Editor’s note: This is a companion article to “Star-Crossed: The Rise and Fall of Anatabloc^®,” an article first published in the September issue of HerbalEGram and then in HerbalGram #104. Both articles address anatabine, a tobacco alkaloid used as an ingredient in products marketed as dietary supplements — notably Anatabloc^® — previously sold by Star Scientific, Inc., now known as Rock Creek Pharmaceuticals, Inc. Since many people in the herb and medicinal plant research and natural products communities are probably not familiar with anatabine, we asked Jay Pierotti, PhD, a chemist with previous professional experience in tobacco analysis, to write a review of the chemistry, toxicology, and animal and human clinical pharmacology of the compound.

Introduction to Alkaloids

Alkaloid-containing plants have been of considerable interest throughout human history. Alkaloids represent a broad category of phytochemicals that generally are defined as nitrogen-containing molecules, which are of particularly important due to their pharmacological activities.[1] Alkaloid classifications can be defined or qualified based on biogenesis from particular amino acids (e.g., ornithine, lysine, etc.), structure (e.g., pyridine, isoquinoline, terpene, purine type, etc.), or biogenic source (e.g., Nicotiana [tobacco] alkaloids), among other schemes.

Archetypal or “true” alkaloids are N-containing heterocycles[2] derived from amino acids. Noteworthy examples of such alkaloids include the following:

(I) Atropine, a tropane alkaloid from plants in the nightshade (Solanaceae) family (e.g., belladonna [Atropa belladonna], Duboisia spp., et al.); (II) Morphine, an alkaloid from the latex of the opium poppy (Papaver somniferum, Papaveraceae); (III) Quinine, an alkaloid from the bark of the cinchona tree (Cinchona officinalis, Rubiaceae). Well-known examples of methylxanthines — terpenoid alkaloids also referred to as protoalkaloids or pseudoalkaloids — are shown in Figure 2:2,³

(I) Caffeine, an alkaloid found in coffee (Caffea spp., Rubiaceae), tea (Camellia sinensis, Theaceae), guarana (Paullinia cupana, Sapindaceae), kola nut (Cola acuminata, syn. C. nitida, Malvaceae), yerba maté (Ilex paraguariensis, Aquifoliaceae), guayusa (I. guayusa), and cacao (Theobroma cacao, Malvaceae); (II) Theobromine from cocoa; (III) Theophylline from tea.

The alkaloids found in Nicotiana species are primarily pyridine-type alkaloids (Figure 4), of which nicotine is thought to be the most abundant in the genus.⁴ Nicotine (Figure 5) and the nicotine-type alkaloids share a common biosynthetic pathway involving a pyridine ring derived from nicotinic acid — a form of vitamin B3.^5,6

Related alkaloids that are found in significantly lower concentrations in Nicotiana species routinely are referred to as “minor” tobacco alkaloids. Among these is anatabine (Figure 6), the advertised active ingredient found in Star Scientific’s Anatabloc^® and CigRx^® products.

Anatabine Chemistry

Anatabine, like the majority of other alkaloids, is a weak organic base. Like nicotine, anatabine is a polar, water-soluble compound that has two nitrogen atoms capable of accepting protons that are responsible for its alkaline properties in aqueous systems. The pKa values[3] — which can be used to evaluate relative basicity — are similar for nicotine and anatabine (see Figure 7). Calculated pKa values for the pyridine and the pyrrolidine nitrogens have been reported as, respectively, 4.23 and 9.13 for nicotine and 4.13 and 8.77 for anatabine.⁴ The differences between pKa values, although small, are sufficient to be resolved by analytical or process separation schemes.^7-10

Related alkaloids that are found in significantly lower concentrations in Nicotiana species routinely are referred to as “minor” tobacco alkaloids. Among these is anatabine (Figure 6), the advertised active ingredient found in Star Scientific’s Anatabloc^® and CigRx^® products.

Anatabine Chemistry

Anatabine, like the majority of other alkaloids, is a weak organic base. Like nicotine, anatabine is a polar, water-soluble compound that has two nitrogen atoms capable of accepting protons that are responsible for its alkaline properties in aqueous systems. The pK_a values[3] — which can be used to evaluate relative basicity — are similar for nicotine and anatabine (see Figure 7). Calculated pK_a values for the pyridine and the pyrrolidine nitrogens have been reported as, respectively, 4.23 and 9.13 for nicotine and 4.13 and 8.77 for anatabine.⁴ The differences between pKa values, although small, are sufficient to be resolved by analytical or process separation schemes.^7-10

Similarly, these chemical differences can have an impact on important reactions such as the formation of carcinogens via nitrosation, i.e., the formation of tobacco-specific nitrosamines.^11,12 At a physiological pH of 7.4, the predominant form of anatabine is the monoprotonated (singly protonated) form (Figure 8), while a smaller amount of the free base (Figure 6) exists as well. The ionization state is an important chemical parameter and one that impacts absorption and metabolism, as the equilibrium of the protonated forms will be shifted by the preferential absorption of one form.^13-15

Occurrence in Tobacco and Tobacco Products

Of the minor alkaloids present in tobacco species, nornicotine and anatabine usually are the most abundant.^16,17 Pakhale and Maru (1998) reported an anatabine concentration of approximately 1.4% of the “alkaloid fraction” (defined as nicotine, nornicotine, anabasine, cotinine, and anatabine) in tested cigarettes.¹⁸ Similarly, Yang et al (2002) reported anatabine levels of approximately 2.4% of the alkaloid fraction (nicotine, nornicotine, anabasine, myosmine, and anatabine) in flue-cured[4] tobacco.²⁰ More recently, Huang and Hsieh (2007) reported a concentration of approximately 1.5% of the total alkaloid fraction (nicotine, nornicotine, anabasine, and anatabine) in selected cigarette tobaccos and 1.2% in cigar tobacco.²¹

In 2008, Liu et al determined that the major form of anatabine in tobacco was the S-(-)-anatabine enantiomer[5] (Figure 6).²³ Interestingly, researchers have proposed that anatabine may be used to detect nicotine from tobacco exposure as opposed to nicotine replacement therapies in which nicotine is the sole alkaloid absorbed.^24,25

Bioactivity

Plant defense

Secondary metabolites such as the nicotine-type alkaloids appear to play an important role in plant defense as their bitter taste and toxicity can deter certain insect species.^26-28 In a study by Steppuhn et al (2004), silencing the gene responsible for nicotine production in N. attenuata (“coyote tobacco”) resulted in a 95% reduction in nicotine and the appearance of anatabine, which was not found in the unmodified plant.²⁹ The authors inferred that the resulting increase in herbivore damage to the modified plants may be due to the lower toxicity of anatabine and the decrease in nicotine and total alkaloids. The possibility of lower anatabine toxicity compared to nicotine also was suggested by Chintapakorn and Hamill (2003) in a similar experiment involving the down-regulation of the gene responsible for nicotine synthesis in N. tabacum.³⁰

Nicotinic acetylcholine receptor (nAChR) activity

Like nicotinef — and nicotinic acid (Figure 9), from which both of its rings are derived^33-35 — anatabine (Figure 6) is thought to be an agonist at nicotinic acetylcholine receptors (nAChRs).^18,36,37 nAChRs are neurotransmitter receptors that activate neuronal and neuromuscular transmissions, which are critical to human health and disease processes.³⁸ In addition to the structural similarity between acetylcholine (the natural agonist of nAChRs) and nicotine-type alkaloids, it has been shown that the pyridine ring, the protonated nitrogen, and the stereochemistry of the molecule are key pharmacophoric factors in nAChR binding.³⁹ Because of nicotine’s generally accepted role in tobacco addiction through activation of nAChRs, studies involving closely-related alkaloids such as anatabine have been of interest to researchers.

In an effort to further understand the mechanisms responsible for the addictive properties of tobacco, Clemens et al (2009) studied the impact of the addition of five minor alkaloids to nicotine dosing.⁴⁰ The researchers found that intravenous infusion of nicotine and anatabine resulted in significantly increased general motor activity in rats over nicotine-only infusions. The authors inferred a possible synergistic effect for anatabine that could play a role in promoting addiction. Results from a similar study by Khalki et al (2012) observing the effects of intraperitoneal doses of nicotine alone versus a tobacco alkaloid extract on rat dopamine release also suggested a possible enhanced addiction potential due to the non-nicotine alkaloids.³⁶

However, more recently, Hall et al (2014) showed that subcutaneous anatabine pre-treatment in rats reduced nicotine self-dosing by 50% at the highest dosing level.⁴¹ Similarly, Mello et al (2014) found that pre-treating monkeys with intravenous anatabine had the potential to significantly reduce nicotine self-dosing without the transfer of addictive behavior to anatabine.⁴² Caine et al (2014) also found that intravenous treatment of rats with anatabine reduced nicotine self-dosing in a dose-dependent relationship.43 As is true of most pharmacological research-based physiological effects on test animals, these intravenous activities do not necessarily correlate with the pharmacology of orally ingested anatabine.

Anti-inflammatory activity

In 2011, Paris et al reported that anatabine reduced Aβ markers for Alzheimer’s disease in cell lines overexpressing wild-type human amyloid precursor protein (APP). An in vivo experiment employing acute intraperitoneal dosing of transgenic mice (that overproduced the human Aβ peptide) showed significantly reduced accumulation of soluble and insoluble Aβ proteins in brain tissue. C-reactive protein plasma levels — an indicator of inflammation — also were significantly reduced.⁴⁴

Paris et al (2013) found that anti-inflammatory activity appeared to be mediated by the regulation of STAT3 (a particular signal transducer and activator of transcription protein) activity using in vitro studies with human blood and in vivo studies with mice following acute intraperitoneal dosing.⁴⁵ STAT3 protein is thought to play a role in promoting inflammation associated with cancer.⁴⁶

In a double-blind clinical trial with 18 men, Jenkins et al (2013) found that anatabine oral supplementation with six to 12 mg/day for 10 days had no measurable effect on non-invasive muscle damage indicators resulting from eccentric isomeric exercise.⁴⁷ In 2014, a similar double-blind trial with 17 men by Jenkins et al also found no benefit from anatabine supplementation (six to 10 mg/day for 10 days) on the muscle stress indicators creatine kinase, lactate dehydrogenase, and myoglobin.⁴⁸ The authors suggested future studies of anatabine supplementation should assess inflammation associated with aging and obesity because the exercise protocols employed did not increase C-reactive protein or TNF-alpha levels — the inflammation markers against which anatabine has been shown to have an effect.^44,45

Paris et al (2013) reported that oral supplementation with anatabine reduced characteristic inflammation and neurodegeneration in mice with experimental autoimmune encephalomyelitis, which is used to model multiple sclerosis.⁴⁹

Also in 2013, Lanier et al reported that seven of 10 human rosacea patients using a face cream containing anatabine (Anatabloc^® Rare Cellular Facial Crème; Star Scientific, Inc.; Glen Allen, Virginia) had improved presentations.⁵⁰ The researchers suggested the possibility of anti-inflammatory activity and, due to the low sample number (n=10) and nature of the study, suggested a larger double-blind study was justified. Using an internet-based survey — which, in general, can produce skewed and unscientific results — of anatabine supplementation for relief of musculoskeletal pain, Lanier et al (2013) found that 82% of 282 respondents reported a benefit for at least one joint-pain condition suggesting possible anti-inflammatory activity.⁵¹

Immunological activity

Caturegli et al (2013) reported that anatabine supplementation in mice with induced autoimmune thyroiditis (as a model of Hashimoto’s thyroiditis) significantly reduced the severity of the disease.⁵² Similarly, in 2014, a double-blind human clinical study of 146 patients with Hashimoto's thyroiditis found a significantly positive immunological effect from anatabine supplementation.⁵³ Anatabine was supplemented in the test group (n=70) with nine to 24 mg/day in lozenge form for three months. Pharmacology and toxicology

The limited data relating to the toxicology and pharmacology of anatabine appear to be from observations in dosing studies and in vitro experiments.^36,40,54-56 Inferences also have been made on anatabine’s toxicity relative to nicotine in the plant studies mentioned above.^29,30 Safety data sheets may list “no data available” or only data for a carrier [solvent] in toxicological information sections.

Riebe and Westphal (1983) found that anatabine dose-dependently induced increased sister-chromatid exchanges, which is a measure of potential mutagenicity, in hamster ovary cells in vitro.⁵⁵ More recently, in 2013, Jenkins et al — noting earlier studies’ findings that acute doses of nicotine impacted heart rate and blood pressure — hypothesized that oral supplementation with anatabine would affect these parameters because of the structural similarities of anatabine and nicotine. However, the researchers found no measurable effect on heart rate or blood pressure at six to 10 mg/day over 10 days in 18 men.⁴⁷

A study by Caine et al (2014) reported that intraperitoneal anatabine pre-treatment in rats produced nicotine-like effects in a dose-dependent relationship, yet had no nicotine-like reinforcing effects, suggesting different pharmacodynamics at nAChR sites.⁴³ The researchers explained that anatabine acts as a full agonist but with lower potency than nicotine and acetylcholine. In an in vitro study, Lanier et al (2013) determined that anatabine had full (albeit weak) or partial agonist activities at different nicotinic receptor sites; the authors also proposed that anatabine inhibited the pro-inflammatory factor NF-κB and could cause apoptosis and necrosis.⁵⁷

Anatabine Sources

Anatabine can be found in numerous Nicotiana species including N. glutinosa³⁴ and N. alata⁵⁸ (Table 1). The compound also has been reported in Lobelia inflata (Campanulaceae)⁵⁹ and D. hopwoodi (Solanaceae)⁶⁰ and can be synthetically created as well.^4,61 A recent patent filed by Rock Creek Pharmaceuticals was issued for the commercial production of anatabine citrate.⁶²

Summary

Animal studies evaluating the effect of anatabine pre-treatment suggest that the compound may reduce nicotine self-dosing, despite the fact that anatabine itself is reported to be a nicotine receptor agonist. There also appears to be some experimental evidence for the anti-inflammatory activity of anatabine using both in vitro and in vivo animal models, but the available data is not conclusive. Few human clinical trials have been conducted on anatabine, but researchers have reported possible anti-inflammatory effects in patients with chronic lymphocytic autoimmune (Hashimoto's) thyroiditis. Additional studies — including pharmacological, safety/toxicology, and human clinical trials — are needed before any firm conclusions can be drawn.

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62. Thomas PT, Kanathkunn DT, Sarveswara RM, Inventors; Rock Creek Pharmaceuticals, Inc. (Gloucester, MA) assignee. Pharmaceutical, dietary supplement, and food grade salts of anatabine 2013.

• [1] According to the International Union of Pure and Applied Chemistry (IUPAC), alkaloids are defined as “[b]asic nitrogen compounds (mostly heterocyclic) occurring mostly in the plant kingdom (but not excluding those of animal origin). Amino acids, peptides, proteins, nucleotides, nucleic acids, amino sugars, and antibiotics are not normally regarded as alkaloids. By extension, certain neutral compounds biogenetically related to basic alkaloids are included.”¹


• [2] According to IUPAC, a heterocycle is a cyclic compound that has atoms of at least two different elements as members of its ring.¹ In the case of anatabine (and related alkaloids) the two elements are carbon and nitrogen.


• [3] The higher the pKa value, the greater the greater the basicity. (i.e., the greater the tendency to accept a proton, H⁺).


• [4] Flue-cured tobacco is also known as “Virginia,” “bright,” or “brightleaf” tobacco and is a common constituent of cigarette and pipe tobacco blends.¹⁹


• [5] Enantiomers are structural mirror images of each other.²² The enantiomer of S-(-)-anatabine is R-(+)-anatabine.


• [6] Nicotine, among other compounds, has been studied extensively with regard to its anti-inflammatory activity via this pathway.^31,32


• [7] As defined by the IUPAC glossary, an agonist is a “[s]ubstance which binds to cell receptors normally responding to a naturally occurring substance and which produces an effect similar to that of the natural substance.”²²