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Pharmacokinetics and Tissue Penetration of a New Carbapenem, Doripenem, Intravenously Administered to Laboratory Animals*

TOSHIHIKO HORI, MASAO NAKANO, YASUO KIMURA and KAZUHISA MURAKAMI

Discovery Research Laboratories, Shionogi and Co., Ltd., Osaka 561-0825, Japan

Abstract. The pharmacokinetic properties of doripenem bacteria including Pseudomonas aeruginosa. Its chemical following 20 mg/kg i.v. infusion were studied in various structure is characterized by a 1ß-methyl group and a laboratory animals. The concentrations of doripenem in sulfamoyl aminomethyl substituted pyrrolidylthio group at plasma, urine and tissue samples were determined by bioassay. C-2. In this study, the pharmacokinetics and tissue Mean AUC0-∞s (Ìgñh/ml) and urinary recoveries (UR, %, 0- penetration of doripenem, administered intravenously to 24 h) were 14.1 and 36.3 in mice, 9.3 and 42.1 in rats, 47.9 laboratory animals, were compared with those of meropenem and 47.6 in rabbits, 78.6 and 83.1 in dogs and 44.1 and 51.0 in and imipenem/cilastatin. Doripenem was remarkably stable monkeys, respectively. In monkeys, with co-administration of against all tested animals and human DHP-Is (4). probenecid, the mean AUC0-∞ of doripenem increased about 2.2 times and urinary excretion was delayed slightly. In mice, Materials and Methods the doripenem level was highest in the blood plasma, followed by the kidney, liver, lung, heart and spleen. These doripenem Antibiotics. Doripenem and meropenem were prepared by Shionogi levels in various tissues rapidly decreased and no accumulation Discovery Research Laboratories, Japan. Imipenem/cilastatin was was observed. Serum protein binding rates (%) of doripenem obtained commercially. were 25.2 in mice, 35.2 in rats, 11.8 in rabbits, 10.2 in dogs, Drug administration. All animals were fasted for 16 to 18 h before 6.1 in monkeys and 8.1 in humans, respectively. the antibiotics were administered; water was given ad libitum. Jcl:ICR male mice (30 to 36 g, 7 weeks old) received an Carbapenems have broad antibacterial activity, particularly intravenous bolus dose (5~100 mg/kg body weight) of the including that against Pseudomonas aeruginosa. They are antibiotics. Samples (n=5) were obtained at 0.083, 0.25, 0.5, 1, 1.5 generally bactericidal at readily attainable blood, tissue and and 2 h (blood samples) and 0.083, 0.25 and 1 h (tissue samples) urine levels (1, 2). These drugs are cleared from the blood after drug administration by jugular incision immediately followed by euthanasia by cervical dislocation. Plasma samples were and largely excreted unchanged in the urine, but are separated by centrifugation at 3,000 x g for 10 min at 4ÆC. The hydrolyzed by dehydropeptidase-I (DHP-I), which is present tissue was removed, weighed and homogenized with 4 parts in the kidney (3). A new parental carbapenem antibiotic, (wt/vol) of methanol-0.1 M MOPS [3-(N-morpholino) doripenem (S-4661) (Figure 1), synthesized in Shionogi propanesulfonic acid] buffer (pH 6) (5:3) using an overhead stirrer. Discovery Research Laboratories, Japan, has an excellent DHP-I was inactivated by methanol. antibacterial spectrum against gram-positive and -negative Jcl:SD male rats (255 to 280 g, 7 weeks old) received an intravenous bolus dose (20 mg/kg) of the antibiotics. Blood samples (n=5) were obtained at 0.083, 0.25, 0.5, 1, 1.5 and 2 h post-dose into heparinized tubes by cardiac puncture following anesthesia with ethyl ether. Plasma samples were separated by centrifugation *Presented in part at the 34th International Congress on at 3,000 x g for 10 min at 4ÆC. In a probenecid study, the rats Antimicrobial Agents and Chemotherapy, Orlando, Florida, received an oral dose (500 mg/kg) of probenecid. U.S.A, in 1994. Kbl:JW male rabbits (2.6 to 2.9 kg, 13 weeks old) received an intravenous bolus dose (20 mg/kg) of the antibiotics. Blood samples Correspondence to: Toshihiko Hori, Infectious Diseases, Discovery (n=5) were obtained at 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4 and 6 h post- Research Laboratories, Shionogi Co., Ltd., 3-1-1, Futaba-cho, dose from the ear artery. Plasma samples were separated by Toyonaka, Osaka 561-0825, Japan. Tel: +81 (6) 6331-8081, Fax: centrifugation at 3,000 x g for 10 min at 4ÆC. +81 (6) 6331-8612, e-mail: [email protected] Pure-bred male beagle dogs (8.3 to 10.2 kg, 8 to 10 months old), from the colony maintained at Shionogi Developmental Research Key Words: Doripenem, pharmacokinetics. Laboratories, received an intravenous bolus dose (20 mg/kg) of the

0258-851X/2006 $2.00+.40 91 in vivo 20: 91-96 (2006)

Figure 1. Chemical structure of doripenem.

antibiotics. Blood samples (n=5) were obtained at 0.083, 0.25, 0.5, 1, 2, 3, 4 and 6 h post-dose from the saphenous and median veins. Plasma samples were separated by centrifugation at 3,000 x g for 10 min at 4ÆC. Female cynomolgus monkeys (2.7 to 4.0 kg) received an intravenous bolus dose (20 mg/kg) of the antibiotics. Blood samples (n=5) were obtained at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6 and 8 h post- dose from the femoral vein. Plasma samples were separated by centrifugation at 3,000 x g for 10 min at 4ÆC. In a probenecid study, the monkeys received an oral dose (100 mg/kg) of probenecid. After drug administration, all the animals were housed singly in Figure 2. Concentrations of doripenem in plasma in laboratory animals stainless-steel metabolism cages which permitted the separate after a single intravenous administration at a dose of 20 mg/kg. collection of urine and feces. The animals were allowed to feed ad libitum during the experimental periods. Urine samples were collected separately under dry ice cooling from each metabolism cage at 0~2 h in mice, 0~2, 2~4, 4~6 and 6~24 h in rats, rabbits and dogs and 0~2, 2~4, 4~6, 6~8 and 8~24 h in monkeys. All system of n-propanol-water (6:4). Plasma samples were mixed with the samples were assayed immediately after each experiment or equal volumes of methanol and the supernatant was separated by stored at –80ÆC until antibiotic assay. Carbapenems are stable at centrifugation. Urine samples were diluted 10 times by volume with –80ÆC for at least 4 weeks in 0.05 M MOPS buffer (pH 6), plasma, 0.05 M MOPS buffer (pH 6). These samples were spotted on a urine and some tissue homogenates of mice. thin-layer plate. After overnight incubation, the active spots were detected by bioautography with E. coli 7437 as the test organism Antibiotic assay. The concentrations of doripenem and related on Mueller-Hinton agar medium. The lower limit of detection in antibiotics in body fluids and tissue homogenates were assayed by this bioautography was about 0.1 Ìg/ml of doripenem equivalent. the band-culture method (5). Escherichia coli 7437 was used as the test organism in Mueller-Hinton medium (Difco). The antibiotic Protein binding. The serum protein binding of doripenem and the concentrations in plasma were calculated from the calibration related antibiotics was determined by ultrafiltration with a curves for the drugs dissolved in normal plasma from the respective micropartition system (6). In preliminary experiments, the amount animals. Urine samples were diluted with 0.05 M MOPS buffer of doripenem to the MPS-3 type ultrafilter (Amicon Corp., (pH 6). The antibiotic concentrations in the diluted specimens and Danvers, MA, USA) was not observed. Just before use, the pH of the tissue homogenate supernatant were calculated from the the plasma was adjusted to 7 using carbonic acid gas. To 0.99 ml of calibration curves for the drugs dissolved in the same specimens. the plasma from various species of laboratory animals and humans The lower limits of assay in doripenem, meropenem and was added 0.01 ml of antibiotic solution (final concentration, imipenem/cilastatin were about 0.02, 0.02 and 0.04 Ìg/ml, 100 Ìg/ml). The antibiotic concentration in the filtrate was respectively. Intra-day precision and accuracy were in the ranges of determined by bioassay to calculate the degree of protein binding. 1 to 13 and 2 to 10%, respectively. The corresponding inter-day values were 0 to 8 and 0 to 13%, respectively, indicating that this Pharmacokinetic analysis. Pharmacokinetic evaluation of the area ∞ assay yielded favorable results. Quality control samples were under the time-plasma concentration curve from 0 h to (AUC0-∞, measured with each analytical run. measured in Ìgñh/ml), and the residence rate constant at steady state (t1/2, measured in h), plasma clearance (CLp, measured in Detection of biologically active metabolites. The biologically active ml/min/kg), distribution volume (Vss, measured in L/kg) were metabolites in the plasma and urine samples were determined by performed by the NONLIN program (7). The concentrations of thin-layer chromatography-bioautography. Precoated cellulose F doripenem and reference antibiotics in the plasma were fitted to a plates (Merck, Darmstadt, Germany) were used with a solvent non-compartment model.

92 Hori et al: Pharmacokinetics of Doripenem in Animals

Table I. Pharmacokinetic parameters of doripenem and reference antibiotics in laboratory animals.

Parameter Urinary a b Antibiotic Animal (n) AUC0-∞ t1/2 Vss CLp excretion (Ìgñh/ml) (h) (L/kg) (ml/min/kg) (%)

Doripenem Mouse (5) 14.1 0.2 0.26 23.7 36.3 Rat (5) 9.3 0.1 0.27 35.7 42.1 Rabbit (5) 47.9 1.0 0.24 7.0 47.6 Dog (5) 78.6 1.0 0.26 4.7 83.1 Monkey (5) 44.1 0.8 0.30 7.7 51.0 Meropenem Mouse (5) 8.2 0.1 0.24 40.5 19.2 Monkey (5) 42.2 0.8 0.25 7.9 34.2 Imipenem/Cilastatin Mouse (5) 19.9 0.2 0.22 16.7 40.7 Rat (5) 19.7 0.2 0.25 16.9 94.5 Rabbit (5) 45.7 0.5 0.20 7.3 38.5 Dog (5) 65.1 0.8 0.31 5.0 63.0 Monkey (5) 58.0 0.7 0.28 5.7 49.4 aAntibiotic was intravenously administered at a dose of 20 mg/kg. bRecovery of antibiotic within the first 2 h after administration for mice and within the first 24 h for other animals.

Results Table II. Mean pharmacokinetic parameters at difference doses of doripenem.

Concentrations in plasma. The concentrations of doripenem Animal (n) Dose AUC0-∞ t1/2 Urinary in the plasma of mice, rats, rabbits, dogs and monkeys given (mg/kg) (Ìgñh/ml) (h) excretion (%) a single intravenous dose of 20 mg/kg are shown in Figure 2. Mouse (5) 5 3.8 0.1 34.3±6.1a The pharmacokinetic evaluations of AUC0-∞, t1/2, Vss and 10 7.6 0.2 33.0±8.9 CLp for each antibiotic are listed in Table I. The mean 20 14.1 0.2 36.3±6.2 AUC0-∞s (Ìgñh/ml) and t1/2 (h) were 14.1 and 0.2 in mice, 50 38.6 0.3 31.8±5.9 9.3 and 0.1 in rats, 47.9 and 1.0 in rabbits, 78.6 and 1.0 in 100 75.3 0.3 40.2±5.6 dogs and 44.1 and 0.8 in monkeys, respectively, and were Monkey (5) 5 14.6±2.1 0.8±0.1 61.4±8.6 10 24.7±3.9 0.7±0.1 63.4±9.0 higher than those of meropenem. The Vss (0.24 to 0.30 L/kg) 20 44.1±8.1 0.8±0.4 51.0±10.6 was similar in all animal species tested. The CLp for mice 50 152±24 1.2±0.3 60.8±14.8 and rats (23.7 to 35.7 ml/min/kg) was higher than that for other animals (4.7 to 7.7 ml/min/kg). aExpressed as mean±standard deviation (n=5) A dose-response study of doripenem was conducted with an intravenous dose of 5 to 100 mg/kg in mice and 5 to 50 mg/kg in monkeys (Table II). In mice and monkeys, a dose-proportionate AUC0-∞ was obtained. Although these Urinary excretion. The recovery levels of doripenem from the t1/2s became slightly longer with increasing dose in mice, the urine in 5 animal species are shown in Table I. Urine was the differences did not appear to be significant. major excretory route for all species, with urinary recovery (%) being the largest in dogs (83.1), followed by that in Distribution in tissues in mice. The concentrations of doripenem monkeys (51.0), rabbits (47.6), rats (42.1) and mice (36.3). and imipenem/cilastatin in tissues are presented in Table III. The urinary recovery of doripenem in dogs exceeded 80% When the concentrations in the tissues were compared 5, 15 and was similar to that of imipenem/cilastatin and slightly and 60 min after administration, the distribution patterns were higher than that of meropenem in mice and monkeys. similar to those of imipenem/cilastatin. The highest concentrations in tissue for doripenem were found in the Effect of cilastatin on pharmacokinetics. The effect of plasma and kidney, followed by the liver, lung, heart and cilastatin, a popular DHP-I inhibitor (3), was observed to spleen. These doripenem levels in various tissues rapidly determine the stability to DHP-I. In mice, the decreased and no accumulation of the drug was observed. pharmacokinetics of doripenem alone and its co-

93 in vivo 20: 91-96 (2006)

Table III. Concentration of doripenem and imipenem/cilastatin in tissues and plasma from mice after intravenous administration at a dose of 20 mg/kg.

Antibiotic Conc. (Ìg/ml or Ìg/g) after: Time (min) Plasma Liver Kidney Lung Spleen Heart

Doripenem 5 46.9±2.3 14.5±1.1 42.2±6.6 11.3±2.5 5.71±0.86 7.77±0.33 15 16.9±3.7 14.8±2.9 16.4±1.8 3.47±0.43 0.99±b 2.52±0.19 60 0.97±0.18 2.04±1.19a 0.80±b NDc ND ND Imipenem / Cilastatin 5 49.0±3.4 10.3±1.2 44.4±4.5 22.2±1.7 6.83±0.83 12.0±1.2 15 20.6±1.8 7.28±0.93 19.3±3.0 10.2±0.6 ND 3.32±1.86a 60 1.99±0.57 ND 2.37±b ND ND ND

Expressed as mean ± standard deviation (n=5) The sensitivities of the plasmic assay for doripenem and imipenem/cilastatin were about 0.02 and 0.04 (Ìg/ml), respectively. The sensitivities of the tissue assay for doripenem and imipenem/cilastatin were about 2.0 and 3.9 (Ìg/g), respectively. an=4, bn=2, cND, not detected.

Table IV. Effect of cilastatin on the pharmacokinetics of doripenema and meropenema in mice and monkeys.

b Animal (n) Antibiotic AUC0-∞ (Ìgñh/ml) Urinary excretion (%)

Alone With combination Ratioc Alone With combination Ratioc

Mouse (5) Doripenem 4.4 8.8 2.0 30.3 66.1 2.2 Meropenem 1.9 8.8 4.6 16.7 68.6 4.1 Monkey (5) Doripenem 24.9 31.0 1.2 62.2 87.3 1.4 Meropenem 16.5 22.5 1.4 40.0 81.7 2.0 aAntibiotic and cilastatin were subcutaneously administered at a dose of 10 mg/kg to mice and intravenously administered at a dose of 10 mg/kg to monkeys. bRecovery of antibiotic within the first 3 h after administration for mice and within 24 h for monkeys. cRatio: alone/with combination.

administration of cilastatin were 4.4 and 8.8 in AUC0-∞ AUC0-∞ increasing and urinary excretion being delayed (Ìgñh/ml), 30.3 and 66.1 in urinary excretion (%), showing slightly. All of these changes were statistically significant in increases with cilastatin (Table IV). In monkeys, the monkeys, but not in rats. These data indicate that renal pharmacokinetics of doripenem alone and its co- tubular secretion plays some part in the renal excretion of administration with cilastatin were 24.9 and 31.0 in AUC0-∞ doripenem in monkeys. (Ìgñh/ml), 62.2 and 87.3 in urinary excretion (%), again showing slight increases with cilastatin. A greater effect on Active metabolites. The plasma and urine of mice, rats, the pharmacokinetic parameters by cilastatin was observed rabbits, dogs and monkeys were analyzed by thin-layer in mice than in monkeys. Compared with meropenem, the chromatography-bioautography to detect plasmic and parameters of doripenem were better in mice and monkeys, urinary metabolite patterns. The chromatograms of plasma showing that doripenem was more stable than meropenem and urine from each animal species showed a spot of to the renal DHP-I of these animals (4). doripenem with an Rf of 0.45, with no active metabolic spots being detected (data not shown). Effect of probenecid. The effect of probenecid was observed to determine whether or not there was renal tubular Serum protein binding. The extent of protein binding of secretion of ‚-lactams (8). In rats, the pharmacokinetic doripenem, estimated by the centrifugal ultrafiltration parameters of doripenem were not changed by probenecid method, is summarized in Table VI. It was approximately (Table V). In monkeys, the pharmacokinetic parameters of 30% in mice and rats and 6-10% in dogs, monkeys and doripenem were changed by probenecid, with the plasma humans, whereas meropenem and imipenem were only

94 Hori et al: Pharmacokinetics of Doripenem in Animals

Table V. Effect of probenecida on the pharmacokinetics of doripenemb in rats and monkeys.

c Animal (n) AUC0-∞ (Ìgñh/ml) Urinary excretion (%)

Alone With combination Ratiod Alone With combination Ratiod

Rat (5) 9.3 9.0 1.0 42.1 41.1 1.0 Monkey (5) 44.1 105 2.2 51.0 51.5 1.0 aProbenecid was orally administered at a dose of 500 mg/kg to rats and 100 mg/kg to monkeys. bDoripenem was intravenously administered at a dose of 20 mg/kg to rats and monkeys. cRecovery of doripenem within 24 h after administration to rats and monkeys. dRatio: alone/with combination.

slightly bound to protein in all species. The extent of plasma Table VI. Protein binding of doripenem, meropenem and imipenem to protein binding of doripenem almost exceeded that of serum protein from laboratory animals and humans. meropenem and imipenem in all of the laboratory animals Animal Binding (%)aof: and humans. Doripenem Meropenem Imipenem Discussion Mouse 25.2 18.9 2.5 Doripenem is a new, injectable 1-‚ methylcarbapenem that Rat 35.2 22.4 4.1 Rabbit 11.8 10.5 0.6 has potent and well-balanced antibacterial activity against Dog 10.2 0.0 0.0 gram-positive and -negative microbes including P. aeruginosa Monkey 6.1 11.7 2.5 (9). When the antibacterial activities of doripenem, Human 8.1 6.1 4.7 meropenem and imipenem were compared, the antibiotic a with the strongest activity was imipenem, followed by Determined by centrifugal ultrafiltration. Antibiotic concentration tested: 100 Ìg/ml. doripenem against gram-positive bacteria, while it was meropenem followed by doripenem against gram-negative bacteria. Against P. aeruginosa, the maximum inhibitory concentration (MIC) values of doripenem tended to be twice The effect of probenecid was observed to determine the as strong as those with meropenem and imipenem. occurrence of renal tubular secretion of doripenem. Renal Doripenem has been found to exhibit excellent therapeutic tubular secretion is thought to play some part in the renal effects in models of systemic and local infections (9), and has excretion of doripenem. Imipenem (3) and meropenem (11) been found to be of good therapeutic value in clinical trials. were influenced by co-administration with probenecid, while In this study, the pharmacokinetic properties of panipenem (12) and biapenem (13) were not. The doripenem were challenged in mice, rats, rabbits, dogs and relationship between tubular secretion, glomerular filtration monkeys. Doripenem was distributed rapidly to almost all and these carbapenem structures could not be determined. the tissues in mice, and the concentration had already Following intravenous administration, doripenem is peaked in each tissue by 5 min after administration. The mainly excreted into the urine. Urinary recoveries of concentrations were greater than the MICs for 90% of doripenem after administration were 36.3% in mice, 42.1% clinically isolated strains of doripenem against the clinically in rats, 47.6% in rabbits, 83.1% in dogs, 51.0% in monkeys most important bacteria, including Staphylococcus aureus, P. and ca. 75% in humans (10), reflecting that doripenem aeruginosa and E. coli (9). The pharmacokinetics of showed comparatively low hydrolysis by DHP-I in dogs and doripenem in plasma AUC0-∞s, t1/2 s and urinary excretions humans (4). in dogs were found to be greater than meropenem and No active metabolites of doripenem could be detected in imipenem/cilastatin. The AUC0-∞ and t1/2 in dogs were the plasma or urine of mice, rats, rabbits, dogs and monkeys comparable to those in healthy subjects given a 1,000 mg as well as humans by the thin-layer chromatography- intravenous dose in a phase I clinical study (10). bioautography (data not shown). The total 24-h urinary excretion accounts for more than The serum protein binding rates of doripenem of mice 70% of the administered dose of the drug in healthy and rats were of medium value and those of the other humans. We found that the drug attained fully adequate animals were low. In dogs, monkeys and humans, the plasma concentrations for favorable therapeutic effects. plasma protein binding ratios of doripenem were the same

95 in vivo 20: 91-96 (2006) as those of meropenem, imipenem, panipenem (14) and 7 Metzler CM, Elfring GL and McEwen AJ: A User’s Manual for biapenem (11). Doripenem in the body fluids and tissues NONLIN and Associated Programs. Upjhon, Kalamazoo, Mich, appeared to be mainly in a free state. 1974. 8 Brown GR: Cephalosporin-probenecid drug interactions. Clin These results, especially its pharmacokinetic properties Pharmacokinet 24(4): 289-300, 1993. and stability to DHP-I, suggest that doripenem without a 9 Tsuji M, Ishii Y, Ohno A, Miyazaki S and Yamaguchi K: In DHP-I inhibitor should be subjected to clinical trials. vitro and in vivo antibacterial activities of S-4661, a new carbapenem. Antimicrob Agents Chemother 42: 94-99, 1998. Acknowledgements 10 Nakashima M, Kato T, Kimura Y, Sasaki S, Konishi M and Oguma T: S-4661, a new carbapenem: VI. Pharmacokinetics We thank M. Kishimoto, S. Matsuda and T. Yamashita for their in healthy volunteers. Abstr. 596, p.32. Program Abstr. 34th excellent technical assistance with the work described in this paper. International Congress on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Orlando, References 1994. 11 Yoshida M., Shiba K, Hori S, Shimada J, Saito A and Sakai O: Basic and clinical evaluation of meropenem. Chemotherapy 1 Harrison MP, Moss SR, Featherstone A, Fowkes AG, Sanders (Tokyo) 40(Suppl. 1): 326-333, 1992. AM and Case DE: The disposition and metabolism of 12 Shiba K, Saito A, Shimada J, Kaji M, Hori S, Yoshida M, Sakai meropenem in laboratory animals and man. J Antimicrob O, Matsumoto F, Imai T and Ueda Y: Fundamental and clinical Chemother 24(Suppl. A): 265-277, 1989. studies on panipenem/betamipron. Chemotherapy (Tokyo) 2 Yamashita N, Kawashima K, Nomura K, Takeuchi H, Hikida 39(Suppl. 3): 362-371, 1991. M and Naruke T: Pharmacokinetics of biapenem in laboratory 13 Masuda T, Yamashita N, Kawashima K, Harada Y and Naruke animals. Chemotherapy (Tokyo) 42(Suppl. 4): 243-250, 1994. T: Mechanism of renal excretion of biapenem in dogs. 3 Norrby SR, Alestig K, Bjornegard B, Burman L, Ferber F, Huber Chemotherapy (Tokyo) 42(Suppl. 4): 210-215, 1994. JL, Jones KH, Kahan FM, Kahan JS, Kropp H, Meisinger MAP 14 Kurihara A, Naganuma H, Hisaoka M, Tokiwa H and and Sundelof JG: Urinary recovery of N-formimidoyl Kawahara Y: Prediction of human pharmacokinetics of thienamycin (MK0787) as affected by coadministration of N- panipenem-betamipron, a new carbapenem, from animal data. formimidoyl thienamycin dehydropeptidase inhibitors. Antimicrob Agents Chemother 36: 1810-1816, 1992. Antimicrob Agents Chemother 23: 300-307, 1983. 4 Kimura Y, Mourakami K, Onoue H, Shimada J and Kuwahara S: S-4661, a new carbapenem: III. Pharmacokinetics in laboratory animals, abstr. 595, p.32. Program Abstr. 34th International Congress on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Orlando, 1994. 5 Kimura Y and Yoshida T: Microbiological assay methods of 6059-S concentrations in body fluids. Chemotherapy (Tokyo) 28(Suppl. 7): 178-188, 1980. 6 D’Costa M and Cheny PT: Ultrafiltrable calcium and Received July 6, 2005 magnesium in ultrafiltrates of serum prepared with the Amicon Revised October 10, 2005 MPS-1 system. Clin Chem 29: 519-522, 1983. Accepted October 13, 2005

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