Protective Effect of N-Acyl Amino Acids (NAAs) on Cephaloridine (CER) Nephrotoxicity in Rabbits

Protective

Effect of N-Acyl Amino Acids (NAAs) on Cephaloridine Nephrotoxicity in Rabbits Yasukuni 'Product

Hirouchi', Akira

Development

Hideo Naganuma', Kamiya3, Ken-ichi

Laboratories 1-2-58

3Department 'Department

of Hospital

Pharmacy

Yukinori Kawahara2, Ryuzo Inui4 and Ryohei Hori5

, 'Analytical and Metabolic Research Laboratories, Hiromachi, Shinagawa-ku, Tokyo 140, Japan

(CER)

Okada',

Sankyo Co., Ltd.,

, School of Medicine, Yamaguchi University, 1144 Kogushi, Ube, Yamaguchi 755, Japan

of Hospital Pharmacy

'Department

, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan of Hospital Pharmacy , Faculty of Medicine, Kyoto University, 54 Shogoinkawahara-cho, Sakyo-ku, Kyoto 606-01, Japan Received

February

18, 1993

Accepted

September

24, 1993

ABSTRACT-The protective effect of N-acyl amino acids (NAAs) against cephaloridine (CER)-induced nephrotoxicity was studied in rabbits. A large single intravenous dose of CER (more than 100 mg/kg) in duced severe proximal tubular necrosis. Simultaneous treatment with several NAAs (at dosages of 100, 200 mg/kg, etc., i.v.), such as N-benzoyl-j3-alanine (NBBA), N-benzoyl-6-aminocaproic acid, and Na,E-dibenz oyl-D,L-lysine, remarkably suppressed the histopathological damage in the kidney induced by CER. NAAs have generally low toxicity in laboratory animals (e.g., the LD50 of NBBA was more than 3,000 mg/kg, i.v. in rats), and NAAs were suggested to be good candidates for reducing the nephrotoxicity of CER and other p-lactam antibiotics. Keywords: N-Acyl amino acid (NAA), Cephaloridine (CER), Proximal tubular necrosis, Basolateral membrane, N-Benzoyl-(-alanine (NBBA)

Some (3-lactam antibiotics,

including cephalosporins,

penicillins, and carbapenems induce nephrotoxicity both in humans and laboratory animals, which has impeded more extensive clinical use of these antibiotics. The renal injury induced by (3-lactam antibiotics is histopathologi cally a limited proximal tubular necrosis (1). Cephalori dine (CER), a widely used cephalosporin antibiotic, also causes renal dysfunction. Several mechanisms for the nephrotoxicity of CER have been proposed (2): 1) high in tracellular accumulation of antibiotics resulting from ac tive secretory transport in tubules, 2) peroxidative decom position of membrane lipids in the renal cortex, 3) mixed function oxidase (MFO) activation, and 4) mitochondria] respiratory toxicity. However, it still remains obscure whether the nephrotoxicity of (3-lactams is derived from a single mechanism or several simultaneous processes. It has already been reported that preloading of some tubular transport inhibitors, such as probenecid, p-amino hippurate (PAH), and benzyl penicillin, prevents CER-in duced nephrotoxicity (3). Despopoulos has also described that the carbonyl and carboxyl groups in the structure are indispensable for interaction with receptors when an or

ganic anion is actively transported via renal tubules (4). A high intracellular concentration of CER has been consi

Table

1.

N-Acyl

amino

acid

derivatives

examined

dered to be closely related to its nephrotoxicity (5), and therefore other compounds that have strongly inhibiting activities against CER tubular transport and thus the pos sibility of reducing CER nephrotoxicity have been investi gated in the present paper. The combination effect of several N-acyl amino acids (NAAs) on CER nephrotoxic ity was investigated in the rabbit. After a screening exami nation on the preventive efficacy of various NAAs against CER-induced nephrotoxicity, it was found that several NAAs (Table 1) showed good potential for preventing CER nephrotoxicity in rabbits. MATERIALS

AND METHODS

Evaluation of nephrotoxicity in rabbits Male Albino rabbits weighing 2.0 3.0 kg were pur chased and acclimated for at least one week before the ex periments. Rabbits were given intravenous doses of CER (75, 100, 150, or 200 mg/kg) with or without NAAs (50, 75, 100, 150, or 200 mg/kg). Cumulative urine samples were collected every day, and their protein and glucose levels were determined. Each rabbit was decapitated under anesthesia (30 mg/kg, pentobarbital) five days after the administration of the drugs, and the kidneys were immedi ately excised and weighed. A slice of the kidney cortex was observed under a microscope, after being fixed with 10010neutral formaldehyde and staining with hematoxy lin-eosin or PAS. Degrees of tubular necrosis were evaluat ed by the following rating system: very slight, very slight ly localized tubular necrosis; slight, very slight to less than 20010of tubular necrosis of the kidney; moderate, 20010to less than 50% of tubular necrosis of the kidney; and se vere, 50010and more of tubular necrosis of the kidney. Analytical Urinary bury-Clark titated by

methods protein was assayed according to the Kings method, and urinary glucose levels were quan a spectrophotometric glucose oxidase kit

(Glucose B-Test; Wako Pure Chemical Industries, Ltd., Osaka). NAAs were determined by a high-pressure liquid chromatograph (LC-6A; Shimadzu Co., Kyoto) equipped with a variable wavelength ultraviolet detector (SPD-6A, Shimadzu Co.). The conditions used for high-pressure liq uid chromatography were as follows: column, Cosmosil 5C18-P 15 cm x 4.6 mm I.D. (Nacalai Tesque Inc., Kyoto); mobile phase, 7-30% acetonitrile, 1010 acetic acid; flow rate, 1.0 ml/min; wavelength, 260 nm; injec tion volume, 10 pl; column temperature, 401C. Materials Cephaloridine

was purchased

from Shionogi & Co.

(Keflodin, Osaka) and Sigma Chemical Co. (St. Louis, MO, USA). Some N-acyl amino acids (N-benzoyl-(3

alanine, N-benzoyl-a-alanine, N-benzoyl-D,L-methionine and Na,e-dibenzoyl-D,L-lysine) and p-aminohippurate were purchased from Tokyo Kasei Kogyo Co. (Tokyo). Other N-acyl amino acids were synthesized by the coupling reaction of amino acids with benzyl chloride in our laboratory. The other chemicals used for the experi ments were of the highest purity available. RESULTS A single intravenous dose of CER (100 mg/kg) induced severe tubular necrosis in the rabbits. This nephrotoxic finding was remarkably improved by concomitant ad ministration of NAAs (Table 2). Compound (Compd.) 1 at 50 mg/kg showed a remarkable preventive effect on the nephrotoxicity induced by a CER dose of 100 mg/kg, but the same dose of Compd. 4 or Compd. 10 seemed in sufficient to prevent the nephrotoxicity. Compd. 1 or Compd. 4 at the dose of 100 mg/kg was remarkably pro tective, while Compd. 11 at the dose of 100 mg/kg was not. A larger dose of Compd. 11, 200 mg/kg, protected the kidney from the damage induced by CER at the dose of 100 mg/kg. Compounds 1, 4 and 10 were more preven tive than Compd. 11 and PAH against CER-induced nephrotoxicity. Against the nephrotoxicity induced by 150 mg/kg of CER, administration of Compd. 2, Compd. 3, Compd. 4, Compd. 5, Compd. 6, Compd. 9 or Compd. 10 at 75 mg/kg inhibited nephrotoxicity. Compd. 1 at 150 mg/kg partially protected the kidney and Compd. 3, Compd. 4, Compd. 5, Compd. 6, Compd. 9 or Compd. 10 at 75 mg/kg also prevented nephrotoxicity. Compd. 1 at 150 mg/kg partially protected the kidney and Compd. 2, Compd. 3, Compd. 4, Compd. 5 Compd. 6, Compd. 7, Compd. 8, Compd. 9 or Compd. 10 at 150 mg/kg remark ably protected the kidney from the damage by 150 mg/kg of CER. Compd. 1 at the dose of 300 mg/kg was remark ably preventive against the nephrotoxicity induced by 150 mg/kg of CER. Against a dose of 200 mg/kg CER, Compd. 9 at 100 mg/kg was not preventive. Compd. 1 or Compd. 7 at 200 mg/kg was not preventive, but Compd. 9 at 200 mg/kg was preventive against the nephrotoxicity induced by 200 mg/kg of CER. These results indicated that equal or larger doses of NAAs to those of CER were required to protect against the nephrotoxicity induced by a 150 mg/kg or higher dose of CER, whereas equal doses of most NAAs showed con siderable effectiveness against the nephrotoxicity induced by 100 mg/kg of CER. These results also suggested that the amount of CER secreted into the proximal tubular cells was so large, after administration of CER at 150 mg/kg or higher doses, that the CER concentration in the

Table

2.

Protective

effect

of NAAs

on CER

nephrotoxicity

in rabbits

Table

2.

continued

Table

2.

continued

tubular cells was not decreased to a nontoxic level. Since a therapeutic dose of CER is only one-tenth of 100 mg/kg, it is considered that the renal damage induced by a CER dose of not more than 100 mg/kg would be completely prevented by the coadministration of NAAs. The histopathological finding of CER-induced

nephro

toxicity is shown in Fig. 1. Severe necrosis of the prox imal tubules was observed five days after a single dose of CER at 100 mg/kg in a rabbit. Nevertheless, there were no changes observed around the glomeruli. The most striking characteristic of CER-induced nephrotoxicity was the extensive localized damage to the proximal tub ules, which

was remarkably

ous administration

prevented

of 100 mg/kg

by the simultane

of Compd

1.

DISCUSSION The intrinsic function of the kidney as an excretory organ results in its inevitable exposure to high concentra tions of not only endogenous but also exogenous com pounds that might induce nephrotoxicity due to direct action. Several drugs, including 'aminoglycoside or cephalosporin antibiotics, are known to cause functional and phathological changes in the kidney. CER nephrotox icity is especially characterized by severe necrosis of the proximal tubules and an obvious increase of urinary pro tein and glucose (Table 2). It was reported that typical features of CER-induced nephrotoxicity are decreased glomerular filtration rate, enzymuria, proteinuria, and glycosuria without hyperglycemia (6), often accompanied

by widespread necrosis of the S2 segment of renal prox imal tubules in both humans and laboratory animals (7). The glycosuria is considered to be derived from a func tional depression in the proximal tubular reabsorption of glucose (8, 9), since the main site of damage by CER was the proximal tubules of the kidney (7, 10, 11). Although multiple doses of CER cause chronic dysfunction not only in the renal tubules but also in the glomeruli, these acute nephrotoxic damages are reversible in many cases. Active accumulation of CER and the interaction of CER with basolateral membranes compared to brush border membranes were found in renal cortical slice preparations (3). Both tubular transport and nephrotoxic ity of CER were prevented in the presence of competitive inhibitors for organic acid transport, e.g., probenecid, PAH, and benzyl penicillin (5, 12-15); and it has been concluded that CER nephrotoxicity is related to the high intracellular concentration resulting from its active trans port (14-18). It also has been recently reported that co administration of a large volume of physiological saline reduced CER-nephrotoxicity (9). These results also sug gest that CER nephrotoxicity will be due to the active transport relating to a sodium ion transporter. In our present study, a PAH dose of 200 mg/kg, i.v. prevented the nephrotoxicity induced by a CER dose of 100 mg/kg, but PAH at 100 mg/kg did not prevent the nephrotoxic ity. Compounds 1 and 4 at the doses of 100 mg/kg were, however, preventive against the CER-induced nephro toxicity. In addition, some NAAs at doses of 100 mg/kg prevented the nephrotoxicity

induced by a CER dose of

Fig. 1. Histological features of rabbit renal tubules following in travenous administration of CER with or without Compd. 1. The renal cortex was observed five days after intravenous doses of CER at 100 mg/kg with or without Compd. 1 (100 mg/kg). The severe tubular necrosis induced by CER alone (upper left) was almost com pletely prevented by coadministration of Compd. 1 (upper right). The lower photo shows the control tissue sample. Renal tissue samples were stained with PAS and observed under a microscope.

150 or 200 mg/kg. There results suggest that the nephroprotective activities of the NAAs examined are more potent than that of PAH due to their higher phys icochemical lipophilicity. NAAs were abundantly secreted from the proximal tub ules, resulting from their high affinity to serum protein (e.g., the protein binding of Compd. 1 was about 95°10), and it is supposed that this pharmacokinetic attribute of NAAs is closely related to the preventive effect against CER-nephrotoxicity in rabbits. Future studies should re veal a more detailed mechanism of the preventive effect of NAAs against CER-induced nephrotoxicity and explain how NAAs inhibit the uptake and accumulation of CER in renal tubules. NAAs should be suitable for clinical use because of their generally low toxicity in animals (e.g., the LD50 of Compd. 2 was more than 3000 mg/kg, i.v. in the rat.). These results strongly suggested that some NAAs will be good candidates for reducing the nephrotoxicity induced not only by CER but also by other ~-lactam antibiotics. Acknowledgments The

authors

Fukushige able

6

7

8

9

10

11

12

13 are

deeply

for preparations

indebted

to

of histological

Dr.

M. Fukami

specimens

and

and their

Dr.

J.

valu

14

advice.

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15

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