Comparison of the Renal Excretory Mechanisms of Cefmenoxime and Other Cephalosporins: Renal Clearance in Rats and Rabbits
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VOL. XXXIV NO. 8 THE JOURNAL OF ANTIBIOTICS 1055 COMPARISON OF THE RENAL EXCRETORY MECHANISMS OF CEFMENOXIME AND OTHER CEPHALOSPORINS: RENAL CLEARANCE IN RATS AND RABBITS IWAO YAtiIAZAKI, YOSHIHIRO SHIRAKAWA and TAKESHIFUGONO Central Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan (Received for Publication April 13, 1981) The renal excretory mechanism of cefmenoxime was compared with those of 6 other cephalosporins (cefotaxime, deacetylcefotaxime, cefotiam, cefazolin, cephaloridine and cefsulodin) in rats and rabbits. In rats, renal clearance corrected for serum protein binding) of cefmenoxime (4.06 ml/minute) was lower than that of cefazolin, similar to that of cefotiam, and higher than those of cefotaxime, deacetylcefotaxime, cephaloridine, and cefsulodin. In rabbits, the value for cefmenoxime (2,519 ml/minute) was markedly higher than those for the other drugs. If the clearance ratio CRr) is more than unity, tubular secretion of a drug is indicated. In rats, the tubular secretion of cefmenoxime equalled the glomerular filtration (CRr--2.17). The tubular secretion of cefmenoxime was lower than that of cefazolin but similar to that of cefotiam. None of the other cephalosporins seemed to be secreted significantly. In rabbits, a large amount of cefmenoxime was secreted (CRP-208). The tubular secre- tion was larger than those of the other cephalosporins. All the drugs except cefsulodin showed significant tubular secretion. A species difference was observed in the CRr of the cephalosporins: the values were generally higher in rabbits than in rats. The use of antibiotics to treat infectious diseases has occasionally been restricted by associated medi- cal conditions, especially renal impairment. Therefore, elucidation of the renal excretory mechanism of antibiotics may provide useful information on the mechanism of renal impairment and is important in determining their safe application, therapeutic potency and duration of action. The renal excretory mechanism of organic acids, including penicillin and cephalosporin antibiotics, comprises glomerular filtration, proximal tubular secretion by anion transport mechanism, and passive reabsorption by nonionic diffusion1). In the present report, the renal clearance of cefmenoxime (SCE-1365, 7;3-[2-(2-aminothiazol-4-yl)- (Z)-2-methoxyiminoacetamido]-3-[(I-methyl-IH-tetrazol-5-yl)thiomethyl]ceph-3-em-4-carboxylic acid), a novel broad-spectrum cephalosporin21, was studied in rats and rabbits. Furthermore, the ratio of tubular secretion to the glomerular filtration of the drug was estimated from the ratio of renal clearance corrected for the plasma protein binding to inulin clearance, and was compared with those of other cephalosporins. Materials and Methods Compounds Cefmenoxime, cefotaxime (7(3-[2-(2-aminothiazol-4-yl)-(Z)-2-nethoxyiminoacetamido]-3-acetoxy- methylceph-3-em-4-carboxylic acid), deacetylcefotaxime (7;3-[2-(2-aminothiazol-4-yl)-(Z)-2-methoxy- 1056 THE JOURNAL OF ANTIBIOTICS AUG. 1981 Fig. 1. Chemical structures of cefmenoxime and other cephalosporins used. .S• X-CONH .N. Z 0 Y CephoIOsporin X Y Z S H2N N N Cefrencxine COOH CH2S N C N NOCH3 CH3 S H2N Cefotcxime N C COOH CH2OCOCH3 NOCH3 S H2N Ceecetyl- COOH CH2OH cefctaxime N C NOCH3 S H2N N N N Cefoticm N CH2 COOH CH2S N CH2CH2N(CH3)2 N N N N N CH2 Cefaz1in COOH CH2S CH3 N 8 S CH2 CH2N Cephcloridine Coo CH CH2N CONH2 Cefsulodin SO3Na COO iminoacetamido]-3-hydroxymethylceph-3-em-4-carboxylic acid), cefotiam (7/3-[2-(2-aminothiazol-4-yl)- acetamido]-3-[[[1-(2-dimethyl)-1H-tetrazol-5-yl]thio]methyl]ceph-3-em-4-carboxylic acid) and cefsulodin (3-(4-carbamoyl-l-pyridiniomethyl)-7(3-(D-a-sulfophenylacetamido)ceph-3-em-4-carboxylate monosodi- um salt) were prepared in our Central Research Division. Cefazolin (7-[l-(1H)-tetrazolylacetamido]-3- [2-(5-methyl-1,3,4-thiadiazolyl)thiomethyl]-3-cephem-4-carboxylic acid; Fujisawa Pharmaceutical, Ja- pan) and cephaloridine (7-[2-(2-thienyl)acetamido]-3-(1-pyridylmethyl)-3-cephem-4-carboxylic acid betaine; Eli Lilly, U.S.A.) were commercial preparations. All, except cefotiam and cephaloridine, were used as the sodium salt. Cefotiam was used as the dihydrochloride and the pH of the aqueous solution was neutralized with Na2CO3. Chemical structures of these cephalosporins are shown in Fig. 1. Inulin (reagent grade; Wako Pure Chemical, Japan) and inulin-[11C]carboxylic acid, labelled at the carboxyl carbon (specific activity 5 mCi/mmole; Radiochemical Centre, Amersham, England), were also used. Animals Male Jcl: Sprague-Dawley rats (Japan CLEA) weighing 192-256 g and male New Zealand White rabbits (Nippon Seibutsuzairyo Center) weighing 2.7 - 3.4 kg were used. All animals were acclimated N VOL. XXXIV NO. 8 THE JOURNAL OF ANTIBIOTICS 1057 to the laboratory environment more than 1 week before the experiment. Rats were fed a diet (CE-2; Japan CLEA) and water ad libitum and rabbits were deprived of a diet (G M-1; Funabashi Nojo, Japan) but not of water of 16 hours before experiment. Measurementent of Renal Clearance a) Rats: The technique of renal clearance was similar to those of GREVEN3, and CARRARA and BAINES4)with slight modification. In brief, each rat was loaded twice orally with 25 ml/kg body weight of saline at 30 and 20 minutes before the operation, anesthetized with 50 mg/kg of sodium pentobarbital (NembutalR; Abbott Labs., U.S.A.) intraperitoneally, and catheterized intratracheally. The left jugular vein, right carotid artery, and both urethrae were cannulated with polyethylene tubes for infusion, blood collection and urine collection, respectively. After the operation, each rat was primed intravenously with 0.5 ml of inulin solution (50 mg/ml in saline) and 1 ml/kg of cephalosporin solution (20 mg/ml). This was followed by an intravenous infusion of a solution containing 9.6 mg inulin, 1 RCi inulin-[14C]carboxylic acid and 6 mg cephalosporin preparation in 12 ml saline at a rate of 12 ml/rat/hour throughout the experiment. After an equilibration period of 2 hours, urine was sampled for three consecutive 30 minutes periods. At the midpoint of each urine collection, 0.5 nil blood was sampled in a heparinized syringe and the plasma was obtained by centrifugation (3,000 rpm x 10 minutes) immediately. The urine and plasma specimens obtained from the animals infused with cefotaxime were frozen immediately on dry ice and kept in a freezer at -20°C, whereas those from the animals infused with the other cephalosporins were kept in the freezer without being frozen on dry ice. b) Rabbits: The technique of determining renal clearance was essentially the same as that used for rats, except that cold inulin was used instead of inulin-[1'C]carboxylic acid. Rabbits were anesthetized with 30 mg/kg of sodium pentobarbital intravenously and tracheotomized. The jugular vein, carotid artery, and both urethrae were cannulated as described above for rats. The rabbits were primed intravenously with 5 ml/kg of inulin solution (15 mg/ml in saline, solution A). This was followed by an intravenous infusion of the solution A at a rate of 18 ml/kg/hour. Two consecutive 30 minute-urine specimens were collected beginning at 2 hours after the start of infusion. The infusion was stopped, and the animals were primed with 1 ml/kg of one of the cephalosporin solutions (20 mg/ml in saline, solution B). This was followed by an intravenous infusion of another solution (1 mg/ml, solution C) prepared with solution A at the same rate mentioned above. Three consecutive 30 minute- urine specimens were collected beginning 30 minutes after the start of the solution C infusion. Three ml of blood specimens were sampled at the midpoint of each urine collection before and after the priming with solution B, and the plasma was separated in the manner described above for rats. The urine and plasma specimens were handled as described above for rats. Measurements of Cephalosporins and Inulin The cephalosporin concentrations in the urine and plasma specimens were assayed using the agar diffusion method. The test organisms used were Proteus mirabilis ATCC 21100 for cefinenoxime and cefotiam, Pseudomonas aeruginosa NCTC 10490 for cefsulodin, Bacillus subtilis ATCC 6633 for cefazolin and cephaloridine, and Proteus r•ettgeri ATCC 9250 for bioautographic determination of cefotaxime and deacetylcefotaxime5) Specimens were diluted more than 30 times with 0.1 M phosphate buffer (pH 7.0) to eliminate substances that might influence the determination. Inulin concentrations in rat urine and plasma were determined based on the radioactivity of inulin- [14C]carboxylic acid using a liquid scintillation counter (Model LSC-653, Aloka, Japan) with dioxane phosphor mixture (naphthalene 1 kg, DPO 120 g, POPOP 3 g, dioxane 7.2 liters and methanol 450 ml). Inulin concentrations in rabbit specimens were determined by the colorimetric technique reported by SCHREINER". Measurementeasurement of Binding of Cephalosporins to Serum Proteins The extent of binding of the cephalosporins to the proteins of rabbit serum was measured by the equilibrium dialysis technique of KONDOet al.11 Solutions of the cephalosporins were prepared so as to give various concentrations with 0.133 M phosphate buffer, pH 7.4, diluted with the same volume of saline. A 0.1 ml aliquot of the cephalosporin solution was added to 4.9 ml of untreated rabbit serum. One ml of the mixture obtained was placed in the inner cell which was divided from 1058 THE JOURNAL OF ANTIBIOTICS AUG. 1981 outer cell with a regenerated cellulose membrane (pore size 15 - 20 A, 0.025 mm thickness), then one ml of the phosphate buffer was placed in the outer cell. Equilibration was carried out for 20 hours at 4°C. A reference experiment was performed by use of the buffer in place of serum. The cephalosporin concentrations of the resultant outer solutions of the cell were determined by high-performance liquid chromatography (ALC/GCP 600 U/4000; Waters Associates, U.S.A.).