The rat model was validated with another agonist
The rat model was validated with another agonist of GPR109A, acipimox. Acipimox has been shown to induce vasodilatation in man, by a mechanism dependent on the release of vasodilator prostaglandins (Edlund et al., 1990, Pontiroli et al., 1992). Acipimox is a weaker agonist of the GPR109A receptor (Lorenzen et al., 2002, Pontiroli et al., 1992, Soga et al., 2003, Tunaru et al., 2003), and thus it is better tolerated than nicotinic acid, with a lower incidence of flushing episodes (Fuccella et al., 1980, Seed et al., 1993). Our results in the rat agree with the reported effects of acipimox in humans. Acipimox is able to induce vasodilatation in the rat, but a higher dose is required to elicit a full response.
The IC50 for the antilipolytic effect of nicotinic L-Stepholidine is easily calculated in conscious rats. However, attempts to evaluate the antilipolytic effects of nicotinic acid simultaneously to vasodilatation proved unsuccessful. Any blood draw during vasodilatation studies resulted in artifacts on the laser Doppler readings (data not shown). On the other hand, barbiturate anesthesia has been shown to disturb the metabolism of free fatty acids (Kaniaris et al., 1975, Renauld and Sverdlik, 1975, Toso et al., 1993), making the interpretation of the effects of GPR109A agonists on lipolysis in anesthetized animals difficult. Thus, the rat model presented limitations, as no direct comparison could be made between the dose of GPR109A agonist resulting in an efficacious inhibition of lipolysis and the dose resulting on the lack of a vasodilatory response. Alternatively, a therapeutic index in the rat can be estimated by the comparison of drug levels associated with the threshold vasodilatation response with those associated with inhibition of lipolysis (IC50).
To develop a model in which lipolysis and vasodilatation are measured in the same animal we explored the utility of the dog. The dog has been used in the past as a model for nicotinic acid inhibition of lipolysis (Lipson et al., 1971a, Lipson et al., 1971b, Pereira, 1967). There are anecdotal references to a visible flushing response in dogs treated with nicotinic acid (Pereira, 1967) but no detailed dose response data are available. Nicotinic acid, dosed either orally (this manuscript) or by subcutaneous injection (data not shown), induces a significant flushing response in dogs, characterized by clear reddening of the ears. Accordingly, we sought to determine whether the conscious dog could be used as a model for both lipolysis and flushing.
We evaluated changes in the redness of the ears, using a handheld spectrocolorimeter (Hom et al., 2001). The measurement of the red/green axis (“a” reading in the L⁎a⁎b⁎mode) allowed for the detection of very slight changes in the redness of the dog's ears. Dogs are easily trained to lie still for the spectrocolorimetric readings. At each time point a blood sample was collected and the color reading performed immediately, the whole procedure lasting less than 1 min. Both nicotinic acid and acipimox induced vasodilatation in the dogs in a dose-dependent manner. The vasodilatation response in the dog shared more similarities to the flushing response described in humans than the response observed in the rat. In the first place, the dogs had a distinct “flush”, characterized by their ears turning visibly red, and they displayed behaviors consistent with the reported tingling/itching reported by humans treated with nicotinic acid. Second, the duration of the flushing response was closer to that observed in man (several hours post-dose) than that observed in rats (less than 30 min). As previously described in man, the flushing response in the dog was dependent on COX activity and mainly mediated by PGD2. Simultaneous measurements of plasma free fatty acids allowed us to calculate the therapeutic indices for nicotinic acid and acipimox on a dose basis.