[Hydrogelexpander as drug delivery system for antibiotics]. / Hydrogelexpander als Medikamentenabgabe-System für Antibiotika.

PURPOSE: The suitability of a high-hydrophilic osmotic self-inflating hydrogel expander consisting of a co-polymer of N-vinylpyrolidone and methyl methacrylate as a drug delivery system for antibiotics to prevent a postoperative infection was investigated in a laboratory setting. METHODS: The dry expanders were incubated in a 0.3 % solution of Ofloxacin or Tobramycin for 24 hours. The completely swollen expander had increased in volume from 0.3 mL to almost 3 mL (adsorbing 2.7 mL of the 0.3 % solution, i. e.,8.1 mg of Ofloxacin or Tobramycin, respectively). Addressing the elimination of both antibiotics, the concentrations in 15 mL elution medium (simulating the volume of the orbit in a newborn baby) were measured after 0.25, 1, 2, 6, 24, 48 and 72 hours of elution. 0.9 % sodium chloride (B. Braun Melsungen, Germany) was used as elution medium. To imitate fluid exchange due to blood perfusion in the surrounding tissue the medium was renewed after every sampling. For each substance 10 expanders were tested. Concentrations of antibiotic were determined by HPLC/UV for Ofloxacin and by using a specific fluorescence-polarisation immunoassay (Abbott TDx) for Tobramycin. RESULTS: Mean concentrations of Ofloxacin at 0.25, 1, 2, 6, 24, 48 and 72 hours after beginning of the elution were 50.2, 46.8, 41.2, 75.4, 88.2, 46.2 and 19.1 µg/mL, respectively. The cumulative amount of Ofloxacin eluted after 72 hours reached 68 % of the loading dose. The corresponding mean concentrations of Tobramycin were 38.8, 48.5, 40.5, 69.8, 88.7, 119.3 and 71.6 µg/mL. The cumulative eluted amount was 88 %. CONCLUSIONS: The investigated hydrogel expanders soaked in 0.3 % antibiotic solution can store and later on release sufficient amounts of Ofloxacin or Tobramycin to produce antimicrobial effective concentrations in vitro in the surrounding environment. This principle, when used in a clinical setting, might help to eliminate post-implantation infection which is one of the major complications in clinical use.

In vitro kinetics of delivery of tobramycin and ofloxacin from osmotic hydro gel expanders.

OBJECTIVE: High-hydrophilic osmotic self-inflating hydro gel expanders are well-accepted for implantation to achieve tissue expansion in defined parts of the body like skin, breast and orbital soft tissue. To prevent post-implantation infections effective antibiotic prophylaxis might be helpful. The suitability of this hydro gel consisting of a co-polymer of N-vinyl-pyrolidone and methyl-methacrylate as a drug delivery system for antibiotics was investigated in a laboratory setting simulating the orbit in a newborn. METHODS: In a first setting the dry expanders were incubated in a 0.3% solution (5 ml) of tobramycin and ofloxacin for 24 h (n = 10 for each substance, adsorbing 2.4 ml of the 0.3% solution, i.e. 7,200 µg antibiotic). Addressing the release of both antibiotics, the concentrations in 15 ml elution medium (0.9% sodium chloride, renewed after every sampling) were measured after 0.25, 1, 2, 6, 24, 48 and 72 h of elution. To simulate the clinical use in a second setting the expanders were dried after incubation in a 0.3% and 0.03% solution of tobramycin (n = 5 for each concentration) before measuring the release. RESULTS: The cumulative amount of tobramycin released after 72 h reached 7,157 µg, i.e. 99% of the initially loaded antibiotic. The cumulatively released amount of ofloxacin was 5,505 µg (76% of loading dose). Main fraction of release (about two thirds) was detected for both antibiotics for a elution period 0 - 24 h. In the periods 24 - 48 and 48 - 72 h the released amount of tobramycin was significantly higher than for ofloxacin. The release from expander dried after loading tobramycin was comparable: The cumulatively released amount of 0.3% and 0.03% incubation solution was 99% and 79% of loading dose, respectively. CONCLUSIONS: The investigated hydro gel expanders soaked in antibiotic solution can store and further on release sufficient amounts of tobramycin or ofloxacin to produce antimicrobial effective concentrations in vitro in the surrounding environment according to the breakpoints reported by EUCAST [14]. This principle, when used in a clinical setting, might help to eliminate post-implantation infection, which is one of the major complications in clinical use.

An in vitro system for the determination of individualized immunosuppression.

The risk of complications of immunosuppressive treatment in organ transplantation increases with the aggregate amount of immunosuppressive medication given to the patient. As the doses of immunosuppressive agents required to achieve comparable effects show considerable variability, methods to assess individual sensitivity toward immunosuppressive regimens are urgently needed. The aim of this study was to develop such an in vitro test system. As immunological model for allogeneic transplantation, individual pairs of recipient-derived lymphocytes and of donor-derived B lymphocytes mimicking HLA expression of cells in the transplanted organ were isolated and assessed in mixed-lymphocyte cultures (MLC). Alloreactivity was readily observed and MLC consisted of CD8(+) and CD4(+) T cells as well as CD56(+) natural killer cells. A proliferation assay to measure the response of individual MLC on the immunosuppression by cyclosporine (CsA) was developed. The concentrations of CsA leading to growth reductions by 50% (inhibitory concentration 50, IC(50)) were found between 110 and 220 ng/mL, which was near the trough whole blood levels for CsA. Accordingly, the IC(90) values (660 to 1760 ng/mL) were near the target values for peak whole blood levels. We believe that these data present a simple and potentially useful in vitro technology that allows for the prediction of individual responses to immunosuppressive therapeutic regimens.

Pharmacokinetic profiling of cyclosporine microemulsion during the first 3 weeks after simultaneous pancreas-kidney transplantation.

The optimal effect of therapy with cyclosporine (CsA) seeks to minimize undesirable side effects while maximizing immunosuppression. This balance, depends on CsA exposure, which may be characterized by the area under the concentration-time-curve (AUC). Therefore, we tested the pharmacokinetic profile of microemulsion CsA as a superior approach to guide clinical immunosuppression after de novo simultaneous pancreas-kidney transplantations. We examined 10 consecutive pancreas-kidney recipients with type 1 diabetes and end-stage renal disease. All patients were treated with a regimen consisting of CsA, mycophenolate mofetil (MMF), and prednisone. Full (9-point) pharmacokinetic studies (C0, C1, C2, C3, C4, C6, C8, C10, C12) were performed on week 1 and during week 3 to examine CsA pharmacokinetic profiles. Mean AUC0-12 of 4431 +/- 2400 microg x h/L at week 1 remained stable at week 3 (5119 +/- 1190 microg x h/L). The C6 sampling time displayed the best correlation with AUC0-12 (r2 = 0.881), followed by C3 (r2 = 0.758). Our preliminary data after simultaneous pancreas-kidney transplantation support the hypothesis that C3 or C6 sampling is a more accurate predictor of the AUC0-12 than C0. The combination of two samplings, namely C3 + C6 (r2 = 0.938) or C2 + C6 (r2 = 0.955) proved excellent prediction of exposure after simultaneous pancreas-kidney transplantation.

Prediction of in vivo drug interaction from in vitro systems exemplified by interaction between verapamil and cimetidine using human liver microsomes and primary hepatocytes.

Emphasis on drug safety is increasing as newly developed drugs become more potent. Interest in the prediction and description of drug interactions is growing accordingly. The study of potential interactions at a very early stage of drug development requires suitable in vitro models that describe drug interactions both qualitatively and quantitatively. The purpose of the work described here was to help assess the predictive value of in vitro drug interaction tests with liver microsomes and hepatocytes by means of the interaction between verapamil and cimetidine. The in vitro inhibition of verapamil metabolism by cimetidine observed during the studies was quantitatively similar to the results reported in published clinical studies after intravenous application. Studies using liver microsome fractions showed that the intrinsic clearances for the formation of various metabolites could be used to predict drug interactions. In addition, work with hepatocyte cultures revealed that an in vitro system covering both phase I and phase II reactions should be included in such studies to permit quantitative prediction of the various metabolic pathways. Both human hepatocyte cultures and human microsomes offer certain advantages for predicting the degree of drug metabolism and interactions in humans at the biotransformation level. Therefore, it seems likely that the simultaneous application of both systems will yield conclusions that most closely approximate the situation in humans.

Up-regulation of cytochrome P450 1A2, 2C9, and 2E1 in chronic pancreatitis.

The oxidative metabolism of xenobiotics is effected mainly by cytochrome P450 enzymes (CYP), which are expressed as a family of genetically related enzymes primarily in hepatocytes. The pancreas is among the extrahepatic tissues expressing CYP, and it has been suggested that intermediates generated by them might be of pathogenetic significance for diseases of the pancreas such as chronic pancreatitis. We studied 10 surgical resection specimens by immunohistochemistry with polyclonal antibodies against recombinant human CYP 1A1, 1A2, 2C9, 2E1, and 3A and used tissues from 11 normal pancreata as controls. In addition, we assayed microsomal preparations for their capacity to metabolize verapamil. In normal pancreata weak to moderate expression of all enzymes was demonstrated immunohistochemically in up to 50% of duct epithelia, acinar cells, and islet cells. In contrast, in chronic pancreatitis an up-regulation was observed, with immunohistochemical positivity in some cases in up to 100% of duct epithelia and acinar cells. The oxidative capacity of microsomal preparations from chronic pancreatitis was higher than that of preparations obtained from control tissues; compared to liver microsomes, however, it was low. The up-regulation of CYP may have pathogenetic significance for chronic pancreatitis. Yet considering the pancreas' capacity for conjugation reactions, conceivably low levels of reactive intermediates could effectively undergo inactivation.

Expression and function of beta-glucuronidase in pancreatic cancer: potential role in drug targeting.

Improvement of non-surgical strategies is a pivotal task in the treatment of pancreatic cancer. Response to treatment with most anticancer agents has been very poor, probably due to insufficient drug concentration in tumor tissue. Increased response rates during chemotherapy might be achieved by dose escalation; however, this approach is often hampered by severe side effects. One strategy to overcome these adverse effects is application of nontoxic glucuronide prodrugs from which the active moiety is released by beta-glucuronidase within or near the tumor. The use of glucuronide prodrugs in pancreatic cancer requires increased expression of the enzyme in the diseased tissue, a problem that has not been addressed so far. We therefore investigated function and expression of beta-glucuronidase in tissue samples from human healthy pancreas (n=7) and pancreatic adenocarcinoma (n=8), respectively. Comparing the ability of tissue homogenates to cleave the standard substrate 4-methylumbelliferyl-beta-D-glucuronide, we found a significantly increased specific beta-glucuronidase activity (P<0.05) in pancreatic cancer (median: 133; 75% percentile: 286; 25% percentile: 111 nmol/mg per h) as compared to healthy pancreas (median: 74; 75% percentile: 113; 25% percentile: 71 nmol/mg per h). Enzyme kinetic experiments with the model prodrug N-[4-beta-glucuronyl-3-nitrobenzyloxycarbonyl] doxorubicin (HMR 1826) demonstrated bioactivation of HMR 1826 by pancreatic beta-glucuronidase. Enzymatic activity was found to be closely related to enzyme contents (r=0.87) as assessed by Western blot analysis. Our data indicate that increased beta-glucuronidase activity in pancreatic cancer seems to be due to an elevated steady-state level of the protein. This may be the basis for new therapeutic strategies in treatment of pancreatic carcinoma by using glucuronide prodrugs of anticancer agents.

Comparison of cyclosporine absorption profiles over a 12-month period in stable pediatric renal transplant recipients.

Evidence suggests that the pharmacokinetic (PK) profile of microemulsion- cyclosporine A (m-CsA) during the 4-hour absorption phase represents an accurate tool to estimate drug exposure. In addition, several reports suggest a close correlation between selected single CsA concentrations at 1, 2, or 3 hours post-dose (C(1), C(2), and C(3)) and the abbreviated area under the curve (AUC)(0-4) among pediatric renal transplant patients. However, it is still unclear whether these PK correlations remain stable and reliable over 12 months posttransplant. In this study, we obtained 4-hour pharmacokinetic profiles (AUC(0-4)) from stable pediatric renal transplant recipients (phase 1) with repeat measurements 12 months later (phase 2). In addition, we evaluated the optimal single sampling point that correlated with the AUC(0-4) during both phases of the study. Over 1 year there was no significant change in the AUC(0-4) of m-CsA in pediatric renal transplant recipients. The mean dose-normalized AUC(0-4) values changed by less than 2.5%, namely, 557 versus 545 ng x h/mL per unit dose, respectively. The C(1) value was the sampling point that showed the best correlation with AUC(0-4); C(0) displayed the weakest correlation. No changes in cyclosporine dosing or glomerular filtration rate estimates were observed throughout the study period. This study demonstrates the stability of drug measurements during m-CsA therapy.

Rapid HPLC assay for verapamil and its metabolites: use for application to in vitro studies.

An improved method using isocratic reversed phase HPLC is presented for the extraction and rapid determination of verapamil and its main metabolites in microsomal preparations and cell culture media. Possibilities for using the method to estimate cytochrome P450 enzymes in microsomal test systems and hepatocyte cultures are described. The studies show that primary hepatocyte cultures are suitable for studying the metabolism and interactions of pharmaceuticals in vitro and could be superior to microsomal systems in many cases.

Up-regulation of ICAM-1 during cold ischemia triggers early neutrophil infiltration in human pancreas allograft reperfusion.

BACKGROUND: Graft pancreatitis is induced by ischemia/reperfusion injury in which neutrophil infiltration is believed to be a crucial early event. This observation suggests the presence of adhesion molecules already at the time of reperfusion. Therefore, this study was performed to evaluate the pattern of ICAM-1 and P-Selectin expression on human pancreas allografts following cold ischemia and reperfusion. PATIENTS AND METHODS: We performed an analysis of pancreas biopsy specimens taken from 13 patients undergoing pancreas transplantation compared with pancreas specimens from 10 patients following resection. Cryostat sections were stained with monoclonal antibodies against CD11b, a neutrophil marker, and the adhesion molecules ICAM-1 and P-Selectin. RESULTS: Extensive infiltration of CD11b-positive cells was detected in venules and capillaries of pancreas allografts after reperfusion (18.38 +/- 0.87) compared with controls (T1 4.22 +/- 0.55) or with tissue specimens at about 10 hours of cold ischemia (2.60 +/- 0.35; P < .001). Similarly, the pattern of P-Selectin showed a moderate expression before organ harvest (1.54 +/- 0.21) and in samples during cold ischemia (1.46 +/- 0.24) followed by a significantly greater number of P-Selectin-positive cells after reperfusion (2.54 +/- 0.18; P = .005). ICAM-1 was only weakly expressed on the surface of the venular endothelium in all controls (0.77 +/- 0.12). In contrast to P-Selectin, ICAM-1 showed prominent up-regulation during cold ischemia (2.23 +/- 0.23; P < .001) with no further increase after reperfusion (2.23 +/- 0.17). CONCLUSION: The data suggested that ICAM-1 was already up-regulated during cold ischemia, possibly representing the mechanism of early neutrophil infiltration observed in human pancreatic ischemia/reperfusion injury.

Biochemical parameters as a measure of food availability and growth in immature rainbow trout (Oncorhynchus mykiss).

1. Rainbow trout held in brackish water (15 parts per thousand) were starved or fed different amounts of food. 2. A significant correlation was found between the growth rates of the different animals and the feed rates. 3. The RNA:DNA ratio in the white epaxial muscle is lowest in starved fish and increases in proportion to the feed rate and individual specific growth rate. The correlations are significant at the P less than 0.01 level. 4. Liver metabolism varies according to food availability. 5. The protein synthesis capacity of the liver (RNA:DNA ratio) and liver somatic index increase as the feeding rate increases. It also correlates significantly with the specific growth rates of the different animals. 6. The intermediary metabolism of the central metabolic organ, the liver, varies in the same way. 7. The activities of the NADPH producing liver enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (IDH) and malic enzyme (ME) increase as the feed rate (and therefore the specific growth rate) increases. 8. G6PDH and IDH activity in the kidney is influenced to a much lower degree by food intake. 9. Summarizing, it can be stated that biochemical parameters can be used to describe comprehensively the metabolic status and growth of rainbow trout.

Effects of temperature, food deprivation and salinity on growth, RNA/DNA ratio and certain enzyme activities in rainbow trout (Salmo gairdneri Richardson).

1. The connection between feeding regime (food deprivation and restricted diet) and thermal acclimation (1-2, 6, 11 and 16 degrees C) was studied in rainbow trout held in diluted seawater (20% S). 2. At 1 degree C, food deprivation effects on all parameters are slight, and on RNA and certain enzymes they are masked by thermal acclimation effects. 3. At a salinity of 20% rainbow trout on a restricted diet and held at 11 degrees C have the highest growth rate. 4. Owing to increasing RNA levels, the RNA/DNA quotient is significantly higher than normal in rainbow trout held at 1 degree C although the fishes do not grow at this temperature. 5. Temperature and feeding both affect the enzymes we studied (liver: G1DH, AspT, arginase, G6PDH, and 6PGDH; kidney: G1DH, AspT, arginase, and Na/K-ATPase; white muscle: AspT and A1T; gill: Na/K-ATPase) differently. Interactions between these two factors also occur in some cases.

Influence of nutrition on biochemical sea water adaptation of the rainbow trout (Salmo gairdneri richardson).

Glutamate dehydrogenase activity in the liver of the rainbow trout increases when the animals are starved for four weeks. Glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase activity in the kidney of rainbow trout kept in sea water (20% S) is significantly higher than in the kidney of rainbow trout kept in fresh water. Gill Na/K-ATPase activity in the rainbow trout is reduced significantly (44%) by starvation for four weeks. Most of the free amino acids investigated in the white muscle of the rainbow trout were present in significantly higher concentrations in animals fed in sea water than in animals fed in fresh water. The concentrations of these amino acids are even higher in the muscle of starved animals held in sea water than in fed animals held in sea water.

Comparison of several approaches of therapeutic drug monitoring of cyclosporin A based on individual pharmacokinetics.

OBJECTIVE: The clinical outcome of patients after organ transplantation is correlated with cyclosporin A (CyA) exposure. It is generally accepted that the area under the concentration-time curve (AUC) provides a reliable means for drug exposure. However, in routine therapeutic drug monitoring (TDM) of CyA, trough levels are mostly used. Currently, a number of different new concepts of CyA-TDM, including approaches such as single, double or triple time-point and abbreviated AUC determinations, have been introduced. The purpose of this study was to compare the predictive value of the different strategies of TDM. METHODS: Calculations were based on 40 individual concentration time profiles after oral administration of CyA to patients who had been included into an ongoing prospective clinical trial. Non-compartmental analysis was used to calculate the AUC0-12h. Multiple linear regression was performed to describe the relationship between the different sets of blood concentrations and the respective AUC0-12h as well as to evaluate their predictive value regarding AUC. Predictive performance was assessed by prediction bias and prediction precision, which were estimated as the mean prediction error and root mean squared error, respectively. RESULTS: When comparing the various combinations of time points, it was found that one-point approaches showed the strongest differences with regard to the predictive value; the associated r2 values differed from 0.203 to 0.792. The two and three time-point approaches showed lower differences - r2 0.802-0.972. The four-point and five-point approaches (r2 0.942-0.982) were the strongest predictors for CyA AUC0-12h. Relative bias ranged from -27.7% to 63.8% and changed significantly when multiple-point predictors were used. In those cases, the predictive performance improved. Considering the predictive performance as well as the smallest bias and highest prediction precision, C3, C1 + C3, C1 + C3 + C6 and C1 + C2 + C3 + C6 were the best predictors. CONCLUSION: The results of this study indicate that in kidney transplant patients a clinically sufficient precise estimation of the CyA AUC is possible using two or three concentration time points.