In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists

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Abstract

The ability of tachykinin NK1 receptor antagonists to inhibit GR73632 (d-Ala-[l-Pro9,Me-Leu8]substance P-(7–11))-induced foot tapping in gerbils was employed as an indirect measure of brain penetration and this was compared with their ability to prevent acute emesis induced by cisplatin in ferrets. (±)-GR203040 ((2S,3S and 2R,3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-yl)-amine), CP-99,994 ((2S,3S)-cis-3-(2-methoxybenzylamino)-2-phenyl piperidine) dihydrochloride), and L-742,694 (2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-4-(5-(3-oxo-1,2,4-triazolo)methylmorpholine) potently inhibited GR73632-induced foot tapping (ID50≤0.85 mg/kg), and acute retching induced by cisplatin (ID50≤0.18 mg/kg). RPR100893 ((3aS,4S,7aS)-7,7-diphenyl-4-(2-methoxyphenyl)-2-[(S)-2-(2-methoxyphenyl)proprionyl] perhydroisoindol-4-ol) was not a potent antagonist of retching (ID50 4.1 mg/kg) or foot tapping (ID50>10 mg/kg). High doses (3–10 mg/kg) of CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-N-[(4-quinolinyl)methyl]-4-piperineamine) dihydrochloride), FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-l-propyl]-N-methyl-N-phenylmethyl-l-3-(2-naphthyl)-alaninamide), and LY303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidinyl)piperidin-1-yl)acetyl)amino]propane) were required to inhibit foot tapping; these agents were not anti-emetic in this dose range. SR140333 ((S)-1-{2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl}-4-phenyl-1 azaniabicyclo [2.2.2]octane; 3–10 mg/kg) failed to inhibit foot tapping or emesis. Affinities for the human and ferret tachykinin NK1 receptor were highly correlated (r=0.93, P=0.0008). Inhibition of foot tapping in gerbils, but not NK1 receptor binding affinity, predicted anti-emetic activity in ferrets (r=0.75, P<0.01). These findings confirm that the anti-emetic activity of tachykinin NK1 receptor antagonists is dependent on brain penetration.

Introduction

Severe nausea and vomiting is a distressing side-effect of cytotoxic drug and radiation therapy which contributes to poor patient compliance and limits the use of aggressive cancer treatment regimens. Cytotoxic drugs elicit an immediate emetic response on the day of therapy (acute emesis) and also protracted vomiting lasting up to 5 days thereafter (delayed emesis). Some relief from acute emesis can be achieved using cocktails of anti-emetic drugs, especially dexamethasone combined with high doses of metoclopramide, but these may themselves elicit unacceptable iatrogenic side-effects (Andrews et al., 1988). The introduction of 5-HT3 receptor antagonists into clinical practice in the early 1990s represented an important advance in the development of well-tolerated drugs specifically designed to prevent the severe emesis caused by antineoplastic therapy (Bunce et al., 1991; Aapro, 1991). However, whilst 5-HT3 receptor antagonists such as ondansetron and granisetron control the acute phase of nausea and vomiting induced by emetogenic cytotoxins in at least 60% of patients, emesis is not fully blocked in the residual population (Aapro, 1991; Yarker and McTavish, 1994), and 5-HT3 receptor antagonists are less effective at preventing the delayed emesis associated with chemotherapy (De Mulder et al., 1990; Butcher, 1993). The clinical utility of 5-HT3 receptor antagonists is further limited by their lack of efficacy against centrally acting emetogens such as morphine and apomorphine (Andrews and Bhandari, 1993).

Recently, tachykinin NK1 receptor antagonists have emerged as an important new class of broad-spectrum anti-emetic agents in preclinical assays of acute emesis. One such compound, the phenylpiperidine CP-99,994, completely abolished cisplatin-induced retching and vomiting in ferrets (Bountra et al., 1993; Watson et al., 1995a). Unlike the limited profile of anti-emetic activity exhibited by 5-HT3 receptor antagonists, CP-99,994 also inhibited retching and emesis in ferrets challenged with a wide range of both peripheral and central emetic stimuli (Bountra et al., 1993; Tattersall et al., 1994; Watson et al., 1995a). The enantioselectivity of the effects of CP-99,994 (Tattersall et al., 1993, Tattersall et al., 1994; Watson et al., 1995a), and the low affinity of this compound for other neurotransmitter receptors in vitro (McLean et al., 1993), confirm the tachykinin NK1 receptor specificity of this anti-emetic activity. The ability of CP-99,994 to block both peripherally and centrally acting emetogens, and the demonstration that direct injection of CP-99,994 into the region of the nucleus tractus solitarius inhibited cisplatin-induced emesis in ferrets (Gardner et al., 1994), suggest that the anti-emetic activity of tachykinin NK1 receptor antagonists is centrally mediated. This proposal has been confirmed recently by the use of a quaternised tryptophan ketone L-743,310, a poorly brain-penetrant tachykinin NK1 receptor antagonist, which was able to prevent cisplatin-induced retching in ferrets when infused directly into the central nervous system (CNS), but not when it was injected intravenously (Tattersall et al., 1996). Thus, the ability of tachykinin NK1 receptor antagonists to block emesis when they are administered systemically appears to be critically dependent on their ability to cross the blood–brain barrier. The central locus of action of tachykinin NK1 receptor antagonists indicates a fundamental difference in the mechanisms underlying their anti-emetic activity from that of 5-HT3 receptor antagonists, which are believed to act mainly in the periphery on vagal afferent fibres (Sanger, 1992), although blockade of central 5-HT3 receptors may also be involved (Higgins et al., 1989).

In addition to the predicted clinical utility of tachykinin NK1 receptor antagonists to control emesis, other possible therapeutic uses suggested from preclinical investigations include rheumatoid arthritis, asthma, migraine, pain and psychiatric conditions (reviewed by Longmore et al., 1995). A large number of structurally diverse antagonists which possess high (nM) affinity for the human tachykinin NK1 receptor have been evaluated in preclinical assays and in clinical trials for these various conditions. These include the recently described morpholine-based antagonist L-742,694 (Hale et al., 1996); the phenylpiperidine GR203040 (Beattie et al., 1995); the piperidineamine CGP 49823 (Vassout et al., 1994); the aryl amino acid derivative LY303870 (Gitter et al., 1995); the N-acylated 3-(3,4-dichlorophenyl)piperidine SR140333 (Emonds-Alt et al., 1993); the triarylperhydroisoindol RPR100893 (Tabart and Peyronel, 1994), and the dipeptide FK888 (Hagiwara et al., 1993). Access of tachykinin NK1 receptor antagonists into the brain is clearly a prerequisite for clinical efficacy in CNS disorders (emesis, pain, psychiatry), but not in conditions associated with inflammation of peripheral tissues. However, only limited information is presently available describing the in vivo pharmacology of these compounds in assays that require brain penetration.

Recently, we described the use of tachykinin NK1 agonist-induced foot tapping in gerbils as a simple in vivo functional assay for CNS penetration of tachykinin NK1 receptor antagonists (Rupniak and Williams, 1994). Like the ferret, the pharmacology of the gerbil tachykinin NK1 receptor appears to resemble that of the human receptor (Beresford et al., 1991). Central infusion of tachykinin NK1 receptor agonists in gerbils elicits a vigorous and readily quantifiable rhythmic stamping or tapping of the hindfeet which can be inhibited by systemic administration of brain-penetrant tachykinin NK1 receptor antagonists (Graham et al., 1993; Vassout et al., 1994; Bristow and Young, 1994; Rupniak and Williams, 1994). In contrast, the poorly CNS-penetrant compound, L-743,310, failed to inhibit foot tapping at a dose which completely blocked chromodacryorrhoea evoked by stimulation of peripheral tachykinin NK1 receptors (Rupniak and Williams, 1994). In the present studies, we have directly compared the ability of the currently available antagonists to block central tachykinin NK1 receptors in vivo, and examine whether activity in the gerbil foot tapping assay is predictive of anti-emetic efficacy in ferrets.

Section snippets

Displacement of 125I-[Tyr8]substance P binding to cloned human tachykinin NK1 receptors and to ferret brain membranes in vitro

Tachykinin NK1 receptor binding assays were performed in intact Chinese hamster ovary (CHO) cells expressing the human tachykinin NK1 receptor using a modification of the assay conditions described by Cascieri et al. (1992). The receptor was expressed at a level of 3×105 receptors per cell. Cells were grown in monolayer culture, detached from the plate with enzyme-free cell dissociation solution (Speciality Media), and washed prior to use in the assay. 125I-[Tyr8]substance P (0.1 nM, 2000

Inhibition of 125I-[Tyr8]substance P binding to cloned human tachykinin NK1 receptors and to ferret brain membranes by tachykinin NK1 receptor antagonists in vitro

Binding of 125I-[Tyr8]substance P to the human tachykinin NK1 receptor expressed in CHO cells was inhibited by all of the tachykinin NK1 receptor antagonists examined in a concentration-dependent and competitive manner. These compounds all exhibited high affinity binding to the cloned human tachykinin NK1 receptor in vitro, with IC50 values in the concentration range from high pM (SR140333, (±)-GR203040, L-742,694, LY303870, FK888 and CP-99,994) to low nM (CGP 49823 and RPR100893; Table 1).

Discussion

The present findings confirm evidence from previous studies (Gardner et al., 1994; Tattersall et al., 1996) that the anti-emetic activity of tachykinin NK1 receptor antagonists is critically dependent upon their ability to cross the blood–brain barrier. The ability of a range of structurally diverse compounds to inhibit tachykinin NK1 agonist-induced foot tapping in gerbils, a centrally mediated behaviour (Rupniak and Williams, 1994), was highly predictive of their ability to prevent

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