United States Patent (19) 11 Patent Number: 4,663,322 Beyer, Jr

United States Patent (19) 11 Patent Number: 4,663,322 Beyer, Jr. 45 Date of Patent: May 5, 1987

54 ANTIHYPERTENSIVE HYPERURETIC AND Sweet, C., et al., Clin. Sci, Mol. Med., 48: 147-151, 1975. SAILURETICAGENT COMBINATIONS Amery, A., et al., New Eng, J. Med., 295: 1198-1199, 76 Inventor: Karl H. Beyer, Jr., P.O. Box 387, 1976. Penllyn, Pa. 19422 Morrison, R., et al., Organic Chemistry, 3rd Ed., Allyn and Bacon, Boston, 1973, p. 817. (21) Appl. No.: 792,236 Chemical Abstracts, 82:80803v (1975) Mendelzon, J., et 22 Filed: Oct. 29, 1985 al., Rev. Argent. Cardiol., 1974, 42(4), 278, 280, 282,284, 289-90). Chemical Abstracts, 88:45134e (1978) Schena, F., et al., Related U.S. Application Data Clin. Ter, 1977, 82(5), 489-94). 63 Continuation of Ser. No. 336,736, Jan. 4, 1982, aban Chemical Abstracts, 85:177484s (1976). Kubo, K., et al., doned. Japan, Kokai 76 52,185, 5/8/76). 51 Int. Cl...... A61K 31/54; A61K 31/495 Benos, D., et al., J. Gen. Physiol, 68(1): 43-63 (1976). 52 U.S. C...... 514/222; 514/255 Primary Examiner-Richard A. Schwartz 58 Field of Search ...... 514/255, 222 Attorney, Agent, or Firm-Oblon, Fisher, Spivak, (56) References Cited McClelland & Maier U.S. PATENT DOCUMENTS 57 ABSTRACT 3,313,813 4/1967 Cragoe ...... 544/.407 Hyperuretic agents of the formula: 3,345,372 10/1967 Hanifin et al...... 544/407 X OTHER PUBLICATIONS R? N Rl (I.) Physicians' Desk Reference, 40th Ed., 1986, pp. 199-1200. y Beyer, K., et al., Pharmacol. Rey, 34: 287-313, 1983. 3 N I R Saito, I, et al., Cardiovasc. Res., 10:149-152, 1976. R N C Beyer, K., Clin. Therap, 1: 425-443, 1978. Zanchetti, A., et al., Am. J. Cardiol, 37: 675-691, 1976. Singh, B., et al., Brit. Med. J., 1: 143-146, 1967. wherein Gombos, E., et al., New Eng. J. Med., 275: 1215-1220, Y is O or NH; 1966. R is OH; NHCONR4R5; or N-C(NR4R5); Kampffmeyer, H., et al., Clin. Pharmacol. Therap, 9: R1 and R2 are hydrogen, amino, or mono- or disubsti 350-354, 1968. tuted amino, provided that Rand R2 may not both be McCaa, R., et al., Adv. Exp. Med. Biol, 130: 227-255, amino or substituted amino; and 1980. R3 is hydrogen or halo; Weber, M., et al., Arch. Int. Med., 137:284-289, 1977. in combination with saluretic agents such as hydrochlo Vaughan, E., et al., Circ. Res., 42: 376-381, 1978. rothiazide; are useful for treating hypertension. Esler, M., et al., Am. J. Cardiol, 36: 708-715, 1975. Morganti, A., et al., Am. J. Cardiol, 43: 600-604, 1979. 16 Claims, No Drawings 4,663,322 1 2 actively secreting and reabsorbing urea in addition to ANTIHYPERTENSIVE HYPERURETIC AND being filtered at the glomeruli and undergoing passive SAILURETICAGENT COMBINATIONS back diffusion. Moreover, it has been discovered that the pyrazinoic This application is a continuation of application Ser. 5 acid derivatives utilized in the present invention are No. 336,736 filed Jan. 4, 1982, now abandoned. capable of inhibiting the active renal tubular reabsorp tion of urea, predominantly; whereas other pyrazinoic BACKGROUND OF THE INVENTION 1. Field of acid derivatives such as the fluoro analog of amiloride the Invention inhibit preponderantly active tubular secretion of urea The present invention is concerned with the combi- 10 without significantly inhibiting active reabsorption of nation of certain pyrazinoic acid derivatives useful as urea. In so doing they lower urea blood levels and in hyperuretic agents and various sailuretic agents; and crease the osmotic concentration of urine as indications with pharmaceutical compositions and methods of of their capability of influencing osmotically modulated treating hypertension in which the active ingredient functions of cells and cell membranes. These may thus employed is a combination of one of said pyrazinoic 15 be called osmoregulatory agents to identify their role in acid derivatives and a saluretic agent. therapy. Combined with various saluretic agents, their In mammals, including man, urea is the principal end usefulness is considered to extend from the management product of nitrogen metabolism. Urea is synthesized in of mild hypertension and pulmonary edema to the neu the body through the intermediation of the ornithine rological symptomatology of malignant or severe hy urea cycle as from the more toxic ammonia and carbon 20 pertension. dioxide. Some twenty-five grams of urea are synthe 2. Brief Description of the Prior Art sized in the human body per day and excreted primarily Cragoe U.S. Pat. No. 3,313,813 describes 3-amino by way of the kidneys. 5,6-disubstituted-pyrazinoyl guanidines and their use as Urea and sodium (chloride) constitute the principal diuretic, natriuretic agents which selectively enhance osmotically active constituents of the body. Whereas 25 the excretion of sodium ions without causing an in this osmotic role is similar for the two, urea and salt, crease in excretion of potassium ions. they differ in important respects. Sodium chloride must Benos et al. in J. Gen. Physiol. 68(1): 43-63 (1976) be obtained from some exogenous source, and the distri describe the effect of amiloride and some of its analogs bution of sodium is essentially limited to extracellular on cation transport in isolated frog skin and thin lipid fluid, including plasma water. Urea is synthetized by the 30 membranes. body in large amounts, and its distribution is both intra However, none of the above references in any way cellular and extracellular-being equivalent to total suggests the use of the particular pyrazinoic body water. (Potassium serves the intracellular osmotic acid derivative hyperuretic agents utilized in the pres role assumed by sodium extracellularly.) ent invention for treating hypertension, eclampsia, ure When excess sodium is consumed, or retained by less 35 mia, and the like, since these references fail to suggest than adequate renal function, iso-osmotic pressure is the hyperuretic activity of said pyrazinoic acid deriva sustained by thirst-stimulated increased fluid consump tion and by fluid retention which may lead to increased tives, and since said derivatives do not have sufficient blood pressure and/or edema. saluretic and antikaluretic activity to be useful in accor In the presence of impaired renal function sufficient 40 dance with the requirements described in said refer to reduce urea excretion, its accumulation may be eCeS, enough to cause increased intracellular as well as extra DETAILED DESCRIPTION OF THE cellular fluid accumulation, if iso-osmotic relationships INVENTION AND PREFERRED are sustained by fluid accumulation. In severe circum EMBODIMENTS stance, uremia may result. It is this intracellular/ex- 45 tracellular accumulation of fluid that is most likely to In accordance with the present invention there is account for the symptomatology as well as the signs of provided a pharmaceutical composition for treating uremia. At sub-uremic elevations of urea and body hypertension and edema comprising a pharmaceutically water levels the symptomatology may well be confused acceptable carrier and a therapeutically effective with salt retention, in which case therapy based on 50 amount of the combination of saluresis or hyperuresis may be used interchangea (a) a compound of the formula: bly-to some extent. (I.) The present state of clinical knowledge recognizes R2 N R that urea is filtered with plasma water at the glomeruli of the kidney and that a portion of that filtered urea 55 y undergoes passive back diffusion in the course of urine 3 N I R formation by the nephron. The passive back diffusion of R N C urea can be reduced by increasing the transit rate, i.e., increasing the urine flow. wherein Increased transit rate can be induced (1) by expand- 60 Y is O or NH, ing body fluid volume, which is self-defeating from a R is OH; NHCONR4R5; or N=C(NR4R5); where therapeutic standpoint; (2) with the aid of an osmotic R* and R5 are each independently selected from the diuretic, e.g., mannitol, which is impractical because of group consisting of hydrogen; C1-10 alkyl, straight or the need to administer large amounts parenterally, or (3) branched chain; aryl C1-4 alkyl; mono- or disubstituted temporarily, by the use of potent saluretic agents at 65 aryl C1-4 alkyl where the substituents are fluoro, chloro, dosages sufficient to alter electrolyte balance. bromo, iodo, or C1-10 alkyl, straight or branched chain; In accordance with the present invention, it has been R and R2 are each independently selected from the discovered that the mammalian kidney is capable of group consisting of hydrogen, amino, and mono- or 4,663,322 3 4. disubstituted amino where the substituents are C-10 consisting of sweetening agents, flavoring agents, color alkyl, straight or branched chain, or C3-8 cycloalkyl; ing agents and preserving agents in order to provide a provided that R1 and R2 may not both be amino or pharmaceutically elegant and palatable preparation. substituted amino; and Tablets contain the active ingredient in admixture with Riis hydrogen; fluoro; chloro; bromo; or iodo; and a non-toxic pharmaceutically acceptable excipients pharmaceutically acceptable salt thereof; and which are suitable for manufacture of tablets. These (b) a saluretic agent selected from the group consist excipients may be, for example, inert diluents, such as ing essentially of ethacrynic acid; furosemide; bumeta calcium carbonate, sodium carbonate, lactose, calcium nide; muzolimine; benzothiadiazines including chloro phosphate or sodium phosphate; granulating and disin thiazide and hydrochlorothiazide; chlorothalidone; 10 tegrating agents, for example maize starch, or alginic quinethazone; metholazone; indacrinone; acetazolam acid; binding agents, for example starch, gelatin or aca ide; ethoxyzolamide; and methazolamide. cia, and lubricating agents, for example magnesium Preferred combinations of the present invention are Stearate, stearic acid or talc. The tablets may be un those wherein for the compound of Formula IY is O or coated or they may be coated by known techniques to NH; one of R1 and R2 is hydrogen or amino and the 5 delay disintegration and absorption in the gastrointesti other is hydrogen; and R3 is hydrogen; and the saluretic nal tract and thereby provide a sustained action over a agent is hydrochlorothiazide. longer period. For example, a time delay material such Particularly preferred compounds of Formula I are as glyceryl monostearate or glyceryl distearate alone or the following: with a wax may be employed. pyrazinoylguanidine; 20 Formulations for oral use may also be presented as 3-aminopyrazinoylguanidine. hard gelatin capsules wherein the active ingredient is The Formula I compounds can be used in the form of mixed with an inert solid diluent, for example calcium salts derived from inorganic or organic acids. Included carbonate, calcium phosphate or kaolin, or as soft gela among such salts are the following: acetate, adipate, tin capsules wherein the active ingredient is mixed with alginate, aspartate, benzoate, benzenesulfonate, bisul 25 water or an oil medium, for example arachis oil, peanut fate, butyrate, citrate, camphorate, camphorsulfonate, oil, liquid paraffin or olive oil. cyclopentanepropionate, digluconate, dodecylsulfate, Aqueous suspensions contain the active materials in ethanesulfonate, fumarate, glucoheptanoate, glycero admixture with excipients suitable for the manufacture phosphate, hemisulfate, heptanoate, hexanote, hydro of aqueous suspensions. Such excipients are suspending chloride, hydrobromide, hydroiodide, 2-hydroxye 30 agents, for example sodium carboxymethylcellulose, thanesulfonate, lactate, maleate, methansulfonate, 2 methylcellulose, hydroxypropylmethylcellulose, so naphthalenesulfonate, nicotinate, oxalate, pamoate, pec dium alginate, polyvinylpyrrollidone, gum tragacanth tinate, persulfate, 3-phenylpropionate, picrate, pivalate, and gum acacia; dispersing or wetting agents may be a propionate, succinate, tartrate, thiocyanate, tosylate, naturally occurring phosphatide, for example, lecithin, and undecanoate. Also, the basic nitrogen-containing 35 or condensation products of an alkylene oxide with groups can be quaternized with such agents as lower fatty acids, for example polyoxyethylene stearate, or alkyl halides, such as methyl, ethyl, propyl, and butyl condensation products of ethylene oxide with long chloride, bromides and iodides; dialkyl sulfates like chain aliphatic alcohols, for example heptadecae dimethyl, diethyl, dibutyl; and diamyl sulfates, long thyleneoxycetanol, or condensation products of ethyl chain halides such as decyl, lauryl, myristyl and stearyl 40 ene oxide with partial esters derived from fatty acids chlorides, bromides and iodides, aralkyl halides like and a hexitol such as polyoxyethylene sorbitol mono benzyl and phenethyl bromides and others. Water or oleate, or condensation products of ethylene oxide with oil-soluble or dispersible products are thereby obtained. partial esters derived from fatty acids and hexitol anhy The pyrazinoic acid derivatives and saluretic agent drides, for example polyoxyethylene sorbitan mono-ole combinations of the present invention possess a high 45 ate. The said aqueous suspensions may also contain one degree of hyperuretic and saluretic/diuretic activity. or more preservatives, for example, ethyl or n-propyl They are of value in the treatment of hypertension and p-hydroxybenzoate, one or more coloring agents, one edema. or more flavoring agents and one or more sweetening For these purposes the combinations of the present agents, such as sucrose or saccharin. invention may be administered orally, topically, paren 50 Oily suspensions may be formulated by suspending terally, by inhalation spray or rectally in dosage unit the active ingredient in a vegetable oil, for example 'formulations containing conventional non-toxic phar arachis oil, olive oil, sesame oil or coconut oil, or in a maceutically acceptable carriers, adjuvants and vehi mineral oil such as liquid paraffin. The oil suspensions cles. The term parenteral as used herein includes subcu may contain a thickening agent, for example beeswax, taneous injections, intravenous, intramuscular, intra 55 hard paraffin or cetyl alcohol. Sweetening agents, such sternal injection or infusion techniques. In addition to as those set forth above, and flavoring agents may be the treatment of warm-blooded animals such as mice, added to provide a palatable oral preparation. These rats, horses, dogs, cats, etc., the combinations of the compositions may be preserved by the addition of an invention are effective in the treatment of humans. antioxidant such as ascorbic acid. The pharmaceutical compositions containing the ac 60 Dispersible powders and granules suitable for prepa tive ingredient may be in a form suitable for oral use, for ration of an aqueous suspension by the addition of water example, as tablets, troches, lozenges, aqueous or oily provide the active ingredient in admixture with a dis suspensions, dispersible powders or granules, emulsions, persing or wetting agent, suspending agent and one or hard or soft capsules, syrups or elixirs. Compositions more preservatives. Suitable dispersing or wetting intended for oral use may be prepared according to any 65 agents and suspending agents are exemplified by those method known to the art for the manufacture of phar already mentioned above. Additional excipients, for maceutical compositions and such compositions may example sweetening, flavoring and coloring agents, contain one or more agents selected from the group may also be present. 4,663,322 5 6 The pharmaceutical compositions of the invention amount of carrier material which may vary from about may also be in the form of oil-in-water emulsions. The 5 to about 95 percent of the total composition. Dosage oily phase may be a vegetable oil, for example olive oil unit forms will generally contain between from about 25 or arachis oils, or a mineral oil, for example liquid paraf to about 500 mg. of active ingredient. fin or mixtures of these. Suitable emulsifying agents may It will be understood, however, that the specific dose be naturally-occurring gums, for example gum acacia or level for any particular patient will depend upon a vari gun tragacanth, naturally-occurring phosphatides, for ety of factors including the activity of the specific com example soya bean lecithin, and esters or partial esters pound employed, the age, body weight general health, derived from fatty acids and hexitol anhydrides, for sex, diet, time of administration, route of administration, example sorbitan mono-oleate, and condensation prod O rate of excretion, drug combination, and the severity of ucts of the said partial esters with ethylene oxide, for the particular disease undergoing therapy. example polyoxyethylene sorbitan mono-oleate. The In accordance with the present invention there is emulsions may also contain sweetening and flavoring further provided a method of treating hypertension agents. comprising administering to a host in need of such treat Syrups and elixirs may be formulated with sweeten 15 ment a therapeutically effective amount of the combina ing agents, for example glycerol, sorbitol or sucrose. tion of Such formulations may also contain a demulcent, a (a) a compound of the formula: preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example as a sterile 20 R2 N Rl (I.) injectable aqueous or oleagenous suspension. This sus pension may be formulated according to the known art Y using those suitable dispersing or wetting agents and IN suspending agents which have been mentioned above. R3 N C The sterile injectable preparation may also be a sterile 25 injectable solution or suspension in a non-toxic parent wherein Y, R, R, R2, and R3have the same meaning as erally-acceptable diluent or solvent, for example as a above; and -- solution in 1,3 butanediol. Among the acceptable vehi (b) a saluretic agent selected from the group consist cles and solvents that may be employed are water, ing essentially of ethacrynic acid; furosemide; bumeta Ringer's solution and isotonic sodium chloride solution. 30 In addition, sterile, fixed oils are conventionally em nide; muzolimine; benzothiadiazines including chloro ployed as a solvent or suspending medium. For this thiazide and hydrochlorothiazide; chlorothalidone; purpose any bland fixed oil may be employed including quinethazone; metholazone; indacrinone; acetazolam synthetic mono- or diglycerides. In addition, fatty acids ide; ethoxyzolamide; and methazolamide. such as oleic acid find use in the preparation of injecta 35 In accordance with the present invention there is still bles. further provided a method of treating edema compris The combinations of this invention may also be ad ing administering to a host in need of such treatment a ministered in the form of suppositories for rectal admin therapeutically effective amount of the combination of istration of the drug. These compositions can be pre (a) a compound of the formula: pared by mixing the drug with a suitable non-irritating 40 excipient which is solid at ordinary temperatures but R2 N R1 (I.) liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are Y cocoa butter and polyethylene glycols. N I For topical use, creams, ointments, jellies, solutions 45 or suspensions, etc. containing the active ingredient are R3 N C-e R employed. For the compounds of Formula I, dosage levels of the wherein Y, R, R1, R2, and R3have the same meaning as order of 25 to 750 mg. per day are useful in the treat above; and ment of the above indicated conditions. For example, 50 (b) a saluretic agent selected from the group consist hypertension is effectively treated by the administration ing essentially of ethacrynic acid; furosemide; bumeta of from about 0.5 to 15 15 mg. of a compound of For nide; muzolimine; benzothiadiazines including chloro mula I per kilogram of body weight per day. Advanta thiazide and hydrochlorothiazide; chlorothalidone; geously from about 1 to about 15 mg. per kilogram of quinethazone; metholazone; indacrinone; acetazolam body weight and especially from about 2 to about 10 55 ide; ethoxyzolamide; and methazolamide. mg per kilogram daily dosage produces highly effec The compounds of Formula I in the combination of tive results. the present invention inhibit urea net reabsorption in a Dosage levels for the saluretic agent component of dose-related manner that includes increased excretion the combinations of the present invention will generally of sodium, chloride and potassium. Their effect is be in accordance with established dosages for these 60 independent of urine flow, and under conditions where agents as independent therapeutics. passive back diffusion is minimized they increase urea The amount of active ingredient that may be com clearance to approximate glomerular filtration rate. bined with the carrier materials to produce a single There is resident in pyrazinoic acid derivatives a dosage form will vary depending upon the host treated variable capability to inhibit secretion of urea into the and the particular mode of administration. For example, 65 lumen of the renal tubule. This is minimal in the pyrazi a formulation intended for the oral administration of noylguanidines of the present invention, which does not humans may contain from 25 to 750 mg. of active agent manifest that effect in Dalmatians; whereas the fluoro compounded with an appropriate and convenient analog of amiloride almost completely inhibits urea 4,663,322 7 8 secretion and fluid excretion even when back diffusion normal and whose extracellular volume is expanded is minimized. In stop-flow experiments, inhibition of carries a greater total expanded volume than if the urea active reabsorption and secretion can be demonstrated. level was normal. What may be considerably more The compounds of Formula I utilized in the present important than the increased total volume is how that invention are active on oral as well as parenteral admin increased "intra-cellular' fluid is distributed within the istration. 3-Aminopyrazinoylguanidine is most effective cells, their membranes, the compartmentalization of promptly, whereas pyrazinoylguanidine effect and their structure. blood level requires perhaps an added hour for maximal In view of the foregoing relationship between urea effect. and salt, it should follow that saluretic diuretic therapy As already indicated, the compounds of Formula I O would be an adequate control of fluid retention in the utilized in the present invention may properly be called absence of hyperuremia. When elevated urea plasma osmoregulatory agents. Osmoregulation, in turn, in concentration co-exists with fluid retention, or hyper volves several interrelated factors. tension that responds to sailuretic agent administration, Salts, as the sum of sodium, chloride and bicarbonate then it would seem rational to induce increased urea ions, supplies some 93% of the 300 milli Osmols/L of 15 excretion-to reduce its blood level. extra-cellular water in humans. Urea supplies about Increased urea blood level in cardiovascular renal 1.3% of that osmotic force, normally. Of the other disease is more common than appreciated. Whereas constituents, only glucose at 1.8% is a factor in osmo increased urine flow increases urea excretion, diuretics regulation. and adrenergic blocking agents are more likely to in Thus, osmoregulation of water volume to sustain 20 crease urea blood levels. In view of this proposed recip isotonicity is by thirst and by the action of the antidi rocal relationship of salt and urea retention, saluretic uretic hormone on the most distal portion of the neph diuretic treatment of hypertension represents only half ron to modulate water reabsorption. Whereas there is of a rational therapeutic measure-combined saluretic an appetite for salt regulation, the kidney seems de hyperuremic therapy. In accordance with the present signed to maximize sodium conservation. The search 25 invention, therefore, use of the pyrazinoic acid deriva for a salt regulating hormone continues, but the only tives of Formula I and the saluretic agents described known "active' sodium reabsorptive mechanisms in the herein makes possible a greater direct control of elec kidney involve the availability of hydrogen ions and/or trolyte and water balance, i.e., homeostasis, and ex the aldosterone-availability modulated availability of pands the utility of present therapy by providing direct potassium for exchange with sodium across the neph 30 regulation of tissue fluid as well as extracellular fluid. ron. Sodium rarely becomes a means for increasing The present invention provides a new and expanded water excretion, unless its reabsorption is decreased by way of looking at the management of electrolyte and the action of natriuretic or saluretic diuretic agents. water balance in terms of osmoregulation. One other homeostatic mechanism, the so-called so Accordingly, the present invention provides for sus dium pump, or sodium-potassium-magnesium-depend 35 tained therapy in the treatment of hypertension; postop ent ATP-ase mechanism common to cell membranes, erative treatment for the relief of tissue accumulation of serves to relegate the osmoregulatory effect of sodium fluids without risk of disturbed acid-base balance; and on fluid volume primarily to extracellular space, or treatment of pulmonary edema. about 25% of total cellular and extra-cellular volume. The following example, which was actually carried Thus, exquisitely small increases in sodium retention are 40 out, will serve to illustrate the hyperuretic activity of capable of expanding extra-cellular volume, thus in the pyrazinoic acid derivatives with and without a salu creasing blood pressure and glomerular filtration rate, retic agent, hydrochlorothiazide. without substantial involvement of cell volume. On the other hand, the osmoregulation of thirst by EXAMPLE the cells of the neurohypophysis is not so sensitive to 45 In Tables I and II are presented the effect of first urea concentration. As ADH influences water reab pyrazinoylguanidine then both pyrazinoylguanidine sorption, so does it alter urea back diffusion by the and hydrochlorothiazide (Table I) and, alternatively, kidney. Conversely, in the course of the human kidney the effect of hydrochlorothiazide then hydrochlorothia normally excreting some 25 grams of urea per day, that zide and pyrazinoylguanidine on excretion of urea and substance serves as the principal osmoregulator of 50 the inorganic electrolytes (Table II) in the dog. Prior to water excretion. the duplicate control 20 minute clearances, creatinine The plasma concentration of urea can be influenced was injected subcutaneously and an isotonic mannitol by both protein intake and by renal tubular reabsorption PO4 buffer venoclysis was begun and continued of a portion of the amount filtered by the glomeruli. throughout the experiment at 1 ml/min. Immediately Thus, a high protein diet normally increases both urea 55 after the second control clearance either pyrazinoyl and water excretion. Low protein intake or reduced guanidine was added to the venoclysis at 3 mg/kg/h glomerular filtration rate decreases urea and fluid out plus 2 mg/kg prime I.V. or hydrochlorothiazide was put. As an approximation, if glomerular filtration rate is added at a rate of 1.5 mg/kg/h plus 1 mg/kg prime. In reduced to half normal, plasma water concentration of either instance, after 30 minutes a succession of three urea will double on a given protein intake. The conse 60 20-minute clearances were conducted. Immediately quence is to expand intracellular volume as well as thereafter, the mannitol infusion was changed to one extracellular by a ratio of 45:25 or 1.8, assuming a con containing both pyrazinoylguanidine and hydrochloro stant osmotic pressure relationship exists between the thiazide at the dosages specified for the first drug phase. two spaces. The reason for this important difference Thirty minutes later, three more 20-minute clearances between the two spaces is that while sodium is essen 65 were performed. Blood and urine were subjected to the tially limited to extra-cellular space, urea is distributed customary analyses. more or less uniformly throughout body water. Thus, If the first drug phase of Tables I and II are com the patient whose urea plasma concentration is twice pared, the typical effects of the two compounds are 4,663,322 9 10 evident. Pyrazinoylguanidine increased urea excretion (a) pyrazinoylguanidine, a pharmaceutically accept and reduced its plasma concentration disproportionate able salt of pyrazinoylguanidine, 3-amino to the increased urine flow. It increased sodium, potas- pyrazinoylguanidine, or a pharmaceutically ac sium and chloride excretion, but the effect of hydro- ceptable salt of 3-aminopyrazinoylguanidine; and chlorothiazide on sodium, potassium, chloride and os- 5 (b) a saluretic agent selected from the group consist molar excretion was approximately twice that of pyrazi- ing essentially of ethacrynic acid; furosemide; noylguanidine. Any marginal effect of hydrochlorothia- bumetanide; muzolimine; saluretic benzothiadia zide on the urea parameters was due to the increase in zines; chlorothalidone; quinethazone; metholazone; urine flow attributable to the greater saluresis. indacrinone; acetazolamide; ethoxyzolamide; and When the two compounds were combined in the 10 methazolamide. second drug phase, the sum of their effects were evident 2. The pharmaceutical composition of claim 1, com in both Tables I and II. The effect on urea was increased prising pyrazinoylguanidine. somewhat by the greater diuresis plus the pyrazinoyl- 3. The pharmaceutical composition of claim 1, com guanidine, and the compounds combined to increase prising a pharmaceutically acceptable salt of pyrazi sodium, chloride, potassium and osmolar output and 15 noylguanidine. urine flow. 4. The pharmaceutical composition of claim 1, com The data discussed above is set out in Tables I and II prising 3-aminopyrazinoylguadine. below: 5. The pharmaceutical composition of claim 1, com TABLE I Effect of pyrazinoylguanidine alone then coadministered with hydrochlorothiazide on the excretion of urea, Na, C, K by the dog. Urine UREA Na C K Wo. P UVAP UV UV UV Utine ml/min pH GFR mg% ml/min CR Leq/min eq/min eq/min mOsm/min Exp. 4141; Dog Wt. 32.3 kg Control phase - isotonic mannitol-PO4 venoclysis at 1 ml/min 0.3 7.0. 78.5 18.5 25.9 0.33 22 8 3 202 0.2 6.9 74.6 18.7 24.6 0.33 23 18 3 189 Pyazinoylguanidine 2 mg/kg I.V. Prime plus 3 mg/kg/h added to mannitol venoclysis at 1 ml/min 0.5 7. 77.0 8.1 34.6 0.45 47 27 20 416 0.4 7.2 718 17.4 38. 0.54 5 30 23 .41 0.5 7.2 74.4 6.9 46.2 0.62 56 36 26 491 Hydrochlorothiazide I.V. Prime 1 mg/kg, 1.5 mg/kg/h added to venoclysis containing pyrazinoylguanidine as above 1.2 7.3 70.6 6.6 SO.8 0.72 140 109 58 .942 13 7.3 73.O. 16.5 54.7 0.75 168 135 68 1.174 14 7.3 75.5 6.5 57.3 0.76 189 155 67 1.267

TABLE I Effect of hydrochlorothiazide alone then coadministered with pyrazinoylguanidine on the excretion of urea, NaCl, K. by the dog. Urine UREA Na Cl K Vol. P UV/P UV UV UV Urine ml/min pH GFR mg% ml/min CR eq/min Leq/min eq/min mosm/min Exp. 4071; Dog Wt. 19.1 kg Control phase - isotonic mannitol-PO4 venoclysis at 1 mi/min 0.2 7.0 65.4 18.2 20.3 0.31 10 9 8 145 0.2 - 7.0 64.2 18.5 21.2 : 0.33 2 O 8 170 Hydrochlorothiazide I.V. Prime 1 mg/kg, 1.5 mg/kg/h added to mannitol venoclysis at 1 ml/min 0.7 7.1, 60.6 8.0 23.0 0.38 58 53 23 .616 0.9 , 66.7 8.1 27.3 0.41 89 78 35 .761 10 7.2. 63.0 18.0 25.2 0.40 107 101 43 874 Pyazinoylguanidine I.V. Prime 2 mg/kg, 3 mg/kg/h added to venoclysis containing hydrochlorothiazide as above 10 7.3 59.8 17.8 28.7 0.48 119 10 49 983 10 7.3 63.9 7.0 38.9 0.61 24 4. 4. 999 0.9 F.3 61.6 6.4 43. 0.70 21 110 36 886 The pyrazinoic acid derivatives utilized in the present prising a pharmaceutically acceptable salt of 3 invention may be prepared in accordance with well aminopyrazinoylguanidine. known procedures, for example those described in U.S. 6. An antihypertensive composition, comprising the Pat. No. 3,313,813. combination: What is claimed is: 65 (a) pyrazinoylguanidine, a pharmaceutically accept 1. An antihypertensive pharmaceutical composition able salt of pyrazinoylguanidine, 3-amino in unit dosage form comprising a pharmaceutically pyrazinoylguanidine, or a pharmaceutically ac acceptable carrier and the combination of: ceptable salt of 3-aminopyrazinoylguanidine; and 4,663,322 11 12 10. The composition of claim 6, comprising a pharma (b) a saluretic agent selected from the group consist ceutically acceptable salt of 3-aminopyrazinoylguani ing essentially of ethacrynic acid; furosemide; dine. bumetanide; muzolimine; soluretic benzothiadia 11. The pharmaceutical composition of claim 1, com 5 prising chlorothiazide or hydrochlorothiazide. zines; chlorothalidone; quinethazone; metholazone; 12. The pharmaceutical composition of claim 1, com indacrinone; acetazolamide; ehtoxyzolamide; and prising chlorothiazide. methazolamide. 13. The pharmaceutical composition of claim 1, com prising hydrochlorothiazide. 7. The composition of claim 6, comprising pyrazi 10 14. The composition of claim 6, comprising chloro noylguanidine. thiazide or hydrochlorothiazide. 8. The composition of claim 6, comprising a pharma 15. The composition of claim 6, comprising chloro ceutically acceptable salt of pyrazinoylguanidine. thiazide. 16. The composition of claim 6, comprising hydro 9. The composition of claim 6, comprising 3 15 chlorothiazide. aminopyrazinoylguanidine. k is : k ak

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