PHAR 505 Exam II: Lecture Review

(2/12) Bruzik Lecture: Chemistry of Lipid Disorders – I – Return of the Bruzer As you may know, Cardiovascular Disease (CVD) accounts for 1 in every 2.8 deaths in the US. Recently in 2010, a massive GWAS project identified 59 loci where CHD and lipid traits were significantly associated. To determine the pathophysiology of these lipid traits, let’s first take a dive into cholesterol. Atherosclerotic Plaques: The deposit of atherosclerotic plaques will decrease the diameter of the lumen within blood vessels. The deposition rate increases with age, severely compromising blood flow. However, symptoms usually do not present until the problem is advanced. Cholesterol Biosynthesis

Carbon Counting Initial: Acetyl-CoA (2C) + AcetoAcetyl-CoA (4C) = HMG-CoA (6C) Enzyme: HMG-CoA Synthase. Formation of HMG-CoA is reversible “The Bottleneck”: HMG-CoA à à Mevalonic Acid (6C) Enzyme: HMG-CoA Reductase, using NADPH cofactor Formation of Mevalonic Acid/Mevalonate is irreversible It is the slowest step, this is where drugs (statins) target “Crucial Intermediate”: ATP dependent phosphorylation of mevalonate -OH coupled with decarboxylation forms crucial Prenyl intermediates Dimethylallyl-PP (5C) Isopentenyl-PP (5C) Oligomer isomerization can interconvert the two prenyl intermediates. Combining them forms Geranyl-PP (10C) “Monoterpene”: Geranyl-PP is by definition an acyclic monoterpene since it has 10 carbons. Addition of another 5C dimethylallyl-PP will: Geranyl-PP (10C), a monoterpene add 5C intermediate Farnesyl-PP (15C), a sesquiterpene X2 + NADPH Squalene (30C) Squalene is 30C linear molecule. Following epoxidation by squalene epoxidase, ring-opening of the epoxide triggers a 1 step series of ring-fusion events, forming 4 fused rings: Lanosterol (30)

- Formation of Lanosterol provides the base structure. It has additional methyl groups that must be trimmed off, oxidation events are required to form the C=C. 15 more steps will produce cholesterol. Accumulation of the late intermediates in between lanosterol and cholesterol is toxic to the cell. Therefore, there are no drug targets following lanosterol synthesis. Accumulation would kill the cell

Cholesterol Fatty Esters - Supply: Esterification of long chain fatty acids (LCFA) may produce two different products depending on the

availability of the required enzymes. High density lipoprotein (HDL) is produced by the donation of an acyl group to cholesterol by ACAT transferase (using Acyl-CoA). If overburdened, excess cholesterol will receive fatty acid groups from Lecithin via LCAT. 80% of cholesterol is synthesized in the liver.

- Demand: Natural catabolism of cholesterol occurs during the formation of bile acids. Bile acid formation involves hydroxylation and reduction of the cholesterol molecule, shortening and polarizing the side chains. This is critical for the function of bile acids, as they are used to emulsify fats by acting as a detergent. Cholic acid may be further modified by amidation using Taurine. Overall, this will reduce the circulating cholesterol

Lipoproteins - Lipoproteins are special lipid carriers responsible for transporting cholesterol

throughout the body. They are structured as spherical agglomerates, with the polar components at the surface to interact with the aqueous environment and a lipophilic core to interact with the cholesterol residues.

- Recognition: Apoproteins on the cell surface function as recognition sites for receptors on peripheral cells, such that the lipoproteins may be taken up

- Density: Lipoprotein density is based on the relative content of protein. Proteins are more dense than fat.

o In terms of protein content and density: HDL > LDL > IDL > VLDL > Chylomicrons. Chylomicrons are very light and float to the surface during a blood sample centrifugation. HDL lipoproteins sink.

Cholesterol Content - Source: The daily summation = 1.6g cholesterol

o Diet (1.2g): The largest contributor towards daily cholesterol, dietary fat in the intestine as taken up by chylomicrons and is brought to the liver.

o De novo synthesis (0.4g) - Transport: As the lipoprotein particles continue to pass through the liver and other tissues, they acquire different

apoproteins. Additionally, their cholesterol content changes following exchange with peripheral cells o VLDL leaves the Liver with endogenous and diet-processed cholesterol to move through the circulation

Low-Density Lipoprotein (LDL) - LDL is the primary transport vehicle for cholesterol in the body. Containing ~1500 cholesterol esters and 500

unesterified cholesterol molecules, the Apo-B100 protein wraps around the LDL particle to support its contents

- LDL Receptor: Present within the liver and on extrahepatic tissues, receptor-mediated endocytosis of LDL by the LDL receptor occurs upon recognition of the Apo-B100 protein. Interaction promotes invagination via the associated intracellular clathrin triskelion coated pit. The LDL receptor structure includes:

o LA repeats: Ligand binding area. There are 7 on each side of the receptor, each with a Ca2+ o EG: Similar to epidermal growth factor, it is a repeat o LY: Highly glycosylated Serine and Threonine residues, used to dissociate the receptor o Alpha Helix: Buried in the membrane o Familial Hypercholesterolemia (FH): Heredity disorder involving a genetic defect in the sequence of LDL

receptor, leading to x5 the normal cholesterol level. Affected individuals rarely live past 20yo, as they have increased risk of atherosclerosis and CHD

- Endosome Activity: LDL Apo-B100 is interacting with the LA repeats at pH = 7. Upon pH change to 5, the 2 Asp (+Cys) residues within the LA repeats become protonated which weakens their association with Ca2 and alters their structure resulting in LDL dissociation and receptor folding. The receptor will recycle back to the membrane.

Strategies to combat high cholesterol in CHD and lipid disorders - Preferred: Although obvious, 75% of overall cholesterol intake is from the diet. The obvious answer would be to

avoid dietary cholesterol, although this would require a change in lifestyle people are not comfortable with - Inhibitors of Cellular Production - Increase catabolism: A certain amount of cholesterol is dedicated to the synthesis of bile acids. Formation of bile

acids requires a lot of energy and cholesterol. Inhibiting the recycling can reduce overall cholesterol (2/14) Bruzik Lecture: Chemistry of Lipid Disorders – II – Back at it again Switches and Feedback Regulation: Responsive mechanisms of cholesterol uptake and metabolism

- Excess Intracellular Cholesterol: High concentrations of cholesterol is associated with having a high concentration of cholesterol metabolites. These metabolites have 2 main regulatory feedback mechanisms:

o (1) Inhibition of HMG-CoA Reductase, thereby inhibiting de novo synthesis of cholesterol o (2) Inhibit LDL Receptor Expression

- Bile Acid Recycling Status: Cholesterol is used during the synthesis of bile acids. Individuals with efficient recycling will effectively inhibit cholesterol synthesis and uptake, since less is needed for bile acid synthesis

- Esterification: In order to be transported within lipoproteins, cholesterol requires esterification to migrate via Cholesterol Ester Transport Protein. Esterification relatively decreases free cholesterol, thereby signaling the need for and synthesis of more cholesterol.

Learning from past mistakes: Development of the early experimental drugs Triparanol and Diazacholesterol demonstrated the toxicity associated with accumulation of late metabolites. Inhibitors of cholesterol biosynthesis late in the biosynthetic pathway led to accumulation of desmosterol in the serum and liver, leading to severe side effects.

- Lesson: Cholesterol synthesis should be stopped at the earlier stages.

Modern Approach: After testing thousands of strains of microorganisms for inhibitors of sterol biosynthesis, Mevastatin/compactin was found in 1972. Due to poor efficacy, it was redeveloped into Lovastatin (1978)

- Target: Inhibition of HMG-CoA reductase decreases de novo cholesterol biosynthesis within cells.

- Biochemical MoA: HMG-CoA +2NADPH !"#$%&')*+,-./0*

Mevalonate. Naturally, reduction of HMG proceeds through a chiral tetrahedral intermediate. The reduction is stereoselective. HMG-CoA reductase catalyzes this slow, irreversible step.

o (1) Hydride Attack: Round 1: A hydride ion (H-) from a NADPH cofactor attacks the HMG carbonyl, producing a short-lived tetrahedral intermediate (sp2àsp3) transition state

§ Transition state sp3 is stabilized by basic activity of a Lys residue (K267) o (2) Hydride Attack: Round 2: CoA dissociates as a leaving group, substantiating concerted collapse of

HMG-O+ into an aldehyde, which is attacked by the second hydride equivalent, producing the reduced alcohol Mevalonate

- Inhibitor MoA: Lovastatin is a transition state analog that functions as a competitive inhibitor of HMG-CoA, but non-competitive for NADPH. This is due to its homology to the transition state, and HMG-like pharmacophore.

o Potency: The Km of HMG for the enzyme is 4µM, Lovastatin is sub-nanomolar (0.1-2.3nM) – Potent! Statin Therapy

- Within the Hepatocyte: Statins directly decrease the synthesis of cholesterol in the liver. As a result, intracellular cholesterol decreases. Homeostatic mechanisms respond by:

o (1) Upregulate synthesis of LDL receptors (mRNAÝ) § Leading to increased removal of B- and E- containing

lipoproteins from circulation (VLDL, IDL) o (2) Reduction in lipoprotein synthesis and secretion of lipoproteins from the liver.

- Systemic Effects o Lowered plasma concentrations of cholesterol-carrying lipoproteins, most prominently LDL. o Removal of remnant particles, such as VLDL and IDL. This is beneficial for hypertriglyceridemia pt. o Benefits are first seen after 1 week of therapy. Maximally at 4-6w.

- Patient Population: Individuals with Familial Hypercholesterolemia (FH) may see a minor benefit in the case of heterozygosity. Homozygous FH patients will not see a benefit.

Clinically Used Statins: The naturally obtained compounds are useful, but often too hydrophobic. Synthetic are improved - Lovastatin: Fungal metabolite + found in Red yeast rice - Pravastatin (Pravachol): While most statin therapies are prodrugs, Pravastatin is the active compound

with Hydroxyl + Carboxyl available to interact. Naturally-derived fungus metabolite. - Atorvastatin (Lipitor): Synthetic, more water-soluble with better bioavailability. Less AE. - Rosuvastatin (Crestor): The most potent of the statins. Sub-nanomolar.

Stain Metabolism - Bioactivation: As previously stated, many of the synthetic statins are prodrugs. Their lactone rings may be opened

to produce the active metabolite by esterases. - Inactivation: CYP3A4 of phase I metabolism hydroxylates statins to prepare them for phase II. - Excretion: Phase II metabolism most frequently involves glucuronidation for clearance in the urine.

Rhabdomyolysis: Rapid breakdown of the skeletal muscle usually due to physical damage of the muscle. Myoglobin is released from the lysed cells. It contains perforin, which is highly viscous and may block capillaries of the kidney potentially leading to renal failure. Cerivastatin (Baycol) led to 52 fatalities as of 2001. It was recalled. Alternative Approach: Interventions targeting cholesterol regulation and catabolism

- Cholesterol is used in the body to produce bile acids. They are oxidized and mixed with bile salts for excretion by the gallbladder to emulsify fats during digestion. Bile salts in the ileum may be sieved with bile acid resin therapy to reduce the amount of recycled cholesterol

- Bile Acid Resins: These resins are inert water-insoluble crosslinked polymers. Have like quaternary ammoniums. o MoA: Resins, such as cholestyramine, bind the negatively charged carboxylate groups of bile salts in the

intestines effectively reducing enterohepatic recirculation of bile acids through sequestration from the lumen. As a result, more cholesterol will be needed for bile acid synthesis to compensate for the loss. Upregulation of 7-a hydroxylase will promote greater cholesterol conversion into bile acids. Through a similar cascade to statins, this reduces the intracellular cholesterol content of hepatocytes, leading to upregulation of LDL receptors, thereby removing LDL and VLDL remnant particles from circulation.

o The Liver’s Response: The liver compensates by increasing cholesterol synthesis. Additionally, patients with elevated triglyceride levels will experience increased hepatic VLDL production, potentially raising serum triglycerides excessively.

o Drug: Cholestyramine: This sequestrant is primarily used to treat hypercholesterolemia. It has been found to slow the progression of coronary atherosclerosis and cause a net increase of coronary lumen

§ Reduces LDL via receptor pathway, and can work in combination with statins! o Drug: Colestipol: Less effective, also reduces LDL via receptor pathway and can be used with Statins.

- Monoclonal Antibodies: Newest and most efficacious therapies in terms of LDL lowering o PCSK9: PCSK9 is a protein responsible for reducing the number of LDL receptors on the cellular

surface. It binds to the EG-F motifs of the LDL receptors to prevent their recycling. o MoA: The mAb bind to and block the action of PCSK9, thereby allowing the LDL receptors to persist on

the cell surface and continue to remove LDL from circulation. There is effectively more LDLR present. o Drugs: Alirocumab (Praluent), Evolocumab (Repatha) o These drugs are great, but they are just too expensive

- Ezetimibe (Zetia) A Selective Cholesterol Absorption Inhibitor o MoA: Ezetimibe inhibits the absorption of biliary and dietary cholesterol from the small intestine without

affecting the absorption of fat-soluble vitamins, TG, or bile acids. Occurs in the Jejunum o Efficacy: Currently, there is controversy over the benefits of ezetimibe therapy. It is suggested to have

lipid-lower properties in monotherapy and synergistic activity with statins. 2008 trials found that the combo product with simvastatin (Vytorin) showed no benefit, whereas 2014 trials show 6.4% reduction of CHD sx compared to simvastatin alone.

- Stanols (Benecol): A margarine spread containing stanol esters. Stanols are not taken up by human cells, and have been shown to lower LDL by up to 15%, and greater benefits are seen when used with statins.

o DATA CHECK: Need to consume >1.3g/daily to observe cholesterol lowering effect - Xenical (Orlistat): A Gastrointestinal/Gastric Lipase Inhibitor

o MoA: b-lactone is attacked by a Ser-residue of GI lipase, forming a covalent adduct that inhibits the enzyme. Normally, GI Lipase hydrolyzes FA-TG to the mono/di-glycerides for chylomicron absorption

- Fibrates: Complex mechanism involving activation of a nuclear transcription factor (PPAR-a), inducing the expression of many genes involved in lipid metabolism. Drugs of this class, such as Gemfibrozil, are indicated for the lowering of TG. They do show increases in HDL, and higher risk of Rhabdo

(2/16) Hellenbart Lecture: Pharmacology and Pharmacotherapy of Lipid Drugs and Disorders Purpose of Treating Hyperlipidemia (HL) 1 in 4 deaths in the US is from Heart Disease

- Left untreated, HL will significantly increase the risk of coronary heart diseases (CHD), including: Coronary Artery Disease (CAD), Cerebrovascular Disease (CVD), Peripheral Vascular Disease (PVD)

- CHD or Hx of MI: Patients diagnosed with CHD or previous history of an event are at 5-7x higher risk of another event.

Pathogenesis of Atherosclerosis - Formation: Over time, fatty streaks in the endothelium develop into atheromas and eventually fibrous plaques.

These plaques have a soft lipid core, but a rough fibrous outer surface. Sufficient perturbation may rupture the plaques, producing microthrombi. Consequently, a clot will develop either fully or partially occluding the blood vessels. Even if these ruptures are stabilized, narrowing of the blood vessel will still develop.

- Complications: Impaired endothelial function may alter the release of Nitric oxide (NO), which significantly induce vasoconstriction, producing symptoms such as chest pain or ischemic-related events.

Lipids and Lipoproteins - Total Cholesterol (TC): Composed of LDL, HDL, and % of TG - Low Density Lipoprotein (LDL): “bad cholesterol”

o Greatest contributor to atherosclerosis. o LDL > 160? 1.5x more likely to have CHD than <130

- High Density Lipoprotein (HDL): “good cholesterol” o Transports cholesterol back to the liver for breakdown and removal o Higher levels are better

- Triglycerides (TG) o No longer a clinically viewed target, due to HDL greatly skewing it. o TG level is a secondary treatment goal, unless ³ 500 which indicates risk of pancreatitis

Relative Level

TC (mg/dL)

LDL (mg/dL)

HDL (mg/dL)

TG (mg/dL)

Healthy < 200 < 100 Higher < 150 Border Unhealthy

200-239 130-159 40s 150-199

Unhealthy ³ 240 ³ 190 £ 40 200-499