Cardiotropic formulation

TECHNICAL FIELD
The present invention relates to cardiology, and more particularly it relates to the preparation of cardiotropic medicaments having a potent effect in severe and refractory conditions of cardiac insufficiency.
BACKGROUND ART
Known in the are is a number of cardiotropic medicaments, in particular a pharmaceutical composition, Ildamen-novodigal (patent No. 3332 Arzneim . U.el,Forsch (Drug Res.),25,Nr.3 (1975), F. Stroman und R. Hempel. Kombination yon Oxyfedrin mit 3-acetildigoxin), which is referenced here as the prior art to a cardiotropic formulation of the invention known under trade name Refrakterin.
The Ildamen-novodigal formulation including 0.2 mg of beta-acetyldigoxin and 18 mg of oxyfedrin is in common use for treatment of a cardiac insufficiency including conditions associated with coronary circulatory failure. Depending on the severity of patient's condition a daily medication of the combination may be increased up to 0.6 mg of beta-acetyldigoxin and 36 mg of oxyfedrin, and the treatment period may be longed for 1 month. The action of the combination of beta-acetyldigoxin and oxyfedrin is based on a positive inotropic synergism. Such combination of beta-acetyldigoxin and oxyfedrin provides a considerable decrease in toxicity of the former, prevents the occurence of bradycardia and arrhythmia thereby increasing the patient's drug tolerance. However, despite wider range over curable and toxic doses of beta-acetyldigoxin combined with oxyfedrin, the gap between them is still small.
The usage of such combination may cause of intoxication, especially in cases of cardiac hypersensivity to glycosides, in patients with myocardial inflammations, particularly in patients with influenza-related or acute isolated myocarditis, and alcohol-drinker's cardiomyopathy.
The usage of beta-acetyldioxin in combination with oxyfedrin at a considerably lower dose than that of the ildamen-novodigal formulation in cardiomyocyte energy-deficient conditions, such as a well-defined heart insufficiency arising from a toxic-allergic myocarditis, may cause non-responsiveness of the myocardial contractile protein system (Tabl.2) which will aggravate the energy-deficient state of cardiac muscle cell (Tab.3).
If such compounds are used individually, the enhancement of myodynamia of contractile proteins are accompanied with a considerable rise in ATP, and yet CP (creative phosphate) decreases in content. (Tabl.3). As a consequence of the deterioration of a metabolic state, there occurs the activation of myocardial and cardiomyocyte degeneration (N. N. Kipshinidze et al., 1983, Materialy nauchnoi sessii NIl klinicheskoi i eksperimentalnoi terapii Minzdrava Gruzii). This phenomenon may be considered to be the basis of cardiac refractory reaction to the conventional methods of treatment.
The object of the present invention is to provide a cardiotropic formulation, which medicative effect together with a low toxicity, will allow one to restore myocardial contractile force and increase the compensation abilities in a well-defined and severe heart failure including cardiac insufficiency with refractory reaction to the conventional methods of treatment and to cut the time for attaining positive results.
DISCLOSURE OF THE INVENTION
The object of the invention is attained by providing a formulation, hereinafter refered to as Refrakterin, distinguished from ildamen-novodigal in that it further includes nicotinamide adenine dinucleotide (NAD), cytochrome C and inosine (riboxin), with oxyfedrin and beta-acetyldigoxin presenting in smaller amounts than that of ildamen-novodigal, at the following weight ratio of ingredients:
______________________________________cardiac glycoside 0.05-0.2B-adrenoceptor stimulant 0.3-3.5nicotinamide adenine dinucleotide 0.5-5cytochrome C 5-15inosine (riboxin) 20-250______________________________________
From the results of our fundamentals follows that severe cardiac insufficiency in various heart diseases at the stage of too far progression of it and in conditions with refractory reaction occuring at myocardial inflammations, alcohol-drinker's cardiomyopathy (and probably at other cardiac diseases) substantially is associated with a simultaneous disturbance of three subcellular systems which are responsible for cardiomyocyte contraction-relaxation including the contractile proteins system, transmembrame Ca transport (coupling the excitation--contraction, regulating the power of contraction and evoking the relaxation of cardiac muscle) and the metabolizable energy system (ensuring an easily available energy source for utilization of it by all cellular systems) (Tabl.1).
Thus, to overcome the problems associated with a refractory reaction and provide a successful recovery of the power failure of the heart a homeostatic state for normal function of the three systems as mentioned above is required, since the activation of one (or two as it is the case of ildamen-novodigal) of such three systems does not cause the normalization of cardiac contractile function, and there occur refractory reaction to the preparation used. In addition, cardiomyocyte structural and functional abnormalities may occur because of the increased (non-compensable) ATP and the aggravation of energy depletion. It is precisely the picture as indicated above which is under observation in myocardial inflammations under ildamen-novodigal treatment, using even much less amounts than that of the original formulation (Tabl. 3).
Thus, the problem directed to overcoming the refractory reaction and attaining a fast, successful reparation of functional activities of the three cardiomyocyte systems is to restore a steady, equilibrium state of functioning of all the above cardiomyocyte systems which are responsible for myocardial contraction-relaxation. In this case, the adequate repair of metabolic activity (in sense of energetics) is of crucial importance, since the activation of function, both the contractile protein system and ionic pumps, give rise to a considerable increase in consumption of energy by cells. Most likely, it is the energy-deficient state which is the basis of the refractory reaction of severe heart failures to the conventional methods of treatment, wherein would take place a one-sided activation of the contractile protein system without the required stimulation of cellular energetic system.
The energy-deficient conditions in any heart disease accompanied with the development of myocardial hypoxia (myocardial infarction, inflammatory and alcoholic cardiac failures, terminal heart insufficiency and the like) are associated not only with the decrease in rate of cellular oxygen transport, and also with considerable losses of cellular cytochrome C, NAD and adenylate nucleotides.
The complex formulation including NAD, cytochrome C, inosine, a cardiac glycoside (.beta.-acetyldigoxin or .beta.-methyldigoxin, or strophanthin K, a contractile protein activator) and a .beta.-adrenoceptor stimulant (oxifedrin or nonachlazin, a cardiotropic sympathomimetic) has overcome the problems associated with a refractory reaction in heart insufficiency and a fast normalization of myocardial structural and functional status.
The usage of the formulation of the invention in toxic-allergic myocarditis with a well-defined power failure of the heart provides the complete restoration (Tabl.4) of normal intracardiac or peripheral hemodynamics even under cardiac hypertension (Tabl 5) This effect is accomplished by a steady equilibrium of functioning of the three cardiomyocyte systems which are responsible for contraction-relaxation reaction. In this case, the following picture develops:
The exogeneous NAD compensates the deficiency of cytosolic and mitochondrial NAD pools. This causes the normalization of NAD-dependent glycolate dehydrogenase activities, glycolytic oxidoreduction and, in turn, ATP production through the glycolytic pathway. In addition, it restores the rate of proton translocation from cytosole to mitochondria (via the malate-aspartate shuttle) for further utilization of it in the electron transport chain.
The replacement of mitochondrial NAD deficiency activates the Krebs cycle NAD-dependent dehydrogenases and regenerates proton translocation from the tricarboxylic acid cycle to the electron transport chain.
In turn, mitochondrial exogenous cytochrome C is incorporated into (on is own place) the electron transport chain. This provides the regeneration (by volume) of electron transport to oxygen and the normal mitochondrial ATP-producing capacity in the oxidative phosphorylation associated with passing the electrons through their transport chain.
The picture described may be fully developed with the proviso that the replacements of NAD and cytochrome C take place at a time.
The replacement of NAD depletion only (by capacity) does not provide the complete restoration of energetic potential of the system since the carrying capacity of the electron transport chain is still low, and the chain is unable to cope with proton flows coming from the Krebs cycle and cytosolic component. By virture of the action of exogenous NAD the level of CP does not increase to the normal (Tabl.6), whereas there is the tendency for a increase in ATP and a decrease in ATP/ADP ratio (Tabl.6,7).
The replacement of cytochrome C losses solely (which is in common use today) also does not provide the complete restoration of the ATP-producing capacity of the energy transfer system (Tabl.6), as the replenishment of cytochrome C losses will not cause the normalization of [NAD]/[NAD.H] ratio (Tabl.8 ) which regulates dehydrogenase activity. In this case, the ADP level goes low, and [ATP]/[ADP] ratio rises above-normal.
The replacement of losses, either NAD or cytochrome C (Tabl.8), does not cause the normalization of the total adenyl nucleotides (Tabl.6, 7). Their total content is still low.
The replacement of the total adenyl nucleotides may be attained by including inosine in the formulation, which promotes de novo adenyl nucleotide synthesis (Tabl. 9).
In addition, inosine enhances considerably the coronary circulation (oxygen delivery to cardiac muscle), and assists in liberating oxygen from oxyhemoglobin of peripheral blood. This, without any doubt, contributes significantly to the restoration of cardiomyocyte ATP-producing capacity that is a matter of great concern at the increasing heart stresses.
Thus, the introduction of exogenous NADs, cytochrome C and inosine into the composition for the replacement of endogenous NAD and cytochrome C losses, and apparently of inosine, makes it possible to restore a homeostatic state of the energy transfer system (normal levels of ATP, ADP, AMP, CP, NAD and NAD.H), its functional capacity,[ATP]/[ADP] and ##EQU1## as along with the absolute content of ATP, ADP, AMP, CP, NAD and NAD.H their ratios are of great importance in regulation of energetic state of the system, that is the case in hand is not a simple summation over the positive effects (each of the agents used activates ATP production to one or another degree), and it means a fine adjustment of such system. It should be noted that normal ATP and ADT are the matter of great concern for the desired structural status of contractile proteins.
In the treatment of cardiac insufficiency after restoration of normal function of the energy exchange system the next problem is to return the contraction ability of the contractile protein system (Tabl.10). This problem is solved by including a cardiac glycoside (Tabl.10) in the formulation for its direct effects on contractile proteins. The basic preparation, Refrakterin, includes .beta.-acetyldigoxin as the cardiac glycoside.
However, in cases of heart hypersensitivity to cardiac glycosides (for example in allergic heart failures, acute isolated myocarditis) a-methyldigoxin (Refrakterin, BM) would be appropriate for use, since the specific activity of .beta.-methyldigoxin is high as that of .beta.-acetyldigoxin by an order of two, with their optimum concentrations being of 10.sup.-8 and 10.sup.-5 M, correspondingly (FIG. 1, Tabl.11).
In case of acute heart insufficiency associated with energy- deficient condition, strophanthin K would be suitable for use (Refrakterin, SK) as the alternative of .beta.-acetyldigoxin because, if chosen at the optimum amount, it acts on energy transformation, both quantitatively (like .beta.-acetyldigoxin) and qualitatively (Tabl.11,14), that is it increases the contraction efficiency. In addition, the range of effective doses for strophanthin K goes far beyond that of .beta.-acetyldigoxin plus .beta.-methyldigoxin. This allows such glycoside to be used effectively in the decreased amounts than that of Refrakterin (FIG. 1).
As .beta.-methyldigoxin chosen at the optimum concentration has effects, like strophanthin K, on contractile protein-transformed energy, both quantitatively and qualitatively, Refrakterin BM is suitable in acute or chronic heart insufficiency which is followed by oxygen- and energy deficiencies.
Finally, the next problem is to provide the normal function of the Ca transport system (Tabl.13). As cardiac glycosides belonging to the foxglove class have no effects directly on the contractile protein system (strophanthin K acts on both systems) the basic formulation (Refrakterin) includes a cardiotropic .beta.-adrenergic receptor stimulant, oxyfedrin, to normalize the function of Ca transport system. Oxyfedrin promotes the intake and release of Ca from sarcoplasmatic reticulum and brings the rate of such processes to the normal values (Tabl.14). In addition, oxyfedrin has a direct effect on the contractile protein system and accelerates glycolysis of phosphorylase due to transformation of it to its active form.
Oxyfedrin has these effects through the activation of adenylate cyclase (Tabl.14a), the inhibition of phosphodiesterase activity (Tabl.14b) and the increase in content of myocardial cAMP.
Moreover, oxyfedrin has effects on the energy-producing system by increasing the ATP level, whereas there occur a considerable decrease in CP (Tabl.3).
Alternatively, oxyfedrin may be replaced with its analogue, nonachlazin (Refrakterin H), with the dose of the latter should be at least 5 times as high as that of the former (Tabl.14 c).
Thus, each ingredient of refrakterin assists in the increase in level of ATP (up to its normal), contraction ability of the contractile protein system and rate of the cardiomyocyte transmembrane Ca transport to a greater or lesser extent. However, the end result of Refrakterin is not the sum of its specific effects, and it appears to be the case of a complex regulation of functional activities of the three cardiomyocyte systems responsible for contraction-relaxation reaction to provide the complete normalization of cardiomyocyte contractile function (even under hypertension) (Tabl.5).
The formulation of the invention is prepared as follows:
NAD (0.5 mg), cytochrome C (10 mg), inosine (80 mg), oxyfedrin (0.3 mg) and beta-acetyldigoxin (0.075) (based on kg of animal body weight) are mixed together and the resultant mixture before its direct usage, is dissolved under agitating in 6 ml of a commercially available normal saline which was heated to a temperature of 38.degree.-40.degree. C. The preparation was stored at 4.degree. C. prior to dilution of it.
The present invention will become more fully apparent from the following Examples of testing the claimed preparation, Refrakterin.

EXAMPLE 1
Medicative effect of Refrakterin in a 3-day TAM
Toxic and allergic myocarditis was induced in rabbits according to the methods of S. V. Andreev and M. V. Sokolov ("Sanogenesis, Meditsina, 1968) by injecting twice intravenously 2 ml of horse blood serum every forth day. Seven days after completion of the last injection 0.5 ml of staphyloccocia toxin were infused.
A medical effect of Refrakterin was recorded after administration of it within a 2-day period at a daily dose of 90.5 mg/g, beginning on the second day from the disease progression.
With a 3-day TAM the administration of Refrakterin within a 2-day period returns the normal status of a peripherial and intracardiac hemodynamics including the normalization of cardiac systolic and diastolic functions (Tabl.4) and their coordination, and the liquidation of blood congestion in lungs and liver. As this takes place, cardiomiocyte functional status will be restored so firmly that heart is capable to cope even with overloads (Tabl.5)
Example II
Medicative effect of Refrakterin in a 10-day TAM
TAM was induced according to-the same procedure as disclosed for a 3-day TAM. A pharmaceutical activity of Refrakterin was recorded after administration of the preparation at a dosage of 90.875 mg/kg within a 5-day period. In these tests for an additional effect on the inflammation process the animals were treated by injection of Refrakterin to them followed by a simultaneous oral administration of acetylsalicylic acid in a dosage of 0.25 mg/kg body weight, beginning on the fifth day from the disease progression. In special experiments aspirin in such dosage has been discovered to have no marked effects on functional activity of any of the three cardiomyocyte systems which are responsible for the contraction-relaxation reaction.
The administration of Refrakterin every day within a five- day treatment period leads to the elimination of peripheral congestion (interstitial fluid in liver, lungs and heart itself decreases to the normal in content), the normalization of cardiac systolic and diastolic functions and their coordination, ensuring normal cardiac work not only at rest (Tabl.4), but under cardiac hypertension as well (Tabl.5)
The Refrakterin treatment of a 10-day TAM results in the coordinated repair of functional activities of the contractile proteins (Tabl.10) and cardiomyocyte transmembrane Ca transport (Tabl.13) with adequate promotion the rate of ATP production as an easily available energy source and energy accumulation as CP (Tabl.15), thereby causing a sharp improvement in cardiomyocyte ultrastructure. In this case it is paticularly remarkable that the restoration of the rate of contraction process and Ca transport with the metabolisable energy support takes place also under cardiac hypertension. Thus, the Refrakterin treatment provides a steady, equilibrium repair of functioning the three cardiomyocytes systems which are responsible for the contraction-relaxation reaction.
Comparative study of effects of Refrakterin and
.beta.-acetyldigoxin-oxyferdin combination
The administration of beta-acetydigoxin in combination with oxyferdin even in considerably decreased amounts than that of ildamen-novodigal (0.15 mg/kg of beta-acetydigoxin+0.6 mg/kg of oxyferdin) for 2 days in both a 3-day- and 10-day TAM causes a sharp increase in myocardial tension which generates the contractile protein system (Tabl.16), with the level of adenyl nucleotides being the same as seen in Control. At the same time the CP content remains at Control level. The administration of such combination in much less amounts (0.05 mg/kg of beta-acetydigoxin +0.2 mg/kg oxyferdin) in allergic myocarditis and adrenaline-caffeine-related heart failure evokes the decrease not only in level of CP, but in ATP as well, due to rising the tension generated by the contractile proteins.
Thus, even a short-term administration of ildamen-novodigal in myocardial inflammations leads to the aggravation of cardiomyocyte energy-deficient state that may cause, eventually, the deterioration of myocardial structure due to the progression of parenchymatous degradation.
Industrial applicability
A cardiotropic formulation of the invention may be used in treatment of severe and refractory conditions of cardiac insufficiency for recovery of the power force of the heart and improvement of myocardial compensation abilities as well as the cutting of time for attaining positive pharmacologic effect.
TABLE 1__________________________________________________________________________State in Systems of Contractile Proteins, Energy Supplyand Ca.sup.2+ transport in a 10-day TAMMechanical parameters of contractionVentricle Parameter Normal (n = 16) TAM (n = 13)__________________________________________________________________________Left Tension, mH/mm.sup.2 2.48 .+-. 0.18 1.57 .+-. 0.13*** Work, nJ/mm.sup.3 21.4 .+-. 3.0 9.0 .+-. 2.0**Right Tension, mH/mm.sup.2 2.44 .+-. 0.20 1.55 .+-. 0.15** Work, nJ/mm.sup.3 19.9 .+-. 3.0 8.3 .+-. 2.0*__________________________________________________________________________Levels of Adenyl nucleotides and creatine phosphate (CP) EnergeticVentricle Group ATP ADP ATP + ADP + AMP ATP/ADP potential CP__________________________________________________________________________Left Normal 2.50 .+-. 0.13** 2.02 .+-. 0.12* 5.59 .+-. 0.18*** 1.35 .+-. 0.12 0.65 .+-. 0.02 3.40 .+-. 0.18*** TAM 1.30 .+-. 0.13 1.49 .+-. 0.15 3.56 .+-. 0.21 1.05 .+-. 0.12 0.57 .+-. 0.02 1.52 .+-. 0.07Right Normal 2.40 .+-. 0.13*** 1.84 .+-. 0.10 5.25 .+-. 0.22*** 1.43 .+-. 0.13 0.65 .+-. 0.01 3.26 .+-. 0.11 TAM 1.67 .+-. 0.12 1.51 .+-. 0.18 3.77 .+-. 0.34 1.27 .+-. 0.10 0.60 .+-. 0.19 1.45 .+-. 0.10__________________________________________________________________________.sup.45 Ca.sup.2+ Binding, Intake and Release from CPF, Mitochondrial CaUptake andCa Content in CPF, mitochodria and cardiac muscle in TAM (nmole .sup.45Ca.sup.+2 per g protein)CPFCa Releasebinding, Ca uptake, min. Ca CaTest 1 min. 3 5 10 15 25 35 45 uptake released__________________________________________________________________________Normal 43.5 .+-. 834 .+-. 945 .+-. 1171 .+-. 1297 .+-. 1356 .+-. 1426 .+-. 1414 .+-. 1345 .+-. 276 .+-.(n = 15) 199*** 50*** 37*** 43*** 27*** 22*** 23*** 19*** 29*** 21**10-day 29.4 .+-. 563 .+-. 608 .+-. 698 .+-. 780 .+-. 829 .+-. 905 .+-. 908 .+-. 938 .+-. 155 .+-.TAM 0.9 25 128 38 39 15 36 23 35 28(n = 7)__________________________________________________________________________ Cardiac CPF Mitochondria muscleMytochondria contentUptake, min. Ca per g Ca per gTest 5 15 30 mcM protein tissue__________________________________________________________________________Normal 116 .+-. 9.0* 188 .+-. 5.7** 210 .+-. 7.0 49.0 .+-. 4.0*** 50.0 .+-. 7.5 2.5 .+-. 0.5*(n = 15)10-day 159 .+-. 19 258 .+-. 23 240 .+-. 8 28.2 .+-. 1.2 33.0 .+-. 3.5 1.12 .+-. 0.2TAM(n = 7)__________________________________________________________________________
TABLE 2______________________________________Effects of oxyfedrin (0.6 mg/kg), beta-acetyldigoxin (0.15mg/kg) and combinations thereof on left/right ventricle tensionin a 10-day TAM (mH/mm.sup.2)Group, VetricleSubgroup Left Right______________________________________Normal 1 2.1 .+-. 0.11 1.98 .+-. 0.14.sup.x(n = 27)10-dayTAMControl (n = 9) 2 0.95 .+-. 0.15 0.88 .+-. 0.14**TREATMENTOxyfedrin 3 4.2 .+-. 0.49 3.90 .+-. 0.42(n = 5)b-Acetyldigoxin 4 4.3 .+-. 0.48 3.8 .+-. 0.19(n = 3)Oxyfedrin + 5 4.93 .+-. 0.4 4.7 .+-. 0.37b-Acetyldigoxin(n = 3)Least significant difference 1-2 <0.001 <0.001between groups (P) 1-3 <0.001 <0.001 1-4 <0.001 <0.001 1-5 <0.001 <0.001 2-3 <0.001 <0.001 2-4 <0.001 <0.001 2-5 <0.001 <0.001 3-4 -- -- 3-5 -- -- 4-5 -- --______________________________________ .sup.x n = 24 **n = 8
TABLE 3______________________________________Levels of adenyl nucleotides and creatine phosphate inallergic myocarditis under effect oxyfedrin (0.6 mg/kg)b-acetyldigoxin (0.15 mg/kg) and combination thereof(.mu.M per g wet tissue)Group ATP ADP AMP CP______________________________________Normal 2.38 .+-. 0.14 1.92 .+-. 0.46 0.92 .+-. 0.11 1.08 .+-. 0.08.sup.+n = 8Control 1.67 .+-. 0.15* 1.80 .+-. 0.14 0.84 .+-. 0.37 0.59 .+-. 0.09*10-dayTAMn = 10Oxy- 2.28 .+-. 0.39 2.39 .+-. 0.27 1.73 .+-. 0.11 0.29 .+-. 0.01*.sup.xfedrin*n = 7b-Acetyl- 2.28 .+-. 0.15.sup.x 2.95 .+-. 0.56 1.24 .+-. 0.31 0.41 .+-. 0.11*digoxinn = 5Oxy- 1.63 .+-. 0.37 2.30 .+-. 0.61 1.15 .+-. 0.34 0.22 .+-. 0.04*.sup.xfedrin +b-acetyl-digoxinn = 4______________________________________ Note: .sup.+ n = 12, comparison to: normal*, Control.sup.x *oxyfedrin 1propanone-[3(2-hydroxy-1-methyl-2-phenyl-ethyl)amino1-(3-methoxyphenyl)
TABLE 4__________________________________________________________________________Intracardiac hemodynamics in TAM after Refrakterin treatment CEI by dp/dt dp/dt Veragood dp/dt CWI, Heart rate, LVP, max EDP, min CI, max mmHg .times. b/sGroup b/min. mmHg mmHg/s mmHg/s mmHg/s s.sup.-1 mmHg/s .times. g g__________________________________________________________________________Normal 228 .+-. 15 71 .+-. 9 1240 .+-. 306 7 .+-. 2 1313 .+-. 281 21.1 .+-. 5.0 547 .+-. 93 6679 .+-. 1365(n = 5)TAMCONTROL3-day 240 .+-. 29 81 .+-. 9 1006 .+-. 122 15 .+-. 4 720 .+-. 95* 15.8 .+-. 3.9 439 .+-. 65 4685 .+-. 203*n = 310-day 202 .+-. 14 49 .+-. 8*.sup.x 1017 .+-. 174 27 .+-. 8*.sup.x 533 .+-. 105* 15.4 .+-. 1.8 409 .+-. 58* 3828 .+-. 989.sup.x+n = 4Refrakterintreatment2 day 218 .+-. 15 84 .+-. 10.sup.x+ 1300 .+-. 210 8 .+-. 2.sup.x * 1180 .+-. 115 20.8 .+-. 1.6 583 .+-. 45.sup.x+ 6038 .+-. 979.sup.x+n = 45 day 244 .+-. 8 69 .+-. 10.sup.x+ 1280 .+-. 366 5 .+-. 2.sup.x+ 1033 .+-. 28.sup.x * 21.5 .+-. 5.4 637 .+-. 186.sup.+ 7378 .+-. 1020.sup.x+n = 7__________________________________________________________________________ Note: Comparison to: normal*; 3day TAM.sup.x ; 10day TAM.sup. *Treatment time: 2 day in 3day TAM, 5 day in 10day TAM
TABLE 5__________________________________________________________________________Intracardiac hemodynamics in TAM after Refrakterin treatmentunder hypertension CEI dp/dt dp/dt, Veragood by max Heart rate, EDP, LVP, max CI, dp/dt CWI, mmHg/sGroup b/min. mmHg/ss mmHg mmHg/s s.sup.-1 mmHg s .times. g mmHg .times. b/s max__________________________________________________________________________Normal 192 .+-. 15 12 .+-. 26 112 .+-. 23 1264 .+-. 504 18 .+-. 3 558 .+-. 85 7010 .+-. 966 953 .+-. 193n = 5TAMCONTROL3-day 210 .+-. 14 27 .+-. 8* 133 .+-. 14 1650 .+-. 27 16 .+-. 4 702 .+-. 92 8277 .+-. 404 1289 .+-. 163*n = 310-day 218 .+-. 19 31 .+-. 2* 141 .+-. 20 1595 .+-. 10 13 .+-. 2* 858 .+-. 255 8333 .+-. 2621 767 .+-. 436*n = 4Refrakterintreatment2 day 200 .+-. 25 16 .+-. 4.sup.x+ 111 .+-. 13 1358 .+-. 110 15 .+-. 3 1360 .+-. 380 8010 .+-. 1050 980 .+-. 85.sup.xn = 45 day 193 .+-. 23 18 .+-. 4.sup.+ 109 .+-. 15.sup.+ 1452 .+-. 269 14 .+-. 2 1766 .+-. 1103 8230 .+-. 1820 1004 .+-. 177.sup.+n = 7__________________________________________________________________________ Note: Comparison to: normal*; 3day TAM.sup.x ; 10day TAM.sup. *Treatment time: 2 day in 3day TAM, 5 day in 10day TAM
TABLE 6__________________________________________________________________________Levels of adenyl nucleotides and CP (mcM/g wet tissue) in myocardial leftventriclein rabbits with a 10-day allergic myocarditis after treatment with NADand cytochrome C ATP + EnergyGroup ATP ADP AMP ADP + AMP ATP/ADP potential CP__________________________________________________________________________Norm (n = 25) 2.5 .+-. 2.02 .+-. 1.07 .+-. 0.07 5.52 .+-. 0.18 1.35 .+-. 0.07 0.65 .+-. 0.02 3.40 .+-. 0.18 0.13 0.12MYOCARDITISControl 1.3 .+-. 1.49 .+-. 0.78 .+-. 0.09*** 3.56 .+-. 0.21*** 1.05 .+-. 0.12* 0.57 .+-. 0.01*** 1.52 .+-. 0.07***(n = 20) 0.13*** 0.16**TREATMENTNAD 2.43 .+-. 2.68 .+-. 0.58 .+-. 0.14** 5.57 .+-. 0.51.sup.++ 1.14 .+-. 0.22 0.65 .+-. 0.04.sup.+ 1.50 .+-. 0.12***0.05 mg/kg 0.22.sup.+++ 0.33**.sup.++(n = 12)Cytochrome C 2.19 .+-. 1.42 .+-. 0.62 .+-. 0.09*** 4.24 .+-. 0.16***.sup.+ 1.56 .+-. 0.09.sup.++ 0.69 .+-. 0.02.sup.+++ 2.07 .+-. 0.22***.sup.+x 55 mg/kg 0.09.sup.+++ 0.05***.sup.xx(n = 9)__________________________________________________________________________ Note: the same symbols as given in Table 2 are used
TABLE 7__________________________________________________________________________Effects of medication of NAD (0.05 mg/kg) and cytochrome C (10/kg) onmyocardial right-ventricularadenyl nucleotides in rabbits with a 3-day allergic myocarditis (mcM/gwet tissue) Statictical Total EnergeticGroup, Subgroup values ATP ADP AMP ATP/ADP Nucleotides Potential__________________________________________________________________________Normal (n = 25) 1 X .+-. m.sub.x 2.40 .+-. 0.13 1.84 .+-. 0.10 0.99 .+-. 0.07 1.43 .+-. 0.13 5.25 .+-. 0.22 0.65 .+-. 0.01MYOCARDITISMyocarditis 2 X .+-. m.sub.x 1.55 .+-. 0.10 1.45 .+-. 0.19 0.83 .+-. 0.13 1.27 .+-. 0.17 3.88 .+-. 0.33 0.60 .+-. 0.02(n = 11)TREATMENTNAD (n = 11) 3 X .+-. m.sub.x 2.06 .+-. 0.12 1.99 .+-. 0.13 0.60 .+-. 0.03 1.07 .+-. 0.11 4.73 .+-. 0.42 0.63 .+-. 0.02Cytochrome C 4 X .+-. m.sub.x 2.00 .+-. 0.28 1.09 .+-. 0.15 0.63 .+-. 0.17 2.14 .+-. 0.28 3.82 .+-. 0.42 0.66 .+-. 0.05(n = 8) P.sub.1-2 0.001 -- -- -- 0.001 0.05 P.sub.1-3 -- -- 0.001 -- -- -- P.sub.1-4 -- -- 0.05 -- -- -- P.sub.2-3 0.01 0.05 -- -- -- -- P.sub.2-4 -- -- -- 0.05 -- -- P.sub.3-4 -- 0.01 -- 0.01 -- --__________________________________________________________________________
TABLE 8__________________________________________________________________________Levels of NAD and NAD-H in myocardial left ventricle in a 10-day TAMafter treatment with NAD (0.5 mg/kg) and cytochrome C (5 mg/kg) (mcM/g ofwet tissue)Groups,Subgroups NAD NAD-H Total NAD-H/NAD__________________________________________________________________________NORMAL (n = 8) 0.68 .+-. 0.02 0.36 .+-. 0.04 1.08 .+-. 0.03 0.50 .+-. 0.036TAMControl 0.47 .+-. 0.05*** 0.41 .+-. 0.05 0.84 .+-. 0.03*** 0.86 .+-. 0.04***(n = 8)TREATMENTNAD 0.68 .+-. 0.04.sup.+++ 0.33 .+-. 0.015 1.03 .+-. 0.03.sup.+++ 0.46 .+-. 0.04.sup.+++0.5 mg/kgCytochome C 0.54 .+-. 0.04**.sup.x 0.48 .+-. 0.09 1.05 .+-. 0.07.sup.+ 0.90 .+-. 0.08.sup.xxx5 mg/kg(n = 6)__________________________________________________________________________ Comparison to: normal*; control.sup.+ ; NAD medication in combination wit cytochrome C.sup.x No symbol not statistically significant. One symbol P < 0.05; two symbols P < 0.01; three symbols P < 0.001
TABLE 9__________________________________________________________________________Levels of rabbit cardiac adenyl nucleotides in experimental 10-day TAMunder effect inosine Left ventricle Right ventricle mcM/g wet tissue Energetic AdT/ mcM/g wet tissue Energetic AdT/GROUP AdT ADP AMP Total potential ADP ATP ADP AMP Total potential ADP__________________________________________________________________________Normal (n = 1) 1 3.23 .+-. 1.49 .+-. 0.71 .+-. 5.36 .+-. 0.73 .+-. 2.23 .+-. 2.75 .+-. 1.47 .+-. 0.74 .+-. 4.89 .+-. 0.68 1.86 .+-. 0.09 0.09 0.13 0.12 0.02 0.18 0.26 0.17 0.03 0.43 0.02 0.18MYO-CARDITISControl 2 1.51 .+-. 1.27 .+-. 0.69 .+-. 3.48 .+-. 0.62 .+-. 1.26 .+-. 1.33 .+-. 1.24 .+-. 0.73 .+-. 3.29 .+-. 0.59 1.15 .+-.(n = 7) 0.09 0.13 0.08 0.20 0.02 0.13 0.12 0.16 0.05 0.26 0.01 0.09Inosinetherapy80 mg/kg 3 2.66 .+-. 1.50 .+-. 0.99 .+-. 5.14 .+-. 0.66 .+-. 1.82 .+-. 2.25 .+-. 1.74 .+-. 0.63 .+-. 4.82 .+-. 0.65 1.41 .+-.(n = 6) 0.13 0.10 0.05 0.17 0.01 0.18 0.43 0.21 0.08 0.29 0.01 0.21160 mg/kg 4 2.99 .+-. 2.36 .+-. 0.98 .+-. 6.20 .+-. 0.65 .+-. 1.25 .+-. 2.23 .+-. 1.85 .+-. 0.96 .+-. 0.96 .+-. 0.63 1.39 .+-. 0.11 0.15 0.16 0.31 0.0 0.12 0.20 0.25 0.06 0.23 0.02 0.30Least P.sub.1-2 <0.001 -- -- 0.001 0.001 0.001 0.001 -- -- -- 0.01 0.05significant P.sub.2-3 <0.001 -- 0.01 0.001 -- 0.05 0.001 -- -- -- 0.01 --difference P.sub.2-4 <0.001 0.001 -- 0.001 -- -- 0.01 -- 0.05 0.01 0.001 --between groups P.sub.1-3 <-- 0.001 -- 0.02 -- 0.02 -- -- -- -- -- --(P) P.sub.1-4 <0.01 -- -- -- -- -- -- -- -- -- -- -- P.sub.3-4 <-- 0.001 -- 0.02 -- 0.001 -- -- -- -- -- --__________________________________________________________________________
TABLE 10______________________________________Effect of Refrakterin on left-ventricular tension generated byPGVM in a 10-day TAM Statictical Tension,Group, subgroup index mH/mm.sup.2______________________________________Normal 1 M .+-. m.sub.x 2.39 .+-. 0.34TAMControl 2 M .+-. m.sub.x 1.37 .+-. 0.15Refrakterin 3 M .+-. m.sub.x 2.31 .+-. 0.26treatment P.sub.1-2 0.05 P.sub.1-3 -- P.sub.2-3 0.05______________________________________
TABLE 11______________________________________Effects of cardiac glycosides on contraction ability and energymetabolism of normal myocardial contractile proteins (.DELTA.H - .DELTA.Q)/Cardiac glycoside P .DELTA.G P/.DELTA.G .DELTA.H .DELTA.Q______________________________________b-Acetyldigoxin 136.5 155 90.2 95.6 10210.sup.-6 Mb-Methyldigoxin10.sup.-8 M 168.8 133.9 189.4 112.6 98.510.sup.-6 M 154.6 86.9 142.1 88.2 100Strophanthin10.sup.-6 M 168.8 150.5 131.7 117.6 110.210.sup.-8 M 149.8 83.0 142.1 90.3 95.6______________________________________ Note: given is percentil to Control (no glycosides) in the Table.
TABLE 12______________________________________Effects of cardiac glycosides on mechnical and thermodynamiccontraction parameters in TAMCardiac glycoside P .DELTA.H .DELTA.H - .DELTA.Q/.DELTA.H______________________________________Control (10-day TAM) 43.4 52.5 77.9b-Acetyldigoxin 10.sup.-6 M 75.6 63.0 76.2Strophanthin K 10.sup.-6 M 75.6 65.7 94.1______________________________________ Note: given is percentil to Control (normal) in the Table.
TABLE 13__________________________________________________________________________Effect of Refrakterin on contractile protein fiblil/mitochondrialCa.sup.2+ transportin treatment of rabbit TAM (mcmole Ca per g protein Ca binding Statis- to CPF Ca Mitochrondrial tical CPF CPF Ca.sup.2+ uptake release Ca.sup.2+ uptakeGroup index 1 min. 3 min. 15 min. 35 min. 45 min. load release 5 min. 15 min. 30__________________________________________________________________________ min.Normal 1 X .+-. m.sub.x 43.5 834 1298 1426 1414 1345 276 122 196 21010-day 1.9 50 37 23 19 29 21 11 8 8 15 15 15 15 15 15 15 15 15 15TAMControl 2 X .+-. m.sub.x 29.4 563 780 905 908 938 155 171 268 269 0.9 25 39 37 23 35 28 17 22 18 10 7 7 7 7 7 7 9 9 9Refrakterin 3 X .+-. m.sub.x 40.4 855 1473 1495 1449 1382 226 140 249 235treatment 2.1 99 142 149 152 167 18 18 41 21 5 6 6 5 6 5 5 5 5 5 P.sub.1-2 0.001 0.01 0.001 0.001 0.001 0.001 0.03 0.01 0.01 0.01 P.sub.1-3 -- -- -- -- -- -- -- -- -- -- P.sub.2-3 0.001 0.01 0.001 0.01 0.01 0.01 -- -- -- --__________________________________________________________________________
TABLE 14__________________________________________________________________________Myocardial CPF Ca.sup.2+ transport in rabbit TAM after oxyfedrintreatment (mcmole Ca per g protein) Ca.sup.2+ uptake Ca releaseGroup 3 min. 5 min. 15 min. 35 min. 45 min. 2 min.__________________________________________________________________________Normal M .+-. m 834 .+-. 50 945 .+-. 37 1297 .+-. 27 1426 .+-. 23 1414 .+-. 19 276 .+-. 21 (15) (15) (15) (15) (15) (15)10-dayTAMControl M .+-. m 563 .+-. 25 608 .+-. 128 780 .+-. 39 905 .+-. 36 908 .+-. 23 155 .+-. 28 (7) (7) (7) (7) (7) (7)TREATMENT P 0.001 0.001 0.001 0.001 0.001 0.01Oxyfedrin M .+-. m 1058 .+-. 97 1108 .+-. 74 1319 .+-. 61 1424 .+-. 42 1438 .+-. 44 332 .+-. 360.6 mg/kg (5) (5) (5) (6) (6) (6) P -- -- -- -- -- -- P* 0.001 0.001 0.001 0.001 0.001 0.001__________________________________________________________________________ P comparisom to normal, P* comparison to Control
TABLE 14a__________________________________________________________________________Effects of nonachlazin and oxyfedrin on cardiac AC activityActivity, pcmole cAMP/mg protein/min.Test Nonachlazin Oxyfedrineconditions Basal 10.sup.-6 M 10.sup.-4 M 10.sup.-6 M 10.sup.-4 M__________________________________________________________________________Prior to 142 .+-. 2 -- -- -- --preincuba-tionn = 4After -- 175 .+-. 3*** 162 .+-. 10** 200 .+-. 15*** 189 .+-. 9**preincuba-tionn = 4__________________________________________________________________________ n number of recurrences Comparison to: basal activity (*) one symbol is P <0.05, two symbols P <0.01, three symbols P <0.001
TABLE 14b__________________________________________________________________________Effects of nonachlazin and oxyfedrin on cardiac PDE activityActivity, pcmole cAMP/mg protein/min.Test Nonachlazin Oxyfedrinconditions Basal 10.sup.-6 M 10.sup.-4 M 10.sup.-6 M 10.sup.-4 M__________________________________________________________________________Prior to 744 .+-. 71 -- -- -- --preincuba-tionn = 4After -- 526 .+-. 47** 408 .+-. 24*** 543 .+-. 59*** 402 .+-. 26***preincuba-tionn = 5__________________________________________________________________________ n number of recurrences Comparison to: original activity (*) one symbol is P <0.05, two symbols <0.01, three symbols P <0.001
TABLE 14c______________________________________Effects of nonachlazin and oxyfedrin on myocardial cAMPlevel in normal rabbit and with TAM Medicinal cAMP,Group agent pcmole/mg protein______________________________________Control n = 5 1 9.5 .+-. 0.06 Nonachalazin 2 12.5 .+-. 0.07 n = 4 Oxyfedrin 3 14.3 .+-. 0.4* n = 410-day n = 4 4 3.4 .+-. 0.2TAM Nonachlazin 5 13.4 .+-. 0.3.sup.+++ n = 4 Oxyfedrin 6 12.0 .+-. 0.4.sup.+++o n = 4______________________________________ n number of recurrences *least significant difference between 1-2 and 1-3 .sup.+ least significant difference between 4-5 and 4-6 .sup.o least significant difference between 1-2 and 1-3 One symbol is P <0.05, two symbols P <0.01, three symbols P <0.001
TABLE 15__________________________________________________________________________Effects of Refrakterin on levels of adenyl nucleotides and CP in a 10-dayTAM, (.mu.cM/g wet tissue) Total Phospho-Group, Statistic nucleo- Energetic rylationsubgroup value ATP ADP AMP Pn ATP/ADP tides potential index CP Creatine__________________________________________________________________________Normal 1 M .+-. m 3.11 2.1 0.67 2.21 1.5 5.88 0.71 1.48 3.26 15.5 0.18 0.44 0.10 0.19 0.09 0.41 0.014 0.21 0.14 1.9 6 6 6 5 6 6 6 5 6 5TAMControl 2 M .+-. m 1.82 1.57 0.84 2.26 1.1 4.24 0.61 2.1 1.69 19.3 0.07 0.07 0.032 0.07 0.08 0.08 0.008 0.37 0.23 2.31 9 9 9 5 9 9 9 5 7 4Refrakterin 3 M .+-. m 3.13 2.4 0.74 2.13 1.43 6.32 0.58 1.56 3.12 18.22treatment 0.08 0.14 0.072 0.15 0.07 0.21 0.006 0.17 0.009 0.77 11 11 11 7 11 11 11 7 7 6 P.sub.1-2 0.001 0.01 -- -- 0.01 0.001 0.001 -- 0.001 -- P.sub.1-3 -- -- -- -- -- -- -- -- 0.001 -- P.sub.2-3 0.001 0.001 -- -- 0.05 0.001 0.001 -- -- --__________________________________________________________________________
TABLE 16__________________________________________________________________________Comparison of Refrakterin effectiveness to Ildamen-novodigal Control 10-day REFRAKTERIN Prior art,Ingredients TAM MI SK Ildamen + Novodigal__________________________________________________________________________Nicotinamide adenine -- 0.25 0.5 0.5 5 5 -- -- --dinunucleotide (NAD)Cytochrome C -- 5 10 10 10 15 -- -- --Inosine -- 20 80 80 80 250 -- -- --Cardiac b-Acetyldigoxin -- 0.05 0.08 -- -- 0.2 0.08 0.15 0.2glycoside b-methyldigoxin -- -- -- 0.008 -- -- -- -- -- Strophanthin K -- -- -- -- 0.03 -- -- -- --b-Adrenoceptor Oxyfedrin 0.3 -- 0.3 -- 0.3 0.3 2.0 -- 0.3 0.6 2.0stimulant Nonachlazin* -- 1 -- 1 -- -- -- 3.5 -- -- --Effect on the Myocardial 50 109 109.5 108.9 112.6 126 121 155 239contractile myofibrilprotein system tensionEffect on CPF Ca binding 67.6 92.9 90 92.6Ca transport CPF Ca uptake 69.8 84.7 102.5 101.7 101 106.9 CPF Ca release 56.0 81.9 110 116.7 Mitochondrial 128 111.9 96.2 58 Ca uptakeEffect on ATP 58.5 95 100.6 -- 95.5 88.6 52.2 69.1energy exchange ADP 74.8 89 114.3 -- 125.4 76.2 86.3 119.8 AMP 125.4 91 110.4 -- 120 60.0 26.3 121.1 ATP/ADP 73.3 82 88.7 -- 100 CP 51.8 98 95.7 -- 96.4 58.3 NADH/NAD 172 125 92.0 -- 105.2__________________________________________________________________________ Note: *Nonachlazin-10{b[1,4diazobicyclo(4,3,0)nonanyl-4-propionyl]2-chlorophenaine dihydrochloride

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Kostin, V. I. (Dep. Pharmacol., Med. Inst., Kemerovo 650029, USSR). Farmakol. Toksikol. (Moscow), 52(6), 49-52 (Russ) 1989.
Karsanov et al. (Repub. Res. Cent. Med. Biophys., Tbilisi, USSR), Izv. Akad. Nauk Gruz. SSR, Ser. Biol., 8(6), 393-9 (Russ) 1982.