Bempedoic Acid a Small Molecule Drug Process and Synthesis, Innovation And /Or Advantages, Development Status And /Or Regulatory| Lupine Publishers Status

  Journal of Surgery| Lupine Publishers

Abstract

Bempedoic acid (ETC-1002), a small molecule drug, promotes low density lipoprotein (LDL) receptor mediated clearance of LDL-cholesterol (LDL-C) by inhibition of adenosine triphosphate citrate lyase (ACL), a mechanism complementary to those of existing lipid-modifying therapies. Bempedoic acid is a pro-drug activated specifically within the liver where it inhibits ACL, a regulatory checkpoint within the cholesterol biosynthesis pathway. By inhibiting ACL, bempedoic acid reduces cholesterol synthesis in liver cells and triggers compensatory LDL receptor upregulation. Inhibiting ACL with bempedoic acid complements other mechanisms targeted by current therapies, resulting in additional lowering of LDL-C, without leading to increases in adverse events (AEs).b In the phase III clinical trials (NCT02666664, NCT02991118) of patients with high cardiovascular risk and elevated LDL-C not adequately controlled by their current therapy, patients are given a daily dose of 180 mg bempedoic acid as an oral tablet, whilst remaining on ongoing lipid-modifying therapy. The present paper describes the process and synthesis, innovation and /or advantages, development status and /or regulatory status of Bempedoic acid.

Keywords:Cardiovascular Disease; Hyperlipidemia; ETC-1002; Low Density Lipoprotein Cholesterol; Synthesis; Statin Associated Muscle Symptoms; Statin Intolerance; Regulatory Status

Introduction

The current standard of care for patients with hypercholesterolaemia is primarily statins which can reduce LDL-C. However, some patients, particularly those with heterozygous familial hypercholesterolaemia, coronary heart disease (CHD), CHD-risk equivalents, and other clinical manifestations of atherosclerotic cardiovascular disease [1] (ASCVD), require additional LDL cholesterol lowering on top of what can be achieved with maximum tolerated statin therapy. Additionally, there are patients who are unable to tolerate statins due to adverse advents such as muscle pain, or increased blood glucose. There is an unmet medical need for patients unable to achieve sufficient reduction in LDL cholesterol with existing treatment options and thus remain at increased risk of cardiovascular disease and the consequences thereof. Bempedoic acid requires activation by a specific enzyme acyl-CoA synthatase (ACSVL1), which is largely restricted to the liver. Therefore, it is believed that unlike statins, myotoxicity is unlikely to occur with bempedoic acid because it does not inhibit cholesterol biosynthesis in skeletal muscle due to the absence of ACSVL1 in these cells. The effect of bempedoic acid is additivenot redundant-to that of statins, because the target of bempedoic acid, ACL, is a distinct regulatory checkpoint on the cholesterol biosynthesis pathway than HMG-CoA reductase, the primary target of statins. Inability to tolerate statins because of muscle symptoms contributes to uncontrolled cholesterol levels and insufficient cardiovascular risk reduction. Bempedoic acid, a prodrug that is activated by a hepatic enzyme does not present in skeletal muscle, inhibits ATP‐citrate lyase [2], an enzyme upstream of β‐ hydroxy β‐methylglutaryl‐coenzyme A reductase in the cholesterol biosynthesis pathway. Statins are widely prescribed for lowering LDL-cholesterol (LDL-C) and reducing the risk of cardiovascular disease. However, many patients are statin intolerant and unable to achieve sufficient LDL-C lowering due to muscle-related side effects. Bempedoic acid (ETC-1002)’s mechanism of action is similar to that of statins, but because it does not inhibit the cholesterol biosynthesis pathway in skeletal muscle, myotoxicity is unlikely to occur. ETC-1002 was first discovered at the original Esperion Therapeutics, which was acquired by Pfizer in 2004 and subsequently spun-out as Esperion Therapeutics in 2008 along with ETC-1002 and other assets. Esperion continues development of ETC-1002 [3], which is currently in phase III trials as an LDL-Clowering agent in patients with hypercholesterolemia. This review discusses the drug candidate’s mechanism of action, effects, safety and clinical data. This paper explaining how the process of synthesis was carried and conclusion of Bempedoic acid offers a safe and effective oral therapeutic option for lipid lowering in patients who cannot tolerate statins.
a) The phase 3 CLEAR (Cholesterol Lowering via Bempedoic acid, an ACL‐Inhibiting Regimen) Serenity clinical trial demonstrates the lipid‐lowering efficacy of bempedoic acid, a firstin‐ class, prodrug, small‐molecule inhibitor of ATP‐citrate lyase, among patients with established statin intolerance and elevated low‐density lipoprotein cholesterol who were receiving stable background therapy.
b) Muscle‐related symptoms contributed to the history of statin intolerance for almost all patients.
c) Although bempedoic acid acts on the same cholesterol biosynthesis pathway as statins, the muscle‐related adverse event rate in CLEAR Serenity with bempedoic acid, which is not activated in skeletal muscle, did not differ from placebo, even among patients who had experienced muscle‐related symptoms while on statin therapy [4].

Development Status And/or Regulatory Designations

Bempedoic acid does not currently have Marketing Authorisation in the EU/UK for any indication. Bempedoic acid or bempedoic acid with ezetimibe in a fixed-dose combination are in phase III clinical trials for the treatment of primary hypercholesterolaemia [5] (heterozygous familial and nonfamilial) or mixed dyslipidaemia in patients who are statin-intolerant, or for whom a statin is contraindicated. Bempedoic acid monotherapy and bempedoic acid with ezetimibe in a fixed-dose combination is also in phase III clinical trials for the treatment of primary hypercholesterolaemia (heterozygous familial and non-familial) or mixed dyslipidaemia in patients unable to reach LDL-Cholesterol goals with the maximum tolerated dose as an adjunct to diet in combination with a statin or statin with other lipid lowering therapies. Esperion Therapeutics was founded in April 2008 by former executives of, and investors in, the original Esperion Therapeutics which was founded in July 1998 and was bought by Pfizer for $1.3 billion in 2004 and then spun out in 2008. ETC-1002 was first discovered at the original Esperion [5], and Esperion subsequently acquired the rights to it from Pfizer in 2008. Esperion own the exclusive worldwide rights to ETC-1002.

Preparation

Bempedoic acid [5,6] was prepared by condensation of 1,5-dibromopentane (I) with ethyl isobutyrate (II) by means of LDA in THF in the presence of DMPU at −78 °C to give ethyl 7-bromo-2,2-dimethylheptanoate (III), which is dimerized with tosylmethyl isocyanide (IV) in the presence of NaH and Bu4NI in DMSO to yield diethyl 8-isocyano-2, 2,14,14-tetramethyl-8-(tosyl) pentadecanedioate (V). Reaction of intermediate (V) with aqueous HCl in CH2Cl2 affords 2,2,14,14-tetramethyl-8-oxopentadecanedioic acid diethyl ester (VI), which is hydrolyzed with aqueous KOH in refluxing EtOH/H2O to provide the dicarboxylic acid ESP-15228 (VII) (1-4). Ketone (VII) is finally reduced by means of NaBH4 in MeOH (1). Scheme 1[6].

7-Bromo-2,2-Dimethylheptanoic Acid Ethyl Ester

7-Bromo-2,2-dimethylheptanoic acid ethyl ester [5]

Under argon atmosphere and cooling with an ice-bath, a solution of lithium diisopropylamide in THF (1.7 L, 2.0 M, 3.4 mol) was slowly dropped into a solution of 1,5- dibromopentane (950 g, 4.0 mol) and ethyl isobutyrate (396 g, 3.4 mol) in THF (5 L) while keeping the temperature below +5 DC. The reaction mixture was stiπed at room temperature for 20 h and quenched by slow addition of saturated ammonium chloride solution (3L). The resulting solution was divided into three 4-L portions. Each portion was diluted with saturated ammonium chloride solution (5L) and extracted with ethyl acetate (2 ‘2L). Each 4-L portion of ethyl acetate was washed with saturated sodium chloride solution (2L), 1 N hydrochloric acid (2L), saturated sodium chloride solution (2L), saturated sodium bicarbonate solution (2L), and saturated sodium chloride solution (2L). The three separate ethyl acetate layers were combined into a single 12-L portion, dried over magnesium sulfate, and concentrated in vacuo to give the crude material (1.7L) which was purified by vacuum distillation. Two fractions were obtained: the first boiling at 88 – 104 °C / 0.6 ton (184.2 g), the second at 105 – 120 °C / 1.4 ton (409.6 g) for atotal yield of 60 %. 1H NMR (300 MHz, CDC1₃/ TMS): δ (ppm): 4.11 (q, 2 H, J = 7.2 Hz), 3.39 (t, 2 H, J = 6.8 Hz), 1.85 (m, 2 H), 1.56 – 1.35 (m, 4 H), 1.24 (t, 3 H, J = 7.2 Hz), 1.31 – 1.19 (m, 2 H), 1.16 (s, 6 H). 13C NMR (75 MHz, CDCI₃/TMS): δ (ppm): 177.9, 60.2, 42.1, 40.5, 33.8, 32.6, 28.6, 25.2, 24.2, 14.3. HRMS (El, pos): Calcd. for CπH₂₂Brθ2 (MH⁺): 265.0803, found: 265.0810.6.18.

2,2,14,14-tetramethyl-8-oxo-pentadecanedioic acid diethyl ester

Under Air atmosphere, to a solution of 7-bromo-2,2- dimethylheptanoic acid ethyl ester⁵ (26.50 g, 100 mmol), tetra-nbutylammonium iodide (3.69 g, 10 mmol) and p- toluenesulfonyl methyl isocyanide (9.80 g, 50 mmol) in anhydrous DMSO (300 mL) was added sodium hydride (4.80 g, 20.5 mmol, 60 % dispersion in mineral oil) at 5 – 10oC The reaction mixture was stiπed at room temperature for 20 h and quenched with ice-water (300 mL). The product was extracted with dichloromethane (3D 100 mL). The combined organic layers were washed with water (200 mL), half-saturated NaCl solution 100 mL), and saturated NaCl solution (200 mL), dried over MgS04, and concentrated in vacuo to get the crude 8-isocyano-2,2,14,14-teframethyl-8-(toluene-4- sulfonyl)-pentadecanedioic acid diethyl ester (36.8 g) as an orange oil, which was used in the next step without purification. To a solution of this crude product (36.8 g) in dichloromethane (450 mL) was added concentrated hydrochloric acid (110 mL) and the mixture was stiπed at room temperature for 1h. The solution was diluted with water (400 mL) and the aqueous layer was extracted with dichloromethane (200 mL). The combined organic layers were washed with saturated NaHC0 solution (2 x 150 mL) and saturated NaCl solution (150 mL). The organic solution was dried over Na2S04 and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, hexanes: ethyl acetate = 11:1) to give 2,2,14,14-tetramethyl-8-oxo-pentadecanedioic acid diethyl ester (12.20 g, 66 % over two steps) as a colorless oil. ^lH NMR (300 MHz, CDC1₃/TMS): δ (ppm): 4.11 (q, 4 H, J – 6.9 Hz), 2.37 (t, 4 H, J – 7.5 Hz), 1.58 – 1.47 (m, 8 H), 1.35 – 1.10 (m, 8 H), 1.24 (t, 6 H, J = 7.2 Hz), 1.15 (s, 12 H). ¹³C NMR (75 MHz, CDC1₃/TMS): δ (ppm): 211.6, 178.3, 60.5, 43.1, 42.5, 40.9, 30.1, 25.5, 25.1, 24.1, 14.7. HRMS (LSIMS, nba): Calcd. for C2₃IL₃O₅ (MH⁺): 399.3110, found: 399.3129.

8-Hydroxy-2.2.14,14-Tetramethylpentadecanedioic Acid

Under nitrogen atmosphere, sodium borohydride (0.06 g, 1.6 mmol) was added to a stiπed solution of 8-oxo-2,2,14,14- tetramethylpentadecanedioic acid (1.18 g, 3.4 mmol) in methanol (50 mL) at 0 °C. The reaction progress was momtored by thin layer chromatography (silica; hexanes: ethyl acetate = 50: 50). Additional sodium borohydride was added after 1h (0.48 g, 13 mmol). After 8 h, the reaction mixture was hydrolyzed with water (50 mL) and acidified with concentrated hydrochloric acid (3 mL) to pH 1. The solution was diluted with water (50 mL) and exfracted with dichloromethane (4 x 25 mL). The combined organic layers were washed with saturated sodium chloride solution (2 x 30 mL), dried over magnesium sulfate, concentrated in vacuo, and dried in high vacuo to give 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid (0.7 g, 60 %) as a very viscous oil. ¹H NMR (300 MHz, CDC1₃/TMS): δ (ppm): 7.42 (br. s, 3 H), 3.59 (br. s, 1 H), 1.65 – 1.00 (m, 20 H), 1.18 (s, 12 H). ¹³C NMR (75 MHz, CDC1₃/TMS): δ (ppm): 184.5, 71.8, 42.1, 40.5, 37.0, 29.8, 25.2, 25.1, 24.9, 24.8. HRMS (FAB): Calcd. for Cι₉H₃₇0₅ (MH⁺): 345.2635, found: 345.2646. HPLC: 83.8 % purity. Keto-substituted hydrocarbons with 11−19 methylene and bis-terminal hydroxyl and carboxyl groups have been synthesized and evaluated in both in vivo and in vitro assays for their potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes as well as for their effects on lipid and glycemic variables in obese female Zucker fatty rats [Crl:(ZUC)-faBR] following 1 and 2 weeks of oral administration. The most active compounds were found to be symmetrical with four to five methylene groups separating the central ketone functionality and the gem dimethyl or methyl/aryl substituents. Furthermore, biological activity was found to be greatest in both in vivo and in vitro assays for the tetramethyl-substituted keto diacids and diols (e.g., 10c, 10g,14c), and the least active were shown to be the bis(arylmethyl) derivatives (e.g., 10e, 10f,14f). Compound 14c dose-dependently elevated HDL-cholesterol, reduced triglycerides, and reduced NEFA, with a minimum effective dose of 30 mg/kg/day. Compound 10g dose-dependently modified non-HDL-cholesterol, triglycerides, and no esterified fatty acids, with a minimum effective dose of 10 mg/kg/day. At this dose, compound 10g elevated HDL-cholesterol levels 2−3 times higher than pretreatment levels, and a dose-dependent reduction of fasting insulin and glucose levels was observed.

Only Keto Compd Described

2,2,14,14-Tetramethyl-8-oxopentadecanedioic Acid [6] (10g).

According to the procedure given for 10f, 9g (8.54 g, 21.4 mmol) was saponified with KOH (85%, 4.53 g, 68.6 mmol) in EtOH (13 mL) and water (5 mL) at reflux for 4 h. The solid product obtained after usual workup was recrystallized from Et2O/hexanes (50 mL/50 mL), affording 10g (4.16 g, 57%) as colorlessneedles. Mp: 82−83 °C. 1H NMR (CDCl₃): δ 11.53 (br, 2H), 2.39 (t, 4H, J = 7.3), 1.60−1.50 (m, 8 H), 1.30−1.20 (m, 8 H), 1.18 (s, 12 H). ¹³C NMR (CDCl3): δ 211.7, 185.0, 42.8, 42.3, 40.4, 29.7, 25.1, 24.8, 23.8.

HRMS (LSIMS, gly): calcd for C₁₉H₃₅O₅ (MH⁺) 343.2484, found 343.2444. HPLC: Alltima C-8 column, 250 × 4.6 mm, 5 μm; 60% acetonitrile/40% 0.05 M KH₂PO₄, flow rate 1.0 mL/min; RI, t_R 6.50 min, 92.6% pure. Bempedoic acid is an oral medicinal product that is in clinical development for the treatment of people with primary hypercholesterolaemia or mixed dyslipidaemia [7-9] with high cardiovascular risk. Abnormal levels of lipids in the blood characterises dyslipidaemia. High levels of cholesterol in the blood (hypercholesterolemia) may be caused by genetic defects as seen in familial hypercholesterolaemia or may occur when genes and other factors such as lifestyle habits interact, as seen in non-familial hypercholesterolaemia. Most people with hypercholesterolaemia have mildly or moderately increased low-density lipoprotein cholesterol (LDL-C) levels (often considered the “bad” cholesterol that may cause blockages of blood vessels). Elevated levels of LDL-C increase the risk of cardiovascular disease, which is responsible for many deaths and disabilities. Bempedoic acid lowers LDL-C via a different mechanism of action and offers the potential advantage of reduced muscular adverse effects when compared to statins which are the current standard of care. Bempedoic acid is being developed for patients at high cardiovascular risk who are unable to reach LDL-C goals with the maximum tolerated dose of statins. The effect of bempedoic acid is additive-not redundant-to that of statins, and if licensed, may offer additional and effective treatment option to use in combination with dietary changes and other lipidmodifying therapies to treat primary hypercholesterolaemia or mixed dyslipidaemia. Bempedoic acid is claimed in U.S. Patent No. 7,335,799 that is scheduled to expire in December 2025, which includes 711 days of patent term adjustment, and might be eligible for a patent term extension period of up to five years. U.S. Patent Nos. 9,000,041, 8,497,301 and 9,624,152 claim methods of using bempedoic acid. PRODUCT: Bempedoic acid.

Bempedoic Acid (Esperion Therapeutics, Inc.)

With a targeted mechanism of action, bempedoic acid is a firstin- class, orally available, once-daily ACL inhibitor that reduces cholesterol biosynthesis and lowers elevated levels of LDL-C by up-regulating the LDL receptor, [10] but with reduced potential for muscle-related side effects. Completed Phase 1 and 2 studies in more than 800 patients treated with bempedoic acid have produced clinically relevant LDL-C lowering results of up to 30 percent as monotherapy, approximately 50 percent in combination with ezetimibe, and an incremental 20+ percent when added to stable statin therapy.

Mechanism of Action

In November 2016, we announced the publication of “Liverspecific ATP-citrate lyase inhibition by bempedoic acid decreases LDL-C and attenuates atherosclerosis,” by Stephen L. Pinkosky, our Associate Director of Translational Research and Biology, et al., in Nature Communications. The paper systematically outlines the experiments and analyses undertaken by us and our collaborators to fully understand the mechanism of action for how bempedoic acid reduces LDL-C, including its specificity for the liver. Bempedoic acid is a prodrug that once activated, inhibits ACL, an enzyme upstream of HMG-CoA reductase [11,12], (the molecular target of statins) in the cholesterol synthesis pathway. Like statins, bempedoic acid decreases cholesterol synthesis in the liver, which results in decreased intracellular cholesterol, up-regulation of LDL receptor activity and increased LDL-C clearance from the blood. Although bempedoic acid and statins both inhibit cholesterol synthesis in the liver, an important differentiating feature is that, unlike statins, bempedoic acid is inactive in skeletal muscle. Specifically, bempedoic acid is a prodrug which requires activation by a specific enzyme, very long-chain acyl-CoA synthetase, or ACSVL1, to convert bempedoic acid to its CoA activated form. This enzyme is present in the liver but not in skeletal muscle. Therefore, bempedoic acid does not inhibit the cholesterol biosynthesis pathway in skeletal muscle, thus providing a mechanistic basis for reduced potential for muscle-related adverse effects. Bempedoic acid has been shown to provide incremental lowering of LDL-C when used in combination with both ezetimibe and statins at all doses [13].

Fixed Dose Combination Bempedoic Acid and Ezetimibe (BA+EZ)

In the second quarter of 2016, the Food and Drug Administration, or FDA, accepted our submission of an Investigational New Drug, or IND, application for the fixed dose combination of bempedoic acid 180 mg and ezetimibe 10 mg, or BA+EZ, which is in development for the same indications as bempedoic acid monotherapy [14,15] (LDL-C lowering and CV risk reduction). We recently completed a bioavailability study and a formulation of BA+EZ has been selected for manufacturing, development and, if approved, commercialization. We expect to announce clinical development and regulatory plans for BA+EZ in the first half of 2017.

Cardiovascular Disease and Elevated LDL-C

Cardiovascular disease, which results in heart attacks, strokes and other cardiovascular events, represents the number one cause of death and disability in western societies. The American Heart Association, or AHA, estimates that approximately 800,000 deaths in the United States were caused by cardiovascular disease in 2013. Elevated LDL-C is well-accepted as a significant risk factor for cardiovascular disease and the CDC estimates that 78 million U.S. adults have elevated levels of LDL-C. A consequence of elevated LDL-C is atherosclerosis, which is a disease that is characterized by the deposition of excess cholesterol and other lipids in the walls of arteries as plaque. The development of atherosclerotic plaques often leads to cardiovascular disease. The risk relationship between elevated LDL-C and cardiovascular disease was first defined by the Framingham Heart Study, [15] which commenced in 1948 to define the factors that contributed to the development of cardiovascular disease. The study enrolled participants [16].

(Esperion Therapeutics, Inc)

a) Licenses

In April 2008, we entered into an agreement with Pfizer pursuant to which we acquired a worldwide, exclusive, fully paidup license from Pfizer to certain patent rights owned or controlled by Pfizer relating to bempedoic acid, and we granted Pfizer a worldwide, exclusive, fully paid-up license to certain patent rights owned or controlled by us relating to development programs other than bempedoic acid. The license to us covers the development, manufacture and commercialization of bempedoic acid. We may grant sublicenses under the license. Under the license agreement, Pfizer is restricted from making, using, developing or testing any of the compounds claimed under the same patents that claim or cover the composition of matter of bempedoic acid. [17], Neither party is entitled to any royalties, milestones or any similar development or commercialization payments under the license agreement, and the licenses granted are irrevocable and may not be terminated for any cause, including intentional breaches or breaches caused by gross negligence [18].

Intellectual Property of Esperion Therapeutics, Inc.

As of December 31, 2016, our patent estate, including patents we own or license from third parties, on a worldwide basis, included approximately 25 issued United States patents and four pending United States patent applications and 23 issued patents and 15 pending patent applications in other foreign jurisdictions. Of our worldwide patents and pending applications, only a subset relates to our small molecule program which includes our lead product candidate, bempedoic acid. Bempedoic acid is claimed in U.S. Patent No. 7,335,799 that is scheduled to expire in December 2025, which includes 711 days of patent term adjustment, and may be eligible for a patent term extension period of up to five years. U.S. Patent Nos. 9,000,041 and 8,497,301 claim methods of treatment using bempedoic acid. We also have a pending U.S. patent application directed to bempedoic acid. There are currently three issued patents and four pending application in countries outside the United States that relate to bempedoic acid [19].

Overall Safety Observations (Esperion Therapeutics, Inc statement)

To date, in completed studies, over 800 patients have been treated with bempedoic acid for periods of up to 12 weeks at maximum repeated doses of 240 mg per day. Bempedoic acid has been safe and well-tolerated with no dose-limiting side effects identified to date in our ongoing or completed clinical studies. No clinical safety trends have emerged to date.

Conclusion

The synthesis of Bempedoic acid was prepared by condensation of 1,5-dibromopentane with ethyl isobutyrate by means of LDA in THF in the presence of DMPU at −78 °C to give ethyl 7-bromo-2,2-dimethylheptanoate , which is dimerized with tosylmethyl isocyanide in the presence of NaH and Bu4NI in DMSO to yield diethyl 8-isocyano-2, 2,14,14-tetramethyl-8-(tosyl) pentadecanedioate . Reaction of intermediate with aqueous HCl in CH2Cl2 affords 2,2,14,14-tetramethyl-8-oxopentadecanedioic acid diethyl ester, which is hydrolysed with aqueous KOH in refluxing EtOH/H2O to provide the dicarboxylic acid ESP-15228 (1-4). Ketone is finally reduced by means of NaBH4 in MeOH (1). Esperion announced new details about its phase 3 program for bempedoic acid, its unique oral, once-daily cholesterol-lowering compound. The company said the phase 3 program would include patients with hypercholesterolemia on any statin at any dose, including those with LDL levels not adequately controlled on current statin therapy in intolerant” patients unable to take even low doses of statins. Bempedoic acid does not currently have Marketing Authorisation in the EU/UK for any indication. Bempedoic acid or bempedoic acid with ezetimibe in a fixed-dose combination are in phase III clinical trials for the treatment of primary hypercholesterolaemia (heterozygous familial and nonfamilial) or mixed dyslipidaemia in patients who are statin-intolerant, or for whom a statin is contraindicated. Bempedoic acid monotherapy and bempedoic acid with ezetimibe in a fixed-dose combination is also in phase III clinical trials for the treatment of primary hypercholesterolaemia (heterozygous familial and non-familial) or mixed dyslipidaemia in patients unable to reach LDL- Cholesterol goals with the maximum tolerated dose as an adjunct to diet in combination with a statin or statin with other lipid lowering therapies.

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Why Vasculitis Probably can be Ameliorated with Magnesium and Antagonists of Ceramides and Platelet-Activating Factor| Lupine Publishers

 Journal of Surgery| Lupine Publishers

Introduction

Vasculitis is characterized as an inflammatory disease of the body’s small blood vessels, particularly in the lungs and kidneys [1-4]. Many other organ regions are usually affected which often induces morbidity and mortality [1-4]. Although the exact causes of vasculitis are not known, it appears to be an autoimmune disease even though physical, chemical injuries and infections can result in vasculitis [1-4]. It is classified as a rare disease in the USA because there are only about 200,000 cases. Although numerous treatments have been advocated, there is no known cure or preventative treatment. Vasculitis often leads to difficulties in breathing and renal shut -down. In addition, vasculitis leads to cardiac malfunctions, cardiac failure and strokes. Vasculitis is clearly more common in the aged [1-4]. Recently, we have found that a few patients that were diagnosed with vasculitis appear to have a magnesium deficiency (MgD), particularly in the serum ionized Mg²⁺fraction [unpublished findings].

Unlike atherosclerosis that takes decades to develop, vasculitis of small and medium sized arterial vessels, as well as microscopic arterioles. venules and capillaries, progresses rapidly, thus producing tissue ischemia via lumen-occlusive intimal hyperplasia and inflammatory syndromes [1-4]. Whether the end result is giant cell arteritis (GCA), polyarteritis nodosa (PAN), Churg- Strauss vasculitis (CSV), Wegner granulomatosis (WG), polymyalgia rheumatica (PR), Behcets disease (BD), or other vascular diseases, invasion of the arterial and microcirculatory walls by macrophages, leukocytes and CD4 T-cells seems to be pivotal [1-4]. In addition, most of these patients appear to demonstrate coagulationopathies. The degrees of luminal stenoses vary from patient to patient. Usually, degradation of the internal elastic laminae (EL) follow suit [1-3]. It has been hypothesized that the latter stenoses are due to concentric growth of the intima which seems to be related to several angiogenic growth factors, e.g., platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) [4]. What stimulates the production of these growth factors is not completely known but is thought to involve activation of nuclear-factor-KB (NF-kB) in the macrophages and leukocytes [4]. As stated, the macrophages and leukocytes clearly play key roles in the development of vasculitis. They are activated by NF-kB to produce a host of cytokines and chemokines which are needed for tissue remodeling and granuloma formation in development of vasculitis [2,4]. What activates the macrophages and leukocytes to induce production of NF-kB is not known.

Why Magnesium Deficiency is Most Likely a Key Player in Development of Vasculitis

While we were routinely investigating the potential role of magnesium deficiency (MgD) in numerous cardiovascular- diseased patients, who presented with coronary arterial diseases, coronary vasospasm, acute myocardial infarctions (AMIs), congestive heart failure , and strokes, we noted that several of these patients had an underlying vasculitis together with significant deficits in serum ionized Mg, but not necessarily total serum Mg levels [5]. More than 50 years ago, two of us found that reduction in the concentration of extracellular free Mg ions (Mg²⁺ ) resulted in vasospasm of coronary, cerebral, and peripheral arterial vessels; the lower the [Mg²⁺ ]0, the more the intense the arterial vasospasm [6-13]. In addition, our laboratories found that microscopic blood vessels in skeletal, cutaneous, and cerebral vascular beds of intact rats, mice, rabbits, guinea- pigs, dogs, and piglets exhibited similar phenomena as dietary Mg intake was reduced over three to 12weeks [14,15]. Moreover, vascular reactivity to circulating humoral and hormonal vasoconstrictor agents (i.e., angiotensin II, norepinephrine, serotonin, numerous peptide mediators, etc.) was intensified when [Mg²⁺ ]0 was reduced; the lower the [Mg²⁺ ]0 , the greater the humoral and hormonal-induced vasoconstriction [6-10,13]. It is important to note, here, that these agents are often present at increased, circulating levels in cases of vasculitis. It is now clear that all cases of vasculitis are associated with increased levels of various cytokines and chemokines (e.g., IFN-alpha, IL-1-beta, IL-2, IL-8, IL-10, IL-4, IL-17, TNF-alpha, MCP-1, among others) which are pro-inflammatory in nature [1-4]. We have found that rats placed on MgD diets for 21days generate all of these pro-inflammatory cytokines and chemokines in the blood, cardiac tissues and arterial vessels [16,17]. Other investigators have also reported finding many of these cytokines and chemokines in MgD animals [18]. Furthermore, we have found that. These MgD animals generate growth factors similar to those found in patients presenting with various forms of vasculitis [4,5]. All of these cytokines, chemokines, and growth factors, we found in the MgD animals, were associated with microvascular wall remodeling and pathological alterations in the postcapillary venules, resulting in reduced lumen sizes, increased vascular reactivity, and adherence of leukocytes and macrophages on the endothelial cell walls [19-21], thus, in many respects, similar to what is seen in vasculitis. Last, but not least, we have found that the MgD state that we produced in the rats resulted in activation of NF-kB in cardiac, cerebral, and peripheral vascular smooth muscle cells [16-23]. In view of our findings, we believe, collectively, it is difficult to dismiss the probable role of MgD in the etiology and sustenance of a state of inflammation and vasculitis. Thus, we recommend that our hypothesis should be tested in two ways [24-26]:

a) Use a Mg²⁺ -ion selective electrode like those we helped to pioneer [27-31], in order to carefully measure the levels of ionized free Mg; and

b) Administer Mg salts, initially, intravenously, then orally, for extended periods of time.

Low Mg²⁺ Induces Leukocyte and Macrophage Sticking, Increased Adhesiveness to Venular Endothelial Walls, and Increased Post-Capillary Permeability in The Microcirculation

Approximately 40 years ago, Ross et al advanced the hypothesis that atherosclerosis is an inflammatory disease brought about by injury to the endothelial surfaces of the macro- and microcirculations [32]. The hypothesis stated that different forms of injury (e.g., ischemic events) will result in numerous dysfunctions in the homeostatic properties of the endothelium, e.g., increases in adhesiveness of macrophages and leukocytes and/or platelets, alteration in the procoagulant properties, formation/release of cytokines/chemokines and growth factors. Usually, inflammation is defined as a response of microcirculatory blood vessels and the tissues they perfuse to infections and damaged tissues which bring cells and host-defense factors/molecules directly from the circulation to all the diverse sites where they are required in order to eliminate/degrade the offending agents [33]. The mediators of the defense mechanisms include white blood cells, macrophages, phagocytic leukocytes, chemokines, antibodies, and complement proteins [33]. The inflammatory process brings these cells and molecules to the damaged or necrotic tissues. During the normal inflammatory process, macrophages, leukocytes, and monocytes migrate across the venous capillary walls through holes in between the endothelial cells due to increases in permeability and move to the site(s) of injury via chemotaxis. This sequence of events is thought to take place in all types of inflammatory events and in developing vasculitis [34]. The normal mediators for these processes to take place are adhesion molecules, cytokines, and chemokines, all of which we have found in patients with different forms of vasculitis and in MgD [5,23].

Probable Contributing Roles of Ceramides and Platelet- Activating Factor as a Consequence of MgD to Etiology of Vasculitis

In the late 1990’s, working with proton-nuclear magnetic resonance spectroscopy (1H-NMRS), and arterial vessels exposed to low Mg²⁺ levels, two of us found an increased synthesis of several sphingolipids (namely, ceramides, sphingosine, and sphingosine-1- phosphate) along with an increased formation of platelet-activating factor (PAF) [35,36]. We and others have reported that many of these sphingolipids (particularly ceramides) and PAF promote vasoconstriction and vasospasm of different types of arterial blood vessels as well as arterioles and muscular venules in the living microcirculation in situ [22-24,26,37-41]. In addition, three of us found that ceramides and PAF cause increases in postcapillary permeability, leukocyte and macrophage adhesion to the endothelial linings of the postcapillary venules, and migration of these latter cell types to the extravascular tissue spaces [41]. What we found, of particular interest, is that vascular smooth muscle cells (of different types), when exposed, in primary cell culture, to low Mg²⁺ caused a synthesis of both ceramides and PAF, which could be selectively inhibited using specific antagonists of ceramides and PAF [42]. More than 30 years ago, Cunningham and colleagues reported that sera from rheumatoid vasculitis patients contained platelet-releasing activity [43]. Two years later, Warren and his colleagues, using a rat model of immune complex vasculitis, found that a receptor blocker of PAF inhibited an Arthus reaction [44]. Sera taken from patients in our hospitals which had an underling vasculitis (of diverse origins), and lowered serum ionized Mg, demonstrated increased levels of both ceramides and PAF [5]. We do not believe these findings are merely coincidental. It is our contention that low Mg coupled to increased cellular and serum levels of ceramides and PAF are causal agents in many types of vasculitis.

Conclusion

Although the exact cause(s) of vasculitis is not known, Mg depletion appears to be a presence in different types of vasculitis. When several of our cardiovascular- diseased patients were admitted to our hospitals, a number of them exhibited an underlying vasculitis coupled with an ionized Mg deficiency along with elevated serum levels of ceramides and PAF. Mg-deficient animals, in our labs, exhibited elevated serum and tissue levels of ceramides and PAF which could be reduced/inhibited with specific antagonists of ceramide and PAF synthesis. Elevated dietary levels of Mg also reduced the synthesis of both ceramides and PAF, at least in experimental animals. Experimental animals fed Mg deficient diets exhibited, in-vivo, inflammatory alterations in the microcirculation similar to those patients presenting with different forms of vasculitis (e.g., elevated cytokines, elevated chemokines, elevated adhesion molecules, elevated tissue levels of NF-kB, along with other substances). In view of these new findings from our laboratories, it is our belief that patients exhibiting vasculitis should be treated with oral Mg supplements along with inhibitors of ceramide and PAF synthesis in order to determine if our hypothesis is valid.

Acknowledgement

Much of our original investigations were supported, in part, by research grants from The National Heart, Lung and Blood Institute, The National Mental Health Institute, The National Institute on Drug Abuse, and The National Institute on Alcoholism and Alcohol Abuse along with unrestricted research grants from several pharmaceutical companies. Some of our studies were initiated while two of us (BMA and BTA) were on the faculty of The Albert Einstein College of Medicine. While our original studies were underway, two of our colleagues passed away, namely Professor Lawrence M. Resnick and Anthony Carella. Both of these outstanding scientists will be sorely missed.

Acknowledgement

Much of our original investigations were supported, in part, by research grants from The National Heart, Lung and Blood Institute, The National Mental Health Institute, The National Institute on Drug Abuse, and The National Institute on Alcoholism and Alcohol Abuse along with unrestricted research grants from several pharmaceutical companies. Some of our studies were initiated while two of us (BMA and BTA) were on the faculty of The Albert Einstein College of Medicine. While our original studies were underway, two of our colleagues passed away, namely Professor Lawrence M. Resnick and Anthony Carella. Both of these outstanding scientists will be sorely missed.

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