WO2009046631A1 - Pharmaceutical composition containing angiotensin converting enzyme inhibitor and b family vitamins and use thereof - Google Patents

Abstract

Pharmaceutical composition contains angiotensin converting enzyme inhibitor (ACEI) and B family vitamins and the use of the composition in treating hyperhomocysteinemia are provided. The combination of ARB and B family vitamins can act on multiple routes and links of hyperhomocysteinemia, reduce the level of homocysteine and inhibit the organism damage effectively caused by elevated level of homocysteine and is better than the single drug, which reavels the significantly synergetic effect of angiotensin II receptor blocker and B family vitamins on the relief of the damage caused by elevated level of homocysteine and the reduction of elevated level of homocysteine.

Description

 Pharmaceutical composition containing angiotensin converting enzyme inhibitor and B vitamin and use thereof

 The present invention relates to the treatment of hyperhomocysteinemia. In particular, the present invention relates to a pharmaceutical composition comprising an angiotensin converting enzyme inhibitor (ACEI) and a B vitamin and its use for the treatment of hyperhomocysteinemia. Background technique

 As early as 1969, people hypothesized that homocysteine (Hcycysteine, Hcy) may be a risk factor for cardiovascular disease. Since then, a large number of studies have accumulated objective evidence that plasma Hcy levels and cardiovascular disease (Cadiovascular disease, CVD) is closely related to the risk of other systemic diseases.

 Ye Deshou et al [Clinical Cardiology Journal, 2005; 21: 536-538] showed that the level of Hcy in Chinese patients with essential hypertension was significantly higher than that in the control group (PO.05): the incidence of high Hcy in the primary hypertension group (Phemia Hcy concentration >15 μΐΊΐοΙ/L) was 36.6%, and the control group was 21.6%. The difference was 15%. The difference was statistically significant (P<0.05), indicating that Hcy was closely related to hypertension. At the same time, plasma Hcy levels can be independent of traditional risk factors such as blood pressure and hyperlipidemia. The increase in plasma Hcy is 5 μπιοΙ/L, which increases the risk of stroke by 59% (OR (odds ratio), 1.59; 95% CI (contribution) Interval), 1.30-1.95), 60% increased risk of deep vein thrombosis (OR, 1.60; 95% CI, 1.15-2.22), ischemic heart disease (including coronary heart disease, angina pectoris, arrhythmia, myocardial infarction, heart failure) And sudden death) increased risk by 33% (OR, 1.33; 95% CI, 1.22-1.46); Hcy level decreased by 3 nd/L, stroke risk decreased by 24%, deep vein thrombosis risk decreased by 25%, ischemic heart disease The risk is reduced by 16%. It indicates that Hcy is closely related to ischemic heart disease, stroke, and deep vein thrombosis [Wald DS, et al. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ. 2002; 325: 1202-1206.] .

 In addition to its important role in the development of CVD, high Hcy is associated with other diseases such as Alzheimer's disease (AD), dementia, cognitive dysfunction, osteoporosis, fracture, depression, Parkinson's disease (PD). , schizophrenia, death, neonatal defects, habitual abortion and other diseases are closely related.

Since the 1990s, a large number of reports have suggested that high plasma Hcy is associated with decreased brain function and the development of AD. In 1998, Clarke et al. diagnosed 164 patients over 55 years old. A controlled study was performed in patients with AD and 148 elderly people of similar age [Clarke R, et al. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol. 1998; 55: 1449-1455.], The results showed that the Cambridge Cognitive Examination (CAMCOG) score and the mini-mental state examination (MMSE) scores of AD patients were significantly lower than those of the control group, while plasma Hcy levels were significantly increased; The most characteristic feature of the study was that 76 patients in the case had histologically confirmed after death. The difference in Hcy concentration between the AD patients after the diagnosis and the control was more significant than that between the 164 clinically diagnosed patients and the control. Seshadri S et al. conducted a prospective trial of 1,092 volunteers with an average age of 76 years. The risk of AD increased by 40% for every 5 mol/L increase in Hcy levels, and it is believed to be at least 8 years old. Hcy levels are associated with the diagnosis of dementia and AD [Seshadri S, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med. 2002; 346: 476-483]; Postrglion et al. Patients not only have high plasma Hcy levels, but their progression is also directly proportional to Hcy levels, so Hcyemia is considered to be not only an independent risk factor for AD, but also associated with the progression and severity of AD [Postiglione A, et al. Plasma Folate, vitamin B12, and total homocysteine tetrahydrofolate reducase gene in patients with Alzheimer's dementia. A case-control study. Gerontolgy, 2001; 47: 324-349.]. More studies have shown that Hcy 7_K can be used independently to predict the upcoming decline in cognitive ability and decline independently of age, gender, education, kidney function, vitamin Β6 level, smoking history and hypertension. Degree of page measurement [Morris MS, et al. Hyperhomocystinemia associated with

J Clin Nutr. 2001; 73: 927-933.]. The possible mechanisms of action of Hcy in AD, dementia or impaired cognitive function include: direct damage of neurons by Hcy; enhancement of neurotoxicity of amyloid Αβ; affecting brain function by impairing microcirculation.

Studies have shown that patients with high Hcy PD have more severe depression and cognitive impairment [OpSuilleabhain PE, et al. Arch Neurol, 2004, 61: 865.]; Xin F et al's study of plasma Hcy levels in hospitalized patients with depression The incidence of Hcy was significantly higher than that of the normal control group. The level of Hcy in the plasma of patients with depression was positively correlated with the degree of depression. As the condition improved, the level of Hcy in the plasma also decreased, indicating that Hcy is closely related to depression [Xin F, et Al. Relationship of Homocysteine and Folic acid with Depression.Heilongjiang Medical J. 2007; 31: 659-662]; In Zhao G et al., plasma Hcy levels were significantly higher in patients with bipolar disorder than in the normal group, indicating that plasma Hcy levels are closely related to bipolar disorder [Zhao G, et al. An association analysis Of methylenetetrahydrofolate dehydrogenase polymorphism, plasma homocysteine levels and bipolar affective disorder. Journal of Psychiatry, 2008; 21: 130-132]. A study by Susser et al. compared Hcy levels in the schizophrenia group and the control group, and divided them into a normal folate level group and a low folic acid level group. It was found that there was a significant difference in Hcy levels in the low folate level group, suggesting a spirit There is an imbalance of Hcy metabolism in schizophrenia [Susser E, et al. Lindenbaum J: Schizophrenia and impaired homocysteine metabolism: a possible association. Biol Psychiatry, 1998; 44: 141]. Studies by Gonzalez S et al have confirmed that high Hcy is closely related to mortality in the elderly population without any chronic disease [Gonzalez S, et al. Homocysteine increase the risk of mortality in elderly individuals. BJ. 2007; 1-7].

The mechanism of damage caused by high Hcy is generally thought to be related to the damage of endothelial cells, oxidative stress, destruction of blood coagulation and fibrinolysis, vascular smooth muscle cell proliferation and direct cytotoxicity. Numerous studies have confirmed that Hcy levels are positively correlated with the damage caused. The Hcy level of healthy people abroad is about 8-10 μιηοΙ/L. Currently, Hcy levels greater than or equal to 10 μηιοΙ/L are defined as high Hcyemia or elevated Hcy [Circulation, 2006; 113: _e 409-e449; Clin Chem Med. 2003; 41: 1392-1403] ₀ Hughes et al found that Chinese males had an average Hcy level of 15.3 μιηοΙ/L and females of 12.2 jimol/L [J Epidemiol Community Health. 2000; 54(1): 31-34.] If the plasma Hcy>14 mol/L is used as the criterion, the incidence of high Hcy in Chinese men is 57%, and that in women is 31%. Studies in Hao et al. also show that Hcy levels in Chinese population are significantly higher, with Hcy greater than or equal to ΙΟμηιοΙ/ L is the standard, the incidence of hyperhomocysteinemia in the south is about 32%, and that in the north is about 58°/. , an average of about 45% [J Nutr 2007; 137: 407~413]. The above data indicate that high Hcyemia is widespread in China, causing serious social harm in many disease areas and must be actively intervened.

Most of the current use of B vitamins, mainly folic acid to reduce Hcy levels, thereby treating high Hcy disease. A meta-analysis showed that folic acid doses were less than 1 mg/d (mean 0.5 mg), 1-3 mg/d (mean 1.2 mg), and greater than 3 mg/d (average 5.7 mg) decreased Hcy levels. The efficacy is comparable [BMJ. 1998; 316: 894-898]. Other studies have shown that taking 0.8 mg of folic acid daily can basically achieve the maximum effect of lowering Hcy levels, while adding vitamin B12 (400 g/day) can only further reduce Hcy levels by 7%, while adding vitamin B6 or increasing Measures such as folic acid dose do not enhance its effect on reducing Hcy levels [Arch Intern Med. 2001; 161: 695-700; Am J Clin utr 2005; 82: 806-812.]. The above results indicate that the use of vitamins or multivitamins has a certain limit in reducing the effect of Hcy levels, and increasing the dose does not improve the effect of reducing Hcy.

 Considering that most clinical studies have used large doses of compound B vitamins (folic acid, B6 and B12), and the previous data suggest that further increases in folic acid doses do not further reduce Hcy levels; meanwhile, studies have confirmed that large doses of folic acid are used in combination. B vitamins may present a safety hazard. In studies such as NORVIT (Norwegian Vitamin Trial) and HOPE2 (Heart Outcome Prevention Evaluation 2), patients are at increased risk of tumor prevalence. Therefore, it is of great medical and social value to further seek ways and drugs that can safely and effectively reduce Hcy levels and antagonize the damage caused by high Hcy.

 Angiotensin-converting enzyme inhibitors (ACEI) are a class of drugs that act by competitively inhibiting angiotensin converting enzyme (ACE). ACE is a non-specific enzyme that, in addition to converting angiotensin I (Angl) to angiotensin II (Angll), also catalyzes the degradation of peptide-like vasodilators such as bradykinin. Therefore, under the action of ACE, the concentration of Angll in the circulation and tissues is increased, and the level of bradykinin is decreased.

 ACEI competitively blocks the conversion of Angl to Angll, thereby reducing circulating and local Angll levels, increasing bradykinin levels, and increasing the release of nitric oxide and vasoactive prostaglandins (prostacyclin and prostaglandin E); At the same time, ACEI can block the degradation of angiotensin 1-7 and increase its level, thereby further expanding the blood vessels and anti-proliferative effects by strengthening the stimulation of angiotensin 1-7 receptor.

Short-term use of ACEI treatment is accompanied by a decrease in the levels of Angll and aldosterone, which lowers the levels of plasma epinephrine, norepinephrine, and vasopressin in the pituitary. Long-term use of ACEI, due to the activation of non-angiotensin-converting enzyme-mediated alternative pathways (such as chymase), Angll and aldosterone levels have returned to pre-treatment trends (aldosterone "escape" phenomenon). Therefore, ACEI increases the levels of bradykinin, angiotensin 1-7, prostacyclin and nitric oxide, which may be the main factors for the persistence of dilated blood vessels and antithrombotic effects. ACEI has been used clinically for many years, HOPE (Heart Outcome Prevention Evaluation), EUROPE (EURopean Trial on reduction of cardiac events with Perindopril in stable coronary Artery disease, European Perindopril for the treatment of stable coronary artery disease Large-scale clinical studies such as event studies), PEACE (Prevention of Events with Angiotensin-Converting Enzyme Inhibition), ANBP2 (Second Australian National Blood Pressure Study) There is no serious safety hazard in ACEI clinical application.

 ACEI is widely used in the treatment of hypertension, congestive heart failure, diabetic nephropathy, etc. Commonly used ACEI drugs include captopril, enalapril, ramipril, lisinopril, benazepril, fosump Lee et al. Summary of the invention

 The object of the present invention is to overcome the deficiency of B vitamins such as folic acid in the treatment of hyperhomocysteinemia, and to provide a pharmaceutical composition containing ACEI and B vitamins in the preparation for effectively antagonizing homocysteine (Hcy) Use of levels of body damage, reduction of Hcy levels, and thus treatment of drugs with high Hcy.

 Hyperhomocysteinemia in the present invention is indicated by elevated plasma homocysteine levels, including body damage due to elevated levels of homocysteine. The damage of the body caused by elevated levels of homocysteine is characterized by vascular smooth muscle hyperplasia, thrombosis, neuronal damage, and microcirculatory disorders. The mechanism is generally believed to be related to oxidative stress, damage to endothelial cells, and destruction of the body. Balance between coagulation and fibrinolysis, causing vascular smooth muscle cell proliferation, cytotoxicity, etc., can eventually cause stroke, deep vein thrombosis, hypertension, ischemic heart disease (including coronary heart disease, angina pectoris, arrhythmia, myocardial infarction , heart failure and sudden death), Alzheimer's disease (AD), dementia, cognitive dysfunction, osteoporosis, fracture, depression, bipolar disorder, Parkinson's disease (PD), spirit Mitosis, death, neonatal defects, habitual abortion and other related diseases.

 The pharmaceutical compositions provided herein comprise one or more of a therapeutically effective amount of an angiotensin converting enzyme inhibitor (ACEI) and a therapeutically effective amount of a B vitamin and a pharmaceutically acceptable carrier.

An angiotensin converting enzyme inhibitor (ACEI) in the composition is selected from the group consisting of enalapril, benazepril, captopril, lisinopril, lei Ramipril, fosinopril, cilazapril, perindopril, quinapril, trandolapril, dirap Delipril, imidapril, temocapril (temocapril) > spirapril, moexipril and alapril. An in vivo active metabolite of an angiotensin-converting enzyme inhibitor, such as a metabolite or a salt of an angiotensin-converting enzyme inhibitor, which is an active component of an angiotensin-converting enzyme inhibitor, or an angiotensin converting enzyme inhibitor. Within the scope of the present disclosure.

 Through experimental studies, the daily dosage of ACEI in the treatment of high Hcyemia in mammals including humans is: enalapril 2.5~40 mg, benazepril 2.5~40 mg, ramipril 1.25~20 Mg, fosinopril 10~80 mg, lisinopril 2.5~80 mg, captopril 12.5~100 mg, quinapril 5~80 mg, cilazapril 1.2~5 mg, Peipu 2~16 mg, delaipril 15~; 120 mg, moxipril 3.75~30 mg, group Dopply 0.5~4 mg, midazolam 2.5~40 mg, sulpiride 3~30 mg Alapril 12.5~: 100 mg. The content of the derivative, precursor, active metabolite or salt of the above substance can be obtained by equivalent conversion of the above substance.

 The preferred daily doses of ACEI drugs are: enalapril 5~40 mg, benazepril 5~40 mg, ramipril 2.5~20 mg, fosinopril 10~40 mg, lisinopril 5~40 mg, captopril 25~100 mg, quinapril 10~40 mg, cilazapril 2.5~5 mg, perindopril 4~8 mg, delaipril 15~60 mg, Moxipril 7.5~30 mg, group Dopply 0.5~2 mg, midazolam 2.5~10 mg, sulpiride 3~15 mg, alapril 25~100 mg. The content of the derivative, precursor, active metabolite or salt of the above substance can be obtained by equivalent conversion of the above substance.

 In the present invention, the B vitamin in the composition is selected from the group consisting of vitamin B1 (vitamin B1), vitamin B2 (vitamin B2), vitamin PP, vitamin B6 (vitamin B6), and vitamin B12 (vitamin B 12 ) biotin (D). One or more of -biotin, vitamin H), folic acid (vitamin B9) and pantothenate (fruit B5) or their derivatives and substances which can release/generate such compounds in vivo.

 The B vitamins are preferably vitamin B6, vitamin B12 and folic acid, more preferably folic acid. Among them, vitamin B6 includes pyridoxine, pyridoxal, pyridoxamine, pyridoxal phosphate, pyridoxamine phosphate, derivatives of the above substances, and substances which can release/produce such compounds in the body.

 Among them, vitamin B12 includes cobalamin, mecobalamin, 5'-deoxyadenosylcobalamin, hydroxocobalamin, cyanocobalamin and other cobalamin derivatives and can release/form cobamine in the body. Substance.

Among them, folic acid includes active metabolites of folic acid, formyltetrahydrofolate, L-methylfolate, folate, folic acid or folate, and substances which can release/form folic acid in vivo. For the treatment of mammals including human hyperhomocysteinemia, the daily dosage of B vitamins is: folic acid 0.2~15mg, vitamin B120.1~2mg, vitamin B60.5~50 mg. The preferred daily dosage is: folic acid 0.2~5 mg, vitamin B121 g~2 mg, vitamin B65~50 mg. The daily dose of folic acid is more preferably 0.4 mg or 0.8 mg.

 When used to treat hyperhomocysteinemia in mammals, including humans, the preferred combination is:

 The ACEI is enalapril maleate or enalapril, and the daily dosage is 5~40mg; the B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2~5mg.

 The ACEI is enalapril maleate, and the daily dose is 10 mg; the B vitamin is folic acid, and the daily dose is 0.8 mg.

 The ACEI is enalapril maleate, and the daily dose is 10 mg; the B vitamin is folic acid, and the daily dose is 0.4 mg.

 The ACEI is enalapril maleate, and the daily dosage is 5 mg; the B vitamin is folic acid, and the daily dosage is 0.4 mg.

 The ACEI is enalapril maleate or enalapril, and the daily dosage is 5 to 40 mg; the B vitamin is vitamin B6, and the daily dosage is 5 to 50 mg.

 The ACEI is enalapril maleate or enalapril, and the daily dosage is 5~40 mg; the B vitamin is vitamin B12, and the daily dosage is l g~2 mg.

 The ACEI is benazepril hydrochloride or benazepril, and the daily dosage is 5 to 40 mg; the B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2 to 5 mg.

 The ACEI is benazepril hydrochloride or benazepril, and the daily dosage is 5 to 40 mg; the B vitamin is B6, and the daily dosage is 5 to 50 mg.

 The ACEI is benazepril hydrochloride or benazepril, the daily dosage is 5~40mg; the B vitamin is B12, and the daily dosage is lg~2mg.

 The ACEI is from fosinopril, the daily dosage is 10~40 mg; the B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2~5 mg.

 The ACEI is from fosinopril, the daily dosage is 10~40 mg; the B vitamin is B6, and the daily dosage is 5~50 mg.

 The ACEI is from fosinopril, the daily dosage is 10~40mg; the B vitamin is B12, and the daily dosage is 1g~2mg.

The ACEI is from ramipril, and the daily dosage is 2.5 to 20 mg; the B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2 to 5 mg. The ACEI is from ramipril, the daily dosage is 2.5~20 mg; the B vitamin is B6, and the daily dosage is 5~50 mg.

 The ACEI is from ramipril, the daily dosage is 2.5~20 mg; the B vitamin is B12, and the daily dosage is l g~2 mg.

 The ACEI is from cilazapril, the daily dosage is 2.5~5 mg; the B vitamin is folic acid, and the daily dosage is 0.2~5 mg.

 The ACEI is self-perindopril, the daily dosage is 4~8 mg; the B vitamin is folic acid, and the daily dosage is 0.2~5 mg.

 In the pharmaceutical composition of the present invention, the active ingredient is an essential component in the composition, wherein one active ingredient is derived from one of an angiotensin converting enzyme inhibitor and the other active ingredient is derived from one or several B vitamins. The content of the active ingredient can be determined according to the daily dosage determined above. The pharmaceutical composition may be in the form of a pharmaceutical preparation, including but not limited to common tablets, bilayer tablets, multi-layer tablets, sustained release tablets, single-chamber controlled release tablets, dual-chamber controlled release tablets, Microporous controlled release tablets, sublingual tablets, orally disintegrating tablets, dispersible tablets, enteric coated tablets, granules, pills, enteric capsules, delayed release tablets, timed/release tablets, ordinary capsules, sustained release capsules Controlled-release capsules, capsules containing pellets or tablets, pH-dependent capsules containing pellets or tablets, oral solutions, films or patches. It should be particularly noted that a pharmaceutical composition containing an angiotensin converting enzyme inhibitor and a B vitamin is made into a tablet or capsule.

 For the treatment of hyperhomocysteinemia, the compounds in the pharmaceutical composition may be administered to the affected individual simultaneously in the same preparation, or may be administered to the affected individual separately. In the case of sequential administration to a diseased individual, the delay in the administration of the second (or additional) active ingredient should not result in a loss of beneficial effects resulting from the combination of the active ingredients. In the case of simultaneous administration to a diseased individual, the compounds in the composition may be present in the same pharmaceutical preparation form, or may be independently present in the same preparation form. If they are independently present in the same formulation, the pharmaceutical composition may be modified in the form of a "combination kit". A "combination kit" is a box-like container containing a combination of drugs in one or more dosage forms and instructions for its use. In the present invention, a combination tablet consisting of one of the angiotensin converting enzyme inhibitors and one of the B vitamins is preferably used.

 Meanwhile, the present invention also provides a pharmaceutical composition comprising an angiotensin converting enzyme inhibitor and one or more of the B vitamins for treating hyperhomocysteinemia, wherein angiotensin is transformed Enzyme inhibitors and B vitamins are as defined above.

As a preferred embodiment, in the composition of the invention: The angiotensin converting enzyme inhibitor is enalapril maleate or enalapril, and the content is 5 to 40 parts by weight; the B vitamin is folic acid or calcium leucovorin, and the content is 0.2 to 5 Wherein, when the ACEI is enalapril maleate, the content is 10 parts by weight; the B vitamin is folic acid, and the content is 0.8 parts by weight.

 The ACEI is enalapril maleate in an amount of 10 parts by weight; the B vitamin is folic acid in an amount of 0.4 parts by weight.

 The ACEI is enalapril maleate in an amount of 5 parts by weight; the B vitamin is folic acid in an amount of 0.4 part by weight.

 A method of treating hyperhomocysteinemia comprising administering to a patient a pharmaceutical composition provided by the present invention.

 As a preferred mode of the present invention, a combination of enalapril and folic acid can be used for the treatment of hyperhomocysteinemia. As a preferred combination preparation, enalapril folic acid compound preparation can significantly reduce the level of homocysteine in rats with hyperhomocysteinemia and protect the damage caused by elevated homocysteine. It is superior to either drug alone and shows statistical differences. Among them, the enalapril folic acid compound preparation was 10 parts by weight of enalapril and 0.8 parts by weight of folic acid.

 As another preferred mode provided by the embodiment of the present invention, a combination preparation of perindopril and folic acid can be used for the treatment of hyperhomocysteinemia. As a preferred combination preparation, the combination of perindopril folic acid can significantly reduce the level of homocysteine in rats with hyperhomocysteinemia and protect against damage caused by elevated homocysteine. It is superior to either drug alone and shows statistical differences. Among them, the perindopril folic acid compound preparation was 4 parts by weight of perindopril and 0.4 parts by weight of folic acid.

 As another preferred mode provided by the embodiment of the present invention, the combination preparation of enalapril and folic acid has an inhibitory effect on the proliferation of vascular smooth muscle cells (VSMC) induced by high concentration of Hcy. As a preferred combination preparation, the enalapril folic acid compound preparation can significantly inhibit the proliferation of VSMC, and the effect is significantly better than either drug alone. Among them, the enalapril folic acid compound preparation was 10 parts by weight of enalapril and 0.2 parts by weight of folic acid.

The pharmaceutical composition of the present invention can be prepared by adding a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients refer to those materials known in the art that can act as a filler or carrier material in tablets, pills, capsules and the like. Usually these substances are approved by the health administration for this purpose. They are also inactive as pharmaceutical agents. Handbook of Pharmaceutical Excipients (A. Wade and

Editor-in-Chief, PJ.Weller, Second Edition, American Pharmaceutical Association, Washington and Pharmacy Press, London Publishing,

1994) Edited pharmaceutically acceptable carriers and excipients. In particular, lactose, starch, cellulose derivatives and the like, and mixtures thereof, can be used as carriers for the active ingredients of the compositions of the present invention.

 The invention is further described in conjunction with the specific embodiments, which are not intended to limit the invention, and any equivalent substitutions in the art in accordance with the present invention are within the scope of the invention. detailed description

 Example 1 : Efficacy of enalapril folic acid in rats with hyperhomocysteinemia

 1, method

 Establishment of animal and high Hcy model: 60 male rats of 250~300g were randomly divided into 6 groups and 10 groups/group. One group was given normal feed as a blank control group, and the other groups (model group, enalapril high dose group, enalapril low dose group, folic acid group and enalapril + folic acid group) were given high methionine feed. For 12 consecutive weeks.

 Grouping and administration: enalapril was administered daily from the enalapril high dose group, the enalapril low dose group, the folic acid group, and the enalapril + folic acid group. 2mg/kg), enalapril (1mg/kg), folic acid (0.08mg/kg), enalapril + folic acid (1mg +0.08mg/kg), the model group and the blank control group were given the same amount of normal saline. , for 8 consecutive weeks. After the last administration, the abdominal aorta was collected for homocysteine (Hcy) (homocysteine (Hcy) detection kit was purchased from Beijing Zhongshan Biotechnology Engineering Co., Ltd.), and nitric oxide (NO) (-nitrogen oxide synthesis) Enzyme (NOS) test kit purchased from Nanjing Bio-Building Bioengineering Institute), superoxide dismutase (SOD) (superoxide dismutase (SOD) (extraction method) test box purchased from Nanjing Bio-Building Bioengineering Institute ), determination of malondialdehyde (MDA) (malondialdehyde (MDA) test kit purchased from Nanjing Bio-Building Bioengineering Institute).

 2, the result

The results showed (see Table 1) that the Hcy level of the model group was significantly higher than that of the normal group. Compared with the model group, the high dose of enalapril and the low dose of enalapril did not significantly improve the level of Hcy; the folic acid group can reduce the level of Hcy; the enalapril + folic acid group can significantly reduce the level of Hcy, which is better than The enalapril high-dose group, the enalapril low-dose group, and the folic acid group showed that enalapril + folic acid had a significant synergistic effect in reducing high Hcy levels. At the same time, the levels of NO and SOD in the model group were significantly decreased, and the level of MDA was significantly increased. The high dose group of enalapril and the low dose group of enalapril had no significant effect on SOD, MDA and NO. Folic acid could increase NO and SOD has no significant effect on MDA. The enalapril + folic acid group significantly increased the levels of SOD and NO, and decreased the level of DA, which was significantly better than the model group and the single drug group, indicating that enalapril and folic acid also antagonize the high Hcy-induced SOD, MDA, and NO damage. Has a significant synergy.

 Table 1 Changes in detection indicators of each group

 Hcy SOD MDA NO Group

(μηιοΙΧ" ¹ ) (U-mL ^-1 ) (nmoLmL" ¹ ) (μπιοΙΧ' ¹ ) blank control group 7.4 ± 1.9** 135.9 ± 17.8** 2.0 ± 0.7** 44.1 ± 15.9** model group 18.9 ± 6.8 59.4±20.1 6.1±2.0 16.7±7.2 enalapril

 18.2±3.6 66.4±16.2 6.0±1.9 18.0±8.9 dose group

Enalapril

 19.2±7.5 63.9±22.2 5.9±1.4 17.6±6.1 dose group

Folic acid group 13.7±2.7* 80.0±21.5* 5.7±1.8 24.5±8.9* enalapril + leaf

10.0±2.2** ^@ 107.9±20.4** ^@@## 3 9±1 37.2±7 5**®® ^##酸组

*Ρ<0.05, **P<0.01, compared with the model group; ^@P <0.05, @@P<0.01, compared with the folic acid group; ^## P<0.01, with the enalapril group (low or high dose) Comparison

 3. Conclusion

 Clinical studies have shown that: B vitamins alone can not effectively treat the body damage, while the use of B vitamins such as folic acid alone to reduce the level of Hcy has a certain limit, the 0.8 mg / d dose of folic acid has basically reached its maximum efficacy, and further increase The dose of B vitamins such as folic acid has a safe and implicit study. The SOD level of the model group is significantly decreased, and the MDA level is significantly increased, indicating that oxidative damage is one of the main damage pathways of hyperhomocysteinemia. One of the most important markers of vascular endothelial injury and dysfunction is the decrease in NO levels. In this study, the level of NO in the model group was significantly reduced, indicating that endothelial cell injury is also an important pathway for high Hcyemia to cause damage.

The data in Table 1 shows that the high dose of enalapril, the low dose of enalapril, and the folic acid monotherapy group are not adequate for the treatment of high Hcy injury; however, the combination of enalapril and folic acid Both of them have significant improvement effects, and their effects are better than those of the single drug group. Meanwhile, the enalapril folic acid compound can synergistically reduce the level of Hcy in this study, and the effect of the effect is significantly different from that of folic acid. The results fully demonstrate that enalapril folic acid acts on multiple pathways and links of hyperhomocysteinemia, effectively antagonizes the damage caused by high Hcy levels, and further reduces Hcy levels, which have an interaction. The pharmaceutical composition provided by the present invention has high safety and is therefore a better choice for treating hyperhomocysteinemia. Example 2: Efficacy of perindopril folic acid in rats with hyperhomocysteinemia

 1, method

 Establishment of animal and high Hcy model: 60 male rats of 250~300g were randomly divided into 6 groups and 10 groups/group. The blank control group was given normal feed, and the high dose of perindopril, the low dose of perindopril, the folic acid group, and the perindopril + folic acid group were given high methionine feed for 12 weeks.

 Grouping and administration: The intraperitoneal administration was started from the 5th week of modeling, and the perindopril high-dose group, the perindopril low-dose group, the folic acid group, and the perindopril + folic acid group were given perindopril daily.

(0.8mg/kg), perindopril (0.4mg/kg), folic acid (0.08mg/kg), perindopril + folic acid (0.4mg+0.04mg/kg), the model group and the blank control group were given separately The same amount of normal saline. For 8 weeks. Blood was collected from the abdominal aorta after the last administration for homocysteine (Hcy) and nitric oxide.

(NO), measurement of superoxide dismutase (SOD) and malondialdehyde (MDA) (detection method is the same as in Example 1).

 2, the result

 The results showed (see Table 2) that the Hcy level of the model group was significantly higher than that of the blank control group. Compared with the model group, the high-dose perindopril group and the low-dose perindopril group had no significant effect on Hcy; the folic acid group could reduce the Hcy level; the perindopril + folic acid group could further reduce the Hcy level, which was better than the culture group. The high-dose Puli group, the low-dose perindopril group, and the folic acid group showed that the combination of perindopril folic acid had a significant synergistic effect in reducing Hcy.

At the same time, the levels of NO and SO in the model group were significantly decreased, and the level of MDA was significantly increased. The high dose of perindopril and the low dose of perindopril had no significant effect on SOD, MDA and NO; folic acid could increase NO, SOD has no significant effect on VEDA. Perindopril + folic acid group significantly increased SOD, NO levels and decreased MDA levels, which was significantly better than the model group and each single drug group, indicating that perindopril and folic acid also have significant synergistic effects in antagonizing high Hcy damage. Table 2 Changes in detection indicators of each group

 Hcy SOD MDA NO Group

(μπιοΙ ' ¹ ) (U.mL ^-1 ) (nmol.mL" ¹ ) (μπιοΙΧ" ¹ ) Blank group 7.4±1.9** 135.9±17.8** 2.0±0.7** 44.1±15.9** Model group 18.9± 6.8 59.4±20.1 6.1±2.0 16.7±7.2

'Pure

 19.1±5.0 67.7±20.8 5.8±1.6 19.2±4.9 dose group

 Perindopril

 19.0±4.4 68.3±22.9 6.1±2.0 18.1±6.1 dose group

 Folic acid group 13.7±2.7* 80.0±21.5* 5.7±1.8 24.5±8.9* Perindopril + leaf

g±i **@@ ^## joy 7±18 5**®® ^## 3.6±1.2**® ^@#酸组

*Ρ<0.05, **Ρ<0.01, compared with the model group; ^@ Ρ<0.05, ^@@ Ρ<0.01 compared with the folic acid group; ^## Ρ<0.01, compared with the perindopril group (low or high dose)

 3. Conclusion

 Clinical studies have confirmed that the use of steroid vitamins alone can not effectively treat the body damage, while the use of folic acid and other steroid vitamins to reduce Hcy levels has a certain limit, the 0.8 mg / d dose of folic acid has basically reached its maximum efficacy, and further increase B vitamins such as folic acid have safety risks.

 In this study, the SOD level of the model group was significantly decreased, and the MDA level was significantly increased, indicating that oxidative damage is one of the major damage pathways of hyperhomocysteinemia. One of the most important markers of vascular endothelial injury and dysfunction is the decrease in NO levels. In this study, the level of NO in the model group was significantly reduced, indicating that endothelial cell injury is also an important pathway for high Hcyemia to cause damage.

The data in Table 2 showed that the high-dose perindopril group, the low-dose perindopril group, and the folic acid monotherapy group had insufficient therapeutic effects on the damage caused by high Hcy levels; however, the combination of perindopril and folic acid was used. All indicators have improved, and the effect is significantly better than the single drug group; meanwhile, in this study, the perindopril folic acid combination can synergistically reduce the level of Hcy, and its effect has a significant statistical difference with the effect of folic acid. The results fully demonstrate that the combination of perindopril folic acid acts on multiple pathways and links of hyperhomocysteinemia, effectively antagonizes the damage caused by high Hcy levels, and further reduces Hcy levels, and the two interact. The pharmaceutical composition provided by the present invention has high safety and is therefore a better choice for treating hyperhomocysteinemia. Example 3: Inhibition of cyanofronic acid on Hcy-induced proliferation of vascular smooth muscle cells (VSMC)

 This work was performed on VSMC cultured in rats to observe the effects of folic acid, enalapril and their combination on Hcy-induced proliferation of VSMC.

 1, materials and methods

 1.1 vascular smooth muscle cell culture

 Take the rat thoracic aorta, according to the Ross method (Ross R. The smooth muscle cell II . Growth of smooth muscle cells in culture and formation of elastic fibers [ J ] . J Cell Biol, 1971, 50 (1): 172- 176) Isolation and subculture. The cells were cultured in DEME medium containing 20% (v/v) calf serum, and the passage 4 cells were used.

1.2 VSMC ³ H-Td R incorporation

The cells were seeded in a 24-well plate at a cell size of 5χ10 ⁵ per well, and cultured in DEME medium containing 20% calf serum for 24 hours. After adherence, the cells were cultured in a DEME medium containing 0.5% calf serum. 24h, then add different drugs to the experiment in the following groups. (1) blank control group: only PBS; (2) Hcy group: Ιθθμπιοΐχ- ¹ Hcy _; (3) enalapril group: Ι.θμιηοΐΐ ¹ enalapril; (4) folic acid group: 0.2 μιηοΐ ·!; ¹ folic acid; (5) Hcy + enalapril group; (6) Hcy + folic acid group; (7) Hcy + folic acid + enalapril group. Each compound group used the same drug dose as the above unilateral group.

After 37 h of 37 kBq ³ H-TdR addition, the reaction was stopped by adding cold PBS. Microporous membrane

(0.25 μηι) The cells were collected and washed, treated with 100 g of ^I / ³ trichloroacetic acid, the filter was placed in a scintillation vial, scintillation fluid was added, and the radioactivity of ³ H was measured on a liquid scintillation counter.

 1.3 Statistical processing

 Experimental data are expressed as Mean±s; inter-group t-test or analysis of variance.

 2, the result

Ιθθμιηοΐχ- ¹ Hcy can significantly increase the dysentery of cultured rat VSMC ³ H-TdR. Folic acid and enalapril can inhibit Hcy-induced VSMC proliferation. Combined use of folic acid and enalapril can further reduce Hcy-induced VSMC ³ H-TdR incorporation, which is superior to single drug, indicating folic acid and Naprilat has a superposition or synergistic effect on inhibiting the proliferation of VSMC caused by Hcy. The results are shown in Table 3.

3. Conclusion The proliferation of VSMC is also one of the important pathological features of high Hcy-induced injury. In vitro cell culture can exclude other interfering factors and specifically observe the protective effect of drugs on Hcy-induced damage. The results showed that both folic acid and enalapril could inhibit the proliferation of VSMC, but the combination of folic acid and enalapril significantly inhibited the proliferation of VSMC, and its effect was significantly better than that of any drug alone. Significant statistical differences indicate that folic acid and enalapril have different pathways and links in delaying/treating the mechanism of Hcy-induced damage to the body, with additive or synergistic effects.

Table 3 Effect of enalapril on proliferation of VCMC Group dose / μιιιοΐχ- ¹ ( ³ H-TdR incorporation)

 Blank control group one 6432±622

Hcy 100 11200±863 ^%%

 Hcy+folate 100+0.2 8820±659*

 Hcy+Ena 100+1.0 8896±759*

Hcy+Ena+folate 100+1.0+0.2 7600±621** ^$

Ena: enalapril; folate: folic acid; % ^% P<0.01 vs control; **P<0.01 vs

Hcy;

 <0.05 vs Hcy+folate; <0.05 vs Hcy+Ena

 Summary: High Hcyemia is associated with a variety of diseases, oxidative damage, cell proliferation, endothelial cell injury, etc. are the main path of injury. At present, folic acid alone or compound B vitamins are not effective in the treatment of clinical high Hcy. The present invention acts on multiple pathways and links of hyperhomocysteinemia, effectively reduces high Hcy levels, and reduces damage caused by high Hcy levels, and High safety will be a better choice and provide a more effective treatment for the treatment of hyperhomocysteinemia. Example 4 Preparation of a compound enalapril folic acid tablet containing 10 mg of enalapril and 0.8 mg of folic acid

 Recipe: Enalapril 10g

 0.8g

 50g

 50g

 10g

 Carboxymethyl starch 30g

Magnesium stearate lg Preparation method: 10 g of enalapril, 0.8 g of folic acid, 50 g of lactose, 50 g of cellulose and 10 g of starch are pulverized and uniformly mixed, and made into a soft material by 10% povidone ethanol solution, granulated, dried, and finished. For the granules, the granules having a water content of about 3% were uniformly mixed with magnesium stearate, and pressed into tablets by a tableting machine. Each of the prepared 1000 tablet tablets contained 10 mg of enalapril and 0.8 mg of folic acid, and the mass ratio thereof was 10:0.8. Example 5 Preparation of compound benazepril containing 10 mg of benazepril and 0.4 mg of folic acid: benazepril

 Folic acid

 Lactose

 Microcrystalline cellulose

 Starch

 Carboxymethyl starch sodium

 Magnesium stearate

The preparation method was the same as in Example 4. Each of the prepared 1000 tablet tablets contained benazepril 10 mg and folic acid 0.4 mg in a mass ratio of 10:0.4.

Claims

Rights request A use of a pharmaceutical composition comprising an angiotensin converting enzyme inhibitor and a B vitamin in the preparation of a medicament for treating hyperhomocysteinemia, wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of Puli, benazepril, captopril, lisinopril, ramipril, fosinopril, cilazapril, perindopril, quinapril, group dopply, dila Puli, midazolam, temocapril, spironolide, moxipril and alapril and their derivatives, precursors, active metabolites or salts; the B vitamins are selected from the group consisting of folic acid, A Calcium tetrahydrofolate, vitamin B6, vitamin B12, vitamin B1, vitamin B2, vitamin PP, biotin and pantothenic acid and derivatives thereof and substances which can release/generate such compounds in the body.  The use according to claim 1, wherein the hyperhomocysteinemia is an injury caused by an increase in homocysteine levels, including oxidative damage, cell proliferation, endothelial cell damage, thrombosis, and the like. Pathological features.  The use according to claim 1, wherein the hyperhomocysteinemia is accompanied by high blood pressure.  The use according to any one of claims 1 to 3, wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of enalapril, benazepril, ramipril, fosinopril, and Lai. Norpley, Captopril, Quinapril, Cilapril, Perindopril, Delapril, Moxipri, Group Dopply, Mipropri, Spirulin, Arap One of the benefits, the daily dosage is: enalapril 2.5~ 40 mg, benazepril 2.5~40 mg, ramipril 1.25~20 mg, fosinopril 10~80 mg, lisino Puli 2.5~80 mg, Captopril 12.5~100 mg, quinapril 5~80 mg, cilazapril 1.2~5 mg, perindopril 2~: 16 mg, delapid 15~ : 120 mg, moxipril 3.75 to 30 mg, group Dopply 0.5 to 4 mg, midazolam 2.5 to 40 mg, spironolide 3 to 30 mg, alapril 12.5 to 100 mg; The vitamins are selected from one or more of folic acid, vitamin B12 and vitamin B6, and the daily dosages are: folic acid 0.2~15 mg, vitamin B12 0.1~2 mg, vitamin B6 0.5~50 mg, the above substances Derivatives, precursors, active metabolite or salt daily dosage may be determined according to the terms of the equivalent thereof.  The use according to any one of claims 1 to 3, wherein: The angiotensin converting enzyme inhibitor is enalapril maleate or enalapril, and the daily dosage is 5~40 tng; the B vitamin is folic acid or calcium tetrahydrofolate, and the daily dosage is 0.2~ 5 mg; the angiotensin converting enzyme inhibitor is enalapril maleate or enalapril, daily dosage 5~40 mg; B vitamins are vitamin B6, daily dosage is 5~50 mg;  The angiotensin converting enzyme inhibitor is enalapril maleate or enalapril, the daily dosage is 5~40 mg; the B vitamin is vitamin B12, and the daily dosage is 0.001~2 mg;  The angiotensin-converting enzyme inhibitor is benazepril hydrochloride or benazepril, and the daily dosage is 5 to 40 mg; the B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2 to 5 mg. The angiotensin-converting enzyme inhibitor is benazepril hydrochloride or benazepril, the daily dosage is 5~40 mg; the B vitamin is B6, and the daily dosage is 5~50 mg;  The angiotensin converting enzyme inhibitor is benazepril hydrochloride or benazepril, the daily dosage is 5~40 mg; the B vitamin is B12, and the daily dosage is 0.001~2 mg; The angiotensin converting enzyme inhibitor is from fosinopril, the daily dosage is 10~40 mg _{; the} B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2~5 mg; The angiotensin converting enzyme inhibitor is from fosinopril, the daily dosage is 10~40 mg _{; the} B vitamin is B6, and the daily dosage is 5~50 mg;  The angiotensin converting enzyme inhibitor is from fosinopril, the daily dosage is 10~40 mg; the B vitamin is B12, and the daily dosage is 0.001~2 mg; The angiotensin converting enzyme inhibitor is from ramipril, the daily dosage is 2.5~20 mg _{; the} B vitamin is folic acid or calcium leucovorin, and the daily dosage is 0.2~5 mg;  The angiotensin converting enzyme inhibitor is from ramipril, the daily dosage is 2.5~20 mg; the B vitamin is B6, and the daily dosage is 5~50 mg;  The angiotensin converting enzyme inhibitor is from ramipril, the daily dosage is 2.5~20 mg; the B vitamin is B12, and the daily dosage is 0.001~2 mg;  The angiotensin converting enzyme inhibitor is from cilazapril, the daily dosage is 2.5~5 mg; the B vitamin is folic acid, and the daily dosage is 0.2~5 mg;  The angiotensin-converting enzyme inhibitor is self-perindopril, and the daily dosage is 4 to 8 mg; the B vitamin is folic acid, and the daily dosage is 0.2 to 5 mg.  The use according to claim 5, wherein the angiotensin converting enzyme inhibitor is enalapril, the daily dosage is 10 mg, the B vitamin is folic acid, and the daily dosage is 0.4 or 0.8. Or the angiotensin converting enzyme inhibitor is enalapril, the daily dosage is 5 mg, the B vitamin is folic acid, the daily dosage is 0.4 mg; or the angiotensin converting enzyme inhibitor is cultured.哚普利, daily dosage is 4 mg, the B vitamin is folic acid, the daily dosage is 0.4 mg. The use according to any of the preceding claims, wherein the pharmaceutical composition can be made into a Tablets, ordinary capsules, granules, sustained release tablets, sublingual tablets, orally disintegrating tablets, dispersible tablets, enteric coated tablets, enteric coated capsules, delayed release tablets, timed release tablets, sustained release capsules, Controlled-release capsules, capsules containing pellets or tablets, pH-dependent capsules containing pellets or tablets, granules, oral solutions, films, patch dosage forms or a combination kit in which the two active components are separately present.  A pharmaceutical composition comprising one or more of an angiotensin converting enzyme inhibitor and one or more of the B vitamins for treating hyperhomocysteinemia, wherein the angiotensin is transformed The enzyme inhibitor is selected from the group consisting of enalapril, benazepril, captopril, lisinopril, ramipril, fosinopril, cilazapril, perindopril, quinapril, Group Dopply, delaipril, midazolam, temocapril, spironolide, moxipriril and/or alapril and their derivatives, precursors, active metabolites or salts; The B vitamins are selected from the group consisting of folic acid, calcium leucovorin, vitamin B6, vitamin B12, vitamin B1, vitamin B2, vitamin PP, biotin and/or pantothenic acid and their derivatives and can be released/generated in the body. The substance of the compound.  The pharmaceutical composition according to claim 8, wherein the angiotensin converting enzyme inhibitor is enalapril and the content is 10 parts by weight, and the B vitamin is folic acid, and the content is 0.4 or 0.8 parts by weight; or the angiotensin converting enzyme inhibitor is perindopril in an amount of 4 parts by weight, and the B vitamin is folic acid in an amount of 0.4 parts by weight.  10. A method of treating hyperhomocysteinemia comprising administering a composition of claim 8 to a patient.