JP2005112835A - Method for producing new medicinal composition and processed food for treating hypertension - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To create a new hypotensive drug based on a new mechanism of action. <P>SOLUTION: By using rats as model animals, the administration of lactoferrin significantly lowers blood pressure dose dependently without affecting the number of heart beats. This hypotensive effect of the lactoferrin which is a part of researches of its physiological activities, is a new finding which has not been described in any of research reports and patent literatures associated with the lactoferrin, and is useful for the blood pressure control of hypertensive patients. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Detailed Description of the Invention

  The present invention relates to a novel pharmaceutical composition for treating hypertension comprising lactoferrin as an active ingredient and a method for producing a novel processed food.

  Hypertension is the largest risk factor for stroke and is also important as a cause of various cardiovascular diseases such as ischemic heart disease, nephrosclerosis, and obstructive arteriosclerosis. The number of hypertensive patients in Japan is 33 million, and in order to reach a healthy retirement with a high quality of life (QOL) at the present time of the aging society, hypertension aimed at preventing severe cardiovascular disease Prevention and treatment are extremely important issues. The number of patients with hypertension is very large, but in fact most patients do not know the cause. Hypertension, for which there is no obvious cause of increased blood pressure in current medical examinations, is called essential hypertension and accounts for more than 95% of hypertension. In other words, it is a case where the cause of hypertension is not found other than genetic predisposition and non-consumption in daily life (excess salt intake, stress, lack of exercise, excessive drinking, obesity, etc.). On the other hand, the remaining few percent have been found to cause blood pressure as a result of clinical tests, and such hypertension is called secondary or symptomatic hypertension.

  In general, if both parents have essential hypertension, about half of the children will have essential hypertension. Genetic predisposition and non-perturbation in daily life are thought to contribute approximately half each to the development of essential hypertension. However, there are large individual differences in the degree of contribution, and the involvement of genetic predisposition may be overwhelmingly large. On the other hand, the involvement of daily life is large, and blood pressure may be completely normalized only by lifestyle improvement. is there.

  Since blood pressure increases with age, and the above-mentioned insomnia of daily life also increases blood pressure, essential hypertension generally develops after middle age, and diabetes and impaired glucose tolerance, one step before that, Alternatively, it is known to accompany adult diseases such as hyperlipidemia, abnormal lipid metabolism, obesity and the like with high frequency. Patients with essential hypertension are about twice as likely to have diabetes compared to those with normal blood pressure, and conversely, those with diabetes are about twice as likely to have hypertension compared to non-diabetics. For these reasons, the genetic predisposition for essential hypertension and the genetic predisposition for diabetes have been considered to be partly common. In addition, it has been pointed out that these predispositions may be associated with predisposition to hyperlipidemia, lipid metabolism abnormality and obesity. In fact, there are many patients with multiple adult diseases such as hypertension, diabetes, obesity, and hyperlipidemia. However, patients with several of these adult diseases have high blood pressure, diabetes, hyperlipidemia, etc. The degree of adult illness is usually not so severe. For this reason, in medical examinations, etc., it is only necessary to be careful and may not necessarily be pointed out as “necessary medical care”. However, it is very dangerous to have multiple of these adult diseases even in mild cases, and the risk of myocardial infarction and stroke is increased several times. For this reason, the case of having these adult diseases together is called multiple risk factor syndrome, also known as “syndrome X syndrome” or “death quartet”, and is very fearful.

  If current medical tests find obvious abnormalities that raise blood pressure, it is called secondary (symptomatic) hypertension. One of them is called renal hypertension, and when the function of the entire kidney decreases due to glomerulonephritis, chronic pyelonephritis, cystic kidney, etc., the function of the renal pelvis, which excretes salt (sodium) and water, decreases and blood pressure increases. . In addition, when blood vessels (renal arteries) that supply blood to the renal pelvis become narrow due to arteriosclerosis, inherent blood vessel abnormalities, or inflammation of the arteries, a substance (renin) that increases blood pressure is released from the kidneys, resulting in hypertension. As a result, blood will flow sufficiently through the kidney, but blood pressure will be too high in other instruments.

  The other is called endocrine hypertension, which occurs due to abnormal secretion of hormones. There are several types of hormones that act on the heart, blood vessels, and kidneys to maintain blood pressure and send blood to important organs to prevent dizziness. However, hypertension develops when these hormones are secreted in excess. Typical diseases include primary aldosteronism, which is caused by excessive secretion of aldosterone, a hormone that maintains the blood pressure by acting on the kidneys and storing salt (sodium) and water in the body, and acts on the heart and blood vessels. There are pheochromocytomas that cause hypertension due to excessive secretion of catecholamines (adrenaline, noradrenaline), hormones that increase blood pressure. However, unlike essential hypertension, these diseases can be cured relatively easily by surgery.

  In Japan, including mild cases, it is considered that about half of the elderly people over the age of 60 have hypertension. The purpose of hypertension treatment is to prevent serious complications resulting from high blood pressure such as stroke, myocardial infarction and cardiac hypertrophy. There are standards around the world on how much blood pressure should be lowered for this purpose. If these are judged comprehensively, it is considered that treatment is necessary when the systolic blood pressure is 140 mmHg or more or the diastolic blood pressure is 90 mmHg or more. However, for younger people under 40 years of age and patients with diabetes that also causes stroke or myocardial infarction, treatment is required if the criteria are a little more severe, that is, systolic blood pressure is 130 mmHg or higher, or diastolic blood pressure is 85 mmHg or higher. is there.

  It is necessary to review lifestyle in order to treat / prevent hypertension. This is because risky hypertension may be resolved by cutting the risk factors that cause high blood pressure. Treatment of hypertension begins with diet and exercise therapy, as do lifestyle-related diseases such as hyperlipidemia, except for those who need to lower blood pressure urgently. The point of diet therapy is salt restriction, calorie restriction, alcohol restriction, etc. The period and degree are determined according to the severity of hypertension. In exercise therapy, so-called aerobic exercise such as walking, jogging, swimming, cycling, etc. is regarded as a preferable exercise for high blood pressure. During exercise, the blood pressure increases temporarily due to the action of the sympathetic nerve, but it is reported that prostaglandins and taurine, which have the effect of lowering blood pressure by continuing exercise, increase, while catecholamines that increase blood pressure decrease. ing.

  In modern society, life without stress is unthinkable. When excessive stress (especially mental stress) or sudden stress is applied, the sympathetic nerves are overstimulated, blood pressure is surprisingly increased, and it is not uncommon for strokes and heart attacks to occur. . Even if it is not an abrupt stress, chronic sustaining stress adversely affects high blood pressure because the excited state of the sympathetic nerve lasts. It is important to acquire stress relief methods such as exercise and hobbies so as not to accumulate stress.

  If the effect is insufficient even after continuing diet and exercise therapy, or if it is necessary to lower blood pressure as soon as possible, treatment with antihypertensive drugs is given. Antihypertensive drugs are not drugs that cure high blood pressure, but are drugs that prevent arteriosclerosis, stroke, heart disease, etc. through blood pressure control. It should not be reduced. Also, just because you are taking medicine, you can't neglect diet and exercise therapy.

  There are five types of therapeutic agents for hypertension, such as calcium antagonists, angiotensin (ACE) converting enzyme inhibitors, angiotensin II (A-II) receptor antagonists, diuretics, and sympathetic nerve inhibitors. Calcium antagonists prevent calcium from entering the smooth muscle cells that make up peripheral blood vessels, dilate blood vessels and lower blood pressure. ACE inhibitors prevent the increase in blood pressure by suppressing the production of A-II that increases blood pressure. The A-II receptor antagonist specifically binds to the A-II receptor, and suppresses the vasoconstriction, fluid retention, and sympathetic excitation that are the effects of A-II. Diuretics act on the kidneys to increase the excretion of sodium and water, and lower blood pressure by reducing fluid volume and circulating blood volume. There are two types of sympathomimetic drugs, alpha blockers and beta blockers. Alpha blockers reduce peripheral blood vessel contraction and lower blood pressure by dilating blood vessels. Beta blockers lower blood pressure by reducing heart rate and cardiac output.

Problems to be solved by the invention

  Antihypertensive drugs vary depending on the type of drug, but side effects such as headache, flushing / hot flashes, malaise / weakness, palpitation, decreased sexual function, and dry cough may occur. Recent antihypertensive drugs are generally said to have few side effects, but antihypertensive drugs require lifelong use, so drugs with fewer side effects and higher antihypertensive effects are desirable. In addition, drugs that exhibit antihypertensive effects with a different mechanism of action from conventional antihypertensive drugs can be valuable alternative treatments for hypertensive patients if existing antihypertensive drugs cannot be used for drug resistance or other reasons. Therefore, new antihypertensive drugs based on new mechanisms of action are the targets of active research worldwide, and biomedical research institutes are fiercely developing. The problem to be solved by the present invention is the creation of a novel antihypertensive drug based on a new mechanism of action.

Means for solving the problem

  In the course of the study to solve the above problems, the present inventors have found that when rats are used as model animals and lactoferrin is administered under anesthesia, the blood pressure is significantly reduced in a dose-dependent manner. This blood pressure lowering action, which is part of the research on the physiological action of lactoferrin, is a new finding that has not been described so far in research papers and patents on lactoferrin.

Lactoferrin is a glycoprotein having a molecular weight of about 80,000 daltons present in mammalian milk, neutrophil secretory granules, tears, saliva, and various mucus. It has the property of chelating two trivalent iron ions in the molecule, and its antibacterial activity is thought to deprive the trivalent iron ion from the growth environment of pathogenic bacteria and make it unavailable for growth. Lactoferrin is a multifunctional protein that exhibits physiologically various actions, antibacterial action so far (e.g. Weinberg, E.D.1995, Acquisition of iron and other nutrients in vivo.Virulence mechanism of bacterial pathogens, 2 nd ed , Edited by JA Roth et al., American Society for Microbiology, Washington D.C. pp. 79-93), antiviral activity (Andersen, J.H., Osbak, S.A., L. and Lor. H., Traavik, T., and Guttenberg, TJ 2001, Lactoferrin and cy. lic lacoferricin inhibit the entry of human fibroblasts. Antivirus Res. 51: 141-149., carcinogenesis-preventing action (Tsuda. Y., Sekine, K., Fujita, K.i., and I200. prevention by bovine lactoferrun and underlying mechanisms --- a review of experimental and clinical studies.Biochem.Cell Biol.80.131-136.), immunostimulation. : A general review.Ha ematologica, 80: 252-267), lipid metabolism improving action (2003. JMS. 81: pp64-67.), analgesic action (Hayashida K., Takeuchi. T., Shimizu, H., Ando. K., and) Harada.E.2003.Lactoferrin enhances opioid-mediated analgesia via native oxide in the spinal cord.Am.J.Physiol.Regul.Comp.R.

Already, the present inventors have shown that lactoferrin (1) has an analgesic effect on rodents, (2) its analgesic effect is counteracted by morphine antagonist naloxone, and (3) has no analgesic effect. without a small amount of lactoferrin in combination with morphine when also enhance 100x analgesia, inhibited by (4) analgesic effects are nitric oxide synthase inhibitor ^{N G -nitro-L-arginine methylester} hydrochloride (L-NAME) but is the, that it is not inhibited in optical isomer ^{N G -nitro-D-arginine methylester} hydrochloride (D-NAME), (5) μ- opioid receptor endorphins showing the analgesic effect bind to body, intrinsic properties such as enkephalin Analgesic peptide (6) When lactoferrin and morphine are continuously instilled into rats and compared with the analgesic effect over time, morphine develops drug resistance within 72 hours and is analgesic. It has been reported that the analgesic action of lactoferrin lasts for a long time without causing drug resistance, while the effect is lost. From the above experimental results, it has been clarified that the analgesic effect of lactoferrin is to increase the production of nitric oxide in ganglia that transmit pain and enhance the effect of endogenous analgesic peptides.

  Endogenous analgesic peptides are known to have an analgesic effect as well as euphoria and sensation similar to those of morphine, and sedate stress. Since one of the causes of hypertension is sympathetic nerve tension / excitement caused by stress, the present inventors have developed sympathetic nerve tension by enhancing the action of endogenous analgesic peptides in vivo. • Estimated that it would calm the excitement and improve hypertension. In fact, as evidence that lactoferrin acts on the cardiovascular system, it is known that its receptor is also expressed in the heart and vascular system (Faucheux. BA, Nillesse. N., Damier. P). , Spik.G., Mouatt-Privent.A., Pierce.A., Levegle.B., Kubis.N., Hauw.J.J., Acid.Y., and et al., 1995. Expression of lactoferrin. (receptors is increased in the mesencephalon of patents with Parkinson disease. Proc. Natl. Acad. Sci. USA. 92: 9603-9607.).

It became clear that it became clear using the thoracic aorta excised from rats and rats as follows. (1) Lactoferrin lowers the blood pressure of rats in a dose-dependent manner (2) Relaxes blood vessels in a vascular endothelium-dependent manner (3) The vasorelaxant effect is because lactoferrin increases nitric oxide production in vascular endothelial cells. Yes, (4) On the other hand, lactoferrin acts on the central nerve and shows a central antihypertensive action depending on the endogenous peptide.

Embodiment of the Invention

    The embodiments of the present invention are mainly oral preparations and injections, but can also be put to practical use in dosage forms such as suppositories, patches, drops, gargles, eye drops, and troches. Lactoferrin is a polymer having a molecular weight of around 80,000 daltons, and is relatively unstable. In the stomach, lactoferrin is easily degraded by pepsin under acidic conditions. For example, an oral preparation is preferably a preparation in which the surface of a lactoferrin granule or tablet is coated with an enteric film that does not dissolve in the stomach but flows into the intestine and dissolves. More specifically, it is as follows. The milk-derived lactoferrin freeze-dried powder has a very low specific gravity and is difficult to compress directly into tablets. It is possible to produce a product by covering the granules produced by the dry method with an enteric coating and filling a certain amount into a hard capsule. In the case of commercialization as a tablet, a lubricant can be added to the granule for tableting, and the tablet can be covered with an enteric coating for commercialization.

Examples of excipients used in producing the enteric preparation of the present invention include lactose, sucrose, maltitol, glucose and other monosaccharides or disaccharides, corn starch, starches such as potato starch, crystalline cellulose, and inorganic substances. Examples include light silica gel, synthetic aluminum silicate, magnesium aluminate metasilicate, and calcium hydrogen phosphate. Examples of the binder include starches, carboxymethyl cellulose (CMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose-sodium salt, and polyvinylpyrrolidone. Examples of disintegrants used for the purpose of disintegrating them into primary particles in the lower gastrointestinal tract include starch, carboxymethylcellulose-sodium salt, carboxymethylcellulose-calcium salt, croscarmellose sodium, and carboxymethylstarch sodium. . As a film agent for enteric coating of tablets and grains, hydroxypropyl methylcellulose phthalate, carboxymethylethylcellulose, cellulose acetate phthalate, methacrylic acid copolymer, zein contained in corn kernel And shellac that dissolves in the alkaline 'region.

The preparation of the present invention generally contains about 0.1 mg to about 100,000 mg, preferably about 1 mg to about 50,000 mg, more preferably about 10 mg to about 10,000 mg once a day as an active ingredient. Or in divided portions can be administered to patients in need of treatment or improvement of condition with the formulations of the invention. The dosage and dosage form can be individually determined according to the age, weight, pathological condition and purpose of administration of the patient to be administered. Examples of lactoferrin include bovine lactoferrin extracted and purified from milk, and genetically modified lactoferrin produced by microorganisms that have been modified with human and other mammalian lactoferrin genes.

    Example 1; Wistar Imamichi male rats weighing 8-9 weeks and weighing 250-330 g were used in the experiments. The rats were inserted with a catheter into the transvenous vein as a drug administration route while maintaining temperature to maintain body temperature under anesthesia with urethane, and a catheter was inserted into the carotid artery for blood pressure measurement. Bovine serum albumin (1500 nmol / kg) or bovine lactoferrin (38.4-1280 nmol / kg) was administered intravenously via a jugular vein catheter. One group contains 5-6 rats. Blood pressure was recorded immediately before administration and maximum blood pressure drop. The maximum decrease in blood pressure is shown 10 seconds after administration with morphine and 90 seconds with lactoferrin. FIG. 1 shows blood pressure (A) and blood pressure change (B) compared to immediately before administration. As is apparent from the figure, lactoferrin lowered blood pressure in a dose-dependent manner.

    Example 2: Wistar Imamichi male rats weighing 8-9 weeks and weighing 250-330 g were used. The rats were inserted with a catheter into the transvenous vein as a drug administration route while maintaining temperature to maintain body temperature under anesthesia with urethane, and a catheter was inserted into the carotid artery for blood pressure measurement. When morphine (133 nmol / kg) or bovine lactoferrin (384 nmol / kg) was administered intravenously, mean blood pressure (FIG. 2A) and heart rate (FIG. 2B) were changed. Six rats were used in the morphine group and seven rats in the lactoferrin group, and the average blood pressure and heart rate are shown as mean ± standard error. As is apparent from the figure, morphine rapidly decreased in heart rate and blood pressure rapidly within 10 seconds after administration, and after that period, blood pressure and heart rate tended to return to pre-dose values. In contrast, the lactoferrin group had no effect on heart rate, and only the blood pressure tended to decrease gradually.

    Example 3 FIG. 3 shows the effect of opioid antagonists, naloxone hydrochloride and naloxone methionide on the antihypertensive effect of lactoferrin and morphine. Morphine (133 nmol / kg) to bovine lactoferrin (384 nmol / kg) were intravenously administered to a rat model prepared in the same manner as in Example 1. For each group, 7 rats were used in the morphine group and 6-7 rats in the lactoferrin group. Naloxone hydrochloride (NLX-H) and naloxone methionide (NLX-M) were also administered simultaneously with lactoferrin or morphine. Intravenously administered naloxone hydrochloride is taken up into the brain across the blood brain barrier, but naloxone methionide does not cross the blood brain barrier. As is apparent from the figure, the antihypertensive effect of lactoferrin is canceled by NLX-H administration but not by NLX-M administration. It was found that the antihypertensive effect of morphine was counteracted by both antagonists. In other words, the action of morphine is clearly peripheral, whereas the central nervous system is involved in the antihypertensive effect of lactoferrin.

Example 4 FIG. 4 shows the effect of the nitric oxide synthesis system on the antihypertensive effect of lactoferrin using the same animal model as in Example 1. FIG. 4A shows the average blood pressure, and FIG. 4B shows the change rate of the average blood pressure in percent. The respective intravenous doses are 384 nmol / kg for lactoferrin, 100 ng / kg for acetylcholine, and 133 nmol / kg for morphine. ^{N G -nitro-L-arginine methyl} ester (L-NAME) and ^{N G -nitro-D-arginine methylester} (D-NAME) were administered intravenous at the same time 50 mg / kg, respectively. These are inhibitors of nitric oxide (NO) synthesis. L-NAME completely counteracted the antihypertensive effect of lactoferrin and significantly increased blood pressure compared to the L-NAME non-administered control. When acetylcholine and morphine were administered at the same time as L-NAME, the increase in blood pressure was significantly suppressed compared to the non-administration control group, but the blood pressure in the L-NAME administration group significantly increased beyond the blood pressure of the non-administration control. However, acetylcholine and morphine show an action to suppress the pressurization due to inhibition of NO synthesis, but do not show an antihypertensive effect. On the other hand, the optical isomer D-NAME that does not inhibit NO synthesis did not inhibit the antihypertensive effect of lactoferrin at all.

    Example 5: FIG. 5 shows the effect of atropine, an apocalaric acetylcholine receptor antagonist, on the antihypertensive effects of lactoferrin and acetylcholine. The animal experiment system used was the same as in Example 1. The antihypertensive effect of lactoferrin is not affected by simultaneous administration of atropine, but acetylcholine, an agonist of muscarinic acetylcholine receptor, completely loses the antihypertensive effect in the presence of atropine. Therefore, it can be concluded that lactoferrin is not a muscarinic acetylcholine receptor agonist antihypertensive.

    Example 6: A rat thoracic aorta was cut to a length of about 5 mm and immersed in a 37 ° C. Krebs-Ringer solution bubbled with a mixed gas consisting of 95% oxygen and 5% carbon dioxide gas. As a result, it was revealed that lactoferrin relaxes the rat thoracic aorta (FIG. 6). This relaxation action is as strong as acetylcholine. Since lactoferrin does not relax the blood vessels in the arterial ring from which the vascular endothelium has been detached, it is certain that the vasorelaxing action of lactoferrin is mediated by vascular endothelial cells. Note that diclofenac, which is a potent prostaglandin biosynthesis inhibitor, does not inhibit the relaxation of blood vessels by lactoferrin, so the action of lactoferrin is not expressed through the promotion of biosynthesis of prostaglandins.

    Example 7 shows the effect of nitric oxide synthesis inhibitor and ATP-sensitive potassium channel inhibitor on rat aorta relaxation induced by lactoferrin. The vasorelaxant action of acetylcholine is completely inhibited in the presence of the NO synthesis inhibitor L-NAME. The vasorelaxant action of lactoferrin is also completely inhibited by L-NAME at any lactoferrin concentration, but is not affected at all in the presence of D-NAME. Therefore, relaxation of blood vessels by lactoferrin is an action mediated by NO. On the other hand, vasodilation by the vasodilator pinacidil is completely inhibited by glibenclamide, an ATP-sensitive potassium channel inhibitor, but it is observed in a dose-dependent manner in the presence of glibenclamide. It became clear that channel opening was not involved.

The invention's effect

    As explained above, lactoferrin acts on the central nervous system to lower blood pressure, while increasing NO production in the peripheral vascular endothelium relaxes arterial smooth muscle and lowers blood pressure by expanding the blood vessel lumen. . Lactoferrin extracted from milk is so safe that it is ideal for the treatment of hypertensive patients who need antihypertensive medication for life.

  Effect of lactoferrin on mean blood pressure in rats   Changes in rat mean blood pressure and heart rate induced by intravenous administration of lactoferrin and morphine   Effects of opioid antagonists on antihypertensive effects of lactoferrin and morphine   Effects of nitric oxide biosynthesis inhibitors on the antihypertensive effects of lactoferrin, acetylcholine and morphine   Effects of muscarinic acetylcholine receptor antagonists on the antihypertensive effects of lactoferrin and acetylcholine   Lactoferrin-induced relaxation of rat thoracic aorta.   Effects of nitric oxide biosynthesis inhibitors and ATP-sensitive potassium ion channel blockers on lactoferrin-induced relaxation of rat thoracic aorta

Explanation of symbols

FIG.

Pre: Before administration of lactoferrin and control bovine serum albumin Post: After administration of lactoferrin and control bovine serum albumin MBP (mmHg): Mean blood pressure of rats (mean value ± standard error)
BSA: bovine serum albumin BLF: lactoferrin extracted from milk P <0.05, P <0.005: statistically significant difference tested by student t-test Change in MBP (%): change in mean blood pressure expressed as a percentage rate

FIG.

○ Solid line; Morphine group ▲ Black square ▼ Solid line; Lactoferrin group A: Change in mean blood pressure, B: Change in heart rate

FIG.

NLX-H: naloxone hydrochloride NLX-M: naloxone methionide * P <0.05 and *** P <0.001, in Student t-test

FIG.

Ach: acetylcholine ^{L-NAME: N G -nitro-} L-arginine methyl ester
^{D-NAME: N G -nitro-} D-arginine methyl ester

FIG.

Atropin: Atropine

FIG.

SNP: nitroprusside sodium salt E (+): with vascular endothelium E (-): vascular endothelium detachment

FIG.

Gli: Glibenclamide (oral diuretic)
Pin: Pinacidil (vasodilator)

Claims (2)

Novel pharmaceutical composition for treating / preventing hypertension and novel processed food containing lactoferrin as an active ingredient Lactoferrin, calcium salt, potassium salt, vitamin C, vitamin E, garlic and its extract, ginkgo biloba and its extract, and ω-3 unsaturated fatty acids such as γ-linolenic acid, eicosapentadecanoic acid, docosahexaenoic acid, Alternatively, a novel pharmaceutical composition and processed food for hypertension treatment / prevention characterized by mixing one or more selected from fish oil, primrose oil, linseed oil and the like containing ω-3 unsaturated fatty acid

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