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PHARMACOKINETICS |
From Clinical Pharmacology (Dr Huang, Dr Lal), Clinical Science (Dr Ostrowitzki, Dr Hill), Biostatistics (Dr Navarro), Clinical Operations (Ms Berger), Regulatory Affairs (Mr Kopeck), Pharma Development Pharmacokinetics and Drug Metabolism (Dr Mau), and Pharmaceutics (Dr Alfredson), Pharma Research, Roche Palo Alto LLC, Palo Alto, California.
Address for reprints: Yue Huang, PhD, Clinical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSREFERENCES |
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Key Words: Levovirin • pharmacokinetics • prodrug • HCV, valine
10%; F. Hoffman-La Roche, Basel, Clinical Pharmacology, unpublished data).5,6
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Levovirin valinate hydrochloride (hereafter referred to as R1518) is a 5'-valinate monoester prodrug of levovirin that exploits a nutrient transporter for enhancement of oral bioavailability (Figure 1). R1518 targets the PEPT1 transporter in intestinal epithelial cells for carrier-mediated drug transport, resulting in an increase in oral bioavailability for levovirin (100%, 87%, and 95% in rat, monkey, and mouse, respectively; Roche Palo Alto LLC, DMPK, unpublished data). R1518 is rapidly hydrolyzed to levovirin prior to portal vein circulation, with low circulating plasma levels (<1% in monkeys).7 The PEPT1 transporter has been extensively investigated as a target for enhancement of transcellular permeation in oral absorption. This transporter is also consistent from Caco-2 to in situ perfusion models and across animal species, affording a target of 3- to 6-fold or even greater increase in exposure for parent compound dosed as the prodrug in humans.8,9
This l-valyl ester prodrug approach has been successfully exploited in on-market nucleoside analogues such as valacyclovir and valganciclovir.10-13 Increased bioavailability is attributed to carrier-mediated intestinal absorption, via the PEPT1 transporter, followed by the rapid and complete conversion from prodrug to the active moiety. The potential advantages of an ester prodrug are (1) a reduction in overall drug load (fewer tablets per day or less frequent administration and lower cost of treatment) and (2) the opportunity to achieve higher exposures of the active drug component to potentially enable enhanced or superior antiviral response.
Two trials, a single-ascending-dose (SAD) as an entry-into-human study followed by a multiple-ascending-dose (MAD) study, were designed to investigate the safety, tolerability, and single- and multiple-dose pharmacokinetics of the drug. More important, from a pharmacokinetic point of view, the primary objective was to determine whether it was possible, using R1518, to achieve plasma concentrations of levovirin greater than those achieved following the oral administration of levovirin. In addition, preliminary information pertaining to the effect of food on pharmacokinetics was explored by studying subgroups of subjects dosed with and without a high-fat breakfast. Female subjects were included to investigate potential gender difference, if any, in the pharmacokinetic disposition of levovirin dosed as R1518.
The pharmacokinetics following single or multiple doses of R1518 reveals an increase in oral bioavailability of levovirin relative to administration of levovirin. By presenting the results of these 2 studies together, with a primary focus on pharmacokinetics, it is believed that these well-characterized clinical data will provide additional evidence for the rationale of improving oral absorption using the l-valyl ester prodrug approach.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSREFERENCES |
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All volunteers provided written, informed consent to participate in the trial. The local Ethics Review Committee (Canterbury Ethics Committee, Christchurch, New Zealand) provided formal approval for the study, which was conducted in accordance with good clinical practice, all local regulatory requirements, and the Declaration of Helsinki.
Study Design
Both studies (SAD and MAD) followed a similar protocol and were conducted at a single clinical pharmacology unit in Christchurch, New Zealand, using a randomized, double-blind, placebo-controlled, parallel-group design.
In the SAD study, a total of 6 dose groups with approximately 7 or 8 subjects per group were studied. In each group, approximately 6 subjects received R1518 and 2 subjects received placebo under fasted conditions. Groups 1 to 5 included only male subjects who received a single dose of 750, 1500, 3000, 4500, or 6000 mg of R1518 or placebo. Group 3 returned 14 days after the initial dose and was given a single dose (3000 mg) of R1518 with a high-fat, high-calorie breakfast (30% fat, 49% carbohydrates, 21% protein, and total calorie content of 400 kcal). Group 6 ran in parallel with group 5 and included only female subjects who were given a single dose of 1500 mg R1518 or placebo under fasted conditions.
In the MAD trial, a total of 5 dose groups with approximately 7 or 8 subjects per group were studied. In each group, approximately 6 subjects received R1518 and 2 subjects received placebo with a moderate-fat meal. Three of the 5 groups included only male subjects who received multiple doses of 750, 1500, or 3000 mg of R1518 or placebo twice daily (BID) for 14 days. The other 2 groups included only female subjects who were given multiple doses of 750 or 1500 mg R1518 or placebo BID for 14 days.
In both trials, dosing began with the lowest dose and was escalated following review of safety and clinical pharmacokinetic data from the preceding dose group. R1518 or matching placebo dosages consisted of a 250-mg- or 1000-mg-strength tablet or a combination of both. Doses were given with 250 mL of water.
In the SAD study, blood samples for R1518 and levovirin plasma concentration determinations were drawn by venipuncture into EDTA tubes immediately before dosing and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hours following the single dose. In the MAD study, blood samples were collected at the following times: predose, 1, 2, 3, 4, 6, 8, and 12 hourson day 1 and predose, 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, and 96 hours following the last dose on day 14. Trough samples were drawn predose in the morning on days 3, 5, 7, and 11. Urine specimens were collected from predose up to 48 hours postdose in the MAD study for urine pharmacokinetic evaluation.
Analytical Methods
Plasma concentrations of R1518 and levovirin were determined by a validated high-pressure liquid chromatography and tandem mass-spectrometry (HPLC/MS/MS) method. The chromatographic conditions included a Zorbax-SB-AQ column (50 x 4.6 mm, 5 µm), Supelco 0.5 µm prefilter. At ambient temperature, the column was eluted with a mobile phase consisting of 0.1% formic acid in water and methanol (1:9) at a flow rate of 0.5 mL/min. Run time per cycle was 6 minutes, with retention times at approximately 2.5 minutes for levovirin and 5.1 minutes for R1518. Ganciclovir was used as the internal standard (ISD). A Perkin-Elmer Sciex API-3000 in the Multiple Reaction Monitoring mode using positive electrospray ionization (4000 V and 450°C) was used to monitor the transitions for levovirin (245.1 
113.1), R1518 (344.1 113.1), and ISD (256.5 152.0). Nitrogen was used as curtain gas (setting: 9), CAD gas (setting: 8), nebulizing gas (setting: 10), and auxiliary gas flow rate at 8 L/min. Dwell time was 100 milliseconds. Collision energy was 15, 29, and 19 eV for levovirin, R1518, and ISD, respectively.
All plasma samples were shipped on dry ice and stored in a freezer at -60°C to -80°C. R1518 and levovirin were extracted from samples using protein precipitation with acetonitrile. After evaporation of the organic solvent, extracts were reconstituted in mobile phase and injected (10 µL) for analysis using LC/MS/MS. The lower limit of quantitation for R1518 and levovirin in plasma was 10.0 ng/mL, with a linear calibration range up to 1000 ng/mL for R1518 and 10,000 ng/mL for levovirin. Samples having an R1518 concentration greater than 1000 ng/mL or levovirin concentration greater than 10,000 ng/mL were diluted with blank plasma for analysis. The intra- and interassay precisions of the method for calibration standards and quality control (QC) samples were 
15% (20% at the lower limit of quantification), expressed as percentage coefficient of variation over the calibrated range. The accuracy of the method was demonstrated by comparing the measured concentration of the calibration standards and QC samples with their theoretical values to be 15% (20% at the lower limit of quantification), expressed as a percentage of deviation from theoretical values.
Frozen urine aliquots were shipped on dry ice to Roche Palo Alto and stored in a -70°C freezer until the concentration of R1518 and levovirin was determined by HPLC/MS/MS using a method similar to the plasma. The lower limit of quantitation for R1518 and levovirin in urine was 50 and 100 ng/mL, respectively, with a calibration range up to 5000 ng/mL for R1518 and 40,000 ng/mL for levovirin. Samples exceeding the upper quantitation limit were diluted with blank matrix for analysis. The intra- and interassay precisions of the method for calibration standards and QC samples were 15% (20% at the lower limit of quantification), expressed as percentage coefficient of variation over the calibrated range. The accuracy of the method was demonstrated by comparing the measured concentration of the calibration standards and QC samples with their theoretical values to be 15% (20% at the lower limit of quantification), expressed as a percentage of deviation from theoretical values.
Pharmacokinetic Evaluation
The plasma concentration-time data for R1518 and levovirin for each subject were analyzed by standard noncompartmental methods using WinNonlin Professional (version 4.1, PharSight Inc). Derived or directly observed pharmacokinetic parameters for R1518 and levovirin included maximum observed concentration (Cmax), time to Cmax (Tmax), terminal elimination half-life (T1/2, computed as the ratio of 0.693 and the negative slope of the least square regression line of the linear terminal portion of the log concentration-time curve), and area under the curve from time 0 to infinity (AUC0-
) or within a 12-hour dosing interval (AUC0-12) in the MAD study, based on the linear trapezoidal rule. In the MAD study, all pharmacokinetic parameters were evaluated for both the first dose on day 1 and the last dose on day 14, with the exception of T1/2 (day 14 only). Time to reach steady state was graphically examined using the trough concentrations collected on days 3, 5, 7, 11, and 14. Steady-state accumulation was calculated as the ratio of pharmacokinetic parameters on day 14 to the day 1 values using Cmax (ARCmax) and AUC0-12 (ARAUC).
Urine pharmacokinetic parameters computed for the MAD study were the percentage of dose excreted in urine from time 0 through 12 hours on day 14 (%Ae) and renal clearance (computed as the quotient of amount excreted in urine during a 12-hour dosing interval divided by plasma AUC0-12 on day 14).
Statistical Analysis
Single- and multiple-dose pharmacokinetic parameters are expressed as arithmetic mean and standard deviation (SD) unless noted. Dose proportionality was evaluated using an ANOVA model (SAS version 8.2; SAS Institute, Cary, NC) with a factor for dose, based on dose-normalized and log transformed Cmax and AUC values. The differences in pharmacokinetics related to gender or food effect was assessed using descriptive statistics.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSREFERENCES |
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Single-Dose Pharmacokinetics
Mean plasma concentration versus time profiles for R1518 and levovirin following the administration of single oral doses of R1518 are presented in Figure 2. Single-dose pharmacokinetic parameters for levovirin and R1518 are presented in Tables II and III, respectively.
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Levovirin reached peak plasma concentration rapidly following oral dosing of R1518 with a Tmax range between 1 and 4 hours (Figure 2). Average Cmax ranged from 7.8 to 38.1 µg/mL, and AUC0- ranged from 70 to 394 µg•h/mL at single-dose levels from 750 to 6000 mg (Table II). Levovirin elimination followed a monoexponential profile with a terminal half-life ranging from 5.7 to 8.3 hours (Figure 2, Table II). Despite increasing values with increasing doses from 750 to 6000 mg (Figure 8), plasma exposure of levovirin demonstrated a statistically significant deviation from dose proportionality (P = .0015 for AUC0-, P < .0001 for Cmax). Within the range of 750 to 1500 mg, however, there was no significant deviation from dose proportionality (P = .37 for AUC0-, P >.99 for Cmax).
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Following oral administration of R1518, the prodrug itself was rapidly absorbed, converted into levovirin, and cleared from the systemic blood. Peak plasma concentrations of R1518 occurred within 2 hours postdose, and the average elimination half-life was estimated to be less than 1 hour across all dose groups (Figure 2, Table III). Plasma exposures of R1518 were much lower than those of levovirin, with relative exposures being less than 6% and 2% based on Cmax and AUC0-, respectively (Table III).
Plasma concentration-time profiles for levovirin and R1518 for the 3000-mg dose group are displayed in Figure 3. In this dose group, male subjects were given the drug under fasted conditions and with a high-fat, high-calorie breakfast. There was no difference in the systemic exposure of levovirin between the fasted and fed states. A descriptive summary of levovirin pharmacokinetic parameters is presented in Table II.
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As shown in Figure 4 and Table II, approximately 40% and 49% higher plasma exposures of levovirin based on average Cmax and AUC0-, respectively, were observed in female subjects dosed with 1500 mg of R1518 compared to plasma exposures in males who received the same dose under the fasted condition. In contrast, similar plasma exposures of R1518, residual prodrug, were observed in men and women.
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Multiple-Dose Pharmacokinetics
Mean plasma concentration versus time profiles for R1518 and levovirin following the administration of multiple oral doses of R1518 are presented in Figures 5 and 6. For better illustration, the 12-hour profiles are grouped by days, following the first dose on day 1 and the last dose on day 14 in Figure 5. Figure 6 illustrates trough concentrations on days 1, 3, 5, 7, 11, and 14. Plasma pharmacokinetic parameters for levovirin and R1518 are presented in Tables IV and V, respectively. Urine pharmacokinetic parameters for levovirin are provided in Table IV.
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For levovirin, plasma concentration profiles and exposures (Cmax and AUC0-12) following the first dose on day 1 of the MAD study were similar to those observed in the SAD study (Tables II and IV). An accumulation ratio of approximately 1.3 to 1.5 was observed based on the day 14 to day 1 ratios of Cmax and AUC0-12 (Table IV). Steady state was attained after about 3 days of dosing (Figure 6). Graphical examination of the data suggested that levovirin exposures increased with increasing doses from 750 to 3000 mg twice daily following a 14-day regimen (Figure 5). No statistically significant deviation from dose proportionality (P > .05) was observed among the dose levels studied, based on pharmacokinetic parameters on day 1 and day 14.
Similar to the observation in the SAD study, R1518 (residual prodrug) plasma levels were low and transient following the administration of multiple doses (Figure 5). Relative exposures (R1518/levovirin) were <2.5% and <1% for Cmax and AUC0-12, respectively, at steady state on day 14 (Table V). Because of the extremely fast disappearance from the plasma, there was no accumulation of R1518 following the 14-day twice-daily dosing regimen (Figure 6).
Analysis of urine samples from the MAD study demonstrated that 75% to 90% of the levovirin equivalent dose was recovered unchanged in urine at steady state on day 14 (Table IV). Estimated renal clearance averaged 5.8 to 6.5 L/h in men and 4.2 to 4.8 L/h in women (Table IV). The amount of R1518 recovered in urine was negligible (<1%).
Results from the women who received doses of 750 or 1500 mg twice daily for 14 days demonstrated approximately 50% higher levovirin mean plasma exposure compared to plasma exposures in men who received the same dose under the same fed condition. No significant difference was observed in the R1518 plasma exposures between men and women. These observations were consistent with the SAD study.
Historic Data Comparison
A comparison was made between levovirin exposures following a 4500-mg single dose of R1518 in the current study (note that 4500 mg R1518 is equivalent to approximately 3195 mg levovirin) and historic data from a 3200-mg single dose of levovirin in a previous study. As shown in Figure 7 and Table VI, the levovirin plasma exposures represented by Cmax and AUC0- following the administration of R1518 were about 12.9 and 7.7 times higher, respectively, than that following dosing of levovirin.
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DISCUSSION
The main objective of the clinical studies was to demonstrate superior systemic exposure of levovirin following the administration of oral dose(s) of R1518, a 5'-valinate monoester prodrug of levovirin, compared to historic data of systemic exposure following the administration of an equivalent or similar dose of levovirin. Historically, the use of amino acid (valine) esters of the antiviral drugs acyclovir and ganciclovir has been shown to improve oral bioavailability of the active drug moiety.9-12 Valganciclovir exhibits an oral bioavailability of ganciclovir of 61%, compared to 6% when ganciclovir itself is dosed orally. Similarly, the oral bioavailability of acyclovir is 54% following dosing as valacyclovir compared to 12% to 20% when acyclovir itself is dosed. The increased oral absorption of these compounds is due to transport by the intestinal dipeptide transporter PEPT1. Transport appears to be stereoselective in that only the l-valine ester of acyclovir is transported while the d-valine ester is not.9
Previously, levovirin has been administered to healthy volunteers and patients in a number of clinical studies. The maximum historic single dose that has been given to humans was 4800 mg, corresponding to the maximum systemic exposures represented by plasma Cmax and AUC of 3.1 µg/mL and 55.3 µg•h/mL, respectively (F. Hoffman-La Roche, Basel, Clinical Pharmacology, unpublished data). A similar level of exposures would have been achieved with doses of R1518 at less than 750 mg. The magnitude of exposure increase at a similar dose is provided in the results of historic data comparison. In principle, saturation in absorption occurs at higher doses at which more significant improvement in bioavailability by the valine ester prodrug would prevail.
It is conceivable that not only the extent but also the rate of absorption was increased following dosing with the valine ester prodrug. Based on the same historic data comparison, this is evidenced by a 1-hour decrease in Tmax and approximately 5-hour shorter terminal T1/2 following the administration of R1518 compared to Tmax and T1/2 following administration of levovirin. The latter argument (shorter T1/2) may be explained with an absorption-rate-limited elimination profile characterized by a longer terminal T1/2 as well as alteration in time and magnitude of peak concentration; the extreme case is also known as "flip-flop" kinetics.
Once absorbed into the systemic circulation, levovirin is primarily excreted in urine as unchanged drug with no metabolites. Maximum urinary recovery is approximately 10% when levovirin was administered orally. In contrast, it ranged between 75% and 90% of levovirin equivalent dose recovered in urine after oral administration of R1518. Assuming the percentage of recovery in urine represents a conservative estimate of oral bioavailability, a 7- to 9-fold increase in oral absorption is estimated for the valine ester prodrug.
In addition to enhanced oral absorption by the valine ester prodrug, instantaneous cleavage of the ester in vivo and extensive conversion to the active moiety are also critical for the prodrug strategy to be successful.8,14 As Beaumont et al pointed out, an ideal ester prodrug should exhibit the properties of resistance to hydrolysis during the absorption phase and rapid breakdown to yield high circulation concentrations of the active component postabsorption.8 Valacyclovir is rapidly converted to acyclovir, with the Cmax of valacyclovir being about 10% and the AUC less than 1% of that of acyclovir.11 In addition, valacyclovir is undetectable in the plasma within 3 hours of an oral dose. The enzyme responsible for hydrolysis of valacyclovir in the rat is located in both the intestine and liver. In the case of levovirin, residual prodrug accounted for only less than 1% of the total AUC of the active levovirin component, and its existence was very transient in plasma, leading to no accumulation following administration of multiple doses of R1518. It is postulated that most of the hydrolysis of prodrug is completed before or during the first-pass process (Roche Palo Alto LLC, unpublished data).
R1518 is a biopharmaceutic class I compound. The lack of effect of a high-fat, high-calorie meal on the pharmacokinetics of R1518 was consistent with the characteristics of a highly soluble and highly permeable compound that results in nearly complete oral bioavailability. Dose-independent pharmacokinetics was observed in both SAD and MAD studies, with slight deviation from dose proportionality in the SAD, in which a broader dose range was tested (Figure 8).
From preclinical and clinical data, levovirin elimination is known to be primarily by renal clearance. Plasma exposure of levovirin following oral administration of R1518 is therefore directly correlated with renal function. In both the single- and multiple-dose studies, levovirin Cmax and AUC were approximately 50% higher in women compared to men who received the same dose levels of R1518. Further exploration of subject demographics and renal function (Table I), using creatinine clearance calculated from the Cockroft-Gault equation,15 suggests that the observed exposure difference may be explained by the fact that in these particular studies, all women were older, lighter, and, as expected, had lower estimated renal function than the men did. This was supported by the urine pharmacokinetic data, in which a similar amount of levovirin was recovered in urine between men and women, although plasma AUC was higher in women than in men. Additional evidence came from the similarity of R1518, the residue prodrug exposure in men and women. Unlike the levovirin plasma exposure, no apparent difference was seen in the plasma exposure of the residue prodrug between men and women who received the same multiple doses of R1518 under the same fed condition. This suggests that the difference in levovirin exposure was not caused by the greater hydrolytic capacity of ester prodrug in women than in men, which would have been supported by lower plasma exposure of residue prodrug in women.
In summary, R1518, the 5'-valinate monoester of levovirin, is a prodrug that results in improved bioavailability and significantly higher plasma concentrations of levovirin than oral administration of levovirin.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSREFERENCES |
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