AU2004242565A1 - Treatment of hypertension with compounds that inhibit the destruction of enkephalins or endorphins - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT(S): Seymour Ehrenpreis

AND

Kenneth Blum ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Nicholson Street Melbourne, 3000.

INVENTION TITLE: "Treatment of hypertension with compounds that inhibit the destruction of enkephalins or endorphins" The following statement is a full description of this invention, including the best method of performing it known to us: Q:\OPER\jrc\2004-2005 Financial Year\Specificalion Filing\ 2553080 div file.doc 31/12/04 TREATMENT OF HYPERTENSION WITH COMPOUNDS THAT INHIBIT THE N DESTRUCTION OF ENKEPHALINS OR ENDORPHINS This application is a divisional of Australian Application No. 25027/00, the entire contents of c which is incorporated herein by reference.

lA FIELD OF THE INVENTION In This invention relates to hypertension or high blood pressure. More specifically, this invention relates to treatment of animals and humans with a substance that inhibits and/or delays the inherent breakdown of a class of substances called enkephalins and/or endorphins that are created and exist within animals. These substances, the enkephalins and endorphins, can lower blood pressure; however, they are rapidly destroyed by endogenous enzymes and ordinarily do not play a major role in controlling blood pressure. However, by protecting the enkephalins and/or endorphins from destruction through the use of enzyme inhibitors, these substances are caused to accumulate, resulting in the- lowering of blood pressure. The amino acid D-phenylalanine (DPA) or the D,L racemic mixture, is such an enzyme inhibitor and is representative of a new class of compounds or drugs which can lower elevated blood pressure by raising body levels of enkephalins and/or endorphins.

DISCUSSION

There are a number of classes of drugs which are currently in use to treat hypertension. These consist of the following: idrugs which modify the adrenergic part of the autonomic nervous system; drugs which dilate blood vessels; diuretics, drugs that inhibit angiotensin converting enzyme (ACE inhibitors); drugs that block calcium channels; and drugs that block angiotensin receptors. Each of these classes of drugs, although highly effective in lowering blood pressure, suffers from a number of very important side effects. Thus the search goes on continuously for better ways to produce anti-hypertensive effects.

The enkephalins and endorphins are peptides which are present in the central nervous system and many other tissues including the blood. Although their main function appears to be control of the pain response, they have many other actions including lowering of blood pressure in some animal species when they are injected 2 0 into the blood stream. However, there are certain drawbacks in using these naturally occurring substances to treat hypertension in humans. They cannot be used orally and they have an additional drawback of having a very short duration of action due to their rapid destruction by a variety of enzymes called enkephalinases or endorphinases.

These enzymes are found everywhere in the body and rapidly destroy any endorphins or enkephalins which are given exogenously or produced endogenously. Therefore, it C, is reasonable to presume that if these enzymes are inhibited by means of compounds or drugs termed enkephalinase or endorphinase inhibitors, thereby preventing these CN1 enzymes from destroying the peptides. the peptides would accumulate in the body and effect a blood pressure lowering action. There are a number of enkephalinase inhibitors which are known at this time, but the only one which has been studied extensively in man is D-phenylalanine. The inventors have previously shown that D-phenylalanine, by virtue of its enkephalinase inhibiting activity, can produce analgesia in animals and man. The inventors have since discovered that D-phenylalanine, by a similar mechanism, is also highly effective in lowering blood pressure in animals and man and this is the basis for the new invention. There have been no other proposals to use this approach for this purpose.

In actual practice, it is expected that the racemic mixture, D,L-phenylalanine would be used for therapeutic purposes because of the considerably lower cost of the racemic mixture as compared with pure D-phenylalanine. It is understood that, as previously described, only D-phenylalanine is the amino acid form which actually inhibits the destruction of the enkephalins. D-phenylalanine and D,L-phenylalanine are long known compounds and listed in the Merck Index.

Use of phenylalanine has been reported from the Faculty of Medicine, Buenos Aires, Argentina (Forsch, 1975; 1976). In the report on treatment of depression, D,L-phenylalanine was administered in quantity of 50 or 100 mg per day for 15 days, and D-phenylalanine was administered in quantity of 100 mg per day for 15 days.

A commercial drug, sold under the Trademark "Deprenon", is available for treatment of depression, by oral ingestion of 3-4 capsules per day. Deprenon's specifications state that each capsule contains: c D-phenylalanine 50 mg Manitol 90 mg _Pervidone 4 mg Magnesium Stearate 3 mg As indicated above, D-phenvlalanine and D,L-phenylalanine also possess I analgesic activity by virtue of the accumulated enkephalins and/or endorphins within Sthe central nervous system. D,L-phenylalanine is currently being sold over the counter in the United States and England as DLPA. For-this purpose, it is Sadministered in divided oral doses of 1-2 grams per day for up to several weeks.

Other inhibitors of enkephalin degradation include D-leucine, bestatin, thiorphan, bacitracin, puromycin, and captopril. These compounds also produce analgesia, as shown primarily in animals, but have not been tested for their effects on blood pressure in animals or man.

A method of choice for evaluating anti-hypertensive drugs before administration to humans is to determine their effectiveness in lowering blood pressure in the spontaneously hypertensive rat (SHR). This genetic variant, at adulthood, develops very high blood pressure systolic in the range of 180 220 mm Hg, diastolic 150 170 mm Hg. A substance which successfully lowers blood pressure in the SHR without causing appreciable side effects would be considered as a good candidate for hypertensive therapy in humans.

D-ribose is a naturally occurring pentose monosaccharide containing a functional aldehyde group and an alcohol group. D-ribose is used by the body in the synthesis of nucleotides and metabolic intermediates such as R-5-P is an important intermediate of the pentose phosphate pathway (PPP) of glucose metabolism (also known as the hexose monophosphate shunt or the phosphogluconate pathway).

D-ribose enters the PPP by being phosphorylated to The PPP results in the formation of NADPH and pentose-based molecules in animal cells. This pathway is especially pronminent in tissues actively carrying out the blosynthesis of fatty acids and steroids from small precursors where reducing power 4 ,1 (NADPH) is needed. The pathway is also active in human and animal crythrocytes.

The NADPH produced is required for preventing the unsaturated fatty acids in the cell membrane from undergoing abnormal reactions with oxygen and for keeping the iron atoms of hemoglobin in their normal ferrous valence state. Pentose molecules g 5 generated by way of the PPP are necessary for the synthesis of nucleic acids, glycogen I and glucose. The biosynthesis of nucleic acids, for example, requires phosphoribosyl-l-pyrophosphate (PRPP) which is derived from ribose. In that regard, ribose is utilized by many different tissues in animals and in man, including the heart c and skeletal muscle.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. IA shows the adenine nucleotide de novo synthetic pathway: phosphate and PRPP are early precursors in this pathway. The interrelationship between the de novo pathway and the degradative and salvage pathways has been demonstrated.

FIG. 1B shows adenine nucleotide synthetic pathways: Pathways 2 and 3 are considered the most active. The two entrance sites of phosphoribosyl pyrophosphate (PRPP) into the salvage pathways is demonstrated.

FIG. 1C shows adenine nucleotide degradation pathways: Degradation of AMP to the diffusable metabolites, adenosine, inosine and hypoxanthine is enhanced during ischemia.

FIG. ID shows blood pressure lowering activity of D-phenylalanine in spontaneously hypertensive rat (tail cuff method).

FIG. IE shows blood pressure lowering activity of D-phenylalanine in spontaneously hypertensive rat (cannulation method).

FIG. 2 shows a dose response to D-phenylalanine for blood pressure lowering in SHR (tail cuff method).

FIG. 3 shows the synergistic effect on blood pressure lowering in SHR by Dphenylalanine and propranolol.

FIG. 3A shows the effect of DPA on blood pressure in normotensive rats (cannulation method).

FIG. 3B shows blockage of anti-hypertensive DPA effects by naltrexone.

FIG. 3C shows reversal of DPA hypotension by naltrexone.

FIG. 4 shows hypotensive effects of DPA in humans.

FIG. 5A shows effects of DPA on blood pressure in SHR.

I 5 FIG. 5B shows effects of thiorphan and actinonin on blood pressure in SHR.

in FIG. 6 shows pharmacokinetics of DPA given orally to a human subject.

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SUMMARY OF THE INVENTION D-phcnylalanine was tested for its anti-hypertensive action in the spontaneously hypertensive rat in the laboratory of a major Japanese pharmaceutical 0 company. They confirmed completely the inventors' observation that DPA at 400 mg/kg gives a significant lowering of blood pressure with a time-course very similar to what the inventors found. Three rats were used. Also tested were two other inhibitors of enkephalin degradation, thiorphan and actinonin. Neither of these were effective in lowering blood pressure even though used at very high doses. In the case of thiorphan this could be explained by lack of penetration into the central nervous system which appears to be the site of action of DPA. We cannot explain the lack of effectiveness for actinonin but it too may not enter the brain in sufficiently high concentration to effect significant inhibition of the enkephalin degrading enzymes.

Thus on the basis of these results, it would appear that DPA is unique among enkephalinase inhibitors in lowering blood pressure, but other EICI inhibitors should not be ruled out.

Data shows the pharmacokinetics of DPA given orally to a human subject at a dose of 2 grams dissolved in 250 ml of water. Phenylalanine of course if present in the blood and the amount present before DPA administration was subtracted from that obtained at each data point. As can be seen, the elimination half life is about 6 h although a significant amount is still present in serum 10 h. after administration.

An important aspect of the present invention is a method for anti-hypertensive therapy in a human. This method comprises administering an effective amount of a substance that inhibits the destruction of enkephalins or endorphins. A preferred 6 inhibitor of endorphinase or enkephalinase is D-phenylalanine, and a preferred mode of administration is oral by tablet or as a dietary component. Such D-phenylalanine Smay be administered as the D-isomer or as part of the D,L-racemic mixture. Such Dphenylalanine is preferably administered in an amount of about 2 to 4 grams daily. In 0 5 some cases the inhibitor may be accompanied by other anti-hypertensive agents such as propranolol or another P blocker.

c, The present invention envisions the use as an anti-hypertensive agent of: D- O phenylalanine; D-leucine or DL-leucine, a combination of D-phenylalanine and D- C"1 leucine; bestatin; thiorphan; captopril: and/or puromycin. These substances may be used individually or in combination. In one preferred aspect, the D-phenylalanine is used in combination with a separate anti-hypertensive agent. One separate antihypertensive agent is a diuretic or blood vessel dilator.

Dietary supplements comprising an inhibitor of endorphin or enkephalin destruction are another preferred aspect of the present invention. These inhibitors of enkephalin or endorphin destruction in one aspect are preferably combined with Dribose. These inhibitors of endorphin or enkephalin destruction are often inhibitors of the appropriate hydrolytic enzymes destroying these substances, i.e. endorphinases or enkephalinases. Other inhibitors that may be used in combination with, or perhaps instead of, D-phenylalanine are hydrocinnamic acid and D,L-leucine. In some cases the anti-hypertensive effects of inhibitors of endorphin or enkephalin destruction may also involve administration of at least one of ferrulic acid, pharmaline, huperzine, at least one of a chromium salt such as, chromium picolinate, chromium nicotinate.

and chromium polynicotinate, Co-enzyme Q, Pycogenol or Hawthorn or Hawthorn extract.

In one important aspect, the present invention provides a treatment of hypertension comprising administering an enkephalinase or endorphinase inhibitor in combination with a diuretic, sympatholytic, direct vasodilator, angiotensin-converting enzyme inhibitor, calcium channel blocker, angiotensin II receptor antagonist, a Ttype calcium antagonist such as nisoldipene, losartin, moxonidine or fenoldopam.

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cK One important aspect of the present invention involves a method for the treatment of hypertension involving administering an enkephalinase and/or S endorphinase inhibitor in combination with a stimulator of increased norepinephrine.

angiotensin 11 antagonists such as clonidine, and adrenergic receptor blockers.

The method of inhibiting hypertension comprising administration of an I enkephalinase or endorphinase inhibitor may also be combined with the Sadministration of an inhibitor of norepinephrine synthesis selected from a group consisting of methyl-p-tyrosine, carbidopa, diethyl, diethyldithiocarbonate. FA163 and Sdisulfiram or other inhibitors ofdopamine-p-hydroxylase.

0 On occasion the administration of enkephalinase or endorphinase inhibitors may also be combined effectively with the administration of a magnesium salt or a chromium salt. Preferred chromium salts include picolinate, nicotinate, chloride, acetate or nicotinic acid-glycine-cysteino-glutamic acid (NA-AA).

In some cases Rhodiola rhosea extract (pharmaline) may be useful in combination with the endorphinase or enkephalinase inhibitors as a method of lowering hypertension. One preferred Rhodiola extract is Salidrosid. Huperzine, Hawthorn berry or Hawthorn berry extract may also be combined with the enkephalinase or endorphinase inhibitors for lowering hypertension. An adrenergicp-blocking agent may be effectively combined with the enkephalinase or endorphinase inhibitors or other substances described herein for an effective hypertension-lowering agent.

An important aspect of the present invention is a pharmaceutical composition for lowering blood pressure, comprising D or D,L-phenylalanine, a chromium salt, Dribose, calcium chelate, L-taurine and L-glycine. In one important aspect, this composition also contains at least one of a brain cognitive enhancing amount of ferrulic acid, pharmaline and huperzine to increase focus, memory or attention.

A preferred pharmaceutical composition of the present invention is one comprising as a daily dose: D or D,L-phenylalanine, I mg to 10,000 mg; chromium salts (picolinate or nicotinate or other salts), 1 microgram to 30,000 micrograms; D- S8 ribose, 100 mg to 10.000 mg: calcium chelate, 10 mg to 3.000 mg; L-taurine, 10 mg to 10.000 mg; and L-glycine, 10 mg to 10,000 mg.

One object of this invention is to provide a new method for reducing blood pressure in animals and man which is safer than currently available means of t 5 achieving this.

Another object of this invention is to provide a new method for producing a longer lasting reduction in blood pressure than is possible by means of currently O available drugs.

N One additional object of this invention is to provide a new method for enhancing the effectiveness of currently available anti-hypertensive drugs, thereby permitting lower doses of such drugs to be used and in this way reducing the incidence of side effects from such drugs.

IESCRIPTION OF ILLUSTRATIVE

EMBODIMENTS

METHODOLOGY

Animal Studies The inventors have evaluated the effectiveness of D-phenylalanine in spontaneously hypertensive rats (SHR) by two methods: 1)By measuring blood pressure directly via an indwelling cannula and 2) By measuring blood pressure by means of a tail cuff.

Method 1: The rats were anesthetized with ether and a midline neck incision was made and the right carotid artery was exposed. A cannula was inserted into the artery and a pressure transducer was attached to the cannula. Blood pressure readings were recorded by means of a polygraph, Baseline blood pressure was recorded after the rat had fully recovered from anesthesia (approximately one hour after cannulation) and ranged between 190 to 220 mm Hg. Drugs were administered via the carotid artery cannula. This method permits measurement of both systolic and diastolic pressure.

Method 2: The rat was placed in a warmed chamber (30°C) and the tail was placed in a cuff which was connected to a blood pressure monitoring device. Blood 9 CN pressure (only systolic) was determined every minute, as well as heart rate. Injections D) of various solutions were made by the intraperitoneal route. This method permits blood pressure measurements to be made for several days.

Drug evaluation: Two different procedures were used: cumulative and single dose. In the single dose studies, one dose of DPA (generally 400 mg/kg) was I administered and the animal's blood pressure was followed for the next 6 h. In the cumulative procedure, 100 mg/kg DPA was administered as a bolus, and when the C1 effect had leveled off an addition 100 mg/kg was given followed by 200 mg/kg of the

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Scompound.

In studying the effect of a combination of drugs, DPA was given at a low dose together with a low dose of the second drug (propranolol) and the blood pressure followed for the next few hours. The doses chosen of each drug separately produced only a very small fall in blood pressure over this time period.

In other studies, two antogonists of endorphins, naloxone and natlrexone were administered to rats treated with DPA. Two procedures were used. In the first, a single dose of either drug (5 mg/kg) was administered followed by the usual doses of DPA as described above. In the second procedure, the naltrexone or naloxone was administered after the DPA had been given at a dose which produced a very significant fall in blood pressure (400 mg/kg). The blood pressure was then followed for the next several hours.

In addition to these studies with the SHR and hypertensive human subjects, DPA was administered to normotensive rats and humans after which their blood pressure was determined.

Enkephalinase inhibitors include a variety of materials, including thiorphan, captoril and puromycin.

Human studies a. Moderately hypertensive subjects.

Three subjects were studied. All subjects were seated in a quiet room for about 30 min before the first blood pressure and heart rates were determined. Systolic and diastolic blood pressures were measured by means of the conventional O 0 sphygmomanometer. Three measurements were made at 5 min intervals or until the blood pressure leveled off. At the same time, pulse rate was taken for I min. After a Cc suitable control period, the subjects were given a solution of 1 or 3 grams of DPA to drink. Blood pressure and heart rate were determined as described above for the next j 5 few hours. In one case, following an initial I gram of DPA, a second dose of I gram of DPA was given 4 h later.

In all cases, blood pressure was checked for one or more days after the initial O dose of DPA was administered.

RESULTS

Animal.studies Although there were some differences in the results with the two methods for measuring blood pressure, it is evident that D-phenylalanine can produce a very significant decrease in blood pressure in the SI-R. Using the tail cuff method, this decrease was apparent at 100 mg/kg (FIG. ID and FIG. or cannulation method (FIG. IE), whereas by the cannulation method the dose required for significant hypotensive (FIG. IA) effect was found to be 400 mg/kg. These results were obtained whether the DPA was administered in a cumulative or single (lose manner. At 400 mg/kg, D-phenylalanine produced a highly significant lowering of both systolic and diastolic blood pressures with no change in heart rate (Table As shown in Table 1 (cannulation data) the initial mean systolic blood pressure was 207.6 mm Hg mm Hg) and within six hours after infusion of DPA, was reduced to 170.8 mm Hg mm Hg). DPA lowered the systolic blood pressure an average of 36.8 mm Hg or 21.5 percent (P is less than DPA's effect on diastolic blood pressure was impressive as well. The mean diastolic blood pressure was initially 184.8 mm Hg (plus or minus 5 mm Hg) and was reduced to 150.2 mm H-g (plus or minus 5 mm Hg) within six hours after infusion. This represents an average decrease in diastolic blood pressure of 34.6 mm Hg or 23 percent. It may be noted that in some instances, D-phenylalanine was able to lower systolic blood pressure of the SHR very close to that of normal rats about 150 mm Hg (Table When DPA was administered along !1 with propranolol, a highly significant potentiation of the hypotensive effect was observed as compared with the effect of each compound given separately (FIG. 3).

This hypotensive effect lasted for several days (Table 2).

Table 1 Time Course for Lowering Systolic and Diastolic Blood Pressure in the Spontaneously Hypertensive Rat by D-Phenylalanine (400 mg/kg) Initial BP 10' 20' 30' I h 2h 6h TRIAL 1 Systolic BP 220 210 200 200 200 192 190 Diastolic BP 220 190 178 180 180 170 170 TRIAL II Systolic BP 220 220 204 202 202 194 192 Diastolic BP 200 200 190 188 186 172 172 TRIAL Ill Systolic BP 218 210 205 194 184 184 182 Diastolic BP 200 190 190 188 164 164 164 TRIAL IV Systolic BP 180 180 170 150 140 130 130 Diastolic BP 160 160 150 136 120 115 115 TRIAL V Systolic BP 200 190 190 182 174 174 160 Diastolic BP 164 160 160 160 158 148 130 Mean systolic BP of Trials I V 207.6 202 193.8 185.6 180 174.8 170.8 Mean diastolic BP of Trials I V 184.8 180 173.6 170.4 161.6 153.8 150.2

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O 12 0 Table 2 Q Long Term Decrease in Blood Pressure Produced in the Spontaneously Hypertensive Rat by D-phenylalanine and D-phenylalanine plus propranolol Change in Blood Pressure in (Systolic) SDrug and Dose Day 1 Day 2 Day 3 Day 4 Day NM g/kg SDPA, 400 27.8 26.6 18.3 DPA, 400 13.0 4.6 DPA, 400 12.9 9.4 2.9 DPA, 200 8.4 16.8 0 Propranolol, 1.5 Propranolol, 5 17.9 DPA, 150 plus Propranolol 5 29.7 DPA. 200 plus Propranolol, 1.5 23.2 3.1 DPA, 200, plus Propranolol, 1.5 26.0 15.8 Similar results were obtained by the tail cuff method. Using this method, it was shown that the hypotensive effect of DPA was still apparent for several days after its administration (Table 2).

It is of great importance that D-phenylalanine had little or no effect on the blood pressure of rats with normal blood pressure (FIG. 3A) (Table This is a very desirable feature of an anti-hypertensive agent, that is, to selectively produce a fall in blood pressure when it is elevated and not to cause marked changes when it is normal.

The same is true for humans (see below). In all instances, the hypotensive effect of D-phenylalanine was not accompanied by a change in heart rate.

O 13 (c Table 3 0 D) Lack of Anti-hypertensive Effect of D-phenylalanine in Rats with Normal Blood Pressure Initial BP 10' 20' 30' Ih 2h 6 h TRIAL 1(140 mg/kg) O Systolic BP 130 132 130 130 130 130 130 V) Diastolic BP 86 84 84 84 86 84 84 N TRIAL II (200 mg/kg) SystolicBP 150 154 154 150 152 154 152 Diastolic BP 86 90 88 88 90 92 TRIAL 111 (400 mg/kg) Systolic BP 124 124 124 122 124 120 122 Diastolic BP 84 86 84 82 82 82 84 TRIAL IV Systolic BP 146 148 146 150 150 146 146 Diastolic BP 86 86 88 90 90 88 88 In another study, animals were pretreated with the specific enkephalin or endorphin antagonist naltrexone for one-half hour followed by infusion of D-phenylalanine. Under these conditions (FIG. 3B). The effects of D-phenylalanine were completely blocked by the naltrexone (Table Alternatively, administration of naltrexone following D-phenylalanine completely reversed the hypotensive effect of the D-phenylalanine (Table Naltrexone itself (FIG. 3C) produced no change in blood pressure. These results indicate that the hypotensive effect of D-phenylalanine is mediated via the endorphin system endogenous enkephalins and/or endorphins) and suggest that other substances which increase levels of enkephalins and/or endorphins could also be used successfully to lower the blood pressure. Another amino acid, D-leucine, has previously been shown to act in a manner similar to D-phenylalanine in blocking degradation of enkephalins.

Table 4 Inhibition by Naltrexone of Anti-hypertensive Effect of D-phenylalanine in SHR A. Effect of naltrexone alone on blood pressure Dose of naltrexone 1 mg/kg plus 1 mg/kg plus 3 mg/kg TRIAL 1 Systolic BP 220 224 222 Diastolic BP 180 180 182 TRIAL II Systolic BP 218 218 220 Diastolic BP 180 178 182 B. Effect of DPA 400 mg/kg following prctreatment with naltrexone 5 mg/kg.

Initial BP 30' 1 h 1.5 h 2 h TRIAL I Systolic BP 220 222 220 220 220 Diastolic BP 180 180 182 180 180 TRIAL 11 Systolic BP 238 240 240 240 240 Diastolic BP 210 208 210 210 210 TRIAL III Systolic BP 224 224 224 220 222 Diastolic BP 200 200 198 196 200 TRIAL IV Systolic BP 220 220 224 220 222 Diastolic BP 200 202 200 198 200 115 STable Reversal of Anti-hypertensive Effect of D-phenylalanine by Naltrexone C DPA 400 mg/kg was followed 30 min later by naltrexone 5 mg/kg Initial BP 30' 1 h 1.5 h 2 h Itf TRIAL I Systolic BP 188 140 194 192 192 SDiastolic BP 168 110 140 144 144 TRIAL II Systolic BP 198 180 200 204 204 CI Diastolic BP 160 140 144 144 144 TRIAL III Systolic BP 228 184 228 224 224 Diastolic BP 180 140 160 180 180 TRIAL IV Systolic BP 226 180 220 224 224 Diastolic BP 182 142 180 184 184 Human results a. Subjects with elevated blood pressure As shown in FIG. 4, DPA caused a pronounced decrease in systolic blood pressure in the 3 subjects studied. When 3 grams of DPA was given, reduction in the blood pressure persisted for many hours and even days (Table On the other hand, I gm of DPA reduced blood pressure for a relatively short period of lime (4 h) when given initially; however, the second 1 gram dose of DPA evidently had a much longer lasting or cumulative effect.

16 0 Table 6 Long Time Decrease in Blood Pressure Produced by D-phenylalanine in Humans ¢m% Systolic Blood Pressure Dose of D-phenylalanine Initial End of Day 24 Hours 96 Hours One Later Later Subject 1 3 grams 154 114 124 128 Subject 2 1 gram plus 1 gram 154 130 145 Subject 3 3 grams 160 142 155 The effect of DPA on diastolic blood pressure in these subjects was fairly minimal. This may be due to the fact that this pressure was fairly close to the normal range and thus would not be expected to be lowered to any great extent.

It should be noted that the fall in blood pressure was not accompanied by any significant change in heart rate.

b. Subjects with "normal" blood pressure As seen in Table 7, DPA at a dose of 2 grams had very little effect on their blood pressure, at most producing a very modest fall.

Table 7 Minimal Effect of D-phenylalanine on Blood Pressure of Young Volunteers with Normal Blood Pressure* Subject Systolic Control Diastolic Systolic DPA Diastolic 1 120 72 120 72 2 120 72 120 72 3 118 62 118 62 4 128 70 125 120 70 115 68 6 125 72 115 68 7 140 82 140 82 8 140 80 137 *Blood pressure measured I h after taking 2 grams of D-phenylalanine.

D-phenylalanine was tested for its anti-hypertensive action in the spontaneously hypertensive rat in the laboratory of a major Japanese pharmaceutical company. They confirmed completely the inventors' observation that DPA at O 17

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400 mg/kg gives a significant lowering of blood pressure with a time-course very Ssimilar to what the inventors found. Three rats were used (see FIG. 5A). Also tested were two other inhibitors ofenkephalin degradation, thiorphan and actinonin. Neither of these were effective in lowering blood pressure even though used at very high doses (see FIG. 5B). In the case of thiorphan this could be explained by lack of

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r penetration into the central nervous system which appears to be the site of action of

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DPA. We cannot explain the lack of effectiveness for actinonin but it too may not enter the brain in sufficiently high concentration to effect significant inhibition of the enkephalin degrading enzymes.

0 Thus on the basis of these results, it would appear that DPA is unique among enkephalinase inhibitors in lowering blood pressure, but other EICI inhibitors should not be ruled out.

Data of FIG. 6 show the pharmacokinetics of DPA given orally to a human subject at a dose of 2 grams dissolved in 250 ml of water. Phenylalanine of course if present in the blood and the amount present before DPA administration was subtracted from that obtained at each data point. As can be seen, the elimination half life is about 6 h although a significant amount is still present in serum 10 h. after administration.

CONCLUSIONS

D-phenylalanine. or D,L-phenylalanine, as an anti-hypertensive agent has the following important features, many of which constitute advantages over other blood pressure lowering drugs currently in use at this time: 1. Lowers blood pressure when it is elevated, not if it is normal; conventional anti-hypertensive drugs can lower blood pressure even if normal.

2. A single dose lowers blood pressure for one or more days in humans.

Conventional anti-hypertensive drugs must be given one or more times a day.

3. Blood pressure lowering is not accompanied by an increase in heart rate, unlike what may occur when conventional anti-hypertensive drugs are given.

0 18 4. D-phenylalanine potentiates the. blood pressure lowering effectiveness of a P blocker such as propranolol permitting lower doses of the latter to be used.

This may also occur with other classes of anti-hypertensive drugs.

D-phenylalanine is essentially devoid of side effects, unlike all other \0 5 anti-hypertensive drugs which have many severe.side effects.

C, 6. Other inhibitors of enkephalin or endorphin degradation may N demonstrate anti-hypertensive action similar to that of D-phenylalanine and thus could O substitute for. or be used together with. D-phenylalanine should the need arise.

D-RIBOSE

Ribose plays a vital role in myocardial metabolism, largely through its participation (as PRPP) in the synthesis of ATP and adenine nucleotides. Ribose bypasses the limiting and critical step in the PPP, the glucose-6-phosphate dehydrogenase (G-6-PDH) reaction, thereby elevating PRPP levels. Elevated PRPP levels can lead to increased myocardial adenine nucleotide biosynthesis which can accelerate replenishment of depleted cardiac adenine nucleotide poolSS. This was demonstrated by Zimmer and Gerlach (1978). They studied the effects of certain penitols and pentoses, including ribose. on the heart of adult female rats. Pentoses and penitols, intravenously injected in a single dose of 100 mg/kg, increased the available pool of PRPP and of the rate of adenine nucleotide biosynthesis of the heart.

The stimulatory effect of isoproterenol on myocardial adenine nucleotide biosynthesis could be further potentiated by ribose and xylitol, but not glucose. The isoproterenolinduced decrease of cardiac adenine nucleotide concentrations could be almost completely prevented by repeated administrations of ribose. Thus, pentoses and penitols in combination with beta-receptor stimulation markedly and quite specifically enhanced adenine nucleotide biosynthesis in the rat heart. The results indicate that the increase in the available pool of PRPP is an important factor for the enhancement of cardiac adenine nucleotide biosynthesis. Moreover, the availability of PRPP and the rate of de novo synthesis of adenine nucleotides in the heart seem to be limited by the flow through the PPP.

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C, The metabolic basis for the effectiveness of ribose is not species specific. The Sactivity of G-6-PDH is in the same order of magnitude in the human heart as in other Sanimal species. Since G-6-PDH is a rate-limiting step in the PPP and thus limits the size of the available PRPP pool as well as adenine nucleotide levels, it appears that the enzymatic basis for ribose's effectiveness the formation of PRPP independent of IN G-6-PDH) is similar in such species as rats, guinea pigs, dogs, and humans (Zimmer Set al., 1983). The effects of ribose on myocardium are discussed in more detail in the section entitled "Ribose Effects on the Myocardium".

O There is information concerning the metabolism of ribose in animal tissues or cells other than the myocardium in the published literature. Segal and Foley (1958) stated that ribose in rat and calf liver homogenates was phosphorylated to phosphate and entered the PPP. Lee et al. (1988) found that supplying adenine or, alternatively, ribose and adenine to livers of starved mice did not improve ATP levels in hepatocytes; by comparison, livers of well-fed animals had high ATP levels. These investigators also reported that supplying these ATP precursors did not improve ATP levels in post-ischemic livers. Zimmer and Gerlach studied the effects of certain penitols and pentoses, such as ribose. on the heart, liver, kidney and skeletal muscle of adult female rats. Pentoses and penitols, intravenously injected in a single dose of 100 mg/kg, induced a considerable enhancement of the available PRPP pool and the rate of nucleotide biosynthesis in the heart but not the liver or kidney 11. De novo synthesis of adenine nucleotides not detectable in skeletal muscle of normal rats became measurable after application of ribose. Similarly, Tullson and Terjung (1991) found that ribose perfusion of endurance-trained rat skeletal muscle increased adenine nucleotide biosynthesis by 3.7 to 4.5 times.

Ribose (as R-5-P) also plays a role in erythrocyte metabolism. In porcine erythrocytes, for example, it was found that ribose was used in the formation of lactate (via R-5-P) and maintained ATP levels in these cells. Dawson et al. (1981) found that ribose (15 mM) alone provided marginally increased 2 3 -diphosphoglycerate (2,3- DPG) maintenance in human blood over control preservative (0.25 CPD-adenine), but ribose with phosphate (10 mM) maintained 2,3-DPG levels above 70% of normal for

I

0 five weeks of storage and two weeks longer than the control. ATP levels were maintained at normal or above for six weeks with phosphate plus ribose or inosine C (1S5mM). 2.3-DPG maintenance has previously been shown to be impaired by phosphate, unless inosine is also present. The ribose and inosine effects on 2,3-DPG s 5 maintenance are not additive. Phosphate also has an enhancement effect on ATP Cr maintenance in the presence of either ribose or inosine.

RIBOSE EFFECTS ON THE MYOCARDIUM Information concerning the effects of ribose on myocardium has come from studies of animal myocardial ATP and adenine nucleotide levels following ischemia.

A persistent consequence of even transient ischemia is a substantial decrement in cardiac energy levels as evidenced by decreased myocardial ATP levels. This decrease in ATP levels is correlated with depressed cardiac function.

ATP and creatine phosphate (CP) are depleted in the myocardium following brief ischemia in the isolated, perfused heart (Swain et al., 1982). The lowering of ATP during ischemia is caused by anoxic inhibition of oxidative phosphorylation; excess ADP which accumulates as a result of catabolized oxypurmes. Pasque et al.

(1982) described this process in detail, as follows: Myocardial AMP and ADP levels initially rise during ischemia as the balance between high energy ATP bond utilization and the mitochondrial ability to rephosphorylate AMP and ADP is disturbed. With prolongation of the ischemic period, however, these levels gradually fall, presumably secondary to the degradation of ADP to AMP, which is, in turn, metabolized to adenosine. Upon reaching the vascular endothelium, adenosine is further degraded to inosine and hypoxanthine.

Myocardial levels of inosine and hypoxanthine rise significantly as ischemia is prolonged. Unlike ADP and AMP, these products of adenine nucleotide degradation diffuse into the vascular space and are rapidly washed out during the post-ischemic reperfusion period. The result is net myocardial purine loss induced by the ischemic insult.

As postischemic reperfusion is initiated, the cellular AMP and ADP that have not been degraded during ischemia are rephosphorylated as the oxygenated 21 reperfusion rejuvenates the mitochondrial machinery and restores the ATP/ADP ratio.

Available adenosine, inosine and hypoxanthine are converted to AMP by the normally rapid salvage pathways. The levels of these diffusable salvage metabolites are quite low, however, because of the washout which occurs during reperfusion. Therefore, t- 5 the salvage pathways are inadequately fueled and, even in combination with the de

NO

t novo pathways, are limited in their ability to rapidly replete AMP levels: Thus, depletion of nucleotide pools occurs during ischemia and, with reperfusion, nucleotide content (and cardiac function) is restored only slowly.

Delayed repletion does not appear to be caused by a defect in mitochondrial synthesis of ATP because CP content is restored rapidly (Swain et al., 1982). The slow repletion of nucleotides, particularly the adenine nucleotides, may be secondary to loss of nucleotide precursors during reperfusion and may result in widespread alterations in myocardial metabolism. The loss of precursors forces the myocardium to rely on de novo synthesis of adenine nucleotides. A postischernic decrement in ATP levels may persist for several days following episodes of myocardial ischemia because the de novo pathways are relatively slow in the myocardium (Mahoney, 1990).

Because of the correlation between decreased ATP levels and depressed myocardial performance, possible interventions into adenine nucleotide degradation and/or biosynthesis of these nucleotides so as to restore myocardial ATP levels have been contemplated, some of which are discussed by Pasque et al. (1982): The repletion of ATP levels during reperfusion following ischemia is dependent upon two factors: the preservation of the cellular machinery necessary to rephosphorylate AMP and ADP, and the availability of AMP and ADP for rephosphorylation. There is evidence suggesting that the ability to rephosphorylate ANW and ADP is preserved in myocardial mitochondria subjected to moderate periods of ischemia. Certainly, the well-documented rapid repletion of CP levels to normal and often supernormal levels upon reperfusion suggests that the machinery is available and functional under these conditions. It is reasonable to conclude that the predominant factor in the inability of the myocardium to completely replete ATP O 22 levels during reperfusion following moderate periods of oxygen deprivation is lack of available AMP and ADP for rephosphorylation to ATP.

¢C Interventions into the adenine nucleotide degradation and synthetic pathways that elevate postischemic ANM levels may be of importance in achieving improved 5 postischemic myocardial recovery. There are two possible sites of intervention. First, by blocking the enzyme 5'-nucleotidase during ischemia, the degradation of AMP to C1 adenosine could be blocked. This would prevent the degradation of AMP and ADP to 0 more diffusable breakdown products. The second point of intervention would be c enhanced replenishment of ANW through the acceleration of salvage and de novo synthetic pathways.

The salvage pathways require less energy than the de novo synthetic reactions and are normally responsible for the majority of the adenine nucleotide synthesis in the heart. However, they are dependent upon the presence of adenosine, inosine and hypoxanthine. On initiation of reperfusion, these metabolites are washed out in large volumes and the salvage pathways rapidly become dependent upon the fixed uptake of the limited supply of purine precursors from the liver.

De novo adenine nucleotide synthesis is particularly responsible for the replacement of the relatively small volume of dephosphorylated adenine nucleotide degradation products which are continually lost during normal cardiac activity. It shares this responsibility with the salvage pathway incorporation of 'new' purine precursors supplied by the liver. Under normal conditions the de novo synthetic pathway is suppressed when purine metabolites are available for fueling the salvage pathways. Following ischemia, suppression of the de novo pathway is released, but this pathway is slow relative to the volume of the postischemic adenine nucleotide deficit. Manipulation of the various rate-limiting steps of de novo synthesis under postischemic conditions could reasonably be expected to further enhance this acceleration.

A biochemical limitation on adenine nucleotide repletion by de novo synthesis is the availability of a primary pentose phosphate pathway substrate, PRPP. In a series of studies using an asphyxia recovery and isoproterenol-induced 23 cardiomyopathy and ATP depletion models, Zimmer (1982) and Zimmer and Ibel S(1983) provided evidence that PRPP availability limits adenine nucleotide synthesis by the de novo and salvage pathways. Furthermore, the direct conversion of adenine to AMP also requires availability of PRPP.

n 5 Zimmer (1983) stated how synthesis of myocardial adenine nucleotides could

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t be stimulated by increasing the availability of PRPP, as follows.

Apart from affecting degradation and transport of adenine nucleotides and their breakdown products, there are basically two ways to stimulate the synthesis of O myocardial adenine nucleotides. The first involves the administration of adenosine, S inosine and adenine, all of which can be used to restore cardiac adenine nucleotides through the salvage pathways, which depend on the availability of 1-pyrophosphate [PRPP] as regards inosine and adenine. The second approach is aimed at enhancing the biosynthesis of adenine nucleotides with ribose. It is based on the fact that the rate of biosynthesis is very low (6 nmole/g/h) compared with the total content of adenine nucleotides, and that stimulation with ribose is possible not only under control conditions but also in such situations as recovery from lack of oxygen, cardiac hypertrophy, and stimulation with catecholamines. The pronounced effect of ribose can be attributed primarily to the increased availability of pyrophosphate, a substrate that appears to be the major limiting factor for biosynthesis of myocardial adenine nucleotides. The stimulation of adenine nucleotide biosynthesis is so considerable that the decline in adenine nucleotides is attenuated or even prevented.

According to Mahoney the "...lack of sufficient PRPP for optimal adenine nucleotide biosynthesis is due to sluggishness..." of the pentose phosphate pathway in the myocardium. It appears that the pentose phosphate pathway is not as active in the myocardium as in other tissues. The slow rate of ATP synthesis is in part due to slow conversion of glucose-6-phosphate to ribose5-phosphate in mammalian heart and skeletal muscle (Tullson and Terjung, 1991: Mauser et al., 1985). This may be due to low levels of two rate-limiting enzymes of the pathway, glucose-6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase (Mahoney, 1990).

24 0 Introduction of ribose would bypass rate-limiting steps in the pentose Sphosphate pathway and lead to generation of PRPP, as pointed out by Pasque et al.

m (1982).

PRPP supplies the ribose-phosphate to all adenine nucleotides, and its \O 5 availability is rate limiting in both salvage and de novo adenine nucleotide synthetic CN, pathways (Figures 2 and PRPP availability is in turn limited by the activity of the hexose monophosphate shunt [pentose phosphate pathway] which supplies the ribose- 8 5-phosphate necessary for PRPP synthesis.

C' Ribose would be converted into ribose-5-phosphate, thus entering the pathway at a point past the rate-limiting G-6-P DH/6-P DGH enzymatic steps and thereby increase PRPP synthesis and in turn de novo adenine nucleotide synthesis. Studies have shown in fact that infusion of ribose into rats accelerate: cardiac adenine nucleotide synthesis, presumably by increasing PRPP levels (Zimmer and Gerlach, 1978; Zimmer et al., 1980).

Ribose has been shown to lead to further stimulation of cardiac nucleotide biosynthesis and promoting the recovery of depressed myocardial ATP levels during recovery from intermittent asphyxic periods, from temporary regional ischemia, and in the non-ischemic myocardium (Pasque etal., 1982; Zimmer., 1980; Ibel and Zimmer, 1986; Zimmer, 1983; Mauser etal., 1985; Zimmer and Ibel, 1984: Clay et al., 1988; Mahoney el al., 1989; St. Cyr el al., 1989).

Data from published studies suggests ribose enhances postischemic contractility and work compared to controls (Pasque et al., 1982; Clay et al., 1988).

Pasque and co-workers investigated the effects of prc-ischemic and post-ischemic ribose perfusions on postischemic myocardial ATP and functional recovery in the isolated working rat heart model. In their study postischemic recovery of myocardial function and ATP levels in isolated, working rat hearts given ribose infusions (250 mg ribose per liter of Krebs-Henseleit buffer) before and after ischemia was improved compared to hearts subjected to the same protocol without ribose administration. The mean percent of functional recovery in control hearts following 15 min of warm ischemia reached values of (mean±SEM) 56.7% 63.5% 65.9% 0

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and 70.5% 4.7% at 2, 5. 10 and 15 min of work following ischemia. Hearts perfused with ribose demonstrated improved mean percent return of function at similar intervals of postischemic work with values of 67.9% 73.7% 3.7%, 81.0% and 84.5% respectively. The differences at the final two in 5 intervals both reached statistical significance (*p<0.0 2 Determinations of myocardial

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It ATP levels pmole/gm of dry weight) made at the end of 15 min of postischemic work were significantly higher (p<0.0 2 in the ribose-treated hearts (18.9 0.7) than in controls (16.3 These investigators concluded that infusion of ribose before and S, after ischemia is a biochemically logical method of improving postischemic 0 myocardial ATP and functional recovery.

The effects of ribose on the pre- and postischemic functional performance of the isolated working heart from 24 month old chronically alcoholic animals was investigated by Clay eial. (1988). This perfusion model permitted the isolated heart to perform work analogous to that of normal physiological load, in a system where systemic pressure and atrial pressure could be altered over a wide range and oxygen loss from the perfusion was at a minimum. There was marked improvement in the performance of isolated hearts taken from alcoholic animals that were perfused with 1.7 mM ribose both before and after a 25 min period of global myocardial ischemia (at 25°C); however, ribose treatment did not greatly affect the performance of hearts of isocaloric, control aged rats. Chronic alcohol consumption significantly affected heart performance, causing a marked reduction in both cardiac and work output. Atier ischemia the work of all hearts was notably decreased; there was no work output in untreated hearts of alcoholic animals. whereas in hearts of alcoholic animals treated with ribose work output was only decreased by Brief ischemia 12 to 15 min) is, however, associated with depletion of myocardial ATP and adenine nucleotide levels (due to delayed resynthesis of adenine nucleotides) (Swain et al., 1982; Reimer etal., 1981) and contractile dysfunction.

The effect of ribose on ATP and adenine nucleotide (AN) levels in male and female (mongrel) dog hearts subjected to brief periods of ischemia was studied by Sami and Bittar (1987). In six dogs, ribose (200 mg/kg/h) was infused for 24 h; in five dogs, 0 26 o saline was infused instead. The dogs were then anesthetized, ventilated and the heart r exposed through left thoracotomy. The LAD was dissected and a snare occludcr eC placed around it. Contractility in the LAD bed was measured with a pair of ultrasonic crystals and a left ventricular catheter. The LAD was briefly occluded for 15 min 'O 5 followed by release of the snare and reperfusion for 60 min. ATP and AN levels were Cr measured before and at the end of occlusion. and during recovery 10, 30, 40. and C, 60 min). Contractility measurements were done similarly. The authors reported that 0 recovery of contractility was significantly improved in the ribose group versus the c, saline group. ATP and AN levels were also higher in the ribose group before ischemia and during reperfusion. The data suggest that by enhancing the resynthesis of adenine nucleotides, and thus ATP. with ribose, contractility recovers at a higher rate after reversible ischemia. In similar fashion, Zimmer and Ibel (1984) reported that continuous intravenous infusion of ribose during recovery from a 15 min period of reversible myocardial ischemia in rats leads to restoration of the cardiac ATP pool within 12 h whereas 72 h are needed for ATP normalization without ribose intervention.

Because of the short-term nature of previous myocardial ischemia experiments with adenine nucleotide synthesis precursors, St. Cyr et al. (1989) determined the effects of ribose infusion in a long-term model of global ischemia in order to identify the precursor which limits myocardial ATP regeneration in the intact animal. Global myocardial ischemia (20 min, 37 0 C) was produced in dogs on cardiopulmonary bypass. With reperfusion either ribose (80 mM) in normal saline or normal saline alone was infused at 1 ml/min. into the right atrium and the animals were followed for 24 h. Ventricular biopsies were obtained through an indwelling ventricular cannula prior to ischemia, at the end of ischemia and 4 and 24 h postischemia and analyzed for adenine nucleotides and creatine phosphate levels. Radiolabeled mnicrosphercs were used to measure myocardial and renal blood flows and no significant difference was found between ribose-treated and control groups. In both groups, myocardial ATP levels fell by at least 50% at the end of ischemia. No significant ATP recovery occurred after 24 h in the control dogs. but in the ribose-treated animals ATP levels 27 rebounded to 85% of baseline by 24 h. Total myocardial adenine nucleotide content S and energy charge also recovered in the ribose group but not in the control animals.

Ribose infusion, therefore, significantly enhanced the recovery of energy levels in the postischemia myocardium in intact animals.

Normalization of diastolic function in rats and dogs subjected to myocardial

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i ischemia was seen with infusion of ribose (Zimmer etal., 1989; Zimmer, 1982; Zimmer, 1983: Ward et al., 1984).

In rats treated with isoproterenol. Zimmer etal. (1980b) found that the Sstimulation of adenine nucleotide biosynthesis by ribose was to such a degree that the 3 decline in cardiac adenine nucleotides was prevented and the development of focal myocardial cell lesions significantly reduced. Based upon thai data, Zimmer examined whether the ribose-induced normalization of myocardial adenine nucleotide levels effects the hemodynamics of nonral and isoproterenolstimulated hearts. He used a micro-pressure transducer catheter to evaluate in vivo cardiac function in adult rats given either a single subcutaneous dose of isoproterenol (25 mg/kg) while receiving continuous intravenous infusion of either physiological saline or ribose (450 mg/kg/h) for various periods of time up to 12 h. Indicators of myocardial metabolism such as the cyclic AMP level, biosynthesis of adenine nucleotides and ATP content were also evaluated. Administration of ribose alone for 5 h in rats not treated with isoproterenol had no inotropic or chronotropic effect. Zimmer concluded that ribose itself does not appear to affect cardiac betaadrenergic receptors or the slow Ca2+ channels, nor does it have any systemic vasoactive influences that might alter left ventricular afterload. However, when ribose was administered to isoproterenoltreated rats, there was an enhancement of isoproterenol's positive inotropic effect concomitant with the normalization of adenine nucleotide and ATP levels.

Zimmer (1983) conducted studies to determine if the impairment of heart function associated with ATP depletion can be prevented by ribose. Adult rats were given isoproterenol (25 mg/kg) by subcutaneous injection simultaneously while the abdominal aorta was constricted to a final diameter of 0.65 nun. Twenty-four h later, these animals were anesthetized and an miniature pressure transducer advanced into 0 28 the left ventrical through the right carotid artery. Heart rate, left ventricular systolic Spressure, maximum rate of increase in left ventricular pressure. ATP and total adenine Cn nucleotides (ATP. ADP and AMP) levels (pmole/g) were determined. All functional parameters and adenine nucleotides were depressed compared to control animals.

5 Rats, unanesthetized and unrestrained, were then given continuous intravenous CN infusions of either 0.9% NaCI or ribose (200 mg/kg) solutions for 25 h (5 ml/kg/h).

C

N The rate of adenine nucleotide biosynthesis was elevated to 14.0±1.3 runole/g/h in O saline treated animals compared to 6.6±0.6 nmole/g/h in the control animals. ATP Cand total adenine nucleotides, however, were significantly lower in saline-treated animals 1 0 and 4.7 pmole/g. respectively) compared to controls (4.4±0.0.7 and 5.8±0.10 pmole/g, respectively). But when ribose was administered, the biosynthesis rate was greatly elevated to 76.7±0.3 pmole/g/h. As a result ATP and total adenine nucleotides were essentially normal in ribose-treated animals (4.2-+0.10 and 5.7-+0.12 pmole/o, respectively). Likewise, the hemodynamic parameters in ribose-treated animals were close to the control values, except for the product of left ventricular systolic pressure and heart rate which was some 10% below control, since heart rate was still slightly reduced. Since ribose itself had no effect on cardiac hemodynamics (Zimmer, 1982), the investigator opined that the improvement in heart function resulted from the normalization of cardiac adenine nucleotides brought about by ribose. The investigator stated that the advantage of ribose over other metabolic interventions is that it does not affect the hemodynamics of the heart with an ultimate change in oxygen demand and that it has no vasoactive properties which may result in afterload alterations.

METABOLISM

Ribose is converted to ribose-5-phosphatc by ribokinase, which can then be utilized in three different ways: a) synthesis of glucose; b) glycolysis (formation of pyruvate); and c) synthesis of nucleotides. In the synthesis of nucleotides, ribose is the substrate for formation of PRPP. PRPP is, in turn. the substrate for de novo synthesis of nucleotides, such as ATP, nucleotide coenzymes, and adenine and 29 hypoxanthine utilization by the salvage pathway as depicted in FIG. IA, FIG. 1B, FIG. 1C (taken from Pasque et al.. 1982). Some of the nucleotides are essential energy _sources for basic metabolic reactions and play important roles in protein, glycogen Ce¢ and nucleic acid synthesis (ribonucleotides and deoxyribonucleotides), cyclic S nucleotide metabolism and energy transfer reactions.

IN While female Sprague-Dawley rats were receiving continuous infusions of Dribose solution (200 mg/kg/h at a rate of 5 ml/kg/h), Zimmer et al. (1989) induced myocardial infarction by ligation of the descending branch of the left coronary artery.

0 The time course of changes in heart function was determined in these closed-chest rats over nine days. Rates of de novo adenine nucleotide biosynthesis were also determined. After two days, these investigators found that the ATP content in nonischemic tissue from ribose-treated animals was higher than that found in animals that had received 0.9% NaCI i.v. infusions. After four days of continuous infusion of ribose, ATP levels were essentially normal. Without ribose, ATP content was still significantly depressed, although it had recovered to a certain extent. The depression of LVSP and LV dP/dtmax was not altered by ribose infusion; however, there was a marked and significant attenuation of the elevation of LVEDP compared to that seen in animals treated with 0.9% NaCI. Since ATP levels were better preserved in nonischemic regions of hearts from animals treated with ribose and this was accompanied by the attenuation of the elevated LVEDP, the investigators concluded that metabolic support of the noninfarcted areas results in improvement of global heart function.

Wyatt and co-workers (1989) examined whether arresting hearts with a cardioplegic solution containing adenosine, hypoxanthine and ribose would result in improved tissue ATP content and left ventricular function after 1 h of global ischemia in dogs supported by cardiopulmonary bypass. Animals with ischemic arrest initiated with a crystalloid cardioplegic solution containing adenosine (100 pmole/1), hypoxanthine (100 [pmole/1) and ribose (2 mmole/1) had significant improvement during postischemic reperfusion. A significant (p<0.05) correlation existed between myocardial ATP content and the recovery of left ventricular function.

The authors concluded that an as anguineous cardioplegic solution containing adenosine, hypoxanthine and ribose maintains myocardial ATP content during Sischemia and reperfusion and enhances functional recovery during the postischemic C period.

While energy in the normal heart is derived principally from fatty acid ,O 5 oxidation, under certain circumstances such as during ischemia, myocardial glycogen CI stores are used and glucose becomes the preferred substrate. In isolated perfused heart c studies, glucose is commonly used as the sole energy source for the heart although Sother substrates such as ribose and xylitol have been used. Mahoney et al. (1989) were interested in examining the role of pentose sugars and polycls in myocardial 0 metabolism. They studied the ability of ribose to serve as the sole added carbon source in a rat isolated working heart model. Rat hearts were extirpated and configured as per the working heart model. Work hearts were perfused for 30 min with Krebs-Henseleit buffer containing 1 mM of one of the following substances: ribose, glucose, pyruvate or xylitol; control hearts were perfused with buffer only.

Pressure, heart rate, myocardial oxygen consumption (MVO), cardiac work, rate pressure product (RPP) and stroke volume were determined. A left ventricle biopsy was done and cardiac ATP, ADP, AMP, creatine phosphate and glycogen levels were determined. Total adenine nucleotide and energy charge (EC) were also calculated.

Hearts perfused with buffer with ribose, xylitol or buffer only did not function as well as hearts perfused with glucose or pyruvate. Hearts perfused with ribose, xylitol or buffer-only eventually were no longer able to perform work; there was no aortic output after about 15 min of perfusion. Changes in hemodynamic parameters were consistent with these findings. The investigators stated that this loss of function resulted from a decrease in generated pressure and lowered stroke volume rather than heart rate suggesting that contractility was primarily affected. ATP, CP, EC and glycogen levels tended to decrease in ribose, xylitol and buffer-only groups, compared to glucose, after 30 min of perfusion. These investigators commented that while ribose or xylitol did not maintain myocardial ATP levels as well as glucose or pyruvate, ATP levels were still at a relatively high concentration compared to that 131 found in postischemic hearts. Yet. cardiac work ceased in the ribose and xylitol groups. They concluded that in the isolated perfused working rai heart ribose or xylitol were not capable of serving as the sole carbon or energy source for these hearts. They stated that neither ribose nor xylitol could be convened to glycolysis tn 5 pathway intermediates rapidly enough to measurably contribute to the generation of

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V" high-energy bonds. Instead, these investigators believe that ribose's role in recovery of postischemic myocardium would seem to be in production of PRPP and repletion of ATP levels.

SZimmer ct al. (1980) reported that, in female rat hearts hypertrophyic, because 0 of aortic constriction and isoproterenol administration, the activity of glucose-6phosphate dehydrogenase and the available pool of PRPP were found to be increased, indicating an enhancement of the flow through the PPP. In triiodothyronine-treated animals, only the cardiac pool of PRPP was elevated. In all three models of experimentally induced cardiac hypertrophy, the enhancement of myocardial AN biosynthesis was potentiated by intravenous infusion of ribose (100 mg/kg). Since ribose gives rise to a considerable elevation of the available PRPP pool, it appears that this is the limiting factor for the increase of AN biosynthesis in hypertrophying hearts.

Constant intravenous infusion of ribose for 24 h prevented the isoproterenol-induced decrease of myocardial ATP levels.

Pliml et al. (1992) investigated the effect of orally-administered ribose on exercise-induced ischemia in stable coronary artery disease. In this study, 20 men with stable but severe coronary artery disease (CAD) underwent two symptom-limited treadmill exercise tests on two consecutive days. One group of 10 randomly chosen patients was given placebo for three days and a second group of 10 was given g/day ribose dissolved in water in four 15-gram doses. In the ribose treated group, the mean time until mm ST-segment depression was significantly greater on day 5 than baseline, whereas there was no significant change in time to ST depression in the placebo group. Two ribose-treated patients did not show ST depression on day 5, so time to the onset of moderate angina was used in the analysis. After treatment, the time to ST depression (day 5) was significantly longer in the group that had received 32 ribose than in the placebo-treated group (mean and 95% confidence interval [CI] 276 [220-331] v. 223 (188-259], respectively; p<0.002). The time to onset of moderate C angina also increased significantly from baseline to day 5 in the ribose group. There was also an increase in this variable in the placebo group but it did not achieve \0 5 significance (p<0.062). There were no significant changes in heart rate, systolic blood 0 C pressure or rate-pressure product from baseline to day 5 in either group.

O RIBOSE INTERACTIONS WITH CARDIOVASCULAR AGENTS Ribose can also prevent the inhibition of cardiac AN biosynthesis by propranolol (Zimmer et al., 1984) Adult rats were treated with isoproterenol mg/kg for 5 h) alone or in combination with propranolol (50 mg/kg for 5 h) while others were treated with propranolol (50 mg/kg for 5 h) alone or in combination with a solution of ribose (450 mg/kg/h). The rate of cardiac adenine nucleotide biosynthesis (nmoles/g/h) was determined in each case. Isoproterenol markedly stimulated cardiac adenine nucleotide biosynthesis that was completely abolished when propranolol was simultaneously administered. Propranolol alone inhibited adenine nucleotide biosynthesis considerably; this effect was prevented when ribose was infused.

Whether ribose retains its stimulating metabolic effects on myocardium when administered in conjunction with the calcium antagonist verapamil and the (Blspecific adrenoceptor blocker metoprolol was studied (Zimmer el al., 1987).

Measurements of functional parameters in closed-chest rats revealed that intravenous administration of ribose (200 mg/kg/h) alone for 24 h had no hemodynamic or vasoactive influence, whereas verapamil and metoprolol infusion of 2 mg/kg/h each for 24 h) induced negative chronotropic and negative inotropic effects. Cardiac output was reduced by metoprolol, but not by verapamil. Ribose did not affect these drug-induced hemodynamic alterations, and verapamil as well as metoprolol did not interfere with the characteristic metabolic effect of ribose, the stimulation of cardiac adenine nucleotide de novo synthesis. The investigators concluded that administration of ribose in combination with these pharmacological agents is compatible.

^13- 3 3

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Zimmer et ul. (1989) also examined the hemodynamic influence of ribose, the d calcium antagonist gallopamil (a methoxy derivative of verapamil), and coenzyme Q 0 on rat hearts subsequent to coronary artery ligation. Myocardial infarction was induced in rats by ligation of the descending branch of the left coronary artery. The i 5 time course of changes in heart function was recorded within the first nine days.

I There was a progressive decline in LVSP, in LV dP/dtmax and in the pressure-rate product. LVEDP was elevated. Cardiac output and stroke volume index were depressed after two days. The ATP content in the non-ischemic region was lower Othan control, but recovered spontaneously toward the normal value within the first four days. Continuous i.v. administration of ribose (200 mg-/kg/hr; 5 mg/kg/h) which stimulates further adenine nuclcotide biosynthesis attenuated the fall and promoted the restoration of ATP in the non-ischemic myocardium within four days after coronary artery ligation. The elevation of LVEDP was attenuated with ribose after two to four days. The calcium antagonist gallopamil administered as i.v. infusion for two days led to a further reduction of all parameters of left heart function, but did not influence the increase in adenine nucleotide and protein synthesis that occurred in the nonischemic heart. Coenzyme Q10 had only slight effects on LVSP, LVIEDP, and LV dP/dtmax, but attenuated significantly the fall in cardiac output and stroke volume index after two days following coronary artery ligation. Thus, all interventions affected differently the infarct-induced changed in heart and circulatory function. An improvement was observed with ribose and with coenzyme Lortet and Zimmer (1989) investigated the functional and metabolic effects of ribose administered to closed chest rats when given in combination with prazosin, verapamil or metoprolol. These investigators found that ribose administration for 24 h at 200 mg/kg/h did not affect heart function in these animals but increased the available pool of PRPP in heart (4-fold) and skeletal muscle (1.7-fold), as assessed by incorporation of 14C-adenine into the adenine nucleotides. The utilization of adenine for AN synthesis, expressed as the ratio of adenine nucleotide radioactivity to tissue extract radioactivity, was 70% in heart and 20% in skeletal muscle under control conditions, and 97% and 88% after 24 h of ribose administration. Ribose decreased 0 34 14C-adenine nucleotide into the adenine nucleotides in kidney, lungs and liver. After 24-h infusion of prazosin (100 (pg/kg/h), The rate and LV dP/dtmax were not changed, but LVSP mean aortic pressure and peripheral resistance were decreased. Cardiac output was enhanced Verapamil (2 mg/kg/h) O 5 and metoprolol (2 mg/kg/h) infused for 24 h decreased the pressure-rate and pressurevolume product of the left ventricle to the same extent Verapamil had no C influence on cardiac output, while metoprolol depressed it Simultaneous 0 administration of prazosin, verapamil or metoprolol with ribose did not affect the c ribose-induced increase in the myocardial PRPP pool. On the other hand, ribose did not alter the characteristic hemodynamic changes induced by these three drugs. The authors concluded that it is feasible to combine ribose with prazosin, verapamil or metoprolol.

EFFECTS OF RIBOSE ON MYOCARDIAL- 20'THALLIUM REDISTRIBUTION Infusion of adenine nucleotide precursors, such as ribose, has been shown to favorably affect 20'Thallium (20'TI) kinetics in animals postischemia. Angello et al.

(1988; 1989) found that, in swine, ribose infusion after transient ischemia modified clearance in both ischemic and nonischemic myocardial regions, resulting in faster 20'TI redistribution.

In one study (Angello el al., 1988) 35, miniature cadmium-telluride (CdTe) radiation detection probes were inserted into the hearts of a total of 17 young pigs, one positioned against the endocardium of the anterior wall and the other against the endocardium of the posterior left ventricular wall. The CdTe probes measured activity continuously. The ribose treatment group consisted of eight animals that received i.v. ribose, 3.3 mg/kg/min for 30 min. The control group consisted of nine animals that received saline in place of ribose. A subtotal LAD occlusion was performed reducing LAD flow to about 25% of baseline based on electromagnetic flow probe readings. Microsphere injections were used to document the flow reduction. After 10 min of occlusion, LAD flow was restored gradually over a 5-10 min period. Thallium was injected during the 10 min of subocclusion. The percent difference in 20'TI activity between the ischemic anterior and non-ischemic posterior O wall during ischemia was comparable. Twenty-five min after the subocclusion was released and intravenous infusion of ribose (3.3 mg/kg/min) or saline was started and continued for 30 min.

Within 5 to 10 min of the initiation of the ribose infusion there was a I) 5 prominent acceleration of the rate of 20'TI washout from the non-ischemic

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I myocardium. An acceleration of the rate of washout from the transiently ischemic Smyocardium also occurred, but to a lesser extent. Within 10 min of initiation or ribose there was essentially no difference in 20'TI activity between ischemic and non-

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ischemic myocardium. From these data, imaging starting 10 min after the initiation of 0 ribose may show significant and near-maximal distribution.

redistribution was defined as the percent decrease in the pretreatment 20'T defect at the end of the 30-min infusion and at 60 min. The ribosetreated animals had significantly greater thallium redistribution at the end of the ribose infusion compared to saline infusion, 48%±1% v. 20%±4% At 60 min there was a small increase in redistribution in the ribose group compared to the end of the infusion but a substantial redistribution in the saline group such that at 60 min after the infusion there was no difference between ribose and saline. There were no differences between groups in heart rate, arterial blood pressure, mean serum glucose, mean serum insulin or in regional myocardial blood flows. The mean decreases in serum glucose between pre-infusion and 60 min post-infusion were 13.8 and 0.0 mg/dl for the ribose and saline groups, not a statistically significant difference.

In the second study, Angello and co-workers (1989) used a porcine model similar to that described above. In this study, ribose or saline was started 20 min prior to the LAD subocclusion. 20'TI was injected during the period of subocclusion (25 min after ribose or saline was started). Treatment was continued throughout the study (2 h).

The ribose group had a 69.9%±6% reduction in the initial 20'TI defect in the ischemic area compared to a 44%±5% in the saline group. This difference was significant The maximum percent difference in the 20'T1 activity between ischemic and non-ischemic myocardium was the same in ribose- and saline-treated

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36 animals. The presence of ribose did not appear to alter the initial myocardial uptake of 20'Thallium. Mean (±SEM) pre-infusion plasma glucose was 148±20 mg/dl in the c ribose group and 127±21 mg/dl in the saline group. At the end of the study these values were 137±18 mg/dl and 113±13 mg/dl for the ribose and saline groups, O 5 respectively. These falls of 11% and 14% were not statistically significant.

1 Ultrasonic crystals were implanted at the epicardial and endocardial surfaces of the ischemic and nonischemic myocardium. In this study in which ribose was started prior to subocclusion of the LAD and continued throughout the study, the percent thickening in the ischemic myocardium in the ribose group may have improved to a greater extent than the saline group. These two studies in young pigs suggest that ribose may alter the washout kinetics resulting in an enhancement of Thallium redistribution.

Two randomized, placebo-controlled clinical trials have evaluated the effects of an intravenous infusion of ribose on thallium redistribution. The objective of the first study by Perlmutter et al. (1991) was to determine if an intravenous infusion of ribose could facilitate 20'TI redistribution after transient myocardial ischemia in patients with coronary artery disease and thus improve the ability to detect jeopardized but viable myocardium. The study was a randomized, placebo-controlled cross-over trial. Patients underwent two 20'TI exercise tests 1 to 2 weeks apart.

A Bruce or modified Bruce exercise protocol was selected appropriate to the patients' exercise capacity. Treadmill exercise was continued until angina or other symptoms occurred. Thallium, 2.0 mCi, was injected at that time and exercise was continued. Within 3 min planar myocardial imaging was begun with a standard gamma scintillation camera with a low-energy all-purpose collimator. The total imaging time was 30 min, 10 min per view.

After exercise the patient received either ribose (3.3 mg/kg/min) for 30 min as a 10% solution or normal saline intravenously. Immediately after completion of this infusion, imaging was repeated. This is referred to as the "I hour image". Imaging actually took place during the period between 60 and 90 min after thallium injection.

Another image sequence was started 4 h after thallium. One to two wk later when the 37 patient was crossed-over to the alternative treatment, ribose or saline, an identical exercise test was carried out to the same heart rate times blood pressure product unless severe limiting symptoms occurred first. The study was single blind. Image interpretation was by an observer blinded to the treatment codes. A computer-assisted S image-processing algorithm assessed interpretation of images. Initial defects were I regions with thallium activity less than 2 standard deviations below the mean value for a group of normal subjects. Reversible defects were defined as having a >12% relative increase in 20TI activity in the region of the defects as seen over >18 degrees K1 of the arc.

0 Seventeen patients with chronic stable angina were enrolled. All patients had known coronary artery disease. This was documented by coronary angiography in 14 and by and ECG abnormalities during chest pain in three. Antianginal therapy was continued throughout the trial while drugs and doses remained constant. Seven myocardial perfusion regions were evaluated per patient: anterior

(ANT),

arteriolateral, apical inferior (INF). inferior-posterior post), posteriolateral and septal (SEP).

At I h there were 50 reversible defects after ribose and 20 after saline. At 4 h there were 57 reversible defects after ribose and 34 after saline. In 13 of the 17 patients, imaging after ribose identified additional reversibility not seen after saline.

At both 1 and 4 h post exercise there were significantly more reversible defects after ribose than saline. There were no clinically significant adverse effects of ribose in this study.

In a second study, Hegewald er al. (1991) worked to determine if ribose infusion after transient myocardial ischemia with 4 h imaging is more sensitive for detecting thallium redistribution than is 24 h delayed imaging without ribose. This study was a randomized, placebo-controlled crossover trial. Patients underwent two exercise tests I to 2 weeks apart. The exercise test protocol was similar to that noted above.

After exercise imaging the patient received either ribose (3.3 mg/kg/min) for 30 min as a 10% solution or normal saline intravenously. After completion of

O

O

c 38 thallium infusion, imaging was carried out at 4 h and 24 h. All patients in the saline group did not have 24-h images One to two weeks later when the patient was crossed-over to the alternative treatment, ribose or saline, an identical exercise test Swas carried out to the same heart rate times blood pressure product unless severe exercise limiting symptoms occurred first. The study was single-blind and image interpretation was by an observer blinded to the treatment codes. Image interpretation procedures were as noted above. Fifteen patients with chronic stable angina and 0coronary artery disease documented by quantitative coronary angiography were randomized. The mean patient age was 62 years and predominately male. Seventythree percent had 2- or 3-vessel disease. Previous Q wave myocardial infarction(s) with an associated wall motion abnormality was present in 40%. Antianginal medication was maintained at constant dosage throughout the study.

There were 57 stress defects on initial post-exercise imaging. Four defects were present on only one study; two had redistribution after ribose and two after saline. Four hours after ribose there were 36 segments with redistribution. Four h and 24 h after saline there were 15 and 18 segments with redistribution, respectively. The differences between 4-h ribose and saline at 4 and 24 h were statistically significant, using McNemar's paired chi-square test.

These two clinical studies demonstrate the potential for ribose to enhance the redistribution of thallium after exercise. There were no clinically significant adverse experiences in these studies.

ORAL RIBOSE ADMINISTRATION Myoadenylate deaminase (MAD) is the rate-limiting enzyme in the purine nucleotide cycle that is biochemically linked to glycolysis and the citric acid cycle (Goebel and Bardosi, 1987). Deficient MAD activity has been reported in association with hypokalemic periodic paralysis, Duchenne's muscular dystrophy and other neuromuscular diseases as well as a primary disease associated with exertional myalgia. The following table from Goebel and Bardosi (1987) Summarizes the diseases in which MAD (MADD) deficiency has been described (noted references are from the reported study): 39 Table 1 Myoadcnylale Dcaminase Deficiency in Association with Other Diseases Disease Duchenne's muscular dystrophy Late onset muscular dystrophy Facioscapulohumoral dystrophy Facial and limb girdle myopathy Congenital myopathy Infantile hypotonia Juvenile spinal muscular atrophy Kugelberg-Welander Adult spinal muscular atrophy Amyotrophic lateral sclerosis Hypokalemic periodic paralysis Malignant hyperthemiia Gout Polymyositis Dermatomyositis Trichinosis Polyarteriitis Systemic sclerosis Lupus erythematosus disseminatus Collagen vascular diseases Sarcoidosis Cardiomyopathy Myasthenia gravis Muscle-eye-brain syndrome Reference Zimmer and Ibel, 1984 Lee et al., 1988 44 Sami and Bittr, 1987 Clay e al., 1988 44 Zimmer and Ibel, 1984 Clay et al., 1988; 44 Zimmer and Gerlach, 1978 Dawson et al., 1981 Zimmcr el al., 1989 Dawson et al., 1981 Zimmer and Ibel, 1984 Lee et al., 1988 Lee e al., 1988 Swain el al., 1982: Zimmer and Ibel, 1984 Lee et al., 1988 Zimmer, 1983 Zimmer, 1982 42 Zimmer and Ibel, 1983 Perlmutter et al., 1991 MAD irreversibly deaminates adenosine monophosphate (AMP) in the purine nucleotide cycle, which plays an important role in skeletal muscle metabolism during exercise. Initial reports of ribose treatment for MADD gave mixed results. Doses of ribose in these initial reports were about 2 grams/day in divided doses. Zollner et al.

(1986) reported successful treatment in a 55 year old patient with MADD with doses of 50-60 grams/day. Four grams were taken before exercise and repeated after 10 to 30 min when muscle pain occurred. Single doses of more than 20 grams led to diarrhea.

In one of the publications from Gross and co-workers (1989) five patients with MAD and 8 healthy volunteers are described. Ribose was dissolved in 400 ml of 0 water and subjects drank 1/2 of this solution every 5 min for 5 h. Diarrhea was Sreported in some instances during oral administration one subject at a dose of 167 mg/kg/h and in the only subject given 222.2 mg/kg/h). Most of the subjects were said Sto report hyperperistalsis but the investigator could not differentiate between the effect of ribose, fasting and water ingestion. It was stated that hyperperistalsis was reported only occasionally during intravenous infusion of doses of 83.3. 166.7 and 222.2 mg/kg/h of ribose for 5 h. Since these were fasted subjects, some hyperperistalsis 0would be expected.

To evaluate the effects of oral ribose on exercise induced ischemia. Pliml el al.

(1992) studied 20 men with documented coronary artery disease. Patients underwent two symptom limited treadmill exercise tests (Bruce protocol) on two consecutive days to produce ischemia resulting in changes in cardiac energy metabolism. Patients whose baseline tests showed reproducibility were randomly allocated to three days of treatment with oral placebo or ribose (60 grams/day dissolved in water administered in four 15 gram doses). Exercise testing was repeated at the end of treatment.

Patients were maintained on usual medications during the study. On the morning of each exercise test, doses were withheld until after the test.

The investigators found the mean time until I mm ST-segment depression was significantly greater after treatment than at baseline with ribose compared to placebo (p<0.002). The mean increase was equivalent to an increase of more than 30% from baseline. Time to onset of moderate angina was evaluated. In the ribose group the time to onset was increased significantly from the baseline to the end of treatment (p<0.00 4 the viable increased in the placebo group, but was not significantly different from the baseline and there was no statistically significant difference between ribose and baseline. The author concluded that in patients with CAD, administration of ribose by mouth for 3 days improved the heart's tolerance to ischemia. The presumed effects on cardiac energy metabolism offer new possibilities for adjunctive medical treatment of myocardial ischemia.

The following examples are included to demonstrate certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that O 41 U the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus C can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or 0C similar result without departing from the spirit and scope of the invention.

Example 1: CI Anti-Hypertensive Compounds Involving Inhibition of Opioid Peptidyl Enzymatic Breakdown and Neurotransmitter Regulation: Combination Therapy 0 Summary: While there are numerous approaches to treat various forms of human and animal hypertension there is still the need for additional medicaments to add to the clinicians weapons to combat the sequelae related to unwanted high blood pressure.

The invention involves the combination of certain inhibitors of "Endorphinase" or "Enkephalinase" or other related inhibitors of enzymes involved in the breakdown of natural opioid peptides. Said inhibitors could be from a group of D-amino acids and their metabolites D-phenylalanine, hydrocinnamic acid, D-leucine etc.) and other precursor amino acids, especially those which effect dopamine synthesis Ltyrosine) as well as herbal-based natural substances (ferrulic acid, pharmaline, huperzine). The addition of chromium salts (picolinate, nicotinate and poly nicotinate etc.) to promote a reduced risk for diabetes and reduced cholesterol levels will be most beneficial. Other important nutrients include co-enzyme Q and pycnogenol and Hawthorn. Additionally, the combination D-phenylalanine, D-leucine or D,L-leucine or other D-amino acids with acetylsalicylic acid to prevent stroke has important prophalatic benefit. The combination of D-amino acids or other similar inhibitors of opioid peptidyl degradation with the following known anti-hypertensive agents is also considered: diuretics 42 0 l.sympaholytics (including central and perphe, l aud'energic receptor blockers C* direct vasodilators S angiotensin-converting enzyme inhibitors 5 e calcium channel blockers angiotensin II receptor antagonists T-type calcium antagonists S* Specific others (Nisoldipine, Losartan, Moxonidine (physiotens- Solvay), Fenoldopam Genes and Hypertension An additional embodiment of this disclosure is the potential of combining the proposed formulae suggested with genotyping for certain known gene polymorphisms and identified alleles human Chromosome 2 [D2S311], tissue pathway inhibitor, the vitronectin receptor alpha- subunit, the alpal chain typel 11 collagen, the alpha-2chain of type V collagen, homeobox D cluster as well as a potential of more than 100 expressed sequence tags which have been recently expressed and have been mapped in the human genome localized and are potential candidates for familial primary pulmonary hypertension and other related hypertensive conditions.

Moreover, another gene which may have a potential role in hypertension may be localized to a micro-satellite polymorphism, D2 S1788, mapped to chromosome 2p21 (approximately 74cM from the tip of short arm) which showed strong evidence of linkage with serum leptin levels. Other genes in the same area include glucokinase regulatory protein (GCKR), and pro-opiomelancortin

(POMC).

Less than 25% of patients with hypertension in the United States have their blood pressure under control, mainly because of inadequate or inappropriate therapy and noncompliance. Approximately one-half of these treatment failures are related to factors such as cost and adverse effects of medication, complex drug regimens, failure of clinicians to fully realize the benefits of anti-hypertensive therapy and lack of patient education. Other major causes of unresponsiveness to anti-hypertensive therapy include "white coat" hypertension, pseudo-hypertension, obesity, volume S43 overload, excess alcohol intake and sleep apnea as well as inappropriate antihypertensive drugs and drug combinations, and unfavorable interactions with prescription and other drugs. In many patients, these factors must be dealt with before blood pressure can be controlled.

It has been calculated that hypertension ranks as the fourth largest mortality risk factor in the world predicting 6% of all deaths. Multiple risk factors for F cardiovascular disease, particularly hypercholesterolemia, are often present in the hypertensive patient. Recent guidelines, ranging from those prepared by the World SHealth Organization/ International Society of Hypertension to those of the three European Societies of Cardiology. Atherosclerosis, and Hypertension, stress the importance of evaluating global risk, based on the presence of all cardiovascular risk factors in an individual or in a group of subjects. It has also been suggested that treatment should aim to correct all modifiable risk factors. Moreover, safety of drug treatment of hypertension can only be seen in relation to efficacy, which has now come to mean not just blood pressure reduction but improvements in hard end-points including mortality.

Moreover, by matching the profile of an anti-hypertensive drug to the clinical and demographic characteristics of the patient risk factors, coexisting diseases, goals of therapy beyond lowering blood pressure), the physician can maximize the efficacy of the regimen and minimize the adverse effects the patient may experience.

Optimal therapy requires a knowledge of the pharmacologic properties of the six broad classes of anti-hypertensive drugs: diuretics, sympatholytics, direct vasodilators, angiotensin-converting enzyme inhibitors, calcium channel blockers and angiotensin 11 receptor antagonists. In this regard, treatment approaches should focus on drugs which do not worsen diabetes mellitus, do not cause dyslipidaemia, or induce potassium loss, while not provoking drowsiness, depression, or being associated with rebound hypertension when medication is stopped.

Single drug therapy for the treatment of hypertension has traditionally been a standard of practice. More recently combination therapy as first-line treatment has gained acceptance both by the medical practice community and the US Food and Oq 44 U, Drug Administration. The advantages of combinations may be a synergistic or additive ant-hypertensive effect, metabolic improvement or both. For example, the m combination of a thiazide-type diuretic and a potassium-sparing diuretic has been quite useful in the past to prevent the need for potassium supplementation. The O 5 combination of 3 -adrenoreceptor blockade and thiazide diuretic results in an additive Santi-hypertensive effect that permits the effective use of very low thiazide doses. The N mechanism of anti-hypertensive effects of each member of the combination are complimentary with increased sympathetic outflow and rcnnin-angiotensin axis activation induced by the diuretic being blunted by beta-l-adrenergic blockade.

Combinations not used as first-line therapy, such as angiotensin converting enzyme inhibitors or angiotensin receptor blockade and a thiazide diuretic, has complimentary antihypertensive mechanisms and have been useful in treating patient groups who do not respond well to converting enzyme inhibitor monotherapy. The combination of a calcium antagonist with diuretic therapy has an additive hypertensive effect as well: however, the complimentary mechanisms are less obvious. Finally, the combination of angiotensin converting enzyme inhibition and calcium antagonist therapy has been useful in selected patients, but again the complimentary mechanism: are less obvious.

As first-line therapy, combinations of diuretics and beta-i-receptor blockers have been useful for achieving increased anti-hypertensive effect with decreased adverse drug effect.

Precursor to Catecholamines (dopamine, norepinephrine) The catecholamines dopamine norepinephrine and epinephrine are all ncurotransmitters. Catecholamines possess two adjacent hydroxyl (01-I) groups on a phenyl ring. In the body, such substances are synthesized from the aromatic amino acid L-tyrosine, which is hydroxylated to L-3, 4 -dihydroxyphenylalanine (L-dopa) by tyrosine hydroxylase. L-tyrosine is actively take up into noradrenergic nerve terminals. L-phenylalanine is a precursor of L-tyrosine (Blum and Kozlowski, 1990; Schwartz et al., 1992).

Tyrosine hydroxylase is located in the cytoplasm of noradrenergic neurons and is the rate-limiting enzyme in the synthesis of norepinephrine. Extensive research has 8 revealed that reduced pteridine cofactor, molecular oxygen and ferrous ions are all required for activity. In the cytoplasm. L-dopa is decarboxylated to DA by L-aromatic amino acid decarboxylase, an enzyme which requires pyridoxal phosphate (Vitamin B6) as a cofactor. The dopamine (DA) is actively taken up into granular in 5 storage vesicles in which the DA is hydroxylated to form norepinephrine by the in enzyme dopamine-p-hydroxylase. This enzyme requires copper, molecular, oxygen and ascorbic acid as a cofactor. In some neurons in the CNS, norepineplirine is further converted to epinephrine by the enzyme phenylethanolamine-N-methyltransferase.

Tyrosine hydroxylase activity is influenced by the following: "end product" inhibition, caused by increased concentration of norepinephrine within nerve terminals which decreases the rate of conversion of L-tyrosine into L-dopa; increased sympathetic activity from the CNS which increases the synthesis of norepinephrine; the angiotensin II mediated increases the rate of norepinephrine synthesis; and agonists clonidine) and blockers phentolamine) of adreno-receptors which change the rate of norepinephrine release by mechanisms involving adrenergic receptors located on the presynaptic terminal.

Inhibitors of the enzymes of norepinephrine synthesis include: methyl-p-tyrosine (inhibits tyrosine hydroxylase); carbidopa (inhibits aromatic amino acid decarboxylase in tissues outside the CNS); and diethyldithiocarbonate, FAI63 and disulfiram (inhibitors of dopamine-l-hydroxylase).

Norepinephrine is stored within the nerve terminal in multiple storage complexes and more than one anatomical location. One form of norepinephrine storage type is a granular complex found within vesicles. in noradrenergic nerve terminals. The granular complex consists of norepinephrine bound to ATP, several proteins collectively called chromogranins, includes dopamine-p-hydroxylase, Mg++, Zn++ and Cu++.

The uptake of DA and norepinephrine into storage vesicles is an active-transport process which requires ATP as an energy source and Mg++ to activate the ATPase enzyme which is Mg++ dependent. This Mg++-dependent 46 aU uptake process of norepinephrine and DA into storage vesicles is a separate and different process from the neuronal uptake process for norepincphrine across the Cc nerve cell membrane, which is an Na.sup.+ /K.sup.+ -ATPase dependent.

The stability of the norepinephrine-ATP-protein- ion storage complex can be 0 5 disrupted by some compounds which act as chelators of This may be linked to the magnesium deficiency sometimes found in chronic cocaine abusers. In this regard, chronic administration of cocaine produces an increase in norepinephrine turnover.

Release of norepinephrine from nerve terminals occurs by a process of exocytosis, which is calcium dependent. whereby a vesicular membrane fuses with the plasma membrane and the vesicular contents, consisting of norepinephrine,

ATP,

dopamine-3-hydroxylase and chromogranins, are released into the synaptic cleft. One mechanism known to control the availability of norepinephrine to postsynaptic receptors operates by means of presynaptic receptors located on the terminal from which norepinephrine is released. The actions of norepinephrine ir. the synaptic cleft are terminated by removal from the synaptic cleft by an uptake system found on presynaptic nerve endings. There are two types of neuronal uptake of norepinephrine--uptake I and uptake II.

Uptake I is energy dependent, requiring ATP which is broken down by a sodium dependent ATPase. This is a high-affinity process, which means that it is efficient at the eliminating low concentrations of norepinephrine from the synaptic cleft. The neuronal uptake system transports norepinephrine into the nerve terminal.

Inside the nerve terminal most of the norepinephrine is taken up into storage vesicles.

Inhibitors of this process include: cocaine, tricyclic anti-depressants, amphetamine and tyramine.

Uptake II involves the accumulation of norepinephrine by nonneuronal tissues.

High plasma levels of norepinephrine derived from stimulation of the adrenal medulla, or intravenous injection of a catecholamine will be removed by uptake into non-nervous tissues such as liver, muscle and connective tissue. The norepinephrine or any other catecholamine diffuses back into the circulation or. more commonly is

I

47 destroyed intracellularly by the enzymes monoamine oxiclase (MAO) and catechol-O-methyltransferase

(COMT).

MAO is found in all tissues which contain mitochondria, and is bound to their outer membranes. MAO is present in liver, brain, nerves, muscles and all actively i' 5 metabolizing tissues. It oxidatively deaminates norepinephrine to 3,

INO

in 4-dihydroxymandelic acid which can then by O-methylated (by COMT) to give rise to 3-methoxy-4-hydroxy-mandelic acid. MAO describes a group of isoenzymes which possess different tissue distributions, substrate specificities, inhibitor characteristics I and physical properties. For example, MAO A has a substrate preference for norepinephrine and 5HT, and is selectively inhibited by clorgyline. MAO P has a substrate preference for dopamine and phenylethylamine, and is selectively inhibited by deprenyl (selegiline). Other well known MAO inhibitors include iproniazid, niacinamide, pargyline, tranclypromine and phenelzine.

COMT is found in large quantity in liver cells. In the CNS, COMT acts on E and norepinephrine which have not been inactivated by neuronal re-uptake.

Pyrogallol, an inhibitor works by blocking the COMT dependent transfer of a methyl group from S-adenosyl-L-methionine to the hydroxyl group at the 3' position of the catechol ring of norepinephrine, E and DA. Dopamine is the precursor of norepinephrine and E, and plays a significant role in the CNS and at some ganglia in the autonomic nervous system.

High intraneuronal amounts of DA inhibits tyrosine hydroxylase by end-product inhibition, thus decreasing the rate of DA synthesis. Furthermore, the rate-limiting step in the synthesis of DA is the conversion of tyrosine to L-dopa by tyrosine hydroxylase. Under normal situations tyrosine hydroxylase is completely saturated with L-tyrosine and thus increase in circulatory tyrosine levels do not increase the rate of DA synthesis. However, this fact changes when there is a deficit in both the amount of DA and when tyrosine hydroxylase is compromised as under the influence of cocaine.

L-dopa is actively taken up into DA neurons in the CNS where it is converted to DA. Following L-dopa therapy there is a significantly increase in the amount of c- 48 DA synthesized and stored. By comparison with the dopaminergic system, there is relatively little increase in the synthesis of norepinephrine following L-dopa.

c treatment.

Dopamine is stored in storage granules where the catecholamine is complexed .C 5 with chromogranins, divalent metal ions and ATP. DA is believed to be released into the synaptic cleft by exocytosis. As with norepinephrine, this is a calcium dependent process and occurs in response to action potentials reaching nerve terminals or to O drugs. The following substances can increase DA release; cocaine, (+)-amphetamine, methylamphetamine, tyramine, amantadine, m-phenmetrazine, phentermine and nomifensine. In addition to causing the release of DA, these compounds can also, to different degrees, inhibit neuronal re-uptake of DA.

After DA is released into the synaptic cleft its action is terminated by a neuronal re-uptake system which is a high affinity, energy-dependent active-transport process. The system is similar to that already described for norepinephrine. Both MAO and COMT are responsible for the transformation of DA to 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA, 3-methoxy-4-hydroxy-phenylacetic acid), respectively. Cocaine. by virtue of blocking re-uptake of DA into presynaptic nerve terminals, prolongs the effect of release DA in the synaptic cleft.

Elevation of brain tyrosine levels results in an increase in L-DOPA synthesis in the brain. L-DOPA in turn is metabolized to dopamine. The synthesis and release of dopamine is elevated following tyrosine administration. Without increasing catecholamine levels, dietary tyrosine increases turnover and release of dopamine and norepinephrine. Stress, cold or certain drugs, induce an increase in nerve firing to lower the levels of catecholamines in the nerve terminals.

L-Phenylalanine is an essential amino acid which is also a precursor for the synthesis of the neurotransmitters dopamine and norepinephrine. These neurotransmitters, as measured by their metabolites, HVA, DOPAC. and MHPH, are significantly altered during periods of intense exercise and physical endurance.

49 L-phenylalanine may be used instead or in combination with L-tyrosine or L-dopa to restore dopamine reserves after depletion by cocaine abuse.

The use of these precursors may be supplemented at appropriate stages of treatment with dopaminergic releasers. blockers, agonists or antagonists, or agents n 5 affecting the reuptake or degradation of dopamine, norepinephrine or epinephrine.

t However, and more importantly, the entire range of dopaminergic activity including C synthesis, and release is regulated to some degree by certain opioid peptides O enkephalins and endorphins). Centrally administered opioid peptides (endorphins and C1 enkephalins) produce elevations in levels of catecholamines in blood plasma in animals and humans (Clouet, 1982). In fact, blockade of presynaptic dopaminergic receptors results in an enhancement of P-endorphin release, showing a unique reciprocal relationship. Compounds that may be used as precursors include Ltyrosine, L-phenyalanine, pharmaline.

Chromium Salts (such as Picolinate, Nicotinate, etc.) Dietary chromium is an essential nutrient whose value in human nutrition has been conclusively documented. Interest in chromium stems from the view that because chromium is an essential trace mineral and a cofactor to insulin, it could play a role in glucose, lipid, and amino acid metabolism by it's potentiating effects on insulin action. Supporting this argument is the observation that chromium deficiency results in impaired glucose tolerance, insulin resistance, elevated blood glucose levels, and symptoms of type 11 diabetes; in addition, adequate amounts of physiologically active forms of chromium can reduce insulin requirements in humans (Kaats et al., 1996).

The National Academy of Sciences has classified chromium as an essential trace mineral and recommends daily intakes of 50 to 200 pg. However, the most reliable studies report that intake among Americans (which is similar for other countries) is suboptimal only 40% of the minimum for women and 60% for men.

There are more than 25 human studies documenting the beneficial effects of supplemental chromium on subjects living at home including improvements in g glucose, insulin, and lipid levels; impaired glucose tolerance: adults with elevated cholesterol levels; insulin and hypoglycemic patients (Mertz. 1992).

c To increase the bioavailability of chromium, several studies have suggested using picolinate acid, a naturally occurring metabolic derivative of tryptophan.

t 5 Picolinate acid appears to combine with trace metal ions in the intestines and blood, K which facilitates the collection and use of essential trace metals (Evans and Bowman, c 1992).

Because deposition of body fat appears to be regulated in part by insulin, improvements in insulin utilization should lead to reductions in fat deposition.

Enhancing the effects of insulin can also have positive effects on muscle tissue because insulin directs amino acids into muscle cells; once amino acids enter the muscle cells, they are assembled into proteins through insulin's effects on the cell's genetic material, that is, DNA and ribonucleic acid. This effect of chromium is important for this work since by doing so it reduces the competition of amino acids like valine or leucine thereby allowing for increased amounts of the amino acid tryptophan (Wurtman, 1982). Insulin also slows the breakdown, or catabolism, of body protein with a net effect of increasing the protein available for building tissue.

Chromium can potentially facilitate the maintenance or addition of fat-free mass (FFM). It has been suggested that if CrP can lower insulin resistance it can improve body composition, as insulin resistance or deficiency results in impaired entry of glucose and amino acids into muscle cells and increased catabolism of muscle protein as well as insulin deficiency's potential to accelerator lipid deposition (Kaates et al..

1996). Other references indicate that insulin resistance may help stabilize body fat in the obese patient, albeit at an obese level, acting much like a "set point" to prevent further weight gain (Eckel, 1992). In general, although animal studies have supported this contention (Liarn et al., 1993), one human study found positive changes in body composition with CrP supplements (Hasten et al., 1992), another reported positive, although not statistically significant changes in body composition (Hallmark el al., 1993), and a third failed to find any positive changes in body composition with CrP supplementation (Clancey el al., 1994). The controversial nature of the literature 1 51 0 reveals that most human studies used small numbers of subjects, and subjects often followed exercise or conditioning programs that could increase the: need for chromium at amounts higher than amounts provided in these studies.

Previous work observing concurrent chromium supplementation and exercise 5 training has been restricted to effects on body weight and composition, with Sconflicting results (Clancy et al., 1994; Evans et al., 1989; Evans et al., 1993; Hallmark el al., 1996; Hasten et al., 1992).

O Chromium Picolinate (CrP) is the most heavily used, studied and promoted Cl chromium compound, but in vitro work suggests that chromium nicotinate may be also viable in the area of weight loss and changes in body composition. In this regard, very recent work by the inventors suggest that the nicotinate salt may be even more important than the picolinate salt (Grant et al., 1997). These data are presented here as an example of the usefulness of Chromium Nicotinate as an addition to the basic composition of matter specified in the aspect of this work.

Chromium Salts and Hypertension: Diabetes Link In this invention it is proposed that various chromium salts in combination with either the D-amino acid of Phenylalanine and/or DL racemate would be a beneficial natural anti-hypertensive formula. The following description is provided to the examiner for review of the mechanisms involved and supporting data.

Dietary trivalent chromium has significant beneficial effects on the insulinsystem often enhancing insulin sensitivity and overcoming glucose tolerance (R.

Anderson, In: Essential and Toxic Trace Elements in Human Health and Disease: An Update, Wiley -Liss, Inc. Pp221-234, 1993). A recent clinical investigation performed in China on patients with diabetes showed a highly favorable response to chromium supplementation i.e. evidence of increased insulin sensitivity (Anderson et al. Diabetes 45 (suppl 124A 1996). Since diabetes and insulin resistance have been associated with hypertension, it is not too surprising that chromium added to feed effectively overcame sucrose-induced elevations of systolic blood pressure in spontaneously hypertensive rats (SHR) [Preuss et al. Clin.Neprology, 47, 325-330, 197).

52 While there were some methodological problems with this paper a rather c low systolic blood pressure for SHR bred animals (average 147 mmhg compared to other SHR's showing a much higher systolic blood pressure as seen in the case for C 5 Ehrenpreis's experiments as depicted in this invention-approximately 189-200 N' mmhg.). The second question. which seems contrary to our view, is the higher than "t baseline blood pressure observed with various chromium compounds in the Prusse et 0 0 al study. The reduction of systolic blood pressure by using our proposed formula would be non-obvious and somewhat unexpected.

With these caveats it is plausible that the use of chromium salts in the diabetic would have very useful benefits especially in combination with D or DLphenylalanine. Interestingly, there is evidence that a substance which is a 37 amino acid hormone secreted by the pancreas, called amylene, maybe associated with hypertension in diabetes mellitus. It is important to note that in various animal models ofindulin resistance including rodents with obesity, glucose intolerance and hypertension, amylene concentration have been reported to be markedly elevated.

This substance has been shown to be co-secreted with insulin and that hyperinsulinaenemic states are associated with hyperamylinaemia. Further evidence to indicate a role of amylene in the genesis of obesity-related hypertension has been suggested by the report in sucrose fed rat (Bhaysar et al. Am. J. Hypertens. 8:55A, 1995). What is important here is that in these animals, the anylin antagonist, AC413, prevented the rise in blood pressure. Moreover, amylene levels are elevated in individuals with essential hypertension (Kautzky Wiler et al. Diabetologia 37: 188- 194, 1994).

It is our proposition that since there is a clear association of hypertension and insulin resistant states and glucose intolerance as reported by a number of investigators (Hayes et al. 40: Diabetolgia 40: 256-261, 1997; Raccah et al. Diabetes Metabolism 25: 35-42, 1999; Barbagalo et al. Diabetes Metabolism 23: 281-294, 1997; Nilsson, Experimental Clin. Endocrin. Diabetes, 105 Suppl. 2: 64-69, 1997; and Fur et al. Arch. Int Med. 147: 1035-1038, 1987) and since, Chromium salts

O

S53 o are known to increase insulin sensitivity and reduce glucose intolerance and has been shown to reduce sucrose induced elevations of systolic blood pressure in SHR, the combination of D- or DI-Phenylalanine and chromium will be most beneficial in diabetes mellitus patients showing associated hypertension. We also believe that this V) 5 combination would be most beneficial in obese patients also presenting with hypertension. The combination exacts low important benefits to these obese patients, l the first being anti-glucose craving and the second being blood pressure lowing, which is important in cases of obesity and associated cardiovascular disease.

rC Taurine Taurine (2-aminoethanesulfonic acid), is a sulfur-containing amino acid that is widely distributed in animal tissues, and has a variety of biological activities (Wright et al. Am.Rev. Biochem. 55: 427-453, 1986). Although much progress has been made in discovering its enigmatic and many side biological effects, scarce insight on the mechanisms of its actions is available.

Taurine has been shown to .reduce arterial blood pressure when directly applied to the CNS by at least two studies (Sgaragli and Pavan, Neuropharmacology, 11:45-56. 1972; Bousquet et al. J. Pharmacol. Exp. Ther. 219: 213-8, 1981). The hypolipidemic and antiatherosclcrotic effects of taurine have been reported in experimental animals, including rats (Hermann, Circ. Res. 7:224-227, 1959; Sugiyama et al. Agric. Biol. Chem. 48:2897-2899, 1986); mice, and rabbits (Petty et al. Eur J. Pharmacol; 180:119-127, 1990; Yamauchi-Takihara et al. Biochem.

Biophys. Res. Commun. 40:679-683, 1986). There are also reports on the effects of taurine on serum cholesterol levels in humans (Truswell et al. J. Artereroscler Res 5:526-532, 165). There are also reports on the effects of taurine on cardiovascular diseases (Yamori et al. In: Yamori and Lenfant (eds) Prevention of Cardiovascular Diseases: An approach To Active Long Life. Amsterdam. Elsevier, pp. 163-177, 1987; Nara et al. J. Cardiovasc. Pharm. 16 (suppl S40-S42, 1990). Previous studies showed that dietary supplementation with taurine affects cholesterol 7alphahydroxylase activity, a rate-limiting effect of bile acid synthesis (Shefer et al. J. Lipid Res. 14:573-580, 1973).

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c 54 SWith regard. to the inclusion of taurine or its synthetic derivatives such as S-2methyltaurine 2 -aminopropanesulphonic acid) in a composition combined with Dphenylalanine, two important papers suggest a potential rationale, but it is unknown and therefore non-obvious that this inclusion would synergize with the blood-pressure i 5 lowing effect of our proposed composition in this invention.

~Work by Braghiroli and associates from Modena, Italy and Georgetown University Medical School found evidence for the arterial blood pressure lowering Sactivity for both taurine and its S-methytaurine enantiomer (20MT). In this first study, the authors found that the hypotensive effect elicited by the i.c.v.

administration of racemate of 2-MT is mainly due to the (S)-enantiomer. The specificity of this effect was proven by the ability of the taurine antagonist TAG (6aninonethyl-4h-methyl-1,2,4 benzohiadiazine 1, 1-dioxide) to prevent it (Braghiroli et al. Pharmacolical Research 34:33-36, 1996).

The second important study was performed by Higeru Murakami and associates from the Medical Research laboratories, Taisho Pharmaceutical Company, LTD in Japan. They evaluated the hypolipidemic effect of taurine in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP).

SHRSP were fed hypercholesteolelemic (HC) diet supplemented with 3% taurine for 50 days, and serum cholesterol was monitored. Cholesterol content and enzymatic activity responsible for cholesterol synthesis and metabolism were also determined in the liver, aorta, and intestine. Taurine prevented increases in the cholesterol level of the serum, liver and aorta induced by HC diet. Several fat deposits of the messenteric arteries induced by a IIC diet were improved by taurine treatment, showing the hypolipidemic and antiatherosclerotic effects of taurine.

Taurine enhanced the activity of cholesterol 7 -alpha-hydroxylase, a rate-limiting enzyme of bile synthesis, and stimulated bile acid production. These results suggest that taurine stimulates bile acid synthesis, which is closely related to the enhancement of cholesterol 7-alpha-hydroxylase activity, and thereby reduces serum cholesterol. In addition, a decrease in the intestinyl acyl CoA cholesterol acyltransferase activity by 0 taurine suggests that the inhibition of cholesterol absorption may also be related to the hypolipidemic effect of taurine.

It is important to note that genetically hypertensive rats such as SHR's and SHRSP's have hypercholesterolemic and fat deposits including cholesteryl esters in the walls in small and medium sized arteries, such as the mesenteric and cerebral Cj arteries, when fed a HC diet for several weeks and these changes are never observed Cin normotensive rats (Yamori et al. Stroke, 7:120-125, 1976; Lowry et al. J. Biol.

0 Chem., 1991: 193:265-275). Additionally, vascular smoothmuscle cells from SHR N and SHRSP have a higher cell prliferation and accumulate mor cholesterol than those from normotensive rats (Yamori et al. Heart Vesicles, 4:94-99, 1988; Maurakami et al. Life Sci. 56:509-520, 1995; Warrick et al. Clin Chem. 28:1379-1388, 1982).

It is interesting to note that Taurine did not have a significant blood pressure lowering effect in the SHRSP rats tested in the above described Murakami (1996) study; once again suggesting a non-obvious inclusion based soley on taurines' controversal effect to lower arterial blood pressure especially in humans.

Cognitive Enhancers The need to add cerain cognitive enhances to typical antihypertensive medications is novel. The rational for this approach is quite compelling. For example the benefits of anti-hypertensive drug therapy for older people have been clearly established. Meta-analysis suggests a 12% reduction in all -cause mortality, a reduction in coronary heart disease and a 36% reduction in stroke. The absolute benefits of treatment are great due to the high incidence of vascular disease among older people. Clinicians may nevertheless have been deterred from initiating treatment because of concerns regarding adverse affects on cognition, mood, functional ability and quality of life. To some, evidence from randomized controlled trials suggests that these concerns are groundless. Interestingly, up to 50% of older people with hypertension may remain untreated: and in over 50% of those who are treated, blood pressure may be inadequately controlled.

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c 56 SRhodiola Rhosea extract (pharmaline) SRhodiola rosea. or Golden Root, is a perennial herbaceous plant of the Orpine (Crassulaceae) family, growing in the Polar Arctic and Alpine regions. In the altai mountains, in Eastern Siberia, Tien-sdhein and in the Far East, the cultivation of Rhodiola rosea has been successfully mastered. It is possible to reproduce it both from seeds and by a vegetative method (Polozhy et al.. 1985; Saratikov and Krasnov, 'v1 1987). The rhizomes contain phenol compounds, among them the most important are Sp-oxyphenylethanol (tyrasol) and its glycoside salidroside determining the biological activity of the Rhodiola preparations (Saratikav el al., 1968). Rhodiola possess stimulative and adaptogenic characteristics. It is thought that this compound improves the ability to perform physical work; reduce fatigue; shorten the recovery period after prolonged muscular workloads; and normalize cardiovascular activity.

During intensive muscular work Rhodiola prevent loss of micurgic phosphates in brain and muscles by optimization of the processes of oxidative phosphorylation, stabilizing the muscular activity of lipids: improving the indicators of metabolism (activation of aminacyl-t-RNA-synthetase) in the skeletal muscles, increase of the RNA content, and increasing the blood supply to the muscles, especially to the brain (Saratikov et al., 1968; Saratikov, 1974). Rhodiola can increase attention span, memory; improve mental work and enhance performed work. The area of the brain involved in this activity is the thalamocortical and posterior hypothalamus (Marina el al., 1973). Various other action have been noted for Rhodiola and include; prevent development of hyper-and hypoglycemia. leukocytosis and leukopenia, erythrocytosis and erythropenia, hypoxia; reduce stress and bring about a cardio-protective action.

The stress-regulative effect of Rhodiola involves it's normalizing effect on the hypophyso-adrenal and opioidergic system. It has also been found that RhPodiola increases the anti-tumor resistance of the organism. It significantly inhibits the growth of experimental tumors, decrease the frequency of their metastases; prolongs the life expectancy of animals with recidivistic tumors, and decrease the outcome of spontaneous tumors (Dementyeva and Yaremenko, 1983). Their is some evidence that Rhodiola also reduces neurosis and fights exhaustion (Saratikov, 1977).

57 o1 U Salidrosid (an extract of Rh odiola) Salidrosid (SAL) at 30 mg/kg prevented disulfiram-induced decrease of Snorepinephrine in the brain of animals. SAL influences brain norepinephrine by virtue of its ability to inhibit the activity of COMT and MAO. SAL does not decrease q- 5 the permeability of the Blood Brain Barrier (BBB) for precursors of the catecholamines and serotonin, and this property makes it useful for the composition, particularly in aspects for the treatment of attention processing disorder.

0 Administration of rhodosine (which contains SAL, aglycone p-tyrosol and rosavin) at C 0.2 mg/kg increases the brain concentration of DOPA. dopamine and 5-IIT in the neocortex and a decrease of the level of norepinephrine in the caudate nucleus in the brain of the intact mice, 30 min after subcutaneous injection others have shown that SAL did not alter the levels of epinephrine (EPI) and DOPA: at a dose of mg/kg, it decreased the content of norepinephrine by 26% and of 5HT, by 15%; at a dose of 100 mg/kg, it decreased the concentration of norepinephrine, DA and 5-IHT by 20, 28, and 23%, respectively. Studies involving the administration of L-dopa mg/kg) and 50 HTP (100 mg/kg) to mice showed that salidrosid (30 mg/kg) increases the rise in exogenous DOPA and serotonin in animals by 26 and 13%, respectively, compared to saline-dopa-5HT-controls. These data indicate that the preparation increased the permeability of the blood brain barrier for the catecholamine precursor.

Moreover, from the research of Petkov (1981) indicates that SAL decreases MOA activity and inhibits COMT activity thereby, slowing the inactivity of Catecholamines by O-methylation and oxidative deamination. Moreover, studies have shown that SAL does not alter the activity of 5-HTP decarboxylase. Consequently, it does not influence the synthesis of serotonin from 5-HTP, but may slow the biotransformation of the amine, by slightly inhibiting MAO. Evidently the increase in the rise of serotonin in the brain in studies involving the combined administration of 5-HTP and SAL is governed by the capacity of the latter to increase the permeability of the blood brain barrier for The literature reveals a number of interactions with Rhodiola and neurotransmiter dynamics. In summary, a decrease of dopamine in the n.accumbens c 58 O (may be due to preferential DA release in this area); an increase of 5-HT in the hypothalamus: an increase of norepinephrine in the hippocampus; and an agonistic C activity of cholinergic receptors has been reported. Certain mechanisms are accepted in neuroscience related to the differential roles of various neurotransmitters in terms C 5 cognition. Cholinergic mechanisms underlie the fixation of memory trace. The noradrenergic system of the brain enhances positive reinforcement. The serotonergic mechanisms are more involved in the process of the consolidation of memory.

0The effects of Rhodiola in rats was studied using several methods of active avoidance with negative and positive reinforcement (Petkov et al., 1986). Using the 0 maze-method with negative (punitive) reinforcement, it has been found that Rhodiola extract in a single dose of 0.10 ml per rat essentially improves learning and retention after 24 h. Significant improvements of the long-term memory is also established in memory tests after 10 day treatment with the same dose of the extract. In a dose of 0.10 ml per rat the Rhodiola extract had a favorable effect on the training processes using the "staircase" method with positive (food) reinforcement as well. In contrast, with other methods used Rhodiola extract in the dose used (0.01 ml per rat) had no substantial effect on learning and memory, showing the inconsistency of this alcoholaqueous extract.

Albino rats were used to study the effects of meclofenoxate and Rhodiola on the memory-impairing action of convulsant electroshock (Lazarova e al., 1986).

While Meclofenoxate administered i.p. in a dose of 100 mg/kg body weight for five days prevented the retrograde amnesia observed after convulsant electroshock upon retention testing on the 3rd and 24th h after the end of the training session. In contrast, once again Rhodiola extract administered orally in a dose of 0.10 ml/rat for 10 days. which in other experimental approaches improved learing and memory, remained ineffective here.

Huiperzine Huperzine is a compound belonging to a class know as acertycholinesterase inhibitors. It has been shown to inhibit the enzyme that is responsible for the breakdown of acetylcholine, an important neurotransmitter, or brain chemical, which 0 59 is believed to be critical in learning and memory. l-luperzine is a naturally occurring compound that was originally isolated from the club moss Huperzine Serratu. It has Sbeen used in Chinese folk medicine and more recently in limited clinical trials conducted in China as a treatment for age-related memory disorders. Results suggest that it improves learning and memory in certain patients. However, these suggested q results have not been substantiated by clinical trials. This natural substance is contemplated for use with the composition of matter claimed in this patent to affect Sattentional processing. In humans the recommended dose to enhance memory is 150 C1 pg daily (the therapeutic range is 1.50 to 1,500 meg daily).

The effects of huperzine A on memory impairments induced by scopolamine were evaluated using a radial maze task and inhibition of cholinesterase in vitro compared with the effects of E2020 and tacrine. Scopolamine (0.2 mg/kg) significantly impaired spatial memory in rats. Huperzine A (0.1-0.4 mg/kg, by mouth E2020 (0.5-1.0 mg/kg, and tacrine (1.0-2.0 mg/kg, could reverse these scopolamine-induced memory deficits. The ratios of huperzine A, E2020 and tacrine for butyrylcholin-esterase:acetylcholinesterase determined by a colourimetric method were 884.57, 489.05, and 0.80, respectively. The results demonstrated that huperzine A was the most selective acetylcholinterase inhibitor, and improved the working memory deficit induced by scopolamine significantly better than did E2020 or tacrine, indicating it may be a promising agent for clinical therapy of cognitive impairment in patients with Alzheimer's Disease (Cheng et al., 1996).

Huperzine A, a novel, potent, reversible, and selective acetylcholinesterase (AChE) inhibitor has been expected to be superior to other AChE inhibitors now for the treatment of memory deficits in patients with Alzheimer's disease. The effects of huperzine A on performance of AF64A-treated rats in the radial maze have been assessed (Zhi et al., 1995). AF64A (2 nmol per side, caused significant impairment in rats' ability to perform the spatial working memory task. This behavioral impairment was associated with a significant decrease in the activity of choline acetyltransferase (ChAT) in the hippocampus. Huperzine A (0.4-0.5 mg kg-1, significantly ameliorated the AF64A-induced memory deficit. These results 60

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N .suggest that AF64A is a useful agent for disrupting working memory processes by altering hippocampal cholinergic function, and such impairment can be effectively ameliorated by huperzine A (Zhi et al, 1995).

SA major component of HuperazonTM is a proprietary extract of the club moss, Huperzia serrata used to treat Alzheimer's. Studies carried out in China indicated that the active substance in this extract Huperzine A. is a promising new treatment for Alzheimer's disease. Other studies indicate that Huperzine A is a superior O acetylcholinesterase (AChE) inhibitor with excellent penetration into the CNS and a remarkable in vivo half-life. Two double-blind clinical trials carried out in China demonstrate that Huperzine A is both safe and effective for the long term treatment of Alzheimer's dementia. In addition to its activity as an AChE inhibitor, recent findings indicate that Huperzine A has other neuroprotective functions: Huperzine A inhibits glutamate-induced cytotoxicity in cultures of rat neonatal hippocampal and cerebella neurons; Huperzine A promotes dendrite outgrowth of neuronal cultures.

Alzheimer's disease is characterized by abnormalities and degeneration of neurons which depend upon acetylcholine and acetylcholine esterase for normal activity and viability. These cells located in the basal forebrain are also implicated in other neurological diseases such as Parkinson's disease. Huperzine A is a potent inhibitor of acetylcholine esterase, superior in activity to Cognex®, the first drug licensed in the USA for Alzheimer's disease and E2020 which was licensed recently by Eisai Pharmaceuticals. In addition, Huperzine A has been shown to protect neuronal cells in culture from death caused by the excitoamino acid glutamate.

Because of the dual pharmacological action of Huperzine A, Huperazon T M provides a unique and important activity for the treatment of attention deficit and senile memory deficits. Toxicology and efficacy studies of Huperzine A show it to be non-toxic even when administered at 50-100 times the human therapeutic dose. The extract is active for 6 h at a dose of 2 pg/kg with no remarkable side effects.

In Alzheimer's disease, double blind controlled studies of over 160 patients, showed significant improvement measured by Weschler scale results, at doses of only 150 pg given twice daily (3-5 pg/kg). In an assessment of patients by their caretakers 0 61 U comparing Huperzine A with a placebo 11 patients on the placebo reported an improvement in clear headedness as compared with 26 patients on the Huperzine A. 8 Spatients on the placebo demonstrated improved memory as compared with 16 on Huperzine A, and one patient demonstrated language improvement as compared with in 5 8 on Huperzine A.

In In comparing the improvement in memory between patients on Huperzine

A

and patients on piracetam, 50% of the patients on piracetam demonstrated improved memory as compared with 85% on Huperzine A, 30% on piraceiam demonstrated N markedly improved memory as compared with 70% on Huperzine A, and 50% on piracetam demonstrated no improvement while only 15% of the Huperzine patients demonstrated no improvement in memory.

Two important characteristics of Huperzine A distinguish it from Cognex and E2020 as well as other experimental compounds in development. Fluperzine A is highly specific for brain acetylcholine esterase (AChE) vs. AChE found elsewhere in the body. This selectivity is believed to be responsible for the relatively low toxicity of the extract. In addition, unlike the two approved drugs for Alzheimer's disease, Cognex® and E2020, Huperzine A has been shown to lack binding to receptors in the CNS that can cause side effects such as the muscarinic receptors MI and M2.

The duration of action of Huperzine A at 3 h is superior to Cognex0 (2 h) and physostigmine (30 min). In behavioral studies of learning and memory enhancement in animals, the difference between amounts of the extract effective for memory and learning and the no-toxic-effect dose (from toxicity studies) was 30-100 fold. These data strongly suggest that Huperzine A can be useful in treating Alzheimer's disease with minimal side effects.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the an that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and O62 scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to g 5 be within the spirit, scope and concept of the invention as defined by the appended c claims.

Example 2: SiHawthorn Berries Categories: Circulatory System 0 Symptoms: Cardiovascular Disease. Circulation, High Blood Pressure, Low Blood Pressure, H-igh Cholesterol, Sleeplessness/Insomnia. Nervousness, Poor Digestion, Overweight Hawthorn berries have been used since the 19th century to support the heart and normalize cardiovascular functions. Today, hawthorn berries are one of the most popular herbs used in Europe. Hawthorn berries appear to work best when taken as a preventative herb, possibly helping reduce the risk of cardiovascular disease.

With its high content of bioflavonoids, hawthorn berries best support the heart- The bioflavonoid substances help dilate and strengthen the walls of blood vessels, relax arteries, and improve circulation of blood to heart muscles.

Hawthorn berries help normalize the heart, either by stimulating or depressing its activity. For this reason, hawthorn berries are used to lower high blood pressure and high cholesterol, as well as increase low blood pressure.

When used to support weight loss programs, hawthorn berries reduce water retention expelling excess salt from the body. Hawthorn berries have also been known to reduce nervous tension, alleviate insomnia and aid digestion.

Take two capsules twice a day with meals, liquid-, take 1/2 teaspoon with water three times a day-

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63 SHawthorn Berry Syrup Slawthorn is a heart and circulatory tonic. It strengthens weak or damaged heart tissue by allowing oxygen to be better utilized by the heart muscle. It has been shown to be a valuable aid for feeble heart action, irregular pulse and preventing V 5 hardening of the arteries. Its anti-spasmodic properties assist in angina pectoris, it is valuable for palpitations, arteriosclerosis, high blood pressure, inflammation of the Sheart muscle, and valvular insufficiency.

Hawthorn Berries (Craraegus oxyacantha) C'1 Long used to treat digestive problems and insomnia, in the late nineteenth century European physicians discovered that the berries from the hawthorn tree were also a cardiotonic- Hawthorn is rich in biofavonoids, compounds that are essential for vitamin C function and that also help strengthen blood and oxygen to the heart. It also lowers blood pressure, thus reducing the work required by the heart to pump blood throughout the body. At the same time, it helps strengthen the heart muscle. It also works as a diuretic, helping to rid the body of excess salt and water, Caution-. Although most hawthorn preparations are safe, this herb is also available in a highly concentrated form that should be used only under medical supervision.

Part Used-. Berries Common Use: Tonic to cardiovascular/ circulatory system, angina pectoralis, heart valve murmur, High blood pressure, arteriosclerosis; Gentle action, may be used safely.

Dosage: 8-12 drops One important focus of the present invention is a method for effecting an antihypertensive therapy in animals which involves the step of administering to the animals a substance that inhibits the destruction ofenkephalins or endorphins. Such a substance is usually an inhibitor of enkephalinase or endorphinase a prototypical substance being D-phenylalanine. Such D-phenylalanine may be administered orally or parenterally and is useful for the lowering of blood pressure in hypertensive humans. In many cases the racemic D,L- phenylalanine may be used for the same

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64 purpose. One effective dosage of D-phenylalanine is administration at about 2 to 4 Sgrams per patient daily. Such administration may be accompanied by numerous C substances, including an adrenergic beta blocker such as propranolol. Analygous antihypertensive results are obtainable by the administration of D-lcucine or D,L- 5 Icucine. This may be in combination with D- or D,L-phenylalanine. In certain cases Sdrug: bestatin; thiorphan; captopril: or puromyein may be used as hypertensive agents, Sperhaps in combination with D- phenylalanine or D,L- phenylalanine administration.

0 In one prefered embodiment, the enkephlinase or endorphinase inhibitors are a component of a dietary supplement. Such dietary supplement might: be complimented with other substances such as D-Ribose. It has been found that hydrocinnamic acid is also useful for inhibiting endorphinase or enkephalinase and controlling hypertension.

In some cases the administration of an endorphinase or enkephalinase inhibitor may be conducted with positive effects in combination with the administration of at least one of ferrulic acid, pharmaline-P, huperzine-H, a chromium salt selected from at least one of chromium picolinate, chromium nicotinate, and chromium polynicotinate.

Other optional inclusions are Co-enzyme, Q Pycogenol and Hawthorn or Hawthorn extract.

Additionally, such enkephalinase or endorphinase inhibitors may be in combination with a diuretic, a sympatholytic, a direct vasodilator, an angiotensinconverting enzyme inhibitor, a calcium channel blocker, an angiotensin II receptor antagonist, a T-type calcium antagonist including, nisoldipene, losartin, moxonidine and fenoldopam.

In certain cases an inhibitor of endorphinase or enkephalinase might be combined with a source of magnesium known to aid in the treatment of hypertension.

Such endorphinase or enkephalinase inhibitors might be in combination with other substances such as Rhodiola rhosea extract (pharmaline) or Salidrosid. Additionally, it may be in combination with Huperzine, Hawthorn berry or Hawthorn berry extract.

Additionally, an adrenergic-beta-blocking agent may also be of value in combination with such endorphinase or enkephalinase inhibitors for the reduction of hypertension.

REFERENCES

The following and previously, mentioned references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Cl Arznieim Forsch, "Use of D-Phenylalanine in Parkinson's Disease", Vol. 26, NR4, 1976.

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Surg., 83:390-398, 1982.

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Claims (31)

1. A method for effecting an anti-hypertensive therapy in a human Scomprising the step of administering an effective amount of a substance that inhibits the destruction of enkephalins or endorphins. in S2. The method of claim 1 wherein the substance is D-phenylalanine and the administration is oral.

3. A method for lowering blood pressure in humans by orally administering an effective amount of D-phenylalanine.

4. The method of claim 3 wherein the D-phenylalanine is administered at a rate daily of about 2 to 4 grams.

5. The method of claim 3 wherein the administration of D-phenylalanine is accompanied by administration of propranolol or other P blocker.

6. The method of claim 3 wherein the D-phenylalanine is supplied as part of a dose of D,L-phenylalanine.

7. The method of claim 5 wherein the D-phenylalanine is administered as D,L-phenylalanine.

8. D-phenylalanine for use as an anti-hypertensive agent.

9. D-leucine or D,L-leucine for use as an anti-hypertensive agent. The use of D-leucine or D,L-leucine as in claim 9 defined further as in combination with D-phenylalanine. 68 1 1. Bestatin for use as an anti-hypertensive agent.

12. Thiorphan for use as an anti-hypertensive agent.

13. Captopril for use as an anti-hypertensive agent.

14. Puromycin for use as an anti-hypertensive agent. The use of claim D,L-phenylalanine administration.

16. The use of claim D,L-phenylalanine administration.

17. The use of claim D,L-phenylalanine administration.

18. The use of claim D,L-phenylalanine administration. 11 in combination with D-phenylalanine or 12 in combination with D-phenylalanine or 13 in combination with D-phenylalanine or 14 in combination with D-phenylalanine or

19. The method of claim 2 in which D-phenylalanine is used in combination with a separate anti-hypertensive agent. The method of claim 19 where the separate anti-hypertensive agent is a diuretic or blood vessel dilator.

21. A dietary supplement comprising an inhibitor of endorphin or enkephalin destruction. 0 69

022. A dietary supplement comprising an inhibitor of enkephalin or endorphin destruction and D-ribose.

23. The dietary supplement of claim 21 where the inhibitor is D- 5 phenylalanine. O

24. The method of claim 1 where the enkephalinase or endorphinase 0 inhibitor selected is from the group consisting of D-phenylalanine, hydrocinnamic CI acid and D,L-leucine. The method of claim 1 further comprising administering at least one of ferrulic acid, pharmaline, huperzine, at least one of chromium picolinate, chromium nicotinate, and chromium polynicotinate, co-enzyme Q, pycogenol and Hawthorn or Hawthorn extract.

26. The method of claim 1 further comprising administering a diuretic, a sympatholytic, a direct vasodilator, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, an angiotensin II receptor antagonist, or a T-type calcium antagonist including, nisoldipene, losanin. moxonidine fenoldopam.

27. The method of claim 1 further comprising administering at least one of a stimulator of increased norepinephrine, angiotensin II, agonists such as clonidine, and an adrenergic receptor blocker.

28. The method of claim I further comprising administering an inhibitor of norepinephrine synthesis selected from the group consisting of methyl-p-tyrosine, carbidopa, diethyl, diethyldithiocarbonate, FA163 and disulfiram or other inhibitors of dopamine-P-hydroxylase.

29. The method of claim I further comprising administering a magnesium The method of claim I further comprising administering a chromium salt.

31. The method of claim 30 where the chromium salt is a picolinate, nicotinate, chloride, acetate or nicotinic acid-glycine-cysteino-glutamic acid (NA- AA).

32. The method of claim I further comprising administering a Rhodiola rhosea extract (pharmaline).

33. The method of claim 1 further comprising administering Salidrosid (a Rhodiola extract).

34. The method of claim 1 further comprising administering huperzine. The method of claim 1 further comprising administering Hawthorn berry or Hawthorn berry extract.

36. The method of claim I further comprising administering an adrenergic- beta-blocking agent.

37. The method of claim I where the enkephalinase or endorphinase inhibitor is D-phenylalaninc.

38. A pharmaceutical composition for lowering blood pressure comprising as a daily dose: D- or D,L-phenylalanine, 1 mg to 10,000 mg; chromium salts (picolinate or nicotinate or other salts), 1 microgram to 30,000 micrograms; D-ribose, 1 100 mg to 10,000 mg: calcium chelate, 10 mg to 3,000 mg; L-taurine, 10 mg to 10,000 mg; and L-glycine, 10 mg to 10.000 rmg.

39. A pharmaceutical composition for lowering blood pressure comprising: D- or D,L-phenylalanine, a chromium salt, D-ribose. calcium chelate, L-taurinc, and L-glycine. O1 The composition of claim 39 further comprising at least one of: a brain cognitive enhancing amount of ferrulic acid. pharmaline, and hupcrzine to increase focus, memory or attention. Dated this 30 th day of December, 2004 SEYMOUR EHRENPREIS AND KENNETH BLUM By Its Patent Attorneys DAVIES COLLISON CAVE