Assessment of the ability of the Privigen® purification process to deplete thrombogenic factor XIa from plasma.

BACKGROUND AND OBJECTIVES: Activated clotting factor FXI (FXIa) has been postulated to play a significant role in thromboembolic events potentially associated with the administration of intravenous immunoglobulin. The purpose of this study was to demonstrate that thrombogenic agents, in particular FXIa and FXI, are depleted or inactivated in Privigen(®) . MATERIALS AND METHODS: The ability of the purification process to deplete FXIa from plasma was studied. All steps of the Privigen(®) production were investigated for potential activation of FXI to FXIa with spiking experiments. RESULTS: Privigen(®) contains no procoagulant activity as determined by FXIa chromogenic assay, non-activated partial thromboplastin time (NaPTT) and thrombin generation assays (TGA, FXIa-like activity). The coagulation times were >200 s in the NaPTT test. FXIa was below the detection limit of 0·14 ng/ml (chromogenic assay) and below the quantification limit of 0·2 ng/ml (TGA). FXIa spiking experiments showed that the analytical methods used can detect traces of procoagulant activity in immunoglobulin samples. FXIa spiking and kinetic experiments during the octanoic acid fractionation step showed that a substantial reduction in FXIa specific activity (by ≥99·9% within 40 min of octanoic acid incubation) was reached already at an early stage of the manufacturing process. These results were confirmed in vivo: in a modified Wessler test, no thrombus was reported. CONCLUSION: The Privigen(®) manufacturing process has the capability to remove thrombogenic factors: octanoic acid precipitation, designed to remove a variety of contaminants during immunoglobulin purification, also removes almost all FXIa from plasma and further purification steps do not activate FXI.

Endothelial cell compatibility of azithromycin and erythromycin.

Phlebitis is a severe local adverse event related to the use of parenteral macrolides. In order to evaluate the effect of azithromycin and erythromycin on human venous endothelial cells, we set up an in vitro model. The intracellular levels of purine nucleotides, as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and guanosine 5'-triphosphate (GTP), were measured by means of high-performance liquid chromatography. Incubation of cells with 2 mg/mL azithromycin and erythromycin resulted in a rapid decline of intracellular ATP from 12.5 +/- 0.9 nmol/million cells to 4.1 +/- 0.3 and 2.6 +/- 0.4 nmol/million cells, respectively, after 60 min. In addition, ADP was extensively depleted from 2.1 +/- 0.17 nmol/million cells to 0.8 +/- 0.09 and 0.8 +/- 0.13 nmol/million cells after 60 min. After exposure of 0.5 mg/mL azithromycin and erythromycin, no significant decline of intracellular high-energy phosphate levels occurred after 20 and 60 min. Based on these results, solutions of azithromycin and erythromycin may not be well tolerated and may cause local adverse reactions even if diluted according to the manufacturer's recommendation.

Impact of Mitomycin-C application time on the scleral Mitomycin-C concentration.

The aim of this study was to determine the effect of varying the application time of Mitomycin-C (MMC) on the scleral concentration of MMC. The sclerae of 14 human donor eyes were used for this study. The episcleral sides of the 4 scleral quadrants of each donor eye were exposed for 0.5, 1, 3 and 5 min to round, 8 mm-diameter sponges soaked with 50 microl of 0.2 mg/ml MMC. After 40-ml irrigation with saline, a central 8-mm diameter scleral disk was punched out, homogenized and analyzed with high performance liquid chromatography (HPLC). The scleral MMC concentrations (microg/g) after 0.5, 1, 3 and 5 min application times were 6.40 (+/-3.38), 9.02 (+/-2.40), 12.31 (+/-3.37), and 13.97 (+/-3.83). The differences of scleral MMC concentration in paired t-tests were statistically significant comparing 0.5 with 1 and 1 with 5 min application. However the effect was relatively small within the range of usual application times (1 to 5 min), and 64% of the MMC was delivered to the sclera within the first min.

Intracellular concentration of the HIV protease inhibitors indinavir and saquinavir in human endothelial cells.

Human vascular endothelial cells may serve as targets and a reservoir for human immunodeficiency virus type 1 (HIV-1). The antiviral activity of HIV protease inhibitors is reported to be related directly to the intracellular amount of the drug. To assess intracellular concentrations of two HIV protease inhibitors, human umbilical venous endothelial cells (HUVECs) were exposed for 3 h and 24 h to 100, 10 and 1 mg/L indinavir and saquinavir. Intracellular drug concentrations and the total drug amount in the supernatant were measured by means of high-performance liquid chromatography (HPLC). Exposure of HUVECs to 10 and 1 mg/L indinavir and saquinavir resulted in undetectable intracellular drug levels in 6 x 10(5) cells/well. Incubation of cells with solutions of 100 mg/L indinavir and saquinavir led to mean intracellular concentrations of indinavir (132 +/- 56 mg/L after 3 h and 150 +/- 29 mg/L after 24 h, respectively) and of saquinavir (96 +/- 10 mg/L after 3 h and 100 +/- 5 mg/L after 24 h) that were comparable to the levels determined for the substances in the supernatant over time (P > 0.001). These data indicate that intracellular concentrations of indinavir and saquinavir correlate well with the extracellular levels. Consequently, measurements of drug concentrations in patient's plasma by HPLC are assumed to be a good means of monitoring the intracellular drug concentration.

In vitro diffusion of mitomycin-C into human sclera after episcleral application: impact of diffusion time.

The purpose of this study was to investigate the impact of different diffusion times of mitomycin-C (MMC) on the intrascleral concentration vs depth profile of MMC in an experimental model. Scleral quadrants of eight human donor eyes were exposed to sponges soaked with MMC for an application time of 1 min. After irrigation with 40 ml saline, we allowed further diffusion of MMC in the sclera for 1, 5, 14 and 29 min until the specimens were further processed. A central 8 mm diameter scleral disk was horizontally dissected with a kryotome at -20 degrees C. MMC concentrations of six layers of 140 microm thickness were analysed by means of high-performance liquid chromatography. The MMC concentrations (microg g(-1)) of layer 1 were: 13.45+/- 5.9 (mean +/- S.D. at 2 min diffusion time), 7.6+/-2.5 (6 min diffusion), 5.6+/-3.1 (15 min diffusion) and 3.6+/-1.7 (30 min diffusion). The corresponding MMC concentrations of layer 6 were: 0.61+/-0.48, 1.47 +/-0.66, 1.83+/-0.42 and 2.98+/-0.97 microg g(-1). The superficial concentration of intrascleral MMC decreased with increasing diffusion time, the deep concentrations increased. After 30 min of diffusion time, equal concentrations of MMC were found in all layers. Even with current low-dose application regimens of MMC the concentrations in the inner side of the sclera rapidly increase beyond the limits of the therapeutic range. Owing to this fast diffusion of MMC, the only means of reducing ciliary body concentrations of MMC is to reduce the dose.

Identification of yeast species by fatty acid profiling as measured by gas-liquid chromatography.

An improved rapid method for the identification of yeasts and yeast-like fungi from clinical sources which is based on fatty acid profiles obtained by gas-liquid chromatography (GLC) is described. The fatty acid profile database is based upon internal standardisation and using the relative retention times and the retention index of the analysed fatty acids. Differentiation between yeast species was achieved by the quantitative and qualitative comparison of measured fatty acid profiles with those in the database. A total of 1024 clinical isolates were analysed by GLC to test the validity of the database. 96.2% of all tested samples were identified correctly to the species level by the improved GLC method.

Intrascleral concentration vs depth profile of mitomycin-C after episcleral application: impact of applied concentration and volume of mitomycin-C solution.

The purpose of this study was to investigate the impact of different concentrations and volumes of Mitomycin-C (MMC) on the intrascleral concentration vs depth profile of MMC in an experimental model. The episcleral sides of scleral quadrants of human donor eyes were exposed for 1 min to sponges (corneal light shield, Merocel Corp.) soaked with MMC. After irrigation with 40 ml saline a central 8 mm diameter scleral disk was horizontally dissected with a cryotome. MMC concentrations of six layers of 140 microns thickness were analysed by means of high-performance liquid chromatography. In Experiment 1 (11 eyes) the sponges were soaked with 50 microliters of 10, 100 and 200 micrograms ml-1 MMC solutions. In Experiment 2 (12 eyes) the sponges were soaked with 10, 30, 50 and 80 microliters of a 200 micrograms ml-1 isotonic MMC solution. In Experiment 1 the MMC concentrations (microgram g-1) of layer 1 were 0.35 (+/- 0.20; 10 micrograms ml-1 group) and 9.22 (+/- 2.92; 200 micrograms ml-1 group). In Experiment 2 the MMC concentrations were 2.57 (+/- 1.17; 10 microliters group), 7.35 (+/- 2.49; 30 microliters group) and 11.67 (+/- 3.25; 80 microliters group). The scleral MMC concentrations were significantly influenced by the applied concentrations (layers 1-5) and by the applied volumes (all layers) of MMC solution. The intrascleral MMC concentration increased linearly with increasing concentration and not linearly with increasing volume of the applied MMC solution. To achieve more predictable scleral concentrations of MMC after trabeculectomy with MMC it seems advisable to control both the concentration and the volume of the MMC solution used to soak the sponge.

Pharmacokinetics of cefpirome during continuous venovenous hemofiltration: rationale for an 8-hour dosing interval.

OBJECTIVE: Cefpirome is a new semisynthetic cephalosporin, primarily eliminated by the kidneys, that requires dosage adjustment in patients with kidney failure. The optimal dosing regimen of cefpirome in patients with continuous veno-venous hemofiltration (CVVH) is unknown. METHODS: Pharmacokinetic properties of cefpirome were investigated in eight anuric patients with acute kidney failure treated by CVVH. All patients received a dosage of 2 g cefpirome every 8 hours after starting the hemofiltration with high-flux polysulfone membranes. Concentrations of cefpirome in plasma and ultrafiltrate were measured by HPLC. RESULTS: Total clearance and hemofiltration clearance of cefpirome were 589.1 +/- 164.5 mL/min and 43.3 +/- 7.8 mL/min, respectively. Serum elimination half-life was 2.36 +/- 0.59 hours. The highest plasma drug concentration was 14.8 +/- 3.2 microg/mL, and it declined to trough levels of 3.1 +/- 0.8 microg/mL at the end of the dosing interval. CONCLUSION: On the basis of previously published pharmacodynamic characteristics of cefpirome and the pharmacokinetic parameters obtained in this study, we calculated a required total daily dose of 2 g every 8 hours to achieve sufficient plasma antibiotic levels to cover the majority of target pathogens. However, this dosage may be insufficient during CVVH for intermediate resistant strains of Pseudomonas aeruginosa.

Serum concentrations of granulocyte-colony stimulating factor in complicated Plasmodium falciparum malaria.

Involvement of neutrophils in the control of blood parasites in malaria has been reported. Both, mononuclear phagocytes and neutrophils are known to be stimulated by cytokines such as TNF-alpha in order to augment the defence potency against the parasites. Previously, it has been shown that serum-G-CSF concentrations are increased in patients with bacterial sepsis. In vitro studies have shown that P. falciparum - infected erythrocytes induce the release of G-CSF by several cells such as endothelial cells and monocytes, however, nothing is known about G-CSF serum concentrations during the clinical course of severe P. falciparum malaria. Thus, it was the aim of the present study to investigate the time course for G-CSF serum concentrations in patients with complicated P. falciparum malaria, and to correlate these values with other mediators of inflammation and hematopoesis. Twenty-six patients suffering from complicated P. falciparum malaria were included in the study, and 20, age and sex matched, healthy volunteers were used as the negative control group. Serum samples for determination of G-CSF were taken on day 0, 7 and 14, and measured by ELISA. We found significantly increased serum concentrations of G-CSF in patients with complicated P. falciparum malaria on day 0, values decreasing to within the normal range by day 7. A significant correlation was found between G-CSF (d0) and procalcitonin, the parasite count, erythropoietin and macrophage inflammatory protein, however no correlation could be shown for the neutrophil count. In conclusion, on the day of hospital admission, elevated serum concentrations of G-CSF were detected in patients with complicated P. falciparum malaria, which might indicate a role of G-CSF in the acute defence mechanism against the parasites.

Intrascleral concentration vs depth profile of mitomycin-C after episcleral application: impact of irrigation.

Mitomycin-C has been reported to cause toxic effects on the ciliary body after episcleral application during glaucoma surgery. We investigated the intrascleral diffusion of mitomycin-C in an experimental model. The episcleral sides of scleral quadrants of 14 human donor eyes were exposed for 5 min to sponges (corneal light shield, Merocel corp., Mystic, CT, U.S.A.) soaked with 200 microg ml(-1)mitomycin-C. After the exposure one of four quadrants was not irrigated and the episcleral sides of three quadrants were irrigated with 40, 100 and 200 ml saline. A 9 mm scleral disk was punched out with a trephine and frozen on a kryotome plate 2 min after the end of mitomycin-C exposure. An 8 mm diameter scleral disk was then cut with a trephine, again frozen on a kryotome plate and then horizontally dissected with a kryotome. For analysis purposes seven cuts of 20 microm thickness were combined to one layer of 140 microm. Six layers could be reproduced and were analysed. The mitomycin-C concentrations of these layers were analysed by high-performance liquid chromatography. A concentration vs depth profile was calculated for each group, and the half-width of concentration was calculated by log-linear regression. The mitomycin-C concentration of layer 1 was 24.51 microg g(-1)(+/-7.52) without irrigation, 13.15 microg g(-1)(+/-4.38) after 40 ml irrigation, 10.29 (+/-3.53) after 100 ml irrigation and 8.4 microg g(-1)(+/-1.62) after 200 ml irrigation. In layers 1-3 the concentration of mitomycin-C was significantly reduced by irrigation (ANOVA). In the deeper intrascleral layers irrigation had no effect on the mitomycin-C concentrations. Between layers 2 and 6 the half-width of the mitomycin-C concentration was 101 microm (no-irrigation group), 141 microm (40 ml irrigation group), 153 microm (100 ml irrigation group), and 164 microm (200 ml irrigation group). Irrigation reduced the mitomycin-C concentration only down to half of the scleral thickness, leaving the deep intrascleral concentrations unchanged.

Homocysteine and laminin are not prognostic markers in patients with septic inflammatory response syndrome.

The aim of this study was to measure plasma homocysteine and laminin concentrations in patients with nonbacteremic systemic inflammatory response syndrome (SIRS) and to compare them with those of a healthy control group. Concerning laminin, significant increased concentrations could be observed in the SIRS group compared to the control group, but for homocysteine, no significance could be observed. In summary, homocysteine and laminin levels are not useful in the prediction of a patient's outcome.

High serum laminin concentrations in patients with Candida sepsis.

BACKGROUND: Laminin, a major component of the basement membrane, plays a critical role in normal cell adhesion and also during tissue invasion of pathogenic microorganisms. MATERIALS AND METHODS: Serum laminin concentrations were determined in 19 patients with Candida albicans sepsis, in 13 patients with bacterial sepsis and in 20 noninfectious controls. RESULTS: Serum laminin concentrations of both, patients with candidal and bacterial sepsis, were significantly elevated compared to the controls (486 ng mL-1 [155-924], median [range]; P < 0.01). Laminin concentrations were significantly higher in patients with Candida sepsis than in patients with bacterial sepsis on day 1 (2565 ng mL-1 [659-6064] vs. 994 ng mL-1 [386-2064]; P < 0.01), day 7 (1594 ng mL-1 [607-4611] vs. 684 ng mL-1 [284-1920]; P < 0.05) and day 14 (1444 ng mL-1 [202-2131] vs. 386 ng mL-1 [180-1658]; P < 0.05). CONCLUSIONS: Laminin serum concentrations might be useful to differentiate nonbacterial, bacterial and fungal etiology.

Penetration and decay of meropenem into the human aqueous humor and vitreous.

The aim of this study was to determine the penetration of intravenously administered meropenem into the human aqueous humor and vitreous. Thirty patients about to undergo cataract surgery and fourteen patients about to undergo vitrectomy received a 2 g dose of meropenem before surgery. Specimens of aqueous humor or vitreous and blood were obtained intraoperatively and analyzed by high performance liquid chromatography (HPLC). The study was designed as a non-randomized prospective trial. Thirty min to 12 hr after administration, mean aqueous humor levels of 13.4 and 1.1 mg/l and vitreous levels between 8.94 and 1.08 mg/l were found, respectively. The peak concentrations are distinctly above the in vitro measured minimum inhibitory concentration of meropenem for 90% (MIC90) of almost all relevant gram-positive and gram-negative organisms, including Pseudomonas aeruginosa and Enterobacteriaceae. With regard to its broad spectrum, high antibacterial activity, and good penetration into ocular fluids, meropenem seems to be an alternative to currently used systemic drugs. Its usefulness in perioperative prophylaxis, as initial therapy after perforating or penetrating injuries, or in the therapy of bacterial endophthalmitis has yet to be proved.

Serum nitrate concentrations in patients with peripheral arterial occlusive disease.

BACKGROUND: Nitric oxide (NO), an endogenous product of L-arginine oxidation, seems to account for the vasodilatatory effect of the endothelium-derived relaxing factor. It was the aim of the present study to measure serum nitrate concentrations, the degradation product of nitric oxide in patients with peripheral arterial occlusive disease (PAOD). PATIENTS AND METHODS: 20 patients with PAOD in Fontaine stage IIb, 10 patients in stage III and IV respectively were included in the study. Serum samples for determination of nitrate were taken at admission after fasting overnight. Nitrate concentrations were determined using a recently developed high performance liquid chromatography which allows direct measurement of nitrate. The control group comprised 14 age and risk factor matched volunteers. RESULTS: We found significantly increased nitrate concentrations in patients with PAOD compared to the control group [stage IIb: 6.65 +/- 1.58 mumol/l; stage III: 6.94 +/- 1.85 mumol/l, stage IV: 7.05 +/- 1.16 mumol/l; control: 4.41 +/- 1.24 mumol/l], however no significance was calculated within the different PAOD groups. There was no association of either diabetes mellitus, hypertension and smoking behaviour with increased nitrate levels. CONCLUSION: These data might indicate that NO might be involved in adaptive vasodilatation already in the early phase of the disease. The source of nitrate in PAOD patients, however, remains unclear.

Cytokines in sepsis due to Candida albicans and in bacterial sepsis.

Cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and tumor necrosis factor-soluble receptor (TNF-sR), and adhesion molecules, e.g. vascular adhesion molecule-1 (VCAM-1) and E-selectin, play an important role in the pathogenesis of bacterial sepsis. Experimental data on cytokine expression during candidaemia are controversial. In this study, plasma concentrations of cytokines and adhesion molecules were compared between patients with sepsis due to Candida albicans and bacterial sepsis. Plasma levels of TNF-alpha, TNF-sR, IL-6, VCAM-1 and E-selectin, were determined in 20 patients with sepsis due to C. albicans, in 20 patients with bacterial sepsis, and in 20 controls on days 1, 7 and 14. On day 1, elevated plasma levels of TNF-alpha, TNF-sR and IL-6 were detected in both sepsis groups compared to controls. On day 1, VCAM-1 levels were higher, and E-selectin levels were lower in patients with Candida sepsis than in patients with bacterial sepsis (p < 0.05). At any time, VCAM-1 levels were significantly greater in patients with Candida sepsis than in patients with bacterial sepsis (p < 0.05). Non-survivors, regardless of the etiology of sepsis, had higher blood levels of IL-6, TNF-sR and E-selectin than survivors. The cytokines, TNF-alpha, IL-6 and TNF-sR, and the adhesion molecules, VCAM-1 and E-selectin, are involved in sepsis due to C. albicans as in bacterial sepsis.

Continuous infusion versus intermittent administration of meropenem in critically ill patients.

This prospective crossover study compared the pharmacokinetics of meropenem by continuous infusion and by intermittent administration in critically ill patients. Fifteen patients were randomized to receive meropenem either as a 2 g iv loading dose, followed by a 3 g continuous infusion (CI) over 24 h, or by intermittent administration (IA) of 2 g iv every 8 h (q8h). Each regimen was followed for a period of 2 days, succeeded by crossover to the alternative regimen for the same period. Pharmacokinetic parameters (mean +/- SD) of CI included the following: concentration at steady state (Css) was 11.9+/-5.0 mg/L; area under the curve (AUC) was 117.5+/-12.9 mg/L x h. The maximum and minimum serum concentrations of meropenem (Cmax, Cmin) and total meropenem clearance (CItot) for IA were 110.1+/-6.9 mg/L, 8.5+/-1.0 mg/L and 9.4+/-1.2 L/h, respectively. The AUC during the IA regimen was larger than the AUC during CI (P < 0.001). In both treatment groups, meropenem serum concentrations remained above the MICs for the most common bacterial pathogens. We conclude that CI of meropenem is equivalent to the IA regimen and is therefore suitable for treating critically ill patients. Further studies are necessary to compare the clinical effects of CI and IA in this patient group.

A simple HPLC method for monitoring mycophenolic acid and its glucuronidated metabolite in transplant recipients.

Mycophenolic acid (MPA) is nowadays in broad clinical use as a substitute for azathioprine. An immunoassay for MPA recently received approval for clinical applications. The high performance liquid chromatography (HPLC) assay for measuring MPA and its glucuronide conjugate (MPAG) we describe here is not only rapid and simple but also extremely sensitive at plasma levels obtained during standard immunosuppressive regimens. The determination of MPAG is possible without any change of the chromatographic conditions (detection wavelength of 214 nm, mobile phase: acetonitrile and 50 mmol/l o-phosphoric acid (50:50, V/V), run time: 15 min). The required equipment is a standard HPLC system including a simple UV-detector. Sample volume of 400 microl is required for both determinations. Detection limit is 0.25 micromol/l for MPA and 5 micromol/l for MPAG. Linearity is excellent for serial dilutions (0.5-25 micromol/l for MPA, 25-500 micromol/l for MPAG) and high accuracies favour the method described. More than 2000 plasma samples tested for MPA in patients after heart transplantation within one year and more than 500 samples for MPAG underline the clinical applicability of this assay.

Short report: total serum levels of the nitric oxide derivatives nitrite/nitrate during microfilarial clearance in human filarial disease.

Nitric oxide (NO) has recently been shown to be cytotoxic to both microfilariae and adult Brugia malayi in vitro and in a murine model, as well against Onchocerca lienalis microfilariae in vitro. We studied the kinetics of nitrite/nitrate, both stable end products of NO, by high-pressure liquid chromatography during microfilaricidal chemotherapy in four filariasis (three Loa loa, and one Onchocerca volvulus) patients. High serum levels of nitrite/nitrate were released during microfilarial clearance and sustained elevated levels were observed six months after chemotherapy, suggesting a role of NO in the elimination of microfilariae in human filariasis.

Ofloxacin clearance during hemodialysis: a comparison of polysulfone and cellulose acetate hemodialyzers.

The pharmacokinetics of ofloxacin were studied in 13 patients with end-stage renal disease during hemodialysis using two different dialyzers: a polysulfone membrane (Fresenius F6) and a cellulose acetate dialyzer (Nissho Nipro FB-150T). All patients received 100 mg ofloxacin orally per day before dialysis. The hemodialysis clearance per square meter surface area was significantly different, with 5.0+/-0.7 L/h and 3.7+/-1.6 L/h, respectively. The serum concentration was reduced by a 3-hour hemodialysis by 49.6%+/-5.8% per square meter surface area and 45.5%+/-4.8% per square meter surface area. The half-life was 4.2+/-1.8 hours and 4.8+/-1.6 hours during the hemodialysis period and 22.8+/-2.2 hours and 23.3+/-1.7 hours between the dialysis sessions, respectively. Comparing polysulfone and cellulose acetate dialyzers, the material of the membrane influences the half-life, the dialysis clearance, and the percentage of drug extracted during hemodialysis. We conclude that the type of dialyzer used has to be taken into account in dosage recommendations for antimicrobial therapy in hemodialysis patients.