Nature of the Uricosuric Effect of Tienilic Acid, a New Diuretic

Kidney International, Vol. 12 (1977), pp. 104—114

Nature of the uricosuric effect of tienilic acid, a new diuretic

Gu LEMIEUX, ANDRE Kiss, PATRICK VINAY and ANDRÉ GouGoux with the technical assistance of ANDREE QUENNEVILLE

Nephrology-Metabolism Division and Renal Laboratory, Hbtel-Dieu Hospital, and the Department of Medicine, University of Mont real ,Montreal,Quebec, Canada

Nature of the uricosuric effect of tienilic acid, a new diuretic. uricosurique n'a été observe chez Ic chien dalmatien. Chez cet Tienilic acid (2,3-dichloro-4-[2.thienylcarbonyl]phenoxyacetic animal Ic rapport Cur/Cc, baisse de 1,15 1,0 aprés l'ad- acid)is a new diuretic with uricosuric properties in man. Experi- ministration d'acide tiénilique. L'acide pyrazinoique adminis- ments were designed in the mongrel dog, Dalmatian coachhound, tré par voie intraveineuse a des chiens batards, charges en urate, rat, guinea pig, rabbit, and chicken to clarify the nature of tienilic avant ou aprCs l'acide tiénilique n'influence pas Ia rCponse uricosu- acid-induced uricosuria. It was demonstrated that tienilic acid is rique. Des etudes de diurèse interrompue montrent que I'acide the most potent uricosuric agent yet reported in the mongrel dog. tiénilique bloque Ia reabsorption nette de l'urate dans le segment Following the iv.administrationof tienilic acid (50mg/kgof body proximal du néphron. II est aussi dCmontré que l'acide tiénilique wt), the ratio of urate clearance over exogenous creatinine clear- deprime de facon importante le transport du PAH aussi bien aux ance (Cur/C)rosefrom 0.41 to 1.0 within 10 mi this effect fortes (20 mg/l00 ml) qu'aux faibles (I mg/l00 ml) concentrations persisting for another 20 mm and declining progressively thereafter plasmatiques. Ainsi, Ia clearance du PAH ne peut pas être utilisée to a value of 0.70 at 100 mm. The effect was the same in urate- pour mesurer Ic debit plasmatique renal si l'acide tiCnilique a etC loaded and unloaded animals. No uricosuric effect was noted in the administrC, a moms que l'extraction rénale du PAH ne soit eva- Dalmatian coachhound. In this animal, mean Cur/Ccrfellfrom luée. Nous avons montrC que l'acide tiCnilique est aussi uricosu- 1.15 to 1.00 following tienilic acid. Pyrazinoic acid given iv, to rique chez Ic rat, bien qu'â un degré moindre que chez Ic chien. urate-loaded mongrel dogs before or after tienilic acid did not Malgré un effet natriurétique considerable l'acide tiénilique a une influence the uricosuric response to the drug. Stop-flow studies in action anti-uricosurique significative chez les animaux qui secre- dogs revealed that tienilic acid blocks net urate reabsorption in the tent l'urate, tels le cobaye, Ic lapin et Ic poulet. Ce travail montre proximal segment of the nephron. It was also demonstrated that que I'acide tiCnilique peut modifier de facon importante soit le flux tienilic acid depresses para-aminohippurate (PAH) transport de reabsorption soit celui de secretion de I'urate. Cette action a lieu markedly both at high (20 mg/l00 ml) and low (I mg/l00 ml) dans le tube proximal et parait Ctre indCpendante de I'activité concentrations of plasma PAH. Thus, PAH clearance cannot be natriurétique du compose. II est probable que Ia drogue entre en used to measure renal plasma flow following tienilic acid unless the competition dans Ia vole commune des acides organiques (PAH) et renal extraction of PAH is estimated. We have shown that tienilic que le transporteur commun a beaucoup plus d'affinité pour l'acide acid is also uricosuric in the rat, albeit to a lesser degree than in tiénilique que pour Ic PAH. the dog. In spite of a considerable natriuretic effect, tienilic acid had a significant antiuricosuric action in urate-secreting animals like the guinea pig, rabbit, and chicken. The present studies dem- Tienilicacid (2, 3-dichloro-4-[2-thienylcarbonylj onstrate that tienilic acid may affect markedly either the reabsorptive or the secretory flux of urate. This action takes place in the phenoxyacetic acid) is a new diuretic with a chemical proximal tubule and appears to be independent of the natriuretic structure (Fig. I) resembling that of ethacrynic acid activity of the drug. It is probable that the drug competes for the [I]. This heterocyclic derivative of phenoxyacetic acid common (PAH) organic acid pathway and that the common carrier has far more affinity for tienilic acid than for PAH. was recently synthetized in France [I]. its natriuretic effect is believed to take place in the cortical diluting Nature de I'effet uricosurique de l'acide tiénilique, un nouveau segment of the distal nephron [2, 3]. In contrast to all diurétique. L'acide tienilique [2,3-dichloro-4(2-thienyl carbonyl) acide phenoxyacetique] est un nouveau diurétique qui posséde des other diuretics, perhaps with the exception of the propriétés uricosuriques chez !'homme. La nature de I'uricosurie organo-mercurials [4], tienilic acid has been shown to induite par l'acide tiénilique a óté étudiée au moyen d'expériences exert a significant and prolonged uricosuric effect in réalisées chez le chien batard, Ic chien dalmatien, le rat, Ic cobaye, le lapin et le poulet. II a été démontré que l'acide tiénilique est Ic plus man [5,6].Such an effect has also been reported in puissant uricosurique connu a l'heure actuelle chez le chien batard. the mouse by the team responsible for its synthesis Après l'administration d'acide tiénilique (50mg/kg)par voie in- [I]. The nature and site of the uricosuric effect of traveineuse Ic rapport de Ia clearance de l'urate a celle de Ia tienilic acid has not been established, One study in créatinine exogène (Cr/Ccr)passede 0,41 a 1,0 en dix minutes. Cet effet persiste pendant encore 20 mm puis decline progressive- man suggested that tienilic acid inhibits urate reab- ment pour atteindre une valeur de 0,70 a 100 mm. L'etl'et est le sorption distal to the site of urate secretion [7], while même que l'animal soit ou non chargé en urate. Aucun effet another suggested inhibition of reabsorption at a pre-

Received for publication June 14, 1976; secretory site in the nephron [3]. The present study and in revised form January 27, 1977. was undertaken in the mongrel dog, the Dalmatian ® 1977, by the International Society of Nephrology. coachhound, the rat, the guinea pig, the rabbit, and

104 Uricosuric eJjèct of tienilic acid 105

(lot 5227 A, ICN Pharmaceuticals Inc., Plainview, NY) was administered iv. as a prime (30 mg/kg), followed by a sustained infusion (0.6 mg/kg/mm) before or after the administration of tienilic acid. Six mongrel dogs not loaded with urate were used as controls and were given an i,v. single bolus of OCH2COOH furosemide (Lasix) (5 mg/kg) over a 5-mm period. Also, ethacrynic acid (Edecrin) (2 mg/kg) was ad- Tienilicacid ministered to another group of five animals loaded Fig. I. Chemical structure of tienilic acid (2, 3-dichloro-4-12- with urate. thienylcarbonylJ phenoxyacetic acid). The experimental protocol used in mongrel dogs to study the effect of tienilic acid on urate excretion was the chicken to clarify the nature of tienilic acid-in- also used in five urate-loaded and four unloaded duced uricosuria. pedigreed Dalmatian coachhounds. Endogenous plasma urate averaged 0.3 mg/lOO ml Methods in nonloaded mongrel dogs, while plasma urate aver- Studies in dog. Fifty-nine experiments were per- aged 3.0 mg/l00 ml in the urate-loaded animals. In formed on 59 mongrel dogs and 18 experiments on 9 the unloaded Dalmatian coachhounds, plasma urate pedigreed Dalmatian coachhounds. All dogs were averaged 0.7 mg/l00 ml and 2.4 mg/lOO ml in urate- female, weighing between 15 and 34 kg. The animals loaded dogs. were anesthetized with sodium pentobarbital (30 In all experiments, exogenous creatinine clearance mg/kg) and intubated. The endotracheal tube was was taken as the measure of glomerular filtration connected to a volume and rate-adjustable respira- rate. Standard PAH clearance was used to measure tion pump (Harvard Apparatus Co., Inc., Millis, renal plasma flow during control periods, but could MA)tomaintain plasma carbon dioxide tension not be used to measure changes in renal plasma flow (Pco2)around40 mm Hg.Heparinizedbloodsam- after the administration of tienilic acid since this drug ples were drawn from the femoral artery. All solu- has a marked depressing effect on the renal extraction tions were delivered to a femoral vein with a Bow- of PAH (see Results). PAH clearance corrected for man-type infusion pump at the rate of 5.0 mI/mm. renal extraction was measured in ten experiments. Adequate and stable urine flow was achieved by in- In order to ascertain that an isotonic solution con- fusing an isotonic solution containing half 5% manni- taining half 5% mannitol and half 0.9% sodium chlo- tol and half 0.9% sodium chloride. Tienilic acid (SKF ride has no significant effect on urate excretion, this 62698) was administered i.v. as a single bolus diluted solution was infused alone at 5 mI/mm continuously in 5% glucose over a 5-mm period at a dosage of 50 during 120 mm to nine mongrel (five urate-loaded mg/kg to 39 mongrel and 9 Dalmatian dogs. This and four unloaded) and nine Dalmatian (five urate- dosage was chosen because preliminary experiments loaded and four unloaded) (previously used) dogs. using a lower dosage (of 5 to 50 mg/kg) showed that Five stop-flow experiments using the left kidney 50 mg/kg induced the optimal effect on urate excre- were performed on five urate-loaded mongrel dogs tion. All clearance periods were of 10-mm duration, before and after the administration of tienilic acid, and samples from 10 periods were collected after the according to a procedure already reported from this administration of the drug. Five mongrel dogs re- laboratory [9]. Inulin-methoxy-3H (New England ceived tienilic acid in the absence of urate-loading. In Nuclear, Boston, MA) (160 tCi) was used as the ten animals, urate-loading was achieved by adding to glomerular marker. Five minutes before the release of the infusion uric acid neutralized to pH 7.40 with the ureteral clamp, 200 mg of PAH was injected. sodium hydroxide. In five dogs, the effect of tienitic Clamping time was 6 mm. Sixty serial urine samples acid was studied during continuous para-amino- of approximately 0.5 ml were collected on each occa- hippurate-loading (plasma PAH, 20 mg/l00 ml) and sion. simultaneous urate-loading. In these five experi- Siudies in rat. Eleven experiments were performed ments, a catheter was introduced in the renal vein by in II female Sprague-Dawley rats weighing between a technique already reported [81 to study the effect of 500 and 600 g. Following pentobarbital anesthesia tienilic acid on renal extraction of PAH. This was i.p. (4 mg/I00 g), the left jugular vein was catheter- also done in five urate-loaded mongrel dogs to study ized for the infusion of 0.06 mI/mm of the isotonic the renal extraction of PA H at low plasma concentra- mixture of half 5% mannitol and half 0.9% sodium tion (I mg/l00 ml). In nine animals, pyrazinoic acid chloride, and containing adequate amounts of 14C- 106 Lemieuxet a! inulin for the measurement of glomerular filtration Tienilic acid was given i.v. as a single bolus of 50 rate. The right carotid artery was also catheterized to mg/kg over 5 mm. Urate excretion was followed at obtain blood samples. The animals were then placed 10-mm intervals during 50 mm following tienilic acid in a temperature controlled cabinet (38°C) and con- administration. Heparinized blood samples were nected to a volume and rate adjustable respirator taken from a polyethylene cannula inserted in the (Harvard Apparatus Co., Millis, MA) following wing artery. Each ureter was catheterized after the cannulation of the trachea. Plasma Pco2 was main- onset of diuresis and fluidization of urine. The cath- tained around 40 mm Hg. The abdominal cavity was eters were tied to the cloacal mucosa to avoid any opened and a catheter (PE-lO) inserted in each ureter. leaks, as previously described [111. The whole surgical procedure took less than 50 mm. Analytical methods. Uric acid in plasma and urine Tienilic acid (50 mg/kg) was administered i.v, after was measured on the Auto-Analyzer by the method two 10-mm control clearance periods, and six 10-mm of Brown [101. The validity of this method for mea- urine collections were obtained following drug ad- surement of urate in plasma and urine in dog, rat, ministration. Five rats were urate-loaded (plasma guinea pig, rabbit, and chicken has been previously urate, 3.2 mg/lOO ml), and six animals were not established in our laboratory [11]. This method in- loaded (plasma urate 0.8 mg/l00 ml). troduces only a slight (7%) error in the measurement Studies in guinea pig. Six experiments were per- of urate in the plasma of rabbits [11]. It was ascer- formed on six female guinea pigs weighing between tained that tienilic acid does not influence the mea- 600 and 900 g. The animals were not loaded with surement of uric acid by this method. Para-amino- urate. The same protocol was used as in the rat, hippurate clearance was determined by the method of except for the rate of administration of the isotonic Bratton and Marshall, as modified by Smith et al mannitol-saline mixture which was administered at [12]. The method of Bonsnes and Taussky [13] was 0.10 mI/mm. used to measure creatinine. '4C-inulin, inulin-me- Studies in rabbit.Fourteenexperiments were per- thoxy-3H, and urate-2-'4C concentrations were formed on 14 New Zealand, white, male rabbits counted with a liquid scintillation spectrometer weighing between 1.6 and 4.2 kg. Light anesthesia (Searle-Nuclear-Chicago, model Isocap-300), as pre- was induced by sodium pentobarbital (15 mg/kg) viously described [9]. No significant difference in administered through the marginal vein of the quenching was noted between samples. Sodium and ear. Adequate body temperature was maintained potassium in plasma and urine were measured by throughout each experiment with a heating lamp. flame photometry. Blood pH and Pco2 were mea- Blood samples were obtained from the carotid artery, sured with appropriate electrodes [11]. Statistical and all solutions were delivered to a jugular vein with analysis was performed using the conventional Stu- a Bowman-type infusion pump at the rate of 2.7 dent's ttestfor paired data, Results are reported in mI/mm. Exogenous creatinine clearance was used to the text as means SEM.Throughoutthe text, the measure glomerular filtration rate. Like in the dog, term "significant" is used to describe a difference standard PAH clearance could not be used to mea- which has a P value of less than 0.05. sure renal plasma flow variations. Six animals received 5% mannitol alone, while eight animals were Results studied during urate-loading with a mean plasma Studies in dog. As shown in Figure 2, the adminis- concentration of 7.50.90 mg/100 ml. In nonloaded tration of tienilic acid to mongrel dogs without urate- animals, plasma urate averaged 0.3 0.02 mg/l00 loading led to a marked increment in uric acid excre- ml. Tienilic acid was given as a slow single injection tion, the ratio of urate over exogenous creatinine of 5-mm duration at a dosage of 50 mg/kg. Five 10- clearance (Cur/Ccr) rising from 0.41 0.06 to a mm clearance periods were obtained after the injec- value near unity (P < 0.01) within 10 mm after the tion of tienilic acid. administration of the drug. This effect persisted for Studies in chicken. Six experiments were performed another 20 mm and declined progressively thereafter on six laying, white, leghorn hens weighing between to a value of 0.700.09 at 100 mm. In these experi- 1.7 and 2.7 kg. Endogenous plasma urate ranged ments, exogenous creatinine clearance did not de- from 5.7 to 43.9 mg/lOO ml (mean, 25.1 6.7 crease by more than 20%, while urate clearance in- mg/lOO ml). The unanesthetized birds were re- creased 2.5-fold at the peak of the uricosuric effect. In strained with outspread wings. In all experiments, the 10 urate-loaded mongrel dogs the results were glomerular filtration rate was measured by inulin similar, Cur/Ccr increasing from 0.64 0.03 to 1.00 clearance using 14C-inulin. The animals were infused 0.05 (P < 0.01) (Fig. 2). This high value was with 5% mannitol at 2.0 mI/mm in the wing vein. unchanged throughout the 100 mm during which the Uricosuric effect of tienilic acid 107

o SKF 62698, 50 mg/kg, no urate-loading (N=5) evident that comparable natriuresis and kaliuresis • SKF 62698, 50 mg/kg, urate-loading (N=10) were achieved with each drug. The fractional excre- AFurosemide,5 mg/kg, no urate—loading (N=6) • Ethacrynic acid, 2 mg/kg, urate-loading (N = 5) tion of sodium (FENa) was greater with furosemide and ethacrynic acid because these agents had a 1.5- Drug greater depressing effect on glomerular filtration rate than tienilic acid. In spite of similar electrolyte excretion, it is evident that only tienilic acid had a marked 00 and continued effect on urate excretion. The lack of . 1.0 effect of either furosemide or ethacrynic acid on urate 0 - excretion is independent of plasma urate concentration since the animals receiving ethacrynic acid were urate-loaded, while those receiving furosemide were 0.5 not. A marked fall in PAH clearance was noted in all the experiments with tienilic acid. This is due to the fact that tienilic acid has a spectacular effect on the renal extraction of PAH. A detailed study performed I I I I I I I —20 0 20 40 60 80 100 at low and high plasma PAH concentrations is shown Time, rn/fl in Table 3. Standard PAH clearance fell by 56% (P < Fig.2. Uricosuric effect of tienilic acid in unloaded and urate-loaded 0.01) one hour after the administration of tienilic acid mongrel dogs. at low plasma PAH concentration and the PAH clearance approached glomerular filtration rate. The clearance periods were obtained. In these experi- 60% fall in PAH extraction (P < 0.01) fully explains ments, creatinine clearance did not change, while the observed fall in PAH clearance. When corrected urate clearance increased markedly. A representative for renal extraction, PAH clearance showed no sig- experiment in a urate-loaded mongrel dog is shown in nificant variation over control for at least 100 mm Table 1. In contrast to tienilic acid, furosemide ad- after the administration of tienilic acid. At high ministered to dogs without urate-loading or ethacry- plasma PAH concentration sufficient to saturate the nic acid given to urate-loaded animals had no effect tubular secretion process, tienilic acid had the same whatsoever on urate excretion (Fig. 2 and Table 2). depressing effect on PAH clearance (P < 0.02), while Sodium and potassium excretion during the adminis- the latter showed no change whatsoever when cor- tration of each diuretic is shown in Table 2. It is rected for renal extraction. Maximal tubular secre-

Table 1. Effect of tienilic acid on urate excretion during PA H-loading in a urate-loaded mongrel dog Plasma Plasma PAH Time PAH urate V Cçr Cur CPAH TPAH extraction CPAHO rn/n mg/IOU ml mg/IOU ml mi/mm mi/mm mi/mm Cur/Ccr mi/mm mg/rn/n % mi/mm Prime; 900 rng of creatinine and 2 g of PA H —75 Infusion: creatinine. 0.2 mg/kg/mm; PAH, 1.0 mg/kg/rn/n; uric acid; 0.3 mg/kg/mm; in half 5% mannitol and half 0.9% sodium chloride at 5 mi/mm —3Oto —20 31.3 2.6 1.1 65.6 36.3 0.55 98.3 10.24 42.8 229.5 —2Oto —10 31.3 3.2 1.2 68.5 37.7 0.55 100.8 10.11 38.0 265.3 —lOto 0 29.9 3.3 1.3 71.9 39.6 0.55 97.0 7.50 41.6 233.2 0 to 5 Tieniic acid, 50mg/kg i.v. Sto 15 30.0 3.8 4.4 55.8 60.1 1.08 54.9 —0.27 25.4 216.1 15 to 25 31.9 3.9 5.5 62.9 68.7 1.09 63.6 +0.22 30.6 207.8 25 to 35 33.0 4.0 4.8 58.1 62.3 1.07 52.7 —1.78 34.6 152.3 35 to 45 30.9 3.8 4.7 59.8 65.6 1.10 65.0 + 1.61 30.2 215.2 4Sto 55 31.3 3.8 4.4 57.2 61.4 1.07 62.9 +1.78 29.2 215.4 55 to 65 31.3 3.7 3.7 60.8 60.0 0.99 67.7 +2.16 31.2 217.0 6Sto 75 31.1 3.8 3.5 56.7 57.8 1.02 62.5 +1.80 30.9 202.3 75 to 85 31.3 4.0 3.0 53.9 51.5 0.96 51.2 —0.85 31.5 162.5 85 to 95 32.3 3.9 3.1 55.1 54.9 1.00 51.5 —1.16 35.0 147.1 95to 105 31.3 3.9 3.0 55.1 54.9 1.00 53.0 —0.66 36.2 146.4 Female mongrel dog no. 101, 19.0 kg, May 18, 1976. =PAHclearance corrected for renal extraction. Other abbreviations used are: C =clearance,Cr =creatinine,Ur =urate, TPAH= transportmaximum for PAH, V =urineflow. 108 Lemieuxet a!

0 tion of PAH was depressed by 80% (P < 0.01)(Table ——CO-r--.')— 0 3). Thus, standard PAH clearance cannot be used to N 6.) >41-H-H+41-41 C) enr—c —0'0en'0enC)-) C)) '000 measure variations in renal plasma flow following NNN N N N N 0. 00N -00 tienilic acid, unless it is corrected for renal extraction. 010 In the five urate-loaded and four unloaded Dalma- N 40 00 0 000 —• enenen enen tian coachhounds, the ratio of Cur/Ccr was slightly 00 ' -H-H-H -4-H-Hi 01 above unity (1.15 0.07) prior to tienilic acid admin- 00 '00\ 000000'0-N '0 'l C C') istration. This value fell to unity (P < 0.01) following 0 the administration of tienilic acid and remained at or N0000—en '0 0'0 en en . Nen —N N l-)NN •0 CO en en 0Ct near unity (Fig. 3). In these experiments, exogenous 0 •0 C- CO 0 -H -ft 41 +-H+H-H41-H-H 0 creatinine clearance did not decrease by more than . C)-) C)-N 0 en 00— 00-0000\ C')0 en N — 0 E C) C')00000 'l- Cli en en 0 15%, but PAH clearance fell by 40% for the reason 0 CO .0 4) C) mentioned previously. In these animals, the natri- 0 —OC00 —00 6.) uretic effect of tienilic acid was similar to that noted 0) 0000 000 000 CO -H-H-H-H-H-H-H CO in the mongrel dogs. 0 C—00—en——o0— CO In the nine mongrel dogs and the nine Dalmatian 0 000do000 000 0 coachhounds, either loaded or unloaded with urate 0 Ci 0 C- 0 N en N Cli — ;C. which received the isotonic mannitol-saline mixture 4) Nr—N 00 C.) >. z 01 —-H-H-H +-41-H-H-H continuously during 120 mm in the absence of admin- 4) — — 'ON N — en00 Q Cl- enCii —000000N 4) CO CO istration of any drug, Cur/Ccr remained unchanged, 0 CO as well as PAH excretion. C.) 0 0 NNN Cfi—N The effect of pyrazinoic acid on the uricosuric ef- 0 000 C) -Hi-H 41+441-H-H-H-H fect of tienilic acid in urate-loaded mongrel dogs is CO 00 00 '0Nt— 0- .Li 00NCCNC/i.. C). CO oodCii 0 C- N00 00 N N shown in Table 4. In the first series of experiments, .0000 o C') pyrazinoic acid was administered prior to and contin- .CO N 00N 00 SO N C") .-.0 040 4) - 0 N00en— CiiNCii'0 en ued after tienilic acid administration. Despite urate- > enenen en en N enen en C') — — —.. -H-H-ft -H+-H-H-H-H 1 loading, it had only a moderate but significant effect 0 6) N N en i '00 C\ CO C)-)C)-) C)-) en N00—05 en00-.00 4) 0 0. en—OS NNSO en C) on urate excretion, Cr/Cc falling from 0.72 to 0.61, 0 CO •0 I-CO a mean fall of 15% (P < 0.01). The uricosuric effect of 40 E N '0 N 0 N N 00 SON en so — 4) tienilic acid was not markedly altered by pyrazinoic C) 00000000000 C) +1-HH+l-+l-l'l-H-H-H-H acid since Cur/Ccr rose significantly from 0.61 to 0.89 N 00'0'ii000 en N 0— en en within 20 mm (P < 0.01). When tienilic acid was 0. 00ren00 0000000 U C) CO administered prior to pyrazinoic acid, the latter also CO C) CO — had no significant effect on Cur/C0r (Table 4). 'ONOO —S000SOCSNen -. N—— — 0N N 0 0(I) The results of the stop-flow studies performed in 4) 01 -H-H-H H-H-H-H-H41-fl-H en Cl-C 0Os en en - '0' N en 0005 en en en '0'— 00500 N 5050 Cii Cli CO five urate-loaded mongrel dogs are shown in Table 5 0 0. 0 and Figure 4, the latter showing a representative ex- C) CO 4)> periment. During the control period, maximal reab- 4) NNS NO 1) 000 N C-I .0 sorption of urate was noted in the proximal segment, 00 -H-H-H -fF41-H-H-H-H-H CO CO 00 Cenho01-0 4) together with maximal secretion of PAH. Following CO 000000 N C- '0 Cli Cii '0 .0 the administration of tienilic acid, the ratio of urine 0. 0 0 to plasma (U/P) urate over U/P creatinine rose from U CO '0—Nen —0-.00 05 N0505 'O C- SO '00 N 00 N li 0 C.) >———NNNN——NNNN 0.41 to 0.75 in the proximal segment (P < 0.01) and CO Q z— -H-l-H+-H+-H-H 4) C) 0 — '0' 0005en CC) L0 N — 'iiN 0500 S0 '.i — — from 0.62 to 0.90 (P < 0.01) in the distal segment, the 0 0. 0000Nli N 5050 Cii Cii '0 latter value being similar to that found during free- F- 6) F- CO 05— N SON '0- 0'0. 0505 0 flow before and after ureteral occlusion. At the same 0000000,000 time, proximal secretion of PAH was markedly inhib- C-) -H-H-H IOU UL) '0'OS00 'O 00 Cii '00 4) ited, the ratio of U/PpAH/U/PCr falling from 5.59 to 0—05000000 N N N N -H— CO 1.78 (P < 0.01), while a value near unity was ob- CO0 CO 6). served at the distal site and during free-flow condi- C-C I 010 tions. 4) 000 E .—000000000C') en'0 C)-)SO N00 05 CO Studies in rat. The results of the 11 experiments I-. '0 4) performed in five urate-loaded (plasma urate, 3.2 0 z 0.50 mg/l00 ml) and six unloaded (plasma urate, 0.8 I >LC (Jricosuric effect of tienilic acid 109

Table3. Effect of tienilic acid on renal hemodynamics and para-aminohippurate (PAH) extraction in the dog5 Low plasma PAH (N =5) High plasmaPAH (N =5)

Time GFR PPAH CPAU EPAK GFR PPAH CPAU EPAH CPAH, T5 mm mi/mm mg/lOOm!mi/mm % mi/mm mi/mm mg/100m1 mi/win % mi/mm mg/mm —10 to —20 60+ 8.50.79±0.1139±28.0 811.9 168±33 69+ 1.120±4.2 117±6.4 55 213 +20 8.8+0.8 Tienilic acid. 50 mg/kg 50 to 60 50 + 5.71.4 + 0.2 61 8.7 33 3.8 197 29 673.6 24 + 2.8 73 + 7.6 30 + 3.4252 + 40 1.8 + 1.8 90 to 100 51 4.61.5 0.2 80 + 10.0 48 + 3.6 163 + II 64 + 3.4 25 2.2 75 6.8 30 + 2.7259 + 42 2.50.9 120 to 130 45 5.01.6 0.3 77 9.6 59 + 2.8128 + 10 59 + 3.4 26 + 2.369 8.0 32 + 2.7220 + 36 1.6 1.2 Valuesare means + SEM. Abbreviations used are: EFAH = renal extraction of PAH, CPAH, = PAH clearance corrected for renal extraction, GFR = glomerular filtration rate, P = plasma, C clearance, TPAH = transport maximum for PAH.

0.14 mg/100 ml) rats are presented in Figure 5.In acid, Cur/Cm fell markedly from 1.32 to 0.220.04 the loaded animals, Cr/Cinrosefrom 0.750.09 to (P < 0.01), indicating abolition of net secretion and 1.00 0.04 (P < 0.05) within 10 mm after the admin- unmasking important net reabsorption. Plasma urate istration of tienilic acid. This effect continued for the rose from 0.600.14 to 0.740.22 mg/l00 ml (P < next 20 mm, falling off to 0.860.07 after one hour. 0.05), a significant increase attributable to urate re- In the unloaded animals, Cr/Cinrosefrom 0.21 tention. This marked inhibitory effect on urate excre- 0,04 to 0.46 0.08 (P < 0.01) within 10 mm and to tion occurred in spite of a significant natriuresis, the 0.54 0.09 (P < 0.01) after 30 mm, decreasing to fractional excretion of sodium increasing from 1.4 0.43 0.07 after 60 mm. Thus, the increment in Cur/Cm following tienilic acid was the same in both groups of animals. Maximal natriuresis was observed 20 mm after the administration of the drug, the frac- 0000 tional excretion of sodium rising from 2.7 0.64% to 3 0 0 4.7 + 0.42% (P < 0.01) in the unloaded animals and PAH from 4.7 1.18% to 9.5 1.41% (P < 0.02) in the 0 0 0 urate-loaded ones. A moderate kaliuretic effect ac- U 'In 00 companied the increase in sodium excretion. 0 Na mm. 0 Studies in guinea pig. Figure 6 shows the results 0 of the six experiments performed on guinea pigs not loaded with urate. During the control period, 0 Cur/Cm averaged 1.32 0.20, indicating net secre- 2 0 tion. Endogenous plasma urate averaged 0.6 0.14 mg/I00 ml. Following the administration of tienilic 000 uIU. SKF 62698 U I 1 .5 - I. I ..51 •_UU.!I 1.0- 0 S.•.•.S •sS C.) 000000 0 00000 0 0 0 0.5- 00 o0 • No urate-loading (N = 4) Oooo o Urate-loading (N = 5) 100%inulin U4 u5 u6 0 5 10 15 20 I I I I —20 0 20 40 60 80 100 Cumulativevolume,rn/ Time,rn/n 00Control •U SKF 62698,50 mg/kg Fig.3. EJj'ct of tienilic acid on urate excretioninurate-ioaded and Fig.4. Effect of tien/lie acid (dark symbols) on urate and PA H nonloadedDalmatian coachhounds. transport in a urate-loaded mongrel dog (VA =uricacid). 110 Lemieuxet a!

Table4. Effect of pyrazinoic acid (PZA) on uricosuric effect of trenilic acid in urate-loaded mongrel dogsa N=4 N=5

Time Pur GFR Pur GFR mm mg/tOO ml mi/mm Cut/Ccr mg/IOU ml mi/mm Cur/Ccr Control —30 to —20 3.2 0.3 73 5.5 0.72 + 0,02 3.1 + 0.2 57± 3 0.54 + 0.04 —20 to —10 3.3 + 0.3 74 6.8 0.69 0.06 3.2 + 0.1 57± 4 0.54 + 0.04 —10 to 0 3.4 + 0.4 71 + 4.0 0.74 + 0.04 3.3 0.2 55 4 0.54 0.07

PZA: prime, 30mg/kg; sustained infusion, 0.6 mg/kg/mm Tienilic acid, 50 mg/kg 10 to 20 3.7+ 0.6 65 + 5.6 0.73 0.03 3.5+ 0.2 55 + 2 1.00 0.07 20 to 30 4.0 + 0.8 71 + 5.2 0.64 + 0.02 3.6 + 0.2 59 3 0.95 + 0.07 30 to 40 4.0 0.8 67 5.4 0.64 0.03 3.7 0.2 54 3 0.96 0.06 40 to 50 4.1 + 0.8 72 7.5 0.62 0.04 3.9 + 0.2 53 + 3 0.97 0.05 50 to 60 4.! + 0.9 81 + 5.7 0.6! + 0.01 3.9 0.3 52 4 0.94 0.06

Tienilic acid, 50mg/kg PZA: prime, 30mg/kg; sustained infusion, 0.6 mg/kg/mm 60 to 70 3.8 0.6 62 + 3.8 0.84 + 0.05 3.8 0.3 56 5.7 0.94 0.04 70 to 80 4.4 + 0.8 76 2.1 0.89 0.07 3.9 64 4.0 0.88 0.06 80 to 90 4.4 + 0.9 75 + 3.2 0.83 0.04 3.7 + 0.6 52 + 3.2 0.93 + 0.08 90 to 100 4.4 + 1.0 74 3.6 0.85 0.07 3.7 0.6 57 + 6.1 0.89 0.06 100 to 110 4.3 1.0 77 5.4 0.84 0.07 3.8 + 0.6 50 + 3.1 0.89 + 0.06 110 to 120 4.4 1.1 64 + 8.5 0.80 0.07 4.0 0.4 54 3.1 0.90 0.06

Values are means + SEM. N=number of animals. Cr/Ccr = ratio of urate clearance to creatinine clearance. GFR = glomerular filtration rate. Pur = plasma urate. 0.65 to 6.0 2.70% (P<0.05). Glomerular filtration PAH was not measured in these experiments. Ten rateremained stable throughout each experiment. minutes after the administration of tienilic acid, gb- Studies in rabbit. Tienilic acid showed no uricosuric merular filtration rate had fallen by 19% and urate properties in either urate-loaded or unloaded rabbits. clearance by 46% in the unloaded animals. The same Cr/Ccr in the unloaded animals averaged 0.79 relationship was found in urate-loaded rabbits. The 0.10, whereas this value was 1.500.10 in the loaded natriuretic effect of tienilic acid was again observed, ones, indicating net secretion (Fig. 7). Following ii- FENa increasing from 5.3 1.32% to 21.5 1.47% (P enilic acid administration, Cur/Car fell by 32% (P < <0.01)in unloaded and from 9.5 1.07% to 32.2 0.01) in unloaded animals and 41% (P < 0.01) in the 2,21% (P < 0.01) in urate-loaded animals. urate-loaded rabbits. In the latter animals, the fall of Studies in chicken. Tienilic acid injected in a wing Cr/Ccr from 1.50 to 0.88—1.10 indicates suppression vein induced a striking reduction in urate excretion of net secretion of urate (Fig. 7). PAH clearance fell (Fig. 8). In these experiments, Cr/Cin fell from 8.1 by 41% (P <0.01) in the unloaded and by 56% (P < + 2.59 to 3.2 0.70 (P < 0.02). Inulin clearance 0.01) in the urate-loaded rabbits, presumably for the remained stable throughout each experiment at 1.4 + same reason as in the dog, but the renal extraction of 0.40 mI/mm. Thus, net secretion of urate fell by 61%.

Table 5. Effect of tienilic acid on renal handling of urate during stop-flow in the dog (left kidney) (N=

Plasma Free-flow Distal Proximal Free-flow

Control: GFR, mI/mm 26.2 3.6 26.0 3.5 U/P urate/U/Pcreatinine 0.644 + 0.019 0.617 0.015 0.412 0.030 0.635 0.030 U/P PAH/U/P creatinine 2.24 0.47 2.87 + 1.27 5.59 +2.43 2.48 + 0.45 UNa, ,nEq/liter 64.7 9.60 12.6 + 1.82 74.6 + 15.0 56.9 + 11.8 P urate, mg/I0Oml 6.8 + 1.5 PPAH. mg/IOOml 4.0 0.02 Tienilic acid, 50mg/kg: GFR,mt/min 24.0 3.2 25.1 3.5 U/P urate/U/Pcreatinine 0.852 + 0.030 0.900 + 0.018 0.750 + 0.016 0.850 + 0.025 U/P PAH/U/Pcreatinine 1.08 0.06 1.9 + 0.27 1.78 0.66 1.20 0.38 UN,mEq/liter 81.5 + 8.93 18.6 3.5 73.4 11.6 77.5 + 8.6 Purate,mg/lOOml 8.8± 1.9 PPAH, mg/IOUml 6.8 + 0.02

Values are means SEM. See preceding tables for abbreviations used. Uricosuric effect of tienilic acid 111

SKF 62698 50 mg/kg 1.4-

1.0 1.2-

0.8 1.0- 0 O.6 0.8- 0 - N' 6 0.4 0.6-

0.2 0.4- U SKF 62698 N=6 50 mg/kg — 0.2- I I I I I I —10 0 10 20 30 40 50 60 Time, mm Fig. 5. Effect of tienilic acid on urate excretion in urate-loaded —10 0 10 20 30 40 50 60 (upper line) and unloaded (lower line) rats. Time,rn/n Fig. 6. Effect of sienilic acid on urate excretion in guinea pigs.

At the same time, plasma urate rose from 25.1 6.73 supported by the fact that tienilic acid inhibits the mg/100 ml to a maximal value of 38.88.68 mg/100 renal tubular transport of para-aminohippurate in ml (P < 0.01) 30 mm after tienilic acid administration this species, even at high PAH plasma concentration. and remained at 36.5 + 9.23 mg/lOO ml at 50 mm. More direct evidence of inhibition of both urate and Thus, suppression of net urate secretion by tienilic PAH transport at the proximal site was obtained by acid led to a rapid and important (55%) increase in our stop-flow studies. One must certainly envisage plasma urate. No PAH was administered to these that both tienilic acid and PAH compete for a com- animals. FENa rose from 9.5 + 1.79% to 50.6 + 2.80% mon pathway for secretion in the proximal tubule, if (P < 0.01) following tienilic acid. tienilic acid can be shown to be secreted. If so, the lack of effect of PAH-Ioading on the uricosuric action Discussion of tienilic acid would further suggest that the com- At the dosage used in the present study, tienilic mon carrier has a far greater affinity for tienilic acid acid is certainly the most important uricosuric agent than for PAH. ever reported in the mongrel dog. The fact that fil- The proximal uricosuric effect of tienilic acid in the tered urate excretion reached 100% following the ad- mongrel dog is most likely independent of the natri- ministration of this drug indicates complete inhibition of net urate transport in the nephron of the mongrel dog. In view of the effect observed in the SKF 62698 mongrel dog, one did not expect tienilic acid to in- o No urate-loading (N = 6) crease urate excretion in the Dalmatian coachhound. • Urate-loading (N = 8) In fact, in Dalmatians with Cur/Ccrratioslightly 1.5- above unity, this ratio fell rapidly toward unity following tienilic acid administration, indicating inhibi- 00 - tion of the slight net urate secretion observed during _-.. 1.0 the control period. Thus, tienilic acid inhibits net 0 - reabsorption of urate in the mongrel dog and net secretion in the Dalmatian. Since bidirectional move- 0.5 ment of urate has been shown to take place in the proximal nephron of the dog [14, 15], it is probable that both reabsorptive and secretory fluxes are inhib- ited by tienilic acid in the mongrel dog. I I I I II I40 The magnitude of the uricosuric response observed —20 —10 0 10 20 30 50 60 in mongrel dogs suggests that tienilic acid affects Time, rn/fl Fig. 7. Effectof tienilic acid on urate excretion in urate-loaded and urate transport mainly in the proximal tubule. This is unloaded rabbits. 112 Lemieuxet a!

SKF 62698, 50 mg/kg least in the dog, tienilic acid is unlikely to exert any effect on urate transport beyond the proximal tubule. 40 - U The i.v. administration of pyrazinoic acid (PZA), a E well known inhibitor of urate secretion in man [24, 30- 25], had only a slight depressing effect on urate excretion in the mongrel dog. Such a slight effect has 20- already been observed by Yu, Berger, and Gutman a- [26]. Tienilic acid exerted its usual pronounced 10- uricosuric effect whether pyrazinoic acid was administered before or after tienilic acid. Thus, in the mongrel dog, the uricosuric effect of tienilic acid is not PZA suppressible. N=6 The control studies demonstrate that in either the 10- mongrel or the Dalmatian coachhound, the continued and prolonged administration of the isotonic mannitol-saline mixture used in this study had no 8- effect whatsoever on urate or PAH excretion, Thus, any effect of tienilic acid on urate and PAH observed 6- in this species can only be attributed to the drug itself. 0 We have demonstrated that tienilic acid is also 0D uricosuric in the rat. This effect is less pronounced 4— than in the mongrel dog. Our results are in agreement with those reported in mice by Thuillier et al [I]. Thus, it is of interest to note that tienilic acid has a 2- uricosuric effect in man [5, 6], mongrel dog, rat and mouse [I], all species where urate reabsorption is preponderant. Our study in the rabbit, guinea pig, and chicken —20 0 20 40 60 adds useful information concerning the mode of ac- Time, mm tion of tienilic acid on urate excretion. Net secretion Fig.8.Effect of tienilicacid on urate excretion and plasma urate in chickens. of urate is readily demonstrable in guinea pigs [27] and in rabbits loaded with urate [28]. The fact that tienilic acid completely abolished net secretion of uretic effect of the drug which apparently takes place urate in guinea pigs and urate-loaded rabbits, while in man in the cortical diluting segment of the distal inducing significant reduction in urate excretion in tubule [2, 3]. This point is well illustrated by our nonloaded rabbits, suggests that in these species, tie- control study with furosemide and ethacrynic acid, nilic acid inhibits urate secretion and has no demon- both loop diuretics [16] which have no effect on urate strable net effect on urate reabsorption. This could excretion in the dog despite a comparable or greater also be taken to indicate that in the rabbit and guinea natriuretic effect than tienilic acid at the dosage used. pig, the secretory flux is more important than in the Although we are certain that in the dog tienilic acid mongrel dog and even in the Dalmatian coachhound. exerts its uricosuric effect in the proximal segment of The present observations in the chicken leave no the nephron, we cannot exclude with certainty an doubt about the ability of tienilic acid to depress additional effect on urate transport in the ioop of urate secretion in this animal. This inhibitory effect is Henle and at a more distal site. According to recent remarkable in view of the fact that pyrazinamide, a micropuncture and microperfusion studies in the rat, potent inhibitor of urate secretion in man [24], Cebus the importance of urate transport in the 1oop of monkey [29], and Dalmatian coachhound [26] has no Henle [17—20] and the distal tubule [20—22] has be- effect on urate excretion in the chicken [30]. The come a matter of controversy. Studies in mongrel striking inhibitory effect of tienilic acid on urate ex- dogs show, however, that urate transport occurs only cretion in the chicken is reminiscent of that reported in the proximal convoluted tubule in this species [15, in this animal for salicylate, and especially probe- 23], while urate secretion in the Dalmatian coach- necid at high dosage [30]. The rapid and striking rise hound does not extend beyond the straight segment in plasma urate concentration when tienilic acid is of the proximal tubule [23]. We therefore feel that, at administered to the chicken is highly confirmatory of Uricosuric effect of tienilic acid 113 the ability of the drug to induce marked suppression 2. LAU K, GOLDBERG M, STOTE R, AGus ZS: Renal effects of of urate secretion. SKF 62698: A new uricosuric diuretic (abstr.). C/in Res The spectacular drop in urate secretion observed in 24:405, 1976 3. STOTE RM, MAASS AR, CHERRILL DA, BEG MMA, ALEXAN- guinea pig, rabbit, and chicken adds support to our DER F: Tienilic acid: A potent diuretic-uricosuric agent. J suggestion that tienilic acid can inhibit either the net Pharmaco/ C/in (Special issue): 19—27, 1976 reabsorptive or secretory flux depending on the mag- 4. FANELLI GM JR, BOHN DL, REILLY SS, WEINER IM: Effects of nitude of each flux in a given animal species. Thus, it mercurial diuretics on renal transport of urate in the chim- panzee. Am J Physiol 224:985—992, 1973 has a uricosuric effect in the mongrel dog and the rat 5. MASBERNARD A, GIUDICELLI CP, KAMALUDIN T: Etude phar- where the secretory flux is probably slight and has an macologique clinique de l'acide dichloro-2, 3-(thiényl-2-céto)- inhibitory effect in animals like the Dalmatian coach- phénoxy-acétique. J Pharmacol Clin (Special issue): 13—18, hound, guinea pig, rabbit, and chicken, in which the 1976 secretory flux of urate is predominant. 6. REESE OG, STEELE TH: Renal transport of urate during diuretic-induced hypouricemia. Am J Med 60:973—979, 1976 Little is presently known of the metabolism of 7. LAU K, STOTE R, GOLDBERG M, AGUS ZS: Mechanism of tienilic acid in the animal species used in the present uricosuria: Studies with ticrynafen (SKF 62698) (abstr.). C/in study. The drug is highly bound to plasma protein in Res 24:405, 1976 mouse, rat, pig, and dog [31]. It is unlikely, however, 8. LEMIEUX G, VINAYP,ROBITAILLE P, PLANTE GE, LUSSIER Y, that the uricosuric effect of tienilic acid in the mon- MARTIN P: The effect of ketone bodies on renal ammonio- genesis. JC/in Invest 50:1781—1791,1971 grel dog, rat, mouse, and even man could be ex- 9. LEMIEUX G, VINAY P, GouGoux A, MICHAUD G: Nature of plained by an effect of the drug on the binding of the uricosuric action of benziodarone. Am J Physiol urate to plasma protein, a mechanism suggested by 224:1440—1449, 1973 Campion, Bluestone, and Klinenberg for other urico- 10. BROWN H: The determination of uric acid in human blood. J suric agents [32, 33]. Many believe that little urate is BiolChem 158:601—608,1945 II. LEMIEUX G, GOUGOUX A, VINAY P, MICHAUD G: Uricosuric bound to plasma proteins at 37°C [34—37]. Simkin effect of benziodarone in man and laboratory animals: A could not demonstrate protein-binding of urate in comparative study. Am J Physiol 224:1431—1439, 1973 mongrel dogs [38]. Furthermore, such a mechanism 12. SMITHHW,FINKELSTEIN N, ALIMINOSA L, CRAWFORD B, could not explain the antiuricosuric effect of tienilic GRABER M: The renal clearances of substituted hippuric acid acid in the guinea pig, rabbit, and chicken. It is more derivatives and other aromatic acids in dog and man. J C/in Invest 24:388—404, 1945 likely that the action of tienilic acid on urate trans- 13. BONSNES RW, TAUSSKY HH: On the colorimetric determina- port is related to its secretion and/or reabsorption in tion of creatinine by the Jaffe reaction. JBiol Chem the renal tubule. This is suggested by the rapid elimi- 158:581—591,1945 nation of the drug in urine (and bile) in mouse, rat, 14. MUDGEGH,CUCCHIJ,PLATTSM,O'CONNELL JMB, BERNDT pig, and dog [31]. WO: Renal excretion of uric acid in the dog. Am J Physiol 215:404—410, 1968 It is evident from the present study that tienilic acid 15. ZIN5 GR, WEINER IM: Bidirectional urate transport limited to has little effect on renal hemodynamics, if it is real- the proximal tubule in dogs. Am J Physio/ 215:411—422, 1968 ized that PAH clearance cannot be used to measure 16. SELDIN DW, EKNOYAN G, SuKI WN, RECTOR FC i: Local- renal plasma flow following its administration, unless ization of diuretic action from the pattern of water and elec- the renal extraction of PAH is also determined. trolyte excretion. Ann NY Acad Sci 139:328—343, 1966 17. KRAMP RA, LASSITER WE, GOTTSCHALK CW: Urate-2-'4C transport in the rat nephron. J C/in Invest 50:35—48, 1971 Acknowledgments 18. GREGER R, LANG F, DEETJEN F: Urate handling by the rat This work was supported by grants from Smith kidney: IV. Reabsorption in the loops of Henle. Pfiuegers Arch Kline & French Canada Limited, the Jeanne Mance 352:115—120, 1974 Foundation, and the Medical Research Council of 19. ROCH-RAMEL F, DIEZI-CHOMETY F, DE ROUGEMONT D, TELLIER M, WIDMER J, PETERS G: Renal excretion of uric acid Canada. The authors acknowledge Misses Denise in the rat: A micropuncture and microperfusion study. Am J Boivin Bouti, Lorraine Marchand, and Pierrette Physio/ 230:768—776, 1976 Fournel for their skilled assistance. 20. DE ROUGEMONT D, HENCHOZ M, ROCH-RAMEL F: Renal urate excretion at various plasma concentrations in the rat: A Reprint requests to Dr. Guy Lemieux, Nephrology-Metabolism free-flow micropuncture study. Am J Physio/ 231:387—392, Division, Hbtel-Dieu Hospital, 3840 St. Urbain St., Montreal, Que- 1976 bec, Canada, H2 W I T8. 21. OELERT H, BAUMAN K, GEKLE 0: Permeabilitätsuntersuch- ungen einiger schwacher organischer Säuren aus dem distalen References Konvolut der Rattenniere. Eur J Physiol 307:178—189, 1969 22. KRAMP RA, LENOIR R: Distal permeability to urate and effects 1. THUILLIER G, LAFOREST J, CARIOU B, BEssINF,BONNETJ, THUILLIER J: Dérivés hetérocycliques d'acides phénoxy-acé- of benzofuran derivatives in the rat kidney. Am J Physiol tiqlues: Synthése et étude préliminaire de leur activité diuré- 228:875—883, 1975 tique et uricosurique.EurJ Med Chem9:625—633,1974 23. ROCH-RAMEL F, WONG NLM, DIRKS JH: Renal excretion of 114 Lemieux et a!

urate inmongreland Dalmatian dogs: A micropuncture study. cokinetic study of 2,3-dichloro 4-(2-thienyl ketol4C) phe- Am J Physiol 231:326—331,1976 noxyacetic acid (Tienilic acid) in animals: 1. Localization, 24. STEELETH,RIESELBACHRE:Therenal mechanism for urate distribution and elimination of 14C Tienilic acid in animals. homeostasisinnormal man. AmJ Med43:868—875, 1967 Eur J Drug MetabPharm1:41—49, 1976 25. GUTMANAB,Yu TF, BERGERL:Renal function in gout: III. 32. CAMPJONDS,BLUESTONER,KLINENBERG.i: Displacement Estimation of tubular secretion and reabsorption of uric acid byuricosuric agents of sodium urate bound to human serum byuse of pyrazinamide (Pyrazinoic acid). AmJ Med albumin. BiochemPharmacol 23:1653—1657,1974 47:575—592,1969 33. BLUESTONE R, CAMPION D, KLINENBERG JR:Halofenate:Its 26.Yu TF, BEROERL,GUTMANAB:Suppression of tubular secre- selection and trial as a primary uricosuric agent. Arthritis tion of urate by pyrazinamide in the dog. Proc Soc Exp Biol Rheum 18:859—862, 1975 Med 107:905—908, 1961 34. YC TF, GUTMAN AB: Ultrafiltrability of plasma urate in man. 27, MUDGEGH,MCALARYB,BERNDTWO:Renal transport of ProcSoc Exp Biol Med84:21—24, 1953 uric acid in the guinea pig. Am J Physiol214:875—879, 1968 35. SHEIKHMl,MtLLER JV: Binding of urate to proteins of hu- 28. POUL5ENH,PRAETORIUSE:Tubular excretion of uric acid in man and rabbit plasma. Biochem Biophys Ada 158:456—458, rabbits. AdaPharmacol Toxicol 10:371—378, 1954 1968 29. FANELLIGM JR, BOHN D,STAFFORD5: Functionalcharacter- 36. FARRELLPC,PoPovicH RP, BABB AL: Binding levels of urate istics of renal urate transport inthe Cebus monkey. Am J ions in human serum albumin and plasma. Biochem Biophys Physiol218:627—636, 1970 Acta243:49—52,1971 30. NECHAYBR,NECHAYL:Effects of probenecid, sodium salicy- 37. FARRELLPC:Protein binding of urate ions in vitro and in vivo late,2,4-dinitrophenol and pyrazinamide on renal secretion of (abstr.).Nephron10:360,1973 uric acid in chickens.JPharmacolExp Ther 126:291—295,1959 38.SIMKINPA:Uric acid binding to serum proteins: Differences 31. DORMARDY,LEVRONJC,ADNOTP.LEBEDEFFT:Pharma- among species. Proc Soc Exp Biol Med 139:604—606, 1972