CN117279642A

CN117279642A - Combination therapy for preventing, inhibiting, treating or reducing aneurysms

Info

Publication number: CN117279642A
Application number: CN202280032401.8A
Authority: CN
Inventors: 才华
Original assignee: University of California
Current assignee: University of California
Priority date: 2021-03-02
Filing date: 2022-03-02
Publication date: 2023-12-22

Abstract

The present disclosure relates to pharmaceutical compositions comprising a folic acid compound and a calcium channel blocker, and methods of treating aneurysms using such pharmaceutical compositions.

Description

Combination therapy for preventing, inhibiting, treating or reducing aneurysms

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application number 63/155,348 filed on day 3, month 2 of 2021 and U.S. provisional application number 63/170,774 filed on day 4, 2021, each of which is hereby incorporated by reference in its entirety.

Government support

The present invention was developed in accordance with government support under grant number HL077440 awarded by the national institutes of health (National Institutes of Health). The government has certain rights in this invention.

Background

Aortic aneurysms are a common and serious vascular disease with a high mortality rate resulting from the death of the patient from sudden rupture of an unpredictable aneurysm. No other treatment options are available other than the surgery correction of the large aneurysms, which is at considerable risk. Abdominal Aortic Aneurysms (AAA) are defined as abdominal aortic distensions exceeding 3cm in diameter, most commonly affecting the infrarenal segment. It is associated with a high risk of death in the event of an aneurysm rupture, resulting in about 20 tens of thousands of deaths worldwide each year. The incidence of AAA in the population older than 65 years is as high as 9%, and meta-analysis of 56 studies showed that the incidence of AAA in the general population is 4.8%. While the most accepted risk factors for AAA include male sex and smoking, other risk factors have also been associated with AAA formation, such as older age, family history, hypertension, and hyperlipidemia. AAA formation mechanisms are complex and involve mainly oxidative stress driven vascular remodeling characterized by matrix degradation and inflammation, resulting in dilation of the abdominal aorta. The only clinical intervention for the treatment of aortic aneurysms is limited to large AAA with a surgical correction size exceeding 5.5cm, and the 30-day mortality of both endovascular aortic aneurysm repair (EVAR) and open surgical repair is high, 1.7-4.7%. There is no oral drug currently available for treating aortic aneurysms of all sizes, including smaller and growing aneurysms, to prevent unpredictable sudden rupture and death.

Thoracic aortic aneurysm is a term used to describe an aneurysm formed at the thoracic region of the aorta, including the ascending thoracic aortic/aortic root region (about 60%) and the descending thoracic aortic region (about 40%). Thoracic aortic aneurysms may be caused by arteriosclerosis (atherosclerosis), elevated blood pressure (hypertension), congenital diseases such as Marfan Syndrome (Marfan Syndrome), or trauma. The prevalence of TAA in the general population is about 4.2%. Similar to AAA, no treatment options/oral medications are available other than surgical correction. Oxidative stress driven vascular remodeling is a shared aneurysmal pathway shared between AAA and TAA.

Cerebral aneurysms develop due to thinning/weakening of the arterial wall. Aneurysms often form at bifurcation or branch in the artery because those sections of the blood vessel are weak and susceptible to vascular remodeling, resulting in aneurysm formation. Cerebral aneurysms are one of the most common cerebrovascular diseases. If the aneurysm is severe, it can develop into a hemorrhagic stroke with cerebral vascular bleeding. Similar to AAA/TAA, no treatment options/oral drugs are available other than surgical correction. Oxidative stress driven vascular remodeling is a shared aneurysmal pathway shared between AAA/TAA and cerebral aneurysms.

Disclosure of Invention

Provided herein are compositions, kits, and methods for preventing or treating an Abdominal Aortic Aneurysm (AAA), a Thoracic Aortic Aneurysm (TAA), or a cerebral aneurysm. In certain aspects, provided herein are pharmaceutical compositions comprising a folic acid compound and a calcium channel blocker. Also provided herein are kits comprising a folic acid compound and a calcium channel blocker.

The methods provided herein include preventing or treating an aneurysm (e.g., an Abdominal Aortic Aneurysm (AAA), a Thoracic Aortic Aneurysm (TAA), or a cerebral aneurysm) by administering a folic acid compound and a calcium channel blocker in combination to a subject in need thereof. In some embodiments, the method comprises ameliorating at least one symptom of or a symptom associated with an aneurysm by administering a folic acid compound and a calcium channel blocker in combination to a subject in need thereof. The symptoms may be, for example, increased superoxide production, increased eNOS uncoupling activity, reduced Nitric Oxide (NO) bioavailability, tetrahydrobiopterin (H ₄ B) Reduced bioavailability, increased vascular (abdominal aorta, thoracic aorta or blood vessels in the brain), increased vascular remodeling, increased elastin degradation (flattening and breakdown), increased vascular inflammation/macrophage infiltration, increased Matrix Metalloproteinase (MMP) activation, increased adventitial hypertrophy, or reduced eNOS function.

Also provided herein are methods of reducing superoxide production, eNOS uncoupling activity, vascular (blood vessels in the abdominal aorta, thoracic aorta, or brain), vascular remodeling, elastin degradation (flattening and decomposition), vascular inflammation/macrophage infiltration, matrix Metalloproteinase (MMP) activation, and/or adventitial hypertrophy in a subject having an aneurysm, comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof. In some embodiments, provided herein are methods of increasing eNOS function, nitric Oxide (NO), and/or tetrahydrobiopterin bioavailability in a subject having an aneurysm by co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

The aneurysm described herein may be an AAA aneurysm, TAA aneurysm, or cerebral aneurysm.

Folic acid compounds can be represented by formula I

Or a pharmaceutically acceptable salt thereof, wherein:

each R ¹ Independently is hydrogen, acyl, ester, amide, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl;

each R ² Independently is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl;

Each R ³ And R is ⁴ Independently is halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl;

m is an integer selected from 0-3; and is also provided with

n is an integer selected from 0-4.

Each R ¹ Independently hydrogen, acyl, ester, amide or alkyl. In some embodiments, each R ¹ Is hydrogen. Each R ² Independently can be hydrogen or alkyl. In some embodiments, each R ² Is hydrogen. M and/or n may be 0.

In some embodiments, the folic acid compound is

Or a pharmaceutically acceptable salt thereof.

The calcium channel blocker may be a dihydropyridine compound. For example, wherein the dihydropyridine compound is represented by formula II

Or a pharmaceutically acceptable salt thereof, wherein:

R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ and R is ¹⁰ Each independently is hydrogen, halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl; and is also provided with

R ⁹ Is hydrogen, acyl, ester, amide, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

R ⁶ May be alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In some embodiments, R ⁶ Is methyl or substituted or unsubstituted phenyl. R is R ⁶ May be phenyl optionally substituted with halogen, haloalkyl, alkyl or nitro.

The dihydropyridine compound may be represented by formula II-a, II-b, II-c, or II-d:

R ⁹ may be hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In some embodiments, R ⁹ Is optionally substituted with halogen, amino, hydroxy, alkoxy, cyano, nitroAn alkyl substituted with a group, acyl, ester, amide, alkylthio, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

In some embodiments, R ⁹ Is that

In some embodiments, R ¹⁰ Is cyano, amino, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl. R is R ¹⁰ May be methyl.

In some embodiments, the dihydropyridine compound is represented by formula II-a-1, II-b-1, II-c-1, or II-d-1:

R ⁵ may be alkoxy, amino, alkyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In some embodiments, R ⁵ Is an alkoxy group optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

In some embodiments, R ⁵ Is that

R ⁷ May be acyl, ester, amide, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In some embodiments, R ⁷ Is that

R ⁸ May be hydrogen, hydroxy, alkoxy, alkylthio, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. R is R ⁸ May be hydrogen, hydroxy, alkoxy, alkylthio, or alkyl optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, R ⁸ Is hydrogen, and is preferably selected from the group consisting of hydrogen,

in some embodiments, the dihydropyridine compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the dihydropyridine compound is

Or a pharmaceutically acceptable salt thereof.

The folic acid compound and the dihydropyridine compound may be in the same composition. The folic acid compound may be folic acid. The calcium channel blocker may be nifedipine. In some embodiments, the amount of folic acid compound administered and the amount of calcium channel blocker administered are therapeutically effective. In some embodiments, the folic acid compound and the calcium channel blocker are administered in a mass ratio of about 10:1 to about 1:10.

The folic acid compound and the calcium channel blocker may be administered in a mass ratio of about 3:1 to about 3:4. Folic acid compounds may be administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, or about 1-1750 mg. In some embodiments, the calcium channel blocker is administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, about 1-1750mg, about 1-2100mg, or about 1-2450 mg. The folic acid compound and the calcium channel blocker may be administered simultaneously or sequentially. In some embodiments, the folic acid compound and the calcium channel blocker are in separate dosage forms.

The aneurysm may be an abdominal aortic aneurysm, a cerebral aneurysm or a thoracic aortic aneurysm. Folic acid compounds and calcium channel blockers may be administered orally.

Drawings

Fig. 1A-1B show that the combination of FA and nifedipine significantly and completely further improved the efficacy of FA in reducing the incidence of aortic aneurysms in a dose-dependent manner. Angiotensin (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of regular diet, folic acid-containing customized diet (FA, 15 mg/kg/day), or customized diet containing FA and different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). Fig. 1A shows the incidence of Abdominal Aortic Aneurysms (AAA) across different groups. The data show that with regular diet, apoE mice infused with Ang II had an AAA morbidity of 85.71%, whereas by oral administration of FA, the AAA morbidity was reduced to 18.75%. The combination of FA with nifedipine at 1.5, 5 or 20 mg/kg/day significantly and completely further reduced AAA morbidity to 12.50%, 11.76% and 0.00%, respectively. * p <0.05, < p <0.001, n=17-35. Fig. 1B shows the actual number of animals examined across different groups and the number of animals developing AAA: apoE, n=34/0; apoe+ang II, n=35/30; apoe+ang ii+fa, n=32/6; apoe+ang ii+fa+nif (1.5), n=24/3; apoe+ang ii+fa+nif (5.0), n=17/2; apoe+ang ii+fa+nif (20.0), n=18/0.

Fig. 2A-2B show that the combination of FA and nifedipine significantly and completely further attenuated the dilation of the abdominal aorta in a dose-dependent manner. Angiotensin (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of regular diet, folic acid-containing customized diet (FA, 15 mg/kg/day), or customized diet containing FA and different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). The dilation of the abdominal aorta was monitored weekly using ultrasound. Fig. 2A shows representative weekly ultrasound images taken from different experimental groups. Fig. 2B shows the grouping data of the ultrasound measurements of the abdominal aortic region. Infusion of Ang II into apo E deficient mice resulted in significant distension of the abdominal aorta (p <0.001 compared to apoE for the same week). Oral administration of FA alone significantly attenuated abdominal aortic dilation (# p <0.01 or # p <0.001 compared to apoe+ang II for the same week). Nifedipine at doses of 1.5 mg/kg/day, 5.0 mg/kg/day and 20 mg/kg/day significantly/completely further attenuated the dilation of the abdominal aorta in a dose-dependent manner compared to the FA alone group (@ p <0.01 or @ p <0.001 for the 1.5 mg/kg/day nifedipine group, @ p <0.01 or @ p <0.001 for the 5.0 mg/kg/day nifedipine group, $ $p <0.001 for the 20 mg/kg/day nifedipine group). Notably, at some point in time, the 5.0 mg/kg/day and 20 mg/kg/day nifedipine groups were significantly more potent than the 1.5 mg/kg/day nifedipine group (p <0.05; p < 0.05% or%% p < 0.001). n=4-19.

Figure 3 shows that the combination of FA and nifedipine significantly and completely further attenuated vascular remodeling in a dose-dependent manner. Angiotensin (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of regular diet, folic acid-containing customized diet (FA, 15 mg/kg/day), or customized diet containing FA and different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). H & E staining data showed that vascular remodeling characterized by elastic degeneration and adventitial hypertrophy, which was further significantly and completely further reduced in a dose-dependent manner by FA in combination with different doses of nifedipine, was significantly reduced in apoE-deficient mice infused with Ang II by oral administration of FA.

Fig. 4 shows that the combination of FA and nifedipine significantly and completely further attenuated elastin degradation in a dose-dependent manner. Angiotensin (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of regular diet, folic acid-containing customized diet (FA, 15 mg/kg/day), or customized diet containing FA and different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). VVG staining data showed that the inboard elastin degradation, characterized by elastin flattening and breakdown, was significantly reduced further in dose-dependent manner by the combination of FA with different doses of nifedipine in apoE-deficient mice infused with Ang II by oral administration of FA.

Fig. 5A-5B show that the combination of FA and nifedipine significantly and completely further attenuated total superoxide production and eNOS uncoupling in a dose-dependent manner. Angiotensin II (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of conventional food, or orally administered folic acid (FA, 15 mg/kg/day) in combination with different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). Fig. 5A shows the total superoxide yield as measured by Electron Spin Resonance (ESR). The data show that the total superoxide yield in the aorta isolated from apoE-deficient mice infused with Ang II was significantly increased, which was significantly eliminated by oral FA administration. The combination of FA with nifedipine at 1.5 mg/kg/day, 5 mg/kg/day and 20 mg/kg/day significantly and completely further attenuated the total superoxide production in a dose-dependent manner. * P <0.01, p <0.001, n=5-7. Fig. 5B shows aortic eNOS uncoupling activity by ESR measurement. The data show that eNOS uncoupling activity reflected by L-NAME suppressible superoxide production is completely diminished by oral administration of FA alone or in combination with different doses of nifedipine. * p <0.05, < p <0.01, n=5-7. A decrease in L-NAME production of superoxide indicates that eNOS is unconjugated to produce superoxide, while an increase in L-NAME production of superoxide indicates that eNOS is conjugated to produce NO.

Figure 6 shows that the combination of FA and nifedipine significantly further improved NO bioavailability in a dose dependent manner. Ang II was infused into ApoE deficient mice and fed regular food, or a mixture of food and FA (15 mg/kg/day), or a mixture of food and FA with different concentrations of nifedipine (1.5 mg/kg/day, 5 mg/kg/day and 20 mg/kg/day). Angiotensin II (Ang II,1000 ng/mg/min) was infused into ApoE-deficient mice of 6-8 months of age for 4 weeks in the presence of conventional food, or orally administered folic acid (FA, 15 mg/kg/day) in combination with different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, and 20 mg/kg/day). Aortic NO bioavailability was determined by Electron Spin Resonance (ESR). The data show that NO bioavailability in the aorta isolated from apoE-deficient mice infused with Ang II is significantly reduced, which is significantly recovered by oral FA administration. The combination of FA with nifedipine at 1.5 mg/kg/day, 5 mg/kg/day and 20 mg/kg/day significantly and dose dependently restored NO bioavailability further. * p <0.05, < p <0.01, < p <0.001, n=6-8.

Figure 7 shows that the combination of FA and nifedipine significantly further improved aortic H in a dose dependent manner ₄ B bioavailability. Ang II was infused into ApoE deficient mice and fed regular food, or a mixture of food and FA (15 mg/kg/day), or a mixture of food and FA with different concentrations of nifedipine (1.5 mg/kg/day, 5 mg/kg/day and 20 mg/kg/day). Determination of aortic H by HPLC ₄ B bioavailability. The data indicate apoE at infusion of Ang IIAortic H in deficient mice ₄ B levels were significantly reduced, which was significantly recovered by oral FA administration. The combination of FA with nifedipine at 1.5 mg/kg/day, 5 mg/kg/day and 20 mg/kg/day significantly and dose dependently further restored H ₄ B bioavailability. * P is p<0.05，**p<0.01，***p<0.001，n＝4-10。

Detailed Description

Oxidative stress plays an important role in the development of AAA. It has been previously determined that a novel role for endothelial nitric oxide synthase (eNOS) in AAA formation is via maintenance of oxidative stress to induce MMP activity and matrix degradation. It has been demonstrated that in the novel model of hph-1 mice infused with angiotensin II (Ang II), eNOS uncoupling mediates AAA formation, with 79% of mice developing AAA within 2 weeks of Ang II infusion and 14% dying from aneurysm rupture (Gao L et al, hypertension (2012)). This is by far the most robust AAA model. Restoration of dihydrofolate reductase (DHFR) expression using oral Folate (FA) administration to re-couple eNOS significantly attenuated AAA formation in hph-1 mice infused with Ang II. Furthermore, a novel role of eNOS uncoupling in AAA development has been observed in apoE-deficient mice infused with Ang II, a well-established classical model of AAA, where oral FA administration is also effective to restore DHFR expression to recouple eNOS, resulting in elimination of aneurysm formation. Furthermore, the inventors have further elucidated that eNOS uncoupling mediates Fbn1 ^C1039G/+ A novel role in Thoracic Aortic Aneurysm (TAA) and AAA formation in Ma Fanzeng Syndrome (MFS) mice, which is targeted with the FA diet to re-couple eNOS to reduce superoxide production and restore NO bioavailability, thereby reducing TAA and AAA formation. Thus, these data further demonstrate the important role of unconjugated eNOS in mediating AAA and TAA formation and demonstrate the general efficacy of FA to attenuate AAA and TAA formation via improved DHFR expression to restore eNOS conjugation activity.

Hypertension is a risk factor for AAA, and AAA patients often have co-existing hypertension. Early studies also investigated the effect of low and high doses of the antihypertensive drug nifedipine (which is a calcium channel blocker) on AAA formation. Although at low doses of nitro-compoundsThe nifedipine has no effect on blood pressure, but both low and high doses of nifedipine can prevent eNOS uncoupling via restoration of DHFR expression and inhibition of NADPH oxidase, thereby attenuating AAA formation. High doses of nifedipine can also reduce blood pressure simultaneously and are therefore particularly valuable for treating patients suffering from both AAA and co-existing hypertension. Nevertheless, neither FA alone nor nifedipine alone is sufficient to completely attenuate AAA formation. The aim of this study was therefore to examine whether the combination therapy of FA and nifedipine has a synergistic effect of enhancing efficacy in inhibiting AAA growth. Thus, apoE-deficient mice infused with Ang II were treated with a combination therapy of FA and different doses of nifedipine (1.5 mg/kg/day, 5 mg/kg/day or 20 mg/kg/day), which exhibited a novel and significantly improved therapeutic effect on AAA formation in a dose-dependent manner. The combination of FA with different doses of nifedipine significantly and completely further attenuated total superoxide production and eNOS uncoupling activity in a dose-dependent manner, leading to significantly and completely attenuated vascular remodeling to reduce aneurysm formation; FA and high dose of nifedipine (20 mg/kg/day) completely inhibited AAA formation to zero incidence. This is accompanied by a dose-dependent further increase in the bioavailability of NO and H ₄ B bioavailability, and recovery of eNOS function/coupling activity. The combination of FA with different doses of nifedipine further significantly and completely eliminated the abdominal aortic dilation determined by ultrasound in a dose-dependent manner, wherein FA-plus-dose nifedipine (20 mg/kg/day) attenuated the abdominal aortic size 100% relative to control levels. These findings very significantly confirm that the novel combination therapy of FA with nifedipine can be used as a robust, first-initiating and most effective oral drug for the treatment of aortic aneurysms.

The present invention is based in part on the following findings: combination therapy of FA and nifedipine reduced vascular remodeling characterized by elastin degradation and adventitial hypertrophy in a dose-dependent manner in the AAA model. The protective effects on AAA morbidity and related pathophysiological changes are due to the potentiating effects of combination therapies on eNOS functional recovery and oxidative stress relief. The combination of FA with different doses of nifedipine was significant in a dose dependent mannerAnd completely further attenuate superoxide production while also completely re-coupling eNOS in a dose-dependent manner to restore NO bioavailability. Combination therapy did further increase eNOS cofactor H ₄ B, while also improving the outcome of eNOS re-coupling. The decrease in eNOS uncoupling can be quantified as eNOS cofactor H ₄ And B. These data indicate that the combination therapy of FA and nifedipine can be used as a novel oral drug for the effective treatment of aortic aneurysms. The role of eNOS uncoupling and oxidative stress in mediating aneurysm formation is the common pathway for AAA, cerebral aneurysms and Thoracic Aortic Aneurysms (TAA) (Gao et al, hypertension (2012), huang et al, redox Biology (2021) and Starke et al, curr Neuroasc Res.2013, month 8; 10 (3): 247-255). Thus, the combination therapies disclosed herein can be used to prevent or treat AAA, cerebral aneurysms, and/or TAAs. Given the common pathway between TAA, cerebral aneurysm and AAA, the combination therapies disclosed herein will show a synergistic effect when administered to subjects with TAA, cerebral aneurysm and/or AAA.

In one aspect, provided herein are pharmaceutical compositions comprising a folic acid compound and a calcium channel blocker.

Pharmaceutical composition

The compositions, kits, and methods of the invention are useful for treating an individual in need thereof (e.g., an individual having an AAA, cerebral aneurysm, or TAA aneurysm). In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When administered to an animal (such as a human), the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.

The one or more folic acid compounds and one or more calcium channel blockers (e.g., nifedipine compounds) may be administered to the subject in a therapeutically effective amount, preferably in the form of a pharmaceutical composition. In some embodiments, the therapeutically effective amount of the one or more folic acid compounds and the one or more calcium channel blockers (e.g., nifedipine compounds) ranges from about 0.01-60mg/kg body weight, about 0.01-45mg/kg body weight, about 0.01-30mg/kg body weight, or about 0.01-15mg/kg body weight of the one or more folic acid compounds, and about 0.01-40mg/kg body weight, about 0.01-35mg/kg body weight, about 0.01-30mg/kg body weight, about 0.01-25mg/kg, about 0.01-20mg/kg, about 0.01-15mg/kg, about 0.01-10mg/kg, about 0.01-5mg/kg, or about 0.01-1.5mg/kg body weight of the one or more calcium channel blockers (e.g., nifedipine compounds). In some embodiments, the ratio of the one or more folic acid compounds to the one or more calcium channel blockers (e.g., nifedipine compounds) administered ranges from about 10:1 to about 1:10. In some embodiments, the amounts of one or more folic acid compounds and one or more calcium channel blockers (e.g., nifedipine compounds) are provided or administered in synergistic amounts or synergistic ratios. In some embodiments, the amount of one or more folic acid compounds administered to a subject is about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, or about 1-1750mg, and the amount of one or more calcium channel blockers (e.g., nifedipine compounds) administered to a subject is about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, about 1-1750mg, about 1-2100mg, or about 1-2450mg. The dosage for treatment may be increased or decreased during a given treatment.

Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions (such as water or physiological buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils such as olive oil, or injectable organic esters). In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes that avoid transport or diffusion through the epithelial barrier, such as injection or implantation), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients may be selected, for example, to affect the delayed release of the agent or to selectively target one or more cells, tissues or organs. The pharmaceutical compositions may be in dosage unit form such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, freeze-dried formulations for reconstitution, powders, solutions, syrups, suppositories, injections and the like. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical application, such as a lotion, cream or ointment.

The pharmaceutically acceptable carrier may comprise a physiologically acceptable agent that acts, for example, to stabilize, increase solubility, or to increase absorption of a compound, such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates (such as glucose, sucrose, or dextran), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of pharmaceutically acceptable carrier, including physiologically acceptable agents, depends on, for example, the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix into which, for example, a compound of the invention may be incorporated. Liposomes, for example, comprising phospholipids or other lipids are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to prepare and administer.

The term "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) Cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The pharmaceutical composition (preparation) may be administered to a subject by any of a variety of routes of administration, including, for example, oral (e.g., drenches in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., patches applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for use in such routes of administration can be found, for example, in U.S. Pat. nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and in the patents cited in such U.S. patents.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, such amounts range from about 0.01% to about 99.99%, preferably from about 5% to about 70%, most preferably from about 10% to about 30% of the active ingredient in one hundred percent.

Methods of preparing these formulations or compositions include the step of associating an active compound, such as a compound of the invention, with a carrier and optionally one or more accessory ingredients. Typically, the formulation is prepared by: the compounds of the invention are homogeneously and intimately associated with liquid carriers or finely divided solid carriers or both, and the product is then shaped if necessary.

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavoring base, typically sucrose and acacia or tragacanth), freeze-dried formulations, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a confectionary lozenge (using an inert base such as gelatin and glycerol, or sucrose and acacia) and/or as a mouthwash, and the like, each containing a predetermined amount of a compound of the invention as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like) for oral administration, the active ingredient is mixed with one or two pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and/or any of the following: (1) Fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) Binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerin; (4) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) Wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) a colorant. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also comprise buffering agents. Similar types of solid compositions may also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like.

Tablets may be prepared by compression or molding, optionally together with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants or dispersants. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Other solid dosage forms of tablets and pharmaceutical compositions, such as sugar-coated pills, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose, other polymer matrices, liposomes and/or microspheres in varying proportions for providing the desired release profile. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved in sterile water or some other sterile injectable medium prior to use. These compositions may also optionally contain opacifying agents and may be those which release the active ingredient only or preferentially, optionally in a delayed manner, at a certain part of the gastrointestinal tract. Examples of embedding compositions that may be used include polymeric substances and waxes. The active compound may also be in microencapsulated form, where appropriate containing one or more of the above excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, freeze-dried for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be admixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

In addition to the active compounds, the ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The spray may additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons (such as butane and propane).

Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms may be prepared by dissolving or dispersing the active compound in an appropriate medium. As discussed above, absorption enhancers may also be used to increase the flux of compounds through the skin. The rate of such flux may be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Any of the compounds or agents disclosed herein may be administered parenterally.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, proper fluidity can be maintained by the use of a coating material, such as lecithin, by the maintenance of the required particle size, in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. Furthermore, absorption of injectable pharmaceutical forms may be prolonged by the inclusion of agents which delay absorption (aluminum monostearate and gelatin).

In some cases, it is desirable to slow down the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The rate of absorption of a drug then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered pharmaceutical forms is accomplished by dissolving or suspending the drug in an oil vehicle.

The injectable depot forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer (polylactide-polyglycolide). Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

For use in the methods of the invention, the active compound may be administered as such or as a pharmaceutical composition containing, for example, from 0.01% to 99.99% (more preferably, from 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

The method of introduction may also be provided by a rechargeable device or a biodegradable device. In recent years, various sustained release polymer devices have been developed and tested in vivo for controlled delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants to provide sustained release of compounds at a particular target site.

The actual dosage level of the active ingredient in the pharmaceutical composition may be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to a particular patient, composition, and mode of administration without toxicity to the patient.

The selected dosage level will depend on a variety of factors including the particular compound or combination of compounds employed or the activity of the ester, salt or amide thereof; route of administration; the time of application; the rate of excretion of the particular compound employed; duration of treatment; other drugs, compounds and/or materials used in combination with the particular compound employed; age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors well known in the medical arts.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian may begin a dose of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect and step up the dose until the desired effect is achieved. By "therapeutically effective amount" is meant a concentration of a compound sufficient to elicit the desired therapeutic effect. It will be generally understood that the effective amount of the compound will vary depending on the weight, sex, age and medical history of the subject. Other factors that affect an effective amount can include, but are not limited to, the severity of the patient's condition, the condition being treated, the stability of the compound, and, if desired, another type of therapeutic agent to be administered with the compounds of the present invention. A larger total dose may be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al (1996) Harrison's Principles of Internal Medicine, 13 th edition, 1814-1882, incorporated herein by reference).

In general, a suitable daily dose of active compound used in the compositions and methods of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

If desired, an effective daily dose of the active compound may optionally be administered in unit dosage form as one, two, three, four, five, six or more sub-doses administered individually at appropriate intervals throughout the day. In certain embodiments of the invention, the active compound may be administered twice or three times per day. In a preferred embodiment, the active compound will be administered once daily.

The patient receiving such treatment is any animal in need thereof, including primates, particularly humans; and other mammals such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, the compounds of the present invention may be used alone or in combination with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benzphetamine, benzathine, betaine, calcium hydroxide, choline, dansyl, diethanolamine, diethylamine, 2- (diethylamino) ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4- (2-hydroxyethyl) morpholine, piperazine, potassium, 1- (2-hydroxyethyl) pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, na salts, ca salts, K salts, mg salts, zn salts, or other metal salts. In some embodiments of the present invention, in some embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutarate, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipate, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+) -camphoric acid, (+) -camphor-10-sulfonic acid, capric acid (capric acid/decylic acid), caproic acid (caproic acid/hexamic acid), caprylic acid (capric acid/octanic acid), carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid (cyclic acid), dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid galactose diacid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid and salts of undecylenic acid.

The pharmaceutically acceptable acid addition salts may also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates may also be prepared. The source of such solvates may be from the crystallization solvent, be inherent in the preparation or crystallization solvent, or be foreign to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and antioxidant agents can also be present in the composition.

Examples of pharmaceutically acceptable antioxidants include: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) Oil-soluble antioxidants such as ascorbyl palmitate, butyl Hydroxy Anisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definition of the definition

Unless defined otherwise herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in connection with the following and the techniques described herein are those well known and commonly employed in the art: chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry.

Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., "Principles of Neural Science", mcGraw-Hill Medical, new York, n.y. (2000); motulsky, "Intuitive Biostatistics", oxford University Press, inc. (1995); loish et al, "Molecular Cell Biology, 4 th edition," W.H. Freeman & Co., new York (2000); griffiths et al, "Introduction to Genetic Analysis, 7 th edition," w.h. freeman & co., n.y. (1999); and Gilbert et al, "Developmental Biology, 6 th edition," Sinauer Associates, inc., sundland, MA (2000).

Unless otherwise defined herein, chemical terms used herein are used according to conventional usage in The art, as exemplified in "The McGraw-Hill Dictionary of Chemical Terms", parker s. Edit, mcGraw-Hill, san Francisco, c.a. (1985).

All of the above and any other publications, patents and published patent applications cited in this application are expressly incorporated herein by reference. In case of conflict, the present specification, including specific definitions, will control.

The term "agent" is used herein to refer to a compound (such as an organic or inorganic compound, a mixture of compounds), a biological macromolecule (such as a nucleic acid, an antibody, including portions thereof, as well as humanized, chimeric and human antibodies and monoclonal antibodies, proteins or portions thereof, e.g., peptides, lipids, carbohydrates), or an extract made from a biological material such as a bacterial, plant, fungal, or animal (particularly mammalian) cell or tissue. Agents include, for example, agents of known structure and agents of unknown structure.

"patient," "subject," or "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals such as humans, primates, livestock animals (including cattle, pigs, etc.), companion animals (e.g., canine, feline, etc.), and rodents (e.g., mice and rats).

"treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as is well understood in the art, "treatment" is a route for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean extending survival compared to the expected survival if not treated.

The term "preventing" is art-recognized and when used in relation to a condition such as local recurrence (e.g., pain), a disease such as cancer, a condition such as heart failure, or any other medical condition is well known in the art and includes administration of a composition that reduces the frequency of symptoms of or delays the onset of symptoms of the medical condition in a subject relative to a subject that does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of cancerous growths detectable in a population of patients receiving prophylactic treatment relative to an untreated control population; and/or delaying the occurrence of detectable cancerous growth in the treated population relative to the untreated control population, e.g., by a statistically and/or clinically significant amount.

The substance, compound or agent may be "administered" to the subject using one of a variety of methods known to those of skill in the art (Administering/administration of). For example, the compound or agent may be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinal, intracerebrally, and transdermally (by absorption, e.g., by a dermal catheter). The compound or agent may also be introduced suitably through a rechargeable or biodegradable polymeric device or other device (e.g., patches and pumps) or formulation that provides for the delayed, sustained or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

The appropriate method of administering a substance, compound or agent to a subject will also depend on, for example, the age and/or physical condition of the subject, as well as the chemical and biological properties (e.g., solubility, digestibility, bioavailability, stability, and toxicity) of the compound or agent. In some embodiments, the compound or agent is administered orally to the subject, e.g., by ingestion. In some embodiments, the orally administered compound or agent is administered in a delayed release or sustained release formulation, or using a device for such sustained release or delayed release.

As used herein, the phrase "co-administration" refers to any administration form of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are effective simultaneously in the patient, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, individuals receiving such treatment may benefit from the combined effects of different therapeutic agents. Any of the compounds or agents disclosed herein may be administered in combination.

A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the desired therapeutic effect when administered to a subject. The complete therapeutic effect does not necessarily occur by administering one dose, and may occur after administration of only a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required by a subject will depend, for example, on the size, health, and age of the subject, as well as the nature and extent of the condition being treated (such as cancer or MDS). The skilled artisan can readily determine the effective amount in a given situation by routine experimentation. As used herein, a "therapeutically effective amount" or "therapeutically effective dose" includes, but is not limited to, an amount or dose that exhibits a synergistic or technical effect when administered (e.g., co-administered) with another compound or agent disclosed herein.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" refers to the case where alkyl groups may be substituted as well as alkyl groups that are unsubstituted.

It will be appreciated that substituents and substitution patterns for the compounds of this invention may be selected by one of ordinary skill in the art to provide chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and those methods described below. If the substituent itself is substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure is created.

As used herein, the term "optionally substituted" means that one to six hydrogen groups in a given structure are substituted with a group of specified substituents including, but not limited to: hydroxy, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH ₂ -O-alkyl, -OP (O) (O-alkyl) ₂ or-CH ₂ OP (O) (O-alkyl) ₂ . Preferably, "optionally substituted" means that one to four hydrogen groups in a given structure are substituted with the substituents described above. More preferably, one to three hydrogen groups are substituted with substituents as described above. It is understood that the substituents may be further substituted.

As used herein, the term "alkyl" refers to a saturated aliphatic group including, but not limited to, C ₁ -C ₁₀ Straight chain alkyl or C ₁ -C ₁₀ Branched alkyl groups. Preferably, the "alkyl" group means C ₁ -C ₆ Straight-chain alkyl group or C ₁ -C ₆ Branched alkyl groups. Most preferably, the "alkyl" group refers to C ₁ -C ₄ Straight-chain alkyl group or C ₁ -C ₄ Branched alkyl groups. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, 4-octyl, and the like. The "alkyl" group may be optionally substituted.

The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbyl C (O) -, preferably alkyl C (O) -.

The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbyl C (O) NH-.

The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbyl C (O) O-, preferably alkyl C (O) O-.

The term "alkoxy" refers to an alkyl group having oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like.

The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, and may be represented by the general formula alkyl-O-alkyl.

The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, the linear or branched alkyl groups have 30 or fewer carbon atoms in their backbone (e.g., C for linear chains _1-30 For branched chains C _3-30 ) And more preferably 20 or moreFew carbon atoms.

Furthermore, the term "alkyl" as used throughout the specification, examples and claims is intended to include both unsubstituted and substituted alkyl groups, the latter referring to alkyl moieties having substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2, 2-trifluoroethyl, and the like.

When used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, the term "C _x-y "OR" C _x -C _y "is intended to include groups containing from x to y carbons in the chain. C (C) ₀ Alkyl represents hydrogen (in the case of a group in the terminal position), and a bond if the group is internal. C (C) _1-6 Alkyl groups contain, for example, 1 to 6 carbon atoms in the chain.

As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, and may be represented by the general formula alkyl S-.

The term "amino" as used herein refers to the following group:

wherein R is ⁹ And R is ¹⁰ Each independently represents hydrogen or a hydrocarbyl group, or R ⁹ And R is ¹⁰ Together with the N atom to which they are attached, complete a heterocyclic ring having 4 to 8 atoms in the ring structure.

The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, e.g., moieties that may be represented by

Or->

Wherein R is ⁹ 、R ¹⁰ And R is ¹⁰ ' each independently represents hydrogen or a hydrocarbon group, or R ⁹ And R is ¹⁰ Together with the N atom to which they are attached, complete a heterocyclic ring having 4 to 8 atoms in the ring structure.

As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5-to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is aromatic, e.g., the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The term "carbamate" is art-recognized and refers to the following groups

Or->

Wherein R is ⁹ And R is ¹⁰ Independently represent hydrogen or a hydrocarbyl group.

As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

The term "carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring in a bicyclic carbocyclic ring may be selected from the group consisting of saturated rings, unsaturated rings, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other rings. Each ring in the fused carbocycle may be selected from the group consisting of saturated rings, unsaturated rings, and aromatic rings. In exemplary embodiments, an aromatic ring (e.g., phenyl) may be fused with a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane, or cyclohexene). Any combination of saturated, unsaturated, and aromatic bicyclic rings, where valence permits, is included in the definition of carbocyclic ring. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo [2.2.1] heptane, 1, 5-cyclooctadiene, 1,2,3, 4-tetrahydronaphthalene, bicyclo [4.2.0] oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3, 4-tetrahydronaphthalene, bicyclo [4.2.0] octane, 4,5,6, 7-tetrahydro-1H-indene, and bicyclo [4.1.0] hept-3-ene. "carbocycles" may be substituted at any one or more positions capable of bearing a hydrogen atom.

The term "carbonate" is art-recognized and refers to the group-OCO ₂ -。

The term "carboxy" as used herein refers to a moiety of the formula-CO ₂ H represents a group.

The term "ester" as used herein refers to the group-C (O) OR ⁹ Wherein R is ⁹ Represents a hydrocarbyl group.

The term "ether" as used herein refers to a hydrocarbyl group that is linked to another hydrocarbyl group through oxygen. Thus, the ether substituent of the hydrocarbyl group may be hydrocarbyl-O-. The ether may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycles and aryl-O-heterocycles. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

The terms "halo" and "halogen" as used herein mean halogen and include chlorine, fluorine, bromine and iodine.

The term "heteroarylalkyl" as used herein refers to an alkyl group substituted with a heteroaryl group.

The term "heteroaryl" includes substituted or unsubstituted aromatic monocyclic structures, preferably 5-to 7-membered rings, more preferably 5-to 6-membered rings, the ring structure of which includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, e.g., the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

The term "heterocycloalkyl" as used herein refers to an alkyl group substituted with a heterocyclyl group.

The terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3-to 10-membered ring, more preferably a 3-to 7-membered ring, the ring structure of which includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heterocyclyl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heterocyclic, e.g., the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl. Heterocyclic groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like.

The term "hydrocarbyl" as used herein refers to a group bonded through a carbon atom that does not have an=o or=s substituent, and typically has at least one carbon-hydrogen bond and a main carbon backbone, but may optionally contain heteroatoms. Thus, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl groups, but substituents such as acetyl (which has an = O substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not considered hydrocarbyl groups. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

The term "lower" when used in connection with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy is intended to include groups in which there are ten or fewer atoms in the substituent, preferably six or fewer atoms. For example, "lower alkyl" refers to an alkyl group containing ten or fewer carbon atoms, preferably six or fewer carbon atoms. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, respectively, whether they occur alone or in combination with other substituents, such as in the recitation of hydroxyalkyl and aralkyl groups (in which case, for example, atoms within an aryl group are not counted when carbon atoms in an alkyl substituent are counted).

The terms "polycyclyl", "polycyclic" and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each of the rings of the ring pair may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring comprises 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

The term "sulfate" is art-recognized and refers to-OSO ₃ H group or a pharmaceutically acceptable salt thereof.

The term "sulfonamide" is art-recognized and refers to a group represented by the general formula

Or->

Wherein R is ⁹ And R is ¹⁰ Independently represents hydrogen or a hydrocarbon group.

The term "sulfoxide" is art-recognized and refers to the group-S (O) -.

The term "sulfonate" is art-recognized and refers to the group SO ₃ H or a pharmaceutically acceptable salt thereof.

The term "sulfone" is art-recognized and refers to the group-S (O) ₂ -。

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be appreciated that "substitution" or "substitution by … …" includes implicit preconditions that such substitution is in accordance with the permissible valences of the substituted atoms and substituents, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo conversion (such as by rearrangement, cyclization, elimination, etc.). As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For suitable organic compounds, the permissible substituents can be one or more and the same or different. For the purposes of the present invention, a heteroatom (such as nitrogen) may have a hydrogen substituent and/or any permissible substituent of the organic compounds described herein which satisfy the valences of the heteroatom. Substituents may include any of the substituents described herein, for example halogen, hydroxy, carbonyl (such as carboxy, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that the substituted moiety on the hydrocarbon chain may itself be substituted, if appropriate.

As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

The term "thioester" as used herein refers to the group-C (O) SR ⁹ or-SC (O) R ⁹ Wherein R is ⁹ Represents a hydrocarbon group.

As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

The term "urea" is art-recognized and may be represented by the general formula

The term "modulation" as used herein includes inhibition or repression of function or activity (such as cell proliferation) and enhancement of function or activity.

The term "pharmaceutically acceptable" is art recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

"pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt suitable for or compatible with the treatment of a patient.

The term "pharmaceutically acceptable acid addition salt" as used herein refers to any non-toxic organic or inorganic salt of any base compound represented by formula I. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include monocarboxylic, dicarboxylic, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, and salicylic acids, and sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Mono-or di-acid salts may be formed, and such salts may exist in hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of formula I are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points than the free base form thereof. The selection of suitable salts will be known to those skilled in the art. Other non-pharmaceutically acceptable salts, such as oxalates, may be used, for example, for isolation of the compounds of formula I for laboratory use, or for subsequent conversion to pharmaceutically acceptable acid addition salts.

The term "pharmaceutically acceptable base addition salt" as used herein means any non-toxic organic or inorganic base addition salt of any acid compound represented by formula I or any intermediate thereof. Illustrative inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of suitable salts will be known to those skilled in the art.

Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereogenic center in their structure. This chiral center may exist in either the R configuration or the S configuration, with the R and S symbols used in accordance with the rules described in Pure appl.chem. (1976), 45,11-30. The present disclosure encompasses all stereoisomers, such as enantiomers and diastereomers, of the compounds, salts, prodrugs, or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 2001/062726.

In addition, certain compounds containing an alkenyl group may exist as Z (cis (zusammen)) or E (trans (entgegen)) isomers. In each case, the disclosure includes mixtures and individual isomers.

Some of these compounds may also exist in tautomeric forms. Such forms are also intended to be included within the scope of the present disclosure, although not explicitly indicated in the above formula.

"prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host after administration to form a compound of the present disclosure (e.g., a compound of formula I). Typical examples of prodrugs include compounds having a biologically labile or cleavable (protecting) group on the functional moiety of the active compound. Prodrugs include compounds that may be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of the use of esters or phosphoramidates as prodrugs of biostable or cleavable (protecting) groups are disclosed in U.S. patent nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. Prodrugs of the present disclosure are metabolized to produce compounds of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in "Design of Prodrugs" editor h. Bundegaad, elsevier, 1985.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful in formulating a drug for pharmaceutical or therapeutic use.

Method

The methods provided herein include preventing or treating an aneurysm (e.g., AAA, cerebral aneurysm, or TAA) by co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof. In some aspects, the method comprises ameliorating at least one symptom or pathology of an aneurysm, comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof. The symptoms are as followsOr the pathology may be, for example, increased superoxide yield, increased eNOS uncoupling activity, decreased Nitric Oxide (NO) bioavailability, tetrahydrobiopterin (H ₄ B) Reduced bioavailability, increased vascular (abdominal aorta, thoracic aorta or blood vessels in the brain), increased vascular remodeling, increased elastin degradation (flattening and breakdown), increased vascular inflammation/macrophage infiltration, increased Matrix Metalloproteinase (MMP) activation, increased adventitial hypertrophy, or reduced eNOS function.

Also provided herein are methods of reducing superoxide production, eNOS uncoupling activity, vascular (blood vessels in the abdominal aorta, thoracic aorta, or brain), vascular remodeling, elastin degradation (flattening and decomposition), vascular inflammation/macrophage infiltration, matrix Metalloproteinase (MMP) activation, and/or adventitial hypertrophy in a subject having an aneurysm, comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof. In some embodiments, provided herein are methods of increasing eNOS function, and Nitric Oxide (NO), and tetrahydrobiopterin bioavailability in a subject having an aneurysm by co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

In some embodiments, the calcium channel blocker is a dihydropyridine compound. In some embodiments, the folic acid compound is folic acid. The calcium channel blocker and the folic acid compound may be in the same or separate compositions. The folic acid compound and the dihydropyridine compound may be in the same composition. The calcium channel blocker may be nifedipine.

In some embodiments, the folic acid compound and the calcium channel blocker are administered in therapeutically effective amounts (e.g., synergistic or expected synergistic amounts).

Folic acid compounds and calcium channel blockers may be present in about 10:1 to about 1:10, or about 100:1, about 95:1, about 90:1, about 85:1, about 80:1, about 75:1, about 70:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 30:1, about 29:1, about 28:1, about 27:1, about 26:1, about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 29:1 the folic acid compound is administered in a mass ratio to the calcium channel blocker of about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 0.95:1, about 0.9:1, about 0.85:1, about 0.8:1, about 0.75:1, about 0.7:1, about 0.65:1, about 0.6:1, about 0.55:1, about 0.5:1, about 0.45:1, about 0.4:1, about 0.35:1, about 0.3:1, about 0.25:1, about 0.2:1, about 0.15:1, about 0.1:1, about 0.05:1, or about 0.01:1. The folic acid compound and the calcium channel blocker may be administered in a mass ratio of folic acid compound to calcium channel blocker of about 3:1 to about 1:3.

The calcium channel blocker and the folic acid compound may be administered in a mass ratio of about 100:1, about 95:1, about 90:1, about 85:1, about 80:1, about 75:1, about 70:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 10:1 to about 1:10, such as about 30:1, about 29:1, about 28:1, about 27:1, about 26:1, about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1, about 0.5:1, about 0:1, about 0.0:1, about 0:0.5:1, about 0.0:1, about 0.0:0.0, about 0:1, about 0:0.0.0.5:1, about 0.0.0.0.0, about 0:1, about 0:0.0, about 0.0.0.0.0, about 0:1, about 0.0.0.0.0.0.0.0.0.5:1, about 0.0.0.0.5:1, about 0.1, about.1, about 0.1, about 0:1). The calcium channel blocker and the folic acid compound may be administered at a mass ratio of calcium channel blocker to folic acid compound of 1:3 or 3:1.

In some embodiments, the folic acid compound is administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, or about 1-1750 mg. The folic acid compound may be present in at least or about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47. 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 1.9 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8, 8.1, 8.8, 8.3, 8.8, 8.9, 8.8.9, 8.8, 8.9, 8.8.9, 8.9 and 8.9. 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.1, 15.2 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3. 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 140, 145 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825. 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530, 1535, 1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590, 1595, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710, 1715, 1720, 1725, 1720, 1735, 1740, 1745, 1750 1755, 1760, 1765, 1770, 1775, 1780, 1785, 1790, 1795, 1800, 1805, 1810, 1815, 1820, 1825, 1830, 1835, 1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890, 1895, 1900, 1905, 1910, 1915, 1920, 1925, 1930, 1935, 1940, 1945, 1950, 1955, 1960, 1965, 1970, 1975, 1980, 1985, 1990, 1995, or 2000 milligrams of total, mg/kg of subject body weight, or mg/kg/day.

In some embodiments, the calcium channel blocker is administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, about 1-1750mg, about 1-2100mg, or about 1-2450 mg. In some embodiments of the present invention, in some embodiments, the folic acid compound may be present in at least or about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46. 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 1.8, 1.7, 0.9 and 0.91; 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8. 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 135, 25, 90, 95, 100, 105, 110, 115, 120, 130, 135 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800. 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140; 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530, 1535, 1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590, 1595, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710, 1715, 1720, 1725, 1730, 1735, 1740, 1745, 1750, 1755, 1760, 1765, 1770; 1775, 1780, 1785, 1790, 1795, 1800, 1805, 1810, 1815, 1820, 1825, 1830, 1835, 1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890, 1895, 1900, 1905, 1910, 1915, 1920, 1925, 1930, 1935, 1940, 1945, 1950, 1955, 1960, 1970, 1975, 1980, 1985, 1990, 1995, 2000, 2005, 2010, 2015, 2020, 2025, 2030, 2035, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085. 2090, 2095, 2100, 2105, 2110, 2115, 2120, 2125, 2130, 2135, 2140, 2145, 2150, 2155, 2160, 2165, 2170, 2175, 2180, 2185, 2190, 2195, 2200, 2205, 2210, 2215, 2220, 2225, 2230, 2240, 2245, 2250, 2255, 2260, 2265, 2270, 2275, 2280, 2285, 2290, 2295, 2300, 2305, 2310, 2315, 2320, 2325, 2330, and so on 2335, 2340, 2345, 2350, 2355, 2360, 2365, 2370, 2375, 2380, 2385, 2390, 2395, 2400, 2405, 2410, 2415, 2420, 2425, 2430, 2435, 2440, 2445, 2450, 2455, 2460, 2465, 2470, 2475, 2480, 2485, 2490, 2495, 2500, 2505, 2510, 2515, 2520, 2525, 2530, 2535, 2540, 2545, 2550, 2555, 2560, 2565, 2570 the amount of mg/kg of the subject 2575, 2580, 2585, 2590, 2595, 2600, 2605, 2610, 2615, 2620, 2625, 2630, 2635, 2640, 2645, 2650, 2655, 2660, 2665, 2670, 2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720, 2725, 2730, 2735, 2740, 2745, 2750, 2755, 2760, 2765, 2770, 2775, 2780, 2785, 2790, 2795, 2800, 2805, 2810, 2815, 2820, 2825, 2830, 2835, 2840, 2845, 2850, 2855, 2860, 2865, 2870, 2880, 2885, 2890, 2895, 2905, 2920, 2925, 2930, 2935, 2940, 2945, 2950, 2955, 2960, 2970, 2965, 2980, 2995, or kg/kg.

The folic acid compound and the calcium channel blocker may be administered simultaneously or sequentially. Folic acid compounds and calcium channel blockers may be administered in combination. The folic acid compound and the calcium channel blocker may be administered in separate dosage forms.

The aneurysm may be an abdominal aortic aneurysm, a cerebral aneurysm or a thoracic aortic aneurysm.

The composition (e.g., folic acid compound or calcium channel blocker) can be administered to the subject systemically, intravenously, subcutaneously, intramuscularly, orally, or topically (e.g., locally to a tumor of the subject).

In some embodiments, the folic acid compound and the calcium channel blocker are administered in combination. In some embodiments, the co-administration comprises administering the folic acid compound and the calcium channel blocker at different times. In some embodiments, the co-administration comprises simultaneous administration of the folic acid compound and the calcium channel blocker. In some embodiments, the co-administration comprises administering the folic acid compound prior to the calcium channel blocker. In some embodiments, the co-administration comprises administering the folic acid compound after the calcium channel blocker.

The compositions disclosed herein (e.g., folic acid compounds or calcium channel blockers) can be administered for any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30 days, at least 60 days, at least three months, at least six months, at least one year, at least three years, at least five years, at least ten years, at least 20 years, at least 30 years, at least 40 years, at least 50 years. The doses may be administered as needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, tricyclically, once daily, twice daily, or three times daily. In certain embodiments, a dose of the composition is administered at regular intervals over a period of time. In some embodiments, a dose of the composition is administered at least once a week. In some embodiments, a dose of the composition is administered at least twice a week. In certain embodiments, the dose of the composition is administered at least three times per week. In some embodiments, the dosage of the composition is administered at least once daily. In some embodiments, a dose of the composition is administered at least twice daily. In some embodiments, a dose of the composition is administered at least three times per day. In some embodiments, each dose of the composition is administered for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least two years, at least three years, at least five years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years.

Toxicity and therapeutic efficacy of the combination of one or more folic acid compounds and one or more calcium channel blocker compounds and compositions thereof according to the present invention can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art. For example, the lethal dose LC50 (the dose lethal to 50% of the population expressed as concentration x exposure time) or LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population) can be determined by methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50. Folic acid compounds and calcium channel blocker compounds that exhibit large therapeutic indices are preferred. While combinations of folic acid compounds and calcium channel blocker compounds that cause toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the treatment site to minimize potential damage to the affected cells and thereby reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Preferred doses provide a range of circulating concentrations including ED50 that are tolerogenic, less toxic or nontoxic. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of one or more folic acid compounds and one or more, for example nifedipine compounds, can be initially estimated from cell culture assays. Dosages may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The level in the plasma may be measured, for example, by High Performance Liquid Chromatography (HPLC). In addition, the dosage appropriate for a given subject may be determined by the attending physician or qualified practitioner based on various clinical factors.

The actual dosage level of the active ingredient in the pharmaceutical composition or agent to be administered may be varied in order to obtain an amount of the active ingredient (e.g., agent described herein) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

The selected dosage level will depend on a variety of factors, including the activity of the particular agent employed; route of administration; the time of application; the rate of excretion or metabolism of the particular compound employed; duration of treatment; other drugs, compounds and/or materials used in combination with the particular compound employed; age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors well known in the medical arts.

A physician of ordinary skill in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a physician may prescribe and/or administer a dose of a compound employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and step up the dose until the desired effect is achieved.

Kit for detecting a substance in a sample

In some embodiments, the invention provides a kit comprising one or more folic acid compounds and one or more calcium channel blockers, optionally packaged in a composition with one or more agents or drug delivery devices, to prevent, inhibit, reduce, or treat an aneurysm in a subject. In some embodiments, the kit comprises optionally one or more folic acid compounds and one or more calcium channel blocker compounds in one or more unit dosage forms, packaged together as a package and/or in a drug delivery device, such as a prefilled syringe. The one or more folic acid compounds and/or the one or more calcium channel blocker compounds may be provided in the form of a pharmaceutical composition. In some embodiments, the one or more folic acid compounds and/or the one or more calcium channel blocker compounds are provided in the form of an oral formulation (e.g., a tablet).

In some embodiments, the kit comprises a carrier, package, or container that can be partitioned to house one or more containers, such as vials, tubes, and the like. In some embodiments, the kit optionally includes an identification description or tag associated with its use or instructions for use. In some embodiments, the kit includes information specified by a government agency regulating the manufacture, use or sale of the compounds and compositions contemplated herein.

Examples

The invention now being generally described, the same will be more clearly understood through the use of the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to be limiting.

Aortic aneurysms are a common and serious vascular disease with a high mortality rate resulting from the death of the patient from sudden rupture of an unpredictable aneurysm. Other treatment options are generally not available other than those that present a significant risk of surgical correction of the large aneurysms. The data indicate that oral administration of FA and nifedipine in combination therapy can be a robust, initial and effective treatment regimen for aortic and other aneurysms.

Example 1: the combination of folic acid and nifedipine significantly and completely further attenuated the host in a dose-dependent manner Aneurysm formation

The applicant has shown that Folic Acid (FA) is re-coupled via eNOS to attenuate superoxide production while at the same time restoring Nitric Oxide (NO) bioavailability, thereby eliminating vascular remodeling, while highly effective in alleviating aortic aneurysm formation (both AAA and TAA) in angiotensin II (Ang II) infused apolipoprotein E (apoE) deficient mice, ang II infused hyperphenylalaninemia (hph-1) mice, and Fbn1 marfan syndrome mice. Nonetheless, FA alone is not sufficient to completely attenuate the development of aortic aneurysms. Here, the therapeutic effect of the combination of FA and nifedipine on aortic aneurysm was examined. Oral administration of FA (15 mg/kg/day) significantly reduced AAA morbidity from 85.71% to 18.75% in apoE-deficient mice infused with Ang II, whereas the combination of FA with different doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day or 20 mg/kg/day) significantly and completely reduced the morbidity in a dose-dependent manner to 12.5%, 11.76% and 0.00%, respectively. The combination therapy also significantly and completely further reduced the dilation of the abdominal aorta as determined by ultrasound in a dose-dependent manner, wherein the combination of FA with 20 mg/kg/day nifedipine reduced the size of the abdominal aorta by 100% relative to the control level. Combination therapy dose dependently eliminates Vascular remodeling characterized by elastin degradation and adventitial hypertrophy. The combination of FA with nifedipine significantly and completely further reduced Electron Spin Resonance (ESR) in a dose-dependent manner, confirming the total superoxide yield and eNOS uncoupling activity, which was accompanied by aortic NOs and H ₄ Further improvement of the dose dependence of B bioavailability.

Oxidative stress has been shown to play an important role in the formation of aortic aneurysms, including AAA and Thoracic Aortic Aneurysms (TAA). Unconjugated endothelial nitric oxide synthase (eNOS) plays a key role in AAA formation by maintaining oxidative stress to induce Matrix Metalloproteinase (MMP) activation and matrix degradation. It was first demonstrated that eNOS uncoupling mediates AAA formation in a novel AAA model, i.e., hph-1 mice infused with angiotensin II (Ang II), with 79% of mice developing AAA within 2 weeks of Ang II infusion, with 14% dying from aneurysm rupture. Hph-1 mice infused with Ang II proved to be the most robust AAA model to date. Restoring the function of dihydrofolate reductase (DHFR) with Folic Acid (FA) to re-couple eNOS significantly attenuated AAA formation in these animals. Furthermore, it has been further demonstrated that eNOS uncoupling plays a novel role in AAA development in apoE-deficient mice infused with Ang II, which is a well-established classical model of AAA, whereas oral FA administration is also effective in restoring DHFR function to recouple eNOS, resulting in elimination of aneurysms. Indeed, knocking out DHFR in mice to promote eNOS uncoupling resulted in excessive AAA formation. It has further been shown that activation of NADPH Oxidase (NOX) isoforms is located upstream of unconjugated eNOS to drive AAA formation. Furthermore, eNOS uncoupling also plays a key role in driving TAA and AAA formation in Fbn1 Marfan syndrome mice (a classical TAA model), which can also be targeted by FA to mitigate the formation of both TAA and AAA (Huang et al, redox Biology, (2021)). Thus, these data further demonstrate a key causal role of eNOS uncoupling in AAA and TAA formation, while also establishing the general efficacy of FA in treating aortic aneurysms in different animal models via restoration of eNOS coupling activity to reduce oxidative stress and subsequent vascular remodeling. Likewise, oxidative stress driven vascular remodeling is a common pathway between AAA/TAA and cerebral aneurysms, and thus FA is equally effective in treating cerebral aneurysms. Nonetheless, FA alone is not sufficient to completely alleviate aortic aneurysm formation.

Hypertension is a risk factor for aortic aneurysms, and AAA patients and TAA patients often have co-existing hypertension. It is also a major risk factor for cerebral aneurysms. Previous studies have investigated the effect of low and high doses of the antihypertensive drug nifedipine, which is a calcium channel blocker, on AAA formation. Low doses of nifedipine have no effect on blood pressure, however, both low and high doses of nifedipine can attenuate AAA formation via DHFR restoration and NOX inhibition, thereby attenuating eNOS uncoupling. High doses of nifedipine can also simultaneously reduce blood pressure and are therefore particularly valuable for treating patients with aortic aneurysms with concomitant hypertension. However, the use of FA or nifedipine alone is not sufficient to completely alleviate the development of AAA. The data presented herein examine whether the combination of FA with different doses of nifedipine enhances the efficacy of treating AAA. apoE-deficient mice infused with Ang II were orally administered FA (15 mg/kg/day) and combinations of FA with increasing doses of nifedipine (1.5 mg/kg/day, 5.0 mg/kg/day, or 20.0 mg/kg/day). Notably, the data demonstrate that the combination of FA with different doses of nifedipine significantly and completely further reduced AAA formation in a dose-dependent manner. Combination therapy was strongly more effective in restoring eNOS coupling activity to increase NO bioavailability and reduce oxidative stress, thereby eliminating vascular remodeling characterized by elastin degradation and adventitial hypertrophy, wherein the combination of FA with nifedipine significantly and completely further reduced AAA morbidity from 18.75% inhibition in FA group (reduced from 85.71% in apoE-deficient mice infused with Ang II) to FA plus 1.5 mg/kg/day, 5.0 mg/kg/day, or 20 mg/kg/day 12.50%, 11.76% and 0.00% of nifedipine group, respectively. The dilation of the abdominal aorta, as determined by echocardiography, was significantly and completely further reduced by combining FA with nifedipine in a dose-dependent manner, wherein the combination of FA with a high dose of nifedipine of 20 mg/kg/day reduced the dilation of the abdominal aortic area 100% relative to the control level.

The combination of FA with nifedipine significantly and completely further reduced the incidence of AAA in a dose-dependent manner

The incidence of AAA was examined in apoE-deficient mice infused with Ang II that underwent oral administration of FA alone or in combination therapy with different doses of nifedipine (1.5, 5 or 20 mg/kg/day). As shown in FIGS. 1A-1B, 85.71% (30 out of 35) of Ang II infused apoE-deficient mice developed AAA after 4 weeks of Ang II infusion. Oral FA administration significantly reduced AAA morbidity to 18.75% (p <0.001, 6 out of 32). The combination of FA with different doses of nifedipine (1.5, 5 or 20 mg/kg/day) significantly and completely reduced the incidence of AAA in a dose-dependent manner to 12.50% (3 out of p <0.05, 24), 11.76% (2 out of p <0.001, 17) and 0.00% (0 out of p <0.001, 18), respectively, compared to the FA alone group, indicating that the combination therapy of FA with nifedipine was significantly more effective in the treatment of aortic aneurysms.

The combination of FA with nifedipine significantly and completely further attenuated the dilation of the abdominal aorta in a dose-dependent manner

To examine the expansion of the abdominal aorta of Ang II infused apoE-deficient mice treated with FA alone or in combination with nifedipine at 1.5, 5 or 20 mg/kg/day, the size of the abdominal aorta was monitored weekly using ultrasound. Fig. 2A shows representative ultrasound images of the abdominal aorta from all experimental groups over 4 weeks, while fig. 2B shows the packet data. Notably, the abdominal aorta of Ang II infused apoE-deficient mice was significantly increased compared to untreated apoE-deficient mice, which was significantly attenuated by oral administration of FA (fig. 2A-2B, compare apoe+ang II, #p <0.01 or # #p <0.001 for the same week). Notably, the combination of FA with 1.5 mg/kg/day, 5 mg/kg/day, or 20 mg/kg/day of nifedipine significantly or completely further attenuated abdominal aortic dilation in a dose-dependent manner, with FA plus 20 mg/kg/day nifedipine groups showing 100% reduced aortic dilation relative to control levels (fig. 2A-2B, @ p <0.01 or @ p <0.001 for the 1.5 mg/kg/day nifedipine group, & gt @ p <0.01 or @ p <0.001 for the 5.0 mg/kg/day nifedipine group, & gt $p <0.01 or $p <0.001 for the 20 mg/kg/day nifedipine group). For some time points (should this still be kept

The combination of FA with nifedipine significantly and completely further attenuated vascular remodeling in a dose-dependent manner

Aortic aneurysm formation is accompanied by extensive vascular remodeling, which is characterized by degradation of the matrix to allow aortic expansion. To examine the extent of vascular remodeling that occurs in the presence of various treatments during AAA, freshly isolated aorta was embedded in paraffin, sectioned and stained with hematoxylin-eosin (H & E). As shown in fig. 3, oral administration of FA significantly attenuated vascular remodeling characterized by elastic degeneration and adventitial hypertrophy in apoE-deficient mice infused with Ang II. The combination of FA with nifedipine at 1.5, 5 or 20 mg/kg/day significantly and completely further attenuated vascular remodeling in a dose-dependent manner

The combination of FA and nifedipine significantly and completely further attenuated elastin degradation in a dose-dependent manner

To assess medial elastin degradation, VVG staining was performed using aortic sections from all experimental groups. As shown in fig. 4, in apoE-deficient mice infused with Ang II, elastin degradation characterized by flattening and breakdown was evident, which was significantly attenuated by oral administration of FA. Importantly, the combination of FA with different doses of nifedipine (1.5, 5 or 20 mg/kg/day) significantly and completely further attenuated elastin degradation in a dose-dependent manner.

The combination of FA with nifedipine significantly and completely further attenuated the total superoxide yield and eNOS uncoupling activity in a dose-dependent manner

eNOS uncoupling plays a mediator role in AAA formation in both new and classical models of hph-1 and apoE deficient mice infused with Ang II and TAA formation in classical models of Fbn1 marfan syndrome mice. Thus, changes in total aortic superoxide production and eNOS uncoupling activity from all treatment groups were examined herein. Freshly prepared aortic lysates were subjected to Electron Spin Resonance (ESR) to determine superoxide production with or without L-NAME (NOS inhibitor). If eNOS is functional and coupled, inhibition of it by L-NAME will increase the superoxide measured, as eNOS is producing NO to scavenge superoxide. However, if eNOS is dysfunctional, uncoupled and producing superoxide, inhibition of it by L-NAME will result in a decrease in the measured superoxide.

As shown in fig. 5A, the total superoxide yield in the aorta isolated from apoE-deficient mice infused with Ang II was significantly increased, which was significantly eliminated by oral FA administration. This represents the molecular mechanism of the protective effect of FA on AAA formation. Interestingly, the combination of FA with different doses of nifedipine (1.5, 5 or 20 mg/kg/day) significantly and completely further attenuated the total superoxide yield in a dose-dependent manner.

As shown in fig. 5B, eNOS uncoupling activity was significantly increased in apoE-deficient mice infused with Ang II. At baseline, the degree of uncoupling of eNOS was minimal in apoE-deficient mice and well compensated. Oral administration of FA reversed eNOS uncoupling activity in apoE-deficient mice infused with Ang II (fig. 5B). The combination of FA with nifedipine at 1.5, 5 or 20 mg/kg/day also completely attenuated the eNOS uncoupling activity, while at the same time the total superoxide production was more effectively eliminated in a dose-dependent manner (fig. 5A-5B). These data indicate that the re-coupling of eNOS and further reduction of total superoxide production are the basis for the enhancement of efficacy in attenuating AAA formation by combination therapy of FA with nifedipine.

The combination of FA and nifedipine significantly further improved NO bioavailability in a dose-dependent manner.

Since FA plus nifedipine re-couples eNOS as described above to reduce superoxide production, aortic NO bioavailability from all experimental groups was subsequently examined using Electron Spin Resonance (ESR). The data show a significant decrease in NO bioavailability in the aorta isolated from apoE-deficient mice infused with Ang II, which was significantly recovered by oral FA administration (fig. 6). Interestingly, the combination of FA with different doses of nifedipine (1.5, 5 or 20 mg/kg/day) significantly further improved aortic NO bioavailability in a dose-dependent manner, while restoring eNOS function to mediate protection against AAA formation.

The combination of FA and nifedipine significantly further improved aortic H in a dose dependent manner ₄ B bioavailability

As an important cofactor for eNOS, H ₄ B deficiency indicates eNOS uncoupling. Herein, aortic H from all experimental groups was determined using HPLC ₄ B bioavailability. As shown in fig. 7, ang II-induced H in apoE-deficient mice was significantly eliminated by oral FA administration ₄ B was absent, while the combination of FA with different doses of nifedipine (1.5, 5 or 20 mg/kg/day) significantly further improved aortic H in a dose-dependent manner ₄ B bioavailability. In conjunction with the above data, these findings strongly suggest that multiple doses of FA in combination with nifedipine proved to be an effective AAA treatment regimen to eliminate oxidative stress and subsequent vascular remodeling via attenuation of eNOS uncoupling activity.

Discussion of the invention

The data presented herein demonstrate for the first time that combination therapy of FA with nifedipine reduces aortic aneurysm formation in apoE-deficient mouse models infused with Ang II, and that nifedipine addition shows a dose-dependent effect in enhancing the efficacy of FA in treating aortic aneurysms. AAA morbidity determined by ultrasound definition of aortic dilation and post mortem necropsy was synergistically, and significantly and completely further eliminated by the combination of FA and nifedipine in a dose-dependent manner. Combination therapy of FA with nifedipine significantly and completely further reduced vascular remodeling characterized by elastin degradation and adventitial hypertrophy in a dose-dependent manner. These protective effects on AAA morbidity and related pathophysiological changes are due to the potentiating effects of combination therapies on eNOS function recovery and oxidative stress relief. FA Combinations of different doses of nifedipine significantly and completely further attenuate superoxide production in a dose-dependent manner, while also completely re-coupling eNOS in a dose-dependent manner to restore NO bioavailability. Combination therapy did further increase eNOS cofactor H ₄ B, while also improving the outcome of eNOS re-coupling. These data strongly demonstrate that combination therapy of FA with nifedipine can be used as a novel oral drug in the treatment of aneurysms, particularly considering the shared common oxidative stress pathway between AAA/TAA and cerebral aneurysms.

Oxidative stress plays a role in the pathogenesis of AAA. Specifically, unconjugated eNOS mediates AAA formation by driving oxidative stress and subsequent vascular remodeling, as shown in the data herein. It has been demonstrated that in hph-1 mice infused with Ang II (a newly established robust AAA model), eNOS uncoupling mediates AAA formation, which can be attenuated by: FA restores endothelial dihydrofolate reductase (DHFR) function/H ₄ B bioavailability to re-couple eNOS. It is also shown herein that eNOS uncoupling mediates sustained oxidative stress and AAA formation in classical AAA models of apoE-deficient mice infused with Ang II, which can be alleviated by oral FA administration. In both hph-1 and apoE deficient mice infused with Ang II, oral FA administration effectively reduced AAA morbidity, prevented abdominal aortic dilation, and reduced maladaptive vascular remodeling. This is mediated by: FA re-couples with eNOS to attenuate superoxide production while restoring NO bioavailability, thereby eliminating oxidative stress by shutting down the enzymatic system of unconjugated eNOS. Furthermore, it has recently been shown that eNOS uncoupling in classical models of Fbn1 marfan syndrome mice plays a similar causal role in TAA formation, wherein the re-coupling of eNOS with FA diet also attenuated TAA and AAA formation. The data from this study further confirm these findings to determine the key role of eNOS uncoupling in aortic aneurysm formation (including AAA and TAA), FA targeting proved to be a highly effective treatment option. Nonetheless, FA alone does not completely attenuate the aneurysm formation. Therefore we propose that FA is in combination with another Combination therapy with the antihypertensive drug nifedipine (which is also effective in alleviating AAA formation as shown in previous studies) will have synergistic and enhanced effects in the treatment of AAA. The role of eNOS uncoupling/oxidative stress in mediating aneurysm formation is a common mechanism for both AAA and TAA. Thus, the combination therapies disclosed herein are useful for preventing or treating TAAs. The combination therapies disclosed herein will also be synergistic when administered to individuals with TAAs. Likewise, it is synergistically effective in treating cerebral aneurysms.

Thus, these data show that apoE deficient mice infused with Ang II were treated with a combination therapy of FA with different doses of nifedipine to examine the potential synergistic and potentiating effects of the treatment on attenuation of aneurysmal formation. The combination of FA with 1.5, 5 or 20 mg/kg/day nifedipine significantly and completely reduced AAA morbidity further to 12.50%, 11.76% and 0.00%, respectively, in a dose-dependent manner, indicating significantly improved efficacy compared to FA alone (18.75%). The combination therapy further eliminates abdominal aortic dilation in a dose dependent manner compared to FA alone group, wherein FA plus 20 mg/kg/day nifedipine attenuated abdominal aortic size 100% relative to control level, again demonstrating a significant further improvement in therapeutic efficacy for treating AAA. Furthermore, vascular remodeling characterized by elastin degradation and adventitial hypertrophy was also significantly and completely further alleviated in the FA plus nifedipine group in a dose-dependent manner.

Combination therapy of FA with different doses of nifedipine significantly and completely further improved the efficacy of FA in attenuating the overall superoxide yield and eNOS uncoupling activity in a dose-dependent manner. While treatment with FA more directly targets eNOS in the coupled state, nifedipine targets seemingly unrelated calcium channel pathways in the cell. In addition to its upregulating effect on DHFR, nifedipine also has an inhibitory effect on NADPH oxidase located upstream of unconjugated eNOS. This differential mechanism may explain the synergistic effect between FA and nifedipine to reduce aortic aneurysm formation via enhanced recovery of eNOS function/NO bioavailability to eliminate the efficacy of oxidative stress and subsequent vascular remodeling. In fact, as an index of eNOS uncoupling activityH of (2) ₄ B deficiency was also significantly further alleviated in a dose-dependent manner by the combination of FA with different doses of nifedipine.

The data disclosed herein strongly demonstrate that the combination of FA with different doses of nifedipine significantly and completely further attenuated aortic aneurysm formation and associated pathophysiology and molecular changes in a dose-dependent manner. Thus, oral administration of FA and nifedipine has proven to be an effective oral medicament for the treatment of aneurysms (including AAA/TAA and cerebral aneurysms) driven by the common pathway of eNOS uncoupling/oxidative stress, wherein compositions containing high doses of nifedipine are particularly suitable for the treatment of aneurysms patients with co-existing hypertension.

Example 5 materials and methods

Animals

Animal use and experimental procedures were approved by the institutional animal care and use committee (Institutional Animal Care and Usage Committee, IACUC) of the university of california, los angeles division (University of California Los Angeles, UCLA). The breeding stock of apoE-deficient mice was purchased from Jackson Labs (Bar Harbor, ME, breed B6.129P2-ApoEtm1 Unc/J) and fed indoors. Animals were housed in ventilated cages and were allowed to drink water and standard food under standard care by laboratory animal medicine department (Division of Laboratory Animal Medicine, DLAM) staff. Experiments were performed using male apoE-deficient mice of 6-8 months of age.

Ang II infusion by micro osmotic pump

Animals receiving Ang II infusion were anesthetized in an isoflurane chamber with 5% isoflurane and then transferred to a nose cone continuously fed 1.5-2% isoflurane to maintain anesthesia. The back area between the scapulae has been cleared of hair and disinfected. Then making a small incision at the cleaned site; an osmotic minipump (Alzet, model 2004) containing Ang II (1000 ng/kg/min, sigma-Millipore, st.Louis, MO, USA) was subcutaneously implanted in mice. Surgical wounds were closed with surgical staples and the animals were allowed to recover in heated cages.

Orally administered Folic Acid (FA) and nifedipine

For groups of animals treated orally with Folic Acid (FA) or a combination of FA and nifedipine at different doses, standard foods were replaced with custom food tablets containing FA alone (15 mg/kg/day) or a combination of FA and nifedipine (1.5, 5 or 20 mg/kg/day) two days prior to implantation of the osmotic minipump for Ang II infusion, and throughout the 4 week Ang II infusion study period.

Ultrasonic detection of abdominal aortic size

The abdominal aortic dilation in each experimental group was monitored using ultrasound. Animals were anesthetized with isoflurane and placed on a temperature control stand. The isoflurane levels were adjusted throughout the experiment to maintain heart rates between 400bpm and 500bpm while maintaining adequate anesthesia in the animals. Hair was removed from the abdomen using depilatory cream, and a preheated ultrasound transmission gel was applied to the abdominal region. An ultrasound probe was placed on the gel to visualize the aorta laterally (Vevo 2100,MS400,30MHz.FUJIFILM VisualSonics,Inc, toronto, ontario, canada). Doppler measurements are used to identify the presence of pulsatile flow in the aorta. By visualizing the aorta immediately adjacent to the left renal artery branch in all animals, consistent positioning of the abdominal aorta for image acquisition is ensured.

Anatomical examination and histological analysis of the abdominal aorta

At the end of the 4 week study period, CO was used ₂ Animals were euthanized. The aorta was removed rapidly from the body, flushed with ice-cold Krebs/HEPES buffer, and connective tissue and fat were cleared. AAA morbidity is determined by ultrasound assessment of abdominal aortic dilation as described above, and direct examination of the postmortem abdominal aorta. For histological analysis, small sections (5 mm) of the abdominal aorta in the upper renal region were removed and fixed overnight in 4% paraformaldehyde, then incubated in 10% sucrose for 24 hours, and then embedded in paraffin. In the case of AAA, the analysis is performed using the center section of AAA. Sections were cut into 5 μm at UCLA Pathology Core and hematoxylin-eosin (H) was performed according to standard protocols&E) Dyeing.

Verhoeff-Van Gieson (VVG) staining

Paraffin-embedded aortic sections were dewaxed by successive washes in xylene (2×), decreasing ethanol from 100%, 90%, 75% to 50%, and distilled water. The sections were stained in Verhoeff solution for 70min and then differentiated in 2% ferric trichloride for 90 seconds. Sections were incubated with 5% sodium thiosulfate for 60 seconds and then counterstained with Van Gieson solution. The sections were then dehydrated with 95% and 100% alcohol and finally washed with xylene. After drying, the plaques were mounted with Permount mounting medium (SP 15-100, thermo-Fisher Scientific, pittsburgh, pa., USA) and images captured with a Nikon TE2000-U fluorescence microscope.

Electron spin resonance assay for aortic superoxide total yield and eNOS uncoupling activity

Aortic superoxide production was determined by Electron Spin Resonance (ESR). Briefly, freshly isolated aorta was homogenized in lysis buffer containing 1:100 protease inhibitor cocktail (Millipore Sigma, P8340), centrifuged at 12,000rpm for 15min, and protein supernatant was collected. After the protein concentration was determined using a protein assay kit (Bio-Rad), 5. Mu.g of protein was loaded into an ice-cold and nitrogen-sparged modified Krebs/HEPES buffer (KHB in mmol/L: naCl 99;KCl 4.7;MgSO4 1.2;KH2PO4 1.0;CaCl2 1.9;NaHCO325; glucose 11.1, naHEPES20) containing diethyl dithiocarbamic acid (5 mmol/L), deferoxamine (25 mmol/L) and superoxide-specific spin trap methoxycarbonyl-2, 5-tetramethylpyrrolidine (CMH, 500. Mu. Mol/L, axxora, san Diego, calif., USA). The mixture was loaded into glass capillaries (Kimble, 71900-50, dover, oh, USA) and the total superoxide yield was determined using an Electron Spin Resonance (ESR) spectrophotometer (eScan, bruker, billerica, MA, USA) in the following manner: the difference was taken in the presence or absence of PEG-SOD (polyethylene glycol-superoxide dismutase; 100U/mL, milliporeSigma, st.Louis, MO, USA). To determine eNOS uncoupling activity, measurements were made by adding L-NAME (N (. Omega. -nitro-L-arginine methyl ester; 10. Mu. Mol/L,80587,Cayman Chemical,Ann Arbor,MI). A decrease in L-NAME production of superoxide indicates that eNOS is unconjugated to produce superoxide, while an increase in L-NAME production of superoxide indicates that eNOS is conjugated to produce NO. The ESR settings used were: a center field, 3480; sweep width, 9G; microwave frequency, 9.78GHz; microwave power, 21.02mW; modulation amplitude, 2.47G;512 point resolution; receiver gain, 1000.

Electron spin resonance measurement of aortic nitric oxide production

Aortic Nitric Oxide (NO) bioavailability was determined by Electron Spin Resonance (ESR). Briefly, freshly isolated aortic rings were spin trapped Fe with freshly prepared NO-specific ²⁺ (DETC) ₂ (0.5 mmol/L) colloids were incubated together in modified Krebs/HEPES buffer in the presence of calcium ionophore A23187 (10 mmol/L) at 37℃for 60min. After incubation, aortic fragments were flash frozen in liquid nitrogen and loaded into finger Dewar (finger Dewar) for analysis using ESR spectrophotometers (esan, bruker, billerica, mass., USA). The instrument used was set as follows: a center field, 3440; sweep width, 100G; microwave frequency, 9.796GHz; the microwave power is 13.26mW; modulation amplitude, 9.82G;512 point resolution; and a receiver gain 356.

Aorta H ₄ HPLC determination of B bioavailability

To determine the aorta H ₄ Level B, freshly isolated aorta at H ₄ B lysis buffer (0.1 mol/L phosphoric acid, 1mmol/L EDTA, 10mmol/L DL dithiothreitol), centrifuging at 12,000rpm for 10min, and then separating the supernatant in acidic (0.2 mol/L trichloroacetic acid, 2.5% I) ₂ And 10% KI) and alkaline (0.1 mol/L NaOH, containing 0.9% I) ₂ And 1.5% KI) solution. After centrifugation, 10 μl of the supernatant was injected into an HPLC system equipped with a fluorescence detector (Schimadzu America Inc, carlsbad, CA, USA). Measurement of H using excitation and emission wavelengths of 350nm and 450nm ₄ Level B.

Statistical analysis

All packet data are presented as mean ± SEM. Statistical analysis was performed using Prism software. Comparison between groups was performed using one-way ANOVA followed by a Newman-Keuls post-hoc test. Statistical significance was set at p <0.05. The AAA morbidity is compared using a list, with a statistical threshold of 0.05.

Incorporated by reference

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

Equivalent scheme

While specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of the specification and claims that follow. The full scope of the invention should be determined with reference to the claims, along with their full scope of equivalents, the description, and such variations.

Claims

1. A kit comprising a folic acid compound and a calcium channel blocker.

2. The kit of claim 1, wherein the folic acid compound is of formula I

Or a pharmaceutically acceptable salt thereof, wherein:

m is an integer selected from 0-3; and is also provided with

n is an integer selected from 0-4.

3. The kit of claim 2, wherein each R ¹ Independently hydrogen, acyl, ester, amide or alkyl.

4. The kit of claim 2, wherein each R ¹ Is hydrogen.

5. The kit of any one of claims 2-4, wherein each R ² Independently hydrogen or alkyl.

6. The kit of claim 5, wherein each R ² Is hydrogen.

7. The kit of any one of claims 2-6, wherein m is 0.

8. The kit of any one of claims 2-7, wherein n is 0.

9. The kit of claim 2, wherein the folic acid compound is

Or a pharmaceutically acceptable salt thereof.

10. The kit of claim 1, wherein the calcium channel blocker is a dihydropyridine compound.

11. The kit of claim 10, wherein the dihydropyridine compound is represented by formula II:

or a pharmaceutically acceptable salt thereof, wherein:

12. The kit of claim 11, wherein R ⁶ Is alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

13. The kit of claim 12, wherein R ⁶ Is an alkyl or aryl group.

14. The kit of claim 13, wherein R ⁶ Is methyl or substituted or unsubstituted phenyl.

15. The kit of claim 14, wherein R ⁶ Is phenyl optionally substituted with halogen, haloalkyl, alkyl or nitro.

16. The kit of claim 11, wherein the dihydropyridine compound is represented by formula II-a, II-b, II-c, or II-d:

17. the kit of any one of claims 11-16, wherein R ⁹ Is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

18. The kit of claim 17, wherein R ⁹ Is hydrogen.

19. The kit of claim 17, wherein R ⁹ Is an alkyl optionally substituted with halogen, amino, hydroxy, alkoxy, cyano, nitro, acyl, ester, amide, alkylthio, carbocyclyl, heterocyclyl, aryl or heteroaryl.

20. The kit of claim 19, wherein R ⁹ Is that

21. The kit of any one of claims 11-20, wherein R ¹⁰ Is cyano, amino, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

22. The kit of claim 21, wherein R ¹⁰ Is cyano, amino or alkyl.

23. The kit of claim 22, wherein R ¹⁰ Is methyl.

24. The kit of claim 11, wherein the dihydropyridine compound is represented by formula II-a-1, II-b-1, II-c-1, or II-d-1:

25. the kit of any one of claims 11-24, wherein R ⁵ Is an alkoxy, amino, alkyl, carbocyclyl, heterocyclylAn aryl or heteroaryl group.

26. The kit of claim 25, wherein R ⁵ Is an alkoxy or amino group.

27. The kit of claim 26, wherein R ⁵ Is an alkoxy group optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

28. The kit of claim 27, wherein R ⁵ Is that

29. The kit of any one of claims 11-28, wherein R ⁷ Is acyl, ester, amide, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

30. The kit of claim 29, wherein R ⁷ Is acyl, ester, amide, carbocyclyl, heterocyclyl, aryl or heteroaryl.

31. The kit of claim 30, wherein R ⁷ Is an ester, heterocyclic or heteroaryl group.

32. The kit of claim 31, wherein R ⁷ Is that

33. The kit of any one of claims 11-33, wherein R ⁸ Is hydrogen, hydroxy, alkoxy, alkylthio, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

34. The kit of claim 33, wherein R ⁸ Is hydrogen, hydroxy, alkoxy, alkylthio, or alkyl optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

35. The kit of claim 34, wherein R ⁸ Is hydrogen,

36. The kit of claim 10, wherein the dihydropyridine compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

37. The kit of claim 10, wherein the dihydropyridine compound is

Or a pharmaceutically acceptable salt thereof.

38. The kit of any one of claims 1-37, wherein the folic acid compound and the dihydropyridine compound are in the same composition.

39. A method of preventing or treating an aneurysm, the method comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

40. A method of ameliorating a symptom of an aneurysm, the method comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

41. The method of claim 40, wherein the symptom is increased superoxide production, increased eNOS uncoupling activity, decreased Nitric Oxide (NO) bioavailability, tetrahydrobiopterin (H ₄ B) Reduced bioavailability, increased vascular (abdominal aorta, thoracic aorta or blood vessels in the brain), increased vascular remodeling, increased elastin degradation (flattening and breakdown), increased vascular inflammation/macrophage infiltration, increased Matrix Metalloproteinase (MMP) activation, increased adventitial hypertrophy, or reduced eNOS function.

42. A method of reducing superoxide production, eNOS uncoupling activity, vasodilation of blood vessels (abdominal aorta, thoracic aorta, or blood vessels in the brain), vascular remodeling, elastin degradation (flattening and decomposition), vascular inflammation/macrophage infiltration, matrix Metalloproteinase (MMP) activation, and/or adventitial hypertrophy in a subject having an aneurysm, the method comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

43. A method of increasing eNOS function, nitric Oxide (NO), and tetrahydrobiopterin bioavailability in a subject having an aneurysm, the method comprising co-administering a folic acid compound and a calcium channel blocker to a subject in need thereof.

44. The method of any one of claims 39 to 43, wherein the folic acid compound is of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

m is an integer selected from 0-3; and is also provided with

n is an integer selected from 0-4.

45. The method of claim 44, wherein each R is ¹ Independently hydrogenAcyl, ester, amide or alkyl.

46. The method of claim 44, wherein each R is ¹ Is hydrogen.

47. The method of any one of claims 44 to 46, wherein each R ² Independently hydrogen or alkyl.

48. The method of claim 47, wherein each R is ² Is hydrogen.

49. The method of any one of claims 44 to 48, wherein m is 0.

50. The method of any one of claims 44 to 49, wherein n is 0.

51. The method of claim 44, wherein the folic acid compound is

Or a pharmaceutically acceptable salt thereof.

52. The method of any one of claims 39 to 43, wherein the calcium channel blocker is a dihydropyridine compound.

53. The method of claim 52, wherein the dihydropyridine compound is represented by formula II:

or a pharmaceutically acceptable salt thereof, wherein:

54. The method of claim 53, wherein R is ⁶ Is alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

55. The method of claim 54, wherein R is ⁶ Is an alkyl or aryl group.

56. The method of claim 55, wherein R is ⁶ Is methyl or substituted or unsubstituted phenyl.

57. The method of claim 56, wherein R is ⁶ Is phenyl optionally substituted with halogen, haloalkyl, alkyl or nitro.

58. The method of claim 53, wherein the dihydropyridine compound is represented by formula II-a, II-b, II-c, or II-d:

59. the method of any one of claims 53 to 58, wherein R ⁹ Is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

60. The method of claim 59, wherein R is ⁹ Is hydrogen.

61. The method of claim 59, wherein R is ⁹ Is an alkyl optionally substituted with halogen, amino, hydroxy, alkoxy, cyano, nitro, acyl, ester, amide, alkylthio, carbocyclyl, heterocyclyl, aryl or heteroaryl.

62. The method of claim 61, wherein R is ⁹ Is that

63. The method of any one of claims 53 to 62, wherein R ¹⁰ Is cyano, amino, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

64. The method of claim 63, wherein R is ¹⁰ Is cyano, amino or alkyl.

65. The method of claim 64, wherein R is ¹⁰ Is methyl.

66. The method of claim 53, wherein the dihydropyridine compound is represented by formula II-a-1, II-b-1, II-c-1, or II-d-1:

67. The method of any one of claims 53-66, wherein R ⁵ Is an alkoxy, amino, alkyl, carbocyclyl, heterocyclyl, aryl or heteroaryl group.

68. The method of claim 67, wherein R is ⁵ Is an alkoxy or amino group.

69. The method of claim 68, wherein R is ⁵ Is an alkoxy group optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

70. The method of claim 69, wherein R is ⁵ Is that

71. The method of any one of claims 53-70, wherein R ⁷ Is acyl, ester, amide, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

72. The process of claim 71 wherein R is ⁷ Is acyl, ester, amide, carbocyclyl, heterocyclyl, aryl or heteroaryl.

73. The method of claim 72, wherein R is ⁷ Is an ester, heterocyclic or heteroaryl group.

74. The method of claim 73, wherein R is ⁷ Is that

/>

75. The method of any one of claims 53-74, wherein R ⁸ Is hydrogen, hydroxy, alkoxy, alkylthio, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl or heteroaryl.

76. The method of claim 75, wherein R is ⁸ Is hydrogen, hydroxy, alkoxy, alkylthio, or alkyl optionally substituted with halogen, cyano, nitro, amino, hydroxy, alkylthio, alkoxy, acyloxy, amido, acyl, ester, amido, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl.

77. The process of claim 76 wherein R is ⁸ Is hydrogen, and is preferably selected from the group consisting of hydrogen,

78. the method of claim 52, wherein the dihydropyridine compound is selected from the group consisting of:

/>

or a pharmaceutically acceptable salt thereof.

79. The method of claim 52, wherein the dihydropyridine compound is

Or a pharmaceutically acceptable salt thereof.

80. The method of any one of claims 52-79, wherein the folic acid compound and the dihydropyridine compound are in the same composition.

81. The method of any one of claims 39-80, wherein the folic acid compound is folic acid.

82. The method of any one of claims 39-81, wherein the calcium channel blocker is nifedipine.

83. The method of any one of claims 39-78, wherein the folic acid compound and the calcium channel blocker are administered in therapeutically effective amounts.

84. The method of any one of claims 39-83, wherein the folic acid compound and the calcium channel blocker are administered at a mass ratio of about 10:1 to about 1:10.

85. The method of claim 84, wherein the folic acid compound and the calcium channel blocker are administered in a mass ratio of about 3:1 to about 3:4.

86. The method of any one of claims 39-85, wherein the folic acid compound is administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, or about 1-1750 mg.

87. The method of any one of claims 39-86, wherein the calcium channel blocker is administered in an amount of about 1-350mg, about 1-700mg, about 1-1050mg, about 1-1400mg, about 1-1750mg, about 1-2100mg, or about 1-2450 mg.

88. The method of any one of claims 39-87, wherein the folic acid compound and the calcium channel blocker are administered simultaneously.

89. The method of any one of claims 39-88, wherein the folic acid compound and the calcium channel blocker are administered sequentially.

90. The method of claim 89, wherein said folic acid compound and said calcium channel blocker are in separate dosage forms.

91. The method of any one of claims 39-90, wherein the aneurysm is an abdominal aortic aneurysm, a cerebral aneurysm, or a thoracic aortic aneurysm.

92. The method of any one of claims 39-91, wherein the folic acid compound and the calcium channel blocker are administered orally.