive stimuli. Interaction of drugs was analyzed using a dose addition model. Both i.t. (1–5 μg) administration of morphine and s.c. (50–250 μg/kg) administration of physostigmine increased the latencies of nociceptive responses in a dose-dependent manner. Two micrograms of i.t. morphine and 100 μg/kg of s.c. physostigmine demonstrated 31.6±10.6 and 34.2±11.4 percentage of maximal possible effect (%MPE), respectively. Simultaneous administration of 1 μg of i.t. morphine and 50 μg/kg of s.c. physostigmine produced a %MPE equal to 84.8±16.9. Thus, combined administration of 1 μg i.t. morphine and 50 μg/kg s.c. physostigmine resulted in a strong, highly significant antinociceptive effect. This effect was much higher than the effect expected if both drugs acted in an additive manner. Supra-additive interaction observed in this study might be a result of simultaneous activation of different neurotransmitter systems involved in nociceptive processing at the spinal as well as at the supraspinal level of the CNS.     

Efficacy of High-Dose Amoxicillin-Clavulanate against Experimental Respiratory Tract Infections Caused by Strains of Streptococcus pneumoniae

The purpose of the present investigation was to determine if the efficacy of amoxicillin-clavulanate against penicillin-resistant Streptococcus pneumoniae could be improved by increasing the pediatric amoxicillin unit dose (90 versus 45 mg/kg of body weight/day) while maintaining the clavulanate unit dose at 6.4 mg/kg/day. A rat pneumonia model was used. In that model approximately 6 log₁₀ CFU of one of four strains of S. pneumoniae (amoxicillin MICs, 2 μg/ml [one strain], 4 μg/ml [two strains], and 8 μg/ml [one strain]) were instilled into the bronchi of rats. Amoxicillin-clavulanate was given by computer-controlled intravenous infusion to approximate the concentrations achieved in the plasma of children following the administration of oral doses of 45/6.4 mg/kg/day or 90/6.4 mg/kg/g/day divided every 12 h or saline as a control for a total of 3 days. Infusions continued for 3 days, and 2 h after the cessation of infusion, bacterial numbers in the lungs were significantly reduced by the 90/6.4-mg/kg/day equivalent dosage for strains for which amoxicillin MICs were 2 or 4 μg/ml. The 45/6.4-mg/kg/day equivalent dosage was fully effective only against the strain for which the amoxicillin MIC was 2 μg/ml and had marginal efficacy against one of the two strains for which amoxicillin MICs were 4 μg/ml. The bacterial load for the strain for which the amoxicillin MIC was 8 μg/ml was not reduced with either dosage. These data demonstrate that regimens which achieved concentrations in plasma above the MIC for at least 34% of a 24-h dosing period resulted in significant reductions in the number of viable bacteria, indicating that the efficacy of amoxicillin-clavulanate can be extended to include efficacy against less susceptible strains of S. pneumoniae by increasing the amoxicillin dose.     

Peripherally acting mu-opioid receptor agonist attenuates neuropathic pain in rats after L5 spinal nerve injury

Studies in experimental models and controlled patient trials indicate that opioids are effective in managing neuropathic pain. However, side effects secondary to their central nervous system actions present barriers to their clinical use. Therefore, we examined whether activation of the peripheral mu-opioid receptors (MORs) could effectively alleviate neuropathic pain in rats after L5 spinal nerve ligation (SNL). Systemic loperamide hydrochloride (0.3–10 mg/kg, s.c.), a peripherally acting MOR-preferring agonist, dose-dependently reversed the mechanical allodynia at day 7 post-SNL. This anti-allodynic effect produced by systemic loperamide (1.5 mg/kg, s.c.) was blocked by systemic pretreatment with either naloxone hydrochloride (10 mg/kg, i.p.) or methyl-naltrexone (5 mg/kg, i.p.), a peripherally acting MOR-preferring antagonist. It was also blocked by ipsilateral intraplantar pretreatment with methyl-naltrexone (43.5 μg/50 μl) and the highly selective MOR antagonist CTAP (5.5 μg/50 μl). However, this anti-allodynic effect of systemic loperamide was not blocked by intraplantar pretreatment with the delta-opioid receptor antagonist naltrindole hydrochloride (45.1 μg/50 μl). The anti-allodynic potency of systemic loperamide varied with time after nerve injury, with similar potency at days 7, 28, and 42 post-SNL, but reduced potency at day 14 post-SNL. Ipsilateral intraplantar injection of loperamide also dose-dependently (10–100 μg/50 μl) reversed mechanical allodynia on day 7 post-SNL. We suggest that loperamide can effectively attenuate neuropathic pain, primarily through activation of peripheral MORs in local tissue. Therefore, peripherally acting MOR agonists may represent a promising therapeutic approach for alleviating neuropathic pain.     

Safety and efficacy of recombinant granulocyte colony-stimulating factor as an adjunctive therapy for Streptococcus pneumoniae meningitis in non-neutropenic adult patients: a pilot study

Twenty-two non-neutropenic adult patients with Streptococcus pneumoniae meningitis received granulocyte-colony stimulating factor (G-CSF) (300-450 Ig/day subcutaneously for 6 days) in addition to cefotaxime plus dexamethasone (9-12 g/day for 10 days and 16 mg/day for 3 days iv, respectively). Patients recovered without evident sequelae in all cases but one (with bilateral hearing deficit). No adverse event was recorded. Improvement of inflammation indices in the cerebrospinal fluid was rapid. The most rapid improvement was seen in glucose concentration, which returned to normal ranges within 24-48 h of treatment. In this study G-CSF administration appeared to be safe and effective; further controlled clinical trials are justified.     

Combination pharmacotherapy with reduced-dose fibrinolytic and platelet GP IIb/IIIa inhibition

Combination fibrinolytic and antiplatelet therapy regimens may provide a means of inducing rapid reperfusion in patients requiring myocardial salvage after an acute myocardial infarction (AMI). This article describes case histories and a therapeutic regimen combining reteplase (5 U + 5 U double bolus) and abciximab (0.25 mg/kg bolus + 0.125 μg/kg/min infusion to a maximum of 10 μg/min for 12 h) for AMI patients before percutaneous coronary intervention (PCI). This medication regimen was used in the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) V clinical trial, for the medical treatment of AMI, resulting in decreased reinfarction rates with similar mortality and intracranial hemorrhage rates as compared to standard fibrinolytic therapy.