Oxotremorine

Initial attempts to treat AD using direct cholinergic agonists were limited by low efficacy and side-effect issues (140—142). Thus trials using RS-86 (25), oxotremorine [70-22-4] (26), arecoline [63-75-2] (27), and pilocarpine [92-32-7] (28) to treat AD were equivocal (Eig. 5). However, the identification of multiple subtypes of muscarinic receptors has stimulated a search for subtype specific muscarinic agonists which may limit side effects while increasing efficacy. 

Oxospiro[pyridazine-5(2H),2 (l H)-quinoxaline]-4-carboxylic acid, 3,4-dihydro-, 3, 33 Oxotremorine toxicity, 1, 120 Oxyberberine... 

FIGURE 4.14 Effects of G-protein on the displacement of the muscarinic antagonist radioligand [sH]-L-quinuclidinyl benzylate by the agonist oxotremorine. Displacement in reconstituted phospholipid vesicles (devoid of G-protein sububits) shown in filled circles. Addition of G-protein (Go 5.9 nmol Py-subunit/3.4 nmol ao-IDP subunit) shifts the displacement curve to the left (higher affinity, see open circles) by a factor of 600. Data redrawn from [14]. 

A dose-response curve to a full agonist is obtained. Shown are data for the dose response to the full agonist oxotremorine (responses as a percentage of the maximal response to oxotremorine) in Table 12.5a. The dose-response curve is shown in Figure 12.6a. 

The dose-response curve after receptor alkylation is shown in Figure 12.6a (open circles). The same function is used to fit the data as employed for the control curve (for this example, Equation 12.5). The parameters of the fit dose-response curves are shown in Table 12.5b. Equiactive concentrations of oxotremorine are calculated according to the procedure given in Section 12.2.1. 

FIGURE 12.6 Measurement of full agonist affinity by the method of Furchgott. (a) Dose-response curve to oxotremorine obtained before (filled circles) and after (open circles) partial alkylation of the receptor population with controlled alkylation with phenoxybenzamine (10 jiM for 12 minutes followed by 60 minutes of wash). Real data for the curve after alkylation was compared to calculated concentrations from the fit control curve (see arrows), (b) Double reciprocal of equiactive concentrations of oxotremorine before (ordinates) and after (abscissae) alkylation according to Equation 5.12. The slope is linear with a slope of 609 and an intercept of 7.4 x 107 M-1. 

Pilocarpine, arecoline and, of course, muscarine itself are naturally occurring muscarinic agonists, while oxotremorine is a synthetic one, which, as its name implies, can cause muscle tremor through a central effect. 

A crude alkaloidal fraction from the stem of Tabernaemontana pandacaqui decreased the motor activity, respiratory rate, induced ataxia, antinociception, and loss of screen grip in rats, suggesting a CNS depression. The extract brought about the prolongation of pentobarbital sleeping time and the oxotremorine-induced salivation, hence possible cholinergic effects (13). 

Both the M2 and M4 receptors are, as indicated above, coupled to Gj pathways and appear to mediate similar responses. The M2 receptor is widely expressed in the CNS but also present in heart and smooth muscle, while M4 is preferentially expressed in the CNS, especially in forebrain. Ablation of the M2 receptor leads to complete loss of muscarinic-agonist-stimulated bradycardia [55]. In the CNS, deletion of the M2 receptor abolishes oxotremorine induced akinesia and tremors [56]. Memory and learning tasks including passive avoidance and working memory are impaired in M2-receptor knockout mice, and there is decreased LTP in hippocampal slices [12],... 

Hirschberg, B. T. and Schimerlik, M. I. (1994) A kinetic model for oxotremorine M binding to recombinant porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells../. Biol. Chem. 269,26127-26135. 

N-methylscopolamine (Costa et al. 1982b Schwab et al. 1983). Animals made tolerant to disulfoton were resistant to the lethal or adverse effects of cholinergic agonists, such as carbachol (Brodeur and DuBois 1964 Costa et al. 1981 Schwab and Murphy 1981) and oxotremorine (Costa et al. 1982b McPhillips 1969a), which are not hydrolyzed by acetylcholinesterase. Tissues from animals tolerant to disulfoton such as the ilea (Foley and McPhillips 1973 McPhillips 1969b McPhillips and Dar 1967) and the atria (Perrine and McPhillips 1970 Schwab et al. 1983), were resistant to the effects of carbachol and/or oxotremorine. Because the uterus and vas deferens have a relatively sparse parasympathetic innervation compared to the ileum and do not receive a steady flow of impulses via this system, these tissues were not as subsensitive to carbachol as the ileum (Foley and McPhillips 1973). Thus, acetylcholine accumulation may be a prerequisite for tolerance development. 

Muscarinic Agonists. Another approach is to use a medication that directly activates subtypes of the acetylcholine receptors, namely, muscarinic receptor agonists. The muscarinic agonists used in the past have not been effective. This includes bethanecol, pilocarpine, and oxotremorine. This line of treatment, however, has not been entirely abandoned, and several medications are currently in testing. 

Other cholinergic agonists have no therapeutic use. Muscarine (4.3) is an alkaloid of the mushroom Anumita muscaria muscarone (4.9) is its semisynthetic analog. Pilocarpine (2.2) is found in the leaves of a shrub and can be used to increase salivation or sweating. Arecoline (4.10) is also an alkaloid, and occurs in the betel nut that is used as a mild euphoriant in India and Southeast Asia. Finally, oxotremorine (4.11) is a synthetic experimental agent that produces tremors and is helpful in the study of antiparkinsonian drugs. 

All five muscarinic receptor subtypes have been detected in the central nervous system. The roles of Mx through M3 have been analyzed by means of experiments in knockout mice. The Mx subtype is richly expressed in brain areas involved in cognition. Knockout of Mx receptors was associated with impaired neuronal plasticity in the forebrain, and pilocarpine did not induce seizures in Mx mutant mice. The central nervous system effects of the synthetic muscarinic agonist oxotremorine (tremor, hypothermia, and antinociception) were lacking in mice with homozygously mutated M2 receptors. Animals lacking M3 receptors, especially those in the hypothalamus, had reduced appetite and diminished body fat mass. 

Whether a toxin is naturally reactive to biological macromolecules or receptors, or requires metabolic activation to produce such a species, e.g. the enzyme-mediated transformation of tremorine (9) to the active parasympathomimetic agent oxotremorine (10), it will usually be subject to chemical or enzymic inactivation in vivo. Interruption of the latter process via appropriate substitution may thus lead to an increase in biological activity or toxicity over that of the parent compound. Perhaps the most striking example of this is provided by the extreme metabolic stability and toxicity of TCDD and the nontoxicity of its de- chloro analogue dibenzodioxin (Table 3). 

Bethanechol, arecoline, oxotremorine, and nicotine have been tried and had no beneficial effect on memory. 

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