VOL. XXXIV NO. 8 THE JOURNAL OF 1055

COMPARISON OF THE RENAL EXCRETORY MECHANISMS OF AND OTHER : 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 (, deacetylcefotaxime, , , and ) 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 and 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--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, 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.). Separation of each cepha- losporin was achieved with a reversed-phase column, It-Bondapak C19(Waters Associates). For elution, 25 % methanol - 0.05 M phosphate buffer (pH 7.0) for cefmenoxime, cefotaxime and cefazolin, 25% methanol - 0.005 M phosphate buffer (pH 7.0) for cefotiam, 10'. methanol - 0.05 M phosphate buffer (pH 7.0) for deacetylcefotaxime and cefsulodin, and 40% methanol - 0.005 M phosphate buffer (pH 7.0) for cephaloridine were used. The extent of binding of a cephalosporin to rabbit serum proteins was calculated by the equation: Per cent bound-(I -[F]/[B]) x 100 where [F] is the cephalosporin concentration in the outer solution and [B] is the cephalosporin concen- tration calculated by subtracting the amount of the cephalosporin in the outer solution from the total amount of the cephalosporin the inner and outer solutions of the reference experiment. For rats, the rates of serum protein binding determined by KOND0 et al.7) using the same technique were used. Calculation of Renal Clearance and Its Correction for Serum Protein Binding Renal clearances of inulin and cephalosporins were calculated from the standard formula, C= [U] XV/[P] where C is the renal clearance (ml/minute), [U] the concentration of inulin or cephalosporins in the urine (ag/ml), V the timed volume of urine (ml/minute), and [P] the concentration of inulin or cephalo- sporins in the plasma (ug/ml). The corrected renal clearance for serum protein binding of cephalosporins (Table 1) was calculated by using [P] instead of [P], where [P']=[P] x (100-binding per cent)/100 Statistical Treatment Statistical significances were evaluated by Student's t-test.

Results

Protein Binding The extent of binding of the cephalosporins to the proteins of rat and rabbit sera is shown in Table 1. The extent of protein binding of cefmen- Table 1. Protein binding rates of cefinenoxime and oxime (86%) was lower than that of cefazolin other cephalosporins in rat and rabbit sera. (>96Y.) but higher than those of cefotaxime, Percent bound') deacetylcefotaxime, cefotiam, cephaloridine, and Cephalosporins cefsulodin (7- 57 %) in rats. In rabbits, on the Ratb) Rabbit other hand, the extent of protein binding of cef- Cefmenoxime 86(20) 99.3 (60) menoxime (99.3 %), cefazolin (98.2 %), and cefo- Cefotaxime 57 (20) 96.7 (50) taxime (96.7%) were higher than those of de- Deacetylcefotaxime 29(20) 27.5 (20) Cefotiam 43 (20) 77.5 (60) acetylcefotaxime, cefotiam, cephaloridine, and Cefazolin 96 (20) 98.2 (100) cefsulodin (12.4-77.5%). Cephaloridine 16(20) 41.5 (60) Renal Clearance in Rats Cefsulodin 7(20) 12.4 (100) a~ Determined by equilibrium dialysis method The results of the renal clearance experiments . Numbers in parentheses denote drug concen- in rats are shown in Table 2. Since cefotaxime tration applied (µg/ml). is known to be metabolized partly in the body to b) Data were obtained from KONDOet al.8) Table 2. Renal clearance of cefmenoxime and other cephalosporins in anesthetized rats.

Corrected for binding') Body wt. CInulin PDrug CDrug Ui)ntg' V°> Cephalosporin Pf-Drug CI'-Drug CR, Pf-Drug, CInulin (No. of animals) (g) (ml/min) 01g/ml) (ml/min) (lIg/min) (leg/ml) (mi/min) (Cf-Drug/CInulin) (Ifg/min) Cefmenoxime (9) 234116 1.96±0.71 70.3+15.0 0.57+0.22 9.76+2.13 4.06:L1.59 2.17+0.89 18.0+ 4.3 37.1 + 9.6 (A) 53.1 ±14.4 0.66+0.33 22.615.8 1.54±0.76 1.23 +0.46 28.5+14.4 33.8+17.2 239±14 1.27+0.58 Cefotaximeb) (9) ~(B)23.0+ 9.3 1.60+0.79 16.3+6.1 2.25+1.10 1.83 +0.63 20.6+12.3 33.1+12.8 Cefotiam (7) 236+ 1 1,75 ±0.85 16.6A- 4.0 2.87+1-02 9.50+2.64 5.03+1.79 3.29+1.39 15.1:L 5.5 44.3 + 6.8 -25 <_3 Cefazolin (5) j 200+ 6 1.48 +0.19 62.0+ 4.8 1.01+0.09 2.50±0.24 .2+2.4 17.3+3.7 .67+0.54 62.3+ 6.1 Cephaloridine (5) 201+ 6 1.91+0.50 39.8+14.8 1.83+0.82 33.4+12.3 2.18+0.98 1 .14=0.45 59.6±11.2 64.8+15.4 Cefsulodin (6) 210+ 5 1.48+0.59 25.7 + 3.0 1.85+0.25 24.0+2.7 1.99+0.27 1.51+0.55 34.6+12.3 46.9± 4.4 Data are expressed in the mean +S.D. a) Subscript f represents the corrected value for serum protein binding of the drug. b) A: cefotaxime, B: deacetylcefotaxime c) UDrug•V: amount of drug excreted.

Table 3. Renal clearance of cefmenoxime and other cephalosporins in anesthetized rabbits.

Corrected for binding') Cephalosporin Body wt. C InuIin PDrug CDrug UDrug'V°> (No. of animals) (kg) (ml/min) (pg/ml) (ml/min) Pf-Drug rug CRf Pf-Drug' Clnnlit, (leg/min) (/'g/ml) (ml/niin) (Cf-Urug/CInulin) (/'g/min) Cefinenoxime (8) 2.96+0.11 11.9 +2.8 49.0+13. 1 17.7+7.0 0.35+0.09 2519+989 208.46 3.95±0.74 803=160 1(A) 45.3+13.0 6.00±1.85 1.50±0.42 182+56 19.6+7.7 14.4±4.6 265+92 Cefotaximeb> (8) 3.07+0.05 9.56±1.1 1(B) 9.20+4.10 67.2±27.8 6.64+2.82 93.1;=41.4 9.73+3.90 63.4+29.8 527+84 Cefotiam (7) 3.03 +0.15 11.5+3.6 41.9 + 7.3 17.8+3.3 9.254-1.60 78.9+14.6 7.24+1.68 111147 744+143 Cefazolin (6) 3.20+0.19 10.611 .8 98.0+15.4 9.89+1.77 1.76+0.27 550+98 52.3 ±10.5 18.7+3.9 948±108 Cephaloridine(6) 3.10+0.13 11.5+1.8 44.0+10.3 14.5+3.4 25.5+5.5 24.6+5.6 2.15_LO.36 289+60 604157 Cefsulodin (6) 2.92-0.14 7.89+1.33 70.1-1-14.2 7.66 _2.45 61.6+11.9 8.75--2.77 1.10+0.23 476+83 521 +141 Data are expressed in the mean+S.D. a) Subscript f represents the corrected value for serum protein binding of the drug. b) A: cefotaxime, B: deacetylcefotaxime. c) Ullrug • V : amount of drug excreted. 1060 THE JOURNAL OF ANTIBIOTICS AUG. 1981 deacetylcefotaxime,a less active metabolite", parameters for both are shown separately here and in Table 3 (rabbit data). The renal clearance corrected for serum protein binding (Cf_,,,.ug)of cefmenoxime was 4.06 ml/ minute, which was lower than that of cefazolin (>25.2 ml/minute), similar to that of cefotiam (5.03 nil/ minute)and higher than those of cefotaxime, deacetylcefotaxime,cephaloridine, and cefsulodin (1.34- 2.25 ml/minute). If the clearance ratio Cf Drug/CInulin(CR1) of a drug is more than unity, tubular secretion of the drug is indicated. The ratio of cefmenoximewas 2.17, indicating that the amount of tubular secretion of the drug was nearly the same as that of glomerular filtration. This ratio was lower (p-0.001) than that of cefazolin (> 17.3), similar to those of cefotiam (3.29), deacetylcefotaxime (1.83) and cefsulodin (1.51), and higher (p<0.05) than those of cefotaxime (1.23) and cephaloridine (1.14). Among the cephalosporins studied, the amount of urinary excretion (UDrug•V)was higher (p<-0.001) than that of glomerular filtration (Pf prug•CInulin)for cefmenoxime,cefazolin, and cefotiam, but not for cefotaxime, deacetylcefotaxime,cephaloridine, and cefsulodin, indicating that these three drugs are excreted signifi- cantly by tubular secretion but the others are not. Renal Clearance in Rabbits There were no significantdifferences between values of inulin clearance before and after the priming administration of any cephalosporin. The renal clearance data of the cephalosporins in rabbits are shown in Table 3. The renal clearance (C1_Drug)of cefmenoxime was 2,519 ml/minute, which was markedly higher than that of cefazolin (550 ml/minute) and those of cefotaxime, deacetylcefotaxime,cefotiam, cepha- loridine, and cefsulodin (8.75,r 182 ml/minute). The clearance ratio (CR1) of cefmenoximewas 208, which was larger (p<0.001) than those of cefa- zotin (52.3), cefotaxime (19.6), deacetylcefotaxime (9.73), cefotiam (7.24), cephaloridine (2.15), and cefsulodin (1.10). The amounts of urinary excretion (UDrug•V) of all the cephalosporins except cef- sulodin were significantly higher (p<0.001) than those of glomerular filtration (Pf-Drug?CInulin),indi- cating significanttubular secretion of all the cephalosporins except cefsulodin. Species Difference The clearance ratios (CRf) of the cephalosporins in rats and rabbits are shown in Fig. 2. The ratios for all the cephalosporins except cefsulodin were significantly higher (p<0.01) in rabbits than in rats, indicating that the proportion of the drugs secreted into tubular lumens was larger in rabbits than in rats.

Discussion It is known that the major route of excretion of the cephalosporins is urinary10-12) However, the precise excretory mechanisms of these drugs have not been determined except in a few instances. Detailed excretory mechanisms of cephaloridine have been studied in relation to nephrotoxicity. This drug has been reported to be excreted into the urine mainly via glomerular filtration with no or only a minute amount of tubular secretion13-16). Cefazolin is considered to be excreted mainly by tubular secretion with a small portion undergoing glomerular filtration, because of its high rate of binding to serum proteinsla13,17).Of the total renal excretion of in rabbits, it has been estimated that 40% is transported into the urine through glomerular filtration and 60% through tubular secretion18. From the CR1 values and the estimated amounts of tubular secretion calculated as (UDrug.V- VOL. XXXIV NO. 8 THE JOURNAL OF ANTIBIOTICS 1061

Fig. 2. Comparison of clearance ratio (CRf) between rats and rabbits.

Cephalcsporin CRf

10 20 30 40

~cefmencxime

208 ' 48

Cefotaxtxime

__, Deacetyl ce foataxme

__Cefazolin

_--Cefsulodin

Data were obtained from Tables 2 and 3. Rat, Rabbit

Difference between rats and rabbits (Stucent't-test) ; N,s. : Not signiricont, 0.01 < P < 0.05, °' 0.001 < P < 0101, *°" P < 0,01

Pf-llrug Clnuun), the degrees of tubular secretion of the cephalosporins used in the present report could be classified as follows. In rats, there are those undergoing (1) high tubular secretion: cefazolin, (2) significant but not high secretion: cefotiam and cefmenoxime, and (3) no significant secretion: deacetylcefotaxime, cefsulodin, cefotaxime, and cephaloridine. In rabbits, there are those undergoing (1) high secretion: cefmenoxime and cefazolin, (2) moderate secretion: cefotaxime, deacetylcefotaxime, and cefotiam, (3) low but significant secretion : cephaloridine, and (4) no significant secretion : cefsulodin. These conclusions, however, are based on the assumption that the cephalosporins are not reabsorbed from the tubular lumen by passive diffusion. If a drug is reabsorbed, the amount of its tubular secre- tion would be greater. YAMANAand Tsw.rr19)have reported that the values of pKa of carboxylic groups in cephaloridine and cafazolin are 1.67 and 2.54, respectively. According to ITAKURA,the values of pKa of cefmenoxime, cefsulodin, and cefotiam are 3.6, 2.6, and 2.6, respectively (personal communication), indicating that a great portion of the drugs exists in the anion form in the urine (pH 5.5 - 8.0) and plasma (pH 7.4). Therefore, it is unlikely that these drugs are reabsorbed by non- ionic diffusion. However, the possibility of reabsorption of the drugs cannot completely be excluded, since an exceedingly higher concentration of the drugs exists in the urine than in the plasma. In fact, there is a report suggesting that cephaloridine is reabsorbed14). A significant tubular secretion of cephaloridine in rabbits was suggested by the results of the 1062 THE JOURNAL OF ANTIBIOTICS AUG. 1981 present study. This finding is in accord with that of TUANOet a1.Yo1and BROGARDet x1.21) These authors estimated that 10- 20% of the urine excretion of cephaloridine in man was by tubular secretion. This estimate was based on the fact that the half-life of the drug became longer and renal clearance was reduced by concomitant administration of probenecid. However, CHILD and DODDS") found no evidence of the secretion of cephaloridine in rabbits. They calculated the clearance ratio on the assumption that cephaloridine was not protein-bound to any significant extent. In the present report, if the protein binding of the drug is disregarded, the same conclusion would be deduced. It might be presumed that, because of their lower renal clearances, highly protein-bound cepha- losporins survive longer in the body than those that are poorly bound. However, in the present study in rats and rabbits direct correlations among the rates of protein binding, values of renal clearance and half-lives in plasma of cefmenoxime and the other cephalosporins were not always observed. For example, in rats, the C1-Drugvalues of cefmenoxime, cefsulodin, and cefotiam were 4.06, 1.99, and 5.03 ml/minute (Table 2), the percentage protein binding was 86, 7, and 43 (Table 1), and the half-lives in plasma were 30, 18, and 18 minutes"-12), respectively. In rabbits, the Cf-Drug values of cefmenoxime and cefsulodin were 2,519, and 8.75 ml/minute, the percentage protein binding was 99.3 and 12.4, and the half-lives in plasma calculated from the data of TSUCHIYAet a1.22)and KITA (personal communication) were 88 and 65 minutes, respectively. These results support those of FUKAYA23' who reported that no correlations exist between the extents of protein binding and plasma half-lives of various and cephalosporins. These phenomena might be explained partly by assuming that although the highly protein-bound cephalosporins (cefazolin and cefmenoxime) have lower glomerular filtration rates than those that are poorly bound (cefsulodin), their compensatory high rates of tubular secretion keep the renal excretions fairly high and the half-lives low.

Acknowledgements The authors are grateful to Messrs. YOSIIIAKIKIMURA, HIDEKAZU NAKAGAWA and KEN'ICHIMAEDA for their technical assistance.

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