Methods for inhibiting inflammatory ischemic, thrombotic and cholesterolemic disease response with methionine compounds

This invention concerns novel methods employing compositions containing as an active antioxidant or antiinflammatory agent the amino acid methionine, and/or one or more related compounds including certain metabolic precursor compounds, for treating or inhibiting inflammatory ischemic, thrombotic and cholesterolemic disease response in a subject. The compounds include the methionine hydroxy analogs, as well as compounds having the structural formula I: ##STR1## and pharmaceutically acceptable N-(mono- and di-carboxylic acid) acyl derivatives and alkyl esters thereof, where n is an integer from 1 to 3.

TECHNICAL FIELD 
This invention concerns novel methods employing antioxidant and 
antiinflammatory dietary or therapeutic compositions containing the amino 
acid methionine (also known as "Met"), and/or one or more related 
compounds including certain metabolic precursor compounds for inhibiting 
inflammatory ischemic, thromboticand cholesterolemic disease response in 
man and animals. 
BACKGROUND OF THE INVENTION 
A variety of efforts have been made over many years to elucidate the 
mechanisms and origins of inflammation and the various forms of disease it 
may cause. Limited success has been achieved in alleviating the symptoms 
of diseases having inflammatory components. Oxidative stress has been 
implicated in many of these diseases, and antioxidant therapy has been 
recommended as one method to alleviate the damage it causes (Cross, et. 
al., Annals of Internal Medicine, 107:526-45, 1987). 
Recently, several investigators have focused on the role of sulfhydryl 
compounds in the mechanism of treatment of some forms of arthritis. 
Cuperus, (Arthritis and Rheumatism 28: 1228-33 1985) showed that 
d-penicillamine, tiopronin, aurothiomalate and aurothioglucose were 
scavengers of the products of activated granulocytes, and Bailey and 
Sheffner (Biochemical Pharmacology 16: 1175-82, 1967) showed that 
acetylcysteine and acetylpenicillamine reduced experimental dermal 
inflammation but that methionine did not. Methionine is known to be 
oxidized to its sulfoxide by granulocytes but not by hydrogen peroxide at 
physiological concentrations. Persons deficient in the enzyme 
myeloperoxidase do not make hypocholorous acid in lymphocytes and appear 
not to suffer unusually from infections. By contrast, persons with 
deficient production of hydrogen peroxide are adversely affected. 
(Stendahl, et al., J. Clin. Invest., 73:366-73, 1984). 
Cuperus, supra, describes a feature of inflammed synovial fluid, such as 
that occurring in arthritis patients, as the accumulation of 
polymorphonuclear (PMN) leukocytes. One function of the leukocytes is the 
destruction of invading elements such as micro-organisms. For this 
destruction, the leukocyte releases hydrogen peroxide and enzymes, e.g., 
myeloperoxidase, into the extracellular fluid. In the presence of hydrogen 
peroxide and chloride ion, myeloperoxidase catalyzes the formation of 
reactive hypochlorous acid (HOCl) which can oxidize tissue components and 
plasma protease inhibitors. Oxidation and subsequent inactivation of these 
protease inhibitors in vivo may lead to unrestrained proteolysis, 
resulting in severe tissue damage. (Weissmann, et.al., Jour. Investigative 
Dermatology, 71:95-9, 1978). 
Several investigators have noted that patients with severe rheumatoid 
arthritis have lower levels of serum SH groups (Hall, Journal of 
Rheumatology 9:593-6, 1982). Ambanelli (Scand. Jour. Rheumatology 
11:203-7, 1982) showed that serum SH groups went up in patients that 
responded to tiopronin therapy. The mechanism of serum SH groups in 
relation to the severity of arthritis has not been established. The 
correlation could be explained by the failure of particular individuals to 
counteract the production of oxidizing substances by immunocytes. 
McKenna, (British J. Rheumatology 25:132, 1986), saw benefit for only 2 of 
15 patients given cysteine methyl ester for rheumatoid arthritis, a direct 
sulfhydryl agent. 
Delrieu, et al., (Revue du Rhumatisme, 55:995-7, Dec., 1988) found no 
statisical difference between treatment and controls in a 24 patient study 
of rheumatoid arthritis using 5 and 10 grams of l-methionine per day for 4 
and 2 months, respectively. Clinical tolerance was good, but 
gastrointestinal distress was encountered by a majority of the patients. 
Gualano (Pharmacology Research Comm. 15:683-96, 1983) showed 
antiinflammatory activity of S-adenosyl methionine but attributed its 
effects to mechanisms of aspirin-like drugs. Davis, (Jour. Am. Pod. Assoc. 
68:24-30, 1978) studied the effects of certain amino acids on inflammation 
measured as edema and found that methionine was not effective in reducing 
edema while cystine was effective. Marcolongo (Current Therapeutic 
Research 37:82-94, 1985) showed beneficial effects of S-adenosyl 
methionine slightly better than ibuprofen in the treatment of hip and knee 
osteoarthritis. Stramentinoli (Am. Jour. Medicine, 83 Suppl 5A:35-42, 
1987) shows that S-adenosyl methionine will inhibit the swelling in 
carrageenin-induced rat paw edema, while l-methionine in equimolar doses 
is completely ineffective. 
Other studies involving the oral administration of S-adenosyl methionine 
have shown that treatment does not increase the blood levels of methionine 
(Baldessarini, et. al., Arch. Gen. Psychiatry, 36:303-7, 1979). In animal 
studies blood level increases of methionine are reflected by parallel 
increases in brain levels of methionine, but a 10 fold increase in brain 
methionine produces only a 50% increase in brain S-adenosyl methionine 
(Rubin, et al., J. Neurochemistry 23: 227-231, 1974). 
In a study of the immunosuppressive activity of D-amino acids, Inoue, et 
al. showed that there was no immunosuppressive effect for d-methionine in 
their mouse assay at a dose of 10 mg per kg body weight. (Japanese J. 
Experimental Medicine, 51:363-6, 1981). 
Regarding acute inflammation, the complement system of the human body (see 
Spector, W. G., Intro. to General Pathology, p. 58-75, Churchill 
Livingstone, New York, 1980) is part of a cascade of enzyme reactions that 
are responsive to external injury in which complement is activated and 
generates peptides known as C3a and C5a which are response-inducing or 
chemotactic for white cells. 
The S-methyl derivative of methionine, S-methyl methionine, also known as 
vitamin U has been shown to have benefit as an anti-ulcer compound and to 
have benefit for allergies. The same benefit is shown for carboxyl esters 
and N-acyl derivatives (Kowa, DT 2821-704). However, in this teaching no 
distinction is made for the d- and l-isomers of S-methyl methionine or its 
derivatives and no claim is made that these compounds act through 
anti-inflammatory mechanisms. 
INFLAMMATORY ISCHEMIA AND THROMBOSIS 
O'Brien (Pathologie Biologie, 32:123-38, 1984) and Munro and Cotran 
(Laboratory Investigation, 58:249-61, 1988) reviewed the evidence for the 
contribution of elastolysis to atherosclerosis including a contribution 
for leukocyte infiltration in eventual plaque development. A more 
significant contribution of immune cells in atherosclerosis was shown by 
Poston and Davies (Atherosclerosis, 19:353-67, 1974) wherein they point 
out the following evidence for etiology of ischemic vascular disease: 
1. The development of rabbit atherosclerosis can be inhibited by 
antiinflammatory and immunosuppressive drugs. 
2. Arthritis can be produced experimentally by immune complexes. 
3. Milk antibodies are increased in persons with myocardial infarction. 
4. Autoantibodies to arterial tissue have been found in ischemic vascular 
disease. 
5. Half of young adults dead from coronary accidents have acute 
inflammatory changes. 
6. Eighty-two percent of young adults with atherosclerotic lesions have 
inflammatory changes in small branches of coronary arteries. 
7. Rabbits (50%) develop aortic lesions after being given a bovine serum 
injection when on a 1% cholesterol diet. 
8. Atherosclerosis develops in coronary arteries of transplanted hearts 19 
months before death of the recipient. 
Leukocytes accumulate in complex thrombi that consist of fibrin and 
platelets (Plow and Edginton, Jour. of Clinical Investigation, 56:30-8, 
1975; and Cybulsky, Chan, Movat; Laboratory Investigation 58:365-78, 
1988). 
Ibuprofen, an antiinflammatory drug, inhibits the response of granulocytes 
to inflammatory mediators and has been shown to reduce the size of infarct 
after experimental ligation of the anterior descending artery. Thus, 
inflammation is implicated in the ischemia and resultant cell destruction 
that occur after blood flow has ceased (Flynn et al.; Inflammation, 
8:33-44, 1984). Cardiac muscle mitochondrial N-Formylmethionyl proteins, 
released after myocardial injury, are chemoattractants for PMN's (Carp; 
Jour. Experimental Medicine, 155:264-75, 1982). Ischemia following 
reperfusion in cardiac arrest or CNS injury results from generation of 
oxygen radicals. PMNs activated by cellular injury can expose injured 
tissues to high local concentrations of oxidants. For a review, see 
Braughler and Hall (Free Radical Biology and Medicine, 6:289-301, 1989) 
and Hall and Braughler (Free Radical Biology and Medicine, 6:303-13, 
1989). 
INFLAMMATORY SYMPTOMS OF SHOCK 
Toxic-shock syndrome (TSS) is caused by a bacterial toxin, and occurs 
mainly in young menstruating women (Schlievert, et al., J. Infectious 
Diseases, 143:509-16, 1981). The PMN may be the common pathway through 
which inflammatory mediators act in septic shock, causing multiple organ 
failure. Aseptic shock, which is mainly reperfusion injury as blood flow 
returns to the area, also depends on the PMN for mediation (Vedder, 
et.al., Perspectives in Shock Research: Metabolism, Immunology, Mediators, 
and Models, Alan R. Liss, p. 181-91, 1989). Endotoxin has been shown to 
stimulated PMNs to release large amounts of toxic oxygen species which 
cause tissue and organ damage (Yamada, et. al., Inflammation, 5:115-26, 
1981). 
LIPID METABOLISM NORMALIZATION 
Heart attacks and strokes are the major killers of man in the industrial 
world (Ross, New Eng. Jour. Med., 314:488-500, 1986). Efforts to reduce 
the incidence have concentrated on altering the diet by reduction of salt 
and fat consumption, especially saturated fat and cholesterol, and by 
increase of exercise with corresponding weight control. Increased 
consumption of bran has been touted as a method to reduce serum 
cholesterol levels, but the phytate in bran can cause zinc deficiency and 
reduced calcium absorption from the diet. (Reinhold, et al., The Lancet, 
Feb. 10, 1973, p. 283-8). High serum concentrations of low-density 
lipoprotein cholesterol is considered a major risk factor for coronary 
heart disease (CHD). Lowering hypertension without concomitant decrease of 
serum cholesterol has little influence on CHD. (Middeke, and Holzgreve, 
Am. Heart J., 116:1708-12, 1988). Nicotinic acid (vitamin B3) is the 
first-line drug for the treatment of patients with primary 
hypercholesterolaemia. Side-effects of the typical daily dose of 3 to 5 
grams a day are flushing, nausea, cramps and diarrhoea (Figge, et al., 
Pharmacotherapy 8:287-94, 1988). 
Truswell et al., (J. Atheroscler. Research, % :526-32, 1965) show that in 
humans administration of 1.5 grams of taurine per day for 15 days produced 
no effect on serum cholesterol concentrations. 
DIETARY DEFICIENCY OF METHIONINE 
Methionine deficiency is not recognized as a disease state in modern 
countries where adequate total protein is generally available. While it is 
recognized that humans, in contrast to most other mammals, cannot utilized 
d-methionine as a source of methionine, it is generally assumed that 
humans can utilized methionine sulfoxide as a source of methionine. The 
only suggestion that methionine sulfoxide might not be nutritionally 
equivalent to methionine is the lack of increase of blood l-methionine 
after administration of l-methionine sulfoxide. Human enzymes have been 
found that can reduce methionine sulfoxide to methionine. 
Methionine is known to be affected by a variety of food processing 
activities. l-Methionine is converted to d-Methionine when proteins are 
heated and a significant amount of the nutritional value of methionine can 
be lost by this mechanism. However, most of the potential loss of 
available methionine occurs through the mechanism of oxidation of 
methionine to methionine sulfoxide. The bleaching of flour is the major 
cause, when during the process of bleaching the chlorine is able to react 
with methionine. When proteins are heated with reducing sugars methionine 
is readily oxidized so that items such as canned peaches are potential 
sources of food with a deficiency of available methionine. More recently, 
as unsaturated fats replace saturated fats in prepared food sources 
additional sources of methionine oxidation occur. For example, the 
unsaturated fats in cake mixes held in a hot warehouse would result in 
oxidation of methionine to its sulfoxide. Published evidence for an 
extensive loss of methionine in food processing as regards human nutrition 
occured in the manufacture of instant oatmeal where the product used in 
nitrogen balance studies apparently had no nutritionally available 
methionine (see Kies, et. al., J. Nutrition, 105: 809-14, 1975). In the 
cooking of several types of beans 40% to 50% of the methionine is not 
available to rats (Sawar and Peace, J. Nutrition, 116:1172-84, 1986). The 
dietary requirement for methionine plus cysteine is based on nitrogen 
balance studies where a total of 800 mg per day is required to bring 50% 
of adults into positive nitrogen balance. No attempt has been made to 
determine the level of methionine that might be optimal for the prevention 
of oxidative damage. 
Discussion 
Methionine has been shown to be a target for the products of stimulated 
polymorphonuclear neutrophils (Tsan and Chen, J. Clin. Invest., 
65:1041-50, 1980). The granular fraction of the PMNs oxidizes methionine 
to its sulfoxide in the presence of peroxide. Peroxide does not oxidize 
methionine to its sulfoxide at normal physiological concentrations. 
Some of the differences measured in the relative effectiveness of 
methionine compounds and other chemicals especially in sulfhydryl reducing 
substances may be attributed to the control mechanisms that operate in 
animals and man to regulate the amounts of these substances wherein giving 
more of a substance does not significantly increase blood and tissue 
levels of that substance. Stegink, (Jour. Nutrition,116:1185-92, 1986), 
showed that 0.5 gm of methionine elevated total blood methionine 2-fold 
for 2 hours with l-methionine but 3-fold for 4 hours with d-methionine. In 
the same study it was shown that methionine sulfoxide administration did 
not result in elevation of blood methionine. This observation suggests 
that methionine sulfoxide is not readily reduced to methionine but it is 
possible that this reduction occurs in tissues where the methionine 
remains sequestered. l-Methionine is an essential amino acid for human 
nutrition. The normal serum level of methionine in man is 15 ppm. 
dl-Methionine is available as a one-a-day food supplement in 500 mg. oral 
tablet form. 
Regarding human nutrition, l-methionine is an essential amino acid whereas 
d-methionine is non-nutritive. For purposes of metabolism, l-methionine 
via S-adenosylmethionine has an important methylating function. In this 
function it loses a methyl group from its sulfur atom to become 
homocysteine. Homocysteine, as is known, when in excess can lead to 
homocysteinuria, and may be heart disease associated (Malinow, et al., 
Circulation 79:1180-88, 1989) and (Olszewski and Szostak, Atherosclerosis 
69:109-13, 1988). Folic acid has been shown to be an innocuous method to 
reduce plasma homocysteine levels (Brattstrom et al., Scand. J. Clin. Lab. 
Invest. 48:215-221, 1988). Administration of 8 grams of l-methionine to 
adult subjects for four days caused a greater than 30% reduction in serum 
folate levels (Conner, et al., PostGrad. Med. J. 54:318-20, 1978). Thus, 
folate should be co-administered whenever methionine is chronically 
consumed. 
Administration of large amounts (5 to 10 grams per day) of l-methionine can 
cause gastrointestinal upset. Many people report a burning sensation in 
the stomach after taking methionine, along with an upset stomach and 
flatulance (Delrieu, et al, Revue du Rhumatisme, 55: 995-7, 1988). Enteric 
coating and timedrelease formulations should avoid the stomach problems 
and allow even elevations of blood methionine for maximum anti-oxidant 
effect. Typical enteric coating agents include cellulose acetate 
phthalate, and other cellulose ethers and derivatives (Johnson, J. C., in 
Sustained Release Medications, Noyes Data Corp, New Jersey, 1980, p.14). 
The Food and Nutrition Board of the U.S. National Academy of Sciences has 
established the Recommended Daily Allowance (RDA) for nutrients for most 
healthy individuals. For a discussion, see The Nurses Guide to Drug 
Therapy, Eisenhauer and Gerald, Prentice-Hall, New Jersey, 1984-5, pages 
584-602, and incorporated here by reference. RDA's include: Vitamin A: 
5000 I.U.; Vitamin B12: 3 mcg.; Vitamin B6: 2 mg; Vitamin B3: 18 mg.; 
Folic Acid: 400 mcg.; Vitamin C: 100 mg.; Vitamin D: 400 I.U.; Vitamin E: 
15 I.U.; Calcium: 800-1200 mg.; Iron: 18 mg.; Selenium: No RDA has been 
established, but the maximum non-toxic suggested dose is 200 mcg. per day; 
Zinc: 15 mg. For a review of life-style risk factors and protective 
factors in the diet, see the article by Bruce N. Ames entitled "Dietary 
Carcinogens and Anticarcinogens", Science, 221:1256-63, 1983, incorporated 
here by reference. 
Different species utilize d-forms of amino acids to different extents. 
Humans and monkeys utilize d-methionine poorly while pigs, dogs, rabbits, 
chickens, rats and mice use d-methionine as a sulfur source fairly well. 
Animals do not metabolize N-blocked-d-methionine as they do 
N-blocked-l-methionine. Some N-blocking groups are not cleaved by enzymes 
that remove common blocking groups such as acetyl groups (Cho, Jour. 
Parenteral and Enteral Nutrition 4: 544-7, 1980; Stegink, Jour. of 
Nutrition 110: 42-9, 1980; Rotruck, Jour. of Nutrition 105: 331-7, 1975). 
The patent to Scheinberg U.S. Pat. No. 4,315,028 describes a method of 
treatment of arthritis employing substituted cysteines. 
The patent to Kowa (DT 2821-704) describes anti-ulcer activity and 
antiallergy benefits for the S-methyl derivative of methionine and 
methionine esters and N-acyl derivatives. The d- and l- isomers of 
S-methyl methionine compounds and derivatives are not distinguished. 
The patent to Damico U.S. Pat. No. 3,952,115 describes foodstuffs 
containing N-acyl l-methionine esters and N-acyl l-cysteine esters. 
d-Isomers are specifically excluded because they are "not nutritionally 
available". 
The patent to Fahim, U.S. Pat. No. 4,711,780, shows the benefit of the 
combination of cysteine with vitamin C and zinc salts in a topical mixture 
for stimulating cell proliferation. The benefit of methionine is claimed 
but not shown. No demonstration of benefit or claim for systemic 
administration is made. 
In view of the widespread incidence of diet-related vascular disease and 
inflammation, a need exists for means, other than diet alone, of 
preventing or inhibiting the incidence and serious consequences of the 
disease. Thus there is a need at present for means of treating disease 
conditions of the kind in which a nutritional deficiency, an inflammatory 
response or abnormal imflammation is implicated. 
It is therefore an object of the present invention to provide methods for 
the prevention, inhibition and treatment of disease conditions of man and 
animals that may be attributable to or result from nutritional deficiency 
and inflammatory responses. 
It is a further object of the invention to provide means for preventing or 
alleviating symptoms of homocysteinuria that may result from excess 
methionine intake. 
These and other objects, features and advantages will be seen from the 
following detailed description of the invention.

SUMMARY AND DETAILED DESCRIPTION OF THE INVENTION 
Our invention is based on the discovery that certain methionine or 
methionine-type compounds in the dl-form or d-form at relatively high, 
well-tolerated doses are potent antioxidant and antiinflammatory agents in 
man and animals. The invention includes means for inhibiting or treating 
conditions predisposing to or presenting as an inflammatory ischemic or 
thrombotic disease response in man and animals. The methionine compounds 
in high daily dosage according to the invention thus may act in vivo to 
inhibit oxidative effects such as the action of hypochlorous acid to 
reduce proteolysis and tissue damage. 
Novel methods for the prevention and treatment of disease conditions of man 
and animals that may be attributable to or result from nutritional 
deficiency of the l-form of methionine, such as lipid abnormalities, are 
also disclosed. 
The inclusion of the amino acids glycine and serine and the vitamins B6, 
B12 and folate for homocysteine normalization are also disclosed. 
For purposes of the invention, one uses at least one methionine-type 
compound selected from the methionine hydroxy analogs and methionine 
compounds having the structural formula I 
##STR2## 
and pharmaceutically acceptable N- (mono- and di- carboxylic acid) acyl 
derivatives and alkyl esters thereof, where n is an integer from 1 to 3. 
Thus, the methionine-type compound (for convenience sometimes referred to 
herein as "methionine" or "methionine compound") may be normethionine 
(n=1), methionine (n=2), homomethionine (n=3), the hydroxy analog, or the 
acyl or alkyl ester derivatives thereof, as defined. Exemplary acyl 
derivatives are the formyl, acetyl, propionyl, and succinyl derivatives, 
of which the formamide, acetamide and succinyl derivatives are preferred. 
Exemplary ester derivatives are methyl, ethyl and isopropyl esters. 
The mechanism underlying the present invention is believed to be that the 
methionine compound acts in vivo to reduce the effect of release by 
polymorphonuclear leuckocytes (PMNs) of hypochlorous acid and other 
oxidants so that systemic oxidation, proteolysis, and tissue damage are 
inhibited. It is believed that the l-form of the methionine compound 
serves to fulfill its essential, recognized nutritional need whereas it is 
the d-form that has a previously unrecognized potent and different action 
at high dosage which is a well tolerated antiinflammatory activity. 
In trials where the antioxidant activity was compared with that of ascorbic 
acid (a known antioxidant substance), methionine, methionine sulfoxide, 
S-Methyl cysteine, and vitamin U (S-Methyl methionine), were mixed with an 
equimolar amount of vitamin C (ascorbic acid) and then titrated with 
sodium hypochlorite. Methionine was three times better (equimolar basis) 
than vitamin C as an antioxidant for HOCl. S-Methyl cysteine had about the 
same level of antioxidant activity as methionine. Methionine sulfoxide and 
vitamin U did not affect the oxidation of vitamin C by sodium hypochlorite 
(as measured by the decrease in ultravio let absorption at 270 
nanometers). Thus, neither S-methyl methionine nor methionine sulfoxide 
are suitable for reducing the effect of hypochlorous acid produced by PMNs 
(neutrophils). 
In contrast to vitamin C which has limited oral uptake of about 100 mg per 
day methionine can be elevated to quite high levels (up to 25 times normal 
levels) in the body by administration of methionine. Spaced administration 
of 1.5 grams of d-methionine results in a 3 fold increase in blood levels 
of methionine. Because d-methionine has a much longer half-life than 
l-methionine and because d-methionine is transported into the brain while 
most otherd-amino acids to not penetrate the blood-brain barrier, it is 
anticipated that when dl- or d-methionine is administered with other 
dietary antioxidants a synergistic effect of overall antioxidant effect 
will be seen. Synergistic antioxidant effects can be detected in humans 
with arthritis by measuring the reduction of blood levels of mixed 
cysteine/homocysteine disulfides. 
To the extent that conditions benefited by the consumption of dl-methionine 
are the result of a dietary deficiency of l-methionine it may be desirable 
to replenish methionine in food products as is currently done for a number 
of vitamins that are also made unavailable by food processing. The 
invention also employs methods for providing methionine in the final 
product for consumption in the amount that provides for replacement of 
unavailable methionine and additional methionine that would accomplish the 
teachings herein where it is desirable to obtain the additional 
antioxidant amount in a normal food item. 
Damico (U.S. Pat. No. 3,952,115) teaches the addition of N-acyl 
l-methionine as a preferred method to reduced undesired effects of 
methionine supplementation. He teaches that the amount of methionine to be 
added to methionine-deficient protein can be determined by amino acid 
analysis in the case of proteins known to be low in methionine content 
such that methionine should be added up to the level characteristic for 
egg protein (an amount recognized by the U.S. Food and Drug administration 
as the upper limit for addition of methionine for commercial foods). In 
the case of proteins for which methionine is lost by food processing such 
as extracted protein of soy bean he teaches that the amount of methionine 
to be added for proper nutrition involves adding methionine derivatives as 
determined by rodent feeding experiments. 
Because of the role that inflammatory cells play in long term tissue damage 
and because of the known dietary correlations of several serious 
inflammatory pain conditions that may be affected by reduced control of 
inflammatory cells, correction of a chronic marginal dietary deficiency of 
methionine and thus improved long term control of inflammatory cells can 
be expected to reduce the severity or incidence of these conditions. As an 
example of the possible contribution of reduced control of inflammatory 
cells, especially PMNs, that may be due to a marginal dietary deficiency 
of available methionine, it has been shown that products of stimulated 
PMNs can cause cellular transformation, a characteristic that has been 
associated with carcinogenesis (Weitzman, et. al., Science, 227: 1231-3, 
1985). Smokers that have been exposed to asbestos have very much higher 
lung cancer rates than exposed non-smokers, and it has been shown that 
smoking oxidizes a methionine residue in alpha1-protease inhibitor, thus 
allowing increased lung proteolysis. Other examples include arthritis and 
migraine headaches. 
It is found according to the invention that methionine, by its systemic 
antioxidant effect, especially d-methionine in humans and N-acetyl 
d-methionine in animals, systemically reduces the activity of immunocytes, 
especially polymorphonuclear neutrophils (PMNs). 
The best method to practice the teachings of described compounds depends on 
the particular conditions being treated and the compositions that are 
required to produce optimal results. In those cases where the methionine 
compound is l-, dl-methionine or a derivative of l- or dl-methionine the 
inclusion of homocysteine affecting amino acids and homocysteine affecting 
vitamins assures adequate conversion of homocysteine to cysteine or the 
methylation of homocysteine to methionine. In addition, in those cases 
where other dietary antioxidants may limit the total benefit to be derived 
from methionine compounds they should be provided with the methionine 
compounds. When methionine compounds are used in the upper portion of the 
dosage range dissolution of the compound in the stomach should be slowed. 
Also, individuals that are more sensitive to gastric upset should be 
provided with slow dissolving compositions to get effective relief. 
The method for treating ischemic or thrombotic disease response in a 
subject preferably comprises administering in the dosage form with the 
methionine compound at least one homocysteine reducing or remethylating 
compound sometimes referred to herein as a homocysteine affecting 
compound. The homocysteine affecting compound is at least one homocysteine 
affecting amino acid or homocysteine affecting vitamin selected from the 
group consisting of glycine, serine, vitamin B12, vitamin B6, and folic 
acid or folate, the compound being present in an amount sufficient to 
enable the systemic conversion of homocysteine to methionine or cysteine. 
The metabolic pathways for such conversion are detailed in: 
Biochemistry--A Case Oriented Approach, Montgomery, Dryer, Conway, 
Spector, eds., Mosby Co., London, IV Ed., 1983, p. 466-70; and: Fleisher 
and Gaull, Clinics in Endocrinology and Metabolism, 3:37-55, 1974; 
incorporated herewith by reference. Background for this is that methionine 
may have an adverse effect when given to subjects with vitamin B12 or 
folate deficiency. This effect is thought to be due to a buildup of 
systemic homocysteine; homocysteine is poorly remethylated in the absence 
or deficiency of vitamin B12 or folate. Also, the vitamin B6 level may be 
too low for the metabolism of homocysteine to cysteine by way of 
cystathionine. Thus, chronic comsumption of excess l-methionine, for 
example, may result in mild homocysteine elevation unless other co-factor 
substances are used or supplemented to stimulate the transformation of the 
excess homocysteine. The buildup is avoided, according to the invention, 
by including at least one homocysteine affecting compound in the dosage: 
vitamin B12 and folate to insure that homocysteine can be systemically 
remethylated to methionine; glycine or serine to insure that homocysteine 
can be reduced by way of cystathionine to cysteine; and vitamin B6 to 
insure that homocysteine can be metabolized to cysteine. The amino acids 
glycine and serine preferably are present in the serving or dosage in an 
amount from 1/5 to 3 times the amount of methionine compound. The 
nutrients, vitamins B12, B6 or folate preferably are present in the total 
daily dosage range of: B12, 0.3 to 30 micrograms; B6, 0.2 to 20 
milligrams; folate, 40 to 4000 micrograms; and combinations thereof. 
INFLAMMATORY ISCHEMIA AND THROMBOSIS 
In one preferred method aspect, the inventive concerns a method for 
inhibiting an inflammatory ischemic, reperfusion ischemic, or thrombotic 
disease response in a subject. Ischemia occurs as well in toxic shock 
syndrome and hypovolemic shock. The method comprises administering to the 
subject a composition in dosage form suitable for oral or intravenous 
administration comprising an effective antiinflammatory amount of at least 
one methionine compound as defined above, preferably at least 0.5 grams 
of methionine content per unit dose, preferably in dl-form, and a 
coagulant inhibitor in an anticoagulant effective amount, preferably at 
least one such inhibitor selected from the group including aspirin (an 
antiinflammatory), dipryidamole (a coronary vasodilator), sulfinpyrazone 
(for its art-recognized uricosuric effect), and dextran (e.g. Dextran 40, 
a blood flow promoter or adjuvant) or other similar art-recognized 
substances for anticoagulation, coronary dilation, uricosuric or blood 
flow promoter effects, and combinations thereof (as detailed, e.g., in The 
Nurses Guide to Drug Therapy, op. cit., p. 510-20, and incorporated 
herewith by reference). 
In another preferred aspect, the method employs a ischemia therapeutic 
antiinflammatory composition in unit dosage form suitable for oral or 
intravenous administration comprising an antiinflammatory effective amount 
of at least one methionine compound as defined above, and at least one 
member from the groups (a) through (e); (a) at least one homocysteine 
affecting amino acid, as defined above, in an amount sufficient to enable 
the systemic conversion, when consumed or administered of homocysteine to 
cysteine, (b) at least one homocysteine affecting vitamin as defined 
above, in an amount sufficient to enable the systemic conversion, when 
consumed or administered, of homocysteine to methionine or cysteine, (c) 
at least one coagulation inhibitor in a symptom relieving amount, as 
defined above, (d) at least one dietary antioxidant in a synergistically 
antioxidants effective amount selected from a group of dietary 
antioxidants including vitamins A,C,E, selenium, or zinc, where the total 
daily dosage range for each is: vitamin A, 500 to 50,000 IU; vitamin C, 1 
to 1000 mg; vitamin E, 1 to 150 IU; selenium, 1 to 200 mcg; zinc, 1 to 150 
mg; and combinations thereof; (e) an inactive excipient that provides 
insolubility in the stomach and solubility in the intestines or excipients 
that make the compounds suitable for systemic administration; and (f) 
combinations thereof. By using agents different in mechanism of action and 
agents having similar mechanisms of action, a synergistic antiinflammatory 
or anticoagulant effect can be expected. 
LIPID NORMALIZATION 
In one preferred method aspect, the invention concerns a method for 
inhibiting cholesterolemic disease response in a subject. The method 
comprises administering to the subject a composition in dosage form 
comprising an effective cholesterol lowering amount amount of at least one 
methionine compound as defined above, and the amino acids glycine or 
serine in a homocysteine lowering amount, as shown above. 
For example, in a case of subject exhibiting an elevated serum cholesterol 
level, it was found that oral administration of a composition containing 
methionine (dl-form) according to the invention for an extended daily 
regimen resulted in lowering the serum cholesterol level from 232 to 196 
mg/dl. In animal experiments where rats are fed large amounts of 
cholesterol, methionine raises blood cholesterol with 25% protein but 
lowers cholesterol on low protein diets. At all levels of protein the 
combination of glycine and methionine lowers cholesterol. However, in 
these animals the combination of methionine and glycine does not cause a 
lowering of triglycerides. (Sugiyama, Agric. Biol. Chem. 49: 3455-3461, 
1985). 
For normalizing cholesterol levels in a human, the methionine compound is 
administrated in a daily oral dose, in the range from 1.0 to 10 grams per 
70 kg of body weight until the cholesterol levels are normalized. The 
glycine or serine compound is coadministered in the range of 1/5 to 3 
times the amount of methionine compound until the homocysteine levels are 
normalized. 
In another preferred aspect, the method employs a dietary or therapeutic 
lipid normalizing composition in unit dosage form or serving, comprising a 
cholesterol normalizing amount of at least one methionine compound as 
defined above and at least one member from the groups (a) through (d); (a) 
at least one homocysteine affecting amino acid as defined above, in an 
amount sufficient to enable the systemic conversion of homocysteine to 
cysteine, (b) at least one homocysteine affecting vitamin as above, in an 
amount sufficient to enable the systemic conversion, when consumed or 
administered, of homocysteine to methionine or cysteine, (c) at least one 
anticholesterolemic such as cholestyramine, clofibrate, gemfibrozil, and 
nicotinic acid, preferably in a per se art-recognized unit dose amount, as 
defined above, (d) an inactive excipient that provides insolubility in the 
stomach and solubility in the intestines; and (e) combinations thereof. 
Because the mechanism of effect of methionine on lipid metabolism is 
different from other agents that lower blood cholesterol a synergistic 
lipid normalizing effect can be expected. 
To the extent that elevated blood cholesterol is the result of a dietary 
deficiency of methionine it may be desirable to replenish methionine in 
food products. The invention also concerns a method for providing 
methionine in the final product for consumption, in the amount of from 
more that 3 grams total methionine but less than about 15 grams methionine 
per 100 grams total protein. The invention also concerns a method of 
combining glycine or serine with methionine compound (n=2, dl- or l-) to 
be added to food products so as to avoid elevated homocysteine. 
PREATION OF PHARMACEUTICAL COMPOSITIONS 
When being utilized as pharmacological agents, the compounds of the 
invention can be prepared and administered in a wide variety of oral and 
parenteral dosage forms. It will be clear to those skilled in the art that 
the following dosage forms may comprise as the active component, one or 
more compounds of formula I, corresponding pharmaceutically acceptable 
salt of any of said compounds, or a mixture of such compounds and/or 
salts. 
For preparing pharmaceutical compositions from the compounds described by 
this invention, inert, pharmaceutically acceptable carriers can be either 
solid or liquid. Solid form preparations include powders, tablets, 
dispersible granules, capsules, cachets, and suppositories. A solid 
carrier can be one or more substances which may also act as diluents, 
flavoring agents, solubilizers, lubricants, suspending agents, binders, or 
tablet disintegrating agents; it can also be an encapsulating material. In 
powders, the carrier is a finely divided solid which is in admixture with 
the finely divided active compound. In the tablet the active compound is 
mixed with carrier having the necessary binding properties in suitable 
proportions and compacted in the shape and size desired. The powders and 
tablets preferably contain from 5 to about 70 percent of the active 
ingredients. Suitable solid carriers are magnesium carbonate, magnesium 
steareate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, 
tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low 
melting point wax, cocoa butter, and the like. The term "preparation" is 
intended to include the formulation of the active compound with 
encapsulating material as carrier providing a capsule in which the active 
component (with or without other carriers) is surrounded by carrier, which 
is thus in association with it. Similarly, cachets are included. Tablets, 
powders, cachets, and capsules can be used as solid dosage forms suitable 
for oral administration. 
Liquid form preparations include solutions, suspensions, and emulsions. As 
an example may be mentioned water or water-propylene glycol solutions for 
parenteral injection. Liquid preparations can also be formulated in 
solution in aqueous polyethylene glycol solution. Aqueous solutions 
suitable for oral use can be prepared by dissolving the active component 
in water and adding suitable colorants, flavors, stabilizing and 
thickening agents as desired. Aqueous suspensions suitable for oral use 
can be made by dispersing the finely divided active component in water 
with viscous material, i.e., natural or synthetic gums, resins, methyl 
cellulose, sodium carboxymethyl cellulose, and other well-known suspending 
agents. 
Topical preparations include dusting powders, creams, lotions, gels, and 
sprays. These various topical preparations may be formulated by well-known 
procedures. See for example Remington's Pharmaceutical Sciences, p. 
773-85, and 1585-602, 17th Ed., 1985, Mack Publishing Co., Easton, Pa. 
Preferably, the pharmaceutical preparation is in unit dosage form. In such 
form, the preparation is subdivided into unit doses containing appropriate 
quantities of the active component. The unit dosage form can be a packaged 
preparation, the package containing descrete quantities of preparation, 
for example, packeted tablets, capsules, and powders in vials or ampoules. 
The unit dosage form can also be a capsule, cachet, or tablet itself or it 
can be the appropriate number of any of these package forms. 
The quantity of active compound in a unit dose of preparation may be varied 
or adjusted from 100 to 1000 mg. according to the particular application 
and the potency of the active ingredient. 
In therapeutic use as pharmacological agents the compounds utilized in the 
pharmaceutical method of this invention are administered at the initial 
dosage of about 10 to about 50 mg per kilogram. A dose range of about 15 
mg to about 30 mg per kilogram is preferred. The dosages, however, may be 
varied depending upon the severity of the condition being treated, and 
compound being employed. Determination of the proper dosage for a 
particular situation is within the skill of the art. Generally, treatment 
is initiated with smaller dosages which are less than the optimum dose of 
the compound. Thereafter, the dosage is increased by small increments 
until the optimum effect under the circumstances is reached. For 
convenience, the total daily dosage may be divided and administered in 
portions during the day if desired. The dosage preferably is in a 
sustained or controlled release form (e.g. an enteric coated or slow 
release dosage form) to insure that the dosage is released in the 
intestine and a uniformly elevated blood level of the methionine compound 
is achieved, as described above. 
The active compounds may also be administered parenterally or 
intraperitoneally. Solutions of the active compound can be prepared in 
water suitably mixed with a surfactant such as hydroxypropylcellulose. 
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, 
and mixtures thereof and in oils. Under ordinary conditions of storage and 
use, these preparations contain preservatives to prevent the growth of 
microorganisms. 
The pharmaceutical forms suitable for injectable use include sterile 
aqueous solutions or dispersions and sterile powders for the 
extemporaneous preparation of sterile injectable solutions or dispersions. 
These various injectables may be formulated by well-known procedures. See 
for example Remington's Pharmaceutical Sciences, Chapter 86, 17th Ed., 
1985, Mack Publishing Co., Easton, Pa. In all cases the form must be 
sterile and must be fluid to the extent that easy syringability exists. It 
must be stable under the conditions of manufacture and storage and must be 
preserved against the contaminating action of microorganisms such as 
bacteria and fungi. The carrier can be solvent or dispersion medium 
containing, for example, water, ethanol, polyol (for example, glycerol, 
propylene glycol, and liquid polyethylene glycol, and the like), 
N,N-dimethylacetamide, suitable mixtures thereof and vegetable oils. The 
proper fluidity can be maintained, for example, by the use of a coating 
such as lecithin, by the maintenance of the required particle size in the 
case of dispersion and by the use of surfactants. The prevention of the 
action of microorganisms can be brought about by various antibacterial and 
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic 
acid, thimerosal, and the like. In many cases, it will be preferable to 
include isotonic agents, for example, sugars or sodium chloride. Prolonged 
absorption of the injectable compositions can be brought about by the use 
in the compositions of agents delaying absorption, for example, aluminum 
monostearate and gelatin. 
Sterile injectable solutions are prepared by incorporating the active 
compound in the required amount in the appropriate solvent with various of 
the other ingredients enumerated above, as required, followed by 
sterilization accomplished by filtering. Generally, dispersions are 
prepared by incorporating the various sterilized active ingredients into a 
sterile vehicle which contains the basic dispersion medium and the 
required other ingredients from those enumerated above. In the case of the 
sterile powders for the preparation of sterile injectable solutions, the 
preferred methods of preparation are vacuum drying and the freeze-drying 
technique which yield a powder of the active ingredient plus any 
additional desired ingredient from a previously sterilefiltered solution 
thereof. 
As used herein, "pharmaceutically acceptable carrier" includes any and all 
solvents, dispersion media, coatings, antibacterial and antifungal agents, 
isotonic and absorption delaying agents and the like. The use of such 
media and agents for pharmaceutically active substances is well-known in 
the art. Except insofar as any conventional media or agent is incompatible 
with the active ingredient, its use in the therapeutic compositions is 
contemplated. Supplementary active ingredients can also be incorporated 
into the compositions. 
It is especially advantageous to formulate parenteral compositions in unit 
dosage form for ease of administration and uniformity of dosage. Unit 
dosage forms used herein refers to physically discrete units suitable as 
unitary dosages for the mammalian subjects to be treated; each unit 
containing a predetermined quantity of active material calculated to 
produce the desired therapeutic effect in association with the required 
pharmaceutical carrier. The specification for the novel unit dosage forms 
of the invention are dictated by and directly dependent on (a) the unique 
characteristics of the active material and the particular therapeutic 
effect to be achieved, and (b) the limitation inherent in the art of 
compounding such an active material for the treatment of disease in living 
subjects having a disease condition in which bodily health is impaired as 
herein disclosed in detail. 
The principal active ingredient is compounded for convenient and effective 
administration in effective amounts with a suitable 
pharmaceutically-acceptable carrier in unit dosage form as hereinbefore 
disclosed. A unit dosage form can, for example, contain the principal 
active compound in amounts ranging from about 500 to about 10,000 mg, with 
from about 1,000 to about 2,000 mg being preferred. Expressed in 
proportions, the active compound is generally present in from about 50 to 
about 500 mg/ml of carrier. In the case of compositions containing 
supplementary active ingredients, the dosages are determined by reference 
to the usual dose and the manner of administration of the said 
ingredients. The daily parenteral doses for lower mammalian subjects to be 
treated ranges from about 1 to 100 mg/kg. The preferred daily dosage range 
is about 2 to 20 mg/kg. 
The invention and the best mode of practicing the same are illustrated by 
the following examples of preferred embodiments of selected compounds and 
their preparation. 
EXAMPLE 1 
______________________________________ 
CAPSULES 
Example 1a 
-d-Methionine 
100 mg, 250 mg or 500 mg 
______________________________________ 
-d-Methionine 500 g 
Lactose USP, Anhydrous q.s. or 
200 g 
Sterotex Powder HM 5 g 
______________________________________ 
Combine the methionine and the Lactose in a twinshell blender equipped with 
an intensifier bar. Tumble blend for two minutes, blend for one minute 
with the intensifier bar and then tumble blend again for one minute. A 
portion of the blend is then mixed with the Sterotex Powder, passed 
through a #30 screen and added back to the remainder of the blend. The 
mixed ingredients are then blended for one minute, blended with the 
intensifier bar for thirty seconds and tumble blended for an additional 
minute. Appropriate sized capsules are filled with 141 mg., 352.5 mg., or 
705 mg. of the blend, respectively, for the 100 mg., 250 mg., and 500 mg. 
containing capsules. 
______________________________________ 
Example 1b 
.sub.-- dl-Methionine 
100 mg, 250 mg or 500 mg 
______________________________________ 
.sub.-- dl-Methionine 
500 g 
Lactose USP, Anhydrous q.s. or 
200 g 
Sterotex Powder HM 5 g 
______________________________________ 
Mix and fill as per Example 1a. 
EXAMPLE 2 
______________________________________ 
TABLETS 
______________________________________ 
The Methionine Compound 250 g 
Corn Starch NF 200 g 
Cellulose, Microcrystalline 
46 g 
Sterotex Powder HM 4 g 
Purified Water q.s. 300 ml 
______________________________________ 
Combine the corn starch, the cellulose and the methionine compound together 
in a planetary mixer and mix for two minutes. Add the water to this 
combination and mix for one minute. The resulting mix is spread on trays 
and dried in a hot air oven at 50 degrees C. until a moisture level of 1 
to 2 percent is obtained. The dried mix is then milled with a Fitzmill 
through a #RH2B screen at medium speed. The Sterotex Powder is added to a 
portion of the mix and passed through a #30 screen, and added back to the 
milled mixture and the total blended for five minutes by drum rolling. 
Compressed tablets of 100 mg., 500 mg., and 1000 mg. respectively, of the 
total mix are formed with appropriate sized punches for the 50 mg., 250 
mg., or 500 mg. containing tablets. 
EXAMPLE 3 
______________________________________ 
SUPPOSITORIES 
Example 3a 
-d-Methionine 
125 mg, 250 mg, or 500 mg per 3 G 
______________________________________ 
.sub.-- dl-Methionine 
125 mg 250 mg 500 mg 
Polyethylene Glycol 
1925 mg 1750 mg 1400 mg 
1540 
Polyethylene Glycol 
825 mg 750 mg 600 mg 
8000 
______________________________________ 
Melt the Polyethylene Glycol 1540 and the Polyethylene Glycol 8000 together 
at 60 degrees C. and dissolve dl-Methionine into the melt. Mold this total 
at 25 degrees C. into appropriate suppositories. 
______________________________________ 
Example 3b 
.sub.-- dl-Methionine 
125 mg, 250 mg, or 500 mg per 3 G 
______________________________________ 
.sub.-- dl-Methionine 
125 mg 200 mg 500 mg 
Polyethylene Glycol 
1925 mg 1750 mg 1400 mg 
1540 
Polyethylene Glycol 
825 mg 750 mg 600 mg 
8000 
______________________________________ 
Prepare as per Example 3a above. 
EXAMPLE 4 
Preparation of Intravenous Formulations 
A solution of from 25 to 50 grams of dl-Methionine is prepared in 1 liter 
of water for injection at room temperature with stirring. The solution is 
sterile filtered into 500 5-ml vials, each of which contains 2 ml of 
solution containing 50 to 100 mg of compound, and sealed under nitrogen. 
In addition, other desired active ingredients, such as those listed above 
for preferred tablet formulations, may be added in appropriate unit dosage 
amounts before the solution is sterile filtered. 
Alternatively, after sterile filtration into vials, the water may be 
removed by lyophilization, and the vials then sealed aseptically, to 
provide a powder which is redissolved in the desired unit dosage 
concentration prior to injection. 
In another preferred aspect, the method employs a ischemia therapeutic 
antiinflammatory composition in unit dosage form suitable for oral or 
intravenous administration comprising an antiinflammatory effective amount 
of at least one methionine compound as defined above, and at least one 
member from the groups (a) through (e); (a) at least one homocysteine 
affecting amino acid from the group of glycine and serine, in an amount 
sufficient to enable the systematic conversion, when consumed or 
administered, of homocysteine to cysteine, (b) at least one homocysteine 
affecting vitamin in an amount sufficient to enable the systemic 
conversion, when consumed or administered, of homocysteine to methionine 
or cysteine, selected from the group consisting of vitamins B12, B6, and 
folic acid where the total daily dosage range for each is: vitamin B12, 
0.3 to 30 mcg; vitamin B6, 0.2 to 20 mg; folic acid, 40 to 4000 mcg, and 
combinations thereof; (c) at least one coagulation inhibitor in an 
anticoagulant effective amount, preferably selected from the group 
including aspirin (an antiinflammatory), dipryidamole (a coronary 
vasodilator), sulfinpyrazone (for its art-recognized uricosuric effect), 
and dextran (e.g. Dextran 40, a blood flow promoter or adjuvant) or other 
similar art-recognized substances for anticoagulation, coronary dilation, 
uricosuric or blood flow promoter effects, and combinations thereof; (d) 
at least one dietary antioxidant in a synergistically antioxidant 
effective amount selected from a group of dietary antioxidants including 
vitamins A,C,E, selenium, or zinc, where the total daily dosage range for 
each is: vitamin A, 500 to 50,000 IU; vitamin C, 1 to 1000 mg; vitamin E, 
1 to 150 IU; selenium, 1 to 200 mcg; zinc, 1 to 150 mg; and combinations 
thereof; (e) an inactive excipient that provides insolubility in the 
stomach and solubility in the intestines or excipients that make the 
compounds suitable for systemic administration; and (f) combinations 
thereof. By using agents different in mechanism of action and agents 
having similar mechanisms of action, a synergistic antiinflammatory or 
anticoagulant effect can be expected.