AU599567B2 - Method of treating memory disorders of the elderly - Google Patents

Description

4 i-AI 39 991 185 PCT WORLD INTELLE L P OPERTY 01 IZATION INTERNATIONAL APPLICATION PUBLISH U9ERW E ENON TERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 85/ 03869 A61K 37/02, 31/28, 31/195 Al (43) International Publication Date: 12 September 1985 (12.09.85) (21) International Application Number: PCT/US85/00325 SU, TD (OAPI patent), TG (OAPI patent).

(22) International Filing Date: 1 March 1985 (01.03.85) Published With international search report.

(31) Priority Application Number: 585,285 With amended claims.

(32) Priority Date: 1 March 1984 (01.03.84) doc ent coals (33) Priority Country: US taendnntt mde U d

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Sectian 49, (71)(72) Applicant and Inventor: ERK, Vernon [US/US]; Cedar Hill Associates, Ste. 2860, 420 Lexington Ave., t to prnting.

New York, NY 10170

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(81) Designated States: AT (European patent), AU, BE (European patent), BR, CF (OAPI patent), CG (OAPI patent), CH (European patent), CM (OAPI patent), DE.

(European patent), FR (European patent), GA (OAPI patent), GB (European patent), JP, LU (European patent), ML (OAPI patent), MR (OAPI patent), NL (European patent), RO, SE (European patent), SN (OAPI 2 SE patent), IpI' TN. OF 1 Title: METHOD OF TREATING MEMORY DISORDERS OF THE ELDERLY I (57) Abstract Method of treatment for memory disorders of the elderly comprising administering manganese-containing pharmaceutical preparations in appropriate ratios with isoleucine, methionine, phenylalanine, tyrosine and valine and in appropriate ratios as between the amino acids used to improve the memory of affected individuals; each of these to be given in cumulative amounts appropriate to the individual patient in a schedule of treatment which varies in amount, frequency and said ratios as it reflects the changing degrees of imbalance of the affected individual with adjustments in metabolic .balance as it approaches that present when loss of memory developed its first manifestations clinically.

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WO 85/03869 PCT/US85/00325 -14- However, as demonstrated, alpha-ketoglutarate is formed -WO 85/03869 PCT/US85/00325 r-1- METHOD OF TREATING MEMORY DISORDERS OF THE ELDERLY BACKGROUND OF THE INVENTION 1. Field of the invention Loss of memory becomes a progressively commoner problem as the individual ages. It occurs in a great many degenerative brain diseases. Degenerative brain disease is frequently associated with disturbances in glucose metabolism.

Such disturbances may be characterized by confusion as well as memory loss. Confused states that are associated with memory loss and also with low blood sugar levels include pellagra, Alzheimer's disease, later stages of Parkinsonism in many cases, Korsakoff's syndrome with Wernicke's in alcoholism and many less well-known syndromes. Kwashiorkor and marasmus, severe forms of protein depletion commonly 00.: lapse into stuporous and c~nfused states in their later 0 stages, and postoperative protein depletion may demonstrate 0 0 similar symptomatology.

The difficulties in explaining and treating these diseases may be taken to indicate how fundamental are the metabolic changes that underlies these frequently terminal neurological events.

o o Any attempt to understand the metabolism of glucose eventually encounters the tricarboxylic acid cycle in the mitochondrion.

The factors involved in maintaining balanced production of high resonant compounds and distribution appears to be involved in each of the above syndromes.

WO 85/03869 PCT/US85/00325 PRIOR ART STATEP-MT -2- 2. Prior Art "Monoamine oxidase is a flavoprotein oxidase of PURPORTED CENTRAL METABOLIC IMPORTAYCE COtNVERTIN.1G TEUROAC!ZIVrE AMIrfES INTTO INACTIVE ALDEHYDES,...The flavin linked monoamine oxidase is localized in the OUTER MITOC!EONDRIAL MEMBRANE OF ANIMALU CELLS. Walsh pp. 402 403.

"tActions: Monoamine oxidase is a complex enzyme system widely distri buted throughout the body. Drugs that inhibit monoamine oxidase in thle laboratory are associated with a number of clinical effects.

Thus it is M TOWT' vn-TFR MAO INH:IBITOR PER S E, O=~R PHARMACO.

LOGICAL ACTIONS, OR AN INTERACTION' OF ROMI is restponsible for the clinical effects observed. Therefore the physician should become familiar with all the effects produced by drugs of th-is class.

POR (Physicians' Desk Reference 1983) p. 1516.

Two classifications of amine oxidases were presented in 1959.

That by Blashko, et al used thfe 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 V ~of their active sites. *99 9cuec "Monoamine oxidase (MAO) has been found in all classes of vertebrates so far examined (1970) maxt'als birds reptiles.

amphibians and teleosts (161). The enzyme occurs in many different tissues particularly in glands, plain muscle, and the nervous system (162).

.WO 85/03869 PCT/US85/00325 -3- In man the parotid and submaxillary glands seem to be the richest source of MAO (163). It also occurs in m6lluscs and plants Kapeller« Adler'...31.

In 1957 iproniazid was introduced for the treatment of depression.

New York Tiaes article June 4, 1981, p. B9. It has been studied extensively S and is a monoamine oxidase inhibitor. However, it has a variety of effects besides the effect on depression. These have frequently posed problems.

The use of these drugs has continued to be empirical. Iproniazid was removed fromthe market becauseof severe liver toxicity. 2t is interesting to note that these drugs exert their beneficial effect in depressed patients anywhere from one to several weeks after treatment is begun.

"'In some instances the improvement may progress to a state of euphoria, hypomania, or even mania. Central stimulatory effects are seen with these drugs in normal individuals as well as in depressed patients." Bevan.

Other effects are orthostatic hypotension, allergic reactions affecting 0 Ithe liver, dizziness, and a number of anticholinergic type symptoms.

*Ce o* C C Disturbances in glucose level have been associated with a variety of diseases. The maintenance of the sugar within normals range is a mntter of bringing the level down in hyperglycemia, and of bringing it up in various kinds of hypoglycemia. Some .e of the hypoglycemias are transient, others longstanding. Once

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developed the hyperglycemia of diabetes mellitus is usually permanent or becomes so. There are instances of hypoglycemia converting to hypergi.ycemia and diabetes mellitus. The latter disease is of universal distribution and a major cause of morbidity and death.

WO 85/03869 PCT/US85/00325 CHEMICAL EFFECTS OF MONOAMINE OXIDASE -4-

"SPECIFICITY

"The enzyme isolated from a number of sources exhibits low specificity. In general, primary, secondary, and tertiary amines, trytamine derivatives and catechol amines are oxidized The enzyme isolated'from human placenta, hcever,will only attack primary amines and with simple alkyl amines increase in chain length results in increased affinity Barman p. 180.

"Inhibition of MAO leads to a very pronounced increase in the levels of norepinephrine in the sympathetic nervous system and of the monoamines serotonin, norepinephrine, and dopamine in the monoamine-containing neurones of the Large amounts of amine now accumulate in the cytoplasm. The storage sites rapidly become filled to capacity with the transmitter. This enhanced accumulation of neuroaines within the neurones is presumed to be the basis for the antidepressant action of the MAO inhibitors. It should be added that the presence in the urine of large amounts of unmetabolized serotonin and 3 0 metltylated catecholamines is characteristic of patients on MAO inhibitor antidepressants. Bevan pp. 183, 184. These urinary compounds indicate clearance of the above amines from the blood and is consistent with an increased turnover rate of increased amounts of each amine.

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

WO 85/03869 PCT/US85/00325 CHEMICAL EFFECTS ON MONOAMINE OXIDASE Halogenated compounds enter the body frequently from the environment. The anaesthetics halothane and methoyyflurane are cases in point. "Incubation of the volatile general anaesthetics halothane or methoxyflurane (labelled with 16C) with hepatic microsomes, NADPH, and oxygen is accompanied by extensive DEC-LOR1NATION.

"Similarly thyroxine and triiodothyronine undergo deiodination by hepatic microsomal enzymes 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 Direct effects of T on isolated mitochondria have been known for some time, but they occur only at 4IGH, UNPHYSIOLOGICAL CONCENTRATIONS and their significance is doubtful. Lash p. 332.

"The actual biochemical mechanism of thyroid hormone action on neura, tissue is poorly understood." is evident that a single regulatory reaction has not been found to explain the multiple a effects of thyroid hormones. 'Although the activities of more than 100 enzymes have been shown to be affected by thyroxine administration it appears that all are not influenced to the same degree. Frisell p. 608 WO 85/03869 PCT/US85/00325 MANGANESE METABOLISM -6- "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 atability of manganese concentrations in the tissues to which earlier reference was made is due to such controlred excretion rather than to regulated absorption. 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 too of manganese into mitochondria and out again. It is a reflection of the mitochondrial pool, which is a very1 labile pool. Marganese is carried in the plasma bound tp protein. Very little of it is cleared I S. 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 *Gee a WO 85/03869 PCT/US85/00325 -7- THE CLEARANCE PATE OF OTHER SMALL IONS, SUGGESTF G THE NOPMAL TRANSCAPILLARY MOVEMENT, the second can be identified with the ENTRANCE OF MANGANESE INTO THE MITOCHONDRIA OF THE TISSUES, AND TIE 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, bein a dynamic mobile state. Underwood p. 185.

54 "The turnover of parenterally administered Mn has been directly related to the level of stable manganc:c in the diet of mice over a wide range A linear relationship between the rate of excretion of the tracer and the level of anganese in the diet was observed and the concentratirn of 5Mn in the tissues was directly related to the level of stale- manganese in the diet. THIS PROVIDES FURTHER SUPPORT FOR THE CONTENTION THAT VARIABLE EXCRETION RATHER THAN VARIABLE ABSORPTION REGULATES THE CONCENTPATION OF THIS METAL IN TISSUES." Underwood p. 185, "Little is known of the mechanism of absorption of manaanese 6 from the gastrointestinal tract, or of the means by which excess dietary calcium and phosphorus reduces manganese availabilty....

Theeffect of variations in dietary calcium and phosphorus on the metabolism of 5Mn in rats has been studied further hb, Lassiter and associates (100). These worker found that the fecal excretion of parenterally administered 54 was much higher and the liver retention lower, on a 1.0% calcium diet than on a 0.64 calcium diet.

WO 85/03869 PCT/US85/00325 -8- It appears, therefore, that calcium can influence manganese metabolism by affecting retention of absorbemanganese as well as by affecting manganese absorption. Variations in dietary phosphorus had no comparable effects on the excretion of intra peritoneally administered 54Mn, nUT THE ABSORPTION OF ORALLY ADMINISTERED 54Mn WAS IIPAIRED. Underwood, p. 196.

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 transnort.

A number of postulates evolved including proton, pho!p ate 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 C 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 operatino 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.

SWO 85/03869 PCT/US85/00325 -9- 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 metabolism 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 aitochondria as sources of high resonant bonds highlights the need for a central control mechanism. Such a 6' S mechanism must collate the energy production of the mitochondria with the energy metabolism qf the cells, organs, and indeed the entire organism. Calcium would seem a logical choice as the modulator of a system interactive between eukaryotic cells and mitochondria This is consisten with the present presentation.

This mechanism or system of control has been called a mechanism of regulation. Listing the sequence of components described includes cation. ATPase pump, Mn, deiodinase, thyroid hormones, monoamine oxidase and amines. ALL ARE FOTD IN CLOSE PROXIMITY IN THE MITOCHONDBIA.

i WO 85/03869 PCT/US85/00325

FORMULATION

ADDENDUM

In order to facilitate the understanding of the use of the method in diabetes mellitus and in hypoglycemic disorders as well as those diseases in which related changes of glucose in body fluids occur, this further addition is written. The MTA (or CMTA) secuence needs to be further discussed in those conditions.

In keeping with the recommendation of Marcus in the J. of the P.O.Soc. September, 1969, Vol. 52. No. 9 on page 559, for facilitating efforts of one skilled in the art to effectively use treatments described, a review of effective amounts of substances to be given is best viewed with an understanding of the underlying chemical systems that have been altered. The mechanisms of 4 regulation here described have inherent differences in the applicable lines of reasoning than are presently associated with the disease states discussed. "Glutamate dehydrogenation may be underscored as a major oxidative reaction in human metabolism in which an amino group, once a pr.rt of many different amino acids, is now converted to free NH/NH3-, 1H 4 OR NH 3 has only two fates: to be reutilized or to be excreted as urea." Frisell, p. 240 (1982).

"The alpha-NH 2 group of glutamate is esnecially vulnerable to oxidative removal by the process of deamination. Particularly in the liver, there is a very active dehydrogenase that is res-onsible for the deaminqtion of glutamate to alpha.ketoglutarate and NH4. The enzyme is present in both the cytosol and the-mitochndria..." Ibid.

WO W85/03869 PCT/US85/00325 This provides for rapid Flutamate breakdonw both in the cytosol and in the mitochondria. It also indicates that the control mechanism for the enzyme ca-n operate not only in the mitochondria but also in the cytoaol. This then relates the dehalogenase of the endoplasmic reticulum to the glutamate dehydrogenase in the cytosol. Similarity is obvious to the debalogenase of the inner membrane of the mitoc hondria which is in close proximity to the glutamate dehydrogenase in the matrix of the mitochondria.

Since the two enzymes are isoenzymes (isozymes), the mechanism for both is inferred to be the same. The dehalogenase in each case is inhibited by mangabhese. As a result thyroxine concentration is greater. Increased thyroxine increases the inhibition of each glutamate dehydrcg3nase.

T7he distance from the endop..asmic reticulunr to the glutamate dehydrogenese in the cytosol has a concentr-tion gradient of T 4 and T 3 The same is true for the distance from the inner membrane to the glutamate dehydracenase in the matrix of the mitochondria. If the distance in each case is too great, concentrations of T4 and T 3 reaching the sites on the glutamate dehydrogenase would be adequate to result in inhibition.

Such inhibition causes an increase in amounts of metabolites required for biochemical syntheses. THIS IS CCNSISTarT WITH THE ANABOLIC FUNCTIONS OF THE- THYROID HO;'1ON'S.

WO 85/03869 PCT/US85/00325 -12- Glutamate dehydrogenase is inhibited when thyroxine occupies the relevant receptor, presumably an allosteric site. Slowing of this enzyme results in less glutamate breaking down. This in turn slows the rate of glutamate formation secondary to transamination of amino acids. With less glutamate there is less formation of glutamine and urea as well. The unaltered amino acids remain at higher levels in the cellular amino acid pools.

While MAO inhibition by thyroxine prevents destruction of K biogenic amines, glutamate dehydrogenase activity inhibition prevents destruction of amino acids. While catecholamines, for instance, are increasing cellular activity, amino acids are becoming available as building blocks for peptide chain synthesis in cells. The two groups 4 of subetsnces are synchronized for the greater metabolism of the cells.

This is reflected in the increase in BMR.

9 There is an optimal range over which these metabolites are readily synchronized. Beyond that imbalances develop. These may be either at the lower or upper activities of normal physiological function. A dog or human with thyrotoxicosis, for instance, may develop hyperglycemia of such magnitude that glucosura occurs. j The attention span can shorten until the subject's normal pattern of behavior becomes less appropriate and disorganized. To maintain health, then, the MTA (or CMTA) sequence must operate within normal limits. Fortunately it appears that these limits usually include a wide range of normal values.

~-rPC WO 85/03869 PCT/US85/00325 -13- "A product of glutamate, gamma.aminobutyric acid (GABA).

is essential for brain function, because it retards transmission of nerve impulses. GABA is produced by the loss of the number one carboxyl group of glutamate:...It may be noted that percent of the total free amino acids of brain can be accounted for by aspartate, glutamate, and their derivatives." Frisell, p. 258 (1982).

The glutamate can be synthesized, of course, from ammonia and alpha-ketoglutarate. The enzyme from beef liver, 1.4.1.3 L-Glutamate: NAD(P) oxidoreductase (deaminating),(glutamate dehydrogenase) Barman 1969 p. 170 Vol. 1, actually functions at a structural conformation conducive to such synthesis.

It is the portal of entry throughout the phyla for ammonia entering into amino acids. This was illustrated by feeding o cattle shredded newsprint and using urea as a source of S nitrogen.

Rode in 1955 at the end of what is one of the most important group of papers ever to appear in the world literature, and especially during this century, reported on nitrogen requirements as follows: "Later, as the minimal human requirements were revealed, the quantities of the essentials decreased, and, in their place, glycine and urea were incorporated in the food. Both of thelatter compounds can be utilized by the growing rat for the puroose in o question No reason exists for doubting that in man they are capable of performing a like function." The structure and results of the e:periments then reported convincingly sustained that thesis.

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WO 85/03869 PCT/US85/00325 -14- However, as demonstrated, alnha-ketoglultarate is formed readily as a poart of the TCA cy-,cle byi isocitrate dehydrogenase.

In fact, alpha-ketoglutarate from glut an5te dehydrogenase ann be thought of stoichiometrically in the above context as a return of the original product to the TCA cycle.

Lehninger, that brilliant analyzer and expositor of the mitochondrion, speaks of glutamate dehydrogenase on p. 4~3q (1970) as follows: "Glut~mate dehydrogenase, probably because of its central role in the transfer of amino groups, is an allosteric enzyme. The beef liver enzyme has a molecular weight of 280,000 and contains a number of apparently identical subunits. The enzyme ASSOCIATED INTO LARGER AGGREGATES OF PAMICLE WEIGHT 2.2 MILLION WHICH ARE ROD-SHAPED. TE EUILIBRIUM1 BET1WEEN THE MONOMERIC AND FOLYVALENT FORMS IS SHIFTED IN 9: ONE DIRECTION OR THE OTHER BY VARIOUS EFFECTORS. The enzyme is inhibited by the effectors ATP, OTP, NADH AND IS ACTIVATED by AD? AND CERTAIN A±41N0 ACIDS.

It is also influenced by the PRESENCE OF THE THYvROID HORMONE THYROXINE and CERTAIN STEROID HORMONES." In this regard, Frisel. on page 240 (1982) remarks: *The thyroid hormone, thyroxine, can also influence the activity of glutamate dehydrogenase but the significance of this action is not yet established." WO 85/03869 PCT/US85/00325 Just before that,-he bad snid, "With rega rd to its macromolecular structure, glutamate dehydropenase conists of SIX IDENTICAL SUBUNITS. As would be surmised, this subunit character of the enzyme gives it the possibility of being subject to allosteric control. Indeed, the dehydrogenase is inhibited by. NAMD, ATP, and GTP, and is stimulated by ADP?, GDP, andy SOME AMINO ACIDS.

Barman, VOL, 1 (1970) pp. 170-171 1.4aj10 is somewhat more specific% *With enzyme isolated from beef liver, GT? and diethylatilbestero. stimulite the oxidation of the following monocarboxylic amino acids: alanine, leucine, isoleucine, methionine Iivaline, norleucine, norvaline and ':.2-aminobutyric acid.

ADP inhibits these oxidations. However, the oxidation of glutamate is inhibited by GT? and diethylstilbesterol but is stimulated by U ADP. These data can be explained in term-s of an ecuilib rium between diffe rent forms U the enzyme WHICH HAVE DIFFERENT RELATINE SUBSTRATE SPECIFICITIES and it is thought that the position of this equilibrium is influenced by modifiers." WO 85/03869 PCT/US85/00325 -16- It can be seen that oxidation of the monocarboxrylic amino acids occurs when the dicarboxylic amino acid glutamate is not being oxidased. The glutamate is hneing oxidized when the monocaroxylic amino acids are not being oxidized.

In table form, Barman'sa data can be presented as follows:

OXIDATION

Stimulate Inhibit 0 Beef liver GTP DES ADP alanine leucine 0 isoleucine 0 methionine 0 valine +0 norleucine 0 2-aminobutyrate 0 L-glutamate 0 Note: For the reverse reaction pyruvate is utilized by frog liver and beef liver enzymes.

In order tounderstand the'diff~rent forms', we will use as standard nomenclature that described by Lehninger (1970) pp. 58-59. It is as follows: "Specific terms are commonly used to refer to different aspects or levela of protein structure. The term p~rimary structure refers to the 'movalent backbone of the polypeptide chain and specifically denotes the sequence of its amino acid residues. S5econdary structure C polypeptide chains, particularly as they occur in fibrous proteins.

WO 85/03869 PCT/US85/00325 -17- The term tertiary structure refers to the manner in which the polypeptide chain is bent or folded to form compact, tightly folded structure of globular proteins (Figure The more general term conformation is used to refer to the combined secondary and tertiary structure of the peptide chain in proteins. The term quaternary structure denotes the manner in which the individual polypeptide chains of a protein having more than one chain are arranged or clustered in space. Most larger proteins, whether fibrous or globular, contain two or more polypeptide chains, between which THERE MAY BE NO COVALENT LINKAGESI (Fig. In general, the polypeptide chains of proteins usually have between 100 .to 300 amino acid units (mol wt 12,000 to 36,000). A few proteins have longer chains, such as serum albumin (about 550 residues) and myosin (about 1,800 i residues), However, any protein having a molecular weight exceeding 50,000 can be suspected to have two or more chains.

S "Proteins possessing more than one chain are known as oligomeric protains; theircomponent chains are called protomers.

A well-known example of an oligomeric protein is hemoglobin, which consists of four polypeptide chains, two identical alphachains and two identical beta-chains. Each chain has about 140 amino acids. The four chains fit together tightly to form a globular assembly OF GREAT STABILITY, despite the fact that THERE ARE NO COVALENT LINKAGES. Oligomeric proteins usually S contain an even number of peptide chains.

WO 85/03869 PCT/US85/00325 There may be anywhere from two to twelve subuni-t chains among the smaller oligomeric proteins to dozens or even hundreds among the larger proteins. Tobacco mosaic vi-rus particles have over 2,000 peptide chains.

"Since oligomeric proteins contain two or more polypeptide chains, which are usually not covalently attached to each other, it may appear im proper or at least ambiguous to refer to oligomeric proteins as "molecules" and to speak of their "'molecular weight.

Hlcwever, in most oligomeric proteins, the separate chains are so tightly associated that the complete particle usually behaves in solution like a simple molecule. Moreover, ALL THE COMPONENT CHAINS OR SUBUNITS OF OLIGOMERIC PROTEINS ARE USUALLY NECESSARY FOR THEIR F~UNCT IONS." To bring this further into perspective in physiological terms and enable u~s to match structural details with observed changes in vital f'unctions, we had best enlarge still fuirther.

upon the "subunits1V' This is discussed on pp. 184;--85 of Lehninger as follows: U "This mechanism for hemoglobin oxygenation is directly applicable to regulatory enzymes. The binding of the first substrate molecule to one subunit of a homotropic enzyme s enhances the binding of a second substrate molecule to a second subunit because there is a conformational change in the first subu-nit which is tr~nsmitted trecharically or 0 sterically to the secord suh~k*1 4t. in all cAses studied to date, regulatory, enzymes have been found to be r?.ther Zarge molecules c!,ntairing subunits; P resmaldy, the e~xisten~ce of WO 85/03869 PCT/US85/00325 -219interacting unitsis necessary for the-r function.

"Note th.-t the term "subunit" IS AMBIGUOUS nd may have Two DIFFE.TNTr AINGS WHE!, APn'ii:D TO OLIGOMERIC PROTEINS. Hemoglobin contains four Structural subunits or protomers, the two alpha and two beta chains, but two-functional subunits, the two alphabeta half molecules.

ISOXTAES

"1Recent reseorch has revealed another way in which the activity of some enzymes may be controlled THROUGH FEATURES OF THEIR MOLECULAR STRUCTURE. A. number of different enzymes have beer found to exist in multiple molecular forms WITHIN A SINGLE SPECIES, or EVM1 WITHIN A SINGLE CELL. Such multiple foxrmi can be detectad and separated by gel electrophoresis of cell extracts; they are therefore distinct moledular Species differing in net electrical charge. Multiple forms :6 a 1:1 within a single species or cellare called isozies (or isoerzymes.

Lnctate dehydrogenase, one of the first ensymes in this class to have been studied extensively, exists in five different major forms, or isozymes, in the tissues of the rat (Figure 9-12). These hAve Oi been obtained in rureform. Although all five isoz-,'mes of lactate j dehydrogenase catalyze the same reaction overall, they have V* DISTINCTLY DIF'L.T K, VALUES for their substrates; the biological Significance of these differences will be described in Chapter and 18. The five isozymde all have the same particle weight, about 134,OOO,and contain four polypeptide chains, each of nol wt 33,500.

WO 85/03869 PCT/US85/00325 When glutamate dehydro,-enase is inhibited, less transamination occurs. This results in less breakdown or amino acids, those which have trarsaminase enzymes. A~though this is true of the inhibition of the polymeric form of glutamate dehydro -enase, the monomeric forms proceed to oxidatively deaminate the three branched chain amino ncids.

Deamination of the methionine chain presumably occurs at the L-homoserirne hydro-lyase (deaminating) conversion to 2-oxo-butyrate. This enzyme is also named homoserine dehydratase with HOH being added dnd N.)and HOF.

being products of the reaction, However, methionine and also 2-aminobutyrate are listed as substrates of glutamate dehydrogenase, presumably in the monomeric form. This raises some question ao to the route of degradation of methionine in its ultimate producti-on of succinyl1 CoA. The cytosol is a thick suspension. The matrix of the mitochondria may be an even thicker suspension with a very high protein acntent. In view of the Male monomers of glutamate dehydrogenase occuzrring in such milieu. it would a be consistent to thfrk of methionine breakdown not involving the B-6 assisted ster in this context. S Lysine and threonine are the two essential amino acids not transaminated. In fact, they are not broken down readil7. Lysine is usdfrfrigognceetoyts the poais a~fd for The overall effect of the above enzymatic steps presumably :e* would be -to increase the demand for thd bnrrchad chain amino acids.

I 085/3869PCT/US85/00325 -21- T.hus, the two aromatic amir,.o acids, pheryla-lanine ard tryptophan re Spared by the inhibition of clutimate dehydrogenase. On th,, other hand, the initial sparing of valine, isoleucine, leucine and methionine is altered subsequently by the monomers of the enzyme oxidatively deaminating them (?methionine). Under those circunstances the sparing effect gives way to increased breakdown and increasee need for these amino Acids. For it ~the present at least, althouigh threoni-ne does degra-de'to some extent I to succinyl Colk, that amino acid does not seem to be involved in either the sparing effect of the polymr-eric form or the increased degradation e~fect of the monomeric form of the inhibited glutamate dehydrogenase.

The lysine is purely ketogenic. Its contribution to the acetyl.CoA pool would seem to be unaltered under these circumistances.

S Alpha-ketoglutar-dte is formed by isocitrate dehydrogenase II in the TCA cycle. This pool is enlarged by the action of glutamate dehydrogenase. Alpha-.ketoglutarate dehydrogenase promptly converts it into succirnyl Cok. The succinyl CoA in turn is corwerted to sueecizie acid by suacinyl CcA synthetase and a molecule of OT? is L formed at the same time from GDP and Pi.

00 Discussing the rate-limiting citrate synthase step in the TCA cycle, Frielhasid "The synse reaction is icceptedt her'efore, as a nonequilibrim reaction and beeomes a major control reaction for the cycle. It is reasonable that the rate of the synthase reaction should be sensitive to the avai~ability of acetyl co. 1; ADDITION, HOWVER, At: INTME~IATE OF T M CYCLE ITSELF, SUCCINrYL coA, cnn INHIBIT C11T1ATE Sn.TKE.SIS BY COMP.7TING W4ITH ACTIVE ACMTTE."1 This suggests that the si-ze of the rools of acety',l CoA WO 85/03869 PCT/US85/00325 -22and suce~inyl CoA can assume prime importance in determining the over-,l rate of the TCA cycle. A number of sulhstances Pre degraded to succinyv CoA. These include: 1. isoleucine and valine via methylmalony. CoA; 2. branched chain tatty acids via propionyl Coa which in turn is changed into methylmnlonyl CoA; 3. methionine and tryptophan via aipha-ketoglutarate to the propionyl CoA.

The methylmalonyl CoA rearranges through the acticn of a mutase to form succinyl CoA. This conversinn requires -12 for the enzyme to be active. The reaction on propionyl CoA itself requires biotn.

4. pyrimidine breakdown products including those from thymine also contribute to the succinl CA pool.

The point has been made in detail before th.pt the polymeric glutamate dehydrogenase feeds into this pool indirect!y through the alpha-ketog'utarate (2-oxo-glutarate). lher, the polymeric for is inhibited, the monomeric fo=.s increase breakdown of the above amir.o acids to produce sucoi-nyl CoA as if replacing that which wp.s lost when the uolyrmeric form, was inhibited.

*0* 0 S-23- The present invention provides for using a method for raising blood glucose levels in degenerative brain diseases, especially of the elderly, in which memory loss and low blood sugar levels commonly occur together. The use of hyperglycemic actions of various amino acids in conjunction with manganese in effective ratios decrease insulin release in chemical hypoglycemia and is used to restore normal levels of glucose to patients with degenerative brain disease occurring with hypoglycemia as part of the syndrome.

According to one aspect of the present invention, there is provided a method of treating memory loss in vertebrates comprising administering to the subject a therapeutically I 15 effective ratio and amount therefor of: I S* at least one amino acid selected from L-valine, Dvaline, L-methionine, D-methionine, L-isoleucine, Disoleucine, their alpha-keto and alpha-hydroxy analogs, and 20 the di- and tri-peptides of the said amino acids, or pharmaceutically acceptable acid addition salts thereof; at least one amino acid selected from L-phenylalanine, L-tyrosine, D-phenylalanine, D-tyrosine, their alpha-keto 25 and alpha-hydroxy analogs, and the di- and tripeptides of the said amino acids, or pharmaceutically acceptable acid addition salts thereof; and a preparation consisting essentially of a manganese 30 compound.

9eo9 900323, gCO16.10CPa39991.ros,23 w WO 85/03869 PCT/US85/00325 HOW TO USE TIL. METH!OD -24- DESCRIPTION OF THE PREFERRED EMBODIMENT The approach to theelderly patient with a memory disorder requires a clear concept of how fragile the patient is and what other diseases may be complicating the clinical picture. When the clinician is satisfied with the general workup the first effort should be to accurately define the status of the glucose metabolism.

These disorders c-n he found through wide areas of the clinical specturm of disease. Thus, multiple small emboli of the br-in from atheromatosis of the blood vessels can produce a general loss of memory comparable to that in Alzheimerls and other diseases. Of all the syndromes that of Alzheimerls is the commonest and involves millions of patients. The downhill course usually lasts some six years on the: 9 average. Alzheimer r-tients have flat glucose tolerance curves that demonstrate hypoglycemia during portions of the testing. The greatest 9.° difficulty with developing a program for Alzheime.r's has come from. the 9 confusion about diagnosis and the constant claims that this or that or some other treatment was effective. It is remarkable for the lack of an effective treatment and for a great number of ineffective treatments. 9..

This confdsion has probably precipitated in large measure the present *9 9 clinical crisis. The false hopes have reflected an unwillingness on the 9 part of those called upon to treat the disease to admit to their true state of knowledge regarding it.

A low blood pressure is characteristic of the disease. The diastolic pressure is uniformly low. The development of slow progressive loss of cells over the anterior and paristal areas of the brain where neurons have the highest o Jygen requirement of any cells in the body is consistent with loss due to recurrent intermittent metDbolic insufficiency and this. conforms well to the finding of flat glucose tilerarce curves.

-7 SWO 85/03869 PCT/US85/00325 In the memory disorders of alcoholism, it is well to recall that hypoglycemia is a common finding in.many alcoholics. The syndrome will respond to vitamin therapy many times if properly sustained. However, many times the alcoholic that develops loss of mmmory is left with serious memory deficits as well.

Patients with the disease frequently go through angry,excited periods of confusion. These are perhaps best explained as due to falling glucose setting off a reactive hypoglyceoia following a burst of adrenalin secretion to overcome theinitial fall in the glucose. Glucose falls, adrenalin is released, glucose rises, insulin is released again. The borderline levels are not sufficient to permit the patient to overcome the confusion when the adrenalin bursts occur in that siturtion as described above.

When a long term GTT of eight hours or less is used it a be necessary to discontinue the test because of the adverse effect on the patient. In order to evaluate the status of the patient what is needed is control of the glucose level, i.e.,controlling thehypoglycemia.

This condition has the same problem. There are many treatments, for the rost part of limited effectiveness and a reliiable effective treatment has not been available before. Now that the glucose level can be raised effectively the first effort should be to restore the glucose level to normal. For that to be used effectively, however, it is necessary to realize that there follows shifts in functionirg of various amino acid pumps. The associated electrole changes that develop with the restoration of the hypoglycemic to the euglycemic may be responsible; the relative amounts of the amino acids being transported change, so that the ratios of aromatic amino acids to one another are altered as well as the ratios of amino acids in other groups.

.1I WO 85/03869 PCT/US85/00325 -26- When the blood sugar is raised, there is a possibility that the blood pressure will. increase as well. This mist bo cnrresc-.ad for ar.d the original blood pressure level established after the glucose level has been stabilized at the desired normal level. For this rason it is useful to raise the blood sugar in small steps, in smn.ll increments.

iThe intervals between such changcs in levels may be a week or more.

SThe general condition of the patient must be considered. However, these medications are cumulative and it is most important that treatment I not be cr-rried past thedesired point. Thus, a stepwise treatment program 11 helps to prevent such a situation arising. This also clearly highlights the need of the clinician to personally supervise the treatment program.

One way to de this is through the use of serial blood sugars before meals and giving the medication at that time when thep-tient will be eating a meal. Such a meal should include high calibre protein. Very likely the differentiation of so:-e of these syndromes is best achieved by restoring-.a normal blood sugar and then reevalu-ting bhe status of the patient.

Because of the changes in firiction of the large neutr-I amino tacid pump which includes many of the essential amino acids, it requires I* 9 9 Ssome time to evaluate where the patient's amino acid balance has come to rest as it were. The nonpolar residues of amino acids are in many instances those of the essential amino acids and this highlights the inense importance i of those amino acids for the synthesis of the active sites of the i^ enzymes. Treatment *hould be directed at giving the patient tin and Treatnent shoul~d be directed at giving the patient tinyr and -27then larger amounts of !-isoleucine u.Ner contrciued circumitances.

The movnr.ts can be ir.croased for a tine, but then must be reduced.

The effort must be to restore the isoleucine,/leu ine ratio to the normal rang Many cases of memory disorder of the elderly are accompanied by changes in hmndwritir.g such as those noted by Chief Jurtice Holmes.

It is best to direct ore's efforts to the isoleucine initially and then later use L-valine In an eff6rt to correct the disturbinces that lecd to the impairments of the handwriting. Always the anounts given should be small and gradually increased and then tapered off as the needs of the patient have been filled. This applies to each of the substances used.

Characteristically lwer blood curves arefound in the degenerative brain diseases of the elderly. Of the diseases mentioned here, all but the atheromatous syndrome S mentioned would be expect.d to fall into t h lower curves.

Even Parkinsonism may be characterized by memory loss, although this is apt to be misjudged because the medications employed are apt to alter the blood sugar levels somewhat.

4The levels in the patient at base level are apt to be low for the glucose.

Example #1 is provided to indicate the treatment S of an individual with low blood sugar values but without a memory loss problem. The patient was approaching retirement age and living an active, fully employed life.

_r 28- The use of valine is essentially related to neuromuscular disorders. These express teemselves diagnostically in alterations in handwriting, which progressively deteriorates in Alzheimer's and Parkinsonism commonly. There is likely to be an associated striking calming effect with its use.

The use of the valine, however, poses a number of metabooic 'considerations. Leucine is formed via the beta-leucine cobalamin-dependent shunt from valine and from branched chain fatty acids. The hypoglycemic effect of leucine can thus be potentiated by valine as a backup source of :leucine. On the other hand, when there is no need for more ::':leucine, this should not be a problem. It illustrates, however :that it may well be best to be sure that the proper range of glucose values,, e.g., 0.0* 0 S* 00 WO 85/03869 Example 1 Patient M.V.

PCT/US85/00325 -29oe o o o

S

Clinical Status: The patient has a long history of a low, flat glucose tolerance test.

Values were especially low a number of years ago. However, they were remaining in the 50 mg range and an attempt was made to change the low level towards the normal range of blood glucose values.

Treatment periods: Initially a weekend in which two S days of treatment were undertaken.

Then at various times the next three weeks.

Treatment period interval: Ranged from one week to two days.

Objective findings: I Blood glucose: Ranged upwards from about 50 milligrams/ 100 milliliters, (meo) to 100 mgo.

II Blood pressure: A Systolic pressure: ranged from 150 to 124 mg Hg (millimeters mercury pressure) B Diastolic pressure ranged from 86 to 110 to 75 mm Hg in that order C Pulse Pressure: ranged from ninety to 34 mm Hg III Pulse: ranged from 70 to 76 Range of medicatoon: Ratios: Mangenese (mg in manganese gluconate) 2 mg+ at one to ten day intervals.

Isoleucine in quarter, half and whole tablet.

500 mg/tablet in time between meals to ten days. (1.6 to 6.7 mg/kg body weight) (0.007 to 0. 27 Objective of Treatment: To bring glucose level to normal range of(lO0-110) to 140-150) m'o Subjective findings: Clinical response: The patient was anxious and upset during part of period associated with a viral upper respiratory infection.

During this interval anxiety was prominent.

An episode of labile blood pressure occurred during the time involved and then drifted down to 124/75 over a two week period The change in blood glacose level occurred easily.

The most striking observation was the level of 90 mgo still present six months later.

The patient continued throughout to feel considerably better from day to day than was his usual pattern.

Claims (3)

1. A method of treating memory loss in vertebrates comprising administering to the subject a therapeutically effective ratio and amount therefor of: at least one amino acid selected from L-valine, D- valine, L-methionine, D-methionine, L-isoleucine, D- isoleucine, their alpha-keto and alpha-hydroxy analogs, and the di- and tri-peptides of the said amino acids, or pharmaceutically acceptable acid addition salts thereof; at least one amino acid selected from L-phenylalanine, L-tyrosine, D-phenylalanine, D-tyrosine, their alpha-keto and alpha-hydroxy analogs, and the di- and tripeptides of the said amino acids and pharmaceutically acceptable acid addition salts thereof; and a preparation consisting essentially of a manganese compound. 00e0 0*0e 0O S S DATED this 23rd day of March, 1990 VERNON ERK by his Patent Attorneys DAVIES COLLISON 11

900323.tQC016. 10t.Pa539991.res.24 J U- iCT/US8 5 0 3 2 INTERNATIONAL SEARCH REPORT International Application No 1. CLASSIFICATION OF SUBJECT MATTER (If several classification symbols apply, Indicate all) 3 According to International Patent Classification (IPC) or to both National Classification and IPC Int. C1.14A61K 37/02 A61K. 31/28 A61K 31/195 US Cl. 424- 177 424/287 424-319 II. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System -Classification Symbols US. CL. 424-177 424-287 424-319 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 6 III. DOCUMENTS CONSIDERED TO BE RELEVANT 14 Category Citation of Document, l a with indication, where appropriate, of the relevant passages 7 I Relevant to Claim No. 1B X US, A, 4,218,474 (Barnish) 1 19 August 1980 X,P US, A, 4,435,424 (Wurtman) 1 6 March 1984 A US, A, 3,865,934 CPlotnikoff) 1 11 February 1975 A US, A, 3,873,296 CAshread) 1 25 March 1975 A US, A, 4,340,592 CAdib 1 i 20 July 1982 I I r I* SSpecial categories of cited documents: 1 later document published after the international filing date doumn deiior priority date and not n confict with the application but document defining the general state of the art which s not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of partcular relevance; the claimed invention filing date cannot be considered, novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an Inventive step which is cited to establish the publication date of another y" document of particular relevance the claimed invention citation or other speal reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined wth one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but In the art, later than the priaor n ty date b laimed document member of the same patent family IV, CERT'FICATION Date of the Actual Completion of the International Search s Date of Mailing of this International Search Report /o I9V, 15 MAY 1985 International Searching Authority i Signature of Authorized Officer ISA/US Form PCTIISA/210 (recond sheet) (October 1981) -T I PCT/US 8 5 o 325 International Application No. FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET N, Chemical Abstracts, Vol. 1979, Shukla, Species variation in manganese induced changes in brain biogenic amines, abst. no. 198507V V.Q OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE to This International search report has not been established In respect of certain claims under Article 17(2) for the following reasons: Claim because they relate to subject matter 12 not required to be searched by this Authority, namely: 2. Claim because they relate to parts of the International application that do not comply with the prescribed require- ments to such an extent that no meaningful International search can be carried out 13, specifically: C btC C C. S C. C Cr VI. OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING Ut This International Searching Authority found multiple inventions in this International application as follows: 1.E As all required additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the international application.

2.M As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the International application for which fees were paid, specifically claims:

3. No required additional search fees were timely paid by the applicant. Consequently, this International search report Is restricted to the invention first mentioned In the claims; it Is covered by claim numbers: As all searchable claims could be searched without effort justifying an additional fee, the international Searching Authority did not Invite payment of any additional fee. Remark on Protest E The additional search fees were accompanied by applicant's protest. E No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (October 1981) International Application No, Ill,. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category* Citation of Document, t1 with indication, where appropriate, of the relevant passages f Relevant to Claim No 's X N, Chemical Abstracts, Vol. 99, 1983, Chandra, Psychiatric illness due to manganese pvisoning, abst. no. 100473U A N, Chemical Abstracts, Vol. 98, 19.83, Kaliman, Biogenic monoamines and their precursors in rats with spontaneous arterial hypertension, abst. no. 195890U X N, The American Journal of Clinical Nutrition, Vol. 6, No. 5, 1958, S.lmon, The Significance of Amino Acid Balance in Nutrition, pages 487-494 i 1 j f boe 1 I I I -0-0oe Form PCT'ISA/210 (extra sheet) (October 1981)