[Research ethics: French regulations and applications in radiation oncology]. / Éthique de la recherche : réglementations françaises et applications en oncologie radiothérapie.

French regulations about research ethics are based on the so-called Jardé law, which defines researches involving human beings. Researches involving human beings require the submission of research protocols to a committee for protection of persons with a precise list of documents to submit for a favourable opinion. This law describes different categories of researches and determines the ethical procedures to apply before setting up a research protocol. This issue of categorisation is central and must be taken into account by researchers from the beginning of the research process. Researches considered as not involving human beings also require a set of ethical precautions focused on patients' information and the collection of their non-opposition (due to the application of the General Data Protection Regulation adopted by the European Parliament). Thus, many regulations exist and they require a real work for researchers to meet these requirements in research ethics. This article aims to summarise French regulations. Selected examples are specifically taken into the field of radiation oncology research.

Absence of placental transfer of pentasaccharide (Fondaparinux, Arixtra) in the dually perfused human cotyledon in vitro.

The synthetic pentasaccharide, fondaparinux, is the first of a new antithrombotic class: selective factor Xa inhibitors. Comparative clinical trials of fondaparinux versus heparins in prevention and treatment of venous thromboembolism are ongoing. Little is known about fondaparinux during pregnancy, as women of child-bearing potential were excluded from clinical trials. No particular safety issue, for either mother or fetus, has been reported for heparins. The objective of this study was to compare in vitro the steady state placental transfer of fondaparinux and enoxaparin at the plasma concentrations reached during acute treatment of venous thromboembolism (1.75 microg/mL and 1 anti-Xa IU/mL respectively), using antipyrine (20 mg/L) as reference. No biological activity was detectable in the fetal venous effluent during perfusion of enoxaparin-antipyrine, fondaparinux-antipyrine or control media. Furthermore, fetal venous samples did not differ significantly from fetal arterial samples. This apparent absence of placental transfer supports further evaluation of fondaparinux in pregnant women.

Diffusion of ofloxacin into human lung tissue.

Ofloxacin 200mg twice daily was administered to 17 patients with pulmonary disorders, which necessitated surgery, during the preceding 48 hours and 200mg was administered 1 hour before the operation. During surgery, blood samples and specimens of healthy and diseased lung tissues were taken simultaneously. Ofloxacin levels were determined by HPLC. The mean values of the tissue concentration/plasma concentration ratio were 3.5 +/- 0.4 for the healthy tissue and 3.9 +/- 0.4 for the diseased tissue. These values reflected good penetration of ofloxacin into both healthy and atelectasic pulmonary parenchyma.

Placental transfer of SR49059 in the human dually perfused cotyledon in vitro.

SR49059 is an antagonist of vasopressin V(1a)receptors currently developed as a tocolytic drug. We investigated the transplacental transfer of SR49059 in vitro using the single pass dually perfused human cotyledon model. Thirteen placentae were collected from normal term pregnancies immediately after delivery. The placental transfer of SR49059 was tested at steady state at three different concentrations (100 ng/ml, 200 ng/ml and 500 ng/ml) along with that of antipyrine 20 mg/l as a reference substance. Concentrations were assayed by liquid chromatography with UV (antipyrine) or mass spectrometry (SR49059) detection. At steady state, the mean+/-s.d. fetal transfer rate of SR49059 was 10.80+/-4.33 per cent, 9.34+/-4.41 per cent, and 11.78+/-3.26 per cent at 100 ng/ml, 200 ng/ml and 500 ng/ml, respectively. The corresponding clearance indices were 0.29+/-0.14, 0.25+/-0.08, and 0.31+/-0.06, respectively. The absence of saturation kinetics is consistent with a passive mechanism of transfer. Moderate amounts of SR49059 are transferred from the maternal to the fetal circulation. The clearance index of SR49059 appeared to be very similar to that of ritodrine, which is currently used as a tocolytic.

Comparative cost of allogeneic or autologous bone marrow transplantation and chemotherapy in patients with acute myeloid leukaemia in first remission.

We compared the direct costs of allogeneic bone marrow transplantation (Allo-BMT), autologous bone marrow transplantation (Auto-BMT) and chemotherapy (Chemo) in 40 adult patients under the age of 55 years with acute myeloid leukaemia (AML) in first complete remission (CR). Fourteen patients were treated with Allo-BMT, 11 with Auto-BMT and 15 with Chemo. These patients, who were part of two cooperative consecutive trials enrolling a total of 196 patients (BGM 84 and BGMT 87 studies), were those who were treated in the CHR Bordeaux and achieved CR after induction chemotherapy. Cost accounting extended over a maximum period of 5 years. The average cost of the procedure for a mean follow-up of 2 years and excluding the cost of treatment after relapse which eventually occurred was significantly higher for the Allo-BMT group (FF 392,724) and the Auto-BMT group (FF 393,461) than for the group that received Chemo (FF 128,947) (p < 0.00001). The average cost for the treatment of relapse was estimated at FF 266,436, irrespective of the previous treatment. The total cost, including the estimated cost of relapse according to its probability of occurring calculated from the original population, was significantly higher for the Allo-BMT group (FF 424,696; p < 0.01) and the Auto-BMT group (FF 505,364; p < 0.0001) than for the Chemo group (FF 304,846).(ABSTRACT TRUNCATED AT 250 WORDS)

A randomized study comparing blood glucose control and risk of severe hypoglycemia achieved by non-programmable versus programmable external insulin pumps.

OBJECTIVE: To compare a non-programmable and a programmable insulin external pump using regular insulin on glycemic stability, the risk of severe hypoglycemia and metabolic control in type 1 diabetic patients. MATERIAL AND METHODS: Ten type 1 diabetic patients were involved in a randomized, crossover study comparing two periods of 3 months with continuous subcutaneous insulin infusion (CSII) either with a non-programmable insulin pump or a programmable insulin pump. Comparisons were made among mean blood glucose values before and after meals, at bedtime and at 2: 00 a.m.; the risk of severe hypoglycemia assessed by the low blood glucose index (LBGI); and HbA1c. RESULTS: Mean average blood glucose (BG) measurements were significantly lower with the programmable in comparison with the non-programmable insulin pump (respectively 157+/-78 vs. 165+/-79, p=0.034). While postprandial values for BG were not different between the two pumps, the use of the programmable pump resulted in a significant decrease in mean preprandial BG levels (140+/-68 vs. 150+/-73 mg/dl p=0.039). Conversely mean BG level was lower at 2 a.m. with the non-prgrammable pump (125+/-81 vs. 134 +/-93 mg/dl, p=0.02) but with a higher incidence of hypoglycemia. Mean LBGI was comparable with the two pumps (3.1+/-8.6 vs. 2.8+/-6.9, p=0.1). There was a 0.2% decrease in HbA1c during the programmable pump period that did not reach statistical significance (p=0.37). CONCLUSIONS: The present study suggests that programmable external insulin pumps, although more complex and more expensive than non-programmable insulin pumps, significantly reduce fasting glycemia during the day without increasing the risk of severe hypoglycemia and are safer during the night.

Passage of S-(+)- and R-(-)-ketoprofen across the human isolated perfused placenta.

Ketoprofen is a chiral non-steroidal anti-inflammatory drug (NSAID) available as a racemic (rac) mixture of S-(+)- and R-(-)-isomers. Its inhibitory effect on prostaglandin biosynthesis resides virtually in the S-form. Interestingly, R-ketoprofen does not undergo substantial metabolic inversion in humans. Though contraindicated during the last trimester of pregnancy, NSAIDs, including ketoprofen, are used as tocolytic agents in some cases. The S/R plasma concentration ratio was reported to average 2.3 in premature neonates whose mothers were given rac-ketoprofen and to be close to 1 in the maternal plasma. Thus, we investigated the placental transfer of rac-ketoprofen in vitro using Schneider's perfused human cotyledon model. Glucosed Earle solutions with and without human serum albumin (HSA) were used. Several maternal perfusates were tested with different rac-ketoprofen concentrations together with 20 mg L-1 of antipyrine as a reference substance. Ketoprofen enantiomers were assayed by a specific HPLC method with derivatization procedure. HSA concentrations in maternal perfusate influenced the placental transfer of ketoprofen enantiomers. In the absence of HSA in the maternal perfusate, the S-(+)/R-(-) concentration ratio was close to 1 in the fetal perfusate. By contrast, this ratio averaged 1.44 after addition of HSA 10 g L-1 on the maternal side. Similar results were found for dialysis experiments using an inert Spectrapor 2 membrane suggesting that the S-(+)-free concentration is superior to the R-(-)-free concentration in the presence of HSA. Direct measurements of the free concentrations by centrifugal ultrafiltration confirmed this hypothesis. Accordingly, the data observed in vivo may result, at least in part, from the stereoselective protein binding of ketoprofen.

Prediction of methotrexate elimination after high dose infusion in children with acute lymphoblastic leukaemia using a population pharmacokinetic approach.

High-dose methotrexate (HD-MTX) with leucovorin rescue is a component of therapy in children with acute lymphoblastic leukaemia. Since MTX toxicity is related to drug exposure, a monitoring of serum MTX concentrations at H24, H48, H72 and until the concentration is less than 0.2 micromol/L is commonly performed. However, a number of patients may reach concentrations of less than 0.2 micromol/L long before the next sampling is scheduled. The aim of our study was to develop a Bayesian method predicting the time at which MTX concentration reaches 0.2 micromol/L in order to decrease the number of samples drawn and to allow for a more rapid patient discharge. Methotrexate population parameters were estimated from a retrospective analysis of 60 infusions in 23 children and MTX concentrations were predicted from an independent set of 20 courses in 14 children with a Bayesian approach using either one (H48) or two (H24 and H48) samples. The following population parameters were obtained using a two-compartment model: CL = 3.51 L/h (inter-individual variability: 66%), Vd = 8.67 L (58%), k12 = 0.0044 h(-1)(105%), k21 = 0.039 h(-1)(25%). Clearance and Vd were found to increase with weight and age respectively. Both sampling schedules tested for the Bayesian estimation enabled accurate prediction of concentrations and provided satisfactory precision despite a small bias. When considering the ability to predict the time at which the threshold was reached, the one-sample (H48) schedule gave the best results. We conclude that a sampling schedule involving only one sample and Bayesian parameter estimation may be able to predict the delay necessary to reach 0.2 micromol/L in each individual.

Determination of levofloxacin in plasma, bronchoalveolar lavage and bone tissues by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method.

The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of levofloxacin in human plasma, bronchoalveolar lavage and bone tissues. The sample extraction was based on a fully automated liquid-solid extraction with an OASIS cartridge. The method used ultraviolet detection set at a wavelength of 299 nm and a separation with a Supelcosil ABZ+ column. The assay has been found linear over the concentration range 0.25-25 microg/ml for levofloxacin in plasma, 1-6 microg/ml in bronchoalveolar lavage and 0.5-10 microg/g for bone tissues and it provided good validation data for accuracy and precision. The assay will be applied to determine the penetration of levofloxacin in human bronchoalveolar lavage (BAL) and bone tissues during pharmacokinetic steady state.

Fluorometric high-performance liquid chromatographic determination of sotalol in biological fluids.

A high-performance liquid chromatographic (HPLC) method for sotalol in biological fluids is presented. Sample preparation involves a simple acid-base double-extraction procedure, and analysis is carried out on a reversed-phase chromatographic system using a muBondapak C18 column. Procainamide was used as the internal standard. As little as 20 ng of sotalol/ml can be detected with good precision by a fluorometric detector. HPLC, which allows more rapid analysis of plasma sotalol levels than does GLC, appears suitable for pharmacokinetic studies as well as for therapeutic drug monitoring.

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review.

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.

Uptake of fluoroquinolones in human monocytes isolated from peripheral blood.

The aim of this study was to develop a technique for separating monocytic cells in suspension from peripheral blood to measure the intracellular penetration of three fluoroquinolones (ofloxacin, ciprofloxacin and sparfloxacin). Mononucleated cells were isolated from the blood on a density gradient with lymphoprep and purified by a specific technique of adhesion and disadhesion on fibronectin. The monocytes were obtained in suspension with 76.8% purity and 97.9% viability. This was a convenient form for measurement of intracellular accumulation by use of the velocity-centrifugation technique. Intra-monocytic penetration of ciprofloxacin, ofloxacin and sparfloxacin was measured at equilibrium after 30-min incubation in the presence of 16 microg mL(-1) antibiotic. The results revealed low intra-monocytic accumulation of ciprofloxacin (intracellular-extracellular = 1.76) and ofloxacin (intracellular-extracellular = 1.42). The penetration of sparfloxacin was significantly higher (intracellular-extracellular = 2.4). This study confirms the important differences between human immunocompetent cells in terms of their ability to concentrate quinolones. It also underlines the importance of monocyte-macrophage cellular differentiation as a determinant of antibiotic penetration.

Uptake of quinolones by in-vitro human monocyte derived macrophages.

We have developed an in-vitro model of monocyte-derived macrophage (MDMphi) to compare fluoroquinolone uptake in monocytes and derived macrophages. Monocyte-derived macrophages were obtained in-vitro by cultivating freshly isolated monocytes for seven days in RPMI 1640 medium, containing foetal calf serum and Rhu granulocyte-macrophage colony stimulating factor. Final suspensions contained 95% viable cells and 63% macrophages. Intramacrophagic accumulation of ciprofloxacin, ofloxacin or sparfloxacin was measured at equilibrium after 30-min incubation in the presence of 16-18 microg mL(-1) antibiotic. The results revealed low intracellular accumulation of ofloxacin in MDMphi (intracellular/extracellular ratio: IC/EC = 1.7). Ciprofloxacin and sparfloxacin uptake was significantly higher. The IC/EC ratios were only slightly increased in macrophages when compared with monocytes under the same experimental conditions. These results suggest that maturation of monocyte to macrophage has only a limited effect on basal quinolone uptake. Monocytic maturation cannot explain the important differences between fluoroquinolone accumulation in monocytes and tissue macrophages. Cell activation may be a greater determinant.

Pharmacokinetic parameters of ciprofloxacin (500 mg/5 mL) oral suspension in critically ill patients with severe bacterial pneumonia: a comparison of two dosages.

The authors determined the pharmacokinetic parameters of a new immediate-release ciprofloxacin suspension in tube-fed intensive care patients with bacterial pneumonia, to compare two dosage regimens: 500 mg b.i.d and 750 mg b.i.d. in this prospective clinical trial. The 20 patients were critically ill and on mechanical ventilation and enteral feeding with bacterial pneumonia. They were randomized to receive two different ciprofloxacin dosages: 500 mg b.i.d (group 1) versus 750 mg b.i.d. (group 2). Blood samples were collected from these patients after reaching steady-state and the pharmacokinetic parameters were determined. The mean (range) serum steady-state concentration at 2 h after enteral administration was: C(max 500) = 2.6 (1.2-4.3) mg/L in group 1 and C(max 750) = 3.5 (1.5-5.9) mg/L in group 2. The mean (range) calculated 12-h area under the serum concentration was high in both groups: AUC(0-12 (500)) = 24.7 (12.9-36.2) mg.h/L in group 1 and AUC(0-12 (750)) = 28.9 (18.3-47.5) mg.h/L in group 2. In conclusion, ciprofloxacin oral suspension was well absorbed via nasogastric route in intensive care patients with severe pneumonia, achieving reliable pharmacokinetic parameters for most of the pathogens and important cost reduction compared to intravenous delivery. However, with less susceptible pathogens such as Staphylococcus aureus or Pseudomonas aeruginosa, higher dosages than 750 mg b.i.d. should be given.

Diffusion of cefepime into cancellous and cortical bone tissue.

The degree of penetration of an antibiotic into the infection site is an important factor in its therapeutic efficacy, particularly in bone and joint infections. In the present study, we examined the bone tissue penetration of cefepime at a dose of 2 g, and the results were correlated to microbiological data to estimate the clinical efficacy of cefepime in bone infections. In this open-label, single-arm, noncomparative study, subjects of similar age, body weight, height and creatinine clearance who were undergoing elective total hip replacement received a single, parenteral 2 g dose of cefepime. Plasma samples were collected simultaneously with bone tissue samples 1.5 hours later, on average, and analyzed by a validated high performance liquid chromatography assay. Ten patients (7 women and 3 men; mean age, 78 years; mean body weight, 57 Kg; mean creatinine clearance, 56 mL/min) were enrolled. The mean +/- SD plasma concentration of cefepime at the time of bone removal was 72.9 +/- 24.4 microg/mL. The mean +/- SD cefepime concentrations were 73.5 +/- 16.2 microg/mL in cancellous bone tissue and 67.7 +/- 17.0 microg/mL in cortical bone tissue. The mean +/- SD ratios of cefepime concentration in bone and plasma (bone/plasma) were 1.06 +/- 0.23 for cancellous bone tissue and 0.87 +/- 0.37 for cortical bone tissue. Cefepime exhibits an excellent diffusion into bone tissue, with concentrations achieved in both cancellous and cortical bone tissue greater than the minimum concentrations required to inhibit the growth of 90% of strains (MIC90) of most of the susceptible pathogens commonly involved in bone infections.

Diffusion of isepamicin into cancellous and cortical bone tissue.

The degree of penetration of an antibiotic into the infection site is an important factor in its therapeutic efficacy, particularly in bone and joint infections. In the present study, we examined the bone tissue penetration of isepamicin at a dose of 15 mg/Kg, and the results were correlated to microbiologic data to estimate the clinical efficacy of isepamicin in bone infections. In this open-label, single-arm, noncomparative study, subjects of similar age, body weight, height and creatinine clearance who were undergoing elective total hip replacement received a single, parenteral 15 mg/Kg dose of isepamicin. Plasma and bone tissue samples were collected a mean 1.3 hours later and analyzed by a high-pressure liquid chromatography method. Twelve patients (3 men and 9 women; mean age, 73.5 years; mean body weight, 53.5 Kg, mean creatinine clearance, 58.5 mL/min) were enrolled. The mean +/- SD plasma concentration of isepamicin at the time of bone removal was 43.0 +/- 10.4 microg/mL. The mean +/- SD isepamicin concentrations were 11.6 +/- 7.1 microg/mL in cancellous bone tissue and 12.0 +/- 7.3 microg/mL in cortical bone tissue. The mean +/- SD ratios of isepamicin concentration in bone and plasma (bone/plasma) were 0.28 +/- 0.14 for cancellous bone tissue and 0.31 +/- 0.20 for cortical bone tissue. The concentrations achieved in both cancellous and cortical bone tissue were greater than the minimum concentrations required to inhibit the growth of 90% of strains (MIC90) of most of the susceptible pathogens commonly involved in bone infections.

Penetration of piperacillin/tazobactam (4 g/500 mg) into synovial tissue.

The degree of penetration of an antibiotic into the infected site is an important criterion for therapeutic success. This is particularly true for bone and joint infections. The association of piperacillin and tazobactam has been widely used in the treatment of serious infections including bone infections, but no study has been devoted to the subject of its diffusion into synovial tissue. Our objective was to quantify piperacillin/tazobactam synovial tissue penetration and to estimate the efficacy of the association against the microorganisms usually encountered in joint infections. In an open-label study, 6 subjects with similar age, weight, height and creatinine clearance, who were undergoing elective total hip replacement, received a single, parenteral, 4 g/500 mg dose of piperacillin/tazobactam. Plasma and synovial tissue samples were collected and analyzed by a validated HPLC method. The mean concentrations of piperacillin and tazobactam 1.5 h after the initiation of infusion were 69.9 +/- 4.9 microg/mL and 7.7 +/- 0.3 microg/mL, respectively, in plasma and 37.1 +/- 2.1 microg/g and 2.8 +/- 0.4 microg/g, respectively, in synovial tissue. The synovial tissue/plasma ratios were 0.5 +/- 0.0 for piperacillin and 0.4 +/- 0.0 for tazobactam. The piperacillin/tazobactam ratios were 9.1:1 in plasma and 13.5:1 in synovial tissue. The concentrations achieved in synovial tissue are above the MICs of most of the susceptible pathogens usually involved in joint infections, which suggests that the piperacillin/tazobactam combination should be effective in the treatment of most joint infections caused by susceptible microorganisms.

Diffusion of oral and intravenous 400 mg once-daily moxifloxacin into lung tissue at pharmacokinetic steady-state.

The degree of penetration of an antibiotic into the infection site is an important factor for its therapeutic efficacy, particularly in respiratory tract infections. In the present study, we examined the lung tissue diffusion of moxifloxacin at a dose of 400 mg administered intravenously or orally once-daily, and the results were correlated to microbiological data to estimate the clinical efficacy of moxifloxacin in lower community-acquired respiratory infections. This was a prospective, randomized, parallel-group trial, open-label, single-center study. Patients undergoing lung surgery for bronchial cancer which necessitates the removal of an anatomical piece of lung tissue were randomized into twelve treatment groups, dependent upon the time of surgery and the moxifloxacin formulation, i.v. or oral, administered. During surgery, one blood sample was taken at the time of tissue collection to determine moxifloxacin plasma concentration. At the same time, tissue samples were taken by pulmonary exeresis. A validated new high performance liquid chromatography assay was used to determine moxifloxacin concentrations in plasma and lung tissue. A total of 49 patients (25 for i.v. administration, 24 for oral administration, 44 men and 5 women, mean age, 61 years, mean body weight, 72 kg, mean creatinine clearance was 84 ml/min/1.73 m2) were enrolled. The mean +/- SD steady-state moxifloxacin ratios between lung and plasma concentrations were respectively: 3.53 +/- 1.89 and 4.36 +/- 1.48 for i.v. and oral administration. The mean steady-state moxifloxacin maximal lung concentrations (Cmax) were respectively 12.37 microg/g and 16.21 microg/g for i.v. and oral administration. Moxifloxacin both intravenously and orally exhibits high penetration in lung tissue, with tissue concentrations far above the MIC90s for most of the susceptible pathogens commonly involved, thus underlining its suitability for the treatment of community-acquired, lower respiratory tract infections.

Mixed pharmacokinetic population study and diffusion model to describe ciprofloxacin lung concentrations.

The pharmacokinetics of ciprofloxacin in plasma and lung tissue at steady-state (500 mg b.i.d.) were studied in 38 patients subjected to lung surgery for bronchial epithelioma. The mean characteristics of the patient population were: age = 60 years (range: 48-70), weight=70kg (47-95), height=165cm (range 160-170) and serum creatinine=85microM (range 62-168). Plasma samples, two for each patient and lung samples, one for each patient, were obtained and analyzed. Seven groups were made according to the time of sampling after ingestion of the 5th dose. A three-compartment model was used to describe ciprofloxacin kinetics in plasma and lung. The non-linear mixed effect model approach was used to estimate the mean and variance of the pharmacokinetic parameters. The mean +/- SD of the estimates (coefficient of variation of interindividual variability as a percentage) were central volume of distribution, 39.45+/-52.47l(133%); steady-state volume of distribution, 145.86+/-97.51l(60%), clearance of influx into lung tissue, 35.83+/-22.57l/h(63%), extrapolated elimination rate constant, 0.173+/-0.25/h and extrapolated elimination half-life, 4.02+/-0.89h. The mean +/- SD ciprofloxacin concentration versus time curve in plasma and lung at steady state was simulated using pharmacokinetic parameters and lung physiological parameters, another approach was studied to model the transport of ciprofloxacin into the lung tissue by diffusion. Ciprofloxacin concentration-time history was obtained both by experiments or simulations. The ciprofloxacin level in the lung tissue followed the ciprofloxacin plasma level with a lag time resulting from the time necessary for ciprofloxacin to diffuse through the lung.

Pharmacokinetic study of doxycycline polyphosphate+ after simultaneous ingestion of milk.

Pharmacokinetic parameters of doxycycline polyphosphate were studied in healthy volunteers after oral administration of a single 200 mg dose of this antibiotic with a breakfast containing or not 200 ml of whole milk. Ingestion of milk had only mild effect upon absorption parameters of doxycycline; only a moderate increase of the lag-time was significant. Elimination parameters of doxycycline were impaired by milk; a decrease of the terminal half-life from 28 h to 15 h, and apparent decrease of the enterohepatic circulation and an increase in total body clearance from 40 to 62 ml/min. were observed.