||||||||||| USOO5153178A United States Patent (19) 11) Patent Number: 5,153,178 Maroko (45) Date of Patent: * Oct. 6, 1992

54) COMPOSITIONS AND METHOD OF 58) Field of Search ...... 514/280, 284, 821, 26; TREATMENT FOR IMPROVING 424/10 CRCULATORY PERFORMANCE (56) References Cited 76 Inventor: Peter R. Maroko, 1765 Garwood Dr., Cherry Hill, N.J. 08003 U.S. PATENT DOCUMENTS 4,761,417 8/1988 Maroko ...... 514/284 *) Notice: The portion of the term of this patent subsequent to Aug. 2, 2005 has been Primary Examiner-Frederick E. Waddell disclaimed. Assistant Examiner-Diane Gardner Attorney, Agent, or Firm-Weiser & Stapler 21 Appl. No.: 226,630 57 ABSTRACT 22 Filed: Aug. 1, 1988 Compositions and methods for increasing the contrac tility of the mammalian heart as shown by a positive Related U.S. Application Data inotropic effect are disclosed, employing protoberbe 63 Continuation of Ser. No. 788,507, Oct. 18, 1985, Pat. rine alkaloids such as berberine, discretine, xylopinine, No. 4,761,417. berrberubine, tetrahydropalmatine, and stepharotine in 51 Int, Cl...... A61K 31/705; A61K 31/44; combination with cardiac such as ouabain, A61K 49/OO , , and . 52 U.S. Cl...... 514/26; 514/280; 514/284; 514/821; 424/10 17 Claims, 17 Drawing Sheets

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5,153,178 1. 2 ence. For further details relating to disorders of the COMPOSITIONS AND METHOD OF TREATMENT heart, reference is made to Harrison's Principles of Inter FOR IMPROVING CIRCULATORY nal Medicine, Thorne, Adams, Braunwald, Isselbacher PERFORMANCE and Petersdorf, McGraw-Hill Book Company, 8th Ed., Part 7, ("Harrison's') Disease of the Organ Systems, This is a continuation of application Ser. No. 788,507, Disorder of the Heart, Chap. 231 through 248, which are filed Oct. 18, 1985, now U.S. Pat. No. 4,761,47. referred to specifically herein and incorporated herein This invention relates to berberine-type alkaloids, by reference. particularly protoberberine drug compositions and their Reference shall also be made herein to the following use in the treatment and diagnosis of circulatory disor 10 clinical books, namely Veterinary Pharmacology and d ders. The compounds of the invention are of special Therapeutics, Jones, Booth and McDonald, Iowa State interest in prophylactic, therapeutic and other applica University Press, 4th Ed., 1977; Physicians' Desk Refer tions to prevent, minimize, control, alleviate, correct, ence "PDR' Medical Economics Company, 36th Ed. remedy and so on, various disorders or symptoms of 1982; and Veterinary Pharmaceuticals and Biologicals, circulatory disorders, including cardiovascular origins 15 “VPB", 1980/1981, Aronson, Harwal Publishing Com or types. The invention relates in terms of subjects (or pany, Media, Pa., 1980. patients) to the field of mammals, i.e. human and veteri The direct cardiac action of drugs may be divided nary fields. into four major areas: (1) an effedt on contractility (ino The invention also encompasses distinct and valuable tropic effect), reflecting alterations in the myocardial embodiments like compositions (and compounds and force-velocity relation at any given initial muscle methods of use) for chronic and acute heart failure and length; (2) an effect on heart rate expressed as an alter other pathologic states that will benefit from an im ation in the rhythmicity, i.e., the frequency of discharge provement in cardiac performance; for the treatment of of normal pacemaker tissue, generally in the sinoatrial shock (cardiogenic and non-cardiogenic shock); for the node; (3) an effect on conductivity, i.e., on the velocity treatment of arrythmias (whether of natural causes or 25 with which the depolarization wave travels through the caused by a drug); for increasing the usefulness of car myocardium and the atrial ventricular conduction sys diac glycosides (like of the digitalis types) including tem; (4) an effect on irritability, i.e. the tendency to broadening their usually limited therapeutic index; for provoke ectopic pacemaker activity, which is depen controlling or correcting A-V (atrio-ventricular blocks, dent on the rate of diastolic depolarization and the to be defined later herein) block in mammals. 30 threshold potential. The unique, unobvious and remarkable aspects of the One of the most serious consequences of all types of invention are quite numerous and will become apparent cardiovascular diseases involves the pathophysiological hereinafter, but at the outset it is most noteworthy that state in which the heart fails in its prime function as a the compounds of the invention are antiarrhythmo muscle acting as a pump. In general, heart failure is the genic, a remarkable utility in and by itself. Another result of severe primary depression of myocardial con aspect, is that the compounds of the invention have a tractility or extreme ventricular hemodynamic over remarkable combination of beneficial properties, such load combined with secondary diminution of the con as, concurrently, having a positive inotropic effect and tractile state. For a description of the basics and disor being antiarrythmogenic. Another unusual aspect is that ders of the myocardial function, especially cardiac con the compounds of the invention are useful both in acute traction see Harrison's, chapter 236. ventricular failure and also in chronic congestive heart Knowledge of the biochemical and physiological failure. These aspects are of course described in greater changes in heart failure has advanced considerably in detail hereinafter. recent years. Unfortunately, the development of phar The invention also relates to other biochemical or macological agents with clinically useful positive prop biomedical applications. The other aspects, to which 45 erties on cardiac contractility (positive inotropic the invention relates, will become apparent to one of agents) has not kept pace. Nearly two centuries have average skill in the art from the teaching herein. passed since W. Withering described the cardiovascular One important, but not the only important field to effects of Digitalis purpurea in 1787. Since then, the which the invention relates is the cardiovascular field, basic treatment of congestive heart failure remains with both in humans and animals. Today cardiovascular 50 the cardiac glycosides as is described in greater detail diseases, which have reached epidemic proportions, herein. Although literally hundreds of cardiac glyco account for a very high proportion of all deaths in the sides have been investigated, not one has been found world, especially in industrialized nations. Approxi with a wide therapeutic index. The most commonly mately one of every five persons has some form of car used cardiac glycosides digoxin and digitoxin, have a diovascular ailment such as heart disease, cerebrovascu 55 very narrow therapeutic index of less than 2, with life lar disease or hypertension. threatening cardiac arrhythmias as the first manifesta Cardiovascular disease not only is fatal but causes tion of toxicity. It is clear that a cardiotonic agent with prolonged suffering and disability in even a larger pro a wide therapeutic index is needed, especially one that portion of the population. In the United States alone, does not have the drawbacks of the glycosides at all or cardiovascular disease was responsible for almost one has them at least to a milder extent. million fatalities in 1979, well over one-half of all re For the treatment of vascular diseases and the phar ported deaths. Almost 5 million persons afflicted with macology of cardiac glycosides, reference is made here cardiovascular disease are hospitalized annually. The to Braunwald, Chapter 16, pages 515-538; Harrison, cost of this disease in terms of human annual costs due chapter 239, pages 1207-1210 and Goodman and to morbidity amount to over 8 billion dollars. Braun 65 Gilman, The Pharmacological Basis of Therapeutics, wald, Heart Disease, A Textbook of Cardiovascular Medi MacMillan Publishing Co., Inc., 5th Ed. which is incor cine, W. B. Saunders Company, Philadelphia, 1980 porated herein by reference, 1975, Section VI, pages ("Braunwald') which is incorporated herein by refer 653–682, ("Goodman'). 5,153,178 3 4. Because the cardiac glycosides are the classical most heart and vascular diseases, especially for a positive common positive inotropic agents, a further discussion inotropic drug. is presented below. There is no teaching or recognition in the prior art of The catecholamines like norepinephrine, isoprotere a berberine alkaloid which is cardiotonic, positive ino nol, dopamine and dobutamine have a limited role in the 5 tropic, and which has the spectrum of cardiovascular treatment of patients with chronic congestive heart properties of the compounds or compositions disclosed failure. They are inactive orally and have a short dura herein. Nor is there such teaching or recognition of the tion of action when given intravenously. Their major therapeutic treatments disclosed herein. There is no use is in the treatment of acute ventricular failure such teaching or disclosure of compounds (or composition as a low cardiac output state after cardiac surgery and 10 s-or methods of use-) like those of the invention shock associated with sepsis or acute myocardial infarc which have the combination of properties disclosed tion. The arrhythmogenic and calorigenic properties of herein or of certain specific properties disclosed herein the catecholamines and lack of activity in patients pre or which naturally flow or result from these properties. treated with beta-adrenergic blocking agents, such as A review of the state of the art shows the following: propranolol further limit the use of these agents. 15 1. Akhter, Sabir, Bhide: "Possible Mechanism of An The positive inotropic activity of glucagon was first tidiarrheal Effect of Berberine', Indian J. Med. Res. demonstrated by Farah and Tuttle more than 15 years 70:233-24, 1979. Possible mechanism of the antidiar ago. Since then there has been interest in the possible rheal effect of berberine was studied. Subjects were role of this agent in the treatment of congestive heart failure. Although the initial clinical studies with gluca- 20 dogs and rats. gon were promising, further evaluation indicated that 2. Creasy, W. A.: "Biochemical Effects of Berber the clinical role of glucagon as a cardiotonic agent was ine', Biochen. Pharmacol. 128:1081-1084, 1979. Berber limited. Glucagon was found to be inactive in patients ine inhibits the biosynthesis of DNA, RNA, proteins with chronic congestive heart failure. and lipids, as well as the oxidation of glucose to CO2 Other drugs which play a role in the treatment of 25 when incubated with S180 cells in vitro. Also tested for heart diseases are the beta-adrenergic blocking agent, inhibitory activity: thalicarpine and d-tetradrine. often used as antiarrhythmic agents. The most com 3. Cohen, H. G. Seifen, E. E., Straub, K. D., Tiefen monly used is propanolol, Goodman, chapter 26 and bach, C., Stermitz, F. R.: "Structural Specificity of the pages 609-704. Recently it was reported that Timolol, a NaK-ATPase Inhibition by Sanguinarine, an Isoquino beta-blocker was approved by the Food and Drug Ad- 30 line Benzophenanthridine Alkaloid", Biochem. Phar ministration ("FDA") as preventive against recurrence macol. 27:2555-2558, 1978. Discussion of structural in patients who had suffered a first heart attack. The specificity of the NaK-ATPase inhibition by sanguina Wall Street Journal, Nov. 27, 1981. rine, an isoquinoline benzophenanthridine alkaloid by Amrinone, a 5-amino-3,4'-bipyridin-6(1H)-one, a comparing the compound to other quinolines and non- and non-catechol in nature, has been 35 isoquinolines including berberine. Berberine is inactive reported to have positive inotropic effects. Farah, with respect to inhibition of NaK-ATPase activity. Alousi, New Cardiotonic Agents: A Search For Digita Study of acridine, acriflarine, chloride, protopine, lis Substitute Life Sci., 1978; 22:1139-48; Alousi, Farah, capaurine, cory cavine oxalate, coralyne chloride, Lesher, Opalka Jr., Cardiotonic activity of amrinone-- corydaline, ethidium bromide, d-tetrahydropalmatine, Win 40680 (5-amino-3,4'-bipyridin-6 (IH)-One)), Circ. 40 boldine, chelidonine, laudanosine, hydrastine, enetine Res. 1979; 45:666-77; DeGuzman, Munoz, Palmer, hydrochloride, nitidine and fagaronine yielded varied Davolos, Alousi, Clinical evaluation of amrinone--a results. new inotropic agent (abst), Circulation 1978; 58 (Suppl 4. Davidson, M. W., Lopp, I., Alexander, S., Wilson, II): 11-183; Benotti, Grossman, Braunwald, Davolos, W. D.: "The Interaction of Plant Alkaloids with DNA. Alousi, Hemodynamic assessment of amrinone, a new 45 II. Berberinium Chloride', Nucl. Acid Res. 2697-2712, inotropic agent, New Engl. J. Med. 1978, 299:1373-7, 1977. Other substances studied include quinacrine, quin LeJemtel, Keung, Sonnenblick, Ribner, Matsumoto, oline methanol, mefloquine, putrescine, coralyne and Davis, Schwartz, Alousi, Davolos, Amrinone: a new proflavine. Systems studied include cows and chickens. non-glycosidic, non-adrenergic cardiotonic agent effec Data suggests that a large portion of the berberinium tive in the treatment of intractable myocardial failure in 50 ring system intercalcates into DNA. man, Circulation 1979, 59:1098-104. 5. Sheppard, H., Brughardt., C. R.: "The Dopamine It was recently reported that an application for a sensitive Adenylate Cyclase of the Rat Caudate Nu NDA has been filed with the FDA for Inocor, a brand cleus. The effect of Aporphines and Protoberberines', of amrinone. Previously, clinical trials had been sus Biochem. Pharmacol. 27:1113-11 16, 1978. Protoberbe pended because the drug promoted blood platelet ag- 55 rines are fairly potent as inhibitors of the DA-cyclase gregation, The Wall Street Journal, Oct. 28, 1981. with little effects on the beta system. Aporphine ana Recent calcium antagonists, like and logues were tested. nifedipine have been proposed to affect coronary perfu 6. Kovar, J., Skursky, L: "Fluorescence Study of sion, primarily by direct action on the coronary vascu Liver-Alcohol-Dehydrogenase Complexes with Ber lature. They too have hemodynamic features which are 60 berine and Other Ligands', Eur, J. Biochem, not desirable. For instance, verapamil is known to re 40:233-244, 1973. Dissocation constant of liver-alcohol duce contractility and may induce atrio-ventricular dehydrogenase-berberine complex was determined. block. Competition study of complex made with NAD, Diuretics are also used for the management of heart NADH, o-phenanthroline, pelargonic and capric acids. failure. Such diuretic therapy is not without complica- 65 Fluorescence of berberine found to be typical for hy tions. Braunwald, page 544 and seq. drophobic probes, indicating that berberine interacts It is evident, from the state of the art, that there is a with a hydrophobic site of the liver-dehydrogenase serious need for a effective drug for the treatment of enzyme. 5,153,178 5 6 7. Kulkarni, S. K. Dandiya, P. C., Varandanu, N. L.: Cryptopine et de la Berberine', Comptes Rendus. Soc. "Pharmacological Investigations of Berberine Sulfate", Biol. 127:1022-1024, 1938. Study of effects of berberine, Japan. J. Pharmacol. 22:11-16, 1972. Berberine sulfate cryptopine and papaverine on blood pressure the ner found to have anti-amoebic action as well as antiacetyl vous system, respiration, the heart and intestine, under choline, anti-histamine and dose-related spasmolytic taken in dogs and rabbits. The three compounds exhib effects in comparison study with vioform and carbar ited comparable effects on blood pressure, respiration sone. Systems tested include rats, rabbits, guinea pigs, and the heart. In the intestine and nervous system, ber frogs and dogs. berine and cryptopine acted similarly, and opposite to 8. Dutta, N. K., Marker, R. P. Rao, N. R.: "Berberine papaverine, which results were attributed to the chemi in Toxin-Induced Experimental Cholera', Br. J. Phar O cal similarity of berberine and cryptopine. In small macol. 44:153-159, 1972. In tests performed in rabbits, doses, berberine had a positive inotropic and chrono berberine found to be both vibriostatic and effective tropic effect on the heart which was suppressed by the against choleragenic toxin. vagus section but was not modified by the injection of 9. Desai, A. B., Shah, K. M. Shah, D. M.: "Berberine or yohimbine. In larger doses berberine de in Treatment of Diarrhea', Indian Pediatr. 8:462-465, 15 pressed heart's contractility. 1971. Berberine found to be an effective tool against 16. Soto, M., Sivori, P. N.: "Accion del Sulfato de acute-gastroenteritis caused by different organisms. Berberina Sobre el Corazon de Maniferos,” Rey, Assoc. 10. Shanbhag, S. M., Kulkarni, H. J., Gaitonide, B. Med. Argent. 47:3062-3068, 1933. B.: "Pharmacological Actions of Berberine on the Cen 17. Soto, M., Sivori, P.N.: "Accion Sobre el Aparato tral Nervous System', Japan. J. Pharmacol. 20:482-487, 20 1970. In tests on mice, rats and cats, berberine found to Circulatorio y Sobre la Respiracion del Sulfato de Ber act as a sedative, potentiating pentobarbitone sleeping berina,” Rey, Assoc. Med. Argent, 47:2492-2501, 1933. time. Berberine found to be devoid of tranquilizing, 18. Chopra, R. N. Dikshit, B. B., Chowhan, J. S.: anticonvulsant and analgesic properties, nor does it "Pharmacological Action of Berberine', Indian J. Med. modify morphine analgesia or barbiturate hyperalgesia. 25 Res. 19:1193–1203, 1932. Study of berberine, its toxicity, ll. Fukuda H., Watanabe K, Kudo T., "Some Obser absorbability by G.I. tract, effect on movement of G.I. vations on the Cardiovascular Effects of 9-Substituted tract, effect on cardiovascular system, vasomotor cen Berberines', Chen. Pharmaceut. Bull 18:1299–1304, ter, respiratory system, urogenital system; and its effec 1970 is a report on a study of certain active principle tiveness against malaria and oriental sore are discussed. extracted from the plant Coptis japonica. This type of 30 Systems studied include cats, dogs, rabbits, frogs and berberine and the 9-substituted alkoxy derivatives were rats. Berberine effects a sharp fall of blood pressure tested for cardiovascular effect in rats, toads and muscle associated with an increase in the volume of the system, preparations. No change in blood pressure from normal intestines and kidneys as well as dilation of blood ves pressure after short transient hypotension (with a mini sels. mum dose of 0.1 mg/kg in rats) was observed. No 35 19. Perkin: "Epiberberine", J. Chem. Soc. change in heart rate was observed (as shown by the 113:503-505, 1918. Nomenclature and structure of ber ECG recordings). The hypotension was attributed to berine and epiberberine. depression or decrease of contractility of the heart. 20. Williams, W. W.: "The Effects of Hydrastis and Where the substitution in the 9- position is -OH, a Its Alkaloids on Blood Pressure'. J. Amer. Med. Assn. transient hypertension is caused. For compounds with 50:26-30, 1908. Study of hydrastine, berberine and hy O-n-C8H17 and O-n-C12H25 substituents no tran drastinine on dogs, turtles and frogs. Discussion on sient (hypo-or hypertension) or other effect on blood drugs' effects on blood pressure, the heart, kidney and pressure were observed. This type of compound gener spleen volume, and respiration. ally had no effect or decreased in blood flow, thus sug 21. Trendelburg, P.: "Bestimmung des Adrenalin gesting a general increase in peripheral resistance. At 30 45 gehaltes in noronalem Blut sowie Abklingen der Wir mg/Kg, (i.p.) this type of berberine extract was lethal. kung einer ennalegen intravenosen Adrenalininjektion 12. Homma, N., Kono, M., Kadohira, H., Yoshihara, mittels physialogisher messmethods.' Arch. Esp. Pathol. S., Masuda, S.: "The Influence of Berberine Chloride Pharmacol. 63:161-176, 1910. on the Intestinal Flora in Infants', Arzneimittel-Forsch 22. Mani, R. S., Noronha, O. P. D.: "Preparation of 11:450-454, 1961. 50 Radioiodinated/131/Berberine,' Radiochem. Radioa 13. Schein, F.T., Hanna, C.: "The Absorption, Dis nal Letters 5:119-123, 1970 Radioioiodinated/131I/- tribution and Excretion of Berberine', Arch. Int. Phar Berberine has been prepared by the carefully controlled macodyn. 126:317-325, 1960. In studies in the rat, ber iodination of berberine sulphate using carrier-free so berine is found to initially concentrate in the animal's dium iodide /131/ and hydrogen peroxides followed pancreas, heart, liver, omental fat and kidney. 55 by ion-exchange purification. 14. Jang, C.-S.: "The Action of Berberine on Mam 23. DAS Gupta, Dikshit: "Berberine in the Treat malian Hearts," J. Pharmacol. Exp. Ther. 71:78-186, ment of Oriental Sore' The Indian Medical Gazette, 1941. Studies on cat, rabbit and dog hearts show that pages 67-70, February 1929. Berberine sulphate inhibits berberine in low and moderate dosages increases heart Leishmania tropica. rate and coronary flow. Little effect on cardiac output 24. Kamat, S. A. "Clinical Trials with Berberine Hy was observed. In large doses it depresses the heart. The drochloride For the Control of Diarrhea in Acute Gas cardio-inhibitory action of acetylcholine is potentiated troentenitis.' J. Assoc. Phys. India 15:525-529, 1967. by small doses of berberine but antagonized by moder Berberine hydrochloride was found to be effective in ate and large doses. Doses of berberine which are antag arresting diarrhea. onistic to acetylcholine are also antagonistic to pilocar 65 25. Skinner, Basil, "The Isomerization of Berberine pine but not to KCl. and Cotarnine Bases in Presence of Alkali.' J. Chen. 15. Mercier, F., Delphaut, J., Blache, P.: "Effets Soc., 823-827, 1950. Study of aqueous and alcoholic Pharmacodynamiques Compares de la Papaverine, de la solutions of berberine. 5,153,178 7 8 26. Bevalot, E, Leboeuy, M., Bouquet, A., Cave, A.: U.S. Pat. Nos. 3,943,251 and 3,903,282 disclose the "Alcaloides des Annonacees: Alcaloides des Ecorces de use of berberine for dilating human eye pupils, Tiges et de Racines de Pachypodanthium confine, En U.S. Pat. Nos. 2,003,204, 2,052,150, 2,486,937 and gler et Diels.” Annales Pharmaceutiques Francaises, Vol. 3,023,147 disclose digitalis derivatives and methods for 35, No. 1-2:65-72 (1977). Study of alkaloid content of 5 their production. stem and root-barks of Pochysodanthium confine. The Japanese disclosure No. 39-21529 discloses 9-sub authors disclose six alkaloids: three tetrahydro stituted berberine derivatives which have a stronger protoberberines, and three, 7-hydroxy aporphines. enterobacterial action than berberine. Japanese disclo 27. Schewe, T., Muller, W. "Hemonung der Atmung sure No. 45-4992 discloses 13-alkyl substituted berber skette durche die Alkaloide Berberinsulfat, Alpinigen O ine derivatives which have antiulcer activity. und Tetrahydropalmatin." Acta Biol. Med. Germ., Vol. French Patent No. 1,568,738 discloses a new process 35, 1019-1021, (1976). Study of alkaloidal inhibition of for the preparation of tetracyclic bases including 2, 3, 9, the NADH oxidase system of electron transfer particles 10, 11- pertomethoxyberberine. in beef heart. British Patent No. 1,265,627 discloses 13-methyl-7, 8, 28. Benotti, J. R., Grossman, W., Braunwald, E., 15 13, 13a-tetradehydro-2, 3, 9, 10-tetramethoxyberberine Davolos, D. D., Alousi, A. A.: "Hemodynamic Assess as an anti-peptic ulcer ingredient. ment of Amrinone', The New England J. of Med., Vol. This list is not represented to be complete but it con 299, No. 25, 1978. Amrinone, a new bipyridine deriva tains those patents and articles which are presently tive, exhibits a positive inotropic action in experimental available. preparations and is effective when administered orally 20 A discussion of cardiac glycosides follows as an intro to dogs. Amrinone was further discussed above. duction to the compounds of the invention and of a 29. Miller, R. R., Vismara, L. A. Williams, D. O., discussion of relevant properties and biological effects. Amsterdam, E. A. Mason, D. T.: "Pharmacological Cardiac glycosides of clinical importance vary in Mechanisms for Left Ventricular Unloading in Clinical structure-and in cardiac effects-depending upon the Congestive Heart Failure. Differential Effects of Nitro 25 botanical source from which they are extracted. Indeed, prusside, Phenotalamine, and Nitroglycerin on Cardiac it has been reported that the terminology employed in Function', Circulation Res. 39:127-133, 1976. the literature is confusing and awkward because the 30. Arentzen, C. E. Rankin, J. S., Anderson, P. A. names refer to botanical origins rather than to exact W., Feezor, M. D., Anderson, R. W.: "Force Fre chemical structures. Goodman, pages 654, 655. It has quency Characteristic of the Left Ventricle in the Con 30 been shown that cardiac glycosides exist in plants as scious Dog', Circulation Res, 42:64-71, 1978. Study of precursors called "native", "natural", or "genuine" three systemic vasodilators, nitroprusside, phenotala glycosides. The three major botanical sources of car mine, and sublingual nitroglycerin. The three produced diac glycosides of clinical importance are digitalis, stro disparate modifications of LV function by their differ 35 phanthus and Scilla (squill). Digitalis purpurea and digita ing alterations of preload and impedance. lis lanata are the recognized source for Digitoxin, U.S. and foreign patents found which discuss berber Gitoxin and Gitalin. Digitalis lanata is the recognized ine type compounds are the following. source for Digotoxin, Gitoxin and Digoxin. Strophan U.S. Pat. No. 3,894,027 discloses a process for resolv thus gratus is the recognized source for ouabain. ing racemic reticuline which is useful as a precursor of 40 Official preparations available for clinical use in the pallidine, sinoacutine, coreximine and the berberine United States are the following: digitoxin, digoxin, la alkaloids. natoside C, deslanoside, acetyldigotixin, powdered digi U.S. Pat. No. 3,884,911 discloses a derivative of ber talis and ouabain. See Goodman, pages 672-676. See the berine formula wherein RCO represents a cinnamoyl PDR, pages 211, 310. group which may be substituted with chloro, acetoxy, 45 Likewise, alkaloids vary considerably depending methoxy, or methylenedioxy which inhibit transplanted upon the origin of the plant. See the Isoquinoline Alka sarcona strain in mice. loids, Chemistry and Pharmacology, Shamma, Aca U.S. Pat. No. 3,865,830 thiophosphamide derivatives demic Press, New York 1972, Chapter 10 and the Chem of isoquinoline alkaloids which have pharmacological istry of Isoquinoline Alkaloids, Tetsuji Kametani, El activity. 50 siver Publishing Co. New York, 1996. U.S. Pat. No. 3,272,707 discloses quinolizine deriva The invention which has several embodiments to tives having tranquilizing, antidepressant and antie which some were referred to above; others are briefly metic activities. described below; others will become apparent as the U.S. Pat. No. 3,420,834 discloses berberine deriva description proceeds. tives having tranquilizing and analgesic properties. The 55 An embodiment of the invention relates to isoquino compound is obtained as a mixture with analogs. line, especially protoberberine alkaloids, their composi U.S. Pat. Nos. 3,426,027 and 3,267,107 disclose ber tions which have a biological effect, especially in the berine derivatives having anticonvulsant, analgesic and treatment of cardiovascular disorders, heart failure, tranquilizing activities. cardiac arrhythmias, shocks of circulatory nature, in U.S. Pat. Nos. 3,910,938, 3,884,911, 3,920,665 and 60 creasing contractility of the heart of mammals, improv 4,033,966 disclose berbine derivatives which inhibit ing aortic blood flow, and decreasing heart rate and transplanted sarcoma strain in mice. systemic arterial pressure, reducing afterload of the left U.S. Pat. No. 4,087,426 discloses berberine and proto ventricle, decreasing preload in the left ventricle, de berberine analogs for treating uterine hemorrhage. crease in calculated vessel peripheral resistance, de U.S. Pat. No. 4,200,629 deals with a process for ex 65 crease in mean arterial pressure and decreasing arrhyth tracting berberine from plants which contain the alka mias. These effects may be obtained to varying degrees, loid. A product results which has activity for inhibiting concurrently or not, with the individual compounds the growth of fungi. (compositions and methods of use) of the invention. 5,153,178 9 10 Of course, contemplated by the invention are compo active such as ouabain which is a stophantic glycoside. sitions using one or more of the compounds of the in However, normally the generic term used (albeit some vention, in which event, certain of the properties or what incorrectly) is digitalis. Indeed, despite the value effects can be synergistic or otherwise. of diuretics and afterload-reducing agents, the glyco With respect to the embodiment of the invention sides remain the principal positive inotropic agents use which relates to the treatment of cardiovascular disor ful in long-term management of patients with conges ders, the protoberberine type alkaloids of the invention tive heart failure. Other drugs, known to act through are useful in the treatment of congestive (or chronic) stimulation of myocardial beta-adrenergic receptors heart failure. Moreover, the protoberberine type alka such as the catecholamines and sympathomimetic loids of the invention are useful in acute heart failure 10 agents, are very potent cardiac stimulants but for the (cardiogenic shock being its more severe manifestation). most part may have serious adverse effects such as This is an important distinction over the digitalis type tachycardia, ventricular irritability and intensification glycosides which are known to be ineffective for treat of myocardial ischemia. Their use is restricted mainly to ment of cardiogenic shock. This is also an important in-hospital intravenous treatment of cardiac shock and advantage of the compounds of the invention in that the 15 they are not given orally for chronic congestive heart treatment of a patient can be continued with the same failure. A major therapeutic advance in the medical drug (one of the compounds of the invention) whereas treatment of congestive heart failure awaits discovery; in traditional therapy with digitalis or others, this is not namely, an effective, non-toxic drug that increases myo possible. cardial contractility without adverse effects such as The present invention relates to protoberberine alka 20 increase in peripheral vascular resistance, decrease in loids and their use both in the pharmacological treat coronary flow arrhythmias and a treatment that is free ment of cardiovascular disorders and as a diagnostic of the adverse effect of digitalis. tool. The compounds of the invention have a strong In accordance with the invention, a group of proto positive inotropic effect, increase the aortic blood flow, berberine alkaloids and derivatives has been discovered decrease the calculated total peripheral resistance of 25 which are found to exert a strong positive inotropic vessels and are free of the drawbacks of digitalis, as has action, decreases peripheral vascular resistance, raises been mentioned above. The compounds also have a cardiac output, and is virtually free of the drawbacks of broad therapeutic index; that is a very small amount is digitalis such as arrhythmias. The compounds of the effective for the desired effect and very large dosages invention have a significantly broader therapeutic ratio are not toxic. 30 than digitalis type cardiac glycosides. The compounds and biologically active compositions For a better understanding of the invention, a review of the invention are useful for the treatment of conges of the pharmacological and cardiovascular properties of tive or chronic heart failure and also acute heart failure digitalis is provided below which will facilitate the and/or cardiogenic shock. Moreover, states of systemic comparison with the protoberberine alkaloids and de arterial hypotension, low cardiac output and shock of 35 rivatives of and used in accordance with the invention. other origins with increased peripheral vascular resis A large number of compounds have as their major tance and/or decreased cardiac output will also benefit pharmacological action the ability to alter cardiovascu from this therapy. lar function. The therapy of cardiac diseases, indepen Another embodiment of the invention relates to the dently of the ethiology of these diseases, is aimed either use of the compounds of the invention on the control of 40 to compensate the mechanical dysfunction of the heart arrhythmias. The compounds and biologically active which is the deterioration of the function of the heart as compositions of the invention are useful in the treatment a pump (and is called heart failure); or treatment of the of cardiac arrhythmias. The scope (and types) of car abnormal electrical stimuli (i.e. arrhythmias) which may diac arrhythmias in which they are active is very ample, cause a life threatening condition. The therapy of the since they are active on arrhythmias provoked by acute 45 latter is antiarrhythmic treatment. The most commonly coronary artery occulusions as well as pharmacologi used drugs are and . The treat cally produced or stimulated or induced arrhythmias by ment of cardiac arrhythmias can also be performed by arrhythmogenic drugs. They may be arrhythmias pro propranolol, a beta-adrenergic blocking agent. Some voked by "reentry' or of the "automatic" types. The other new antiarrhythmic agents are being either intro compounds of the invention are effective both in pre 50 duced or tested now. To treat the former condition (i.e. venting and in interrupting (including arresting or stop any form of heart pump failure) digitalis and certain ping) arrhythmias. They are effective on ventricular other structurally closely allied drugs have in common arrhythmias and on supraventricular arrhythmias. In a specific and powerful action on the myocardium that the former category, they were active on premature is unrivaled for the treatment of congestive heart fail ventricular ectopic beats, bigemini, ventricular tachy 55 le. cardia and ventricular fibrillation. In the latter cate More recently vasodilators have been proposed (per gory, they were active in atrial fibrillation and atrial haps to overcome or compensate a principal shortcom paroxysmal tachycardia. The effect of the compounds ing of digitalis), to treat heart failure in a different man of the invention as antiarrhythmogenics is a remarkable ner, i.e. by reducing the afterload and thus improving effect. performance not through a positive inotropic mecha With respect to cardiac disorders, depression of ven nism. These agents also reduce the preload (generally tricular function is the principal cause of heart failure, measured by left ventricular end diastolic pressure or and improvement of myocardial contractility by means pulmonary wedge pressure) which is generally elevated of cardiacally active glycosides have been the keystone in patients with with congestive heart failure. in the management of this pathologic condition in hu 65 It is known that the main pharmacodynamic property mans and animals. These glycosides are typically repre of cardiac glycosides like digitalis is its ability to in sented by digitalis glycosides since these are the most crease the force of myocardial contraction. The benefi commonly used, although other glycosides are equally cial effects of the drug in congestive heart failure-in 5,153,178 11 12 creased cardiac output; decreased heart size, venous frequently provokes abnormal rhythms due to its low pressure, and blood volume-are explained on the basis therapeutic index. Practically all types of existing ar of increased contractile force, that is, a positive inotro rhythmias have been reported to appear as a result of pic action. Because digitalis often significantly slows the digitalis intoxication: atrial premature beats, atrial tach ventricular rate in atrial fibrillation, it was believed that 5 ycardia with block, nodal premature beats, junctional the main effect of the drug was to slow the heart rate. tachycardia, ventricular premature beats isolated or in However, it is reasonably well-established now that different cyclical patterns being bigemini the most fre digitalis is principally effective in congestive heart fail quent, ventricular tachycardia, "bidirectional' ventric ure, in contrast to non-congestive heart conditions, ular tachycardia, finally ventricular fibrillation. It in regardless of cardiac rhythm and it is thought that relief 10 creases the duration of the P-R interval which when is not by virtue of cardiac slowing but by its direct exaggerated is a toxic manifestation and constitutes first action to increase the force of myocardial contraction. degree A-V block. Thereafter, there may occur second Cardiac glycosides are known to exert their inotropic degree A-V block, Wenckebach or Mobitz type, and stimulation by increasing the rate at which tension or the advanced 2nd degree A-V block may evolve to force is developed. It is also known that the positive 15 complete (3rd degree) A-V block. inotropic effect of digitalis is not dependent on cate The protoberberines of the invention do not show an cholamine liberation or potentiation, for this has been increase in the duration of the A-V interval which is the demonstrated in chronically reserpinized dogs and in basis of all the more advanced (and serious) A-V blocks. the presence of beta-adrenergic blocking agents. It will be also shown that the protoberberines did not It is believed that the action of digitalis on the "car 20 increase spontaneously neither the A-H nor the H-V diac tone' is due to the fact that digitalis does increase intervals which constitute the A-V interval while digi the tone of the muscle during systole, that is, the force talis typically prolongs the A-H interval. Digitalis very of systolic contraction is increased. But it is known that characteristically depresses the S-T segments and short digitalis does not increase the resistance to stretch of the ens the corrected QT intervals (QTc). The protoberbe ventricles in diastole, not even in high concentrations, 25 rines of the invention contrary to digitalis (and to beta Digitalis is known to reduce the size of the failing heart adrenergic agonists) increase the QTc intervals and do because it increases the contractile power. not depress the S-T segments. More recent studies have shown that digitalis in Many other side effects may occur with digitalis, the proves performance at any given filling pressure of the most common being those related to the gastrointestinal heart. Digitalis increases cardiac output in patients with 30 system: anorexia, nausea, vomiting, diarrhea. Neurolog congestive heart failure and with "latent" heart failure, ical side effects are well known including coma. that is in subjects who had heart disease but were not Other drawbacks from the use of digitalis are head yet in overt failure. But it has also been reported that in ache, fatigue, malaise and drowsiness. Other neuropsy normal subjects digitalis causes an increase in total pe chiatric effects have been noted. Vision has often been ripheral vascular resistance, mean arterial blood pres 35 reported to be blurred. Experimental observation in sure, reduction in forearm blood flow and increases in animals suggests that digitalis preparations increase venomotor tone. This is caused by combination of a blood coagulability and heparin (in sodium or calcium moderate, direct vasoconstrictor action on both arterio form) has been recommened to be used to reduce the lar and venous smooth muscle and a centrally mediated toxicity of digitalis and ouabain. increase in sympathetic tone. This opposes the effects of 40 Digitalis, as had been noted above, is of little hemo increased cardiac contractility by increasing impedance dynamic or clinical benefit in cardiogenic shock but is to ventricular ejection. The two effects thus counteract usually moderately effective in pulmonary edema and each other, and there is essentially no change in cardiac milder forms of heart failure secondary to myocardial output. As explained further below, the compounds of infarction. Digitalis has variable effects on angina pec the invention differ markedly and advantageously in 45 toris, reducing this symptom in the presence of cardio that respect. In contrast to normal subjects, in conges megaly and heart failure but tending to increase it in tive heart failure, varying degrees of sympathetically their absence. mediated peripheral vasoconstriction already exist as For more information regarding the pharmacology of part of the compensatory response. Thus, in patients digitalis and other similar cardiac glycosides, reference with congestive heart failure, cardiac output increases, 50 is made to "Goodman' pages 653 et seq. there is a reduction of peripheral resistance, an increase An important problem arises in the diagnosis and in blood flow, and a reduction of venomotor tone. Fre treatment of digitalis intoxication. Digitalis is often used quently there is no increase in arterial pressure. The in situations where the toxic effect of the drug are diffi effects of digitalis upon arterial pressure in patients with cult to distinguish from the effects of the cardiac dis congestive failure are variable. 55 eaSt. Digitalis is known to exert a negative chronotropic The choice of the digitalis glycoside is important. action, which in part is a vagal effect and in part is due The two main criteria in selection are speed of on-set to a direct action on the sinus pacemaker. The apparent and duration of cardiac action. Yet all of the digitalis suppression of pacemaker activity which may take products have approximately the same narrow thera place following high doses of digitalis is probably due peutic index. That is, the therapeutic dose is approxi not to the arrest of the pacemaker but rather to a sino mately 50-60% of the toxic dose. For maintenance atrial block related to a depression of conduction. This therapy, prolonged duration of action is often desirable is the reason why in patients with second degree atrio such as afforded by digitoxin; when more rapid elimina ventricular block, digitalis may be harmful by inducing tion is required, then digoxin or are consid complete (third degree) block. The increase in the A-H 65 ered. Where emergency therapy and rapid on-set of interval is a well documented property of this drug action may be imperative ouabain, deslanoside and di which is the cause of these atrioventricular blocks. In goxin may also be used for this purpose. Digoxin is the patients who need this therapy for heart failure, digitalis most common preparation used in the United States. 5,153,178 13 14 Digitalis is reported to be the fourth most frequently which are antiarrhythmics that improves cardiac per prescribed drug in the United States. But it also has one formance and minimize heart failure. Generally, drugs of lowest margins of safety. A most serious shortcoming that are positive inotropic do not possess antiarrhyth of digitalis is its narrow therapeutic range which is mic properties and antiarrhythmic drugs generally do about 40 to 50%. It has been reported that there is no 5 not possess positive inotropic properties. "non-toxic' . A compound which is both a positive inotrope while Recently the first randomized trial of the effects of concurrently being antiarrhythmic, is, as far as could be digoxin was conducted in 25 patients with heart failure determined, not known. Conversely, an antiarrhythmic (Lee D. C. S., Johnson, R. A., Bingham, J. B., Leahy, which concurrently is a positive inotrope, is, as far as M., Dinsmore, R. E., Goroll, A. H., Newell, J. B., O could be determined, not known. Thus, such a com Strauss, H. W. and Haber, E. Heart Failure in Outpa pound where both of these advantageous properties are tients. A randomized trial of digoxin versus placebo. coupled is not known. In addition, the invention encom New England Journal of Medicine, 306:799-705, (1982). passes interrupting (also stopping) and preventing digi It was found that based on a clinical scoring system, talis-induced arrhythmias and concurrently improving fourteen patients improved while eleven did not im 15 the contractility of the mammalian heart, another prove. Only patients with a third heart sound showed unique aspect of the invention. improvement independent of the severity of the heart In one aspect of the invention, the administration is failure. It is also of interest that there was a failure to performed on a patient having congestive heart failure. increase the ejection fraction (done by multigated nu In, another aspect it is performed on a patient under clide ventriculography) in these patients. 20 cardiogenic shock. In another aspect of the invention, It is evident from the above discussion of the effects the therapy is given sequentially to a patient under of digitalis that the drug does have a number of impor cardiogenic shock and thereafter for cardiac mainte tant shortcomings. nance therapy. In situations where a patient may be Since the compounds of and used in accordance with under therapy with a compound of the invention for this invention are different from digitalis and allied 25 congestive heart failure and then is subjected to conges cardiac glycosides, the above description of these tive heart failure, appropriate therapy with a compound glycosides is useful to set the compounds of the inven of the invention may be continued with a compound of tion apart and show their differences over the prior art. the invention. Another embodiment of the invention It is an object of this invention to provide a class of encompasses the administration of the compounds of compounds which have a combination of advantageous 30 the invention together with digitalis to have additive properties over digitalis (and other cardiac glycosides). action (or synergistic) on the positive inotropic effect It is a further object of the invention to provide a new and on treatment of heart failure. class of compounds which may be designated generi Another embodiment of the invention encompasses cally as protoberberine alkaloids which are positive the administration of the compounds of the invention inotropic agents which are accompanied with other 35 together with digitalis to increase further contractility desirable properties. Other objects of the invention to treat heart failure and to prevent digitalis induced have been referred to above and others will become arrhythmias, thus improving the therapeutic ratio of apparent from the following disclosure. digitalis and allowing a higher dose with less toxic (ar It is another object of the invention to provide a rhythmic) effects. berberine-type compound (and pharmaceutical compo Another embodiment is the administration of a con sitions which include same) which act on the mammal pounds of the invention with the main objective to treat cardiac system differently or have different effects, than arrhythmias to prevent their occurrence or to interrupt reported previously with natural or other "berberine'. (or stop) them in the acute or chronic phases, both of A composition aspect of the invention comprises a supraventricular and ventricular origins in all patients, biologically or pharmaceutically acceptable carrier and 45 especially in those patients who will benefit by the he as the active component thereof, one or more of the modynamic effects of the compounds of the invention, isoquinolines alkaloids which are positive inotropes and such as the positive inotropic effect or decrease in pe have one or more of the therapeutic properties de ripheral vascular resistance and increase in cardiac out scribed (and the biologically acceptable salts of these put; or in those patients in which the negative inotropic compounds). The invention discloses also preferred SO effects of the commonly used antiarrhythmic agents is compositions. disadvantageous or contraindicated. The versatility of In a method aspect of the invention, the invention the compounds of the invention and the unusual aspects relates to a therapeutic method for increasing cardiac of the invention are well illustrated by the above. contractility, preferably accompanying this effect by The compounds of the invention have a combination one or more other therapeutic effects improving aortic 55 of beneficial properties generally associated or de blood flow, decreasing heart rate, decrease in arterial scribed as properties or effects on the cardiovascular pressure, decrease in afterload of the left ventricle, de system of mammals, both humans and animal. These crease in the preload of the left ventricle, decrease in properties may include a positive inotropic effect which diastolic arterial pressure, decrease in total peripheral can to varying degrees, in accordance with the inven resistance and freedom of arrythmias which comprises tion, be associated (or accompanied by) other beneficial administering at least one compound of the invention to effects on the cardiovascular system like decrease in an animal (e.g. a mammal), preferably a human (male or diastolic arterial pressure, decrease in total peripheral female) in a dose sufficient to cause a positive inotropic resistance and others described above. effect. Thus, as can be seen, the invention does encom Other effects of the compounds of the invention are pass several embodiments, particularly the compounds 65 physiologically distinct from the other beneficial prop of the invention that improve cardiac performance and erties; for instance the antiarrythmic effect, the capabil minimize heart failure while they also are antiarrhyth ity of controlling, minimizing, alleviating or preventing mics. Also the invention encompasses compounds circulatory shock in a mammal (such shock having as its 5,153,178 15 16 symptom a decrease on blood flow and/or oxygen sup ply). -continued In a further aspect of the invention it has been discov FIGURES ered that the compounds of the invention control atrial Nurnber Title arrhythmias and reverses atrial fibrillation to sinus Conscious Dog VI Effect of Berberine Hydrochloride rhythm. Atrial fibrillation is detrimental to cardiac per on Left Ventricular dP/dt in Heart formance and also increases the risk of embolism. Failure The traditional therapy has been electric shock, quin VIII Effect of Berberine on End idine and digitalis, all not as satisfactory as the therapy Diastolic Pressure (LVEDP) in O Heart Failure now made available here. IX Effect of Berberine Hydrochloride Other objects are or will become apparent from the on Total Peripheral Resistance in disclosure. Heart Failure X Effect of Berberine Hydrochloride TABLES AND FIGURES on Cardiac Output (CO) in Heart Failure The following are the Tables and Figures which 15 X Effect of Berberine Hydrochloride further illustrate the invention. on Stroke Volume (SV) in Heart Failure XI Effect of Berberine Hydrochloride on "Frank-Starling Curve" in Heart ABLES Failure. Comparison of CO to LVEDP Number Title XIII Effect of Berberine Hydrochloride I LV dP/dt (Protocol M) on "Frank-Starling Curve" in Heart II Aortic Fow (Protocol M) Failure. Comparison of SV and df/dt (Protocol M) LVEDP IV Heart Rate (Protocol M) XIV Abnormalities of Circulatory w Left Atrial Pressure (Protocol M) Function. Schematic V Left Venticular End 25 Representation from "Braunwald" Diastolic Pressure (Protocol M) XV ECG Wave and Intervals. Schematic VII Systolic Pressure Representation from "Braunwald" (Protocol M) XV Effect of Continuous Intravenous WEEI Diastolic Pressure Administration of Berberine (Protocol M) Hydrochloride on PR interval In IX Effect of Ouabain on Aortic flow 30 Anesthetized Dogs. and peripheral resistance XVI Effect of Continuous Intravenous X Multiple Infusion of Berberine in Administration of Berberine a Dog Prepared as in Protocol M Hydrochloride (0.7 mg/kg/min) on Systolic Pressure QRS Interval in Anesthetized Dogs. (Protocol RK) XII Diastolic Pressure (Protocol RK) 35 XIII Comparison of Berberine. The compounds of the invention are berberines, more Berberrubine Tetrahydropalmatine, specifically protoberberines, as further described and Corexinine XIV Effect of Berberine on Ejection herein. The term protoberberine also herein includes Fraction the protoberberines known as tetrahydroprotoberbe XV Effect of Berberine (0.2 rines and retroprotoberberines, quaternery amonium mg/Kg/min) on Ejection Fraction protoberberine dihydrobeberines, tetrahydrop (as done by MUGR) in Conscious Dogs with Acute Left Ventricular seudoberberines and dihydroprotoberberines. In the Failure protoberberine alkaloids of the invention, substituents XV Effects of Berberine and Ouabain can be present at C-2 and C-3 and either at C-9 and -10 on Hemodynamics in Dogs with Acute or at C-10 and -ll position. In certain instances, a hy Heart Failure 45 droxyl or methoxyl may be present at C-1. A methyl XVI Effect of Berberine, Ouabain and the Combination of Both Peak LV group can be found at C-13, while in other cases an dP/dt (numbers are percent alcoholic hydroxyl is located at C-13 or at C-5. increase compared to before The preferred compounds of and used in the inven treatment) tion may be represented by the following Formula I. 50

FIGURES Number Title I Changes in LV dP/dt max During 55 Continuous Intravenous Infusion of Berberine Hydrochloride I Changes in Pulse Pressure During Continuous Infusion of Berberine Hydrochioride III Changes in Aortic Flow During Continuous Infusion of Berberine Hydrochloride IV Changes in Heart Rate During Continuous Infusion of Berberine Hydrochloride in which: V Effects of Continuous Intravenous 65 R1 and R2 may be the same or different and represent Administration of Berberine Hydrochloride hydrogen, hydroxy, preferably a lower alkyl like WI Effect of Berberine Hydrochloride methyl or a lower alkoxy like methoxy or ethoxy, aryl on Ejection Fraction on the oxy containing generally no more than 12 carbons but 5,153,178 17 18 preferably 6 or 7 carbons like phenyloxy or benzyloxy, or when taken together R1 and R2 form an alkylene FORMULA II dioxy like a methylene dioxy group; R3 represents hydrogen, hydroxyl, preferably a lower alkoxy like methoxy or ethoxy or an aryloxy as de scribed for R1 or R2; R4 represents hydrogen or preferably a lower alkyl like methyl; R5 represents hydrogen, preferably a lower alkyl like methyl, aryl like phenyl or arylalkyl (preferably alkyl where the alkyl preferably a lower alkyl) like benzyl; (-)-CANADINE ((-)-TETRAHYDROBERBERINE) Re and R7 may be the same or different and represent It will be noted that it is also within the contempla hydrogen, hydroxy, preferably a lower alkoxy like me- 15 tion of the invention that biological metabolite(s) result thoxy or ethoxy, aryloxy containing no more than 12 ing from the compounds of the invention, are also carbons but preferably 6 or 7 carbons like phenyloxy or within the invention, which metabolites are responsible benzyloxy, carbamoyl-O(CO)NR13R-14 when R13 and for the positive inotropic effect and at least one of the R14 are the same or different and represent hydrogen, beneficial properties disclosed here. The definition of preferably lower alkyl like methyl or ethyl, or aryl like 20 compounds of the invention includes such metabolites. phenyl, or when taken together Rs and R7 form an Within the general formula of compounds of the alkylene group like a methylene dioxy group; invention, certain classes are identifiable which are R8 represents hydrogen, hydroxyl, preferably a lower specifically interesting from the objectives of the inven tion. These are the classes which have a methylene alkoxy like methoxy or ethoxy, aryloxy containing no 25 dioxy group on ring A; those having one or two lower more than 12 carbons but preferably 6 or 7 carbons like alkoxy substituents on ring D, those having a lower phenyloxy or benzyloxy; alkyloxy and a hydroxyl group on ring D, those having R9 represents hydrogen, preferably a lower alkyl like one or more, especially two lower alkoxy (like me methyl, ethyl, lower alkyl substituted with methyl like 30 thoxy) substituents on both rings A and D and wherein CH2OH, or hydroxyl; R10 is hydrogen, or the class represented by R1 and R7 R10 represents hydrogen or an alkyl, like lower alkyl, being concurrently lower alkoxy, like methoxy while like methyl; R2 and R8 are concurrently hydroxyl. R11 represents hydrogen, hydroxyl, preferably lower The invention also contemplates adding to the ring(s) or elsewhere or replacing one or more of the above alkoxy like methoxy or ethoxy; 35 named substituents by one or more halogens, like chlo R12 represents hydrogen or preferably lower alkyl rine or other therapeutically beneficially active substitu like methyl; and tents which enhance or otherwise potentiate one or R13 represents -CHO or an hydroxy alkyl, like hy more of the desired properties of the compounds of the droxy methyl. 40 invention. Likewise, any of the above substituents can Ring B may be unsaturated between carbons 5 and 6. be an ester, an amido, amino, or other functional group Ring C may be saturated between carbon 8 and the which will have a desired therapeutic effect. Such a nitrogen or it may be saturated between carbon 13 and group may be inert, decrease one of the therapeutic 14 to yield a tetrahydroberberine. Other degrees or effects (if this is desired) or a side effect (if one is noted 45 which may be less desirable) or increase a particularly positions of the saturation or unsaturation are possible. desirable one. All such compounds are intended and are The isomers are considered within the generic for contemplated and in the compounds of the invention. mula and the specific embodiment contemplates either It will be noted that certain subgroups or classes of the d, 1-racemic mixture of the compounds or the spe compounds are preferable if certain therapeutic effects cific di- or 1-resolved form of the compound. It is con- 50 are specially sought after, whereas other classes or sub templated that all forms of these racemates must be classes will be preferred if other therapeutic effects are always of the same potency in all respects. especially sought after. Since the compounds of the A- in the formula represents a biologically accept invention have several types of therapeutic effects it able, especially a therapeutically acceptable anion such will be apparent to one skilled in the art that the selec 55 tion of the special species will depend in a certain mea as to form salts of the compounds illustrated including sure on what effect is sought after most. the quaternary ammonium salts and the addition salts of It will also be noted that the compounds of the inven organic or inorganic acids. Illustrative acids are inor tion (or the composition thereof) are used in a amount ganic strong acids like sulfuric, nitric, ethanedisulfonic, which brings about a positive beneficial therapeutic phosphoric, mono-di- or tri-organicacids like citric, 60 effect. These amounts will vary depending on the effect acetic, lactic, tartaric, sulfanic, succinic, fumaric, ma sought and on the particular species used. It will also leic, hydrochloric, hydrobromeric, aliphatic or aro depend on whether the compound is used for prophy lactic or curative or maintenance therapy purposes; the matic acids like benzoic and numerous others. first reason usually calling for a smaller amount, and the Following traditional nomenclature the rings are is last, intermediate amounts. One skilled in the art, such identified from A to D and the numbering of the carbon as a physician or a veterinarian, will readily be able to atoms as illustrated for canadine ((-)-tetrahydroberbe prescribe the appropriate dosage for the particular pur rine) below in Formula II. pose. Since the compounds of the invention have such a 5,153,178 19 20 remarkably wide therapeutic ratio and low toxicity, the when used for treatment of cardiovascular or other latitude available for appropriate dosage is large. disorders. Amounts as modes as 0.0001 mg/Kg of body weight of A preferred compound of the invention is berberine a compound of the invention can give a positive thera which has the properties described herein. For best peutic effect; generally amounts over 1 g/Kg may be 5 reproductibility of properties, the synthetic, non-natu unnecessary, unless the clinician or physician deems it rally occurring protoberberine products are preferred. advisable. It is evident that the mode of administration Preferably, the protoberberines which have a purity of influences also the dosage administered whether it is for at least about 90%, most preferably at least about 99% instance by injection, oral or infusion, and if by inges pure. tion or whether subcutaneous, intravenous or other- 10 A class of particular interest are the dehydroberbe wise. None of these considerations will present an rines which are not naturally occurring, i.e. synthetic, undue expermentation or problem for the physician or especially of a purity of over about 90%, more prefera veterinarian. bly of at least 99% purity. A particularly favored spe The description of the synthesis of many of the com cies is the dehydroberberines (synthetic) of at least 90%, pounds of the invention can be found in the literature. 15 preferably at least 99% purity. A species used was the Examples of these include the synthesis of copistine (A. hydrate (2.5H2O). The anhydrous form is reported to Klasek et al, Tetrahedron Lett., p. 4549 (1969)), berber have a molecular weight of 371.8. ine (Kametani et al, J. Chem. Soc. (c) p. 2036 (1969)) and It is to be noted that the premium placed on lack of palmatine (Feist, Dschu, Arch. Pharm., 263, 301 (1925)) side effects, purity, and other desirable properties dis where the C-ring is aromatic and there are 2, 1 or no 20 cussed above (and herein) is specially desirable when methylenedioxy groups present in the molecule. Where human species are treated, whereas such strict require the C-ring has been reduced, representative examples of ments may be somewhat less necessary when animal synthesis include those for 1-tetrahydrocopistine (Kla species are treated, especially of the non-edible type. sek et al, Tetrahedon Lett. p. 4549 (1969)), cariadine Typically bovine, equine, feline and swine and other (Russell, J. Am. Chem. Soc. 78, 3115 (1936)), tetrahy- 25 animals are contemplated. dropalmatine (Brasher, Dietta, J. Org. Chem., 26, 2231 Included in the terms used above, (i.e. berberine, (1961)) and coreximine (Kametani et al., J. Chem Soc C, protoberberine, dehydroberberine, etc.) are the biologi 112 (1968). General syntheses for the aromatic proto cally, and especially the pharmaceutically or therapeu berberines are disclosed in U.S. Pat. No. 3,910,938 and tically acceptable salts of the above. Illustrative salts are for the tetrahydro compounds in U.S. Pat. Nos. 30 described elsewhere herein. Salts generally used quite 3,272,707 and 3,426,027. The synthesis of tetrahydropal satisfactorily were the hydrochlorides, the sulfates and matine methiodide, a typical quaternary amimonium others. salt is described by Narosimham and Bhide, Chem. Ind. Most preferred species are the berberrubine(s) (for (London), p. 621 (1969). Among the acceptable salts are mula III below), berberine (formula IV below), tetrahy the sulfate, nitrate, phosphate, citrate, acetate, maleate 35 dropalmatine (formula V, below) and coreximine (for lactate, tartrate, succinate, chloride, bromide, iodide, mula VI) below. benzoate and the like. The salts are prepared by meth ods known in the art. Of interest, if further details with respect to synthesis FORMULA III or other aspects are needed for one skilled in the art, 40 reference is made to Shamma Isoquinoline Alkaloids Chemistry and Pharmacology, Academic Press, New York, 1972 ("Shamma") and Tetsuji Kametani, The Chemistry of Isoquinoline Alkaloids, 1969, Hirokawa OHis Publishing Co, Tokoy Elsiver Publishing Co., New 45 York, (especially chapter 10), both books being incor OCH3 porated herein by reference. Berbertubine Amongst the isoquinoline compounds of particular interest are the following classes: the protoberberines FORMULA IV and retroprotoberberines, the protopines which are 50 identifiable by R9 representing an oxygen as illustrated in Shamma, chapter 18; the rhoedines and papaverru bines, the homoprotoberberines, as shown in Shamma, chapter 27. For purposes of this invention, the term berberine is generic to proto- and to homoberberines, 55 OCH3 It is also contemplated in accordance with the inven OCH3 tion that certain chemical functions or structures may be combined with the protoberberine structure of the compound of the invention to give compounds of prom FORMULAW ising cardiovascular, in particular, positive inotropic 60 propertiesor the other desired effects disclosed herein. It is contemplated for instance, that one or more sugars (glycoside) be linked to the protoberberine structure. In this manner the resulting compound is capable of hav ing enhanced potency and duration of action whether 65 OCH3 or not caused by increasing solubility. The sugar residue is capable of enhancing the stability of the protoberbe OCH3 rine alkaloid in the biological system, e.g. in a patient, Tetrahydropalmatine 5,153,178 21 22 -continued glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. These compositions FORMUAW can also contain adjuvants such as stabilising, preserv CH3O ing, wetting, emulsifying and dispersing agents. As is known in the art, they can be sterilized, for HO N example by filtration through a bacteria-retaining filter, by incorporation of sterilising agents in the composi tions, by irradiation or by heating. They can also be manufactured in the form of sterile solid compositions OCH3 10 which can be dissolved in sterile water or some other sterile injectable medium immediately before use. OH As has been discussed above, the percentages of ac Corexinine tive components in the said composition and method for increasing cardiac contractility and the other properties The favored isomer of tetrahydropalmatine is d-tet 15 rahydropalmative but the d, l, mixture or 1-tetrahy can be varied so that a suitable dosage is obtained. The dropalmatine is also useful. The favored isomer of dosage administered to a particular patient is variable, coreximine is (-)-corexinine but the (-)- or depending upon the clinician's judgment using the fol (-)-corexinine is likewise useful. lowing as criteria: the route of administration, the dura Typical compounds of and used in accordance with 20 tion of treatment, the size and condition of the patient, the invention are the following: berberrubine, berberine the potency of the active component and the patient's (umbellatine), d-tetrahydropalmatine, discretine, response thereto. An effective dosage amount of active xylopinine(1-norcoralydine), stepharotine, capaurimine, component can thus only be determined by the clinician capaurine, ophiocarpine, dehydrothalictrifoline, dehy considering all criteria and utilizing his best judgment drocorydaline, thalictricavine, thalictrifoline, (base II), 25 on the patient's behalf. Like criteria shall guide the isocorybulbine and alborine, and others disclosed veterinarian. herein. It is within the contemplation of the invention as The salts are generally a more convenient form for already referred to above, to selectively substitute or use. In practice the use of the salt form amounts to use add to the rings, selected substituents which favorably of the base form. Pharmaceutically or biologically ac 30 influence the desired properties of the compounds and ceptable salts are salts whose anions are relatively in compositions used in the instant invention. nocuous to the animal organism in dosages used so that In summary, what is contemplated by the invention the cardiotonic or other desirable effect of the free base are those protoberberines, preferably of the one of the is not vitiated by the effects of the anions. Mixtures of the compounds of the invention and of 35 classes described which have a positive inotropic action their salts may also be used as is contemplated within (both in congestive (or chronic) heat failure and in car the invention. It is not within the contemplation of the diogenic shock in mammals (human or animals) (female invention that one skilled in the art should be able to or male), which inotropic action is preferably (but not avoid the spirit and scope of the invention by the use of necessarily), accompanied to at least to some degree, by a different salt or a different compound. 40 one or more of the following cardiovascular effects: In clinical practice said compound or salt thereof will vasodilation or at least no increase in calculated periph normally be administered orally or parenterally in a eral vascular resistance, no increase in heart rate, in wide variety of dosage forms. crease in blood flow in the aorta, decrease of aortic Solid compositions for oral administration include pressure caused by peripheral vasodilation, reduction of compressed tablets, pill, powders, and granules. In such 45 afterload and freedom of arrhythmias and antiarrhyth solid compositions, at least one of the active compounds mic effect. is admixed with at least one inert diluent such as starch, A preferred class of compounds are those which are calcium carbonate, sucrose or lactose. These composi antiarrhythmogenic, whether or not that effect is ac tions may also contain additional substances other than companied by other beneficial therapeutic effect like inert diluents, e.g., lubricating agents, such as magne 50 positive inotropic effect. sium stearate, talc and the like. Preferred are those compounds which have a wide As is known in the art, liquid compositions for oral therapeutic index and those which differentiate them administration include pharmaceutically-acceptable selves by one or more therapeutic properties from digi emulsions, solutions, suspensions, syrups and elixirs talis glycosides. containing inert diluents commonly used in the art, such 55 as water and liquid paraffin. Besides inert diluents such A typical compound of the invention, such as the compositions may also contain adjuvants, such as wet type of berberine used, possesses a combination of ad ting and suspending agents, and sweetening, flavoring, vantageous properties. Berberine, in subjects without perfuming and preserving agents. According to the latent or overt heart failure, increases myocardial con invention, the compounds for oral administration also tractility and accelerates the blood flow in the aorta. include capsules of absorbable material, such as gelatin, Berberine also decreases the heart rate and diastolic containing said active component with or without the aortic pressure by peripheral vasodilation. addition of diluents or excipients. Digitalis is known to contract blood vessels, increas As is known in the art, preparations according to the ing peripheral vascular resistance; also to cause cen invention for parenteral administration include sterile 65 trally mediated increase in sympathetic tone. In con aqueous, aqueous-organic, and organic solutions, sus trast, the berberine used does not cause peripheral vas pensions and emulsions. Examples of organic solvents cular constriction but causes a decrease in calculated or suspending media are propylene glycol, polyethylene peripheral resistance. 5,153,178 23 24 Berberine causes mean arterial pressure to decrease placed to record left atrial, left ventricular and aortic because the diastolic pressure is decreased. With digita pressures. Descending aortic blood flow was measured lis, arterial pressure is increased. with an electromagnetic flowmeter. Heart rate (HR) In further contrast to digitalis, berberine is virtually was calculated using electrocardiographic recording, free of cardiac side effects noted above for digitalis. lead aVF. All parameters were recorded on a Gould Berberine does not cause any type of arrhythmia within polygraph. the dosages administered. There is no premature ven The dogs were randomly assigned either to the con tricular beats or atrio-ventricular block. trol or to the treated group. Six of the dogs were in the Berberine is also different from beta-adrenergicago control group and these dogs were infused with a pla nists; these increase contractility but also increase heart 10 cebo, namely the vehicle used for the drug tested. rate. The invention therefore provides an important The berberine used was a synthetic berberine (of a new class and use of positive inotropic agents. purity of 99%), HCl salt and 2.5 H2O. Apart from and distinct from their cardiovascular Berberine (as the hydrochloride salt) was adminis inotropic effect, the compounds of the invention-and tered to six anesthesized dogs (barbiturate). The dose their compositions-are also useful in the treatment not 15 was 0.7 mg/kg/min. intravenously infused during 5 only of cardiac shock, but also in certain stages of other minutes. This dose was repeated after 45 minutes. types of shock (due to other initiating causes), like ana Systolic arterial pressure was maintained at a con phylactic shock (for which epinephrine remains a pri stant rate using a reservoir full of blood under constant mary therapeutic agent), hemorrhagic shock, non pressure connected to the femoral artery. Whenever the hemorrhagic oligemic shock, distributive shock, septic 20 arterial pressure was not maintained constant in the shock, or shock due to metabolic, toxic or endocrine work reported (and allowed to drop) appropriate refer failures. These types of shock are generally the conse ence is made herein to that fact. quences of diminished blood volume, i.e. decreased Systemic arterial pressure was maintained at a con cardiac output and decrease systemic arterial pressure stant rate in order to obtain accurate dR/dt since this generally accompanied by vaso or venoconstriction and 25 parameter is of extreme importance in these studies. tachycardia. Thus, the compositions of the invention by However, as a consequence, the vascular peripheral its positive inotropic effects and/or decrease in periph resistence, when described as a function of mean aortic eral constrictions, provide an appropriate therapy to pressure, was higher than if the pressure would be al alleviate or control such shock situations. lowed to fall with the administration of the compound. The compounds of the invention also are useful as 30 Therefore, the reduction calculated total peripheral cardiac imaging agents. The compounds of the inven resistence is underestimated. Also dP/dt is less in tion can be used for studies of the heart, anatomically creased when bradycardia (lower heart rate) occurs and and functionally. This effect of the compounds of the since heart rate was allowed to fall (which is the effect invention will be further discussed below. of the drug), the increase in dP/dt reported here is The pharmacological and cardiovascular data of the 35 actually underestimated as compared to preparations in compounds of the invention was obtained following the which heart rate would have been maintained constant. protocols described below, which are generally of the The tables which follow (tables I through IX) are standard traditionally accepted types. briefly summarized below. Certain terms are commonly used to describe the Table I (Protocol M) shows the left ventricular maxi mechanical properties of cardiac muscle. For definition mum rate of pressure change (LV dR/dt) measured in reference is invited to Braunwald, pages 431-452, mmHgxsect. which is incorporated herein by reference. Table II (Protocol M) shows aortic blood flow veloc The usefulness of the compounds of the invention as ity measured in mlxml. positive inotropic agents, (and other uses as disclosed) is Table III (Protocol M) shows the maximum rate demonstrated by effectiveness in standard pharmaco 45 change of aortic flow velocity (i.e. maximum flow ac logical test procedures, for instance in causing a signifi celeration) (maximum dF/dt) measured in mlx min-2. cant increase in the cardiac contractile force in the Table IV (Protocol M) shows the heart rate (HR) anesthesized dog with virtually no increase or decrease calculated from the recordings of lead aVF of the elec in heart rate and blood pressure. Protocols used are trocardiogram measured in beatX min. described herein or by reference to standard medically SO Table V (Protocol M) shows the mean left atrial accepted publications. pressure (LAP) values measured in mmHg. The term "contractility' or "inotropic state' has a Table VI (Protocol M) shows the left ventricular different meaning than performance. end-diastolic pressure (LVEDP) measured in mmHg. The term "contractility" is useful to identify a change Table VII (Protocol M) shows the systolic aortic in contractility (or inotropic state) of the heart as an 55 pressure (SAP) measured in mmHg. alteration in cardiac performance that is independent of Table VIII (Protocol M) shows the diastolic aortic changes resulting from variations in preload or after pressure (DAP) measured in mmHg. load. When loading conditions remain constant, an im Table IX shows the effect of ouabain on aortic flow provement in contractility will augment cardiac perfor and calculated peripheral resistance. mance (a positive inotropic effect) while a depression in 60 Table X (Protocol M simile) shows hemodynamic contractility will lower cardiac performance (a nega changes in a dog following repetitive infusions (16 suc tive inotropic effect). The basal level of contractility is cessive infusions, over five minutes of 3.5 mg/Kg of reduced in chronic myocardial failure. berberine) measurements taken at fifteen minute inter The experimental work was carried out as follows: vals at constant pressure. Twelve healthy adult mongrel dogs (20-30 kg) of both 65 For further details on the statistical method used sexes were anesthetized with intravenous sodium pento here, see Snedecor, G. W. and Cochran, W. F.: Statisti barbital (25 mg/kg). A left thoracotomy was performed cal Methods, 6th ed., Ames, Iowa State University through the fifth intercostal space. Catheters were Press, 1967. 5,153,178 25 26 In Tables I through IX the results of these experi the goal of the treatment is to increase the blood flow ments are presented. The first two lines presents the which as a consequence of heart failure is reduced. studied parameter in absolute values, the third line rep For the role of aortic blood flow in the assessment of resenting the "t' values used in Student's t test for cardiac performance, see Braunwald, chapter 14, pages group observation, between the treated and control 472-484, incorporated herein by reference. groups. The fourth line presents the level of signifi It is to be noted that since the systolic arterial pres cance. The next four lines give the values as changes (A) sure of the dogs was maintained constant, the data refer from the value before treatment to the value after treat ring to aortic flow suggests berberine is not a vasocon ment. The third set of values represent the changes in strictor. In contrast as has been noted herein before, it percent. 10 has been reported that digitalis and like cardiac glyco The last two sets of two lines represent the paired sides have a vasoconstricting effect in the non-failing t-test values and the derived p values obtained by ana heart. lyzing the data from before the infusion of the drug (last Table III shows the rate of aortic blood flow velocity set of two lines) or the placebo (next to last set of two change per unit of time, i.e. acceleration of flow. The lines), in the subsequent time interval (i.e. after infu 15 parameter dR/dt is a measure of the acceleration of sion). Therefore, each dog serves as his own control, blood flow in the aorta and is measured in ml/sec. It is thus increasing the statistical value of the tests. In the a parameter that indirectly indicates ventricular con control group it shows whether the value changes with tractility. The heart expels the blood with more vigor time. Most importantly it shows whether berberine since with stronger contractions the acceleration of the caused any changes in the treated dogs. 20 blood is faster in the aorta. As is observed from the Table I represents dP/dt maximum of the left ventri Table in the six control dogs, no significant change in cle. The dF/dt values at constant systolic left ventricu the dF/dt is seen but in the berberine-treated dogs a lar pressure assess cardiac contractility. An increase in significant change in df/dt occurred (p<0.01). This dP/dt indicates positive inotropic drugs. can be observed from the last four lines of Table III. The table summarizes the data of maximum dP/dt in 25 mmHg/sec. Both groups of dogs started at the same The increase in the acceleration of blood flow was in level of dR/dt. A small increase in the dR/dt in the the order of 65% (comparison of first and last columns). control group was observed. In the treated group a Table IV shows the data obtained on the heart rate much more marked increase was observed. (HR) expressed in beats/minutes. In the control group As can be observed from the table, the difference 30 (next to last set of two lines), no significant change in between the control group and the berberine-treated the heart rate occurred; in the berberine-treated group group at the end of the experiment was 50%, which is the change was significant. The data shows a reduction statistically significantly higher. The difference in in HR of 9 beats/min. This decrease in HR is important dP/dt, 2009-132 mmHg/sec in the control group and since if the increase in contractility were due to adrener 2974-121 mmHg/sec in the berberine treated group 35 gic stimuli, heart rate would have increased (thus it is shows that this index (dP/dt) of myocardial contractil neither a compound that acts like a catecholamine nor it ity was augmented by 50%. Both differences between acts by releasing catecholamine or increasing their con the two groups as well as in the increase in this index of centration in blood or myocardial tissue by other contractility in the berberine treated group at each time means). Moreover, heart rate, per se, does influence interval when compared to its value before administra 40 dP/dt. Increases in HR augment dP/dt while fall in HR tion of berberine was statistically significant with the p reduces dR/dt. Thus, normally a decrease in HR is level of less than 0.01; thus there is a chance smaller associated with a decrease in dP/dt. The marked in than 1 per 100 that this difference is attributable to crease in dip/dt of the compounds tested accompanied random occurrence. with fall (rather than increase) in HR demonstrated The increase in myocardial contractility shown is a 45 again the potency of berberine. very remarkable for a drug which is not a beta-adrener Table V shows the mean left atrial pressure (LAP). gic agonist since it is not dependent on catecholamine The initial blood pressure was low (i.e. normal) averag increase in catecholamines in the blood or in the myo ing 1 or 2 mmHg, which is as expected in good prepara cardial tissue. tions of non-failing hearts. Under these circumstances Table II shows the data relative to the peak aortic 50 no significant change can be expected and there was no blood flow velocity in the descending aorta measured change in LAP. with an electromagnetic flowmeter. Stroke volume (the Table VI shows the analysis of the left ventricular amount of blood ejected by the left ventricle) is the area end diastolic pressure (LVEDP). LVEDP was normal, and the flow curve when measured in the ascending averaging 3 mmHg, which is as expected in good prepa aorta. The performance characteristics of this flow 55 rations without heart failure and similarly to mean left meter have been described by McDonald in Flow Meters atrial pressure (Table V), it was not expected to change. for Pulsatile Flow in Blood Flow in Arteries by McDon Indeed no change was shown in that parameter. ald, Williams & Wilkins, Baltimore, 1974, pages In Tables V and VI, no paired t-test analysis was done 209-237. In the data analysis, 0 blood flow was assumed since it would have been redundant. at the end diastole. Table VII shows an analysis of the aortic systolic As is observed from the last four lines in the control pressure. As described above, it was maintained at con group, peak blood flow velocity did not change signifi stant rate. Therefore this parameter showed no change. cantly; in the berberine-treated group, peak blood flow Table VIII is a record of the diastolic aortic pressure velocity increased significantly. The increase was (DAP). In the control group, diastolic pressure did not nearly 50%. 65 change (84 mmHg and 82 mmHg). In the berberine The increase in blood flow is the most sought after treated group, the DAP fell from 84 to 62 mmHg. This effect in patients with heart failure since blood flow drop of 20 mmHg is statistically significant. It shows indicates an increase in cardiac performance. Indeed, that there was a peripheral vasodilatation. 5,153,178 27 28 The change in diastolic pressure in the non-failing will be observed to result in a reduction on myocardial heart contrasts with the effect of digitalis type cardiac oxygen consumption and less myocardial injury than glycosides which in the non-failing heart produce vaso the control. Other species of the preferred groups de constriction or increase systemic vascular resistance. As scribed above show similar behavior and therapeutic shown in the above data, as a result of this vasodilation, effect. an influx of blood from the reservoir to the dogs took Table X shows hemodynamic change in a dog fol place. lowing repetitive infusions administered over a period In 11 dogs at the end of the above-mentioned 90 of 5 minutes and the interval between the infusions was minute experiments, ouabain 0.03 mg/Kg was given around 15 minutes. The infusion of this high dose of intravenously in a bolus (Table IX). As shown in Table O berberine shows the good therapeutic index of berber IX, the administration of ouabain did not change signifi ine and especially the fact that no arrhythmias oc cantly diastolic aortic pressure but decreased signifi curred, that aortic flow continued to be augmented, that cantly the flow in the descending aorta by 15% from diastolic pressure (i.e. peripheral resistance) continue to 1195-89 to 1018.93 ml/min (P<0.01). Thus, the cal be low, that left ventricular dP/dt and aortic dr/dt culated peripheral resistance increased by 18% 15 assessing ventricular contractility continued to be in (P<0.02). This shows that contrary to berberine which creased and that heart rate decreased. Therefore, these acted favorably reducing peripheral resistance and in have no reversal of effects (biphasic response) when the creasing aortic blood flow, digitalis increased periph drug was given. eral resistance and reduced aortic blood flow. This data also indicates that if the blood pressure Both vasodilation and increase in contractility in 20 would not have been maintained constant by blood prove cardiac performance. The former by decreasing infusion, there would have been a decrease in arterial peripheral resistance and therefore afterload, and the systemic pressure. Therefore, the peripheral vasodila latter by increasing contractility. The action of both tion can be appreciated by the fall of the diastolic arte will, therefore, augment cardiac output and the delivery rial pressure which also is commonly expressed as the of blood to the organs which is the main function of the 25 heart. widening in the pulse-pressure (PP). PP is the value Drugs that increase contractility generally increase which is the difference between systolic and diastolic myocardial oxygen consumption. In addition, tachycar pressures. dia and increase in afterload increase further oxygen It is noteworthy that the berberine-treated dogs did consumption. The increase in contractility does not 30 not develop any type of arrhythmias during the experi necessarily increase oxygen consumption if heart size is mental period. Specifically the dogs did not exhibit at the same time reduced significantly (Laplace Law). premature ventricular beats or any degree of atrio ven See Maroko, P. R., Braunwald, E., and Ross, J. Jr.: The tricular blocks. Digitalis and other allied cardiac glyco metabolic costs of positive inotropic agents. Chapter 33, sides are known to stimulate arrhythmias of different , Corday, E. and Swan, H. J. C., 35 types, as has been described above, Editors, Baltimore, Williams & Wilkins Publishing This work shows that berberine increases the classi Company, 1973, pp. 244-250, which is incorporated cal indices of heart contractility such as dF/dt and hereby reference. Since the increase in contractility due dF/dt by at least 50% and increased considerably aortic to the compounds of the invention was found to be flow. Berberine also reduced the heart rate, another accompanied by a decrease in afterload (i.e. decreased desirable result. In addition, berberine decreased the peripheral resistance) and a reduction in heart rate, the calculated peripheral resistance. These results indicate effect of the compounds of the invention on oxygen that berberine is an important and potent positive ino consumption will be more favorable than that of beta tropic agent and increases cardiac performance by both adrenergic agonists that increases heart rate or that do its positive inotropic effect and vasodilation. not reduce peripheral resistance (as digitalis). This de 45 The mechanism of berberine is dissimilar to that of sirable property of the compounds of the invention beta-adrenergic agonists, which while increasing con assumes added importance in patients with ischemic tractility, increase heart rate also. While berberine is heart disease and specifically in patients with acute similar from this point of view to digitalis type cardiac myocardial infarction in whom an increase in myocar glycosides, it differs from digitalis for instance, since it dial oxygen requirement may be extremely deleterious 50 does not provoke arrhythmias, a most undesirable toxic if not fatal. effect of effect of digitalis. Berberine differs also from To verify this additional advantageous therapeutic digitalis because it reduces peripheral resistance in the property, in pilot experiments, it was shown that when non-failing heart. consecutive occlusions were compared, berberine (0.2 Table X shows the results of the administration of 0.7 mg/kg/min) caused less myocardial injury (as reflected 55 mg/kg/min up to a dose of 56 mg/Kg, in one dog. It is by epicardial ST segment elevations) than a control noted that the dp/dt increased by 350% after the eighth occlusion and much less than when isoproterenol was dose and df/dt by 450%. No arrhythmias were ob infused during an infusion. These changes were due to served. This data confirmed by other data, suggests that lower myocardial oxygen consumption during the oc berberine has an excellent therapeutic index, in contrast clusion with berberine treatment. In addition, the infu to digitalis. sion of berberine in dogs without coronary artery occlu When the corresponding experiments are run using sion resulted in a reduction in myocardial oxygen con coreximine like results are obtained. There is an increase sumption despite the increase in contractility (dP/dt). in dP/dt, peak blood blow, acceleration of blood flow This is quite an advantageous therapeutic combination and a decrease in the heart rate and diastolic pressure. of properties which further distinguish the compounds 65 Equivalent results are obtained when tetrahydropal of the invention from the conventional drugs. matine is used. Likewise coreximine, tetrahydropalmatine and ber When berberubine is used in the preceeding experi berrubine administration to dogs (in varying dosages) ments like results are obtained. 5,153,178 29 30 Likewise, typical compounds which come within the sure was maintained constant in order to verify the generic formula of the compounds of the invention as effect of berberine on dP/dt. The present data (Tables disclosed above, produce like therapeutic effects in the XI-XII) show that in addition to the positive inotropic preceeding experiments. effects (increase in dP/dt and dF/dt) that aortic pres The compounds of the invention have, as has been sure (mean, systolic and diastolic) were reduced when shown above, positive inotropic effects and an in blood pressure was not maintained constant. This hypo proved performance by increasing aortic blood flow. tensive effect was accompanied by another favorable Furthermore, it was shown that they produce a distinct effect which is a reduction in heart rate. systemic prolonged and continuous arterial hypoten In 4 groups of dogs instrumented as in the last series sion. This hypotensive effect is due to peripheral vaso 10 of experiments (Protocol RK) the effects of increasing dilation and obviously not to a negative inotropic effect. dose response on systemic arterial pressure was exam Compounds of the invention are therefore of significant ined. The only difference from that protocol was that utility. only one dose of berberine (as opposed to two) was In the treatment of heart failure, the reduction in administered. afterload is an important and frequently used therapy in 15 The effects on the measured parameters is dose re the treatment of patients with heart failure. Afterload sponsive. In four groups of dogs, the changes in blood has been defined, when applied to the intact ventricle, pressure were analyzed. The dosage administered was as the tension, force, or stress (force per unit cross-sec 7.5, 15, 30 and 70 mg/Kg. It was observed that gener tional area) in the ventricular wall, after the onset of ally blood pressure drop was most marked in the first 5 shortening, and it is a key determinant of the quantity of 20 minutes. After 90 minutes following the injection (when blood ejected by the ventricle. The two main parame the experiment was discontinued), the average fall in ters that change afterload in accordance with Laplace's means arterial pressure in the group which received 15 Law are left ventricular wall tension and arterial pres mg/Kg was 19 mmHg; in the group that received 30 sure, the former being a function of its diameter, wall mg/Kg it was 37 mmHg and in the group that received width and shape. See Braunwald, page 438-439. 25 70 mg/Kg it was 63 mmHg. This shows the gradual The following tables report the experiments showing response with increase of dosage of the drug. No pri the effects of the berberine on systemic arterial pressure, mary arrhythmias were observed in the dogs over the when this parameter is permitted to vary. time and dosage administered. Table XI shows systolic pressure; and Heart rate was also dose-dependent decreasing after Table XII shows diastolic pressure. 30 administration of 7.5, 15, 35 and 70 mg/Kg of berberine The protocol was the same as described above except by 20, 16, 40 and 103 beats/min., respectively. that in these dogs, contrary to the previously reported In conclusion, it was demonstrated that both changes, series, the systolic arterial pressure was not maintained that is, decrease in heart rate and blood pressure, are constant. Also, the dogs were instrumented only with proportional to the dose of berberine administered. an electrocardiogram (with lead a VF being constantly 35 In one dog using protocol M simile, 240 mg/Kg of recorded), and polyethylene cannula in aorta intro berberine HCl was administered and dP/dt increased by duced through the carotid artery to measure aortic 75%, aortic flow by 67%, heart rate fell from 150 to 114 pressures, and another cannula in the jugular vein for beats/min., diastolic aortic pressure fell from 92 to 67 drug administration. Average heart rate (measured by mmHg, and left atrial pressure fell from 8.5 to 7.5 continuously monitored electrocardiographic lead mmHg. No arrhythmias were noted, even at that higher aVF) was reduced from 146-4 (means - 1 S.E.M.) to concentration. This data shows that higher doses of 132-7 mmHg (p<0.1) after the first 45 minutes. After berberine also favorably affect the dP/dt and aortic the second 45 minutes heart beat rate was further re blood flow and other characteristics without induction duced to 124-6 mmHg (p<0.01). of arrhythmia. This shows the good therapeutic index Mean systemic arterial pressure fell significantly from 45 of this compound and shows the lack of biphasic re 114-7 mmHg to 87-5 mmHg (p<0.005) at 5 minutes sponse of LV dP/dt. to return to and stabilize in the 90's and 100's. It was When this type of experiment was repeated with after 10, 15, 30 and 45 minutes, respectively, 93-4, tetrahydropalmatine, a first dose of 3.5 mg/Kg was 925, 101-7 and 101-7 mmHg. After the second given without maintaining the pressure constant and dose, the pressure fell significantly to 94-4 (p<0.025) 50 arterial pressure fell from 142/115 to 118/90 mmHg. mmHg at 5 minutes, to stabilize thereafter in the mid When the second infusion was given with constant 90's. It was respectively at 10, 15, 30 and 45 minutes pressure, dP/dt increased by 17%. 95-6, 95-5, 97-5 and 97-5 mmHg, respectively. Tetrahydropalmatine has, like other compounds of Thus after 90 minutes the mean pressure fell signifi the invention a favorable effect on the contractility of cantly from 114-7 to 96.5 mmHg (p<0.025). 55 the heart and on aortic blood flow. In Table XI, the systolic and in Table XII, the dia Likewise comparable results are obtained when the stolic pressure respectively changed in the same direc experiments are repeated using coreximine. tion to the mean arterial pressure. The most marked Similar values are obtained when berberine, represen decrease was in the diastolic pressure. The pulse pres tative compounds which come within the generic for sure (systolic minus diastolic pressure) increased from mula groups listed herein and disclosed above produce an average of 28 before berberine administration to 35 like beneficial therapeutic effect. mmHg at the end of the experiment. This increase in Berberine is well tolerated and no noteworthy side pulse pressure is attributable to the reduction in periph effects were noted in the various experiments done in eral resistance and/or increase in contractility. dogs. The LD50 for rats when injected intravenously in These experiments show the beneficial effect of re 65 a bolus was about 55 mg/Kg. In cats the LD50 was peated dose of berberine given intravenously on sys about 55 mg/Kg. It is noted though that higher dosages temic arterial pressure and heart rate. In the experi are administerable depending on the method (and ments described earlier (Tables I-X) the arterial pres speed) of administration and the particular species of 5,153,178 31 32 the compounds of the invention some of which have Therefore, group b, c and d were used to analyze the even or expected to have even a lower LD50. It is also various effects of continuous infusion of berberine and known that certain animals, like the equine species or its dose response and groups e and f and g used to exam swine species will tolerate higher dosages without ine the effects of these three other protoberberines. undue adverse effects. 5 Moreover, their effects were compared to the berberine To examine the effect of varying doses of protoberbe treated dogs with the same dose (i.e. Group c). rine on the hemodynamic parameters, the following FIGS. I, II, III and IV show that left ventricular protocol (Protocol Z) was carried out: maximal dP/dt, aortic pulse pressure, aortic blood flow Mongrel dogs weighing between 15 and 25 kg were and heart rate did not change significantly in the control anesthetized with intravenous sodium thiamylal (20 O dogs (group a). mg/kg initially with additional doses given as needed). The effect of the infusion of 0.7 mg/Kg/min of ber The dogs were intubated endotracheally, and respira berine (group b) is shown in comparison to control tion was maintained by a Harvard respirator. The chest (group a) as well in comparison to dogs that received was then opened in the fourth left intercostal space and 0.2 (group c) and 0.02 (group d) mg/Kg/min in the the heart suspended temporarily in a pericardial cradle. 15 same figures (I-IV). These findings will be discussed Aortic pressures were recorded through a polyethyl below. ene cannula introduced through the carotid artery (Sta FIG. V., however, summarizes specifically the effects tham P23 Db pressure transducer) and electrocardio of the infusion of berberine 0.7 mg/Kg/min during the gram (lead aVF) were recorded throughout the experi 35 minute period of the experiments. The data are pres 20 ented as a per cent change from before infusion. It ment on a polygraph (Gould Instruments). Another shows that berberine infusion resulted in an increase in polyethylene cannula was placed in the left atrium for LV dP/dt of 35% after 5 minutes and that thereafter the measurement of atrial pressure (Statham P23 Db pres increments in LV dP/dt were smaller being the increase sure transducer). An electromagnetic flowmeter (Bi at 35 minutes of 43.2-7.0% (P<0.001). Thus, it seems otonix Laboratories) was placed around the ascending 25 that when a continuous infusion of 0.7 mg/Kg/min is aorta and the aortic flow was recorded on a polygraph, given, the dose in the first 5 minutes is responsible for Two catheters were inserted into the left ventricle most of the increase and thereafter it reaches nearly a through a femoral artery. plateau. The plateau may be due to the progressive fall The first ("pigtail”) served as a reference, while the in heart rate; otherwise, it may be expected that dP/dt second (Millar microtip transducer catheter) was con 30 would have increased further. The aortic pulse-pressure nected through a Millar transducer (Millar Instruments, (i.e. systolic minus diastolic pressures) widened (i.e. Houston, Tex.) to the polygraph, where both left ven increased) progressively throughout the experiment and tricular pressure curves were recorded simultaneously. is directly and linearly proportional to time (and to the The left ventricular end diastolic pressure was recorded dose infused). After 35 minutes it increased significantly separately through a Millar catheter. The first deriva 35 by 125-19% (P<0.001). This remarkable increase in tive of the left ventricular pressure (dP/dt) was ob pulse pressure is due to the fall in the diastolic pressure tained by electronic differentiation of the left ventricu since the systolic pressure was maintained constant. lar pulse recorded through the Millar catheter. Thus, it shows the intense and progressive effect of the A reservoir of blood was connected with the femoral compound as an agent reducing afterload and a periph artery in order to maintain constant left ventricular eral vasodilator. pressure during the experiment. In this protocol, simi Peak aortic blood flow, measured in the ascending larly to protocol M, systemic arterial pressure was aorta increases progressively and is linearly propor maintained constant in order to have accurate measure tional to time (and total dose injected). After 35 minutes ments of dP/dt which represent well left ventricular flow increased significantly by 73%. The increase in contractility when the systolic pressure is maintained 45 stroke volume (calculated by planimetry) was of constant. However, calculated total peripheral resis 169-36% (P<0.005) over the control and the increase tance will be higher (an underestimation of the fall). in cardiac output was 75-29% (P<0.005) over the After stabilization of hemodynamics as reflected by control. Calculated total peripheral resistance after 35 continuously monitored parameters for 15 minutes, the minutes decreased by 46.8-9.3% (P<0.001). Thus, it substances to be studied were infused intravenously 50 became apparent that berberine (0.7 mg/Kg/min) acted using a Harvard infustion pump. Registrations were very favorably because it increased contractility and made every 5 minutes at a paper speed of 200 reduced peripheral resistance and consequently aug mm/second, while all the parameters were recorded mented markedly the aortic blood flow. constantly at a speed of 2 mm/second. The dogs were Moreover, heart rate was reduced by 32-3% randomly assigned to several groups: 55 (P<0.001). This is, per se, a favorable effect since it Group a) Control-in these dogs a solution of 5% dex renders the heart performance more efficient especially trose in water was infused in the same quantity as in when tachycardia is initially present as in many patients the berberine treated animals (n = 7). with heart failure. The fall in heart rate decreases myo In all berberine, berberrubine coreximine and tetrahy cardial oxygen consumption which benefits the balance dropalmatine groups the compounds were infused at of oxygen of the myocardium. It shows also that the constant rate for 35 minutes and the data analyzed at 5 increase in contractility is probably even greater than minute intervals. reported here since the fall in heart rate, per se, has a Group b) Berberine 0.7 mg/Kg/min (n = 10). negative inor tropic effect in the anesthetized dog. Fi Group c) Berberine 0.2 mg/Kg/min (n=7). nally, the fall in heart rate suggests that the effect of Group d) Berberine 0.02 mg/Kg/min (n = 7). 65 berberine is not similar to that of a beta-adrenergic Group e) Berberrubine 0.2 mg/Kg/min (n = 7). agonist. Group f) Tetrahydropalmatine 0.2 mg/Kg/min (n=4). All these effects (increase in contractility, decrease in Group g) Coreximine 0.2 mg/Kg/min (n = 8). afterload, peripheral vasodilation, increase in aortic 5,153,178 33 34 blood flow and reduction in heart rate) are beneficial in all increased the dP/dt values in similar magnitude the treatment of any condition requiring the improve (from 32 to 44%). Berberine did increase it mildly more ment of performance of the heart and especially if that than the others. The fall in diastolic arterial pressure (as has to be done with a minimum increase in expenditure reflected by widening in the pulse-pressure (“PP") was of energy (i.e. of oxygen). shown in all drugs. It was noted to be more than twice As shown in FIG. I, the effects of 0.2 and of 0.02 as large with berberine than with the other three drugs. mg/Kg/min of berberine are also statistically highly Heart rate ("HR") dropped with all compounds; it fell significant in increasing contractility (LV dP/dt). When approximately 16-17% with berberine, berberrubine all curves (group b, c, and d) are analyzed together and coreximine and by 5.5% with tetrahydropalmatine. there is a clear dose response with dp/dt increasing O Stroke volume ("SV') increased significantly with ber proportionally and linearly to dose given. With the berine, tetrahydropalmatine and corexinine; just higher dose there is a tendency to reach a plateau when slightly (and not statistically significantly) with berber the increase reaches 40%. It is very important to note rubine. With a larger dosage of 0.5 and 0.7 that even the low dose of 0.02 mg/Kg/min resulted in a mg/Kg/min., stroke volume increase will be noticeable highly significant increase in dP/dt which was 5 with berberrubine. 35.8-8.2% (P<0.0005) after 35 minutes. This also Consequently, calculated peripheral resistance was shows again the very good therapeutic ratio. In one reduced significantly by over 30% with berberine and additional dog, berberine was infused in even a small tetrahydropalmatine, while coreximine and berberine concentration of 0.002 mg/Kg/min and dP/dt in did not show that property to that pronounced degree. creased by 20%. 20 Thus, when it is desired to maximize increase in con The increase in pulse-pressure indicates the fall in tractility without vasodilation, the compounds of diastolic aortic pressure which signify peripheral vaso choice are coreximine and berberrubine, whereas ber dilation and afterload reduction. The dose response berine and tetrahydropalmatine are the compounds of here is also evident and again even the infusion of 0.02 choice when vasodilation and contractility both are mg/Kg/min (group d) increased pulse pressure by 25 desired concurrently. 18.5-10%. This analysis shows that in all of the measured param The increase in stroke volume was also dose depen eters the changes were directionally the same, though dent. Again, this reflection of better cardiac perfor of different magnitude. Thus, for example tetrahydro mance was elicited even with the dose of 0.2 palmatine is a compound of choice when decrease in ng/Kg/min (group c) increasing significantly aortic heart rate is not as necessary; berberrubine and corexi flow by 53.6-17.7% (p<0.01). mine will be compounds of choice when lesser effect on Finally, the fall in heart rate was also dose dependent. pulse pressure is desired. However, at the smaller doses there was no significant Traditional drugs for the treatment of acute heart fall in heart rate. failure such as the cathecholamines and other sympa From these experiments it can be concluded that 35 thomenetic amines which are known to exert a positive berberine acts very favorably on the measured hemody inotropic effect by interacting with myocardial (beta 1) namic parameters with the view of its application in adrenergic receptors such as dopamine, dobutamine or patients in whom heart performance should be im isoproterenol are traditionally administered not only in proved such as chronic congestive heart failure, acute cardiogenic shock but in "low output syndrome' situa heart failure in all its types of manifestations such as tions such as by infusion during and after cardiac sur cardiogenic shock and pulmonary edema, in low car gery. Braunwald, pages 553/557. diac output syndromes and in non-cardiogenic shock To determine further the effects of berberine on pa syndromes or diseases in which it is clinically advanta tients, experiments were carried out on conscious dogs geous to augment cardiac output, increase cardiac con (Protocol F) and the measurement of left ventricular tractility especially when this property and peripheral 45 performance was assessed atraumatically using the mul vasodilation may be sought after such as in shocks of tiple gated radionuclide ventriculography (MUGR) non-cardiogenic origin. similarly to what is done in patients. The multiple gated Moreover, it shows that the effect of berberine on the radionuclide ventriculography is used in patients to different favorable parameters occurs at different doses assess their cardiac (i.e. left ventricular) performance by and is differently dose related. Thus, the fall in heart 50 analyzing in an atraumatic manner, the ejection fraction rate occurs later (i.e. in the higher dose) than increases (EF) of the left ventricle. This technique substituted the in LV dR/dt, increases in aortic flow or fall in aortic analysis of ejection fraction by contrast angiography diastolic pressure. Also the fall in peripheral resistance, since the latter can be done only in the catheterization the increase in pulse-pressure and the increase in aortic laboratory with the patient submitted to the introduc flow continue to change even in the highest doses that 55 tion of catheters into his arteries and left ventricular were given in these experiments while LV dip/dt at the cavity as well as an injection of a radio-opaque dye. higher doses but not in the smaller doses tends to reach MUGR consists in using red blood cells labelled with a plateau. Thus, it may be possible to tailor the amount stannous radioactive technetium. These red blood cells of berberine to the specific condition of the patient. The can be labelled either in vivo or in vitro prior to injec same is done with coreximine, tetrahydropalmatine and tion. The blood that is now emitting gamma radiation berberrubine. Likewise with other representative spe can be detected by an external gamma camera. This cies of the compounds of the invention, like "tailoring' device can sum many cycles of the heartbeat obtaining is attractive therapeutically. an average cycle. Then this cycle is divided in time The comparision of berberine, berberrubine, tetrahy intervals so that, as an example, the end systolic and end dropalmatine and coreximine is shown in table XIII, 65 diastolic frames can be identified. where all were given at a dose of 0.2 mg/Kg/min Ejection fraction is the end distolic minus the end (group c, e, f and g). The results show that berberine, systolic volumes divided by the end diastolic volume. It and berberrubine, tetrahydropalmatine and coreximine indicates therefore the pumping effectiveness of the left 5,153,178 35 36 ventricle since it measures the fraction (or the percent if eraged. After discontinuation of the infusion again four multiplied by 100) of the volume of blood that is ex radionuclide ventriculographies were obtained in the pelled from the left ventricular cavity. next thirty minutes and averaged. Berberine infusion Experiments were carried out in trained conscious significantly increased EF in the dogs with acute left dogs. 40nCi of pertechnatate were used to label red ventricular failure from 27+3% to 40-3% (p<0.001). blood cells that were then injected intravenously. This increase of nearly 50% showed a very potent ef MUGR was done during infusion of placebo (5% dex fect of this drug. After the discontinuation of berberine trose in water) and during berberine intravenous infu infusion EF fell somewhat but continued to be signifi sion (0.7 mg/Kg/min during 5 minutes). The images cantly elevated for the next half an hour when the ex were acquired using fixed density of the left ventricle. 10 periment was terminated. They were measured in quadruplicate and averaged. These results demonstrate the highly significant in The external gamma camera used was Ohio Nuclear provement both biologically and statistically of the (model VIP 560). heart failure by the administration of berberine in a Table XIV is an example of one such dog. The ejec conscious dog. tion fraction (EF) was 31.5, before any treatment. After 15 When these experiments are repeated with other the 5, 15, 30 and 45 minutes of infusion of dextrose (in representative series of the invention, like improve control) it was 39, 34, 34 and 28%, respectively. Thus, ments on EF are observed. With tetrahydropalmatine, before the infusion of berberine it averaged 33-2%. corexinine and berberrubine like results will be ob After the infusion of 0.7 mg/Kg/min of berberine for 5 served. minutes, the EF was 46, 47, 44 and 38%, respectively; 20 This data (together with other reported and devel averaging, therefore, 44-2%. This increase was statis oped data) supports the conclusion that berberine is tically significant (p<0.01). It was an increase by 33% useful in congestive heart failure since EF is a sophisti versus the pre-berberine period. A second infusion of cated and commonly used clinical parameter to evalu the same dose of berberine resulted in EF of 46, 37, 40 ate performance of the heart which is the main goal of and 38%, being on the average 40+2% which is also 25 treatment of patients with heart failure. The increase in different from the pre-berberine period (p<0.05). EF, which was done without heart failure, strongly Another example is shown graphically (FIG. VI). suggests the effect to be even more marked with pa This dog received an infusion of 5% dextrose in water tients with heart failure. Finally, the experiments with for 15 minutes during which time the ejection fraction acute left ventricular failure confirm its usefulness. was stable around 50%. Thereafter, an infusion of ber 30 It has been further discovered that berberine is an berine 0.7 mg/Kg/min was started and continued for ideal agent for scanning the heart. This is performed in the next 15 minutes. The ejection fraction increased a known manner by attaching a gamma ray-emitting continuously from 50 to 54 to 65 and finally to 70% marker such as iodine, bromine or technitium to berber after 15 minutes; i.e. an increase of 40%. ine. In that respect, berberine has the advantages of These two dogs are excellent examples of the potency 35 such known scanning agents such as thallium (or potas of berberine that increased ejection fraction in these 2 sium analogs) commonly used today. dogs by 33% and by 40%, respectively. These increases It is known in the field of cardiac examination that to are remarkable under any circumstance but more so obtain a scan of a cardiac silhouette after intravenous since they were obtained a) in the conscious dog and injection of a radioisotope (such as in this case the therefore with its reflexes intact and without any possi 40 gamma ray-emitting marked berberine), the area of the ble cardiodepressant effect of anesthesia and b) because cardiac silhouette occupied by the labelled intracardiac they were obtained in a dog without failing heart. In the blood pool is comparable with the cardiac silhouette on non-failing heart situation, here shown, a drug has to be the chest roentgenogram (the difference being the wall much more potent in order to increase ejection fraction thickness). This property can be used for the detection than in a situation of heart failure. 45 of the pericardial infusion in which case the X-ray films Nevertheless, to verify the response of dogs with will show a much wider silhouette, The intravenous acute heart failure with this sophisticated technique, injection of a small bolus of a gamma emitter such as this condition was provoked by previous embolization 99mTc pertechnetate permits rapid visualization of the into the left coronary artery (as described below in heart, great vessels and other vessels by an angiocintilla protocol HV). Thereafter, the effect of berberine infu 50 tion camera. This technique is used in a variety of ac sion was analyzed after embolization. Thus, in one dog quired or congenital cardiac lesions. Such radioisotopes before embolization (i.e. before acute left ventricular are used for myocardial imaging. Typical radioactive failure) EF was 47%. After embolization and before substances as 43K (potassium), 81. Rb (rubidium) or berberine administration EF fell to 27%. Berberine was 201T (thallium) concentrate in normal myocardium. infused in a rate of 0.2 mg/Kg/min for 30 minutes and 55 Such myocardial imaging is known and is described for MUGR was repeated after 2, 15, 22 and 28 minutes of instance in Harrison's, Chapter 234, pages. 1153 and seq. infusion. EF improved clearly from 27% to 35, 37, 39 which is incorporated herein by reference. and 38% respectively. In another dog EF before embo This property of berberine differentiates it and is lization was 55% after embolization it fell to 39%. The another advantage over digitalis since digitalis is not a administration of berberine 0.2 mg/Kg/min restored 60 suitable marker for the heart but rather for the liver. EF to 60, 62, 55, 55% at 2, 15, 20 and 30 respectively. Preferential concentration of berberine in the heart Six dogs were studied using this technique (Table rather than in other thoracic organs or the liver is a sine XV). Their average left ventricular ejection fraction qua non for a good imaging agent. Berberine level mea (EF) fell from 55+% to 27-h%2 (p<0.001) after embo surements were done spectrophotometrically. The ex lization showing that they had now left ventricuar fail 65 periment consisted of injecting 3.5 mg/Kg of berberine ure. The thirty minute infusion of berberine (0.2 (SO4) intravenously and killing the rats after 15 minutes, mg/Kg/min) was administered. During this period four 90 minutes, 6 hours, 24 hours, 48 hours and 1 week. radionuclide ventriculographies were obtained and av Each group consisted of 5-6 rats. A control group (no 5,153,178 37 38 berberine administration) was used to determine the 5 minutes. Administration of microspheres was discon absorbance of the different organs at that wave length. tinued when LV dP/dt had been reduced by 30% or The data showed that the concentration in the tissue of more when measured 4 minutes after the last dose. All rat heart was 16-2, 19+3 and 13-3 ug/g of berberine dogs received prophylactic antiarrhythmic treatment at 15 min, 90 min and 6 hours respectively, falling to with intravenous , 1.5 mg/kg body weight as a zero thereafter. It was released rapidly from the heart bolus 20 min after the end of embolization, followed by thus showing no significant tendency to accumulate in a continuous infusion of 45 ug/kg/min. that organ. Its concentrate was 1, 1 and 3 ug/g in the Forty-five minutes were allowed to elapse to verify lungs and 0.2 and 0 in the skeletal muscles or in the the stability of the preparation. If LV dR/dt or left blood at the same time intervals. Also in the liver it was O venticular end diastolic pressure had changed by 20% 0, 6, and 2 ug/g showing an excellent ratio of concentra or more between 30 and 45 minutes after embolization, tion between the heart and each one of the above-men the dogs were excluded from this study and not ran tioned tissues. domized. Forty-five minutes after the end of emboliza To verify the effects of berberine in the situation of an tion, the dogs were assigned randomly to three groups: acute left ventricular failure (Protocol HV) the follow 5 a) Berberine-treated group, 0.2 mg/kg/min, dissolved ing was carried out. in 60 ml of a solution of 5% dextrose in water adminis Experiments were carried out in 21 mongrel dogs of tered in a constant infusion during 30 minutes; b) oua either sex with body weight between 19 and 39 kg. bain-treated group, 0.03 mg/kg was administered in 60 Anesthesia was induced with sodium pentobarbital, 25 ml of a 5% solution of dextrose in water during 30 mg/kg of body weight intravenously, and small supple 20 minutes; and c) Control group, administration of 60 ml mentary doses were given as necessary to maintain a of a 5% solution of dextrose in water during 30 min. constant level of anesthesia. The dogs were placed in A paired Student t test (two-tailed) was used for the left lateral position and ventilation was maintained statistical analysis of changes in hemodynamic variables through a cuffed endotracheal tube with a volume-con within each treatment group, and a Student t test for trolled respirator (Harvard Apparatus Co., Millis, 25 group observations (two-tailed) was used for statistical Mass.). Polyethylene cannulae were placed into the analysis of differences between treatment groups. All jugular and femoral veins for intravenous infusions and data are presented as mean -1 standard error. drug administration. Electrocardiograms (lead a VF) The successive injections of microspheres into the were monitored continuously throughout the experi left main coronary artery caused a gradual reduction in netS. 30 LV dR/dt and increase in left ventricular end diastolic A polyethylene cannula was introduced into the aorta pressure. The average number of doses used for induc through the left carotid artery to record arterial pres tion of acute heart failure was 7.70.4 which corre sure. (Statham P23 ID transducer, Statham Instruments, sponds to about 6.3x105 microspheres. The emboliza Inc. Oxnard, Calif.). A Millar microtip transducer cath tion was terminated when LV dip/dt was reduced by eter (Millar Instruments, Inc., Houston, Tex.) was intro 35 37.3% from 2988-152 mmHg/sec to 1823-65 duced into the left ventricular cavity through the femo mmHg/sec (p<0.01, n=21), left ventricular end dia ral artery to measure left ventricular pressures. For stolic pressure increased by 145-16% from 5.5-to.2 adjustment of zero level, left ventricular pressure was mmHg to 12.5–0.6 mmHg (p<0.001, n=21), means also measured through the fluid-filled lumen in the Mil systemic arterial pressure decreased by 15.3% from lar catheter (Statham P23 ID transducer). The maximal 126-5 mmHg to 106-i-4 mmHg (p<0.001, n=21), rate of rise of left ventricular pressure, LV dP/dt, was heart rate decreased by 27-5 beats/min, from 1635 recorded by means of a differentiator. All these parame beats/min to 135-6 beats/min (p<0.001, n=21), car ters were recorded continuously on a polygraph (Gould diac output decreased by 23.2% from 3.24 L/min to 2800, Gould Instruments, Cleveland, Ohio). Cardiac 2.41+0.10 L/min (P<0.01, n=21), and total peripheral output was determined by the thermodilution tech 45 vascular resistance increased by 154-5% from nique, using a thermistor catheter in the pulmonary 3228-153 dyn.sec.cm-5 to 3702-270 dyn.sec.cm-5 artery and calculated by a cardiac output computer (p<0.01, n=21). (Elecath, Electro-catheter Corp., Rahway, N.J.). The The coronary artery embolization induced ST seg coefficient of variation for duplicate CO measurements ment elevation and T wave changes consistent with was 5%. In addition to the parameters directly ob acute global myocardial ischemic injury. After the end tained, total peripheral vascular resistance was calcu of embolization, LV dip/dt and left ventricular end lated from the ratio of aortic diastolic pressure and diastolic pressure did not change significantly and were cardiac output. Stroke volume was calculated by divid completely stable in all but three dogs after 30 minutes. ing cardiac output by heart rate. Heparin (Invendex) Twenty one dogs were randomly assigned to berbe (5000 IU) was administered to avoid clotting of the 55 rine-treated (n = 7), ouabain-treated (n = 7), or control catheters. (n=7) groups. No significant differences were found for To induce acute left ventricular failure, initially the any of the hemodynamic variables among the three left main coronary artery was catheterized with a Jud randomized groups (Table XVI). The infusion of ber kin coronary artery catheter (7F, ISCI Cardiology and berine increased LVdP/dt and reduced left ventricular Radiology Products, Billerica, Mass.) under fluoro end diastolic pressure within 2.5 minutes. At the end of scopic control. It was introduced through a femoral the 30-minute infusion, LVdP/dt increased by 28-E8% artery, and polystyrene microspheres (3M Company, from 1572-80 mmHg to 2012-95 mmHg (p<0.005, St. Paul, Minn.) with a diameter of 52.5-4.0 pum Table XVI), and was significantly higher than in the (meant-SD) were injected into the coronary artery. control group (FIG. VII). Left ventricular end diastolic The microspheres were used in a concentration of 1 mg 65 pressure decreased by 38t4% from 13.3-0.8 mmHg (i.e., 12,000) per ml saline. The first dose of microsphere to 8.1+0.5 mmHg (p<0.001, Table XVI), and was solution was 0.5 ml/kg body weight, and all subsequent significantly lower than in the control group (FIG. doses were 0.25 ml/kg body weight, administered every VIII). Systolic arterial pressure was not significantly 5,153,178 39 40 changed by berberine (Table XVI). Diastolic and mean Berberine HCl Infusions. Comparison with a control arterial pressure were significantly although only group. slightly reduced by berberine (Table XVI). There was Thus, while berberine increased left ventricular con an increase in pulse pressure from 28-2 mmHg to tractility (LVdP/dt), decreased left ventricular end 33-4 mmHg. Heart rate fell by 8 beats/min. (Table diastolic pressure, decreased peripheral resistance and XVI). Total peripheral vascular resistance increased in clearly improved left ventricular function, digitalis the control group, but in contrast it decreased by (ouabain) was completely ineffective. This indicates 19-2% from 4915-559 dyn.sec.cm 5 to 4004-496 that berberine can be given successfully in such circum dyn.sec.cm-3 in the berberine-treated group (Table stances when digitalis is not effective such as in acute XVI). Thus it was significantly lower than in the con 10 heart failure. In the cases in which cardiogenic shock is trol group (FIG. IX, p<0.05). In the control group, the most typical example, berberine similarly to beta cardiac output and stroke volume fell from 1.77+0.28 adrenergic agonists was very effective. Moreover, it liters/min to 1.540.24 liters/min and from 2.91.82 was reasonable to assume that when berberine improves ml/beat to 11.9-1.7 ml/beat, respectively. In contrast, left ventricular function to the same extent as beta ad in the berberine-treated group, cardiac output changed 15 renergic agonists, this improvement occurs with a from 1.760.15 liters/min to 1.93-0.16 liters/min and smaller expenditure of oxygen, i.e., with smaller eleva stroke volume from 14.6+ 1.9 ml/min to 17.32.7 tion of myocardial oxygen consumption, or even with a ml/min. Thus, cardiac output and stroke volume were decrease in oxygen consumption. This was confirmed significantly higher at the end of infusion in the berbe for berberine as shown above. Contributing, but not rine-treated dogs (FIGS. X, XI). 20 solely responsible for this property, will be (in the case In the control experiments, the left ventricular func of berberine), the lack of tachycardia, the decrease in tion curves (see Abnormalities of Circulatory Function) afterload (lower arterial pressure), and the decrease in are shifted downwards to the right, indicating further preload (lower left ventricular end diastolic pressure) deterioration of left ventricular function. The relation and the probable reduction in left ventricular cavity 25 dimensions. It should be emphasized that the fall in ship between changes in left ventricular end diastolic LVEDP due to the infusion of berberine was quite pressure and changes in cardiac output and stroke vol marked which is of great importance in the clinical ume from before to 30 min after the start of berberine situation of heart failure. These results indicate there infusion are shown in FIGS. XII and XIII in the form of fore that beside its effect of increasing contractility, of ventricular function diagrams. In contrast to the control 30 decrease afterload and peripheral resistance and of in group, during berberine infusion left ventricular end creasing cardiac output, berberine is also a potent pre diastolic pressure is reduced simultaneously with an load reducing agent. increase in both cardiac output and stroke volume, shift Other typical compounds of the invention perform in ing the curves upwards and to the left, indicating in the same way. Coreximine, tetrahydropalmatine, ber provement in ventricular function. 35 berrubine with certain variations also shown are compa Protocol HV was performed in accordance with that rable preload reduction. published in Smiseth, Mjos and Refsum,: Hemodynamic These experiments (which were conducted in closed effects of the B-adrenergic Receptor Agonist Pirbuterol chest animals) show that berberine is not only effective during Acute Ischemic Left Ventricular Failure in in the non-failing heart but that indeed it is also very Dogs. Acta Pharmacol. Toxicol. Scand. (Suppl. IV) pg. 40 effective in the acute left ventricular failure situation 53, 1981, which is the most crucially intense and life threatening Quabain did not change any of the hemodynamic circumstance in the gamut of cardiac failures. It shows parameters from before to 30 minutes after the start of that its effect is clearly superior to ouabain and probably drug administration (Table XV), comparable to beta adrenergic agonists over which it 45 may have the advantage of influencing the determinant FIGURE LEGENDS of oxygen consumption in such a way as to consume less FIG. VII. Changes in Left Ventricular dP/dt oxygen. This last property may be of great advantage in (LVdP/dt). Comparison between berberine control patients in whom the shock syndrome or any other form groups. (*= p <0.01 for comparison between the of acute or chronic heart failure is due to ischemic heart groups). 50 disease and more particularly acute myocardial infarc FIG. VIII. Changes in Left Ventricular End Dia tion. In these circumstances the increase in myocardial stolic Pressure (LV EDP) by berberine Hol. oxygen consumption may cause a worsening of the ** = p <0.01 for comparison between the groups). balance between oxygen supply and demand provoking FIG. IX. Changes in Total Peripheral Vascular Re at its more extreme cases myocardial tissue injury and sistance (TPR). (*=p <0.05 for comparison between 55 necrosis (see Maroko, P. R. and Braunwald, E., "Ef the groups). fects of Metabolic and Pharmacologic Intervention on FIG. X. Changes in Cardiac Output (CO) by Berber Myocardial Infarct Size Following Coronary Occlu ine HCL. See test, FIG. 4. (*=p <0.01 for comparison sion", Circulation 53 (Suppl. I): 1-162-I-168, 1976, which between the groups). is incorporated herein by reference). FIG. XI. Changes in Stroke Volume (SV). In summary, these experiments in the model of acute (** = p <0.05 for comparison between the groups). left ventricular failure demonstrate the following FIG., XII. Relationship Between Mean Changes in sought out properties: a) Lack of tachycardia. Indeed Cardiac Output (CO) and left ventricular end diastolic average heart rate was reduced by 8 beat/min. b) De pressure (LVEDP) from before to 30 min after berber crease in afterload and in calculated total peripheral ine HCL infusion. Comparison with a control group. 65 resistance, showing that this compound is an afterload FIG. XIII. Relationship between Mean Changes in reducing agent and a peripheral vasodilator. c) De Stroke Volume (SV) and Left Ventricular End Dia crease in preload showing that in addition berberine is a stolic Pressure (LV EDP) from before to 30 min after preload reducing agent. d) Increase in left ventricular 5,153,178 41 42 dP/dt showing that berberine increases left ventricular The duration of the QRS complex (0.04 to 0.10 s) contractility. This result is more impressive in this set of reflects the time required for depolarization of ventricu circumstances because aortic pressure was permitted to lar muscle. Block in a bundle branch prolongs it. An change, there was a modest fall and lower pressures approximation of the refractory period of the ven artificially underestimate contractility measured by tricules may be obtained by measuring the Q-T interval maximum dP/dt. In other words, the real increase in (from the onset of the QRS to the end of the T wave). contractility is more than assessed by maximal dp/dt The Q-T interval is rate dependent and may be affected which is here reported. by numerous pathophysiologic influences. See FIG. Other typical compounds of the invention give com XV. parable results. Tetrahydropalnatine, coreximine and O berberrubine perform in an equivalent manner. FIG. XV FIG. XIV (from Braunwald FIG. 16-2 page 512) The waves of the electrocardiogram-P, QRS, T, shows the relationship between left ventricular end and U-are indicated. The measurements of the P-R diastolic pressure (LVEDP) and cardiac index (left interval, QRS complex, S-T segment, and QT interval ordinate) or stroke volume (right ordinate) in a normal 15 are identified on the right. (N) and a failing heart (F) of a man. The upper limit of It has been found in accordance with the invention normal of LVEDP (12 mm Hg) and lower limit of following protocol Z, that berberine does not increase normal of CI (2.2-CI liters/min/m2) are shown, as are the P-R interval (FIG. XVI). This was so even when the values associated with congestive symptoms administered i.v. for 60' (0.7 mg/Kg/min) or a total of (LVEDP 20 mm Hg) and with impaired perfusion (2.2 42 mg/Kg. No manifestation of toxicity were observed. liters/min/m2). A and A' represent the operating points In contrast, digitalis is known to increase the P-R at rest of a hypothetical patient with heart failure and of interval. This is known to be related to causing atrio a normal person, respectively. Reduction of physical ventricular blocks. Braunwald, page 249 and Harrison's, activity allows the failing heart to meet the demands of page 1209, Goodman, pages 665-666. the metabolizing tissues. 25 FIG. XVII shows that the QRS complex or interval This Fig. shows diagrammatically the relationship is not increased (i.e. virtually remains unchanged) over between LVEDP and cardiac index (left ordinate) or the period of i.v. administration. Antiarrhythmic drugs stroke volume (right ordinate) in a normal (N) and a like quinidine and procainamide may prolong the QRS failing heart (F). Viewed differently, the effect of ber interval, which is not a desirable effect. berine is to cause curve O through A of FIG. XIV 30 Berberine causes an overall increase in QTc interval. (failing heart) to return to normal to the curve shown by This effect is similar to quinidine and procainamide. OA' (normal). This result with berberine on the This indicates an antiarrhythmic effect for berberine. LVEDP is particularly significant. Digitalis is known to shorten the QTc interval. The analysis of the "Starling Curve" in our previous Thus, the data showed that berberine, while increas related experiment in protocol HV (FIGS. XII and XIII 35 ing the QTc, does not prolong the QRS interval. It and table XIII) that berberine and the other compounds shows also that in the dosage used, the drug is not toxic. of the invention improve left ventricular function and In contrast, digitalis is known to increase P-R and to that this improvement is markedly superior to that decrease QTc. achieved by digitalis. This is also demonstrated in FIG. When dP/dt is increased by 43% (in experiment in XIV. protocol Z) QTc increased by an average of 16%. For a more complete understanding of the electrocar The antiarrhythmic properties of the compounds of diographic data and findings, especially on waveforms, the invention are further illustrated below. durations and intervals, the following may be informa Experiments were carried out to analyze the electro tive. physiologic properties of berberine (protocol TE). In The P wave of atrial depolarization is normally the 45 this protocol closed-chest dogs were anesthetized with initial wave of activity during the cardiac cycle. Ven intravenous pentobarbital (30 mg/Kg). Electrode cath tricular muscle depolarization is represented by the eters were placed, under fluoroscopy, in the high right QRS complex. A Q wave is an initial negative wave; an atrium, right ventricle, and in the aorta near the non R wave is an initial positive wave or a positive wave coronary aortic cusp to record His bundle potential. In following a Q wave; and an S wave is a negative deflec 50 addition aortic pressure (Statham gauge) and surface tion following an R wave. A QRS complex having a Q ECG were obtained. All parameters were recorded on wave which returns to the base line but does not pro an Electronic for Medicine polygraph (Model DR 8), duce a positive wave is labeled a QS complex, and a with a paper speed of 100mm/sec. The recordings were second positive wave is labeled R". The T wave repre obtained before berberine treatment and after five suc sents ventricular muscle repolarization, and is some 55 cessive five minute infusions of 0.2 mg/Kg/min of ber times followed by a small wave, the U wave. Repolari berine in each one of the dogs. At each one of these time zation of atrial muscle is represented by the Ta (or T) intervals recordings were done with the "spontaneous' wave, which occurs during the P-R interval and QRS normal sinus rhythm and with atrial and ventricular complex. The interval between the end of the QRS extrastimuli were given during the normal sinus as well complex and the onset of the T wave is the S-T seg as during the pacing phases of the experiment to deter ment. The interval between the P wave and the QRS mine the refractory period of the atria, atrio-ventricular complex is the P-R (or P-Q) interval, measured from the node and of the His-Purkinje system and ventricle. onset of atrial depolarization (P) to the onset of ventric Moreover, sinus node recovery time was measured. ular depolarization (Q). The duration is 0.12 to 0.20s in The results showed that berberine in this dose did not the adult. Since AV nodal activation begins before the 65 change the A-H, H-V and QRS intervals and that atrial end of depolarization of atrial muscle, the P-R interval refractory period did not change. Ventricular effective may be used as a rough approximation of AV conduc refractory period was increased by 11%. Sinus node tion time. recovery did not change. QTc changed by 13%; heart 5,153,178 43 44 rate decreased by 8%. These experiments show again controlled or negated this objectionable effect is a clear the potential antiarrhythnic properties of the com demonstration of the effectiveness of an antiarrhythmo pounds of the invention. When coreximine is substituted genic effect. in the above experiments comparable results are shown; It should be noted that both in the digitalis model of likewise for tetrahydropalmatine and berberrubine. arrhythmias and the aconitine model of arrhythmias in Thus the compounds of the invention have the prop which the experiments were done with the compounds erty to reduce, arrest or interrupt arrhythmias. This of the invention are classical models to study antiar property was studied in different ways in order to in rhythmic drugs. This, the observation that a drug pre clude arrhythmias produced by different causative vents, interrupts, reduces or abolishes arrhythmias in agents like drugs known to cause arrhythmias. This, the 10 these models is taken by the knowledgeable or one antiarrhythmic effect of the compounds of the invention skilled in the field to which the invention pertains not were studies in models where arrhythmias were pro only to show their specific effectiveness against these ducted by digitalis, by aconitine and by coronary artery drugs, but as their overall anti-arrhythmic properties. ligation. In the compositions (or mixture) of the invention The effect of the compounds of the invention is quite 15 discussed above, the amount of the compound of the unexpected in that a biologically active drug which is invention issued is the smallest amount necessary to positive inotropic does not normally have an antiarr cause a positive inotropic effect and the amount of arr hythmogenic effect. The implications of this finding are hythmia-causing drug can be any concentration (or quite significant. Without enumerating all of the impli proportion relative the composition of the invention) cations (the others being apparent to one of average 20 and either in a concentration generally acceptable with skill in the art), it will be seen that this use of the com out causing arrhythmia or in such an amount as nor pounds of the invention significantly increases the possi mally are considered arrhythmogenic. bilities of use of digitalis-type cardiac glycosides both in For instance digitalis is administered intravenously, the human and veterinary field. The cardiac glycoside typically incrementally; the protoberberine may be ad can be used with the compounds of the invention in the 25 ministered incrementally before, simultaneously or after dosage in which the cardiac glycoside are commonly the administration of digitals or the protoberberine may used with the result that there is a decrease in the risk of be infused at a steady rate during the administration of arrhythmias attributable to the cardiac glycoside espe the digitalis. The amount of digitalis thus administered cially when they are administered over a longer period may be less than is usual, the same amount or more than of time; or perhaps, more importantly, cardiac glyco 30 usual depending on the circumstances and condition of sides like digitalis can now be used in accordance with the patient. the invention in a dosage in which the cardiac glycoside For maintenance therapy it is in the range of about normally is toxic (or likely to cause arrythmia) or has 0.001 to 50 mg/Kg, for the compounds of the invention other adverse cardio-vascular effects). Thus, the com depending on the judgment of the physician's recom pounds of the invention significantly broaden the thera 35 mendations. peutic index of cardiac glycosides. This is a new and A study was carried out to examine the effects of the unexpected utility which provides a real incentive and simultaneous administration of berberine on digitalis removes a serious concern which traditionally existed (i.e. ouabain) produced arrhythmias. In dogs which with the cardiac glycosides, were dosed simultaneously with berberine and ouabain For instance, it is generally accepted that ouabain has (constant infusion) ventricular ectopic activity (VEA) an average digitalizing dose from 0.30 to 0.50 mg when and ventricular tachycardia (VT) appeared significantly administered i.v., deslanoside 0.80 mg i.v.; digoxin 1.25 later than in dogs which were dosed only with ouabain. to 1.50 mg orally and 0.75 to 100 mg i.v.; digitoxin 0.70 This demonstrates that these ventricular arrhythmias to 1.20 mg orally and about 1.0 mg i.v. Dosages in ex appear only with a higher dose of ouabain and that cess are generally considered exposing the human pa 45 berberine significantly and markedly reduces the occur tient to risk. (see, Braunwald, page 523). Similarly, in rence of these life threatening ventricular arrhythmias. veterinary medicine corresponding dosages are consid The data show that berberine protects the dogs from ered exposing the animal to risks of arrhythmias. Ac developing ventricular arrhythmias. This property may cordingly, an aspect of the invention encompasses a be of extreme clinical importance because it suggests compound of the invention in conjunction with an arr 50 that by giving berberine, the therapeutic ratio of digital hythmic-causing drug, even in a concentration where is-like drugs can be increased and that patients may be such drug would cause arrhythmia were it used alone able to receive higher doses of digitalis with a lesser risk on the mammal. of having the most feared toxic effect, that of ventricu The term "in conjunction' means not only in a mix lar arrhythmias. ture with a compound of the invention but means any 55 The experiments were conducted in closed-chest combination which when in the biological system of the anesthetized dogs which were randomized to three mammal permits the compounds of the invention and groups. All received ouabain 0.03 mg/Kg as a bolus the arrhythmic effect to take place causing the drug to intravenously and thereafter a continuous infusion with act on and in the same biological environment. a constant rate of 0.0006 mg/Kg/min until death. The In the therepeutic method sense, it is contemplated first group did not receive any other intervention and that all or any part of the compounds of the invention be served as control. The second group received simulta administered to the mammal before, together with or neously berberine 1 mg/Kg intravenously as a bolus and after the arrhythmia-causing drug, or any variation then 0.2 mg/Kg/min as a continous infusion. The third thereof. group received berberine only after the appearance of It should be noted in this conjunction that aconitine is 65 VEA in a frequency of 50% of more of all systoles. a drug which is considered generally not acceptable any In the controls, the time of appearance of 25% of longer because of its pronounced arrhythmic effects. VEA, 50% of VEA and VT was respectively 22-4.7 The fact that a compound of the invention effectively min, 23.9+4.7 min and 30.1+6.5 min and in the dogs 5,153,178 45 46 treated simultaneously with berberine it was 66.5-14.6 34-15% and 16-7% while in the dogs that received min (p<0.0005), 66.7-15.3 min (p<0.005) and berberine in addition to digitalis the diastolic pressure 96.115.1 min (p<0.005). This demonstrated that the fell by -9-5%, -13-9%, -13-10% and simultaneous treatment with berberine prolonged the -2011%. All changes were statistically significant. time of appearance of VEA and more importantly of Since it was demonstrated above that the administra VT by around 300% which is clinically extremely im tion of digitalis and berberine has obvious advantages . portant and statistically highly significant. from an antiarrhythmic point of view, the effect of the This data was analyzed also from the point of view of association of ouabain and berberine on contractility how much digitalis has to be given before provoking (LV dP/dt) will be examined. the arrhythmia. Thus, for example, in the control situa O In anesthetized closed chest dogs the systolic arterial tion it was necessary only to administer 0.04 ng/Kg of pressure was maintained constant with a reservoir full ouabain to provoke VT, while in the berberine treated of heparinized blood (as in protocol "M" and "Z" de dogs the dose was significantly higher, i.e. 0.088 scribed above) and a "pigtail" and Millar catheter were mg/Kg. introduced into the left ventricular cavity (as in proto Also in these experiments the frequency of occur 15 col "Z" and "HV', described above). All data was rence of any arrhythmias was analyzed (VEA more recorded on 8 channel polygraph (Gould Instruments) than 25%, VT or death). It was found that at 30 minutes with a paper speed of 200 mm/sec when necessary and in the control, twelve dogs had arrhythmias and four at 2 mm/sec constantly. did not, while in the berberine treated one had arrhyth In seven dogs ouabain 0.03 mg/Kg given intrave mia and eight did not. This is a highly significant differ 20 ence (p<0.005) by Fisher Exact test). When analyzed at nously as a bolus, resulted in a progressive increase in 45 minutes, in the control group fourteen had arrhyth dP/dt after 5, 10, 15, 20, 25 and 30 minutes by 17.2%, mias and two did not, while in the treated group three 24.3%, 34.4%, 36-4% and 44.6% and 49.7% of had arrhythmias and six had not. This is also highly the initial value of peak LV dP/dt respectively. Then significant (p<0.02 by Fisher Exact test). When ana 25 berberine (0.7 mg/Kg/min) was infused for the next 30 lyzed after 60 minutes, all sixteen controls has arrhyth minutes and LV dP/dt further increased to 87.10%, mias while in a treated group three had arrythmias and 82-13%, 87-13%, 95-16%, 96- 16% and 94.14% six did not. This is highly significant (p<0.005 by of the initial LV dP/dt respectively. The increases in Fisher Exact test). Finally, when analyzed after 90 min dP/dt when only berberine (0.7 mg/Kg/min) without utes, in the control group all had arrhythmias and in the 30 digitalis were given was at 5, 10, 15, 20, 25 and 30 min treated group four had arrhythmias and five did not. utes 30.4%, 36.3%, 39-3%, 42-5%, 39.5% and This also is highly significant (p<0.001 by Fisher Exact 40.7%. Thus, the increase in maximal LV dp/dt was test). both statistically and biologically highly significant When berberine treatment started only after the ap when the effect of the two drugs was compared to the pearance of 50% of VEA, the time of appearance of 35 effect of each one of them alone (Table XVII). It is sustained VT was 30.16.5 minutes in the control noteworthy that the increase in contractility was more group, and 84.5+13.8 minutes in the berberine treated than the summation of the effects of both when given group (p<0.005). This demonstrates that berberine separately. This suggests that the effects are more than when given when VEA are already frequent, will delay additive but synergistic. by more than 250% the appearance of sustained VT. Coreximine, tetrahydropalmatine or berberrubine Also when analyzed by the amount of ouabain that perform in the same manner. provoked VT it was 0.048 mg/Kg in the controls and Other select compounds of the invention also per 0.080 mg/Kg in the treated dogs. formed markedly well as antiarrhythmias. Moreover, the effects of intravenous bolus injection Therefore, the usefulness of the compounds of the of berberine (0.4 to 1 mg/Kg) was studied (in compari 45 invention is markedly increased because: son to dextrose 5% injection). They were administered 1) Berberine, perse, (and the other compounds of the when the dogs exhibited VT and were considered effec invention) increases contractility and cardiac perfor tive if they succeeded to revert VT into sinus rhythm mance with an excellent therapeutic ratio. within five minutes. With the injection of placebo (dex 2) Berberine (and other compounds of the invention) trose) there was no effect in any of the twelve instances. 50 increases the therapeutic ratio of digitalis when the two With berberine injection 30 out of 14 were effective. compounds are given together, permitting the adminis This is statistically highly signilificant (p<0.005) and tration of digitalis type cardiacglyoside in an amount biologically (clinically) of extreme importance. greater than ever given before with less toxic side ef In the above experiment, other typical species of the fects (ventricular ectopic beats, ventricular tachycardia, compounds of the invention are tested and give compa 55 ventricular fibrillation). rable results. Berberrubine, tetrahydropalmatine and 3) The combination of drugs has additive or synergis coreximine likewise gave satisfactory results showing tic properties in the treatment of contractility. their antiarrhythmic and antiarrhythmogenic proper 4) The compounds of the invention like berberine ties. (and the other compounds of the invention) can coun It may also be noteworthy that in two berberine dogs teract the undesired vasoconstrictor effect of digitalis ventricular fibrillation reverted without any other ther type cardiacglycoside on the peripheral vessels. apy. This is remarkable and suggests an increase in 5) The compounds of the invention like berberine threshold for ventricular fibrillation. (and the other compounds of the invention) is an effec Berberine can counteract the vasoconstrictor effect tive . It is effective in abolishing of digitalis on the peripheral vessels. This is shown by 65 premature ventricular beats and in supressing estab the observation that the diastolic pressure in the control lished ventricular tachycardia, an extremely severe and group which received only digitalis rose after 10, 20, 30 life threatening arrhythmia, by administration before, and 60 minutes respectively by 31E9%, 16-til 1%, during, or after administration of digitalis type cardiac 5,153,178 47 48 glycoside, and is expected to reverse ventricular fibrilla a bolus). In the controls seventeen out of twenty-four tion. rats died within the first fifteen minutes, while in the Moss et al (Circulation 64, No. 6, 1981, p.< 1150) berberine treated group only six out of twenty-two rats suggests that digitalis therapy increases the early post died which is biologically and statistically highly signif hospital mortality of myocardial infarction patients icant (x2 = 8.7, p<0.01). with combined electrical and mechanical dysfunction. This experiment demonstrates the effectiveness of The antiarrhythmic activity of berberine (and the other berberine in preventing arrhythmic death after coro compounds of the invention) and its synergistic activity nary artery occlusion when the arrhythmias are caused with digitalis type cardiacglycoside shows that treat by the ischemic insult or injury to the myocardium. ment with berberine (and the other compounds of the 10 This experiment shows that berberine is effective not invention) or the combination of berberine (and the only in “automatic' but also in the "re-entry' type of other compounds of the invention) and digitalis type arrhythmias. The clinical application is to patients with cardiacglycoside is beneficial in such cases. ischemic heart disease and more specifically to those To examine the effects of berberine on arrhythmias with acute myocardial infarction. The administration of produced by intravenous infusion of aconitine in fifteen 15 the compounds of the invention is thus useful in patients dogs after development of VEA, of at least 50% of the prone to arrhythmias due to coronary artery obstruc beats or more, the dogs were given berberine intrave tion or coronary spasm or myocardial infarction of any nously as a bolus (1-5 mg/Kg) or 5% dextrose in water. etiology as well as ischemic sudden death. In the treated group, regression of the arrhythmias to It was unexpected that 50% of the rats that were normal sinus or supraventricular rhythm occurred in 20 monitored by electrocardiography died due to ventricu 78% of the dogs, but in the controls it occurred only in lar fibrillation and 50% died with complete A-V blocks, 20% which is statistically (p<0.05) and biologically which suggested that berberine did not only prevent significant. It is noteworthy that in all but one of these death from fibrillation but also due to A-V blocks. successful experiments, there was a regression of VT to When specific species of the compounds of the inven normal sinus rhythm and that in two cases ventricular 25 tion are used individually in the above work, like per fibrillation reverted to normal sinus rhythm. Thus, ber formance was observed. With berberrubine, tetrahydro berine was effective also in this model of ventricular palmatine, and coreximine, like antiarrhythmic control arrhythmias. When berberine is substituted by other is observable under the same experimental conditions. typical species of the compounds of the invention, com Another embodiment of the invention is the use of the parable results are obtained. 30 compounds of the invention in the control of mammal To verify the effect of berberine on supraventricular shock, such shock having as its clinical symptom or arrhythmias in another group of dogs, the topical appli manifestation, a decreased cardiac output. The most cation of aconitine (0.04%) solution to the atria pro important consequence of shock is decrease of blood voked either atrial ectopic beats, atrial tachycardia or flow to the vital organs such as brain, heart and kidneys. atrial fibrillation. The injection of intravenous berberine 35 Thus, commonly the most obvious result of treatment in a bolus (1-5 mg/Kg) was effective in abolishing these of shock are evaluated by urinary output. arrhythmias in contrast to the injections of placebo (5% Regardless of etiology, circulatory shock is a condi dextrose in water). This shows that besides being effec tion which has as a manifestation diminished blood flow tive in abolishing the most severe and life threatening and insufficient tissue oxygenation resulting therefrom. ventricular arrhythmias, it also abolished atrial ectopics, Shock is related initially to reduced blood volume, de atrial tachycardia and interrupted atrial fibrillation creased cardiac output or change in peripheral resis which are frequently of clinical importance to the well tance. being of the patients and when abolished can improve The etiology of circulatory shock can be classified the hemodynamics and decrease the risk of emboliza into several categories: hypovolemic shock, septicemic, tion. The other typical species of the compounds of the 45 anaphylactic, hemorrhagic, neurogenic vasculogenic invention performed in like manner. shock, and cardiogenic shock and others, all of which Another series of experiments were done using the rat are contemplated within the scope of the invention. model of acute coronary artery occlusion. In these ex Hypovolemic shock results from a significant reduc periments the effect of berberine was examined on mor tion in blood volume. The reduction frequently arises as tality. Thus, Sprague-Daley rats (Charles River) weigh 50 a result of major tissue trauma such as ruptures of the ing over 300 grams were anesthetized with ether, their liver or spleen or severance of a major artery. chest opened in the 5th left intercostal space their heart Vasculogenic shock results from vascular changes everted and the left main coronary occluded with a frequently caused by bacterial infection and bacterial surgical stitch. Thereafter the heart was repositioned toxins associated therewith. Endotoxins (such as LPS) and the thorax closed with a purse string suture, as 55 are especially capable of inducing shock. described in more detail by us previously (Maclean, Cardiogenic shock results from abnormalities in car Fishbein, M. C., Braunwald, E. and Maroko, P. R., diac pumping capacity. The compounds of the inven Long Term Presentation of Ischemic Myocardium tions are effective in relieving, preventing or control After Experimental Coronary Artery Occlusion. J. ling (to varying degrees) and arresting these various Clin. Iny. 61, pages 541-551, 1978 and also in Maclean, forms on non-cardiogenic shocks in addition to their D., Fishbein, M. D., Maroko, P. R. and Braunwald, value in the control of cardiogenic shock. Hyaluronidase-Induced Reductions. In Myocardial In For a discussion of circulatory shock in veterinary farct Size. Science, 194, pages 199-200 (1976)). The and human medicine, attention is invited to Jones elal, mortality in rats weighing over 300 mg was greater than pages 583 to 593, and to Braunwald, chapter 18, both of in smaller rats. 65 which are incorporated by reference. After finishing the surgical procedure, the rats were In noncardiogenic shocks the goal of the therapy is to randomized into controls (injection of 5% dextrose in restore systemic arterial pressure with adequate blood water) and berberine treated (1 mg/Kg intravenously as flow to the important organs. In many shock syndromes 5,153,178 49 SO especially when they progress for several hours, a pe At this time, i.e., two hours and fifteen minutes after ripheral vasoconstriction appears which aggravates the administration of LPS, 1000 ml of 5% of dextrose in shock syndrome by reducing blood and oxygen supply water was administered to all dogs during the next to the tissues distal to the constriction. (See Rushmer, forty-five minutes. This was done since in the treatment Van Citters and D. Franklin-DeFiuitia and classifica of all hypovolemic shocks (which include the endotoxic tion of various forms of shock, Shock Pathology and shock) the treatment has to include augmentation of Therapy, Bock, K. D. Academic Press 1962, pgs. 1-22) intravascular volume with infused fluids. by R. C. Hehei, R. H. Ditzman, G. J. Motsay, C. B. The systemic systolic arterial pressure increased to Beckman, C. H. Shafney, "Growth of the Concept of 107-5 mmHg in the controls, but increase more to Shock and Review of Status and Knowledge', pages O 127-6 mmHg in the berberine treated dogs (p<0.02). 377-409, Steroids and Shock. J. T. Glenn, University Thus, while in the control dogs the final systolic pres Park Press, 1974. sure was significantly lower than initial (i.e., before LPS Moreover, there is a depression in myocardial con infusion) pressure 140-7 vs 107-5 mmHg (p<0.001), tractility. (See, Myocardial Depressant Factor and Circu in berberine treated dogs the systolic pressure after latory Shock, A. M. Lefer, Klinische Wochenshrift 15 treatment was not statistically different from that before 52:358-370, 1974 and see Left Ventricular Performance shock 136-10 and 127-6 mmHg (T=1.01, p>0.40). in Endotoxic Shock in Dogs, W. G. Guntheroth, J. P. Thus the treatment with berberine and 5% dextrose in Jacky, S. Kawabori, J. G. Stevenson, A. H. Moreno, water restored the systolic arterial pressure while this Amer. J. Physical 242:H172-H176, 1982). treatment without berberine did not do so. 20 Peak left ventricular dP/dt was in the treated group Accordingly, the effects of berberine were studied in 3742-305 mmHg which is significantly higher the model of endotoxic shock in dogs, in which the (p<0.001) by approximately 50% than its dP/dt before endotoxic shock correspond to that provoked by gram shock, 2565-166 mmHg. This shows that whey sys negative bacteria with injection of lipo polysacharides tolic arterial pressure (and systolic LV pressures) were (LPS). 25 restored the dP/dt was 50% more than in the normal, The usefulness of the compounds of the invention in before shock state, which indicates on the one hand controlling shock is illustrated further as follows. again, the positive inotropic properties of berberine and, Intravenous administration of endotoxin (LPS), 5 mg on the other hand, that the complete restoration of the produced an immediate and precipitous fall in arterial systolic pressure that occurred in treated dogs (but not pressure. In dogs which had been given endotoxin treat 30 in the controls) should be credited this positive inotro ment with berberine increased pulse pressure, systolic pic effect of berberine. pressure and dP/dt resulted. No cardiac arrhythmias or Urinary output during the second hour of shock (be conduction defect was observed. In contrast, the con fore berberine or placebo treatments) and the third hour trol dogs showed a gradual decrease of dP/dt, systolic of shock (when the dogs were treated with either ber pressure and pulse pressure. ECG showed sinus brady 35 berine or placebo) were compared in each dog. This cardia followed by idioventricular rhythm. analysis is of crucial importance since it would indicate The response to the following 5% dextrose in water the restoration of effective organ perfusion which in the infusion in the berberine treated dogs was much more case of the kidneys results in urinary output. In the marked as far as increase in systemic arterial pressure control dogs the urinary output continued to decrease and left ventricular dP/dt. and in the third hour urinary output was less by approx Nineteen closed-chest barbiturate anesthetized with imately 50 ml than in the second hour, i.e. -51.7-c32.9 artificial respiration dogs were studied. All measure ml/hour. In contrast, the berberine treated dogs urinary ments were done by the technique described before output increased from the second (pre-treatment) to the LPS. 5 mg was given and resulted in a precipitous fall of third (treatment) by 30.5-14.3 ml. This difference is arterial systolic pressure to 56-4 mmHg and diastolic 45 biologically important and statistically significant (p pressure to 33-4 mmHg from their original 0.025). When the dosage is increased to twice as much, 138.8/106-8 mmHg. After two hours the dogs were even faster and more pronounced improvements are randomized to controls (given as placebo 5% of dex observed. trose in water) and berberine treated dogs which were The series of experiments is repeated using tetrahy treated to the minimal dose that will result in an eleva 50 dropalmataine and comparing it with LPS. Like posi tion of systemic systolic arterial pressure by at least 5 tive results are obtained and the endotoxic shock cause mmHg. This small dose 0.053-0.008 mg/Kg/min was by LPS is controlled. Parallel experiments with repre then used throughout the experiments. In the dogs that sentative series of the compounds of the invention gives received placebo within fifteen minutes systolic pres comparable results, including the berberrubine and sure continued to fall and decreased further to 50-6 55 corexinine. mmHg while in the berberine treated it rose to 66t4 When the dosage is increased to twice as much, even mmHg. This is statistically highly significant. At the faster and more pronounced improvements are ob same time peak left ventricular dP/dt fell in the control served. dogs to 1050-145 mmHg/sec while it increased to The embodiment of the invention wherein the corn 2062-204 mmHg/sec in the berberine treated group pounds of the invention are useful in the treatment of which is statistically highly significant. The pulse pres hypovolemic shock (including endotoxic shock) in a sure (systolic minus diastolic pressures) did not change mammal is very noteworthy. That the compouds of the in the control group but increased significantly from invention are also capable of restoring systolic arterial 24-2 to 32-2 mmHg (p<0.005) in the treated group. pressure after shock underscores the potency and effect Thus, it was demonstrated that in the endotoxic shock, 65 even of their positive inotropic effect. A compound the infusion of very low doses of berberine resulted in a which is a positive inotrope is not necessarily a com very marked increase in contractility and improvement pound which corrects or alleviates shock, nor for that in hemodynamics. matter restores systolic pressure after shock. 5,153,178 51 52 In conclusion, berberine treatment was highly benefi so. Continuous monitoring is maintained so that the cial in treatment of this type of shock syndrome by infusion may be stopped when the desired effect is increasing contractility, imposing (or restoring normal) achieved. Thereafter a maintenance dosage is adminis arterial pressure and ameliorating organ perfusion as tered or whatever dosage is advisable to maintain an reflexited by urinary output. There were also no notice adequate level of improvement which may be adminis able toxic side effects such as arrhythmias. tered orally or by injection. These therapeutic measures When given orally in variable doses (200-1000 are known to one skilled in the art and need not be mg/Kg) to rabbits and cats improvements in systemic detailed more here. arterial pressure and left ventricular dP/dt comparable As shown from the data above and elsewhere, the to those obtained when the compounds of the invention O compounds of the inventions have a therapeutic index were given intravenously. The effects were observed significantly broader than that of digitalis. Digitalis has after approximately thirty minutes. Like results are a therapeutic dose approximately 50 to 60% of the toxic obtained when the compounds of the invention are dose. administered in the dosage necessary for the desired Variations in onset of action or speed of action and effects to dogs. 15 duration is likely to be observed between the various From this and other data, there is indirect evidence compounds of the invention. that berberine typically is quite effectively utilized dur The invention also as discussed above contemplates ing the first 24 hours and yet that its effect is not tran the use of the compounds of the invention in veterinary sient but extends over several hours. This utilization of applications. What has been disclosed herein above and berberine and other compounds of the invention has 20 hereinafter is in accordance with the invention applica favorable implications for oral administration. It is a ble to the veterinary field, e.g. the treatment of animals akin to a retard effect. It means that the administration as well as to humans. To amplify more specifically to of the compounds of the invention need not be as fre those knowledgable in the veterinary arts it will be quent, under appropriate circumstances. noted that cardiac are also useful in the ther The protoberine alkaloids of the invention are admin 25 apy of animals for congestive heart failure. See Veteri istered in the manner most appropriate under the cir nary Pharmaceutical and Biologicals 1980/1981, Horwal cumstances. Administration may be oral, subcutaneous Publishing Company for instance pages 16 16/120, 122, or intramuscular injection. The compounds may be 176, 177, (“VPCB'), which is incorporated herein by administered by intravenous route, parenterally. It has reference and Veterinary Pharmacology and Therapeu been noted that berberine is soluble (among others) in 30 tics, Jones, et al., Iowa State University Press., 4th Ed. sugar solutions so that it may be often well-administered 1977, pp. 519–527 ("Jones et al"). by intravenous route. The compounds of the invention therefore are valu Preferred is oral administration to patient in need of able in animal species treatment, e.g. equine, bovine, cardiac therapy whether it is in the non-failing or failing pigs and swine, feline, canine and aviano as well as heart. 35 others to replace, or to be used in conjunction with (as The dose in which the compounds of the invention described in accordance with the invention), cardiac are to be administered depends of course as discussed glycosides commonly used in veterinary therapy, like above on the circumstances of the patient, in particular, digitoxin, gitoxin, gitalin, ouabain, and others. whether or not it is a situation where cardiac arrest or The dosages in which they are used are described failing heart is occurring or whether it is in a non-failing 40 elsewhere herein. For other details, one skilled in the art heart situation for therapeutic purposes. In either situa is referred to Jones et al. which is incorporated herein tion, the dose is that minimum dose which shows a by reference, especially chapter 24, Cardiac Drugs. beneficial effect. This may be observed by the measure The compounds of the invention are also useful as ment of the symptoms which are clinically significant. arryhytmic drugs, as described herein above, to replace Thereafter the dose may be such as to sustain the 45 or supplement those like quinidine, procainamide or beneficial effect on an hourly or daily basis such as propanolol, lidocaine, and diphenylhydantoin, conven follows. Under appropriate circumstances where sus tionally used in veterinary sciences. For other drugs taining therapy is desired, as little as 0.001 mg/kg or which alleviate or suppress arryhythmia, see VPCB, even less may be administered. Generally amounts over page 527. The compounds of the invention are therefore 5 g/kg are not necessary since they may be uneconomi 50 useful to replace or in conjunction with such antiarr cal unless very fast action is necessary in emergency hythmia compounds like the phenothiazines such as situations. In those cases a theraputically sufficient promazine, chlorpromazine and promazine (acetyl amount should be administered. Where severe cardiac promazine) and others which are known to reduce ar decompensation is present and the patient has not yet rhythmias in animals. However these drugs, because of received any protoberberine alkaloid of the invention it 55 their narrow therapeutic index or low effectiveness or may be given immediately followed by treatment at the objectionable side effects, have not been adequately appropriate rate. Optimal benefit dosage is maintained satisfactory. and the maintainence dose can be adjusted correspond Cardio-stimulatory and vasoactive drugs are used in ingly, that is usually from 0.01 to 0.2 mg/kg per day. the treatment of circulatory shock in animals. Drugs When an adequate level of improvement is observed the 60 commonly used like sympathetic agonists (norepineph dosage may be reduced. rine, epinephrine or isoproterenol) levarterenol, ste When it is desired by the physician that beneficial roids and others described in Booth et al.) have short results be obtained in minutes rather than in hours, comings. The compounds of the invention provide intravenous medication is indicated. The compound of valuable new therapeutic agents in that respect. A par the invention may be dissolved in sterile water or some 65 ticular malignant form of circulatory shock observed in other sterile medium such as 5 percent dextrose in water horses afflicted with the acute colitis (colitis X) syn or saline. The administration may be all at once or alter drome lends itself to treatment with the compound of natively infused gradually over the period of an hour or the invention particularly well. 5,153,178 53 54 All of the uses, including controlling various forms of The above illustrations are not intended to limit the shock like hypovolemic or vasculogenic shock de invention thereto. Variations and changes of the param scribed above, are of course equally applicable for the eters, the specific compounds of the invention used and various compounds of the invention or the various ani other variables will become evident to those skilled in mal species. the art without departing from the spirit of the inven Animal health care and the use of drugs for such care, tion including its equivalents in all respects and terms maintenance and therapeutic purposes have and are including methods, compounds, compositions etc, continuing to assume greater importance in the country. which are all intended to be encompassed herein. The scientific literature in veterinary sciences attests to I claim: the importance of animal care with drugs. The com O 1. A therapeutic positive inotropic composition for pounds of the invention are particularly useful as the increasing contractility of the heart as shown by a posi user describes because of their wide spectrum of prop tive inotropic effect which comprises a biologically erties but especially in edible animals because of wide acceptable carrier and in an amount effective to cause a therapeutic range and low toxicity. positive inotropic effect, a cardiac glycoside selected 15 from the group consisting of ouabain, digoxin, digi The treatment of these disorders in the veterinary toxin, desianoside and digitalis and a protoberberine field on animals species is especially important when the alkaloid selected from the group consisting of berber animal being treated is of great value as is the case with ine, discretine, xylopinine, berberrubine, tetrahydropal a thoroughbred horse or a valuable show animal. matine and stepharotine and the pharmaceutically ac The actual determination of the numerical therapeu 20 ceptable salts thereof, the composition being effective tic e.g., cardiotonic dosage definitive or advisable for a to cause such a positive inotropic effect. particular compound of the invention is readily ob 2. The therapeutic composition of claim 1, wherein tained according to the above-described standard test the protoberberine alkaloid is berberine. procedures by technicians versed in pharmacological 3. The therapeutic composition of claim 2, wherein test procedures, without any need for any extensive 25 the cardiac glycoside is ouabain. experimentation. The percentages of active component 4. The therapeutic composition of claim 2, wherein in the composition of the invention and method for the cardiac glycoside is digoxin. increasing cardiac contractility and for the other benefi 5. The therapeutic composition of claim 2, wherein cial effects obtained by the compounds of the invention, the cardiac glycoside is digitoxin. may be varied so that a suitable dosage is obtained. The 30 6. The therapeutic composition of claim 2, wherein dosage administered to a particular patient is variable, the cardiac glycoside is deslanoside. depending upon principally on the symptom or disease 7. The therapeutic composition of claim 2, wherein sought to be alleviated, the clinician's judgement, using the cardiac glycoside is digitalis. as criteria: the route of administration, the duration of 8. A therapeutic method of increasing contractility of treatment, the size and condition of the patient, the 35 the mammalian heart as shown by a positive inotropic potency of the active component and the patient's re effect which comprises administering to a mammal in sponse thereto. The dosage administered depends, of need thereof in an amount effective to cause a positive course, also on whether or not the patient is suffering of inotropic effect, a composition which comprises a bio congestive heart failure or acute heart failure or if the logically acceptable carrier and a protoberberine alka compound is given to interrupt or to prevent a certain 40 loid selected from the group consisting of discretine, type of arrhythmia. The higher dosages are of course xylopinine and stepharotine and the pharmaceutically preferred to interrupt or cause the cessation of the ar acceptable salts thereof, in conjunction with a cardiac rhythmias. An effective dosage amount of active com glycoside selected from the group consisting of oua ponent can thus be determined by the clinician, consid bain, digoxin, digitoxin, deslanoside and digitalis, caus ering all traditional criteria and using of course, his best 45 ing a positive inotropic effect over a wider therapeutic judgement on a patient's behalf. It will depend also index than by administration of the cardiac glycoside what species is being treated humans or animal, and alone. what species of animal is being treated. 9. The therapeutic method of claim 8, wherein the It is important to note as is apparent from the above protoberberine alkaloid is administered intravenously. 50 10. The therapeutic method of claim 8, wherein the that the properties in the compounds of the invention cardiac glycoside is ouabain. are separable and not connected, i.e., there are com 11. The therapeutic method of claim 8, wherein the pounds of the invention which possess one of the thera cardiac glycoside is digoxin. peutic or prophylactic properties disclosed and not the 12. The therapeutic method of claim 8, wherein the other properties, whereas in other compounds two or 55 cardiac glycoside is digitoxin. more properties will be present concurrently, though 13. The therapeutic method of claim 8, wherein the (as discussed and disclosed) not necessarily to the same cardiac glycoside is deslanoside. degree or intensity. In short, the properties are not 14. The therapeutic method of claim 8, wherein the always associated and indeed should be considered cardiac glycoside is digitalis. disassociated from each other. 15. The therapeutic method of 8, wherein the cardiac It is also within the spirit of the invention that the glycoside and the protoberberine alkaloid are adminis compounds of the invention be used together or not, in tered periodically. conjunction (as defined above) with each other or not. 16. The therapeutic method of claim 8, wherein the As is evident from the disclosure, the invention de protoberberine alkaloid is administered in a dosage in scribes and contemplates, of course, both the prophy 65 the range of about 0.001 to about 0.2 mg/kg per day. lactic and the therapeutic use of the compounds of the 17. The therapeutic method of claim 8, further com invention, illustratively, with respect to the antiarrhyth prising discontinuing the administration. mic properties. k s k