WO1998030222A1

WO1998030222A1 - Use of diadenosine polyphosphates for reducing blood pressure

Info

Publication number: WO1998030222A1
Application number: PCT/US1998/000546
Authority: WO
Inventors: Richard H. Hilderman; M. Michael Swindle
Original assignee: Clemson University
Priority date: 1997-01-13
Filing date: 1998-01-13
Publication date: 1998-07-16
Also published as: EP0969848A1; AU5821898A; CA2277479A1

Abstract

A process for reducing blood pressure is disclosed. The process includes the step of administering to a patient one or more adenylated dinucleotides. Particular examples of adenylated dinucleotides that may be used in the process of the present invention include diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, and diadenosine hexaphosphate. By reducing blood pressure, the composition of the present invention can be used to treat a patient during hypertensive emergencies, can be used to reduce bleeding, and can be used to treat acute congestive heart failure.

Description

USE OF DIADENOSINE POLYPHOSPHATES FOR REDUCING BLOOD PRESSURE

The present invention is based on a provisional application, filed January 13, 1997 and having U.S. Serial No. 60/035,819.

Field of the Invention The present invention generally relates to a composition and process for reducing blood pressure. More particularly, the process of the present invention is directed to administering a therapeutic agent to a patient for reducing arterial blood pressure during hypertensive emergencies, during surgery to reduce bleeding, and for the treatment of acute congestive heart failure.

Background of the Invention Blood pressure refers to the force exerted by the blood against the walls of the blood vessels. Blood pressure is created by the rhythmic contraction of the heart in combination with peristaltic waves of contraction in the walls of some blood vessels. In common medical usage, blood pressure refers specifically to the pressure of the blood in the arteries, measured in millimeters of mercury (mmHg) .

Blood pressure varies from one individual to another and in the same person from time to time. Blood pressure is at its highest during contraction of the heart, which is referred to as systolic pressure. Blood pressure is at its lowest, on the other hand, during relaxation of the heart muscle, which is referred to as the diastolic pressure. At normal levels, systolic pressure in a human male is typically about 120 mm Hg, while diastolic pressure is typically about 80 mm Hg.

Blood pressure in humans should be routinely checked and carefully monitored for abnormalities. Pressures at levels higher or lower than normal pose serious health risks. For instance, high blood pressure, which is also referred to as hypertension, is one of the principal causes of atherosclerosis, heart disease, and kidney disease. High blood pressure is caused by the constriction or narrowing of the arteries. When the arteries are constricted, the heart must pump more forcefully in order to move the blood into the capillaries. During rapid and dramatic increases in blood pressure, herein referred to as hypertensive emergencies, steps should be taken immediately to reduce the pressures before any permanent damage occurs to the heart or any internal organs. Thus, those skilled in the art have been searching for various pharmaceutical drugs capable of reducing blood pressure, especially during hypertensive emergencies. Currently, sodium nitroprusside is clinically used during such emergencies. Sodium nitroprusside is a rapid- acting vasodilator, active on both arteries and veins. A vasodilator refers to an agent that causes blood vessel dilation, thus reducing blood pressure. Specifically, sodium nitroprusside relaxes the vascular smooth muscle that surrounds peripheral arteries and veins. Sodium nitroprusside is more active on veins than on arteries. Dilation of the veins promotes peripheral pooling of blood and decreases venous return to the heart, thereby reducing left ventricular pressure and pulmonary capillary wedge pressure. Dilation of the arteries, on the other hand, reduces vascular resistance, systolic arterial pressure and mean arterial pressure. Sodium nitroprusside is particularly well adapted for use during hypertensive emergencies because, once injected into the blood stream, the drug has a quick hypotensive effect, typically reducing blood pressure within a minute or two after infusion. Further, sodium nitroprusside dissipates rapidly after infusion is discontinued. Besides being used during hypertensive emergencies, sodium nitroprusside is also used to reduce blood loss during and after major surgical procedures. Sodium nitroprusside is also used for the treatment of acute congestive heart failure. Unfortunately, the use of sodium nitroprusside does have its disadvantages and drawbacks. For instance, once injected into the body, sodium nitroprusside reacts with hemoglobin producing cyanide as a side product, which is toxic and poisonous to humans. Cyanide, when present in sufficient concentrations, can prevent the blood from delivering oxygen to various parts of the body, leading to metabolic acidosis, oxygen depletion, confusion, and death. Consequently, sodium nitroprusside can only be administered to patients within a small range of dosages, can only be administered for short periods of time, should not be administered repeatedly, and cannot be used on patients particularly susceptible to cyanide. Another disadvantage to using sodium nitroprusside is that the tissues of the body have a tendency to absorb the drug during use. Later, after infusion has discontinued, the drug is released by the tissues, causing a hypotensive state when not desired. This effect is referred to as a rebound phenomenon.

Besides having toxic side effects and exhibiting rebound phenomenon, sodium nitroprusside is a labile and very unstable compound. For example, sodium nitroprusside degrades when exposed to light and cannot be stored for long periods of time. Thus, sodium nitroprusside must be handled very carefully prior to and during use.

In view of the above problems experienced with sodium nitroprusside, a need exists for a pharmaceutical agent capable of reducing blood pressure for use during hypertensive emergencies and during other situations when a hypotensive agent is needed.

Summary of the Invention The present invention recognizes and addresses the above noted drawbacks and deficiencies of the prior art. Accordingly, it is an object of the present invention to provide a therapeutic agent for reducing blood pressure. It is another object of the present invention to provide a composition and process for reducing blood pressure during hypertensive emergencies.

Another object of the present invention is to provide a composition containing an adenylated dinucleotide for decreasing blood pressure.

Still another object of the present invention is to provide a composition and process for reducing blood pressure with improved safety.

Another object of the present invention is to provide a composition for reducing blood pressure that is naturally occurring.

Another object of the present invention is to provide a composition for reducing blood pressure that is stable and can be stored for extended periods of time.

Another object of the present invention is to provide a composition for reducing blood pressure that can be used during hypertensive emergencies, and during and after surgical procedures to reduce bleeding.

A further object of the present invention is to provide a lead compound for use in the development of novel therapeutic agents for reducing blood pressure and treating hypertension emergencies.

These and other objects of the present invention are achieved by providing a process for reducing blood pressure comprising the step of administering to a patient a composition containing an adenylated dinucleotide. The adenylated dinucleotide is administered to the patient in a pharmaceutically effective dosage sufficient to reduce the blood pressure of the patient. More particularly, the composition of the present invention is preferably administered to the patient intravenously, but it is believed that the composition can also be administered via other routes, i.e., subcutaneously and intramuscularly. Beside an adenylated dinucleotide, the composition can contain a liquid carrier, such as saline, plasma, or dextrose. In one preferred embodiment, the adenylated dinucleotide is an α,ω-adenine dinucleotide, such as diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, or diadenosine hexaphosphate. The composition can contain a single adenylate dinucleotide or a mixture of different adenylated dinucleotides.

In most applications, the adenylated dinucleotide is administered at a dosage level of from about 50 micrograms per kilogram body weight per minute to about 700 micrograms per kilogram body weight per minute, and more particularly between about 100 micrograms per kilogram body weight per minute to about 500 micrograms per kilogram body weight per minute. Because adenylated dinucleotides have been discovered to work very rapidly at reducing blood pressure and because recovery time after administration of the drug has ceased is also very rapid, the composition of the present invention is particularly well suited for use during hypertensive emergencies. The drug is also well adapted for use during and after cardiovascular surgery to reduce bleeding, and for use in treating acute congestive heart failure.

Other objects, features and aspects of the present invention are discussed in greater detail below.

Brief Description of the Drawings A full and enabling disclosure of the present invention, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying Figures, in which:

Figure 1 is a graphical representation of the results obtained in Example 1; Figure 2 is a graphical representation of the results obtained in Example 1 ; and

Figure 3 is a graphical representation of the results obtained in Example 1.

Figure 4 is a graphical representation of the results obtained in Example 4.

Figure 5 is a graphical representation of the results obtained in Example 4.

Figure 6 is a graphical representation of the results obtained in Example 4. Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention. Detailed Description of the Preferred Embodiments It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary constructions.

In general, the present invention is directed to a composition and process for reducing blood pressure. The process is directed to administering to a patient a composition containing an adenylated dinucleotide, which has been found to be a hypotensive agent rapidly and significantly reducing the mean arterial pressure in the body.

The composition is particularly well suited for use during hypertensive emergencies, during and after cardiovascular surgery in order to reduce bleeding, and for treating acute congestive heart failure. Once injected into the body, adenylated dinucleotides according to the present invention cause dilation of the arteries. This hypotensive effect occurs very rapidly, typically within ten seconds of administration. Once administered, mean arterial pressure is reduced and cardiac output is increased without significant effects on heart rate, right ventricular pressure, blood gas levels, or electrocardiogram morphology. Further, because adenylated dinucleotides are rapidly degraded by the body, the effects of the drug typically disappear within five minutes after infusion is ceased.

Adenylated dinucleotides, which were discovered in the 1950 *s, are naturally occurring molecules found in bacteria, yeast and animals, including humans. Specifically, adenylated dinucleotides are found in a diverse number of biological tissues including platelets, hepatocytes, adrenal chromaffin cells, and the central nervous system. The particular adenylated dinucleotides found well suited for use in the process of the present invention are referred to as the ,ω-adenine dinucleotides, which are also referred to as bis (5-adenosyl)polyphosphates.

The adenylated dinucleotides of the present invention consist of two adenosine moieties linked via their 5 position by a chain of two or more phosphates. As used herein, individual adenylated dinucleotides will be abbreviated as follows:

Ap_xA wherein x represents the number of phosphates in the connecting chain between the two adenosine moieties. The chemical formula for the adenylated dinucleotides is as follows:

Specific adenylated dinucleotides believed to be effective in reducing blood pressure are Ap₃A (diadenosine triphosphate) , Ap₄A (diadenosine tetraphosphate) , Ap₅A (diadenosine pentaphosphate) , and Ap₆A (diadenosine hexaphosphate) . In one preferred embodiment, Ap₄A is incorporated into the composition of the present invention.

Although naturally occurring, adenylated dinucleotides can be easily synthesized. For instance, adenylated dinucleotides are commercially available from Sigma Chemical Company in St. Louis, Missouri. In pure form, adenylated dinucleotides generally are available as a powder.

In accordance with standard methods well known to those skilled in the art, adenylated dinucleotides can be used as a lead compound to create analog compounds for use in the process of the present invention. Analog compounds possess similar biological functions, but differ in their chemical structure. Such compounds may be created by substituting different functional groups, such as methyl, acetyl, amino or hydroxyl groups, for those already present on the parent compound. Additional functional groups may also be added to the parent adenylated dinucleotide. The resulting compounds may exhibit more desirable therapeutic properties, or be more pharmacologically active, than the parent molecule. Standard texts such as "PRINCIPLES OF MEDICINAL CHEMISTRY," William O. Foye, Thomas L. Lemke, and David A. Williams, editors, 4th Ed., 1995, may be consulted to produce such compounds, without undue experimentation.

When administered to a patient according to the process of the present invention, an adenylated dinucleotide is combined with a carrier and injected into the arterial system. Examples of carriers that may be combined with an adenylated dinucleotide powder to form a solution include saline, plasma or dextrose. In general, any suitable carrier may be used that allows the drug to be administered intravenously without any adverse effects.

Treatment with adenylated dinucleotides can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the particular patient and the route of administration. The route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal), or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous or intraperitoneal) . Specific adenylated dineucleotides can be administered alone, or can be coadministered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) , such as sterile suspensions or emulsions. Adenylated dineucleotides may be administered in admixture with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard pharmaceutical texts, such as "REMINGTON'S

PHARMACEUTICAL SCIENCE," 17th Ed., 1985, may be consulted to prepare suitable preparations.

As mentioned above, the effective dosage and route of administration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the host as well as LD₅₀ and other screening procedures which are known.

When injected intravenously into the blood stream, a solution containing an adenylated dinucleotide only takes a few seconds to be distributed throughout the body. The dosage level that is administered to a particular individual will vary depending upon a number of factors. For instance, the dosage level will depend on the age and size of the patient, the patient's condition, the reason for administering the drug, and the amount of reduction in blood pressure that is desired. For most applications, it is believed that a dosage level between about 50 micrograms (μg) per kilogram (kg) of body weight per minute to about 700 μg per kg body weight per minute will be adequate regardless of the application. More particularly, in most applications, it is believed that the dosage level will be between 100 μg per kg body weight per minute to about 500 μg per kg body weight per minute. The composition of the present invention containing an adenylated dinucleotide is administered to a patient until the hypertensive emergency is ceased or until the desired effect has been obtained. As described above, it has been discovered that adenylated dinucleotides administered in accordance with the present invention induce a rapid dose dependent depression of the mean arterial pressure without significant effects on vein dilation, right ventricular pressure or heart rate. Once the drug is injected, reduction in blood pressure can occur within ten seconds, reaching a maximum effect within three minutes. Recovery to baseline upon cessation of the infusion is also very rapid, typically taking less than two minutes. Adenylated dinucleotides have also been found to increase cardiac output by relaxing the heart muscle, thereby increasing the volume of the heart chamber. Of particular advantage, adenylated dinucleotides have the above described effect on the circulatory system, without significantly affecting any other hemodynamic factors. The mechanism by which adenylated dinucleotides reduce blood pressure according to the present invention remains unknown. However, AP₄A has been shown to modulate a number of biological processes including blood vessel tone. This action of AP₄A may be due to activation of a second message system, since evidence has shown that extracellular AP₄A initiates a redistribution of intracellular calcium, an ubiquitous second messenger.

Recent data suggest that AP₄A acts as a vasodilator by inducing endothelial cells to release nitrous oxide (NO) through the eNOS pathway. Nitric Oxide Sythase (NOS) generates NO from the terminal guanidino nitrogen of L-arginine, yielding citrulline as a by-product. Two classes of NOS have been identified; one of which is a constitutive calcium-dependent isozyme that is found in vascular endothelium (eNOS) . A transient elevation in extracellular calcium promotes calcium binding to calmodulin and, once formed, this calcium-calmodulin complex activates eNOS. After the production of NO catalyzed by the action of eNOS, NO relaxes blood vessels by diffusing across the endothelial cell membranes and binding to iron in the heme of vascular smooth muscle guanylate cyclase, thereby activating the enzyme to generate cGMP. It is the action of cGMP, believed to occur through several mechanisms, which elicits smooth muscle relaxation.

Consistent with the above hypothesis, the inventors have demonstrated that AP₄A induces the release of NO from endothelial cells in a dose- dependent fashion. Adenylated dinucleotides are particularly well suited for use during hypertensive emergencies and during and after cardiovascular surgery to reduce bleeding. Adenylated dinucleotides are naturally occurring, are non-toxic, and can be administered in broad ranges. The drug does not undergo a rebound phenomenon and is completely stable and storable.

The present invention may be better understood with reference to the following examples:

EXAMPLE 1 The following tests were performed in order to demonstrate that an adenylated dinucleotide (Ap₄A) rapidly and significantly reduces the mean arterial pressure and is rapidly degraded once administration is discontinued.

Two domestic swine, weighing 13 to 18 kilograms were pre-anesthetized with ketamine, given intramuscularly, and placed under general anesthesia. Swine were selected for this experiment because they share many similarities with humans in cardiovascular function and morphology. The swine were ventilated at 17 to 20 cm H₂0, 12 to 16 breaths per minute, and 10 to 15 ml/kg tidal volume. Blood gases were stabilized at a ph of 7.36 to 7.42, pC0₂ of 44.1 to 47.3, p0₂ of 112 to 118 at 36.4 to 37.1°C body temperature. Once a baseline was established for mean arterial pressure, right ventricular pressure and heart rate, infusion of a solution containing Ap₄A dissolved in physiologic saline using an IMED infusion pump was initiated for 3 minutes at each of the following rates: 100 μg/kg/min, 150 μg/kg/min, 200 μg/kg/min, 250 μg/kg/min, 300 μg/kg/min, 350 μg/kg/min, and 700 μg/kg/min.

Administration of successive increments of the composition was delayed between dosages until cardiovascular parameters had returned to the established baseline. The infusion was administered in the precava at the entrance to the right atrium. Blood pressure was continuously monitored with catheters placed in the aortic arch via the carotid artery and in the right ventricle via the external jugular vein. Figures 1, 2 and 3 graphically represent the results obtained during the test. As shown in Figure 1, in both animals, the mean arterial pressure (MAP) was depressed in a dose dependent fashion without significant effects on right ventricular (RV) pressure. Specifically, Figure 1 illustrates the relative percent of baseline values for each dose at the end of the three minute infusion.

Figure 2 shows the percent change in mean arterial pressure (MAP) for each dosage level, during each three minute infusion. As shown, once infusion was started, the adenylated dinucleotide began reducing blood pressure within ten seconds and reached maximum effect after three minutes. Figure 3 illustrates the change in mean arterial pressure (MAP) after infusion of the adenylated dinucleotide at 700 μg/kg/min was discontinued. As shown, recovery time to baseline was approximately two minutes. In fact, it was observed that for all dosages, the recovery time was two minutes or less.

During the experiment, significant cardiac arrhythmias and changes in electrocardiogram (EKG) morphology were not noted. EXAMPLE 2

Two additional swine were anesthetized and prepared as described in Example 1. In this Example, however, more extensive monitoring was performed on the animals. Specifically, a Swan- Ganz catheter was placed in the right ventricle via venesection of the right femoral vein. The carotid artery catheter was also advanced into the left ventricle. In addition to the measurements made in Example 1, cardiac output was determined using the thermodilution method with a CO computer. Using fluoroscopy, the Swan-Ganz catheter was passed into a small branch of the pulmonary artery and, by using standard balloon inflation and pullback techniques, the following pressure measurements were made: pulmonary capillary wedge, pulmonary artery, right ventricle, right atrium, and inferior venacave.

A solution containing the Ap₄A adenylated dinucleotide dissolved in saline, was infused for thirty minutes at a rate of 200 μg/kg/min and for ten minutes at a rate of 700 μg/kg/min in one of the animals. In the second animal, the solution was injected such that Ap₄A was administered at a rate of 200 μg/kg/min for fifteen minutes and, following the infusion, for fifteen minutes at a rate of 700 μg/kg/min. For both animals, pressure values were measured at baseline, half way through the infusion for each rate, at the end of the infusion, and following a return to baseline values five minutes after stopping the infusion. The results obtained in this Example were similar to Example 1. Steady state hemodynamics were achieved within three minutes after starting infusion at either dosage rate. The systemic arterial pressure decreased twelve to twenty percent at 200 μg/kg/min and thirty to forty percent at 700 μg/kg/min, without any significant alteration in venous pressures, heart rate or blood gas values. The CO was improved at both dosages, eighteen to twenty percent at 200 μg/kg/min and twenty-seven to thirty-two percent at 700 μg/kg/min. Simultaneously, the ejection fraction increased approximately ten percent. Systemic vascular resistance was reduced twenty-eight to thirty-six percent at all dosages in both pigs. All values returned to approximate baseline values within five minutes post infusion. All other hemodynamic values were not significantly affected.

EXAMPLE 3 A 20 kg male Yucatan miniature pig was used in an acute experiment to develop a model of acute renal hypertension and to test the effects of an adenylated dinucleotide on the condition.

The pig was anesthetized as described in Example 1 and catheters were placed in the internal jugular vein and carotid artery to monitor peripheral blood pressure. An ear vein was used to administer a 0.9 percent sodium chloride solution at a rate of 20 ml/kg/hour following an IV bolus. The external jugular vein was cannulated for the administration of the compound.

The kidneys of the pig were surgically approached through a mid-line celiotomy. A left nephrectomy was performed, reducing the mean renal blood pressure in the right kidney from 45 mmHg to 11 mmHg using a suture ligature, while monitoring the pressure via a needle attached to a transducer line in the distal segment of the artery. The systemic mean arterial pressure rose from 63 mmHg to 83 mmHg over the course of an hour.

Infusion of a solution containing Ap₄A and saline at an Ap₄A dosage level of 200 μg/kg/min reduced the mean arterial pressure to 63 mmHg.

Apparent catecholamine induced compensation raised the mean arterial pressure to 74 mmHg a minute later. This rise was rapidly followed by a steady state mean arterial pressure of 68 mmHg. After five minutes, the dosage rate was changed to 700 μg/kg/min, which resulted in a mean arterial pressure decrease to 55 mmHg. The pressure remained in a steady state for the five minute duration of the infusion. Except for the time during which the heart attempted to compensate in the initial stages of the infusion, the heart rate was not significantly different from the baseline. The mean arterial pressure rose to 82 mmHg within one minute of stopping the infusion.

This Example particularly demonstrates how an adenylated dinucleotide administered according to the process of the present invention can be used for treating hypertensive emergencies, such as renal hypertensive emergencies.

EXAMPLE 4

The following Example demonstrates that AP₄A induces the release of nitrous oxide (NO) from endothelial cells in a dose-dependent fashion.

NO was measured using the ISO-NO Mark II Isolated Nitric Oxide Meter. To measure NO, bovine arterial endothelial cells (BAEC) were grown in 35 mm gelatin-coated petri dishes. Cells were washed three (3) times with Krebs-Henseleit buffer [10 mM Hepes (pH 7.4), 120 mM NaCl, 4.6 mM KCI, 1.5 mM CaCl₂, 0.5 mM MgCl₂, 1.5 mM NaH₂P0₄ , 0.7 mM Na₂HP0₄ , 10 mM glucose], resuspended in 2.0 ml of Krebs- Henseleit buffer and incubated for sixty (60) minutes at 37°C in a humidified atmosphere of 95% air-5% C0₂. The cells were then washed three (3) times with Krebs-Henseleit buffer and resuspended in 2.0 ml of Krebs-Henseleit buffer containing 10 U/ml of superoxide dismutase.

To measure NO release, the sensor probe was inserted vertically into the petri dish containing BAEC, so that the tip of the probe was dipped about 1 mm into the solution. The dishes were not capped and all measurements were performed at room temperature. The data was collected for 60 min. (5 samples/sec) and analyzed using the DUO 18 2 Channel, 18-Bit Data Recording System.

The total nmoles of NO were determined after 60 min. of incubation by integration of the curve. Signal to background ratio was 475 (+/-50) . Basal levels of NO varied from 0.3 to 0.4 nmoles/min/10⁶ cells, and were zeroed out prior to starting the experiments with the addition of AP₄A. Various concentrations of AP₄A, ranging from 0.5 to 2.5 μM, were added to the cells, and the amount of NO released was measured.

Figure 4 graphically represents the results obtained during the test. As shown in Figure 4, NO was released in a linear fashion with an AP₄A concentration ranging from 0.5 to 2.5 μM. NO release reached a maximum of 0.84 nmole/min/10⁶ cells with a AP₄A concentration of 3.75 μM.

In subsequent experiments, the effect of L- arginine (L-Arg) , the biosynthetic precursor of eNOS, and L-NMA and L-NNA, competitive inhibitors of eNOS, were determined on AP₄A induced NO release. BAEC were grown in 35 mm gelatin-coated petri dishes, as described above. Cells were incubated for 60 minutes at 37°C with varying concentrations of L-NMA or L-NNA, prior to the addition of 6 uM AP₄A. Additional cultures were incubated for 60 minutes at 37°C with 100 μM L-NNA and varying concentrations of L-Arg, prior to the addition of 6 μM AP₄A. NO release was recorded and analyzed as described above. As shown in Figure 5, both L-NMA and L-NNA analogs inhibit the release of NO in a dose dependent fashion. Approximately 50% of NO released is inhibited at 10 and 22 μM by L-NMA and L-NNA, respectively. The ability of L-Arg to overcome the inhibitory effect of L-NNA is demonstrated in Figure 6, where variable amounts of L-Arg and a fixed amount of L-NNA were incubated with BAEC for 60 minutes at 37°C, prior to inducing NO synthesis with AP₄A. L-Arg, as expected, effectively reverses the L-NNA inhibition of NO release. These data are consistent with AP₄A inducing NO release through the eNOS pathway.

These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.

Claims

WHAT IS CLAIMED IS;

1. A method for reducing blood pressure in human or animal patients comprising administering to said patient a therapeutically active dosage of a composition comprising an adenylated dinucleotide for a time effective to decrease the blood pressure in said patient.

2. A method according to claim 1, wherein said blood pressure is decreased by reducing mean arterial pressure and increasing cardiac output, without significantly affecting vein dilation, right ventricular pressure, and heart rate.

3. A method according to claim 1, wherein said adenylated dinucleotide comprises a naturally occurring adenylated dinucleotide.

4. A method according to claim 1, wherein said adenylated dinucleotide comprises a synthesized adenylated dinucleotide.

5. A method according to claim 1, wherein said adenylated dinucleotide is an ,╧ë-adenine dinucleotide selected from the group consisting of diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, diadenosine hexaphosphate and mixtures thereof.

6. A method according to claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier being chosen from the group consisting of plasma, saline, dextrose and mixtures thereof.

7. A method according to claim 1, wherein said adenylated dinucleotide comprises diadenosine tetraphosphate.

8. A method according to claim 1, wherein the therapeutic dosage is in a range of about 50 micrograms per kilogram of body weight per minute to about 700 micrograms per kilogram of body weight per minute.

9. A method according to claim 1, wherein said composition is administered via a mucosal or a parenteral route.

10. A method according to claim 1, wherein said composition is administered to a patient at a dosage and for a time sufficient to reduce bleeding.

11. A method according to claim 1, wherein said composition is administered to a patient at a dosage and for a time sufficient to treat congestive heart failure.

12. A therapeutic composition for reducing blood pressure in a patient comprising an adenylated dinucleotide and a pharmaceutically acceptable carrier.

13. A therapeutic composition as defined in claim 12, wherein said adenylated dinucleotide comprises a naturally occurring adenylated dinucleotide.

14. A therapeutic composition as defined in claim 12, wherein said adenylated dinucleotide comprises a synthesized adenylated dinucleotide.

15. A therapeutic composition as defined in claim 12, wherein said adenylated dinucleotide is an ,╧ë-adenine dinucleotide selected from the group consisting of diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, diadenosine hexaphosphate and mixtures thereof.

16. A therapeutic composition as defined in claim 12, wherein said pharmaceutically acceptable carrier is chosen from the group consisting of plasma, saline and dextrose.

17. A method for reducing blood pressure in human or animal patients comprising administering to said patient a therapeutically active dosage of a composition comprising an adenylated dinucleotide and a pharmaceutically acceptable carrier, wherein said adenylated dinucleotide is an ,╧ë-adenine dinucleotide selected from the group consisting of diadenosine triphosphate, diadenosine tetraphosphate, diadenosine pentaphosphate, diadenosine hexaphosphate and mixtures thereof.

18. A method according to claim 17, wherein said therapeutically active composition is administered in a dosage range of from about 50 ╬╝M/kg/min to about 700 ╬╝M/kg/min.

19. A method according to claim 17, wherein said therapeutically active composition comprises a naturally occurring adenylated dinucleotide.

20. A method according to claim 17, wherein said therapeutically active composition comprises a synthesized adenylated dinucleotide.

21. A method according to claim 17, wherein said therapeutically active composition is administered to a patient at a dosage and for a time sufficient to reduce bleeding.

22. A method according to claim 17, wherein said composition is administered to a patient at a dosage and for a time sufficient to treat congestive heart failure.

23. A method according to claim 17, wherein said adenylated dinucleotide comprises diadenosine tetraphosphate.