Jpn. J. Oral Biol., 31: 89-94, 1989.

ORIGINAL

Hypocalcemic action of the several types of salicylic acid analogues

Yuzo Kato, Kazuhisa Nishishita, Hideaki Sakai, Masahiro Tatsumi and Kenji Yamamoto

Department of Pharmacology, Nagasaki University School of Dentistry, 7-1 Sakamoto-machi, Nagasaki 852, Japan

〔Accepted for publication: July 6, 1988•l

Key wards: Aspirin/salicylic acid/hypocalcemia/structure-activity relationship

Abstract: The present study was performed to see the structure-activity relationships on the aspirin- induced hypocalcemia. Several kinds of salicylic acid (SA) analogues administered orally with a stom-

ach tube. In general, the drugs were suspended in the 2% CMC solution. At the scheduled times after the treatment, 60 ƒÊl of the blood was collected to determine the level of calcium. Aspirin, sodium salt of o-hydroxybenzoic acid (Na-salicylate), sodium salt of m- and p-hydroxybenzoic acid (HBA), 2,5-

dihydroxybenzoic acid (DHBA), PAS sodium dihydrate (PAS-Na), salicylamide (SAM) and 2 % CMC control were used. Hypocalcemia was induced by aspirin and Na-salicylate but not by m-and p-HBA-

Na. In addition, DHBA and PAS caused hypocalcemia when they were administered intravenously but not orally. These results suggest that the carboxyl group must be adjacent to the hydroxyl group on the benzene ring to induce this type of hypocalcemia and that the SA structure would be able to

induce hypocalcemia, even in the presence of the additional third substituent on the same ring. On the comparison between aspirin-DL lysine (water soluble aspirin) and SA-DL lysine, SA-DL lysine, which is not an inhibitor of PG synthetase, was more effective on the hypocalcemic action than ASP-DL

lysine. The phenomenon was observed at the stage especially immediately after the intravenous injection, when the acetyl group may be more responsible to acetylate the PG synthetase in the aspirin-

DL lysine group. The present results seems to be consistent with the previous hypothesis that PGs are not involved in the process of aspirin-induced hypocalcemia in the rat.

pocalcemic effect is separated from the anti- Introduction pyretic-analgesic and antiinflammatory effects. Aspirin is widely used as an antipyretic, For the purpose, several drugs were admin- analgesic and antiinflammatory drug and also istered to experimental animals to demonstrate as an inhibitor of platelet aggregation in drug-induced hypocalcemia. Since the p.o. vivo1). To prescribe more safely on clinical administration of indomethacin2) and ketopro- treatment, we must have much data on the fen5), both of them are the potent analgesic side effects of aspirin. antiinflammatory agents, did not decrease the Hypocalcemia was induced dose-dependently blood-calcium level, the hypocalcemic action after the administration of aspirin2,3). A simi- seemed to be a different phenomenon from lar phenomenon was demonstrated by sodium the other useful effects. salicylic acid4,5). Another report demonstrat- The present study was performed to obtain ed that hypophosphatemia also has been ac- basic data on the relationships between the companied by hypocalcemia following the in- chemical structure and hypocalcemic effect af- travenous injection of sodium salicylate6). ter administration of drugs, especially, several It is important to see whether or not the hy- types of salicylic acid analogues. 90 Jpn. J. Oral Biol., 31: 89-94, 1989.

been known that PAS is readily absorbed and Materials and Methods that DHBA is poorly absorbed from the gastro-

Male rats of different strains were used. intestinal tract9). Therefore, each drug was

In the first experiment, the conventional rats dissolved in distilled water and administered

of the Wistar strain, and in the second and the intravenously with the same dose of 1.11

last experiment, the SPF rats of the Sprague- mmol/kg body weight to the non-fasting rats

Dawley strain were used, respectively. under light ether anesthesia. Physiological

Throughout the experiments, animals (5 to 7 saline was given as a control at the same

weeks age) were housed in wire-bottomed volume (0.2 ml/100 g of body weight).

cages (4 or 5 animals per cage) under a 12 hr In the last experiment, hypocalcemic effects light cycle for at least 5 days prior to use. were compared between aspirin-DL lysine They were given a standard diet (CE-2, (ASP-DL lysine) and salicylic acid-DL lysine Nippon Clea) and tap water ad libitum. Dur- (SA-DL lysine). Since ASP-DL lysine on ing the experimental period, the room temper- the market (Venopirin, Green Cross Co.

ature was kept at 22•Ž•}1•Ž and the rela- Ltd. Osaka Japan) contains calcium chloride

tive humidity at 55 to 75%. In the first exper- as stabilizer, ASP-DL lysine without calcium iment, animals were fasted overnight (from was prepared10) for the present experiment. 5:00 PM) to increase the efficiency of Preparation of ASP-DL lysine and SA-

drug absorption and then given food again 6 DL lysine hr after drug administration. Each drug was Solution A: 812 mg of DL lysine (free suspended in a 2 % CMC (Na-carboxymethyl base) was dissolved in 7 ml of physiological cellulose) solution and administered with a saline stomach tube, since the route of administra- Aspirin mixture: 1 g of aspirin was mixed tion of aspirin is practically always oral in with distilled water. When the Solution A the clinical use. The control rats were given was added to the Aspirin mixture in an ice the 2% CMC solution at the same volume bath, aspirin dissolved gradually becoming (1 ml/100g of body weight). The adminis- the solution of ASP-DL lysine. The solution tration of each drug was performed between was made up to 20 ml with physiological sa-

10:00 AM and 12:00 noon to minimize line. ASP-DL lysine solution was kept in changes due to diurnal variations in the value the ice bath till immediately before use to

for plasma calcium7,8). Aspirin (2.22 mmol/ avoid hydration of the aspirin. SA-DL lysine

kg), sodium salt of o-hydroxybenzoic acid solution was prepared by the same procedures.

(Na-salicylate, 2.50 mmol/kg), sodium salt of Each drug was administered intravenously m-and p-hydroxybenzoic acid (HBA, 2.50 with a dose of 1.11 mmol/kg body weight.

mmol/kg) or 2% CMC solution was admin- DL lysine solution was given as a control

istered (single dose) to 5 groups of 8 rats at the same volume (0.4 ml/kg of body

each. In addition, 3 types of salicylic acid(SA) weight). At the scheduled times after the

analogues were given to the other groups treatment, about 60 ƒÊl of blood was collected

of rats. Salicylamide (SAM), the amide of with a heparinized glass tube from the cut

SA, is one of the antipyretic analgesics. Para end of the tail of the unanesthetized rats.

aminosalicylic acid sodium salt (PAS-Na) After centrifugation, 20 ƒÊl of plasma was used

has a bacteriostatic effect but not an antipy- for the determination of total calcium in plas- retic analgesic one. 2,5-dihydroxybenzoic ma using an atomic absorption spectropho-

acid (2,5-DHBA, Gentisic acid) is one of tometer11). Since the blood was sampled sev-

the the metabolites of salicylic acid and has eral times within the short experimental pe-

an analgesic anti-inflammatory effect. Those riod, a minimum volume of blood was col-

drugs were administered with the equivalent lected only for the calcium determination to

dose of aspirin (2.22 mmol/kg). minimize the loss of body fluid. Throughout

In the second experiment, sodium salts of the experiments, each drug was administered SA analogues, m-HBA-Na, p-HBA-Na, PAS- immediately after the blood sampling for the

Nay and 2,5-DHBA-Na, were used. It has 0-hr group. Statistical analyses were per- Y. Kato. et al: Salicylic acid analogues and hypocalcemia 91

Table 1-a Effect of aspirin and three types of hydroxybenzoate administered orally on the plasma calcium level.

Table 1-b Effect of three kinds of the salicylic acid analogues administered orally on the plasma calium level.

Experiments employed the same procedures as described in Table 1-a. Dose of each drug: 2.22 mmol/kg of body weight. Values shown represent mean values and SD (in parentheses) for 6 male rats in each group. No significant difference is found in each group from control.

formed using student's t test. der the non-fasting condition. Both m- and p-HBA-Na, also, did not change the calcium Results level in plasma. On the contrary, a slight The hypocalcemia following the admin- but significant decrease was observed in both istration of aspirin and sodium salicylate is groups of PAS-Na and 2,5-DHBA-Na (Ta- shown in Table-1 a. The plasma calcium ble 2). level decreased with time after the p.o. As shown in Fig. 1, DL lysine solution did administration of both aspirin and sodium not have an effect on the plasma calcium salicylate and reverted to the normal range level. On the other hand, both ASP-DL lys- at 24 hr. On the other hand, both groups ine and SA-DL lysine significantly decreased of m-and p-HBA-Na and other 3 drugs, SAM, plasma calcium level when compared with the PAS-Na, 2,5-DHBA, did not show any signif- control throughout the experimental periods icant change from the CMC control (Table (from 30 min to 6 hr). Between the 2 groups 1 a, 1 b). of rats, the value of the ASP group was After the intravenous injection, the plasma not different from that of SA group except calcium level was maintained throughout the 30 min after the administration. In the peri- experimental period in the control group un- od (from 0 to 30 min), the net decrease of 92 Jpn. J. Oral Biol., 31: 89-94, 1989.

Table 2 Effect of salicylic acid analogues administered intravenously on the plasma calicium level.

logues used in the present experiments are shown in Fig. 2. Hypocalcemia was induced by aspirin, SA and sodium salicylate but not by m-and p-HBA-Na. These observations suggest that the carboxyl group must be adja- cent to the hydroxyl group on the benzene ring to induce this type of hypocalcemia. The same structure-activity relationship has been known in the antipyretic-analgesic effect and demonstrated in the inhibition of dentine formation of rats12). SAM has not the carboxyl group and no effect on plasma calcium level, probably due to not being metabolized to SA in vivo13) Saito et al. has prescribed that SA ana- logues showing hypocalcemia were compounds finally metabolized SA14). Also, the content of SA in plasma was correlated with the con- tent of calcium in plasma after the p. o. ad- Fig. 1 Hypocalcemia induced by aspirin-DL lys- ministration of aspirin (unpublished data). ine (open circle) and salicylic acid-DL The concentration of SA in blood may be lysine (closed circle). Each treatment was administered intravenously (1.11mmol/kg). involved in the mechanism of the phenome- DL lysine solution (triangle) was given non. In addition to these facts, the influ- as a control with the same volume (0.4 ence of the third substituent added to the SA ml/100g). Values shown represent mean structure is complicated. Both DHBA and values and SD for 10 rats in each group. PAS-Na caused hypocalcemia when they were Significantly different from control: ap< administered intravenously but not orally. 0.001, b0.001excretion types of SA Discussion metabolites13). Therefore, the hypocalcemia The chemical structure of the SA ana- induced by both PAS-Na and 2,5-DHBA- Y. Kato. et al: Salicylic acid analogues and hypocalcemia 93

Fig. 2 Chemical structures of several types of salicylic acid analogues used in the present experiment. HBA: hydroxybenzoic acid, SAM: salicylamide, DHBA: dihydroxy- benzoic acid, PAS: p-aminosalicylic acid

Na may not result from the SA in blood. Thus, ASP-DL lysine administered intrave- Probably, SA structure would be able to in- nously has been expected to inhibit the PG duce hypocalcemia by an unknown mechanism, biosynthesis most effectively, especially at the even in the presence of the additional third early period of the experiment. On the con- substituent. trary, SA-DL lysine, which is not an inhibitor In general, aspirin is rapidly hydrolyzed of PG biosynthetase, was more effective on before it enters the circulation after the p.o. the hypocalcemic action than ASP-DL lysine administration15). After the i.v. injection of in the present experiment (Fig. 1). ASP-DL lysine, aspirin is also rapidly hydro- These results seem to be consistent with lyzed becoming salicylate in blood. Recently, the previous hypothesis2,5 that the inhibition Saegusa16) demonstrated that, after the i.v. of PG biosynthesis may not be involved in the injection of Venopirin (1.0 mmol/kg and 2.5 process of aspirin-induced hypocalcemia. mmol/kg), the concentration of aspirin de- The mechanism of action, however, is not creased with time and that it was undetectable clear, yet. Additional work is required to even 2 hr after the injection. It has been see the mode of action of salicylates and to known that the ability to acetylate the pros- obtain more data on the adverse effects of taglandin (PG) synthetase (cyclooxygenase) is aspirin. an important mode of action of aspirin17).

抄 録:aspirin投 与 に よ る 実 験 的 低Ca血 症 の 発 現 機 序 を 解 明 す る 目 的 で, salicylic acid(SA)に 類 似 し

た 化 学 構 造 を 持 つ 薬 物 を 用 い て 構 造 活 性 相 関 に 関 す る 基 礎 的 実 験 を 行 っ た.薬 物 の 一 部 を2%CMC水 溶 液

と 混 和 し,一 晩 絶 食 さ せ た ラ ッ トに,そ れ ぞ れaspirin(2.22 mmol/kg),o-,m-,p-HBA・NA(hydroxy-

benzoic acid,2.50 mmol/kg)の 用 量 で 胃 内 投 与 した 。 そ の 後,経 時 的 に60μlの 採 血 を 行 い 血 漿Ca値 を 測

定 し た 。 絶 食 は6時 間 群 の 採 血 後 に 解 除 した.低Ca血 症 は, aspirinとo-HBA・Na投 与 群 に お い て 従 来 の 報 告 どお り に 確 認 さ れ た 。 一 方,OH基 がCOOH基 に 対 し て,そ の 他 の 位 置 に あ る よ う なHBAの 構

造 異 性 体 に お い て は,低Ca血 症 が 発 現 しな か っ た 。 ま たCOOH基 が ア ミ ド化 し たsalicylamideも 低Ca

血 症 を 発 現 しな か っ た 事 か ら,ベ ン ゼ ン 核 上 にCOOH基 とOH基 がorthoの 位 置 関 係 に あ る 事 が,こ の

現 象 の 発 現 に と っ て 必 須 で あ る と思 わ れ た.こ の 条 件 を 満 た し て,し か も第3の 置 換 期 が 同 じベ ン ゼ ン 核 上 94 Jpn, J. Oral Biol., 31: 89-94, 1989.

に 存 在 す る 薬 物 で も,aspirinに 比 し て 程 度 の 弱 い 低Ca血 症 が 発 現 し て い た 。 次 にaspirinとSAを そ れ

ぞ れDL lysineに 溶 解 し て 静 脈 内 投 与 し,低Ca血 症 を 発 現 さ せ た と こ ろ,最 初 の30分 に お け る反 応 性 は

aspirinの 方 が 弱 く,以 後,両 群 間 に 差 が な か っ た 。aspirin-DL lysineを 静 注 す る と 初 期 に はcyclooxyge-

nase を 効 率 よ く ア セ チ ル 化 す る の で, PGの 関 連 し た 生 体 の 反 応 に 関 し て は,経 口 投 与 のaspirinよ り速

効 性 で あ る と報 告 さ れ て い る.し か し本 実 験 で は 前 述 の ご と く,静 注 後 初 期 に は か え っ てaspirin-DL lysine の 方 がSA-DL lysineよ り も 低Ca血 症 の 進 行 は 遅 延 し た の で, aspirin由 来 の 低Ca血 症 の 発 現 に ア セ チ

ル 化 反 応 は 関 与 して い な い の で あ ろ う と考 え られ る 。 封 上 の よ う に 今 回 の 実 験 結 果 は,aspirin由 来 の 低Ca

血 症 は 生 体 内 で のPGの 作 用 と は 切 り離 し て 考 え る べ き 現 象 で あ る と い う従 来 の 報 告 を 支 持 す る も の で あ る

と 考 え る 。

1952. References 10) Yamanouchi, K., Iwai, M., Murashima, R. 1) Irino, O., Saitoh, K. and Ohkubo, K.: An- and Yokoyama, K.: Metabolism and excre- ti-platelet actions of salicylates; In vivo, ex tion of Venopirin in rabbits and rats-experi- vivo and in vitro effects of choline salicylate. ments by the use of carboxyl 14C labelled Folia phantacol. japan. 86: 17-23, 1985. aspirin-. In: Venopirin medical literatures (in Japanese with English abstract) (1) pp. 46-55, The Green Cross Co., Osaka, 2) Kato, Y., Senzaki, H. and Ogura, H.: As- Japan, 1983. (in Japanese) pirin-induced hypocalcemia in the rat. Toxi- 11) Trudeau, D.L. and Freier, E.F.: Deter- col. Appl. Pharmacol. 64, 64-71, 1982. mination of calcium in urine and serum by 3) Ueno, K., Masumura, H., Shimoto, Y., Kita- atomic absorption spectrophotometry (AAS). gawa, H., Yokoyama, A., Sakai, T. and Clin. Chem. 13: 101-114, 1967. Saito, H.: Species differences in hypocalce- 12) Sonobe, Y.: Studies on the inhibitory ac- mia induced by acetylsalicylic acid. Res. Co- tion of sodium salicylate on dentine forma- mm. Chem. Path. Pharmacol. 36: 383-393, tion in rat incisor. Jpn. J. Oral Biol. 23: 1982. 879-892, 1981. (in Japanese with English 4) Kato, Y. and Ogura, H.: Effect of the non- abstract) steroid antiinflammatory drugs on the exper- 13) Flower, R.J., Moncada, S. and Vane, J. imental ectopic calcification in the rat. R.: Analgesic-antipyretics and anti-inflam- Bone Metabolism 14: 29-34, 1981. (in Japa- matory agents; Drugs employed in the treat- nese with English abstract) ment of gout. In: Goodman and Gilman's 5) Ueno, K., Masumura, H., Kitagawa, H., The Pharmacological Basis of Therapeutics Naminohira, S., Saito, H. and Sakai, T.: (Goodman Gilman,. A., Goodman L.S., Hypocalcemic effect of acetylsalicylic acid in Rail, T. and Murad, F. eds.) 7 th ed., pp. rats. J. Pharm. Dyn. 4 229-231, 1981. 674-715, Macmillan pub. New York, 1985. 6) Yamada, S. and Morohashi, T.: Effect of 14) Saito, H., Yokoyama, A., Takeno, S., sodium salicylate on renal handling of cal- Sakai, T., Ueno, K., Masumura, H. and cium, phosphate and magnesium. Folia Kitagawa, H.: Fetaltoxicity and hypocalce- pharmacol. japan. 88: 395-401, 1986. (in mia induced by acetylsalicylic acid analogues. Japanese with English abstract) Res. Comm. Chem. Path. Pharmacol. 38: 7) Talmage, R.V.: Effect of fasting and par- 209-220, 1982. athyroid hormone injection on plasma 45Ca 15) Packham, M.A.: Mode of acetylsalicylic concentrations in rats. Calcif. Tiss. Res. acid. In: Acetylsalicylic Acid. (Barnett, H. 17: 103-112, 1975. J.M., Hirsh, J. and Mustard, J.F. eds.) pp. 8) Shinoda, H. and Seto, H.: Diurnal rhythms 63-82, Ravan Press, New York, 1982. in calcium and phosphate metabolism in 16) Saegusa, T.: The effects of aspirin DL- rodents and their relations to lighting and lysine and sodium salicylate on dentine for- feeding schedules. Miner. Electrolyte Metab. mation in the rat incisor. Jpn. J. Oral Biol. 11: 158-166, 1985. 30: 559-571, 1988. 9) Arnott, D.G. and Doniach, J.: The ef- 17) Roth, G.J., Stanford, N. and Majerus, P. fect of compounds allied to resorcinol upon W.: Acetylation of prostaglandin synthe- the uptake of radioactive iodine (131I) by the tase by aspirin. Proc. Nat. Acad. Sci. 72: thyroid of the rat. Biochem. J. 50: 473-479, 3073-3076, 1975.