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Document Type and Number:
WIPO Patent Application WO/1985/003873
Kind Code:
Method of treatment of hypertension and toxemia of pregnancy in vertebrates. It comprises the administration of manganese-containing pharmaceutical preparations in appropriate ratios with tryptophan to lower the blood pressure. These are given in amounts appropriate to the individual subject in a schedule of treatment which varies in amount, frequency and said ratios, as it produces a cumulative effect, reflecting the changing blood pressure of that affected individual adjusting back to within the normal range of blood pressure.

Application Number:
Publication Date:
September 12, 1985
Filing Date:
March 01, 1985
Export Citation:
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International Classes:
C07F13/00; A61K31/195; A61K31/28; A61K31/40; A61K33/00; A61K38/00; A61K38/04; C07K5/04; (IPC1-7): A61K37/02; A61K31/195; A61K31/40
Foreign References:
Other References:
J. Med. Chem., Vol. 25, 1982, SAFDY, Tryptophan Analogues. I. Syntesis and Antihypertensive Activity of Positional Isomers, pages 723-730
CHEMICAL ABSTRACTS, Vol. 90, 1979, SHUKLA, Species Variation in Manganese Induced Changes in Brain Biogenic Amines, Abst. No. 198507v
CHEMICAL ABSTRACTS, Vol. 98, 1983, KALIMAN, Biogenic Monoamines and their Precursors in Rats with Spontaneous Arterial Hypertension, Abst. No. 195890u
CHEMICAL ABSTRACTS, Vol. 99, 1983, CHANDRA, Psychiatric Illness due to Manganese Poisoning, Abst. No. 100473u
The American Journal of Clinical, Vol. 6, No. 5, 1958, SALMON, The Significance of Amino Acid Balance in Nutrition, pages 487-494
See also references of EP 0181342A4
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1. A method of treating hypertension in vertebrates and other organisms which comprises orally administering to an affected subject in an antihypertensively effective ratio an effective amount therefor of at least one of (a) com¬ prising Ltryptophan, Dtryptophan, and their alphaketo and alphahydroxy analogs and acetylLtryptophan and acetylDtryptophan and the dipeptides and tripeptides of the aminoacids or a pharmaceutically acceptable acid addi¬ tion salt thereof and an effective nonlethal amount there¬ for of (b) a preparation consisting essentially of a man¬ ganese compoundo.
2. A method of treatment of toxemia of pregnancy in Placentalia which comprises administering to an affected subject in an antihypertensively and antieclamptically effective ratio an effective amount therefor of at least • one of (a) comprising Ltryptophan, Dtryptophan, and their alphaketo and alphahydroxy analogs and acetyl Ltryptophan and acetylDtryptophan and the dipeptides and tripeptides of the aminoacids or a pharmaceutically acceptable acid addition salt thereof and an effective nonlethal amount therefor of (b) a preparation consist¬ ing essentially of a manganese compound. TITUTE SHEET.


1. Field of the invention

Essential hypertension is one of the most general of the -diseases occurring in western civilization. It is commonly associated with changes in lipid metabolism and atheromatosis. The changes in atherosclerosis produce the principal causes of arterial occlusion and death in hypertensives, e.g., coronary heart disease, cerebro- vascular accidents, renal insufficiency and intermittent claudication.

The chemical changes that occur in hypertension in humans occur in other species. The level of " blood pressure directly reflects changes in the metabolism of biogenic amines. The increased pressures encountered result from a relative preponderance of ca echolamines, especially norepinephrine. The oxidative processes involved in the modulation of catecholamine levels and the levels of other biogenic amines are directly involved in maintaining the blood pressure within normal limits.

Toxemia of pregnancy is characterized by hypertension occurring during pregnancy. The pathological changes occurring in the placenta and fetus directly relate to the vasoconstriction occurring as a result of the hypertension.


"Monoamine oxidase is a flavoprotein oxidase of PURPORTED CENTRAL METABOLIC IMPORTANCE- CONVERTING NEϋROACTIVE AMINES INTO INACTIVE ALDEHYDES_ ...The flavin linked monoamine oxidase is localized in the OOTER MITOCHONDRIAL MEMBRANE OP ANIMAL CELLS. Walsh pp. 402.403. "Actions: Monoamine oxidase ' is a complex enzyme system; widely dxstri bated throughout the body. Drugs that inhibit monoamine oxidase in the laboratory are associated with a number of clinical effects. Thus it is UNKNOWN WHETHER MAO INHIBITOR PER SE, OTHER PHARMACO LOGICAL ACTIONS. OR AN INTERACTION OF BOTH is responsible for the clinical effects observed. Therefore the physician should become familiar with all the effects produced by drugs of this class. PDR (Physicians' Desk Reference 1983) p. 1516.

Two classifications of amine oxidases were presented in 1959. That by Blashko, et al used the response to carbonyl inhibitors to distinguish between the activities of the various amine oxidase. That by Zeller, et al, used semicarbazide inhibitors. The use of inhibitors to classify amine oxidases reflected difficulties encountered in purifying these enzymes and studying the structure of their active sites.

"A. Occurence

"Monoamine oxidase (MAO) has been found in all classes of vertebrates so far examined (1970) : mammals birds reptiles, amphibians and teleosts (161) . The enzyme occurs in many different tissues particularly in glands, plain muscle, and the nervous system (162)

In man the parotic and submaxillary glands seem to be the richest source of MAO (163)« It also occurs in molluscs and plants (-> ) _, " Kapeller-Adler p. 31•

In 1957 iproniazid was introduced for the treatment of depression. New York Times article June , 1981, p. B9„ It has been studied extensively and is a monoamine inhibi¬ toro However, it has a variety of effects besides the ef¬ fect on depression. These have frequently posed problems. The use of these drugs has continued to be empirical. It is interesting to note that these drugs exert their bene¬ ficial effect in depressed patients anywhere from one to several weeks after the beginning of treatment. "In some instances the improvement may progress to a state of eu¬ phoria, hypomania or even mania. Central stimulatory ef¬ fects are seen with these drugs in normal individuals as well as in depressed patients." Bevan. Other effects are orthostatic hypotension, allergic reactions affecting the liver, dizziness, and a number of anticholinergic type symptoms=

"Isopropylnorepinephrine, the most potent cardiac stimulant in the Pharmacopeia, is useful in the immediate treatment of heart block." As to actions, "Isopropylnor¬ epinephrine acts almost exclusively on beta-adrenergic receptors." Vevan p. l θ. This drug, which is modified norepinephrine is useful in the treatment of bronchial asthma0 However, it must be administered by sublingual tablet or oral inhalation to avoid excessive VAS0C0N- STRICTION elsewhere.

es. 'ϋ * _,_ * l »«__-__»_*_ *• > _."f3" " j' u' •-j__£<__ S___.H. EET

Our- interest in isoproterenol (isopropylnorepinephrine) is related to the demonstration of vasoconstrictor lesions by

Selye and other members of the Montreal group i__ 1953.

Rona et al summarized as follows: "It is shown that isoproterenol...is capable, when administered subcutaneously to the rat, of producing gross and microscopic myocardial necrosis. There is a close correlation between the dose injected and the degree of severity of the necrosis, which makes possible the production of standardized myocardial lesions.

"On the basis of the localization and histological characteristics of the myocardial necrosis produced by this drug and the similarity to.ischemic necrosis produced by other techniques we tentatively conclude that the lesions in both cases have a common pathogenetic mechanism."

In toxemia of pregnancy, vasoconstriction, hypertension and fluid retention are the three features of the disease that are constantly present. "All these lesions may be explained on the basis of widespread vasoconstriction, the cause of -which constitutes the problem of numerous investigators. Studies on patients under a ' variety of experimental conditions indicate that the vasoconstrictor factor is more likely humoral than neurogenic". Netter p. 237. There are the progressive changes of hypertension in the mother with toxemia, and a very high percentage of women with hypertension experience toxemia of pregnancy. The incidence increases as the prospective mother falls into an older and older age group.



"The enzyme isolated from a number of sources exhibits low specificity. In general, primary, secondary, and tertiary amines, tryptamine derivatives and catechol- amines are oxidized (1,5) • The * enzyme isolated from human placenta, however, will only attack primary amines and with simple alkyl amines increase in chain length results in increased affinity (7)**" Barman p. 180.

"Inhibition of MAO leads to a very pronounced increase in the levels of norepinephrine in the sympathetic nerv¬ ous system and of the monoa ines serotonin, norepine¬ phrine, and dopa ine in the monoamine-containing neurones of the CNS..o.Large amounts of amine now accumulate in the cytoplasm. The storage sites rapidly become filled to capacity with the transmitter. This enhanced accumu¬ lation of neuroamines within the neurones is presumed to be the basis for the antidepressant action of the MAO inhibitorso ...It should be added that the presence in the urine , of'large amounts of unmetabolized serotonin and 3-0- methylated catedholamines is characteristic of patients on MAO inhibitor antidepressants." Bevan pp. 183, 13*--..

These urinary compounds indicate clearance of the above amines from the blood and is consistent with an in¬ creased turnover rate of increased amounts of each amine.

"The flavoprotein responsible for the oxidative deami- nation of the catecholamine (monoamine oxidase) is found in a wide variety of tissues and is located primarily in the outer membrane of mitochondria." Frisell p. 628.

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Halogenated compounds enter the body frequently from the environment. The anaesthetics halothane and methoxyflurane are cases in point. "Incubation of the volatile general anaes-

16 thetics halothane or methoxyflurane (labelled with Cl) with hepatic microsomes, NADPH / and oxygen is accompanied by extensive


"Similarly thyroxine and triiodothyronine undergo deiodination by hepatic microsomal enzymes (8)." 3acq p. 577. "Dimino and

Hoch (1972) found a considerable enrichment of iodine in liver mitochondria of rats injected with- T 4 . These mitochondria were more dense than those of untreated animals and and appeared to contain iodine TIGHTLY BOUND TO THEIR INNER MEMBRANES (9) _

...Direct effects of . on isolated mitochondria have been known

4 for some time, but they occur only at HIGH, ONPHYSIOLOGICAL CC)NC_Ξr-ITRATIONS and their significance is doubtful. (9)" Lash p. 332. "The actual, biochemical mechanism of thyroid hormone action on neural tissue is poorly understood." It is evident that a single regulatory reaction has not been found to explain the multiple effects of thyroid hormones. tt Although the activities of more them 100 enzymes have been shown to be affected b^ thyroxine administration it appears that all are not influenced to the same degree. (10).' ' Frisell p. 608


"The early studies of Greenberg (65) with radiomanganese indicated only 3-4% of an orally administered dose is absorbed in rats. The absorbed manganese quickly appeared in the bile and was excreted in the feces. Experiments since that time with several species including man indicate that manganese is almost totally excreted via the intestinal wall by several routes. These routes are interdependent and combine to provide the body with an efficient homeostatic mechanism regulating the manganese levels in the tissues (16, 90,129). The relative stability of manganese concentrations in the -tissues to which earlier reference was made is due to such controlled excretion rather than to regulated absorption. (27)." Underwood p. 184.

It is important to realize that each of these tissues in the intestinal tract are actually using the same system to take in and to dispose of manganese. What is being described above is the flow of manganese into mitochondria and out again. t is a reflection of the itochondrial pool, which is a very labile pool. Manganese is carried in the plasma bound to protein. Very little of it is cleared by the kidneys.

"Injected radiomanganese disappears rapidly from the bloodstream (23, 90). Borg and Cotzias (28) have resolved this clearance into three phases. The first and fastest of these is identical to •

THE CLEARANCE RATE OF OTHER SMALL IONS, SUGGESTING THE NORMAL TRANSCAPILLARY MOVEMENT, the second can be identified with the ENTRANCE OF THE MANGANESE INTO THE MITOCHONDRIA OF THE TISSUES, AND THE THIRD AND SLOWEST COMPONENT COULD INDICATE THE RATE OF NUCLEAR ACCUMULATION OF THE ELEMENT.... The kinetic patterns for blood clearance and for liver * uptake of manganese are almost identical indicating that the two manganese pools - BLOOD MANGANESE AND LIVER MITOCHONDRIAL MANGANESE - RAPIDLY ENTER EQUILIBRIUM. A high proportion of the body manganese must, therefore, be in a dynamic mobile state_ Underwood p. 185_

"The turnover of pa enterally administered J - _-Mn has been directly related to the level of stable manganese in the diet of mice over a wide range (27) _ A linear relationship between the rate of excretion of the tracer and the level of manganese in the diet was observed and the concentration of J Mn in the tissues was directly related to the level of stable manganese in the diet, THIS PROVIDES FURTHER SUPPORT FOR THE CONTENTION THAT VARIABLE EXCRETION RATHER THAN VARI¬ ABLE ABSORPTION REGULATES THE CONCENTRATION OF THIS METAL IN TISSUES." Underwood p. 185.

"Little is known of the mechanism of absorption of manganese from the gastrointestinal tract, or of the means by which excess dietary calcium and phosphorus reduces manganese availability„ ... The effect of variations in die¬ tary calcium and phosphorus on the metabolism of ^ Mn in rats has been studied further by Lassiter and associates (100). These workers found that the fecal excretion of parenterally administered -' Mn was much higher and the liver retention lower, on a 1.0% calcium diet than on a 0.64- calcium diet." Ibid 186.


It appears, therefore, that calcium cam influence manganese metabolism by affecting retention of absorbed manganese as well as by affecting manganese absorption. Variations in dietary phosphorus had no comparable effects On the excretion of intra- peritoneally administered 54 Mn, BUT THE ABSORPTION OF ORALLY ADMINISTERED 54 Mn WAS IMPAIRED. Underwood, p. 136. ; During 1970 a rash of books drew attention to energized translocation or transport and to the changes in conformation of the membranes of the mitochondria. There were extensive correlations devised with the mitochondrial oxidative phosphorylations. By 1975 some of this was discounted by claims that many solutes crossed the mitochondrial membranes without active transport. A number of postulates evolved including proton, phosphate and other mechanisms for these transfers.

In muscle and nervous tissue there are differences of sixty millivolts or more between the inner and outer surfaces of cell membranes. A Ca/Mg pump explains a wide variety of data. There seemed initially to be good data for high resonant phosphate compounds activating the cation pumps of mitochondria. Such a pump is affected by changes in concentration of- calcium and it is' also modulated by magnesium. Mn goes in and out of mitochondria

readily. It does so by active translocation and in the company of alkaline earth metal cations. Other metals participate but to a lesser degree. A Ca/Mg pump operating in tandem with Na/K ATPase pumps not only fits the cell membrane, but it also would have a place in the mitochondrial scheme of things.

It has long been suggested that mitochondria represent primitive bacteria originally ingested when cells developed phagocytic functions. The effective oxidation processes of the ingested cells are cited as the cause of the symbiosis developing. The corollary of that suggestion is the need that developed to correlate flow of high resonant compounds between the original cell and the mitochondria. This theory suggests that metabolic disease might well occur at the site of such a complex metabolic adjustment between the metabo¬ lism of two different cells» This mechanism of regulation is consistent with that theory.

The added point must be made that the high efficiency ascribed to mitochondria as sources of high resonant bonds highlights the need for a central control mechanism.. Such a mechanism must collate the energy production of the mito¬ chondria with the energy metabolism of.the cells, organs, and indeed the entire organism. Calcium would seem a logi¬ cal choice as the modulator of a system interactive between eukaryotic cells and mitochondria. This is consistent with the present presentation.

This mechanism or system of control has been called a mechanism of regulation. Listing the sequence of compon¬ ents described includes cation, ATPase pump, Win, deiodinase, thyroid hormones, monoamine oxidase and amines. ALL ARE FOUND IN CLOSE PROXIMITY IN THE MITOCHONDRIA.


The valence electrons are the ones in the outer orbitals. At each level, the effort is to achieve a filled group of orbitals. Thus, the chloride atom is likely to add an electron its outer set of orbitals and carry an eextra electron when it is structured as an ion. Thus it mimics the electron cloud of argon.

The calcium atom has a filled 4s Λ grouping but tries to get rid of those two electrons to form Ca ion which then mimics the argon electron cloud. The electron clouds of group VIII A belong to the inert gases and thus are all filled.

Group VII B is between the alkaline earths and the halogens. At the fourth row, the smallest of this group lies between calcium which is next to the left end of that row and bromide which is next to the right end of that row. It is the fifth transition metal to the right of calcium and the tenth metal to the left of bromide. Bromide has two of the s electrons in the fourth series and five * of the p electrons in the fourth level. Iodide has the same distribution of s and p at the fifth level. In addition, it has the full ten of the d electrons in the fourth level. Since there is a spacial over lapping of the orbitals of the two levels , it illustrates the opportunities for similarities in the electron clouds that surround the nuclei of atoms when elements ,are larger with a larger number of orbitals.

These considerations of structure relate the first element of the VIIB group to calcium on the left end of its row and to iodine on the other end of the next row, i.e., in size. In other words it has strong similarities to calcium and to iodine. When viewed at the atomic level of size it would appear similar to calcium and it would appear similar to iodine. Three is the quantum for its d electrons. A quantum number is the numerically defined symbol designed to indicate the 'energy* of.the electron. Energy in reality is the total movement of the electron. The quantum numbers of the electrons indicate their move¬ ment and on occasion we shall refer to this as 'resonance*. Resonance infers the amount of movement one attributes to an electron in its orbital.

Electrons, then, are defined by their positions relative to the position of the nucleus of an atom or in the case of molecules relative to the position of the nuclei of the atoms around which an orbital extends. We may indicate position by P and the change in position by delta P. Defining the position by P and the change in position by delta P enables us to measure the pathway of the electron from some arbitrarily selected site and thus comparing its movement with that of the other entities in its atomic environment.

At the atomic level, manganese looks like a calcium ion when both of lose their outer electrons and conform to the inert gas Argon.

Iodine looks like manganese. Both are solids at room temperature and both have outer electrons of a group of two electrons and a group of five electrons. Except for the halogens, THIS OUTER CONFIGURATION IS FOUND ONLY IN MANGANESE. Except for some considerations of spin, the atoms of iodine and manganese should have very pro¬ nounced similarities. These are reflected in the SCB radii, and manganese is the element that would be expect¬ ed to most closely approximate the specificity require¬ ments of the deiodinase enzyme located at the inner mem¬ brane of the mitochondria.

The salts of manganese are found nearby inside the mitochondria. These salts occur there along with the salts of calcium, strontium and magnesium. Active trans¬ location of manganese into the mitochondrial matrix by high resonant ATP conforms well to the calcium in the active translocation of divalent cations. Manganese also con orms well to the active site of the inner membrane enzyme which selectively removes the iodine atoms from the 3 and position of the distal phenyl rings of thy- ronine in the thyroid hormones T^ and T-


...— ' .—* _r * **


Manganese-containing pharmaceutical preparations decelerate the rate of oxidation of biogenic amines. The increased levels of amines resulting cause higher levels of activity in most cells in the organism. Shifts in biological parameters such as blood pressure occur. By combining the use of the manganese with tryptophan the precursor of serotonin there occurs a lowering of blood pressure.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the invention, there are manganese-contain compounds provided that are adapted for use in the system of oxidation of amines in biological systems. It is the overall concept of the subject invention to providethese compounds to control the rates of amine oxidation.

In order that the invention may be more easily understood, the following example will now be given, though by way of illustration only, to show details of the ' formulation of the invention and the clinical test results obtainable using such formulations.

In this method hypertension " is best evaluated initially by the clinician personally whenever any treatment is undertaken. This is dictated by the changes in dosages that sometimes occur very, quickly. It is desirable that the patient should be taken off all drugs when beginning treatment. In order to evaluate the actual pressures beingexperienced, it is necessary to know if there are any drug effects involved.

Because of the cumulative nature of the medication, 'as if filling up a hole*, treatment should be begun with small amounts and worked up to maximum dose. Then, almost as quickly, over a period of days, it may be necessary to decrease the dosage again. The manganese may be considered to modulate the tryptophan in its action as a precursor.

Example #1

Patient S.S. Postmenopausal

Long history of hypertension.

Overweight, moderately.

Active. Treatment periods: Ranged from one day to five weeks.

Treatment: manganese content in mg in manganese gluconate. tryptophan in one hundred mg tablets.

Treatment period interval: four month period during spring and summer. Objective findings:

Blood pressure

A. Systolic pressure: ranged from 122 to l6θ mm. Hg pressure (mm. mercury)

B. Diastolic pressure: ranged from 70 to 90 millimeters Hg

C. Pulse pressure: Ranged from Z to 70 mm Hg

(= difference between systolic and diastolic)

Pulse: ranged from 6k to 78/minute.

Range of Medication: Manganese mg 1 to 3» usually 2 mg Tryptophan: none to 150 mg, usually 50 to 100 mg medication given about two out of three *blood pressure checks" . Tryptophan: range of 0.67 to 2 mg kg body wt. Manganese: range of 0.0l mg to 0.04 mg kg B.W.

Objective of treatment: Maintain blood pressure within normal range (e.g., diastolic 90 or below) Maintain proper ratio of affective tone and blood pressure control

Subjective findings:

I Affect (emotional tone): tended to deteriorate when blood pressure control maintained almost entirely with tryptophan. Restored by increased use of manganese.

II Patient's opinion of effect of treatment: tended to be more favorably evaluated when more of the man¬ ganese was given, although in such small amounts. Summary:

Clinical response: On a very limited intake of the two medications, frequently none being given when the blood pressure was checked, for a four month period satisfactory control of blood pressure.

The manganese was given about sixty per cent of the time, the tryptophan somewhat more.

The tryptophan ratio to manganese, Tr/mn ranged from l-ty_L to 150/1 (mg)