A. Cloning of cDNAs Encoding Hydroxy-carboxylic Acid Receptors

The HCA₃ receptor (GPR109B) was the first hydroxy-carboxylic acid receptor whose cDNA was cloned and analyzed (Nomura et al., 1993). In search of new putative leukocyte chemotactic peptide receptors, Nomura et al. (1993) used degenerate oligonucleotide primers representing conserved cDNA regions encoding transmembrane domains of the human receptors for interleukin-8, complement factor 5a and N-formyl peptides to perform polymerase chain reactions on a human monocyte cDNA library. Full-length clones were isolated from the same cDNA library using the obtained polymerase chain reaction amplificate. One of the cDNAs encoding a new G-protein-coupled receptor was called HM74 (HCA₃/GPR109B), and the receptor was shown to be expressed in monocytes and neutrophils. It was also noted that the HCA₃ sequence differed considerably from that of leukocyte chemotactic peptide receptors, suggesting that this receptor had a different ligand.

The HCA₁ receptor (GPR81) was first found by a database search in which expressed sequence tag and high-throughput genomic sequences databases were analyzed for homologies to sequences of various known G-protein-coupled receptors using the TBLAST algorithm (Lee et al., 2001). The HCA₁ receptor cDNA was then cloned from a human bacterial artificial chromosome genomic clone localized to chromosome 12q. Lee et al. (2001) noticed the high homology to the HCA₃ receptor (GPR109B, HM74) and also observed that the genes encoding HCA₁ and HCA₃ receptors are near each other on the same human bacterial artificial chromosome.

The HCA₂ receptor (GPR109A) was originally identified in mice in a search for genes differentially expressed in interferon-γ (INF-γ)/tumor necrosis factor-α (TNF-α)-stimulated macrophages (Schaub et al., 2001). Schaub et al. (2001) used a subtractive hybridization strategy to identify cDNAs present in a cDNA library generated from the murine macrophage cell line ANA-1 stimulated for 16 h with INF-γ and TNF-α but absent from cDNA libraries derived from untreated ANA-1 cells or from IFN-γ stimulated embryonic fibroblasts. The expression of the HCA₂ receptor in response to INF-γ and TNF-α was verified subsequently in various mouse macrophage cell lines, and the murine HCA₂ receptor was therefore designated PUMA-G (protein up-regulated in macrophages by IFN-γ). Expression of HCA₂ could also be induced in the spleens of mice after infection with Listeria monocytogenes. Human and rat HCA₂ receptors were cloned after the discovery of HCA₃ as a low-affinity receptor for nicotinic acid during a search for novel paralogs of the HCA₃ receptor (Soga et al., 2003; Wise et al., 2003).

B. Sequence Alignment and Phylogenetic Tree

The HCA₁ receptor (GPR81) has been shown to be expressed in humans and rodents, and, based on genomic sequences of various species, seems to be present in most mammals as well as in fish (C. Kuei, J. Yu, J. Zhu, J. Wu, T. Lovenberg, and C. Liu, manuscript in preparation). Likewise, the HCA₂ receptor (GPR109A), which exhibits substantial homology to the HCA₁ receptor (approximately 50% amino acid sequence identity), is present in mammalian species. HCA₂ and HCA₃ receptors are highly homologous, being 95% identical on the protein level (see Figs. 1 and 2). The receptors differ by 15 amino acids that cluster in the first and second extracellular loops as well as in the outer parts of transmembrane regions 2 and 3. In addition, the HCA₃ receptor has an extended C terminus containing 24 additional amino acids. In contrast to HCA₁ and HCA₂ receptors, the HCA₃ receptor is not present in rodents. The analysis of available genomic sequences shows an ortholog of the human HCA₃ receptor gene only in chimpanzees, whereas lower primates, such as rhesus monkeys, do not carry a gene encoding an HCA₃ receptor. Thus, the HCA₃ receptor is obviously the result of rather recent gene duplication. Several single-nucleotide polymorphisms in the coding regions of genes encoding HCA₂ and HCA₃ receptors occur with heterozygosity ratios of 0.14 to 0.46 (Zellner et al., 2005). It is not clear, however, whether any of these variations alters the physiological or pharmacological functions of the receptor proteins.

C. Deorphanization of Hydroxy-carboxylic Acid Receptors

D. Proposed Nomenclature

In the past, various names were given to the receptors HCA₁ (GPR81), HCA₂ (GPR109A), and HCA₃ (GPR109B) (see Table 1). After the identification of the HCA₂ receptor (GPR109A) as a receptor for nicotinic acid, this receptor family has been called the “nicotinic acid receptor family” or “niacin receptor family.” However, the name nicotinic acid receptor family is not appropriate because nicotinic acid (niacin) is not an endogenous ligand and because only HCA₂ (GPR109A) and neither HCA₃ (GPR109B) nor HCA₁ (GPR81) is a receptor for nicotinic acid. Endogenous ligands for all three members of the family have been described (see section II.C). Because all these endogenous ligands are hydroxy-carboxylic acids, we propose that this receptor family be called the “hydroxy-carboxylic acid (HCA) receptor family,” which currently has three members: HCA₁, HCA₂, and HCA₃ (see Table 1).

The G-protein-coupled receptors most closely related to the HCA receptor family are the orphan receptor GPR31 and the 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) receptor OXER1 (Fig. 1 and 2) (Zingoni et al., 1997; Hosoi et al., 2002; Bjarnadóttir et al., 2006). An arginine residue in transmembrane helix 3, which has been proposed to represent an anchor point of the carboxylic group of HCA receptor ligands (Tunaru et al., 2005), is conserved not only among HCA receptors but also in GPR31 and the 5-oxo-ETE receptor (Fig. 1). These homologies suggest that GPR31 and OXER1 ligands may structurally resemble HCA receptor ligands. Although endogenous ligands for GPR31 are unknown, OXER1 binds not only 5-oxo-ETE but also, with lesser affinity, 5-hydroxy-eicosatetraenoic acid and 5-hydroperoxy-eicosatetraenoic acid (Hosoi et al., 2002; Jones et al., 2003; Brink et al., 2004). Thus, the fact that OXER1 is activated by a polyunsaturated fatty acid substituted in the 5-position with an oxo, hydroxy, or hydroperoxy group resembles HCA receptors also with regard to its agonistic ligand.

A. HCA₁

The HCA₁ receptor was originally reported to be expressed in human pituitary tissue (Lee et al., 2001). However, this has never been confirmed. Meanwhile, evidence has been provided that the receptor is primarily expressed in adipocytes (Wise et al., 2003; Ge et al., 2008; Jeninga et al., 2009; Liu et al., 2009; Ahmed et al., 2010). In humans, the HCA₁ receptor was found to be most abundantly expressed in brown adipose tissue with also rather high expression in white adipose tissue (Liu et al., 2009). Similar expression patterns were found in mice and in rats. In addition, receptor expression was markedly induced during differentiation of 3T3-L1 preadipocytes (Ge et al., 2008; Jeninga et al., 2009; Liu et al., 2009). Very low levels of HCA₁ receptor expression were seen in several other tissues. However, it is not clear whether this is due to specific expression of the HCA₁ receptor or to the presence of adipocytes in these tissues. It is noteworthy that in both mouse and human models, it could be shown that thiazolidinediones acting via the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) increase the expression of HCA₁ receptor mRNA 4- to 5-fold (Jeninga et al., 2009). This effect was shown to be mediated by the binding of the PPARγ/retinoid X receptor heterodimer to a functional PPAR-response element present in the proximal promoter of the HCA₁ receptor gene (Jeninga et al., 2009). Jeninga et al. (2009) provided additional data suggesting that the antilipolytic effect of thiazolidinediones is mediated in part through the up-regulation of the HCA₁ receptor in adipocytes.

B. HCA₂

The receptor of HCA₂ shows highest expression levels in white and brown adipose tissue in both human and mouse (Soga et al., 2003; Tunaru et al., 2003; Wise et al., 2003). During the differentiation of 3T3-L1 preadipocytes, expression of the HCA₂ receptor is induced 1 to 2 days earlier than expression of the HCA₁ receptor (Ge et al., 2008; Jeninga et al., 2009). Similar to HCA₁, the expression of the HCA₂ receptor is increased by PPARγ activation (Jeninga et al., 2009). Because there is no functional PPAR-response element in the promoter of the HCA₂ receptor gene, it has been suggested that the PPAR-response element present in the proximal promoter of the HCA₁ receptor gene controls the HCA₂ receptor gene promoter (Jeninga et al., 2009).

Apart from adipocytes, expression of the HCA₂ receptor is also found in various immune cells, including neutrophils, macrophages, dendritic cells, and epidermal Langerhans cells (Schaub et al., 2001; Benyó et al., 2005; Maciejewski-Lenoir et al., 2006; Kostylina et al., 2008) but not in mouse and human B and T lymphocytes or in human eosinophils (Schaub et al., 2001; Kostylina et al., 2008; Tang et al., 2008). HCA₂ receptor expression was not detected in immature bone marrow neutrophils, indicating that induction of HCA₂ receptor expression occurs during the late stages of terminal differentiation of neutrophils (Kostylina et al., 2008). Expression of the HCA₂ receptor in macrophages can be increased by treatment of cells with IFN-γ, TNF-α, lipopolysaccharide, and CpG oligodeoxynucleotides, whereas IFN-α, IFN-β, granulocyte macrophage–colony-stimulating factor, interleukin-1, interleukin-12, and interleukin-18 had no effect (Schaub et al., 2001). In primary human monocytes and adhesion-differentiated macrophages, the HCA₂ receptor was up-regulated by hypoxia (Knowles et al., 2006).

Expression of the HCA₂ receptor has also been reported in the apical membrane of epithelial cells of the small and large intestine. Expression in intestinal epithelial cells seems to be reduced in colon cancer as well as under germ-free conditions (Thangaraju et al., 2009; Cresci et al., 2010). In addition, retinal pigment epithelial cells express the HCA₂ receptor, which appears primarily localized to the basolateral membrane of these cells (Martin et al., 2009). On the mRNA level, HCA₂ receptor expression has been shown in primary human keratinocytes (Maciejewski-Lenoir et al., 2006; Tang et al., 2008); however, immunohistochemistry failed to demonstrate HCA₂ receptor expression in keratinocytes (Maciejewski-Lenoir et al., 2006). Expression of the HCA₂ receptor in keratinocytes has recently been shown by genetic and functional approaches (Hanson et al., 2010), and strong HCA₂ receptor expression has also been described in the human epidermoid carcinoma cell line A431 (Maciejewski-Lenoir et al., 2006; Zhou et al., 2007). Similar to macrophages, expression of the HCA₂ receptor in keratinocytes and keratinocyte cell lines is induced be IFN-γ (Tang et al., 2008).

C. HCA₃

Expression of the HCA₃ receptor seems to be very similar to that of the HCA₂ receptor. The HCA₃ receptor is expressed in adipose tissue (Soga et al., 2003; Tunaru et al., 2003; Wise et al., 2003), and this expression can be increased by activation of PPARγ (Jeninga et al., 2009). In addition, the HCA₃ receptor is expressed in various immune cells, including neutrophils, monocytes, and macrophages (Nomura et al., 1993; Yousefi et al., 2000; Ahmed et al., 2009a; Irukayama-Tomobe et al., 2009). Expression in neutrophils has been shown to be stimulated by granulocyte macrophage–colony-stimulating factor (Yousefi et al., 2000), whereas expression in primary human monocytes and phorbol 12-myristate 13-acetate-differentiated THP1 and U937 monocytic cell lines can be increased by hypoxia (Knowles et al., 2006). Evidence has also been provided for the expression of the HCA₃ receptor in epithelial cells of the colon (Thangaraju et al., 2009).

A. Structure-Activity Relationships for the HCA₂ Receptor

2. Pyrazoles.

One compound, pyrazole-3-carboxylic acid (Table 3, R1 = R2 = H), was a high-efficacy partial agonist in the rat [³⁵S]GTPγS binding assay (relative intrinsic activity of 85%) with submicromolar affinity (K_i = 0.59 μM) in the rat spleen radioligand binding assay (Lorenzen et al., 2001). This finding was taken as the starting point for a synthetic program, both in academia and industry. Partial agonists may display tissue selectivity on the basis of differences in receptor expression in different tissues. This was thought to be potentially beneficial in the case of the HCA₂ receptor, as the side effect of flushing might be separated from the desired action in dyslipidemia. In the 1980s, Seki et al. (1984) had reported on substituted alkylpyrazoles as hypolipidemic agents; logically, Lorenzen et al. (2001) hypothesized that such compounds might act via the HCA₂ receptor. Van Herk et al. (2003) prepared two series of alkyl- and benzyl-substituted pyrazole-3-carboxylic acid derivatives (Table 3). Partial agonism was maintained throughout the series, and some compounds had affinity values in the same range as nicotinic acid. This was particularly true for the propyl- and butyl-substituted derivatives as well as the 3-carbon atom ring-closed compound, with K_i values of 0.14, 0.072, and 0.16 μM, respectively, and relative intrinsic activities of 70, 81, and 56% in rat tissue. The R1 = 3-chlorobenzyl derivative had quite acceptable affinity (K_i = 0.50 μM), modest potency in the [³⁵S]GTPγS binding assay,, and a relative intrinsic activity of 39%. A more extended series of pyrazoles, along the same vein of either alkyl or benzyl substitution, was disclosed by Gharbaoui et al. (2007), corroborating the findings by Van Herk et al. (2003). The authors identified the 3-fluorobenzyl R1-substituted pyrazole as slightly more active than the 3-chlorobenzyl derivative in a cAMP assay. Introduction of a fluoro substituent on position R2 (Table 3) was tolerated when in combination with an alkyl substituent on R1 (Skinner et al., 2007b). In that same publication, it was shown that the carboxylic acid could not easily be replaced by a similarly acidic tetrazole moiety, because most pairs differed 1 to 2 log units (or even more) in activity. Later, Semple et al. (2008) reported on one particular exception, in which the carboxylic acid/tetrazole switch yielded a partial agonist with substantial activity on the cloned human and mouse receptors in a cAMP assay. In mice, this compound [3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydrocyclopentapyrazole (MK-0354)] (Fig. 4a) was as active as nicotinic acid in reducing the amount of plasma free fatty acids in vivo and had quite favorable pharmacokinetic properties, but did not cause vasodilation in the mouse ear, a surrogate marker for flushing. This differential behavior might indeed be due to the partial agonistic nature of the compound, although other explanations such as biased, ligand-directed signaling may be equally feasible (Richman et al., 2007; Walters et al., 2009). MK-0354 appeared very selective, in that it did not show activity in a panel of more than 120 other proteins, including the human ether-a-go-go-related gene channel. Imbriglio et al. (2009) extended on the scaffold of MK-0354 by introducing fluorinated phenyl substituents, the 2,3,5-trifluoro variant of which (Fig. 4b) was 2- to 3-fold more potent than nicotinic acid in vitro. It displayed an acceptable pharmacokinetic profile in vivo and, like MK-0354, caused no vasodilation in the mouse ear. Similar derivatives, now with a carboxylic acid function, were reported by Schmidt et al. (2009) and Imbriglio et al. (2010). One typical racemate example (Fig. 4c) and its most active enantiomer were as active as nicotinic acid in both the [³H]nicotinic acid and [³⁵S]GTPγS binding assays. Boatman et al. (2008) (Fig. 4d) reported a further derivatization of MK-0354 with a cyclopropane extension.

View this table:

• View inline
• View popup

TABLE 3

Affinities (K_i values in radioligand displacement study), potencies (EC₅₀ values in [³⁵S]GTPγS binding assay), and relative intrinsic activities (RIA; [³⁵S]GTPγS binding assay) of pyrazole-derived partial agonists for the rat nicotinic acid receptor

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Chemical structures of bicyclic pyrazole MK-0354, a highly selective partial agonist for the HCA₂ receptor, and two later follow-up compounds.

3. Acifran Analogs.

Acifran (Fig. 3) was developed in the early 1980s as a lipid-lowering agent, but structure-activity relationships were not reported. Mahboubi et al. (2006) synthesized and evaluated a small number of acifran analogs to study phosphorylation of extracellular signal-regulated kinase (ERK) 1/ERK2 in CHO cells expressing the human HCA₂ receptor and to explore the triglyceride-lowering potential in fructose-fed rats. First, they identified the S-enantiomer of acifran as the active principle. Second, the introduction of a para-fluor substituent on the phenyl ring (Fig. 5a) preserved activity in the in vivo animal model, whereas other modifications were not allowed. Third, there was little selectivity with respect to the HCA₃ receptor. A further, more extensive study was performed by Jung et al. (2007). Other substituents on the phenyl ring were tried; a 3-chloro (Fig. 5b) or -bromo substituent yielded slightly higher potency than acifran in a cAMP assay of the human HCA₂ receptor, as was the case for the introduction of a similarly substituted thiophene moiety instead of phenyl (Fig. 5c). Again, little selectivity, if any, was observed with respect to the human HCA₃ receptor. Some of the analogs were resolved into their individual stereoisomers, showing that the (+)-isomer was invariably the biologically active principle.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Novel acifran analogs. *, chiral center.

4. Anthranilic Acid Derivatives.

Anthranilic acid derivatives emerged from high-throughput screening campaigns at a number of companies. Merck has been most prolific in bringing these results into the peer-reviewed public domain. Shen et al. (2007a) reported on the optimization of an early hit (Fig. 6a) with an EC₅₀ value of 6 μM in the [³⁵S]GTPγS binding assay, focusing on pharmacokinetic aspects, including the so-called serum shift. Although not addressed in all publications, the anthranilic acid derivatives, unlike the pyrazoles discussed above (Schmidt et al., 2009), seem to be prone to high plasma protein binding with a strong negative impact on the in vivo activity of the molecules. This was simulated by measuring the apparent affinities in the [³H]nicotinic acid binding assay in the absence and presence of 4% human serum. Serum shifts (IC₅₀₊/IC₅₀₋) of 1000-fold or more seemed to be were typical for some of the anthranilic acid derivatives. A typical example is the biphenyl compound (Fig. 6b) with high affinity in the [³H]nicotinic acid binding assay in the absence of serum (IC₅₀ = 4 nM) but with a serum shift of 8000. Changing one of the phenyl groups to a heterocyclic moiety as in Fig. 6c improved this profile (IC₅₀ = 10 nM; serum shift = 40). This compound was tested in vivo, showing a favorable profile of a strong reduction of FFA levels in mice after oral dosing but failing to induce vasodilation in the mouse ear as a measure for the flushing side effect. The introduction of a piperazine ring as a linker between the aromatic fragments within the molecules yielded a series of urea analogs as in Fig. 6d (Shen et al., 2007b). This compound had an IC₅₀ value of 140 nM in the [³H]nicotinic acid binding assay and an EC₅₀ value of 470 nM in the [³⁵S]GTPγS binding assay, displaying high-efficacy partial agonism (83% of the maximal nicotinic acid response). Its serum shift was not reported. A similar derivative without the hydroxyl group on the quinoxaline moiety was tested in vivo. After intraperitoneal administration, it reduced free fatty acid levels in mice at least as efficaciously as nicotinic acid and did not induce any vasodilation. Building further on the bicyclic quinoxaline moiety, the Merck researchers developed tricyclic derivatives as well (Shen et al., 2009). A typical example (Fig. 6e) was tested in vivo in rats rather than mice. Although not endowed with optimal pharmacodynamic and pharmacokinetic properties, the compound reduced free fatty acid levels as well as nicotinic acid, but its effect was longer lasting. Vasodilation in the rat ear was less than with nicotinic acid, yielding a therapeutic index (defined as EC_{50, vasodilation}/EC_{50, free fatty-acid suppression}) of 8 compared with 1 for nicotinic acid. In that study, it was also demonstrated that partial hydrogenation of the anthranilic acid phenyl ring yielded compounds that retained activity on the HCA₂ receptor. This finding was more elaborately explored in another article from the Merck group (Raghavan et al., 2008). The tetrahydro analog (Fig. 6f) had 2 nM affinity in the [³H]nicotinic acid binding assay, an EC₅₀ value of 18 nM in the [³⁵S]GTPγS binding assay, and very acceptable pharmacokinetics in mice, whereas a value for the serum shift and in vivo efficacy data were not presented. The authors concluded that the tetrahydro variants of anthranilic acid derivatives might fare better than the parent molecules because they seemed to yield improved oral bioavailability and better cytochrome P450 profiles. A recent publication (Shen et al., 2010) describes the discovery of MK-6892 (Fig. 6g), which resulted in a preclinical candidate from the Merck anthranilic acid effort. It was also found (Ding et al., 2010; Schmidt et al., 2010) that the cyclohexene ring system in such compounds can be further substituted (e.g., Fig. 6h).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

Anthranilic acid derivatives and related compounds as high-affinity HCA₂ receptor full agonists.

5. Fumaric and Other Acids and Their Esters.

A mixture of fumaric acid esters is on the market in Germany for the treatment of psoriasis. It has recently been shown that both the monomethyl (MMF) and monoethyl (MEF) esters of fumaric acid (Fig. 7), but not fumaric acid itself, have micromolar affinity for the HCA₂ receptor (Tang et al., 2008). MMF induced a concentration-dependent Ca²⁺ signal in transfected cells with an EC₅₀ value of 9.4 μM (nicotinic acid, 2.0 μM). MEF was approximately 3-fold less potent than MMF with an EC₅₀ value of 26 μM, whereas dimethylfumarate was inactive. In a [³H]nicotinic acid radioligand displacement assay, somewhat lower K_i values were obtained: for MMF, 0.98 μM; for MEF, 1.3 μM. A number of “simple” acids were tested by Ren et al. (2009) in several HCA2 receptor assay formats. The two most potent compounds were trans-cinnamic acid and para-coumaric acid (Fig. 7), with IC₅₀ values in a [³H]nicotinic acid binding assay of 36 and 58 μM, respectively (nicotinic acid, 0.1 μM). In the [³⁵S]GTPγS binding assay both compounds were somewhat less active, with EC₅₀ values of 240 and 310 μM, respectively. It is noteworthy that in a similar assay on the HCA₃ receptor trans-cinnamic acid was also active (EC₅₀, 180 μM), whereas para-coumaric acid was not. Oral administration of trans-cinnamic acid to wild-type mice led to a significant reduction in plasma free fatty acid levels, whereas the compound was without effect in HCA₂ receptor KO animals.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 7.

Fumaric acid esters and aromatic acids as agonists for the HCA₂ and HCA₃ receptor.

6. Pyridopyrimidinones.

Peters et al. (2010) reported on a different and quite intriguing scaffold from which HCA₂ receptor agonists were derived. The pyridopyrimidinones (Fig. 8) can be regarded as derivatives of nicotinamide (Fig. 3), a compound shown to be inactive at HCA₂ receptors. Nevertheless, submicromolar affinity and potency were observed in this series, although the compounds reported so far suffer from poor pharmacokinetics.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 8.

Pyridopyrimidinones as a new class of selective HCA₂ receptor agonists.

7. Pyrazolopyrimidines as Allosteric Agonists.

Shen et al. (2008) discovered another series of agonists for the HCA₂ receptor with intriguing pharmacological activity. The chemical structure of the compound that was most thoroughly described is represented in Fig. 9. When tested alone it stimulated [³⁵S]GTPγS binding to the human receptor to 75% of activation levels achieved with nicotinic acid, classifying it as a partial agonist. Its potency was higher than for nicotinic acid, with EC₅₀ values of 0.12 μM for the allosteric compound and 1.0 μM for nicotinic acid in this assay format. It is noteworthy that the presence of the pyrazolopyrimidine (500 nM–1 μM) shifted the concentration-effect curve of nicotinic acid significantly to the left, when measured at the level of cAMP in intact CHO cells expressing the human receptor, suggestive of its allosteric enhancing capability. In a radioligand binding assay, the pyrazolopyrimidine dose-dependently increased rather than displaced specific [³H]nicotinic acid binding to 400% of control levels with an EC₅₀ value of 0.17 μM, yet another token of its nature as an allosteric enhancer. In general, such compounds might be used to increase the potency and/or intrinsic activity of the natural ligands for the HCA receptors. This would provide a rather physiological way of intervening with receptor function, especially if the compounds were pure allosteric enhancers without agonist activity when tested alone.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 9.

Allosteric agonist for the HCA₂ receptor.

B. Structure-Activity Relationships for the HCA₃ Receptor

Because the HCA₃ receptor occurs only in higher primates, the development of (rodent) animal models is hampered, which may explain the limited medicinal chemistry efforts so far. Despite the high (>95%) homology between the two receptors, nicotinic acid is in fact very selective for the HCA₂ receptor. Acifran, however, is not very selective for the two receptors, with slightly lower micromolar affinity for the HCA₃ receptor (for example, Mahboubi et al., 2006; Ren et al., 2009). The most extensive structure-activity study with acifran analogs (Jung et al., 2007) showed that an ethyl rather than a methyl substituent at the chiral center in acifran (see Fig. 3) provided some (5-fold) selectivity for the HCA₃ receptor, whereas all other compounds were slightly selective for the HCA₂ receptor. Ren et al. (2009) identified ortho-coumaric acid (Fig. 5) as ∼20-fold selective for the HCA₃ receptor in a [³⁵S]GTPγS binding assay. EC₅₀ values for this compound were 70 and 1270 μM for the HCA₃ and HCA₂ receptors, respectively, whereas the isomer para-coumaric acid was inactive at the HCA₃ receptor but not at the HCA₂ receptor (see section V.A).

The first exclusive search for HCA₃ receptor ligands was reported by Semple et al. (2006). In a screening campaign using a forskolin-stimulated cell line expressing the human HCA₃ receptor, they discovered a benzotriazole compound (Fig. 10a) with r = isopropyl as a 400 nM hit, reducing the cAMP production; its activity was confirmed in an antilipolysis assay with human subcutaneous adipocytes. Further exploration of the R-substituent led to a number of other potent HCA₃ receptor agonists [e.g., R = 2-butyl (EC₅₀ = 330 nM)], R = CH(CH₃)CH₂OCH₃ (EC₅₀ = 200 nM) being the most potent compound. None of the compounds displayed any activity on the HCA₂ receptor. A subtle change to R = propyl instead of isopropyl yielded significantly less activity (EC₅₀ = 7.4 μM).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 10.

Various classes of HCA₃ receptor ligands.

The same research team discovered that some 4-amino- 3-nitrobenzoic acids, used as intermediates in the synthesis of the benzotriazoles, also displayed significant activity at HCA₃ receptors and selectivity over HCA₂ receptors (Skinner et al., 2007a). A wider range of these acids, now substituted at the 4-amino group (Fig. 10b), was synthesized and tested in the cAMP whole-cell assay mentioned above. Three compounds displayed full agonist responses with EC₅₀ values below 100 nM, the most potent (R₁ = propyl, R₂ = H) with an EC₅₀ value of 30 nM. In the same publication, it was hypothesized that a pyridine ring could substitute for the nitro-aryl moiety, avoiding the nitro group as a possible toxicophore. Indeed, the resulting 6-amino-substituted nicotinic acids (Fig. 10c) proved active as HCA₃ receptor full agonists when tested in the cAMP whole-cell assay. The structure-activity relationships between the two series were very similar. Again, the n-propyl-substituted compound was the most active one (EC₅₀ = 51 nM). Double substitution of the amino group, however, was less favored than in the 4-amino-3-nitrobenzoic acids.

The synthetic efforts were extended to the pyrazole carboxylic acids as a template for the HCA₃ receptor (Skinner et al., 2009). Not surprisingly, the same substituted amino group was introduced to the pyrazole ring system (Fig. 10d). Here, double substitution to yield a tertiary amine was particularly explored, providing a typical pattern of thiophene derivatives, one of which (Fig. 10e) had high potency with an EC₅₀ value of 3 nM and more than 1000-fold selectivity with respect to the HCA₂ receptor. It is noteworthy that replacing -S- with -O- to yield 3′-furanyl substitution rendered the compound at least 1000-fold less active at the HCA₃ receptor.

A. G-Protein Coupling

The sensitivity of nicotinic acid-induced effects to the action of pertussis toxin (islet-activating protein) pointed to G-proteins of the G_i/G_o-family as the G-proteins coupled to the nicotinic acid receptor (Aktories et al., 1983). After the identification of the HCA₂ receptor as a nicotinic acid and ketone body receptor and the discovery of ligands activating HCA₁ and HCA₃ receptors, it has been shown in numerous models that the effects mediated by all three receptors are inhibited by pertussis toxin (Soga et al., 2003; Tunaru et al., 2003; Wise et al., 2003; Cai et al., 2008; Ge et al., 2008; Ahmed et al., 2009a; Irukayama-Tomobe et al., 2009; Liu et al., 2009). Thus, HCA₁, HCA₂, and HCA₃ receptors are G_i/G_o-coupled receptors.

B. Downstream Signaling

Consistent with the fact that all three receptors are G_i/G_o-coupled, agonists of HCA₁, HCA₂, and HCA₃ receptors have been shown to inhibit adenylyl cyclase activity and thereby to decrease cAMP levels in various cells after heterologous expression of the receptors as well as in primary adipocytes (Soga et al., 2003; Tunaru et al., 2003; Wise et al., 2003; Richman et al., 2007; Ahmed et al., 2009a, 2010; Liu et al., 2009). In adipocytes, a decrease in cAMP results in an antilipolytic effect because cAMP is the major intracellular regulator of lipolysis by stimulating cAMP-dependent kinase to activate lipolytic enzymes (Duncan et al., 2007). Also, in human neutrophils as well as in the human epidermoid cell line A431, activation of the HCA₂ receptor has been shown to result in decreased cAMP levels (Zhou et al., 2007; Kostylina et al., 2008). Kostylina et al. (2008) suggested that the decrease in cAMP levels induced by activation of the HCA₂ receptor in neutrophils induces apoptosis through a reduction in phosphorylation of the proapoptotic protein Bad via cAMP-dependent protein kinase.

In immune cells, activation of G_i results in stimulation of phospholipase C β-isoforms, most likely through the release of G-protein βγ-subunits (Exton, 1996). Consistent with this, HCA₂ and HCA₃ receptors, which are expressed in neutrophils, macrophages, and other immune cells, have been shown to mediate ligand-induced increases in free intracellular Ca²⁺ concentrations in a G_i/G_o-dependent manner (Benyó et al., 2006; Tang et al., 2006; Kostylina et al., 2008; Ahmed et al., 2009a; Irukayama-Tomobe et al., 2009). It has been suggested that the increase in intracellular Ca²⁺ concentrations mediated by HCA₂ receptors in response to nicotinic acid results in the activation of Ca²⁺-sensitive phospholipase A₂ and subsequent formation of prostanoids (Benyó et al., 2005, 2006; Tang et al., 2006). This would well explain the rapid formation of prostanoids such as prostaglandins D₂ and E₂ in response to nicotinic acid (Morrow et al., 1989; Cheng et al., 2006; Maciejewski-Lenoir et al., 2006). Alternatively, phospholipase A₂ can also be phosphorylated and activated by ERK, which in turn can be activated through HCA receptors (Tunaru et al., 2003; Richman et al., 2007; Ahmed et al., 2009a; Liu et al., 2009; Walters et al., 2009). This effect is dependent on β-arrestin, and activation of ERK via the HCA₂ receptor has been shown to involve the recruitment of β-arrestin to the plasma membrane (Walters et al., 2009). In addition, Walters et al. (2009) showed that upon HCA₂ receptor activation, β-arrestin-1 binds to cytosolic phospholipase A₂ (PLA₂) and activates the enzyme. This results in the release of arachidonic acid and provides an alternative mechanism for the stimulation of PLA₂-dependent prostanoid formation through HCA receptors in immune cells. The HCA₂ receptor-mediated activation of PLA₂ and subsequent formation of prostanoids such as prostaglandins (PG) D₂ E₂ in epidermal cells is a critical mechanism underlying the nicotinic acid-induced flushing reaction (see section VII).

C. Receptor Desensitization

There is ample evidence that some of the effects mediated by the HCA₂ receptor are subject to desensitization (Stern et al., 1991). Consistent with this, nicotinic acid-induced flushing mediated by the HCA₂ receptor as well as nicotinic acid-induced increases in intracellular Ca²⁺ concentrations are showing desensitization within minutes (Benyó et al., 2005; Kostylina et al., 2008). However, whether these desensitization phenomena are due to effects on the receptor itself or are at the level of downstream signaling processes is not clear. For heterologously expressed HCA₁ and HCA₂ receptors, internalization of the receptor in response to ligand application has been described previously (Richman et al., 2007; Liu et al., 2009; Li et al., 2010). In the case of heterologously expressed HCA₂ receptor, internalization is dependent on the type of ligand. The full agonist nicotinic acid induced internalization, whereas a partial agonist (which, unlike nicotinic acid, did not induce ERK phosphorylation) had no effect (Richman et al., 2007). Internalization of heterologously expressed HCA₂ activated by nicotinic acid was shown to occur in a manner dependent on G-protein-coupled receptor kinase 2 (GRK2) and arrestin 3, whereas the recycling of the internalized receptor was independent of endosomal acidification (Li et al., 2010).

A. HCA₁

Because the HCA₁ receptor is almost exclusively expressed in adipocytes and because it mediates lactate-induced, G_i-dependent inhibition of adenylyl cyclase activity, the obvious biological role of the HCA₁ receptor is the inhibitory regulation of adipocyte lipolysis (Cai et al., 2008; Liu et al., 2009; Ahmed et al., 2010). The classic situation leading to elevated plasma lactate levels is intensive exercise during which the anaerobic degradation of carbohydrates to lactate is the major energy-providing pathway (Brooks and Mercier, 1994). It is plausible that under conditions of intensive exercise, when free fatty acid oxidation is strongly reduced, lactate would exert an inhibitory effect on the fatty acid release from adipocytes (Issekutz and Miller, 1962). Although lactate can inhibit lipolysis(Fredholm, 1971; Boyd et al., 1974; Cai et al., 2008; Liu et al., 2009; Ahmed et al., 2010), there is no proof for this concept, which has been rather controversial (Trudeau et al., 1999). When wild-type and HCA₁ receptor-deficient mice were trained to exercise at an intensity that resulted in plasma lactate levels sufficient to activate the HCA₁ receptor, no difference between the plasma concentrations of free fatty acids could be observed between wild-type and HCA₁ receptor-deficient mice (Ahmed et al., 2010). Therefore, lactate and its receptor do not seem to critically contribute to the regulation of lipolysis under conditions of intensive exercise.

Adipocytes are another source of lactate, in that they can convert more than 50% of the metabolized glucose to lactate, a process stimulated by insulin and glucose uptake (DiGirolamo et al., 1992). The liver takes up the lactate released from adipocytes and uses it for gluconeogenesis and glycogen synthesis. Because insulin-induced glucose uptake results in a severalfold increase in extracellular lactate levels in adipose tissue (Jansson et al., 1994; Qvisth et al., 2007; Ahmed et al., 2010), the hypothesis was formulated that lactate, acting through the HCA₁ receptor, contributes to the inhibition of lipolysis induced by insulin. In fact, studies in HCA₁ receptor-deficient mice and adipocytes clearly showed that insulin-induced inhibition of lipolysis and insulin-induced decrease in adipocyte cAMP were strongly reduced in the absence of HCA₁ (Ahmed et al., 2010). Thus, lactate acting through HCA₁ functions in an autocrine and paracrine fashion to mediate insulin-induced antilipolytic effects (Fig. 11). Mice lacking HCA₁ show a reduced weight gain under high fat diet, indicating that this mechanism may contribute to the increase in body weight under hypercaloric diet. Thus, blockade of the HCA₁ receptor may be a strategy for treating obese patients.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 11.

Functions of the recently deorphanized receptors HCA₁, HCA₂, and HCA₃. The lactate receptor HCA₁ mediates the short-term anabolic effects of insulin on adipocytes and thereby helps to store energy after feeding (A). In contrast, HCA₂ and HCA₃ receptors are involved in the long-term regulation of lipolytic activity, being receptors for the ketone body 3-hydroxy-butyrate (HCA₂) and the β-oxidation intermediate 3-hydroxy-octanoate (HCA₃). In situations of increased β-oxidation rates (e.g., during starvation), 3-hydroxy-butyrate and 3-hydroxy-octanoate plasma levels are increased and result in the inhibitory regulation of lipolysis via HCA₂ and HCA₃ receptors, respectively, in the form of a negative feedback loop (B). Thereby, HCA₂ and HCA₃ receptors help preserve energy stores during starvation. AC, adenylyl cyclase; TG, triglycerides; HSL, homon-sensitive lipase; ATGL, adipocyte triglyceride lipase; FFA, free fatty acids; PKA, cAMP-regulated protein kinase.

B. HCA₂

Although the HCA₂ receptor is an important target for the drug nicotinic acid, its biological role is instead related to the ketone body 3-hydroxy-butyrate, which activates the receptor with an EC₅₀ of approximately 700 μM (Taggart et al., 2005). Under normal conditions, 3-hydroxy-butyrate plasma levels are 50 to 400 μM, but they increase after an overnight fast to 1 to 2 mM and reach 6 to 8 mM during prolonged starvation (Owen et al., 1969). Because the formation of 3-hydroxy-butyrate in the liver depends on the delivery of free fatty acids induced by lipolysis in adipocytes, the activation of the HCA₂ receptor by 3-hydroxy-butyrate at millimolar concentrations and the subsequent antilipolytic effect represent a classic negative feedback mechanism that controls the lipolytic rate during starvation (Senior and Loridan, 1968) (Fig. 11). In isolated adipocytes from wild-type mice 3-hydroxy-butyrate inhibits lipolysis, an effect that is not seen in adipocytes from HCA₂ receptor-deficient mice (Taggart et al., 2005), further supporting the notion that the HCA₂ receptor functions as a metabolic sensor that regulates lipolytic activity during starvation to avoid excessive triglyceride degradation.

The HCA₂ receptor agonist nicotinic acid has been used for decades as an antidyslipidemic drug to prevent and treat atherosclerosis (Meyers et al., 2004; Carlson, 2005). Its therapeutic potential is well established (see section VIII). Several mechanisms have been proposed to account for the antiatherosclerotic effects of nicotinic acid (Gille et al., 2008; Kamanna and Kashyap, 2008; Digby et al., 2009). Nicotinic acid reduces low-density lipoprotein (LDL) cholesterol, triglyceride, and lipoprotein(a) plasma levels and simultaneously increases levels of HDL cholesterol (Carlson, 2005). Among the mechanisms that have been proposed to account for the antidyslipidemic properties of nicotinic acid is the activation of HCA₂ receptors expressed on adipocytes. The antilipolytic effect of HCA₂ receptor activation on adipocytes results in a decreased release of free fatty acids from fat cells. This in return reduces the supply of free fatty acids to the liver, leading to a reduced synthesis of triglycerides and very-low-density lipoprotein as well as to a subsequent drop in LDL cholesterol levels (Carlson, 1963). How nicotinic acid increases HDL cholesterol levels is less clear. The decrease in triglyceride content of apolipoprotein B-containing lipoproteins may result in a decreased exchange of triglycerides for cholesteryl esters from HDL particles mediated by the cholesterol transfer protein, eventually leading to increased HDL cholesterol levels (Kontush and Chapman, 2006; Offermanns, 2006; Joy and Hegele, 2008). This mechanism is supported by the observation that HDL-cholesterol elevation in response to nicotinic acid depends on the presence of cholesterol transfer protein (Hernandez et al., 2007; van der Hoorn et al., 2008). Whether the HCA₂ receptor also mediates the increase in HDL-cholesterol levels in response to nicotinic acid, however, remains unclear (Bodor and Offermanns, 2008; Kamanna and Kashyap, 2008). Because mouse lipoprotein metabolism is considerably different from that of humans, genetic mouse models are not helpful in analyzing this question. Selective antagonists of the HCA₂ receptor tested in appropriate models may eventually be helpful in answering this question.

The physiological role of the HCA₂ receptor in immune cells, such as macrophages or neutrophils, that express the receptor remains currently unclear. Given that plasma levels of 3-OH-butyrate, the endogenous ligand of HCA₂, are highly elevated under starving conditions, one may speculate that the HCA₂ receptor expressed on immune cells mediates anti-inflammatory effects that could be advantageous under conditions of starvation. In this respect, it is interesting to know that HCA₂ expression in macrophages is up-regulated by various cytokines and activators of innate immunity (see section IV).

There is good evidence that expression of the HCA₂ receptor in epidermal Langerhans cells and keratinocytes is responsible for one of the major unwanted effects of nicotinic acid, the flushing reaction. Flushing results from a cutaneous vasodilation associated with a sensation of tingling and burning that causes many patients to discontinue nicotinic acid therapy (Benyó et al., 2005, 2006; Maciejewski-Lenoir et al., 2006; Gille et al., 2008; Kamanna et al., 2009). The induction of a flush reaction by nicotinic acid or the antipsoriatic drug monomethyl fumarate is mediated by HCA₂ and shows a biphasic increase in dermal blood flow (Hanson et al., 2010). The first phase is induced via activation of HCA₂ on Langerhans cells, and HCA₂ on keratinocytes is responsible for the late phase of the response. Whereas Langerhans cell-mediated flushing involves cyclooxygenase-1, PGD₂, and PGE₂, keratinocyte-mediated late-phase flushing involves cyclooxygenase-2 and PGE₂ (Benyó et al., 2005; Cheng et al., 2006; Paolini et al., 2008; Hanson et al., 2010). The expression of HCA₂ in various cells of the epidermis and the marked epidermal and dermal effects resulting from the activation of HCA₂ has raised the question whether this receptor system has any physiological or pathophysiological role in the skin, besides its pharmacological function. HCA₂-mediated prostanoid formation in Langerhans cells and keratinocytes, for instance, may underlie some of the many forms of skin reactions in which alterations or traumata in the epidermis result in dermal vasodilation and various dermal sensations. It will be interesting to explore in the future the potential physiological and pathophysiological role of HCA₂-mediated cellular effects in the epidermis.

The HCA₂ receptor has also been shown to be expressed in intestinal epithelial cells where the receptor may respond to butyrate, which is present in millimolar concentrations in the gut lumen. It has been suggested that HCA₂ thereby functions as a tumor suppressor and anti-inflammatory receptor (Thangaraju et al., 2009).

C. HCA₃

The biological role of the HCA₃ receptor seems to be quite similar to that of the HCA₂ receptor with regard to the regulation of adipocyte lipolysis. The plasma concentration of the β-oxidation intermediate 3-hydroxy-octanoate, which activates HCA₃ receptors at micromolar concentrations, increases substantially under conditions of increased free fatty acid oxidation during fasting, during diabetic ketoacidosis, in various mitochondrial fatty acid β-oxidation disorders, and under the influence of a ketogenic diet (Costa et al., 1998; Jones et al., 2002; Ahmed et al., 2009a). Similar to HCA₂, the HCA₃ receptor mediates a negative feedback mechanism under these conditions to counterbalance prolipolytic stimuli (Ahmed et al., 2009b) (Fig. 11). The fact that the HCA₃ receptor is found only in higher primates suggests that the optimal regulation of lipolytic activity under conditions of increased free fatty acid release during starvation and other extreme conditions represents an important mechanism to use the energy stored in form of triglycerides economically and to avoid a waste of energy. The advantage of having additional regulatory mechanisms under these conditions was apparently sufficient to drive the evolution of additional lipolysis-controlling receptors in higher primates. Whether HCA₃ has potential biological roles outside the adipose tissue is currently not known.

A. Nicotinic Acid Alone

Nicotinic acid has been used in clinical practice for more than half a century now, ever since Altschul et al. (1955) pioneered its use as an antidyslipidemic drug in man. This finding was followed-up by a number of further clinical studies that all established the usefulness of nicotinic acid as a “broad-spectrum” lipid-modulating drug (for a recent review, see Carlson, 2005; Guyton, 2007), albeit with predominantly dermatological side effects (flushing). In a well defined early long-term trial, nicotinic acid treatment had no effect on total mortality compared with placebo, although it showed a modest benefit in lowering the incidence of recurrent myocardial infarction (Coronary Drug Project Research Group, 1975). It is noteworthy that 9 years after conclusion of this trial, mortality in the nicotinic acid-treated patient group was a significant 11% lower than placebo (Canner et al., 1986). The authors concluded that this delayed benefit of nicotinic acid, occurring after discontinuation of the drug, may relate to the early favorable effect of nicotinic acid on reinfarction incidents and/or may be the result of the cholesterol-altering effect of nicotinic acid.

B. Nicotinic Acid with Antiflushing Strategies

The use of generic nicotinic acid in its crystalline form has always been associated with the common side effect of flushing, which had a strong negative impact on patient compliance. Strategies such as taking tablets during meals and/or taking acetylsalicylic acid concomitantly often render the flushing bearable; in addition, it seems to wane over time.

C. Nicotinic Acid in Combination with Other Lipid-Altering Drugs

Many later clinical trials were designed to evaluate the use of nicotinic acid in combination with other lipid-altering drugs, particularly HMG-CoA reductase inhibitors (“statins”). On many occasions, a significant reduction of coronary events and mortality was observed. With statins emerging as a first-choice strategy for lipid lowering in the 1990s, it was only logical to examine the combined effects of nicotinic acid with one of the early HMG-CoA reductase inhibitors, simvastatin, in a double-blind trial with 160 patients with established coronary heart disease and low HDL cholesterol plasma levels observed for 3 years (Brown et al., 2001). The mean levels of LDL and HDL cholesterol were unaltered in the placebo group, but changed by −42 and +26%, respectively, in the simvastatin-plus-nicotinic acid group. This was manifested clinically by a strong reduction in the frequency of a first cardiovascular event (24 versus 3%). Although the ideal control group receiving simvastatin alone was not tested, the reduction in cardiovascular events in patients treated with simvastatin and nicotinic acid was much larger than would have been expected from a treatment with simvastatin alone.

An important surrogate endpoint in many trials is the impact on the size of atherosclerotic lesions, which is not altered very much on statin monotherapy (Guyton, 2007). In all clinical trials, addition of nicotinic acid to statin therapy resulted in a reduced development of atherosclerotic lesions. For instance, in the ARBITER 2 study (Taylor et al., 2004), patients with coronary heart disease who were treated with statins and had low levels of HDL cholesterol received nicotinic acid as a supplementary drug regimen. The primary endpoint was the change in common carotid intima-media thickness after 1 year. After this period, compared with values at the start of the study, the thickness was significantly increased in the placebo group but unchanged in the patients treated with nicotinic acid. The overall difference in thickness progression between the nicotinic acid and placebo groups was not statistically significant, however (Taylor et al., 2004). A similar, more recent study (ARBITER6-HALTS) compared the addition of either nicotinic acid (extended release) or ezetimibe to statin monotherapy (Taylor et al., 2009). Niacin proved superior to ezetimibe on the basis of the same primary clinical endpoint of the mean common carotid intima-media thickness after 14 months of treatment. A total of 363 patients were enrolled, but after 208 patients had completed the study, it was decided to halt the trial because the use of ezetimibe led to a paradoxical increase in atherosclerosis, despite the significant (further) reduction in LDL cholesterol levels. In the nicotinic acid-plus-statin group, a significant regression of both mean and maximal carotid intima-media thickness was observed, in combination with a significant 18% increase in HDL cholesterol levels.

Two cardiovascular endpoint trials are currently under way to test whether the addition of nicotinic acid to statins has a beneficial effect on the progression of cardiovascular disease, incidence of major cardiovascular events, and cardiovascular disease-associated mortality. The Atherothrombosis Intervention in Metabolic Syndrome with low HDL/high triglycerides and Impact on Global Health Outcomes (AIM-HIGH) study compares simvastatin and extended-release nicotinic acid with simvastatin monotherapy in 3300 patients at high risk suffering from cardiovascular disease, low HDL cholesterol, and high triglyceride levels (http://clinicaltrials.gov/ct2/show/NCT00120289). The Heart Protection study 2: Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2-THRIVE) compares simvastatin plus extended-release nicotinic acid plus the prostaglandin D₂ receptor 1 antagonist laropiprant with simvastatin monotherapy in more than 20,000 patients with coronary heart disease (http://clinicaltrials.gov/ct2/show/NCT00461630). The results of these trials are expected to be reported between 2011 and 2013.

D. Fumaric Acid Derivatives

The use of fumaric acid esters for the treatment of psoriasis goes back to case reports in Germany as early as 1959 (Schweckendiek, 1959). A small randomized, double-blind, placebo-controlled study involving 39 patients showed that oral administration of a combination of fumarate esters (the monoethyl- and dimethyl analogs) led to a significant reduction of body surface affected with psoriasis with an average of 21% at the start of the study to 6.7% after 16 weeks. Almost all patients suffered from flushing and diarrhea (Nugteren-Huying et al., 1990). Prolonged use of fumaric acid esters up to 14 years did not seem to cause unacceptable or unbearable side effects, composed mostly of flushing, diarrhea, and lymphocytopenia (Hoefnagel et al., 2003). In a recent study, a retrospective analysis of close to 1000 patients treated in over 150 centers for at least 2 years yielded similar results (i.e., sustained clinical efficacy and an acceptable safety profile) (Reich et al., 2009). Among the many suggested molecular mechanisms of action, the fumarate esters have been shown to interact with the HCA₂ receptor, which may in fact cause the flushing. A similar preparation (BG-12, consisting of dimethylfumarate only) was tested in a double-blind, placebo-controlled clinical phase IIb study in more than 250 patients with relapsing-remitting multiple sclerosis. Treatment with BG-12 (240 mg three times daily) significantly reduced the mean total number of new brain lesions by more than 70% compared with placebo. Again, adverse events included abdominal pain and flushing (Kappos et al., 2008). Given the expression of HCA₂ receptors in various immune cells and the reported beneficial effects of fumaric acid esters, which are able to activate HCA₂ receptors, it is tempting to speculate that HCA₂ receptors may be interesting targets for anti-inflammatory or immunosuppressive therapeutic approaches.

E. Clinical Candidates

A partial agonist for the HCA₂ receptor, MK-0354 (Fig. 4), was evaluated in both phase I and II clinical studies. The effects of single and multiple doses of MK-0354 (300 mg–4 g) were evaluated in two phase I studies conducted in healthy men. In a phase II study, the effects of MK-0354 (2.5 g once daily) on lipids were recorded during 4 weeks in 66 dyslipidemic patients. In the phase I study, it was noticed that a single dose (300 mg) of MK-0354 reduced free fatty acid levels comparable with a 1-g dose of extended-release nicotinic acid, which did not alter after 7 days of administration. In the phase II study, 2.5 g of MK-0354 produced little flushing; however, no clinically meaningful effects on lipids, especially on HDL levels, were observed (Lai et al., 2008).