Method and formulation of stimulating nitric oxide synthesis

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DESCRIPTION

Method and formulation to stimulate the synthesis of nitric oxide.

Background

This invention relates, in general, to compositions for treating cardio-, cerebro- or renovascular diseases with associated hypertension as well as cardio-, cerebro- or renovascular diseases without hypertension, in particular, it relates to a unique formulation used in the treatment of these diseases and their symptoms, where an endogenous biological source of nitric oxide (L-arginine) and a stimulator of nitric oxide synthase (NOS), particularly nitroglycerin, they are mixed prior to administration to form a mixture that is useful in treating nitroglycerin tolerance.

Description of Related Art

For several decades, nitroglycerin has been administered to humans as a vasodilator agent in the treatment of cardiovascular disease. Nitroglycerin or glyceryl trinitrate is an organic nitrate ester that, when administered to a subject, is biologically converted to nitric oxide (NO), which is a pharmacologically active metabolite. NO, for example, activates soluble guanylate cyclase in vascular smooth muscle cells which, in turn, increases cyclic guanosine monophosphate (cGMP) producing vasorelaxation (Waldman et al., 1987, Cyclic GMP synthesis and function, Pharmacol. Rev. , 39, 163) and finally produces vasodilation and a reduction in blood pressure. However, the efficacy of nitroglycerin is greatly reduced because the recipient of therapeutic nitroglycerin administration rapidly develops a tolerance to the beneficial effects of nitroglycerin. Therefore, the initiation of tolerance to nitroglycerin significantly limits the therapeutic value of nitroglycerin because increasing dosages has little or no effect on vasorelaxation or vasodilation. (Bogaert, M., 1991, Clinical relevance of tolerance to nitrovasodilators, J. Cardiovas. Pharmacol. 17 (Suppl. 3), S313; and Unger, P., et al., 1991, Tolerance to intravenous nitrates, J. Cardiovasc. Pharmacol. 17 (Suppl. 3), S300). The precise mechanism of tolerance to nitroglycerin is unknown. Theories explaining tolerance include the following: the sulfhydryl stores necessary for direct biotransformation of nitroglycerin to active nitric oxide are depleted by excess nitroglycerin substrate. (Boesgaard, S., et al., 1991, Nitrate tolerance: effect of thiol supplementation during prolonged nitroglycerin infusion in an in vivo rat model, J. Pharmacol. Exp. Ther. 258, 851); nitroglycerin reduces the activation of vascular guanylate cyclase (Henry P. J., et al., 1989, SNitrosothiols as vasodilators: Implications regarding tolerance to nitric-oxide-containing vasodilators, Br. J. Pharmacol. 98, 757); or that during tolerance to nitroglycerin the degradation rate of cGMP can be increased (Axelsson, KL, et al., 1987, Nitrate tolerance from a biochemical point of view, Drugs 33, 63).

Recently, it has also been shown that nitric oxide is formed enzymatically as a normal metabolite from arginine in the vascular endothelium to provide an important component for the formation of endothelium-derived relaxation factor (EDRF). Macrophages and neurons have also been shown to produce nitric oxide in the body as a component of their cell-killing and / or cytostatic function.

More recently it has been established that a family of enzymes called NOS form nitric oxide from L-arginine, and the nitric oxide produced is responsible for endothelium-dependent relaxation and activation of soluble guanylate cyclase, of neurotransmission in the central and peripheral nervous systems, and activated macrophage cytotoxicity (Sessa, William C., 1994, The Nitric Oxide Synthase Family of Proteins, Review, pp. 131-143).

Nitric oxide synthase exists in many different isoforms including a constitutive form (cNOS) and an inducible form (iNOS). The constitutive form is present in normal endothelial cells, neurons, and some other tissues. Nitric oxide formation by the constitutive form in endothelial cells is believed to play an important role in the regulation of normal blood pressure. The inducible form of nitric oxide synthase has been found to be present in activated macrophages and is induced in vascular smooth muscle cells, for example, by various cytokines and / or microbial products. In sepsis or cytokine-induced shock, overproduction of nitric oxide by the inducible form of nitric oxide synthase plays an important role in the life-threatening hypotension that is observed.

As described above, the conversion of L-arginine to nitric oxide is enzymatically catalyzed by NOS and the resulting by-product is L-citrulline. Although initially described in endothelium, as described above, NOS activity has now been described in many cell types. The brain, endothelium, and macrophage isoforms appear to be products of different genes that have approximately 50% amino acid identity. Brain and endothelial NOS have very similar properties, the main differences being that brain NOS is cytosolic and endothelial enzyme is primarily a membrane-associated protein.

Functionally, the constitutive form of nitric oxide synthase (cNOS), which is the predominant synthase present in the brain and endothelium, can be active under basal conditions and can be further stimulated by increases in intracellular calcium that occur in response to agonists. mediated by calcium ionophores or receptors. CNOS appears to be the "physiological" form of the enzyme and plays a role in a diverse group of processes.

ES 2 236 716 T3 biologicals. Certain in vitro studies suggest that nitric oxide synthase activity may be regulated in a negative feedback manner by nitric oxide itself. In the cardio-, cerebro-, or renovascular circulation, the main target for constitutively produced nitric oxide is soluble guanylate cyclase located in vascular smooth muscle, myocardium (myocytes), and coronary vascular smooth muscle.

In the presence of a normal substrate, nitric oxide is preferentially obtained by nitric oxide synthase. However, in the absence of L-arginine, brain nitric oxide synthase is believed to generate the free radicals superoxide and hydrogen peroxide. This property of nitric oxide synthase has important potential implications for neurotoxicity and pathophysiological states such as ischemia.

Unlike the constitutive form of the enzyme, the inducible calcium-independent form was only initially described in macrophages. It is now known that induction of nitric oxide synthase can occur in response to appropriate stimuli in many other cell types. These types include both cells that do not normally express a constitutive form of nitric oxide synthase, such as vascular smooth muscle cells, and cells such as myocardial cells (Levine, B, et al., 1990, Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med. 323: 236-241) that express considerable levels of the constitutive isoform.

INOS exhibits negligible activity under baseline conditions, but in response to factors such as lipopolysaccharides and certain cytokines, expression occurs over a period of hours. The induced form of the enzyme produces much greater amounts of NO than the constitutive form, and the induced NOS appears to be the "pathophysiological" form of the enzyme, because high concentrations of NO produced by iNOS can be toxic to cells. INOS induction can be inhibited by glucocorticoids and some cytokines. Relatively little is known about the post-transcriptional regulation of iNOS. The cytotoxic effects of NO are probably largely independent of guanylate cyclase and cyclic GMP formation.

Most of the research in this area has focused on inhibitors of iNOS stimulation using various derivatives of L-arginine. However, little research has been done on cNOS stimulation and its effect on nitroglycerin tolerance. Nitroglycerin tolerance has continued to frustrate the healthcare community because to date there is no effective way to stimulate physiological NO production above the basal tolerance or resistance of nitroglycerin to maintain the beneficial effect of nitroglycerin administration during prolonged periods.

In the art an effective method is needed to treat cardio-, cerebro- or renovascular diseases with hypertension and their symptoms as well as cardio-, cerebro- or renovascular diseases without hypertension and their symptoms to solve the basal resistance-tolerance of nitroglycerin.

Summary of the invention

The term "subject" is used herein to refer to any mammal, including humans, in which nitric oxide formation from arginine occurs. Compositions of the present invention for use in subjects contemplate prophylactic use as well as curative use in therapy of an existing condition. The term "native NO", as used herein, refers to nitric oxide that is produced via the biotransformation of L-arginine or the L-arginine dependent pathway. The term "end points", as used herein, refers to clinical events encountered in the course of treatment of cardiovascular disease, up to and including death (mortality).

It is an object of this invention to provide compositions for treating pharmacological tolerance to nitroglycerin.

It is another object of this invention to provide compositions for preventing, treating, arresting or ameliorating disease states that benefit from the biotransformation of L-arginine into endogenous nitric oxide or "native" nitric oxide.

It is another object of this invention to provide a formulation having a combined arterial and venodilator effect.

It is another object of this invention to provide compositions that ameliorate or prevent tachycardia and prevent or treat ischemia.

It is another object of this invention to premix L-arginine and nitroglycerin to achieve a synergistic effect to treat tolerance to nitroglycerin by increasing or maximizing the ability of nitroglycerin to produce "native" nitric oxide, and reducing clinical end points, including mortality. .

It is another object of this invention to provide compositions that prevent reperfusion injury in subjects who have undergone a sudden restoration of blood flow.

It is another object of this invention to provide the blend or blend formed to reduce L-arginine dosage requirements and the corresponding deleterious consequences of volume overload.

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It is another object of this invention to provide a mixture of nitroglycerin and L-arginine for the treatment of hypertension, hypertensive heart disease, coronary heart disease, including angina, myocardial infarction and sudden death, and a wide range of cardiovascular diseases (heart failure , stroke and peripheral vascular disease) and renovascular ischemia / hypertension.

These and other objects of this invention are provided by one or more of the embodiments provided below.

One aspect of the present invention provides a therapeutic composition for use in the prevention or treatment of a disease in a subject by vasodilation or vasorelaxation, comprising a venous dilator in admixture with an arterial dilator.

Another aspect of the present invention provides a therapeutic mixture comprising a mixture of L-arginine and a nitric oxide synthase agonist.

In one embodiment of the invention, therapeutically effective amounts of L-arginine and a cNOS agonist are mixed together prior to administration to a subject.

In another embodiment of the invention, therapeutically effective amounts of L-arginine and nitroglycerin are combined at a physiologically acceptable pH prior to administration.

In another embodiment, compositions for treating hypertension in a subject by vasodilation or vasorelaxation comprise: selecting a subject with hypertension; administering to said subject an anti-hypertensive formulation comprising a mixture of a venous dilator and an arterial dilator; obtain periodic measurements of the subject's blood pressure; and continuing administration of the formulation until a desirable blood pressure or therapeutic effect is detected in the subject. The desirable blood pressure in a hypertensive subject should ultimately be within the following ranges: systolic pressure preferably in the range of 95-180 mmHg, more preferably in the range of 105-165 mmHg, and even more preferably in the range of 120 at 140 mmHg; and the diastolic pressure preferably in the range of 55-115mmHg, more preferably in the range of 65100mmHg, and even more preferably in the range of 70-90mmHg, with the most preferred range being 7585mmHg. Under no circumstances should the systolic pressure be below 95 mmHg.

Another embodiment is to provide compounds for preventing or treating cardiovascular disease in a non-hypertensive subject by vasodilation or vasorelaxation comprising: selecting a subject; administering to said subject a formulation comprising a mixture of a venous dilator and an arterial dilator, wherein the venous dilator is a combined non-dependent and endothelium-dependent source of nitric oxide (i.e., nitroglycerin) and said arterial dilator is a dependent source nitric oxide endothelium (L-arginine); obtain periodic measurements of vasorelaxation in the subject; and continuing administration of the formulation until a desirable state of vasorelaxation or a desirable therapeutic effect is detected in the subject. A desirable state of vasorelaxation is, for example, a reduction in systolic pressure of about 20 mmHg and a reduction in diastolic pressure of about 10 mmHg. Under no circumstances should the systolic pressure be lowered below 95 mmHg.

Another embodiment is to provide compounds for treating hypertension in a subject by vasodilation, comprising: selecting a hypertensive subject; administering to said subject an anti-hypertensive formulation comprising a mixture of L-arginine and nitroglycerin; obtain periodic measurements of blood pressure in the subject; and continuing the administration of the anti-hypertensive formulation until a desirable blood pressure is detected in the subject.

Another embodiment is to provide compounds for stimulating cNOS in a subject comprising: selecting a subject; administering to said subject a formulation comprising a mixture of L-arginine and nitroglycerin, to maximize the production of "native" NO to treat tolerance and reduce end points including mortality.

Brief description of the drawings

Fig. 1 is a schematic representation of nitric oxide production illustrating the proposed L-arginine dependent and independent pathways.

Fig. 2 is a bar graph illustrating the cNOS stimulatory effect of combined administration of Larginine and nitroglycerin in rat aorta.

Fig. 3 is a bar graph illustrating the absence of the cNOS stimulatory effect of the combined administration of L-arginine and SNP in rat aorta.

Fig. 4 is a human dose study demonstrating the absence of tachycardia during administration of the formulation described herein.

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Detailed description of the preferred embodiments

Combining L-arginine with nitroglycerin prior to administration has been found to exceed the level of resistance or tolerance normally established when nitroglycerin is administered alone. It is believed that NOS can be stimulated by nitroglycerin and that the premix with L-arginine has a beneficial synergistic effect that may be due to a complex or coordinated formation between nitroglycerin and L-arginine. An excess of L-arginine provides an additional substrate for stimulated nitric oxide synthase that catalyzes the biotransformation of Larginine into nitric oxide.

Such stimulation of NOS in the presence of excess L-arginine can be used to prevent, treat, stop or ameliorate any disease or condition that may be positively affected by NO production. Such conditions include cardio-, brain- and renovascular diseases with hypertension and their symptoms as well as cardio-, brain- and renovascular diseases without hypertension. The mixture is particularly useful for subjects who need native NO production. The application of such a mixture is beneficial for: (1) chronic stable angina; (2) unstable angina; (3) acute myocardial infarction; (4) hibernating myocardium; (5) stunned myocardium; (6) limitation of ventricular remodeling after myocardial infarction and subsequent risk of congestive heart failure; (7) prophylaxis of a recurrent myocardial infarction; (8) prevention of sudden death after myocardial infarction; (9) vasospastic angina; (10) congestive-systolic heart failure observed in association with items 1-6 above; (11) congestive-diastolic heart failure observed in association with points 1-10 before and 12-15 after; (12) microvascular angina observed in association with items 1-11 above and items 15 and 16 below; (13) silent ischemia observed in association with points 1-12 above and points 15 and 16 later; (14) reduction of ventricular ectopic activity observed in association with points 1-13 above and with point 15 posterior; (15) any or all of the above 1-14 ischemic myocardial states associated with hypertensive heart disease and impaired coronary vasodilator reserve; (16) blood pressure control in the treatment of hypertensive crisis, perioperative hypertension, uncomplicated essential hypertension, and secondary hypertension; (17) regression of left ventricular hypertrophy observed in association with items 15 and 16 above; (18) prevention and / or regression of epicardial coronary atherosclerosis observed in items 1-17 above; (19) prevention of restenosis after angioplasty; (20) prevention and / or amelioration of free radical mediated reperfusion injury in association with items 1-19 above; (21) use of the combination in the prevention of myocardial injury during cardioplegic arrest in the course of coronary bypass or other open heart surgery, that is, the use of the combination as a cardioplegic solution; (22) after transplant cardiomyopathy; (23) renovascular ischemia; (24) cerebrovascular ischemia (transient ischemic attack (TIA) and stroke).

Fig. 1 is a schematic illustration showing the proposed nitrovasodilator-induced mechanism of action on both a generator cell and a target cell and their interrelationship. It appears that the mechanism of action of nitroglycerin or glyceryl trinitrate (GTN) is dependent on L-arginine and independent of Larginine and this implication has achievable effects in relation to the development and treatment of tolerance to nitroglycerin and reduction of clinical end points and mortality. One type of generator cell is an endothelial cell, but it can also be an endocardial cell or a coronary endothelial cell; and a corresponding type of target cell is a vascular smooth muscle cell, but it can also be a myocardial (myocyte) cell. The cells of the vascular smooth muscle are located mainly in the veins, arteries and coronary arteries. The following discussion will focus on nitrovasodilator-stimulated smooth muscle and myocyte relaxation where nitric oxide synthase is cNOS, the constitutive form of nitric oxide synthase, generator cells are endothelial cells, and target cells are smooth muscle cells. vascular. This illustration is not intended to imply any cellular relationship between the various sites of action, but rather is intended to illustrate their functional relationship.

In fig. 1, NO production can be due to a variety of sources and mechanisms that are discussed in detail in Ignarro, (Louis J. PhD., 1991, Pharmacology of Endothelium-Derived Nitric Oxide and Nitrovasodilators, The Western Journal of Medicine, p. 51-62) which is incorporated herein in its entirety by reference. In the L-arginine-independent or non-endothelium-dependent pathway, the activation of guanylate cyclase (GC) by nitric oxide (NO) depends on the type of nitrovasodilator used. Inorganic nitrite (NO₂) is loaded and only limited amounts can infiltrate the cell, but intracellular nitrite can be converted to NO. Lipophilic organic nitrate esters (R-OH) are converted to NO by reactions facilitated by acid thiol (R-SH). Snitrosothiols (R-SNO) are labile intermediates that decompose spontaneously and produce NO. It is believed that one of the mechanisms by which thiols enhance the action of nitroglycerin and reverse nitroglycerin tolerance to some degree is through the direct reaction between thiol (R-SH) and nitroglycerin (GTN) to form the labile intermediate S-nitrosothiol (R-SNO), which decomposes as previously described (R-SH + GTN R-SNO is not shown in fig 1). Nonenzymatic formation of exogenous NO is believed to occur with thiol sources such as cysteine, dithiothreitol, N-acetylcysteine, mercaptosuccinic acid, thiosalicylic acid, and methylthiosalicylic acid. Nitrates such as isosorbide dinitrate and isosorbide 5'mononitrate can also be used to produce NO, as they are simply commercially available intermediates for the known L-arginine independent pathway. Nitroprusside ((CH)₅-FeNO) form No after degradation and does not depend on thiol. GTP is guanosine triphosphate; HONO is nitrous acid; Meth. Blue is methylene blue; R-ONO is organic nitrite esters; and R-SS-R represents a disulfide. In the L-arginine independent pathway, the glyceryl trinitrate (GTN) reaction is represented by R-ONO₂ and it is believed that it requires a certain reserve of thiols, such as a sulfhydryl containing enzyme, to generate NO, and it was initially thought that intracellular thiol deficiency results in tolerance to the pharmacological actions of nitroglycerin. However, this does not explain tolerance, since dependent thiols

ES 2 236 716 T3 of exogenous doses do not result in reversal of tolerance to nitroglycerin (Fung HL, 1988, Journal of Pharmacology and Experimental Therapeutics. 245: 2, 524-30) although they may exert a beneficial effect as independent donors of NO instead of facilitating the spontaneous release of nitric oxide. (Munzel T., MD, et al., 1994, What Causes Nitroglycerin Tolerance? Clinical Cardiology. 20 No. 9: 40-47).

However, for the first time it is hypothesized in this paper that nitroglycerin tolerance may involve a secondary pathway, or indeed this "secondary pathway" may be the primary pathway. This "secondary pathway" is the L-arginine dependent pathway or the endothelium dependent pathway shown in FIG. 1. As seen in Fig. 1, the generating cell is known to have several receptor-mediated agonists such as the endothelial B receptor (ET_B); acetylcholine (Ach); substance P (SP), histamine (H); arginine vasopressin (AVP); bradykinin (BK); adenosine triphosphate (ATP); prostaglandin F_2nd (F_2nd); oxytocin (OT); and the calcium ionophore (A23187) that stimulates the production of NOS. However, until now it has not been speculated that nitroglycerin may have the dual role of agonist for NOS and prodrug for the sulfhydryl-mediated L-arginine independent pathway.

Nitroglycerin was previously thought to have no effect on the biotransformation of L-arginine into "native" nitric oxide, but it is now believed that nitroglycerin or a complex or coordinate of nitroglycerin (GTN complex in Fig. 1) with L -arginine has a stimulatory effect on cNOS. The mechanism is not well understood, but it appears that the new combination of nitroglycerin and L-arginine before administration may have a hitherto unexpected synergistic effect on cNOS stimulation which may be due in part to a new complex formulation that serves as a release system for unprocessed nitroglycerin. On the other hand, cNOS stimulation may be the result of a cNOS that has a unique receptor site for the complex or it may be due to nitroglycerin being in an equilibrium state of dissociation with L-arginine. Administration of the two in combination also provides a suitable substrate for cNOS processing of L-arginine, since L-arginine will be added in excess.

It seems that some complex or coordinate is formed between L-arginine and nitroglycerin when the two are mixed. This is shown in Table I, where the coordinate was studied using 300 MHz Bruker NMR. The studied samples consisted of the following: sample A, a concentrated standard (100 mg of L-Arg in 0.5 ml of D₂OR); sample B, a concentrated mixture (100 mg of L-Arg plus one tablet of nitrostat in 0.5 ml of D₂OR); sample C, a diluted standard (one drop of sample A in 1.0 ml of D₂OR); and sample D, a diluted mixture (13 mg of L-Arg plus 3 tablets of nitrostat in 1 ml of D₂OR). These samples were compared and combined on a computer to determine if a complex had formed. The addition of nitroglycerin to L-arginine resulted in a change in chemical shifts for the L-arginine multiplet ad 1.9 and the triplet at 33.2, the signals studied most rapidly. This change is shown in table I.

TABLE I

Signal analysis 33.2

<td></td><td>Signal Frequency</td><td></td>

<td>sample C (Hz)</td><td>sample D (Hz)</td><td>Change</td>

<td> 979,032</td><td> 980,119</td><td>1,087 Hz</td>

<td> 972,107</td><td> 973,281</td><td>1,174 Hz</td>

<td> 965,272</td><td> 966,364</td><td>1,092 Hz</td>

<td>signal 31.9</td><td></td><td></td>

<td></td><td>Signal Frequency</td><td></td>

<td>sample C (Hz)</td><td>sample D (Hz)</td><td>Change</td>

<td> 582,392</td><td> 584,513</td><td>2.121 Hz</td>

<td> 572,108</td><td> 577,287</td><td>2,179 Hz</td>

<td> 573,365</td><td> 575,607</td><td>2,242 Hz</td>

<td> 567,231</td><td> 569,348</td><td>2,117 Hz</td>

<td> 565,698</td><td> 568,118</td><td>2,420 Hz</td>

<td> 559,425</td><td> 561,673</td><td>2,248 Hz</td>

The change in proton chemical shifts in L-arginine in the presence of nitroglycerin is a strong indicator that a complex of the substances in solution is being formed to form a different intermediate of the two independent substances. This is further confirmed by the fact that the shift was not concentration dependent. In this way, it can be fairly concluded that L-arginine and nitroglycerin do not act independently in solution, but rather are somewhat involved in the formation of a complex that changes the chemical environment of the L-arginine protons and that it can be detected using high resolution NMR spectroscopy. This may explain the only beneficial NO release system that solves

ES 2 236 716 T3 the resistance-tolerance threshold previously observed in the administration of nitroglycerin alone. However, the beneficial effect may simply be due to the simultaneous administration of L-arginine and a cNOS stimulator.

The combination of L-arginine and nitroglycerin can also result in a combined arterial and venous dilator effect. Used alone, nitroglycerin is primarily a venodilator and produces a rapid increase in heart rate due to its venous reserve, while L-arginine on the other hand, when used alone, is primarily an arterial dilator. Therefore, the combination of the two results in a balanced arterial and venodilator effect, counteracting the tendency of one or the other to produce tachycardia, which is adverse for ischemia in an evolving myocardial infarction. This is suggested by preliminary data obtained in dog studies and is most notable in the data shown in Table II. The data in Table II were generated by administering L-arginine at 5 cc per minute, where L-arginine was at a concentration of 10% w / v (g / ml) and nitroglycerin was administered at 3.38 jug / kg / minute by intravenous (IV) administration over a period of five minutes. The dog was a beagle weighing 13.6 kg. When administered in combination, the relative concentrations and dosages remained the same. BP is blood pressure (systolic / diastolic in mmHg); MAP is mean arterial pressure (mmHg), CO is cardiac output (liters / min); TPVR is total peripheral vascular resistance (dynes * sec / cm³); ATPVR is the change in total peripheral vascular resistance (%); and HR is heart rate (beats per minute).

TABLE II

Canine Study

<td>Agent Before L-arginine</td><td>BP 130/75</td><td>MAP 93.3</td><td>CO 1.44</td><td>ÍTPVR) (64.8)</td><td>HR 105</td><td>ATPVR 31.6%</td>

<td>Later</td><td> 105/55</td><td> 71,7</td><td> 1,62</td><td> (44,3)</td><td> 102</td><td></td>

<td>Before</td><td> 105/60</td><td> 75,0</td><td> 1,63</td><td> (46,0)</td><td> 104</td><td></td>

<td>Nitroglycerine</td><td></td><td></td><td></td><td></td><td></td><td> 24,5%</td>

<td>Later</td><td> 70/40</td><td> 50,0</td><td> 1,44</td><td> (34,7)</td><td> 105</td><td></td>

<td>Before</td><td> 105/60</td><td> 75,0</td><td> 1,56</td><td> (48,1)</td><td> 102</td><td></td>

<td colspan="2">Nitroglycerin + L-arginine</td><td></td><td></td><td></td><td></td><td> 16,8%</td>

<td>Later</td><td> 70/40</td><td> 50,0</td><td> 1,60</td><td> (31,3)</td><td> 98</td><td></td>

Observing the effect on CO or cardiac output, it can be seen that after administration of Larginine alone, the increase in cardiac output is due to the effect of L-arginine as mainly an arterial dilator; and the reduction in cardiac output observed with nitroglycerin alone is mainly due to a venous dilator effect; while the combination produces a substantially balanced arterial and venous dilating effect (a change in cardiac output of only 0.04 (1.60-1.56)). Hence, the absence of a tendency to tachycardia (that is, no evidence of activation of the baroreceptor reflex).

Another beneficial mechanism of the combination relates to the fact that, used alone, nitroglycerin has only a minimal beneficial effect in limiting reperfusion injury with patients who have had recent heart attacks and an abrupt restoration of blood flow. The same is observed in patients undergoing reestablishment of blood flow after coronary bypass operations to remove the bypass pump. This form of reperfusion injury is considered to be mediated by the generation of free radicals after reperfusion and preliminary data especially in cats show that L-arginine administered alone limits the production of free radicals. (Weyrich, AS, PhD., Et al., 1992, The Role of L-Arginine in Ameliorating Reperfusion Injury After Myocardial Ischemia in the Cat. Circulation. 86: 279-288). Therefore, the combination would be likely to limit reperfusion injury relative to nitroglycerin used alone.

Another beneficial effect of the use of the combination over each one used alone refers to the fact that the studies carried out with volunteers so far with l-arginine alone reveal that it is a weak vasodilator in terms of the dosage requirements (600 ml / time as reported by Nakaki T., et al., 1990, L-arginine Induced Hypotension. The Lancet, p. 696). Patients who have unstable coronary syndromes and myocardial infarction with or without the complication of congestive heart failure are prone to volume overload with IV fluid administration. Therefore, combining nitroglycerin with L-arginine could markedly limit the dosage requirements for total L-arginine and thus the risk of developing congestive heart failure. This could also be important in patients who have compromised kidney function and are prone to acidosis and kidney failure with large volumes of L-arginine.

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The main combination to be used will be a mixture that includes therapeutic concentrations of L-arginine and nitroglycerin in water. Any pharmaceutical grade L-arginine will suffice and should preferably be diluted to 2.5-60% w / v (g / ml), more preferably 5-45% w / v (g / ml), even more preferably between a 7.5 and 30% w / v (g / ml), even more preferably 10-15% w / v (g / ml) and even more preferably 10% w / v (g / ml) of Larginine. Typical expected doses will be 30 g of L-arginine in sterile water (total volume 300 ml). L-arginine is ultimately expected to be in a hydrochloride salt: L-arginine base ratio of about 10: 1 to about 25: 1, and even more preferably in a 15: 1 hydrochloride salt to base ratio. at about 20: 1, with the most preferred ratio of hydrochloride salt to base being 15: 1. In this example, the hydrochloride salt will comprise 28 to 29 g and 1 to 2 g of L-arginine will be in base form. The nitroglycerin to be combined with the L-arginine is expected to have a concentration dependent on the mass of the subject in kg and that the dosing time is preferably in the range of 0.1 jug / kg / minute to about 5 jug / kg / minute, more preferably in the range of 0.2 jug / kg / minute to about 4 jug / kg / minute, even more preferably in the range of 0.5 jug / kg / minute to about 3 jug / kg / minute, even more preferably in the range of 0.75 jug / kg / minute to about 2 jug / kg / minute, and even more preferably about 1 jug / kg / minute. Therefore, depending on the IV volume, the time of administration, and the subject's weight, nitroglycerin will be added in an amount sufficient to obtain the desired range (ie, 1 pg / kg / minute). If a transdermal system is used, the nitroglycerin release should preferably be between 0.2 mg / h and 1 mg / h, more preferably between 0.3 mg / h and 0.8 mg / h, and even more preferably between 0, 4 mg / h and 0.6 mg / h. The package is expected to contain lyophilized L-arginine in a glass bottle to which nitroglycerin and sterile water can be added to give 30 grams of L-arginine and 1 to 960 milligrams of nitroglycerin diluted to a full volume. with 300 cc sterile water. Alternatively, nitroglycerin, L-arginine and water can be added in sterile glass bottles adjusted to a physiological pH. The pH, upon reconstitution in water, should preferably be in the range of about 5-8, more preferably in the range of 6-7.5, even more preferably in the range of 7 to 7.5, and even more preferably should be about 7.4, which is the physiological pH, to avoid the current problem that is present in the solutions that require the limitation of the pH of 5.6 to avoid the bacteriological growth in periods of prolonged rest when they are transported in solution.

Nitroglycerin dose may vary based on future studies on the effect of the combination ratio on heart rate. Furthermore, although the discussion focuses on intravenous administration, buccal, intracoronary, intramuscular, topical, intranasal, rectal, sublingual, oral, subcutaneous, or patch administration, alone or in combination, also applies. Due to its compatibility, the combination of L-arginine and nitroglycerin in patches may be the most common use, as is currently the use of nitroglycerin alone in the form of patches. The feasibility of the patch technology is confirmed by the L-arginine solubility test in Tridil ™. The solubility test demonstrated the following: without the addition of water, approximately 170 mg of L-arginine will dissolve in 1.0 ml of Tridil ™ (5 mg of nitroglycerin / ml); A clear colorless mixture was obtained when 2500 mg of L-arginine hydrochloride, 1.0 ml of Tridil ™ and 2.8 ml of deionized water were combined at 30 ° C with gentle stirring and then cooled to room temperature (approximately to 24 ° C); and a very thick, yet pourable suspension was obtained when 2500 mg of L-arginine, 1 ml of Tridil ™ and only 0.5 ml of deionized water were combined. These results suggest that L-arginine and Tridil ™ have a high degree of solubility compatibility and could therefore be easily incorporated into current patch delivery technology.

The following illustrates the mechanism of action described above and the treatment of cardiovascular, brain, and renovascular diseases:

Example 1

It was recently discovered that dogs treated to achieve a basal nitroglycerin effect could additionally respond by co-administration of nitroglycerin and L-arginine in water in a manner similar to that commonly observed clinically with the addition of sodium nitroprusside (SNP) to nitroglycerin. ; however, compared to SNP, L-arginine combined with nitroglycerin had much more favorable hemodynamic effects. Compared to SNP, vascular resistance was reduced by 50%, cardiac output doubled, and contractility increased. This led to the hypothesis that the combination of L-arginine and nitroglycerin was generating EDRF rather than SNP, which is known to directly produce nitric oxide.

As there is still debate about whether EDRF is identical to nitric oxide, it was hypothesized that EDRF, not being identical to NO, would explain the difference in the hemodynamic effect. To explain the extra EDRF it was hypothesized that nitroglycerin, in addition to being a prodrug for nitric oxide, was also an agonist for the activation of cNOS and that the speed limitations of L-arginine in the canine model could be explained by a supply-demand decoupling in L-arginine uptake, particularly in disease states such as hypertension, hyperlipidemia or arteriosclerosis in which endothelial cells are involved, which is considered an active transport process with potential rate limitations that can possibly be overridden by passive diffusion of excessively administered L-arginine. Hence it is reasonable to combine L-arginine with nitroglycerin for the treatment of nitrate resistance and tolerance. To test this hypothesis, the effects of exposure of intact rat aorta to nitroglycerin combined with L-arginine in aqueous solution were studied and the results were compared with the results obtained with SNP combined in an aqueous solution with L-arginine. The effect of the combination of L-arginine and nitroglycerin is shown in figure 2. The clinical preparations were as follows:

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Preparation of animals

Eight Sprague-Dawley rats were used in this nitroglycerin study and two were used in the SNP study. After removing the aorta from each rat, the aorta was cleaned and cut into 5 segments. Segments were randomized to minimize variation in baseline values. After this, the segments were incubated in Earl's saline at 37 ° C.

Treatment protocol

Nitroglycerin group - one of the five extracted segments served as a control to assess the integrity of the endothelium (baseline activity). The other four received 50 µmol of L-arginine. After 30 minutes, 1 ml of IBMAX (50 µmol) was added to the 5 segments to prevent any further degradation of cGMP by phosphodiesterase (IBMAX is isobutyl methyl xanthine). The 5 segments were treated as follows: A - control - basal activity; B is the L-arginine group - 50 // mol of L-arginine added to the basal group; C is the nitroglycerin group - 5 // mole of nitroglycerin in 50 // mole of L-arginine; D is nitroglycerin + N^G-nitro-L-arginine methyl ester (L-NAME a known inhibitor of NOS function) - 5 // mol of nitroglycerin + 0.5 mmol of L-NAME and 50 // mol of L-arginine; and E is the L-NAME group - 0.5 mmol of L-NAME and 50 µmol of L-arginine. After 50 minutes, each segment was removed and placed in 500 µl of 0.1 N HCl. They were left for one hour, after which they were removed and weighed.

Cyclic GMP Assay

For the determination of cGMP 400 µl of HCl solution remaining after the purifications were removed, weighed and transferred to gamma flow tubes and the cyclic GMP was determined by radioimmunoassay. Data interpretation

A. Control - Basal. This represents the activity of the cGMP initially that was generated based on soluble guanylate cyclase activation NO sources, ie the baseline level.

B. L-arginine group. This represents the activity of the cGMP generated by L-arginine and EDRF (endogenous or "native" NO production).

C. Nitroglycerin group. (L-arginine plus nitroglycerin). CGMP activity represents the sum of B (Larginine) plus nitroglycerin induction of cNOS and subsequent EDRF produced in addition to nitric oxide from nitroglycerin by the L-arginine independent pathway (prodrug effects).

D. L-NAME group. L-arginine (L-arginine plus nitroglycerin plus L-NAME). Represents cGMP activity from enzymatic conversion of nitroglycerin alone, as L-NAME used in excess inhibits NOS-derived EDRF from all sources.

E. L-arginine + L-NAME - represents cGMP activity due to non-nitric oxide sources that activate soluble guanylate cyclase and was subtracted from all measures to eliminate the effects of cGMP activation due to non-nitric oxide agents. they are NO (atrial natriuretic factor, etc).

From all this it is evident that: the total NO of nitroglycerin is CB; NO from the enzymatic degradation of nitroglycerin to NO equals DE; stimulation by EDRF (NOS) of nitroglycerin = (CB) - (DE).

SNP group

A second group of two rats was examined as above, with the exception that in this group nitroglycerin was substituted for SNP in the treatment protocol. These results are shown in figs. 3 A ', B' and E 'which correspond exactly to A, B and E of FIG. two. C 'is equal to 50 // mol of L-arginine plus 1 // mol of SNP and represents the activity of the cGMP produced by the stimulation by L-arginine of the production of EDRF plus any activation of cNOS by SNP plus NO from of SNP by non-enzymatic conversion. It does not appear that SNP requires any sulfhydryl groups, but rather forms NO and cyanide as a by-product in a non-enzymatic manner. D 'is SNP plus L-NAME - represents the activity of cGMP generated by non-enzymatic conversion of SNP to NO alone, ie exogenous or "non-native" NO. SNP total NO = C'-B '; Total NO of SNP obtained by non-enzymatic conversion = D'-E '; SNP EDRF by NOS activation = (C'-B ') - (D'-E').

Results

Figures 2 and 3 summarize these results with a representative bar graph of the respective detected picomoles of cGMP / 100 mg wet tissue. Although not shown in fig. 2, when nitroglycerin and LNAME were combined in the absence of L-arginine, similar results were obtained with respect to cGMP production. Both in fig. 2 as in fig. 3, the bar labeled NOS is the amount of "native" NO produced, which is the total NO minus the NO produced via the L-arginine independent pathway.

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Nitroglycerin resistance - tolerance has frustrated cardiologists and pharmacists since 1888. (Stewart DD, 1888, Remarkable Tolerance to Nitroglycerin. Philadelphia Polyclinic. 172-5). These results confirm the hypothesis obtained in fig. 1 and clarify the mechanism of tolerance to nitroglycerin. An additional nitroglycerin activation site is believed to be cNOS in endothelial cells. Under conditions leading to tolerance, the agonist effect of nitroglycerin on cNOS induction leads to a depletion of L-arginine in endothelial cells secondary to rate limitations in the active transport kinetics of the L-arginine pump. as can be seen in fig. 1. This creates a decoupling situation between supply and demand in the membrane uptake stage and explains why arginine is rate limiting in the canine model. This may also explain why a nitrate-free interval is required during nitroglycerin administration. This is believed to be necessary for endothelial cells to be able to replenish deficient L-arginine by active transport. It is believed that by adding L-arginine to nitroglycerin EDRF can be generated, and in the process a significant reduction in mortality and clinical end points can be obtained over the use of nitroglycerin alone or in combination with SNPs or others. donors of exogenous NO.

The fact that veins are more sensitive to exogenous NO (and more likely also to “native” NO) compared to arteries, explains why at low doses, nitroglycerin is primarily a venous dilator compared to PNS, which it is a balanced arteriovenous dilator. This explains why at 37 micrograms / h nitroglycerin becomes arterial, because at this level all potential EDRF is produced and the NO prodrug conversion takes place as the last source of nitric oxide generated by nitroglycerin. This last source of NO generated from prodrug conversion is equivalent to NO from SNP and generates a similar arterial effect.

EDRF may not be identical to NO and is possibly the precursor (L-OH-NO half-life 350 seconds) for NO. This would explain the failed attempts to substitute SNP for nitroglycerin in clinical situations, such as unstable angina and acute myocardial infarction (Flaherty, JT, MD, 1983, Comparison of Intravenous Nitroglycerin and Sodium Nitroprusside in Acute Myocardial Infarction. American Journal of Medicine, 53-60.); since EDRF has better anti-ischemic actions and using SNP would not produce EDRF, SNP would not lead to the beneficial effects on mortality that could be achieved with nitroglycerin. Another beneficial effect of EDRF produced by stimulation of cNOS with nitroglycerin may be due to the ability of EDRF to function as a free radical scavenger with respect to exogenous NO. (Zembowicz A., et al., 1991, Nitric Oxide and Another Potent Vasodilator are Formed from N^G-hydroxy-L-arginine by Culture Endothelial Cells. Pharmacology. Proc. Natl. Acad. Sci. United States 88: 11172-76). In a reperfusion injury, a free radical scavenger (possibly EDRF) is needed to absorb the free radicals that arise, which occurs with L-arginine and nitroglycerin, but not with SNP, a non-native source of NO. This can be explained because the intermediate EDRF with SNPs would not be expected. Tolerance is established and the beneficial effect of nitroglycerin is lost because EDRF is no longer being produced or at least until the rate limiting step is passed by adding L-arginine substrate. This provides an additional beneficial mechanism for the combination or complex because it refers to the fact that used alone, nitroglycerin soon loses its beneficial effect in limiting reperfusion injury with patients who have had recent heart attacks and a sudden restoration of flow. blood. The same is observed in patients who are experiencing a re-establishment of blood flow after coronary bypass operations to remove a bypass pump. This form of reperfusion injury is considered mediated by the generation of reperfusion free radicals and preliminary data, especially those obtained in cats, show that L-arginine administered alone also limits free radical production. Therefore, the combination would likely limit reperfusion injury relative to nitroglycerin used alone.

These results indicate the formation of a new drug combining nitroglycerin with excess L-arginine to take advantage of the passive diffusion abrogation mechanism of the transport pump across the endothelial cell membrane as a treatment for resistance-tolerance to nitroglycerin. Such a formulation has applications including hypertension, hypertensive heart disease, coronary heart disease (angina, myocardial infarction, and sudden death), cardiovascular diseases (congestive heart failure, stroke, peripheral vascular disease), cerebrovascular ischemia (TIA), and renovascular ischemia.

Another potential utility of this complex is to independently produce EDRF as seen here in the rat and canine aorta results, which will be of great value as a treatment for nitroglycerin tolerance without additional toxicity and without the drawbacks of administration of nitroglycerin alone, as currently used. The method of administration would not change.

It appears that the L-arginine-nitroglycerin mixture stimulates cNOS selectively and does not induce iNOS. This is confirmed by the following:

1. INOS induction generally leads to irreversible vascular collapse and death. The classic example is endotoxic shock. This was not observed in the present studies.

two. The induction of iNOS is associated with a positive feedback mechanism to increase the transport of L-arginine into the endothelial cell with iNOS. (Lind, DS, MD, 1993, Endotoxin Stimulates Arginine Transport in Pulmonary Artery Endothelial Cells. Surgery; 114; 2; pp. 199-205). Therefore, supplementing the administration of L-arginine would only accelerate the tendency of vascular collapse.

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3. In states in which iNOS induction is not initially present, the administration of nitroglycerin and L-arginine, alone or in combination, does not lead to irreversible vascular collapse. Both nitroglycerin alone and in combination produce dose-dependent hypotension, which is reversible after discontinuation of exposure to the respective drugs.

With respect to paragraph 2 above, in the iNOS induction states described above, it is believed that the development of tolerance to nitroglycerin may be an opposite effect of nitroglycerin on the membrane pump, that is, a negative feedback mechanism on active transport of L-arginine across the membrane. This can be a factor that leads to the development of tolerance.

With respect to paragraph 3 above, iNOS induction may be a common feature of all vascular shocks, including hemorrhagic and cardiogenic shock. Similarly, advanced stages of congestive heart failure with poor performance syndrome (borderline cardiogenic shock) may be associated with cytokine production (tumor necrosis factor) and iNOS induction. Care must be taken in future use of L-arginine in combination with nitroglycerin in these states, in the same way that care is now exercised when using nitroglycerin alone in patients who are hypotensive.

An 8 hour infusion has been performed in a normal human volunteer using a wide series of nitroglycerin concentrations ranging from 12.5 mg / 250 ml total volume to 100 mg / 250 ml total volume of 10% Larginine, and Most importantly, the absence of previously presented tachycardia with L-arginine or nitroglycerin alone has been discovered. Additionally, at 2.5 times the currently approved Larginine exposure dosages (75 g total) there was no indication of metabolic acidosis from the HCl present in the currently approved L-arginine formulation. This study is summarized below.

Example 2

The following study is a dose variation study in normal human volunteers for intravenous nitroglycerin combined with L-arginine. The aim of this study is to examine the combined administration of intravenous nitroglycerin with 10% L-arginine (aqueous) for the following:

1. Reflex tachycardia (activation of the baroreceptor reflex).

two. Hypotensive activity (therapeutic effect).

3. Metabolic disorders-metabolic acidosis.

Four. Electrocardiographic abnormalities with prolonged infusion.

The patient studied in this dose range study was a 47-year-old normotensive white male with no previous history of illness or hospitalization and without chronic medications.

The materials used in this study consisted of the following:

1. Tridil brand of intravenous nitroglycerin (5 mg per cc).

two. 10% L-arginine in water (R-Gene^TM-KABI).

3. Normal saline solution.

Four. Five sterile 150cc vacuum sealed bottles.

5. Two Ivac pumps including a 3-way spigot for alternate drug and saline infusions.

6. A Propac heart monitor.

7. A Spacelabs 2000 24-hour blood pressure monitor

8. A Holter recorder model No. 2011 Cardionostics Dural-Lite.

The patient's preparation consisted of a pretreatment with 40 mg of Pepcid (famotidine-MERCK) and 50 mg of benadryl the night before. The administration of 50 mg of benadryl was repeated the morning of the study. This was done to block the Hj and H receptors.₂ of any possible activation by L-arginine.

On the morning of the study, a baseline EKG was obtained along with complete blood count (CBC) and serum chemistry. Following this, the 24-hour Holter monitor, ambulatory blood pressure monitor, and Propac were attached. The blood pressure monitor was calibrated against Propac and a discrepancy of approximately 20 mmHg of systolic blood pressure and 10 mmHg of diastolic blood pressure was observed in the left arm in front

ES 2 236 716 T3 to the right respectively. Next, an IV line was established in the left foot in the left saphenous vein with an 18-gauge angiocatheter. An initial maintenance infusion with saline was started at KVO (maintenance open vein) rate. Following this, six rapid dose response titrations were performed over the next eight hours and are shown in FIG. 4 with 1/4 (bottle n ° 1), 1/2 (bottle n ° 2), and nitroglycerin of total concentration in 10% L-arginine (bottle n ° 3). This was followed by an infusion of full strength nitroglycerin in water without L-arginine (bottle # 4). This was followed by an infusion of 10% pure L-arginine without nitroglycerin in 10% L-arginine (bottle # 5). Finally, an infusion consisting of double concentration nitroglycerin in 10% L-arginine (bottle # 6) was administered. Nitroglycerin full concentration was defined as 50 mg of nitroglycerin in a total volume of 250 cc of 10% L-arginine in water or water alone (bottle # 4).

With each infusion, the initial rate was 25 cc per hour. After this, the infusion was doubled to 50 cc per hour. This was increased by 50 cc per hour every 5 to 10 minutes until a total infusion rate of 300 cc per hour was achieved. During these infusions, blood pressure and heart rate data were recorded every two minutes by Propac before increasing the infusion rate as described above. During bottle changes, the infusion was switched to normal saline at 100 ml per hour. At the beginning of each infusion, an estimated 10 ml of "dead space" was removed from the infused remaining in the pre-bottle by displacing the first 10 ml at full speed. The 25 ml sequence was then restarted as described above.

After the final infusion, a repeat of the serum chemistry, CBC and EKG was obtained.

For each infusion, the averages of the systolic and diastolic blood pressures of the right arm were calculated. Similarly, the average heart rate was also calculated. These means were obtained by taking each individual reading obtained every 2 minutes, adding them and dividing by the period in which the infusion had occurred (not including measurements between infusions during bottle changes).

The results are summarized in fig. 4. In fig. 4, SBP means systolic blood pressure, DBP means diastolic blood pressure, and HR means heart rate. There does not appear to be any evidence of reflex tachycardia with the relationship between nitroglycerin and L-arginine used in fig. 4. There was a dose-dependent reduction in blood pressure along with a trend toward dependence on nitroglycerin concentration. There was no evidence of metabolic acidosis developing secondary to L-arginine infused over a prolonged period up to the total dose of 75 g administered over 8 hours. There was no indication of arrhythmia. There were no indications of electrocardiographic abnormalities. This clearly indicates that the administration of the combined L-arginine / nitroglycerin does not have the adverse consequences seen with L-arginine or nitroglycerin when administered alone.

The foregoing description of the invention is illustrative of the preferred embodiments of the invention currently contemplated by the inventor. However, it will be apparent that the above description of the invention is not to be construed in a limiting manner, there being various equivalent systems and ways of carrying out the present invention. For example, it is contemplated that L-arginine comes from commercially available products such as R-Gene^TM or any other source of pharmaceutical grade L-arginine, and nitroglycerin can be obtained from a variety of delivery systems well known in the art for nitroglycerin alone, for example: tongue sprays such as Nitrolingual spray^TM (0.4 mg / measured dose of Poulenc Rorer); transdermal systems such as Minitran ™ (0.6 mg / hour from 3M); topical ointments such as Nitro-Bid ointment^TM (2% from Marion Merrel Dow), as well as tablets and patches (currently using the commercial patch product called Tridil^TMof Du Pont). This list is not exclusive at all, but is simply a representation of the diversity of nitroglycerin delivery systems that could easily be modified to be a delivery system for the combination of L-arginine and nitroglycerin. All that is needed are compatible systems for the simultaneous release of nitroglycerin and L-arginine.