Chapter 3Niacin: Vitamin and Antidyslipidemic Drug

Elaine L. Jacobson, H. Kim, M. Kim, and M.K. Jacobson

Abstract Niacin is defined collectively as nicotinamide and nicotinic acid, bothof which fulfill the vitamin functions of niacin carried out by the bioactive formsNAD(P). In the last few decades numerous new enzymes that consume NAD(P)as substrates have been identified. The functions of these enzymes are emergingas exciting paradigm shifts, even though they are in early stages of discovery. Therecent identification of the nicotinic acid receptor has allowed distinction of thedrug-like roles of nicotinic acid from its vitamin functions, specifically in modu-lating blood lipid levels and undesirable side effects such as skin vasodilation andthe more rare hepatic toxicities. This information has led to a new strategy for drugdelivery for niacin, which, if successful, could have a major impact on human healththrough decreasing risk for cardiovascular disease. Understanding the many othereffects of niacin has much broader potential for disease intervention and treatmentin numerous diseases including cancer.

Keywords Cardiovascular diseases · Clinical trial · HDL · LDL · Modulating bloodlipids · Niacin · Nicotinamide · Nicotinic acid · Risk for triglycerides · Vasodilation ·Topical delivery strategy

3.1 Niacin’s Multifaceted Metabolism

Nicotinic acid and nicotinamide, collectively referred to as niacin, were identi-fied as compounds that prevent and cure the dietary deficiency disease, pellagra(Elvehjem et al. 1938). Since then the identification of functions of niacin and it’sactive metabolites, NAD(H) and NADP(H), in cell metabolism constitute a largemetabolome in and of itself [Fig. 3.1, reviewed in (Hassa et al. 2006; Ziegler 2000)].

Both nicotinic acid and nicotinamide function as vitamins, supporting thebiosynthesis of NAD(P)(H) (Fig. 3.1). The de novo pathway from tryptophanto NAD is not functional in most human tissues and may not be significant in

E.L. Jacobson (B)Division of Medicinal Chemistry, Department of Pharmacology and Toxicology, Collegeof Pharmacy and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA

Niadyne Development, Inc., Tucson, AZ, USAe-mail: jacobse@pharmacy.arizona.edu; elaine.Jacobson@pharmacy.arizona.edu

37O. Stanger (ed.), Water Soluble Vitamins, Subcellular Biochemistry 56,DOI 10.1007/978-94-007-2199-9_3, C© Springer Science+Business Media B.V. 2012

38 E.L. Jacobson et al.

Fig. 3.1 The vitamin functions of niacin

humans, at least under partial dietary restriction (Fu et al. 1989). NAD+/NADHplay crucial roles in energy generation and in regulating numerous enzymes ofenergy metabolism, while NADPH is essential for reductive biosynthesis and main-tenance of the antioxidant status of the cell. In more recent decades, numerousenzyme activities have been identified that consume NAD+ or NADP+ as a substratefor post-translational modification of proteins and to synthesize small signalingmolecules. Multiple classes of mono-ADP-ribosyl transferases have been identi-fied that have specificity for unique proteins at a specific amino acid side chain, e.g.,arginine, cysteine, histidine, etc. While the chemistry and biochemistry of thesemodifications have evolved, understanding the biological impact of these modifica-tions is an early science. Multiple poly (ADP-ribosyl) polymerases (PARPs) havebeen discovered and shown to consume NAD+ to regulate many cellular functionsthat involve assembly and disassembly of protein-nucleic acid complexes, includingDNA repair complexes, mitotic spindles, telomeres, etc. NAD+ dependent proteindeacetylases (SIRTs) have emerged more recently with seven putative gene productsidentified to date in human tissues. These enzymes have been implicated in manycellular regulatory events from effecting longevity to regulating specific enzymes ofenergy metabolism such as glutamate dehydrogenase (Haigis et al. 2006). Finally,ADP-ribosyl transferases that catalyze an internal transfer within the ADP-ribosemoiety to form the small signaling molecules, cyclic ADP-ribose (cADPR) andphospho-cyclic-ADP-ribose (Pc-ADPR) appear to effect numerous regulatory roles,many of them involving calcium signaling, however the functions of these enzymesalso are poorly understood. The enzymes/pathways mentioned here and noted in

3 Niacin: Vitamin and Antidyslipidemic Drug 39

Fig. 3.1 have been briefly mentioned only to illustrate the large number of functionsniacin effects as a vitamin. It is important to note that the recent discovery of thenicotinic acid receptor separates the vitamin functions of these two forms of niacinfrom drug-like functions, since only nicotinic acid can bind the receptor [reviewedin (Offermanns 2006)].

3.2 Niacin’s Potential and Pitfalls as an Antidyslipidemic Drug

Blood levels of different lipoprotein classes are strongly associated with the risk ofcardiovascular diseases leading to death by heart attack and stroke. Thus, the man-agement of blood lipid levels is an area of enormous significance to public health.As an example, Fig. 3.2 shows data from the Framingham Heart Study documentingthat elevated high density lipoprotein cholesterol (LDL) and insufficient high den-sity lipoprotein cholesterol (HDL) are independent risk factors for coronary arterydisease (CAD). Most public health messages over the past 15 years have focusedon the link between LDL and cardiovascular disease, but the medical community isincreasingly realizing that both LDL reduction and HDL elevation are critical forreducing the risk of cardiovascular disease.

The importance of HDL to public health is illustrated further by the fact thatnearly 40% of males and 15% of females in the United States have HDL valuesbelow 40 mg/dL. These data along with the findings that a 6% increase in HDL cantranslate to 22–29% decreases in coronary events and deaths illustrate the enormouspublic health significance of identifying a safe and effective therapy to elevate HDL.The importance of blood lipids to public health has led to the development of inten-sive therapy to modify blood lipids. Table 3.1 provides an overview of agents used

Fig. 3.2 Risk of CHD as a function of blood cholesterol

40 E.L. Jacobson et al.

Table 3.1 Drugs for the management of blood lipids

Drug class % ↑ in HDL % ↓ in LDL % ↓ in TG

Statins 5–6 25–28 11–15Bile acid sequestrants 3–4 9–18 5–10Cholesterol absorption inhibitors 1 15–18 5–10Fibrates 6–18 4–11 21–35Nicotinic acid 26–32 17–18 27–35

and the range of effects of different agents that have been compiled from manydifferent clinical studies. Nicotinic acid is the oldest drug known to modify serumlipids and it is the most effective drug currently available for elevation of HDL.The statin drugs are effective in reducing LDL but they do not effectively raiseHDL. In addition to its ability to raise HDL, nicotinic acid also is effective inreducing cardiovascular risk by lowering serum LDL and triglycerides (TG), whichalso are a risk factor in CAD. So why is nicotinic acid not the most widely useddrug? It is because compliance is poor due to its side effect profile of skin flushingand associated puritis. Furthermore, many individuals with hepatic dysfunction arecontraindicated for oral niacin therapy and other oral lipid modification therapiesincluding statin therapy. Standard protocols indicate that nicotinic acid, combinedwith diet and exercise, should be the first agent used in an attempt to lower LDL,lower TG, and raise HDL. However, the side effect of flushing frequently drivespatients with low HDL to seek alternatives.

Currently, there are two sources of nicotinic acid available to elevate HDL levels.First, it can be obtained as over the counter (OTC) formulations in a variety ofdifferent forms. The second source is a slow release formulation approved by theUS Food and Drug Administration as a once per day dose under the trade nameNiaspan. The Niaspan formulation of nicotinic acid decreases vasodilation but stillhas a very significant side effect profile including skin flushing. Niacin also becameavailable in 2002 as Advicor, a combination therapy that contains a statin, lovastatin,along with Niaspan.

The issue of a side effect profile that limits compliance with therapy is partic-ularly significant for the treatment of a risk factor such as low HDL because mostpatients on therapy are outwardly healthy and thus an individual who feels fineis reluctant to take a medication that makes one feel uncomfortable. A recentlypublished study (LaRosa and LaRosa 2000) estimates the discontinuation rate ofpatients placed on oral nicotinic acid therapy at 46%. While the number of patientscurrently on nicotinic acid therapy is difficult to estimate, it is reasonable to assumethat the side effect profile of oral nicotinic acid limits use to far less than 10% ofthe number of individuals who could benefit from this therapy. We describe belowthe strategy for development of a new technology for the elevation of HDL andreduction of LDL and TG by transdermal delivery using a nicotinic acid prodrug,lauryl nicotinate. This technology is designed to achieve the stated endpoints with-out the limiting side effect profile and to provide lipid modulation therapy tothose whose hepatic dysfunction contraindicates oral therapies, because transdermaladministration greatly reduces the liver first pass effect.

3 Niacin: Vitamin and Antidyslipidemic Drug 41

3.3 Niacin’s Unique Effects on Blood Lipids

Nicotinic acid is known to inhibit lipolysis in adipose tissue leading to decreases inplasma levels of free fatty acids, decreases in lipoprotein Lp(a), very low-densitylipoprotein cholesterol (VLDL or TG) levels, and LDL, while it increases HDLup to 35%. However, it is not completely clear how nicotinic acid increases HDL.A growing interest in the ability of nicotinic acid to inhibit cAMP in adipose tis-sue due to the Gi-mediated inhibition of adenlyl cyclase (Aktories et al. 1980) andthe ensuing hypothesis that nicotinic acid acts as an agonist of a Gi-coupled recep-tor ultimately resulted in the discovery of the nicotinic acid receptor (Soga et al.2003; Tunaru et al. 2003; Wise et al. 2003). The potential mechanisms by whichnicotinic acid may function through this receptor as noted in the model of Fig. 3.3include a decrease in TG (VLDL) through decreased cAMP effects on lipolysis(yellow adipocyte, Fig. 3.3), leading to reduced cholesterol ester transfer protein(CETP) mediated exchange of TG and cholesterol esters between Apo-B contain-ing lipoproteins (VLDL, LDL) and HDL, with the end result being increased HDL(grey box, Fig. 3.3) [reviewed in (Offermanns 2006)].

Recent studies demonstrating that nicotinic acid modulates leptin andadiponectin release and down stream signaling as depicted in Fig. 3.3 support alter-nate mechanisms for affecting HDL metabolism to provide cardioprotective effects.Here, leptin signals via the leptin receptor (LR), a member of the Type I cytokinereceptor family that mediates intracellular signaling via the activation of associated

Fig. 3.3 Niacin receptor mediated signaling

42 E.L. Jacobson et al.

Jak family tyrosine kinases (Myers 2004). The LR recruits the signal transducerand activator of transcription 3 (STAT3, green box, Fig. 3.3), which in turn inducesimportant positive effectors of leptin action. The peroxisome proliferator-activatedreceptor alpha (PPAR-..) has been shown to be a key proximal mediator of thelipopenic action of leptin (Lee et al. 2002), mediating pleiotropic effects such asstimulation of lipid oxidation, alterations in lipoprotein metabolism, and inhibi-tion of vascular inflammation. PPAR-.. binds the hypolipidemic fibrates to increaseplasma HDL dependent on expression of apoA-I (Fruchart et al. 1999). Furthermore,PPAR- activation induces cholesterol removal from human macrophage foam cellsthrough stimulation of the ATP binding cassette transporter 1 (ABCA1) path-way (Chinetti-Gbaguidi et al. 2005). Functional interactions between apoA-I andABCA1 are necessary for the initial lipidation of apoA-I. The expression of hepaticscavenger receptor class BI (SR-BI) is modulated also by leptin (Lundasen et al.2003). Thus, leptin appears to be important in integrating the transport of cholesterolfrom extrahepatic tissue to the liver since HDL lipidation, mediated by ABCA1,and delipidation, mediated by SR-BI, are modulated by leptin. Functional SR-BI iscritical to the lipid and apolipoprotein composition of HDL and its absence is asso-ciated with increased susceptibility to atherosclerosis. It also has been suggestedthat decreased hepatic uptake and catabolism of HDL-ApoA-I may contribute toincreased HDL, however this mechanism is not likely to be mediated via thenicotinic acid receptor as it is not expressed in the liver (Soga et al. 2003; Tunaruet al. 2003; Wise et al. 2003). In addition to modulation of HDL, Fig. 3.3 also showsadditional cardioprotective effects mediated by the niacin receptor on NF-kB andthe blocking of inflammatory responses known to be very important in CAD (pur-ple box, Fig. 3.3) and effects via leptin and adiponectin illustrated in the blue box ofFig. 3.3. Many studies of the mechanisms that cause diseases of the artery now indi-cate that inflammation leading to oxidative stress is a major factor in atherosclerosisand that the ability of HDL to prevent oxidative damage to LDL is crucial to theability of HDL to decrease the risk of arterial diseases (Kontush et al. 2003). HDLis comprised of multiple fractions classified as HDL2 and HDL3, each of which canbe further separated into sub fractions designated as HDL3a, b, c, etc.

Many studies now suggest that it is the HDLC3 subfraction that reduces the riskof arterial disease progression and protects LDL from oxidation. Taken together,these observations provide compelling evidence that the goal of therapy to raiseHDL should include a focus on the HDL3. Here we overview the evidence for thisconclusion. Recent clinical studies have evaluated the relative risk of progressionof coronary artery disease to HDL subfractions concluding that risk reduction isassociated with the HDL3 and not with HDL2. Yu et al. (2003) examined the riskof incident coronary heart disease in more than 1,700 patients over a 9 year fol-low up in the Caerphilly coronary heart disease study. The odds ratios demonstratethat HDL3 shows a strong association with risk reduction while HDL2 did not. Thestudy of Syvanne et al. that was derived from the Lopid coronary angiography trial(Syvanne et al. 1998) evaluated the progression of atherosclerosis in 372 subjects.Like the Caerphilly study, this study demonstrated a strong risk reduction associ-ated with HDL3. But in contrast to the Caerphilly study, this study actually detected

3 Niacin: Vitamin and Antidyslipidemic Drug 43

an increased risk of disease progression associated with HDL2. Additional evidencesupporting the role of HDL in antioxidant functions is derived from recent biochem-ical studies that have specifically implicated HDL3 as an important protective factorby which HDL achieves risk reduction through prevention of accumulation of oxi-dized LDL particles. In this case, the small, dense HDL3 particles conferred thegreatest protection against LDL oxidation. The order of protection and size fromsmallest to largest is HDL3c > HDL3b > HDL3a > HDL2a > HDL2b (Yoshikawaet al. 1997) (Kontush et al. 2003). Studies of the fibrate class of drugs provide fur-ther evidence implicating HDL3 as being crucial to CAD risk reduction. These drugsreduce the risk of CAD, and they also have been shown to raise HDL3 but not HDL2(Sasaki et al. 2002). Finally, evidence supporting a role of HDL3 in modulating riskrelates to promoting efflux of excess cholesterol from cells in the arterial wall, trans-porting it back to the liver for elimination, a process known as ‘reverse cholesteroltransport (RCT)’. Nascent apoA-I containing HDL particles interact with periph-eral cells and acquire lipid through a transport process facilitated by ABCA1. RCThas been implicated in atheroprotective role of HDL but its efficacy had not beendemonstrated in acute coronary syndromes. However, a recent study has reporteda rapid reduction of atherosclerosis with intravenous administration of a recombi-nant apoA-I Milano (apoA-IM) (Nissen et al. 2003). ApoA-IM is a variant of thenaturally occurring apoA-I that was discovered in residents of an Italian villagecharacterized by low rates of cardiovascular disease and longer life spans. ApoA-IM differs from naturally occurring apoA-I in that it has a different amino acidcomposition. Interestingly, carriers of apoA-IM are characterized by predominant,heterogeneous HDL3 and a marked reduction of HDL2, indicating that apoA-IMhas a major effect in HDL particle interconversion and that a shift in HDL subfrac-tions from HDL2 to the smaller, more dense HDL3 confers cardioprotection (Kimet al. 2004).

3.4 Making a Good Old Drug Better

With the goal of enhancing the potential and ameloriating the pitfalls of niacin, wehave synthesized, characterized, and screened niacin derivatives as potential pro-drugs for transdermal delivery. The objectives in searching for effective transdermalprodrugs were to control the rate of delivery in order to keep niacin concentrationbelow the threshold for vasodilation and to decrease the dose required by avoid-ing first pass metabolism. An overview of the strategy to deliver nicotinic acidtransdermally to the blood circulation is shown in Fig. 3.4. The relative lipophilicityof a number of niacin derivatives was determined using the octanol/water partitioncoefficients. Partition coefficients were then related to the rates of partitioning of thederivatives from the stratum corneum to the epidermis following topical applicationusing intracellular NAD as a biomarker of delivery. Prodrug candidates from methylnicotinate through octyl nicotinate failed screening tests because they partitioned ata rate rapid enough to cause vasodilation at the site of application, since follow-ing conversion to nicotinic acid they exceeded the threshold for vasodilation. Decyl

44 E.L. Jacobson et al.

Fig. 3.4 Overview of transdermal delivery strategy using niacin derivatives

nicotinate showed variable effects on vasodilation. Finally, lauryl nicotinate waschosen as the lead candidate because it did not cause vasodilation, yet demonstratedsystemic delivery of nicotinic acid in animal models as evidenced by achievingtissue saturation at a skin site distal to the site of application.

The feasibility of modulating blood lipids by transdermal delivery of niacin wasthen demonstrated in an animal model. Wild type mice are severely limited as ananimal model for lipoprotein metabolism as their lipoprotein profile is more than80% HDL. Transgenic mice carrying human lipoprotein metabolism genes havebeen used to overcome this limitation for the study of lipoprotein metabolism. Forproof of principle studies, transgenic mice carrying the human genes for CETP andapoB 100 were studied. An oral dose of 0.75% nicotinic acid in the drinking waterwas compared to topical application of lauryl nicotinate doses that corresponded innicotinic acid equivalents to 12.5 and 25% of the oral dose. Decreases in LDL andTG observed in both topical dosages and in the orally dosed animals were nearlyidentical, demonstrating that transdermal delivery of nicotinic acid to modify bloodlipids is feasible. This led to completion of preclinical toxicology and pharmacologystudies whose outcomes supported further drug development.

3.5 Progression to Clinical Trials

A Phase I clinical trial was conducted in a double blind, randomized, placebo con-trolled dose escalation design. Healthy males ranging in age from 18 to 72 yearsof age were recruited. Subjects enrolled in the trial were required to have HDLlevels below 40 mg/dL, which represent the low end of normal HDL ranges.Each treatment group had nine subjects, six receiving a cream containing 20%lauryl nicotinate, three receiving a placebo control cream. The daily dosages for

3 Niacin: Vitamin and Antidyslipidemic Drug 45

topical application were 1.5, 3.0, 6.0, and 9.0 ml of cream that corresponded toapproximately 150, 300, 600, and 900 mg of nicotinic acid equivalents. Fastingblood samples were collected at each treatment visit for lipid analysis and safetymonitoring. After 1 week of application of placebo cream, dosing was initiated withthe lowest dosing group and moved to the higher dose groups based on the lackof any adverse events that would dictate not initiating treatment of the higher dosegroup. Treatment was for a period of 8 weeks, which is about 50% of the treatmentperiod required to see maximum pharmacodynamic effects on blood lipids whenniacin is administered orally.

The safety of lauryl nicotinate was assessed by the reporting of adverse eventsand by analysis of blood chemistry parameters. There were no instances of systemicskin flushing or puritis in the study. There were no severe adverse events related todrug. Blood chemistry, hematology, and urine parameters also were monitored toassess safety. Oral nicotinic acid therapy has been associated with increased fastinglevels of blood glucose, uric acid, and the liver enzymes aspartate aminotransferase(AST) and alanine aminotransferase (ALT). In this study, lauryl nicotinate treatedsubjects did not show a statistically significant elevation in blood glucose, uric acid,AST, or ALT. In addition, there were no statistically significant different valuesbetween lauryl nicotinate and placebo subjects in any of the other blood chemistry orhematology parameters that included alkaline phosphatase, total bilirubin, inorganicphosphate, hemoglobin, hematocrit, platelet count, WBC count, and RBC count.In summary, the data indicate that lauryl nicotinate is both safe and well toleratedwith no side effects that would limit compliance with the therapy.

The primary endpoint of the Phase I clinical trial was assessment of safety andany side effects not related to safety that might limit compliance with therapy.However, blood lipid profiles were determined to provide preliminary pharmaco-dynamic information that could prove useful for follow up studies. The statisticalpower of the pharmacodynamic information obtained in this study was limited byseveral factors that included (i) lack of information on an optimal dosing sched-ule, (ii) a limited number of subjects in the study, and (iii) inherent variability ofthe laboratory determination for HDL. Despite these limitations, useful informationwas obtained. Blood lipid parameters were determined at baseline and following4 and 8 weeks of treatment. The data demonstrate a statistically significant increasein both total HDL of approximately 10% and in the ratio of HDL to total cholesterolof approximately 16%. As discussed above, compelling evidence is accumulat-ing that indicates that a desired goal of nicotinic acid therapy is elevation of theHDL3c subfraction. Thus, we Fig. 3.5: HDL subfraction distributions following8 weeks treatment analyzed HDL subfractions. The data are shown in Fig. 3.5for the lowest (1.5 ml) dosing group and demonstrate that lauryl nicotinate treat-ment preferentially increases the HDL3 subfraction. Indeed, the smallest and mostpotent antioxidant subfraction, HDL3c is elevated an average of 47%, and the nextmost potent antioxidant subfraction HDL3b is elevated by an average of 19% rel-ative to placebo treatment. The results of lauryl nicotinate treatment reported hereare in sharp contrast to results from studies with oral niacin, which increases theHDL2 subfractions (Morgan et al. 2003). An important quantitative note regarding

46 E.L. Jacobson et al.

Fig. 3.5 HDL-C subfractiondistributions following 8weeks treatment

percentage changes in cholesterol fractions should be made here. Since HDL iscomprised of the smallest particles containing the least cholesterol, it is likely that a10% increase relative to the total cholesterol pool represents much more than a 10%increase in total number of HDL particles. Reporting changes in percent cholesterolwithin HDL in this case may represent an under estimate of efficacy, especiallyif the smaller particles of the HDL fraction are the most efficacious. In summary,this phase 1 clinical trial demonstrated that application of a cream containing laurylnicotinate was well tolerated and at doses 10% of effective oral doses resulted instatistically significant elevation of HDL and the ratio of HDL to total cholesterol in8 weeks.

Clearly, the functions of niacin are many (Fig. 3.1). The recent identification ofthe niacin receptor introduces a new phase of discovery regarding the effects ofthis vitamin and drug. The potential positive impact on human health of modulatingniacin dependent metabolism is immense if its metabolic processes can be furtherunderstood to unleash its potential in a selective manner without side effects. Suchefforts could have a major effect on both cardiovascular diseases as well as others.

Acknowledgments The research described herein was supported in part by the National Institutesof Health and Niadyne, Inc. ELJ and MKJ are principles in Niadyne, Inc. and conflict of interestmanagement is conducted by the University of Arizona Board of Regents.

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