CA1277903C - Method of treating hypoglycemia in vertebrates - Google Patents

Abstract

ABSTRACT

The invention relates to a method of raising blood glucose in vertebrates comprising administration of manganese-con-taining pharmaceutical preparations in appropriate ratios with isoleucine, methionine, phenylalanine, tyrosine and valine and in appropriate ratios as between the amino acids used to increase the blood glucose level of the vertebrate having the blood sugar level raised to within normal limits;
these to be given in cumulative amounts appropriate to the individual patient in a schedule of treatment which varies in amount, frequency and the said ratios, all of which reflect the changing degrees of imbalance as the affected individual adjusts the glucose level to within normal limits.

Description

~2~79C3 ~. ~ , .

A METHOD OF TREATING HYPOGLYCEMIA IN VERTEBRATES
BACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to the raising of the blood sugar level in vertebrates. It relates to the compounds that can be used to avoid excessive release of insulin by the beta cells of the Islets of Langerhans in hypoglycemia, i.e., when the glucose level falls below the normal parameters of concentration.
The invention is directed to providing preparations that will prevent excessive release of insulin.

Hypoglycemia is an increasingly common condition throughout life. It is present at all ages, but is apt to become more common from the teens into the twenties and presents a frequent problem in the thirties and forties.

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12779~3 INTRODUCTION

This is a method for changing the rates of oxidation of neuroactive amines and other biogenic amines by monoamine oxidase. Increasing activity of the enzyme results in lower levels of these amines. Decreased activity results in higher levels of these amines. Many of the affected amines fulfill the specificity requirements of receptor sites on the membranes of cells. When such a receptor site is occupied the activity of a membrane pump changes in many cases. Production of "second messenger" molecules such as cAMP and cGMP at the inner surface of the membrane occurs in most cases. These cyclic compounds commonly activate protein kinases. The protein kinases are enzymes possessing phosphorylase activity.
When protein kinases are activated in the nucleus of the cell production of RNA becomes possible and eventually protein synthesis. When protein kinases are activated in the cytosol such actions as glycogenolysis become possible.
The effect is determined by how the cell is built when differentiated. The structure of the cell determines what specialized function it will serve when protein kinases are activated.
So many of the amines oxidized by monoamine oxidase affect these various cells that most of the major functions of the body are affected. This may result in profound alterations in the vital functions of vertebrates and other organisms. Imbalance between vital functions are defined as diseases if morbidity or risk to life occurs.

8~1S~ -2-Healthy individuals have both optimum levels of these amines and appropriate ratios between these amines. Many metabolic diseases in the human are associated with changes in the levels and ratios of these amines. The rates of oxidative deamination of these amines by monoamine oxidase constitutes part of a MECHANISM OF REGULATION imbalance of which produces many of the diseases of vertebrates.

The following abbreviations and definitions are in the approximate order in which the words are introduced.

Neuroactive Capable of affecting nerve cell function.
Biogenic amine An amine originating in a biological system.
The term is generally applied to an amine associated with some physiological action.
Specificity The exactness with which a molecule must be structured in order to occupy a receptor site of a protein or peptide.
Receptor site A molecule, usually a protein, which has an active site in it. Occupation of the active site may, for instance, activate or inactive an enzyme.
Active site Region of enzyme surface which interacts with the substrate molecule. (Lehninger) Second messenger A compound formed on the inside of the cell membrane, e.g., cAMP, cGMP., when the first messenger occupies a receptor site.

sd/sp -3-First messenger A compound which occupies a receptor site on the outer surface of a cell membrane, e.g., adrenalin.
Membrane pump Any of many protein complexes incorporated into the membranes of cells by which smaller molecules, ions. or atoms are pumped in and out of cells or their compartments by activity of enzymes in the protein complex.
Cytosol The fluid suspension within cells exclusive of organelles such as the nucleus and mitochondria.
Glycogenolysis The breakdown of glycogen.
RNA Ribonucleic acid cAMP Adenosine monophosphate cGMP Guanosine monophosphate Glycogen A starch like compound occurring in the cells of vertebrates and other organisms.
Differentiation The formation of a new cell type from a simpler cell type and the preparation of tbe new cell structurally to carry out a new function.
Vital Pertaining to life.
Disease A definite morbid process having a characteristic train of symptoms, It may effect the whole body or any of its parts. Dorland 23rd edition p. 303.
As here used, an imbalance in vital processes sufficient to interfere with maintenance of life support sy~tems and to reduce vitality and to produce risk to life.

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1277~3 Morbid L. morbidus; sick Pertaining to disease or affected with disease. Dorland 23rd edition. p. 856.
echanism of Regulation As here used, a system of interacting enzymes whose combined actions serve to regulate the levels of molecules involved in maintaining life supporting functions.
ertebrate An organism possessing a spinal column.

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`` ~27~9~3 2. Prior Art "Monoamine oxidase is a flavoprotein oxidase of pur-ported CENrRAL METABOLIC Ih~ORTANCE CONVERTING NEU~OACTIVE
AMINES INTO INACTIVE ALDEHYDES The flavin linked mono-amine oxidase is localized in the OUTER MITOCHONDRIAL MEM-B~ANE oP ANIMAL CELLS Walsh pp 402, 403 "Actions Monoamine oxidase is a complex enzyme sys-tem widely distributed throughout the body. Drugs that in-hibit monoamine oxidase in the laboratory are as~ociated with a number Or cllnical e~fects Thus, it is UNKNOWN
WH~THER ~AO INHIBITOR PER SE, OTHER PHAR~ACOLOGICAL ACTIONS, OR AN INTERACTION OP BOrH IS respon8ible ~or the cllnical e~ect- ob~er~ed. There~ore, th- phyrician should become familiar with all the e~rects produced by drugs in this cla-s. PDR (Physicians' Desk Re~er-nc- 1983) p. 151~
Two ola~si~ication~ o~ am~n- oxidases wer- presented ~n 1959 That o~ 81ashko, et al u~ed the reYponse to carbo-nyl inhibitors to distinguish between the activities Or th~
various amine oxidase That Or Zeller, et al, used semi-carbazide inhibitors The use Or inhibitors to classify ths amine oxidases reflected difficulties encountered in purirying these enzymes and studying the structure Or their active sites "A Occurenco Monoamine oxidase (MAO) has been found in all classes o~ vertebrates so ~ar examined (1970) ~ammals, birds, reptiles, amphibians and teleosts (161) ~ne enzyme occurs in many different tissues, particularly i ~lands, plain muscle, and the nervous system (162) 1Z7~9l~3 In man the parotid and submaxillary ~lands seem to be the richest source of MAO (163). It also occurs in molluscs and plants (4).~ Kapeller Adler p. 31.
In 1957 iproniazid was introduced for the treatment of depression. New York Times article June 4, 1981, p. B9.
It has been studied extensively 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 em-pirical. Iproniazid was removed from the market because of severe liver toxicity. It 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 Or euphoria, hypomania, or event mania. Central stimulatory ef~ects are seen with these drugs in normal individuals as well as in depressed patients." ~evan. Other effects are orthostatic hypotension, allergic reactions affecting the liver, dizziness, and a number of anticholinergic type symp-toms.

MAO Monoamine oxidase. MAOI Monoamine oxidase inhibitor.

Inhibitor A compound or atom which when it occupies the active site of an enzyme prevents the usual chemical reaction(s) from taking place there.
Mitochondria Membrane surrounded organelles in the cytoplasm of aerobic cells which contain respiratory enzyme systems. (Lehninger~
The 'powerhouse' of the cell. An organelle engaged in the production of high resonant 10 compounds by oxidation of compounds from all the major foods.
Depression (L. depressio: de down + premere to press) Dorland 23rd edition p. 366. 3. A lowering or decrease of functional activity. 4. Absence of cheerfulness or hope; emotional dejection.
Euphoria (Gr. The power of bearing easily) Ibid p. 480.
In psychiatry an abnormal or exaggerated sense of well being.
Hypomania (hypo. + Gr. mania madness) Mania of a moderate type. Ibid p. 651.
Mania (Gr. madness) Ibid p. 795. 1. A phase of mental disorder characterized by an expansive emotional state, elation, hyperirritability, overtalkative~ess or flight of ideas, and increased motor activity;
specifically, the mania of manic depressive psychosis.
Orthostatic hypotension Lowered blood pressure when the person changes from a supine X sd/sp -8-- \

to an erect position. Ibid p. 654 Anticholinergic Blocking the passage of impulses through the parasympathetic nerves; parasympatholytic.
Ibid p. 99.
CHEMICAL EFFECTS OF MONOAMINE OXIDASE
"SPECIFICITY
"The enzyme isolated from a number of sources exhibits low specificity. In general, primary, secondary, and tertiary amines, tryptamine derivatives and catecholamines 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 nervous system and of the monoamines serotonin, norepinephrine, and dopamine in the monoamine-containing neurones of the CNS....Large amounts of amine now accumulate in the cytoplasm.
The storage sites rapidly become filled to capacity with the transmitter. This enhanced accumulation of neuroamines 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-methylated catedholamines is characteristic of patients on MAO inhibitor antidepressants."
Bevan pp. 183, 184.
These urinary compounds indicate clearance of sd/sp -9-the above amines from the blood and is consisten't with an increased turnover rate of increased amounts of each amine.
IlThe flavoprotein responsible for the oxidative deamination 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.

sd/sp -9A-~2779Q3 he following abbreviations and definition5 are ln the approximdte order in which th~ words are introduced.

Affinity Attraction.
Antidepressant Tending to overcome a feeling of depression.
Flavoprotein An enzyme having a riboflavin (82) compound as - a coenz~me.
sy~pathetLc nervous system A dlvision of the autonomic nervous system.
Catecholamine3 ~ne group of neuroactive amines, e.q., dopamine, norepinephrlne, and epinephrine.

~ ~2779U3 CHEMICAL EFFECTS ON MONOAMINE OXIDASE

Halogenated compounds enter the body frequently from the environment. The anaesthetics halothane and methoxyflurane are cases in point.
"Incubation of the volatile general anaesthetics halothane or methoxyflurane (labelled with 16Cl) with hepatic microsomes, NADPH, and oxygen is accompanied by extensive DECHLORINATION."
"Similarly thyroxine and triiodothyronine undergo deiodination by hepatic microsomal enzymes (8)." Bacq p. 577.
IlDimino and Hoch (1972) found a considerable enrichment of iodine in liver mitochondria of rats injected with T4. These mitochondria were more dense than those of untreated animals and appeared to contain iodine TIGHTLY
BOUND TO THEIR INNER MEMBRANES (9). ...Direct effects of T4 on isolated mitochondria have been known for some time, but they occur only at HIGH, UNPHYSIOLOGICAL CONCENTRATIONS
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".
"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 degrçe.
(IO)." Frisell p. 608.

sd/sp -11-~"~` 1 ~ ~N~3 ADVANTAGE', OVER THE PRIOR ART

Leyden Webb maintains that a NORMAL STATE of a biological system should be regarded as a perfect balance of complex chemical and functional activities, the possibilities depending on a precise coordination of the rates of these various processes. The incorporation of an inhibitor into a biological system leads to an alteration of the rates of some of these processes and thus to a disturbance of the perfect balance.
Usually only the results of the imbalance induced by the inhibitor can be determined. Thus, if the inhibitor upsets the balance to the detriment of the organism, it is generally termed a poison. On the other hand, when the change of balance favors the survival of the total system, the inhibitor may be regarded as a drug. Expeller Adler p. 50.

l. The invention provides for the use of the natural components of the 'mechanism of regulation' of which monoamine oxidase is a component part. This makes it possible to distinguish between clinical effects due to MONOAMINE OXIDASE lNHIBITION per se and OTHER PHARMACOLOGIC
ACTIONS.
In fact, it provides a control against which other drugs can be tested, such as those presently in use as monoamine oxidase inhibitors.
2. The invention provides a method of knowing , ~ sd/sp -12-. .

~ 1Z;~X9~3 what substance is producing what effect.

3. When natural substances are administered in the use of this method they have cumulative effects.
This dictates a sequential decrease in the frequency with which these substances are to be given.
Eventually treatment may, in fact, be required only very infrequently if at all.
Cumulative (L. cumulus, heap). Increasing by successive additions, the total being greater than the expected sum of its parts. Dorland 23rd Edition p. 337 Bouvier '~ol.l 1914. Rawle's revision p. 737 gives the meaning in legal terms similarly, with the sense of heaping up, building a mound, as of cumulative evidence which is that which goe6 to prove what has already been established by other evidence. Both of these, the medical and the legal, are wide of the mark intended here.
Ac used here it is in the sense of filling in a depression or trench not in the sense of building a mound.
DESCRIPTION OF THE NOVELTY OF THE INVENTION

The MTA sequence is a central mechanism of regulation which maintains vital functions within the commonly encountered parameters of normal individuals. It is present in vertebrates and other organisms. The complexity of the MTA sequence sdtsp -13-.

varies as the complexity of the organism. Thus, some active amines present in man are not found in lower or~anisms.

Imbalance of active amines in the MTA sequence results in changes in vital functions. Balance is restored by restoring the structures of the MTA sequence. This comprises administering effective amounts of at least one of the precursors of active amines decreased in the MTA sequence, and comprises administering effective amounts of the deiodinase inhibitor.
The substances administered are cumulative as structures of the MTA sequence are restored. The amounts of these natural substances are administered:
a. with decreasing frequency and b. in decreasing amounts.
Considerable intervals may elapse before administration of effective amounts of these substances require repeating.

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q9~3 DESCR I PT I ON OF THE PROCESS
The administration of manganese compounds results in inhibition of deiodinase enzyme and an increase in the concentration of T4 and T3.
The increased concentrations of T4 and T3 increase the inhibition of monoamine oxidase and glutamate dehydrogenase.
The levels of dopamine and norepinephrine and serotonin and other biogenic amines increase as well as those of amino acids.
Cells with receptor sites for these transmitters and other active amines have an increased probability of having those receptor sites occupied.
A great many cell types in the body are activated.
Activation of a cell type is accompanied by more metabolic activity and protein synthesis.
In carrying out the process for which the cell type has been differentiated and programmed there is an increase in oxidation.
This increase in the use of oxygen can be measured indirectly by determining the basal metabolic rate (BMR).

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"Many investigations have been carried out with the goal of identifying some specific enzyme catalyzed re'action in oxidative metabolism that is controlled by the thyroid hormone. In early investigations it was found that thyroxine stimulates respiration and uncouples oxidative phosphorylation when added to isolated mitochondria. Moreover, thyroxine also promotes the respiration-dependent swelling of mitochondria.
However such effects REQUIRE EXCESSIVELY HIGH CONCENTRATIONS
OF THYROXINE and it is now doubtful whether they represent true endocrine effects of the hormone.... The molecular basis for the mode of action of thyroxine is still a challenging puzzle." Lehninger p. 413.

To understand Lehninger's statement about thyroxine stimulating respiration and uncoupling of oxidative phosphorylation when added to isolated mitochondria it is necessary to provide the following sequence of definitions, many of which are his.

Mitochondria Membrane~surrounded organelles in the cytoplasm of aerobic cells which contain respiratory enzyme systems. (Lehninger) Oxidation The loss of electrons from a compound: an oxidizing agent is an electron acceptor. Ibid.

ADP Adenosine diphosphate ATP Adenosine triphosphate Oxidative phosphorylation The enzymatic phosphorylation of ADP to ATP which is coupled sdlsp -16-1~ 3 to electron transport along the respiratory chain to oxygen (in mitochondria). Ibid.
Energy Movement.
Coupled reactions Two chemical reactions which have a common intermediate and therefore a means by which energy can be transferred - from one reaction to the other. (Lehninger) We can now substitute movement of electrons for energy in the above definition.
Common intermediate A chemical compound that is common to two chemical reactions, as either a reactant or a product. Chemical energy may be transferred from one reaction to another via such a common intermediate. (Lehninger) Similarly we can now substitute electron movement for chemical energy in the above definition.
Uncoupling agent A substance (example 2,4-dinitrophenol) which can uncouple phosphorylation of ADP from electron transport; the energy is then released as heat. (Lehninger) Chemistry The exchange and/or sharing of electrons between the outer orbitals of atoms.
Heat The movement of molecules and atoms. It is calibrated in terms of the rate of movement. At room temperature the skin sensors registers no change, for instance.

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~ 3 When the rate of movement of molecules and atoms in the skin increases, an increased number of impulses are transmitted to the brain where a feeling of hotness is experienced referable to the original site.
When the rate of movement decreases, the brain interprets a feeling of coldness in the site of origin of the impulses.
Electron dynamics The study of the movement of electrons.

It may be restated as a study of the energy of electrons. A more specific presentation permits the total movement of an electron to be defined as the energy of that electron. However, reversing the statement, the energy of the electron is the total movement of the electron. This, in effect, renders the word ~energy~ redundant whenever movement and change in movement is stated for the electron in a system.
Respiration The oxidative breakdown and release of energy from fuel molecules by reaction with oxygen in aerobic cells. (Lehninger) Electron transport The movement of electrons from subs~rates to oxygen catalyzed by the respiratory chain during respiration. (Lehninger) Looking at respiration, that definition can be restated as the release of sd/sp -18-~ ~2~79~3 movement of electrons from molecules reacting with oxygen in aerobic cells.
Electron carriers Enzymes such as flavoproteins and cytochromes which can gain and lose electrons reversibly;
the respiratory chain consists of a series of electron carriers. (Lehninger) In order to follow the specific movements of electrons it is necessary now to address the other terms used by Lehninger with regard to energy. Thus:
0 Free energy That component of the total energy of a system which can do work under conditions of constant temperature and pressure. (Lehninger).
We may substitute total movement for total energy.
Work When a force acts against resistance to produce motion in a body the force is said to do work.
WEAST 49th edition 1968-1969. When we analyze this we find work being defined in terms of the displacement, i.e., change in position, delta P of a body. Work is being defined in terms of movement of a body.
We can then state that free energy becomes that component of total movement which is available to move another body. In other words, for work to be done, there must be movement from an area of high rate of movement to be applied to an area of low rate of movement.
We may derive from the definition of free energy the sense that a compound with electrons having sd/sp -19-~277~.3 high rates of movement can transfer some of that movement to electrons in compounds where the electrons have lower rates of movement.
Hydrolysis The cleavage of a molecule into two or more smaller molecules by the addition of a water molecule. (Lehninger) Excited state That energy-rich state of an atom or molecule existing after an electron has been moved from its normal stable orbital to an outer orbital having a higher energy level, as the result of the absorption of light energy (Lehninger). We may substitute resonance rich for energy-rich indicating the type of movement of an electron in an orbital, i.e., curvilinear.
Similarly we may use the term higher resonance level for higher energy level, again indicating the movement of the electron. Finally we may use the term light photon movement for light energy absorbed during the photoelectric effect. In each instance we are able to define the term energy as a kind of movement of the electron.
Low-energy phosphate compound A phosphorylated compound having a relatively low standard free energy of hydrolysis. (Lehninger) High-energy pho~phate compound A phosphorylated compound X sd/sp -20-~ - , 12~7~

having a highly ~egative standard free energy of hydrolysis. Ibid.
We may state this as a relatively small amount of total movement available and the second as a relatively large amount of total movement available to move another electron or electrons.
High energy bond A bond that yields a large (at least 5Kcal/mole) decrease in free energy upon hydrolysis under standard conditions.
(Lehninger) This can be restated to say that a large amount of resonant movement (at least SKcal/mole) becomes available on hydrolysis under standard conditions to move electrons or other bodies. It is important to recall that heat is the movement of molecules and atoms and that movements of electrons can be transferred to other entities within the molecule and thus change the movement of the nucleus or nuclei.
The high resonance phosphates, then, are ideal for producing changes in the movements of compounds with which they interact sd/sp -21-- 1277~ 3 by transferring the movements of their electrons to those of the interacting compound.
"It has been demonstrated that thyroxine stimulates the synthesis of protein by microsomes and that this stimulation is SECONDARY TO AN EFFECT ON THE MITOCHONDRIA (Sokoloff et al, JBC, 238, 1432. 1963)." U.S. Dispensatory p. 1186 26th edition 1967.
"It is evident that a single regulatory reaction has not been found to explain the multiple effects of thyroid hormones....The question of the "primary" site of action of the hormone remains unanswered." Duncan (Bondy ed.) p. 769 1969.
"In hyperthyroidism the elevated metabolic rate is reflected in increased glycogenolysis, a hyperglycemia, and glucosuria. The clinical syndrome for this condition is called TOXIC DIFFUSE GOITER, or EXOPHTHALMIC GOITER, OR Grave's DISEASE." Frisell p. 609. "The basal metabolic rate (BMR) meagures oxygen consumption in the basal state and is expressed as percentage of values found in normal individuals of the same age, sex, and body surface area (13.)" Harrison p. 449. The correlation between the catecholamine effect and monoamine oxidase activity is clear. Similarly the correlation between thyroid effect and catecholami~e effect is clear. In essence Exophthalmic goiter Goiter accompanied by bulging eyes sdtsp -22-~2779l~3 due to mucoprotein accumulation behind the eyes.
Toxic diffuse goiter Goiter in which there are diffuse changes throughout the enlarged gland.

Phosphate bond energy A term used to denote the decline in free energy as one mole of a phosphorylated compound undergoes hydrolysis to equilibrium at pH
7.0 and 25 , in a 1.0 M solution.
WE may state this as a decline in the electron movement during hydrolysis, movement presumably transferred to water.
The same effect is being discussed when thyroid and monoamine oxidase effects upon neurotransmitter amines are being discussed.

Thus far the control of transmitter amine levels by monoamine oxidase oxidative deamination has been traced to the availability of thyroid hormones T4 and T3. The next step is the inhibition of deiodination in order to have a high enough concentration of these thyroid hormones to effectively inhibit the monoamine oxidase in the outer membrane of the mitochondria. We have noted that large amounts of T4 and T3 have been demonstrated tightly bound to the inner membrane of the mitochondria. These were sd/sp -23-studies conducted outside the body. The concentrations required for the activities to be demonstrated were said to be too high, to be nonphysiological. The enzyme that occurs in the mitochondria is a dehalogenase similar to that in the endoplasmic reticulum. The specificity of these enzymes include bromide chloride, and iodide, but with fluoride there is less activity. When we consult the periodic table, these comments are of interest. The four halogens named are nonmetals. The remaining one astatine is a metal by this classification.
METALLIC RADII
The metallic radium may be defined as one half the distance between the atoms of a metal in the metallic close packed crystal lattice tin which the metal exhibits a coordination number of 12). Metallic radii are generally some 10 to 15% larger than the single bond covalent radii (see Table 2.1) but at the same time are considerably smaller than the nonbinding van der Waals radii. The metallic bond (see Chapter ,) that governs the proximity of metal atoms to one another in the metallic crystal is NOT localized between any two pairs of atoms as is the covalent bond. This results in the two metal atoms not being drawn quite as close to each other in the metallic crystal as they are in forming a covalent bond. At the' same time, however, the bonding forces are appreciable in governing interatomic distances as evidenced by the comparatively much larger van der Waals radii for the same atom. METALLIC RADII ARE QUITE OFTEN REFERRED to sd/sp -24-~27'-~9~

as ATOMIC RADII in reference tables." Demitras, et al pp. 37,38. Iodine is classified as the largest honmetal element, the one with the greatest mass.
The fifth postulate of Dalton's atomic theory has been stated as "The combining proportions mentioned in the laws of definite and multiple proportions are related to the relative mass of atoms.~ Blumberg & Stanley p.
49. The periodic law used by Mendeleev to construct the Periodic Table in a form that was finally quickly accepted can be stated thus "The properties of the elements vary in a periodic way with an increase in atomic number."
Ibid. p. 206. Thus, both properties of mass and the number assigned to an element are related to the properties of the element.
PERIODIC TRENDS IN ATOMIC RADII
"Whether one considers the covalent radii, metallic radii, or van der Waals radii, significant trends appear as one traverses a period of the periodic table or compares these properties in a group. THE IMPORTANCE OF THE RADII
LIES IN THE FACT THAT THE SIZE OF THE ATOM as much as any other single property of the atom determines its PHYSICAL
AND CHEMICAL PROPERTIES. The chemical reactivity of an atom is a function of the ability of the atom to lose, gain, or share electrons, and this ability is very largely controlled by the electrostatic attractive force exerted by the nucleus on the valence electrons. In turn, the attractive force is governed largely by the distance of the valence electrons from the nucleus, i.e., the radius.

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lZ7791~3 At the same time many physical properties such as density are dependent on the radii of the atom." Demitras, et al, pp. 38, 40.
VALUES OF RADII
In table 2.1 of Demitras et al, the radii of the elements in group VIIA read as follows:
Halogens: Single bond covalent radii (SBC R).
F 0.64 Cl 0.99 Br 1.14 I 1.33 At 1.45 These are nonmetals except for the last so that no metallic radii are given.
In table 2.4 van der Waals radii are listed as follows;
F 1.35 Cl 1.60 Br 1.95 I 2.15 Group VIIB
The radii of these transition metals are listed as follows:
SBC RAdius 1st 1.17 2nd 1.27 3rd 1.28 Metallic radius 1.27 1.35 1.37 respectively for the same elements in order of increasing size.

sd/sp -2SA-THE VALENCE ELECTRONS ~2~903 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 extra electron when it is ~tructured as an ion. Thus, it mimics the electron cloud of argon.
The calciwm atom ha~ a filled 452 grouping but tries to get rid Or those two electrons to form Ca++ ion which then mimics the argon electron cloud. The electron clouds Or group VIII A belong to the inert ga~es and thu~ are ~11 ~illed.
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 Or that row.
It is the fifth transition metal to the right Or calcium and the tenth metal to the left of bromide. Bromide has two Or the 8 electrons in the fourth æeries and five Or the p electrons in the fourth level. Iodide has the Qame - distribution of 8 and p at the fifth level. In addition, it has a full ten of the d electrons in the fourth level.
Since there is a special overlapping Or 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.

,-~277903 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 o~ the next row, i.e., in 3ize. In other words it has strong similarities to calcium and to iodine. When viewed at the atomic level Or size it would appear ~imilar to c~lcium and it would appear similar to iodine. Three is the quantum for its d electrons. A guantum number is the numerically defined symbol designed to indicate the 'energy' of the electron.
Enorgy in reality i8 the total movement o~ the electron.
The quantum numbers Or the electrons indicate their move-ment and on occacion we 3hall rerer to this as 'resonance'.
Resonance inrers the amount Or movement one attributes to an electron in its orbital.
Electrons, then; are defined by their positions relative to the position Or the nucleus Or an atom or in the case o~ molecule~ relativo to the position Or the nuclei Or the atoms around which an orbital extend~. We may indicate position by P and the change in position by delta P. Derining the position by P and the change in position by delta P enable~ us to measure the pathway Or the electron from some arbitrarily selected ~ite and thus comparing it~ movement with that Or the other entities in its atomic environment.
At the atomic level, manganese look~ like a calcium ion when both of lose their outer electron~ and conform to the inert gas Argon.

~ ~7 ~ 27r7~ 3 Iodine looks like manganese. Both are solids at room temperature and both have outer electrons of a group Or two electrons and a group of five electrons. Except for the halogens, THIS WTER CONFIWRATION IS FOUND ONLY
IN MANGAMESE. 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-ment8 o~ the deiodinase enzyme located at the inner mem-brane Or the mitochondria.
The salt8 Or manganese are round nearby inside the ~itochondria. These salts occur there along with the salts of calcium, strontium and magnesium. Active trans-location Or manganese into the mitochondrial matrix by high resonant ATP conforms well to the calcium in the active translocation Or divalent cations. Manganese also conforms well to the active site of the inner membrane enzyme which selectively removes the iodine atoms from the 3 and 5 position of the distal phenyl rings of thy-ronine in the thyroid hormones T4 and T3 WHEN MANGANESE OCCUPIES THE ACTIVE SITE OF THE
DEIODINASE THE ENZYME IS PREVENTED FROM REMOVING IODINE
FROM THE THYROID HORMONES AND THE MOLECULES OF THE THYROID
HORMONES INCREASE IN CONCENTRATION. Thus, when the man-ganese enters the nonpolar pocket of the active site it inhibits the deiodinase.

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- , ~
lZ779~3 MANGANESE METABOLISM
"The early studies of Greenberg (65) with radio manganese 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. It is a reflection of the mitochondrial 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

sd/sp -29---" 1.2779~3 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 body man anese must, therefore, be in a dynamic mobile state.
Underwood p. 185.
"The turnover of parenterally administered 54Mn 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 54Mn in the tissues was directly related to the level of the stable manganese in the diet.
THIS PROVIDES FURTHER SUPPORT FOR THE CONTENTION THAT
VARIABLE EXCRETION RATHER THAN VARIABLE ABSORPTION REGULATES
THE CONCENTRATION OF THE METAL IN TISSUES." Underwood p. 18S.
"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 reduce manganese availability....The effect of variations in dietary calcium and phosphorus on the metabolism of 54Mn in rats has been studied further by Lassiter and associates l100). These workers found that the fecal excretion of parenterally administered 54Mn was much higher and the ~! sd/sp -30-liver retention lower on a 1.0~ calcium diet thah on a 0.64 calcium diet. It appears, therefore, that calcium can 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 intraperitoneally administered 54Mn, BUT THE ABSORPTION OF ORALLY ADMINISTERED 54Mn WAS
IMPAIRED. Underwood. p. 186.
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 membrane 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 CalMg 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 sdlsp -31-~` 1~9~

with Na/K ATPase pumps not only fits the cell me~brane, 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 f low 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 beween 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 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 mitochondria with the energy metabolism of 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 consistent 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 X sd/sp -32-amines. ALL ARE FOUND IN CLOSE PROXIMITY IN TH~ MITOCHONDRIA.
Of these seven components we may concentrate on the small molecules; the enzymes would be PDO for phosphorylase deiodinase and oxidase. The small molecules give MTA
for manganese, thyroid hormone, and amine. If calcium were added at the beginning when cation pumps are discussed as part of the mechanism of regulation, then the letters might be CMTA. The word 'sequence' affords a description of event.
Here we will use the designation 'MTA sequence', and if there is reason to emphasize calcium or divalent cations other than that of manganese 'CMTA sequence' can also be referred to in the textual material.
The formulation here discussed as 'MTA sequence' has been provided in keeping with the suggestions presented in the article by Marcus in the J. of the P.O. Soc. September, 1969, Vol. 52, No. 9 on page 559. It is an attempt to insure that one skilled in the art will be able to reproduce the re6ults described in a disclosure or advanced in a constructive reduction to practice. It has been felt that treatments are greatly facilitated by insight into this complex biochemical mechanism.

sd/sp -33-i27~9~!3 "On the 18th of February, 1969, Paul Langerhans defended his thesis about the cell clusters in the pancreas which later came to bear his name. Exactly 100 years after that remarkable observation, representatives from the main centers of islet research met in Umea, Sweden, for a second international symposium on ~The Structure and Metabolism of the Pancreatic Islets.""
The hypoglycemic effects of leucir.e and of arginine discussed at that symposium were reported in Vol. 16 of Wenner-Gren Center International Symposium Series (1970) Pergamon Press Limited, ed. Falkner, Mellman, and Taljedal.
This was some fifteen years ago.
Unger, et al discussed the regulation of glucagon release in vivo. On page 147 they state, " In man arginine infusion elicits a parallel rise in insulin and glucagon during which glucose concentration rises to an average peak of 16mg~o." Then, they further remark, "In genetic diabetes, however, the glucagon response is intact but the beta-cell response is reduced; the result i8 hyperglycemia with an average peak rise of 45 mg~o.
One could postulate that if alpha-cell function were reduced without symmetrical impairment of beta-cell function, the administration of arginine would result in hypoglycemia, and, indeed, experiments in dogs in which all of the pancreas save the uncinate process had been resected support this supposition..."

i277~Q~
.

Hellerstro~ et al discuss the effects of amino acids on oxygen consumption of the beta-cell in relation to insulin release. Valine and arginine were without effect, "...whereas a marked respiratory enhancement was noted with either alanine or leucine."
Stork, et al studied beta-cell respiration in the presence of sulfonylureas. The latter "...strongly depressed" leucine degradation in the beta-cells.
Malaisse, et al demonstrated that gut extract "...also significantly enhanced the stimulant effect of leucine,"
on insulin secretion.
Another paper by Unger, et al led them to conclude~
"This suggests that the magnitude in the hyperglycemic response to arginine is determined by the glucagon con-centration in relation to the concomitant insulin level, rather than to glucagon concentration alone."
The last paper of the symposium was "The Pancreatic Beta-Cells in the Pathogenesis of Human Diabetes Mellitus~
by Cerasi and Luft. Their summary reads as follows~
"The findings of a delayed and decreased insulin response to glucose infusion in healthy monozygotic twin sibs of patients with diabetes mellitus makes it very likely that this defect in insulin release is a major, genetically determined pathogenetic factor involved in the development of diabetes mellitus. In a non-selected population of subjects with normal glucose tolerance, the 3 ~

12779~)3 frequency of such a defective insulin response to glucose is around 20%, indicating that the prediabetic state is rather common in a general population.
"The biochemical abnormality responsible for the de-fective insulin release in prediabetics is not known.
Evidence is presented in the present work indicating that the adenyl cyclase system of the beta-cell might be involved in this abnormality, since insulin response can be normalized in prediabetics by pretreatment of the subjects either with theophylline or with large doses of human growth hormone, both agents known to result in an elevation of the intracellular level of cyclic 3,5-AMP."
The conference is summarized by B.A. Houssay. This gives the most advanced status of the study of diabetes mellitus and the lowering of blood glucose at that time.
Despite this knowledge and the continuing study of it, a dependable, continuing modulation of insulin release in NIDDM (Type II) diabetes mellitus by the use of these naturally occurring substances was not achieved.
From these observations of the effects of under-secretion of insulin in producing diabetes mellitus, it is easy to conceive of the opposite situation in which there exists a lowered blood sugar from a relative excess of insulin secretion with a resulting excessive drop in blood glucose, i.e., hypoglycemia.

..., ..-......
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~.2779~.~
, .
SUMMARY OF THE INVEN~ION

The present invention provides a method for raising blood glucose in vertebrates with hypoglycemia. The use of amino acids having hyperglycemic actions in various ratios each with the other and each with manganese in effective ratios decreases insulin release in chemical hypoglycemia.
The present invention differs in its relation to effective amounts given in that these amounts are constantly changing, so that there is a pattern of changing requirements as to frequency, amount and individual requirements.

3 ~

''' 1 ~ 9~ 3 DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with this invention, patients with hypoglycemia have usually been treated with exercise and with a diet such as one low in sugars and high in protein. In addition, a variety of hormonal and other agents claimed to produce a rise in the blood sugar level have also been used. The above program and medication can for the most part be continued as this method is initiated. It will, however, be desirable to discontinue any hormone used in the previous treatment in order to determine base values before initiating treatment.
Following an introductory period of observation, the hyper-glycemic agents named in the invention may be used stepwise.
The introductory period permits evaluation of the fasting blood sugar.
A fasting blood sugar is taken when the hyperglycemic agent is to be introduced. The desired amount of the agent is then given with food. A day later or after a suitable time period a subsequent fasting blood sugar determination is made to detect any change in blood glucose.
For example, isoleucine in the amount of 125 mg may be given initially. If there is no change, this should be increased stepwise to 250 and 500 mg or higher values until a drop in the blood glucose can be correlated with the amount given.

~ 3 The less direct actions of valine, methionine, phenylalanine and tyrosine are best explored after the treatment schedule has been undertaken.
A small dose of manganese of one or two mg (calculated as manganese content in manganese gluconate) can be given separately. Both the glucose level and the subjective response of the patient are monitored-, If these initial amounts are well tclerated, the amount can be increased by increments of two to three mg at a time up to doses of five and ten mg or more.
Once the upper amount tolerated has been determined, these levels are likely to decrease rapidly. There is no need to activate special methods of disposing of manganese by giving much larger amounts.
The amount of manganese reached for positive correlation with a drop in the glucose level is likely to fall progressively over a few weeks or days. The amounts required sometimes decrease rapidly and are spaced out over longer and longer intervals. It is best to be prepared to promptly drop to lower amounts at the earliest indication that the amount required is decreasing.
Combinations of manganese and isoleucine may app~ar to have synergistic effects. This reflects the different mode of action of each. The manganese may appear to have a modulating effect on the action of isoleucine.

3 ~

As amounts are adjusted downwards, increase the interval between treatments as well as decreasing the amounts given.
This is the 'cumulative' effect of pharmaceutical agents, which may be described as being similar to 'filling a hole'.
This use of the word cumulative must be distinguished from that in legal phraseology. The legal definition follows the heaping up or piling up definition of the word cumulative.
The clinician must be aware o~ the i~plications of the above.
It is best that the substances be administered personally during the initial period of treatment. The period of adjustment varies from patient to patient. It is unw~se to provide the patient initially with a standard daily dosage on the assumption that the effects will be predictable.
As the blood sugar rises, any other agents used may be discontinued. No effort should be made to force the glucose level above the normal range, which may be estimated in the human at 110 to 140 milligrams/100 milliliters of blood, i.e., between 110 to 140 mg~0.
Hyperglycemic effects produced in such patients by the use of aents which cause a stress reaction, e.g., cortisol, which cauæes the breakdown of protein should be avoided.
~he overall effectsof these substances may prove to be quite adverse and are to be avoided. In accordance with this, any form of stress, such as injury and infection, are prone to produce variations in the amounts of isoleucine and/or manganese required. No manganese should be given during fever.

.. ~/ ~, ~Z779~3 Example # 1 Patient M.V.
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, the~ were remaining in the ~0 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 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 ab~ut 50 milligrams/
100 milliliters, (mg~O) to 100 mg~O.
II Blood pressure~
A Systolic pressure2 ranged from 150 to 124 mg Hg (millimeters mercury pressure) B Diastolic pressure ranged from 86 to 110 to 7~ 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.
~00 m ~tablet in time between meals to ten days. (1.6 to 6.7 m ~kg body weight) (0.007 to 0. 27 Objective of Treatment~ To bring glucose level to normal range of(100-110) to 140-150~ mg~O
Subjective findingsl The patient was anxious and upset during part of period associated with a viral upper respiratory infection.
During this interval anxiety was prominent.
Clinical response~ An episode of labile blood pressure occurred during the time involved and then drifted down to 124/7~ over a two week period The change in blood ~ucose level occurred easily.
The most striking observation was the level of 90 mg~O still present six months later.
The patient continued throughout to feel considerably better from day to day than was his usual pattern.

4"
.... .. .

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~ " 12779Q3 Example # 2 Patient T.S midlife History of headaches Recurrent peptic ulcer with occasional activity treated with tagamet.
Treatment periods: varied from one to two weeks to two consecutive days Treatment: After fbs (fasting blood sugar) and blood sugar before meals manganese gluconate manganese calculated in milligrams, mg Days 5, 15, 16, 18, 39 and 54 1 mg to 2mg given.
Isoleucine separately or with manganese gluconate in amounts of one-fourth to one tablet, i.e., 125 to 500 mg Treatment period interval: Initially at two to ten days;
^ then two to fifteen days thereafter.
Objective findings:
I Blood glucose: Increased from about 60 milligrams/100 milliliters i.e., from 60 to 100 mg% (milligrams per cent) over a seven week period.
II Blood pressure:
A Systolic (upper) from 154 to 116 mm Hg (millimeters mercury) B Diastolic from 92 to 78 sd/ml ` -42-91!33 C Pulse pressure = difference between systolic and diastolic = from 62 to 40 III Pulse: from 66 to 80/minute Range of medication:
Ratios: isoleucine to manganese = 63/1 in mg to 250/1 About 2 mg/kg to 8mg/kg isoleucine About 0.03 mg/kg to 0.09 mg/kg body weight Objective of treatment: To restore fasting blood sugar (fbs) to normal range of values (100 to 110) to (140 to 150) mg% without headaches developing Subjective findings: Patient appeared to feel better subsequent to the course of treatment.
Clinical course correlated with a slow drop of 25 to 30 mg% over about a four month period. Affect good.
Clinical response: Satisfactory Course of treatment uneventful sd/ml -42A-"` ~ Z77~

Example 3 Patient M.A. mid-thirties Returned after ten year interval. Under dietary treatment for reactive hypoglycemia.
Avoided crowded areas.
Did only local driving.
Treatment periods: Each for one or two days consecutively.
Treatment: blood sugar drawn fasting and before meals and hyperglycemic agent given 2 to 3 timestday with food manganese gluconate 1-2 mg/treatment period.
Treatment period interval: increased from 2-3 weeks to 3-5 months.
Blood sugar: increased from 65-70 mg% to 85-90mg%.
Objective of treatment: to restore blood sugar to normal range of fasting values, i.e., 90 to 110 mg%.
Clinical Response: Normal range of blood sugar - patient symptom-free full-driving schedule sd/ml -43-

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition for treating hypoglycemia, comprising:
(a) a compound selected from the group consisting of L-methionine, D-methionine, L-isoleucine, D-iso-leucine, an alpha-keto analog, an alpha-hydroxy analog, a dipeptide and a tripeptide of said amino-acids, a pharmaceutically acceptable acid addition salt of said amino-acids, analogs and peptides, and a mixture thereof, (b) a compound selectd from the group consisting of L-phenylalanine, D-phenylalanine, L-tyrosine, D-tyrosine, an alpha-keto analog, an alpha-hydroxy analog, a dipeptide and a tripeptide of said amino-acids, a pharmaceutically acceptable acid addition salt of said amino-acids, analogs and peptides, and a mixture thereof; and (c) a nonlethal, pharmaceutically acceptable, physiologically tolerable, pharmacokinetically appropriate amount of an Mn compound:
wherein components (a) to (c) are present in effective amounts and ratios to render the composition antihypoglycemically active.