The novel NK1 receptor antagonist MK–0869 (L–754,030) and its water soluble phosphoryl prodrug, L–758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets

Neuropharmacology 39 (2000) 652–663 www.elsevier.com/locate/neuropharm

The novel NK1 receptor antagonist MK–0869 (L–754,030) and its water soluble phosphoryl prodrug, L–758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets F.D. Tattersall a,*, W. Rycroft a, M. Cumberbatch a, G. Mason a, S. Tye a, D.J. Williamson a, J.J. Hale b, S.G. Mills b, P.E. Finke b, M. MacCoss b, S. Sadowski b, E. Ber b, M. Cascieri b, R.G. Hill a, D.E. MacIntyre b, R.J. Hargreaves a a

Department of Pharmacology, Merck, Sharp and Dohme, Neuroscience Research Centre, Terlings Park, Harlow, Essex, CM20 2QR, UK b Merck Research Laboratories, Rahway, NJ, USA Accepted 15 September 1999

Abstract The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK–0869 (L–754,030) 2–(R)–(1–(R)– (3,5–bis(trifluoromethyl)phenylethoxy)–3–(S)–(4–fluoro)phenyl–4–(3–oxo–1,2,4–triazol–5–yl)methylmorpholine and its water soluble prodrug, L–758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK–0869 and L–758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK–0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5–HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK–0869 (4–16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK–0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK–0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK–0869 and its prodrug, L–758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.  2000 Elsevier Science Ltd. All rights reserved. Keywords: MK–0869 (L–754,030); Ferret; Cisplatin-induced emesis; Vomiting

1. Introduction The use of 5–HT3 receptor antagonists such as ondansetron (Zofran), tropisetron (Novaban) and granisetron (Kytril) has revolutionised the treatment of emesis induced by anti-neoplastic chemotherapy in humans. In the clinic all the 5–HT3 receptor antagonists so far tested have a similar profile of anti-emetic action and although their dosing regimens vary there is no difference in their

* Corresponding author. Tel: +44-1279-440000; fax: +44-1279440390. E-mail address: david [email protected] (F.D. Tattersall).

anti-emetic efficacy (see review by Gregory and Ettinger, 1998). The 5–HT3 receptor antagonists have been shown to decrease markedly the initial acute phase of emesis that occurs in the 24 h period after administration of antineoplastic drugs such as cisplatin, but they are only poorly effective as monotherapy against the delayed phase of emesis (⬎24 h) which occurs both in humans (De Mulder et al., 1990; Tavorath and Hesketh, 1996) and experimental animals such as ferrets (Rudd and Naylor, 1994). Attempts have been made to enhance the anti-emetic activity of the 5–HT3 receptor antagonists by varying the dosing intervals, routes of administration and including dexamethasone in the treatment regimen (Gralla, 1995) but these measures have met with limited success.

0028-3908/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 7 2 - 0

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The 5–HT3 receptor antagonists are believed to act by a predominantly peripheral site of action, in ferrets, preventing the stimulation of the abdominal vagal afferent fibres by 5–HT released from the enterochromafin cells of the gut by cytotoxic agents (Sanger, 1992). This probably explains why the 5–HT3 receptor antagonists are ineffective against the emesis induced by the centrally acting stimuli apomorphine and morphine in experimental animals (Andrews, 1994). There is thus still a need for anti-emetic agents that can provide both longer and more complete protection against druginduced emesis. Recently, preclinical studies have shown that human type selective tachykinin neurokinin1 (hNK1) receptor antagonists such as CP–99,994, CP–122,721, GR203040 and GR205171 (Bountra et al., 1993; Tattersall et al. 1993, 1994; Gardner et al. 1995, 1996; Gonsalves et al., 1996) antagonised the retching and vomiting response induced in ferrets by the chemotherapeutic agent cisplatin and centrally acting emetogens such as morphine and apomorphine. Watson et al. (1995) showed that CP– 99,994 attenuated emesis evoked by the gastric irritant copper sulphate in dogs and Gardner et al. (1996) and Grelot et al. (1998) have demonstrated the anti-emetic activity of GR205171 in Suncus murinus and pigs respectively. Thus the anti-emetic activity of NK1 receptor antagonists is not restricted to ferrets. This broad spectrum of activity displayed by the NK1 receptor antagonists is probably due to their central site of action (Tattersall et al., 1996) which is believed to be in the vicinity of the nucleus tractus solitarius (NTS) in the dorsal vagal complex (DVC). The DVC comprises the NTS, area postrema and dorsal motor nucleus of the vagus. This complex is an ideal locus of action for antiemetic agents since, at the NTS, the vagal afferents from the gastrointestinal tract converge with inputs from the area postrema and other important brain regions believed to be important in the control and integration of emesis (Leslie and Reynolds, 1992; Tattersall et al., 1996). Thus the NK1 receptor antagonists may provide broader protection against a variety of emetogens in humans than that produced by 5–HT3 receptor antagonists. The preclinical development of the 5–HT3 receptor antagonists used ferrets to identify their potent activity against cytotoxic chemotherapy-induced emesis and the anti-emetic activity of this class of compounds was confirmed in the clinic, suggesting that this species would appear to be relevant for experimental studies of chemotherapy-induced emesis (see review by Veyrat-Follet et al., 1997). In the present studies we have therefore profiled the anti-emetic activity of the novel NK1 receptor antagonist MK–0869 (L–754,030) and its more water soluble phosphoryl prodrug L–758,298, which was developed to facilitate intravenous dosing since it has enhanced aqueous solubility when compared with MK– 0869. Two protocols were used. Initially the ability of

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MK–0869 and L–758,298 to inhibit the acute emetic response to cisplatin in a four hour observation period was assessed. However this experiment probably only allows assessment of how NK1 receptor antagonists will perform in the acute phase (up to 24 h) of cisplatininduced emesis in the clinic. In a second protocol the ability of orally dosed MK– 0869 to inhibit not only acute but also delayed emesis induced by cisplatin in ferrets was determined during a 72 h observation period following cisplatin administration. It has been shown using this protocol that the NK1 receptor antagonist CP–99,994 inhibits both acute and delayed phases (Rudd et al., 1996b) but that ondansetron, whilst it has good activity against the acute phase, is only poorly active during the delayed phase (Rudd and Naylor, 1994; Singh et al., 1997) similar to the situation encountered in the clinic. Interestingly the concomitant use of dexamethasone with ondansetron led to an enhancement of the anti-emetic activity in ferrets (Rudd et al., 1996a; Rudd and Naylor, 1996) similar to the effects of co administration of these agents in the clinic (Tavorath and Hesketh, 1996) suggesting that this protocol has relevance to studies concerned with the development of anti-emetics for use in humans. We have also examined if prevention from the delayed phase of emesis is dependent on the prevention of the acute phase of emesis by starting dosing with MK–0869 at 24 h after cisplatin administration when the acute phase of emesis has already occurred. The synthesis of MK–0869 has been reported elsewhere (Hale et al., 1998). Its structure, together with that of its prodrug, are shown in Fig. 1. The data below summarise the preclinical anti-emetic activity of MK–0869 and its prodrug, L–758,298 in ferrets and were used to support the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy (Navari et al. 1998, 1999; Van Belle et al., 1998).

2. Methods 2.1. Binding studies The binding affinities of both MK–0869 and L– 758,298 for cloned human and rat NK1 receptors in intact CHO or COS cells were determined using the methods described by Cascieri et al. (1992) and for ferret NK1 receptors using synaptosomal membranes purified from brain cortex by discontinuous sucrose gradient centrifugation (Cascieri et al., 1985). Affinities for human NK2 and NK3 receptors were also determined after expression in CHO cells as detailed by Sadowski et al. (1993). In order to examine further the selectivity of these compounds, the affinities at over 90 other neuro-

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Fig. 1. Structures and summary of binding affinities for MK–0869 its prodrg L–758,298.

transmitter receptor and ion channels were determined by Panlabs DiscoveryScreen (Bothell, WA, USA). 2.2. In vivo studies All experiments were carried out under the authority of the UK Animals (Scientific Procedures) Act, 1986 and complied with its associated guidelines. 2.2.1. Acute emesis studies Individually housed male ferrets (1.0–2.0 kg body weight) were used in these studies. During experiments they were housed individually in cages with ad libitum access to food and water. The experimental methods used were essentially as reported previously by Tattersall et al. (1996). Briefly studies were carried out as follows.

2.2.2. Surgery To facilitate administration of substances intravenously, a polythene catheter was acutely inserted into the left jugular vein under a brief period of halothane anaesthesia (5% for induction, 3% for maintenance: O2 carrier). After injection the catheter was removed, the wound closed and the animal was allowed to recover from anaesthesia in its home cage. Typically the ferrets were moving around the cage within 15 min. 2.2.3. Acute: intravenous and oral studies To examine the anti-emetic activity of test compounds after intravenous administration, ferrets were injected with MK–0869, L–758,298, ondansetron (0.1–3 mg/kg i.v.) or drug vehicle followed 3 min later by cisplatin (10 mg/kg i.v.).

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In oral pretreatment studies ferrets were dosed by gavage with MK–0869 or drug vehicle. Sixty minutes later ferrets were injected with cisplatin (10 mg/kg i.v.) under halothane anaesthesia as described above.

Data are expressed as the total number of retches and vomits occurring in the 240 min observation period and in some of the studies as the cumulative number of retches occurring with time in 5 min bins.

2.2.4. Acute: combination studies with ondansetron or dexamethasone In order to investigate if the anti-emetic activity of NK1 receptor antagonists could be augmented by combined treatment with ondansetron or dexamethasone, ferrets were dosed with MK–0869 (0.1 mg/kg i.v.) and either ondansetron (0.1 mg/kg i.v.) or dexamethasone (20 mg/kg i.v.). These doses of each compound were chosen since they were determined in the preliminary experiments and from published data (see Table 1 and also Rudd et al., 1996a) to have only minimal effects on emesis when given alone. Groups of animals were also treated with appropriate drug vehicles (see Drugs section). Three minutes after these pretreatments, cisplatin (10 mg/kg i.v.) was injected. In all experiments the ferrets were continuously monitored by trained observers during recovery from anaesthesia and the numbers of retches and vomits occurring in the 240 min following the cisplatin injection, together with the time they occurred, were recorded. Due to its cytotoxic properties, all animals given cisplatin were used only once and humanely killed with an overdose of sodium pentobarbitone at the end of the 240 min observation period.

2.3. Delayed emesis studies Male ferrets (1.2–2.4 kg) were used in these studies. All animals were housed individually with food and water available ad libitum. Fresh food was presented to the ferrets at approximately 4.00 pm each day when the cages were also cleaned out and the animals inspected. Three experiments were conducted: 2.3.1. Experiment 1: single dosing of MK–0869 Ferrets were orally dosed with MK–0869 (4, 8 or 16 mg/kg) or drug vehicle. Two hours later cisplatin (5 mg/kg i.p.) was administered. The animals were observed for the next 72 h for retching and vomiting. 2.3.2. Experiment 2: daily dosing with MK–0869 Ferrets were orally dosed with MK–0869 (1, 2 or 4 mg/kg) or drug vehicle. Cisplatin (5 mg/kg i.p.) was administered 2h after this initial dose of MK–0869. Subsequently, at 24 h and 48 h after the first oral dosing treatment the ferrets were administered with the same dose of MK–0869 or vehicle as they had received initially. The animals were observed continuously for the next 72 h for retching and vomiting.

Table 1 The anti-emetic activity of MK–0869, L–758,298 and ondansetron in acute cisplatin-induced emesis in ferrets during a 4 h observation period. Significant differences from the vehicle-pretreatment group are shown as *P⬍0.05 (one-way ANOVA followed by Dunnett’s test (BMDP statistical analysis package) Treatment mg/kg MK–0869 p.o. Methocel (veh) 0.3 1 3 MK–0869 i.v. PEG 300 (veh) 0.1 0.3 1 3 L–758,298 i.v. Water 0.1 0.3 1 3 Ondansetron i.v. Water 0.1 0.3 1 3

Mean retches (±sem)

Mean vomits (±sem)

Protected/Tested

154.8±14.5 48.5±9.8* 20.0±8.3* 2.8±1.7*

22.3±2.1 7.3±2.3* 3.8±1.8* 0.5±0.3*

0/4 0/4 0/4 2/4

100.0±24. 89.5±25.9 5.3±3.3* 0±0* 0±0*

12.7±3.8 12.7±3.7 1±0.7* 0±0* 0±0*

0/6 0/4 0/4 4/4 4/4

164.3±41.3 115.8±29.4 26±4.6* 2±2* 0±0*

20.3±4.8 15.8±3.2 4.3±0.3* 0.5±0.5* 0±0*

0/4 0/4 0/4 3/4 4/4

144.2±32.6 109.8±21.7 56.2±17.1* 4.8±3.9* 0.5±0.5*

15±2.8 14.6±2.8 7.8±2.5 0.6±0.6* 0.25±0.25*

0/5 0/5 1/5 3/5 3/4

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2.3.3. Experiment 3: treatment of an established emetic response This study was carried out simultaneously with Experiment 2 (see above). The ability of MK–0869 to prevent the delayed phase of emesis (which occurs on days 2 and 3) when retching and vomiting had already occurred on day 1 was examined. Ferrets were dosed with cisplatin (5 mg/kg i.p.) and observed for retching and vomiting. Twenty-four and 48 hours later (after the acute phase of emesis had occurred) ferrets were dosed with MK–0869 (4 mg/kg p.o.) and observed continuously for retching and vomiting. In all delayed emesis experiments, animals were observed continuously for the whole 72 h of experimentation by trained observers who recorded any retching and vomiting behaviour together with the time at which it occurred. At the end of this the ferrets were killed with an overdose of anaesthetic. 2.4. Data analysis For the acute emesis experiments the data were analysed as the total number of retches and total number of vomits in the 4 h observation period and differences from vehicle pretreated ferrets assessed using a one-way ANOVA followed by Dunnett’s test. In delayed emesis experiments the data are shown as the number of retches+vomits occurring in 6 h time bins for the whole of the 72h observation period. Data were analysed for the total numbers of retches+vomits occurring on each day and differences from vehicle pretreated animals assessed using a one-way ANOVA followed by Dunnett’s test. In both acute and delayed emesis experiments, significant differences from vehicle pretreated animals were considered to be when P⬍0.05. 2.5. Drug preparation MK–0869 (L–754,030) 2–(R)–(1–(R)–(3,5–bis(trifluoromethyl)phenylethoxy)–3–(S)–(4–fluoro)phenyl–4– (3–oxo–1,2,4–triazol–5–yl)methylmorpholine (Fig. 1) and its water soluble phosphoryl prodrug L–758,298, were synthesised in the Medicinal Chemistry Department of Merck Research Laboratories, Rahway, New Jersey, USA. In acute (4 h) emesis studies, MK–0869 was dissolved and diluted in polyethylene glycol average molecular weight 300 (Sigma) for i.v. injections or suspended in Methocel (0.5% w/v) for oral studies. L– 758,298 powder was stored at ⫺80°C and dissolved in water immediately before i.v. administration. A dosing volume of 1 ml/kg was used in both i.v. and oral studies using MK–0869 and L–758,298. Ondansetron (Zofran injection 2 mg/ml; Glaxo) was injected at 1.5 ml/kg for a 3 mg/kg dose but at all other doses as a 1 ml/kg solution with dilutions made in water. Dexamethasone (Decadron Injection Shock Pak; Merck Sharp and

Dohme) was administered as a 20 mg/ml solution as supplied. In delayed emesis studies MK–0869 was dissolved using a vehicle of 10% ethanol: 60% propylene glycol: 30% water. Cisplatin was used a 1 mg/ml sterile clinical supply obtained from David Bull Laboratories (10 ml/kg dosing volume for acute studies or 5 ml/kg for delayed emesis studies giving 10 or 5 mg/kg respectively). All doses of drugs are expressed in terms of the free base.

3. Results 3.1. Binding studies The affinities of MK–0869 and L–758,298 at NK1, NK2 and NK3 receptors are shown in Fig. 1. A summary of the binding profile and selectivity of MK–0869 has been previously reported by Kramer et al. (1998) and Hale et al. (1998) but a brief summary of the data is included below. In radioligand binding assays, MK–0869 is 3000-fold selective for the human cloned NK1 receptor versus the human cloned NK3 receptor and ⬎50,000-fold selective over the human cloned NK2 receptor. In a range of assays at other human cloned G–protein coupled receptors, MK–0869 retained ⬎50,000-fold selectivity for the human cloned NK1 receptor. MK–0869 was inactive in human monoamine oxidase A and B assays and at human serotonin 5–HT1A, 5–HT2A, 5–HT2c, 5–HT3, 5– HT5, 5–HT6, and 5–HT7 receptors (IC50⬎3 µM). In the PANLABS panel of radioligand binding screens using native animal tissues, MK–0869 inhibited [3H]substance P binding to native NK1 receptors in rat submaxillary gland; there were no significant interactions of MK–0869 with any other native animal G–protein coupled receptors or ion channels examined in the PANLABS screen. MK–0869 was inactive in monoamine uptake site (NE, 5–HT, DA) counterscreens using human and animal tissues (IC50⬎3 µM). These data suggest that MK–0869 exerts selective activity at NK1 receptors. The prodrug, L–758,298 was 1000-fold selective versus the endothelin A receptor, and is ⬎2500-fold selective versus the other receptors tested. 3.2. Acute emesis MK–0869, L–758,298 and ondansetron (0.3–3 mg/kg i.v.) dose dependently and significantly (P⬍0.05) inhibited the retching and vomiting response to cisplatin (10 mg/kg i.v.: Table 1). Inhibition of emesis was complete at 1 and 3 mg/kg i.v. for MK–0869 and 3 mg/kg i.v. for L–758,298. MK–0869 (0.3–3 mg/kg p.o.) significantly inhibited the retching and vomiting (P⬍0.05)

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with two out of four animals completely protected at 3 mg/kg p.o. When the data from each treatment group were expressed as the mean cumulative numbers of retches occurring in 5 min epochs there was a marked difference between the profile of activity of both MK–0869, and its prodrug L–758,298, when compared with the 5–HT3 receptor antagonist ondansetron. MK–0869 suppressed the emetic activity with flattening of the cumulative numbers of retches occurring with time whereas the predominant action of the 5–HT3 receptor antagonists was to increase the latency to retching but with time the antiemetic activity appeared to wear off and the numbers of retches occurring approached that seen in ferrets that had been pretreated with drug vehicle (Fig. 2). Similar results were seen with vomiting (data not shown). When a submaximally effective dose of MK–0869 was combined with either ondansetron or dexamethasone there was an enhancement of the anti-emetic

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activity (Fig. 3). Both retching and vomiting were significantly inhibited by combined treatment with MK– 0869+dexamethasone or MK–0869+ondansetron (P⬍0.05: only data for retching shown) but when these compounds were given alone they were without significant effect (P⬎0.05). One out of the six animals given the combined treatment of MK–0869+dexamethasone was completely protected for the whole 4 h observation period. Combined treatment with ondansetron+MK–0869 completely prevented retching and vomiting in 3 out of 6 ferrets whereas one out of six ferrets had no emesis following ondansetron alone. 3.3. Delayed emesis studies 3.3.1. General behaviour of ferrets The ferrets used in these experiments spent most of the 72 h experimental period curled up, apparently either

Fig. 2. Effects of MK–0869, L–758,298 and ondansetron on the profile of the retching response induced by cisplatin (10 mg/kg i.v.). Values shown are the mean cumulative numbers of retches in 5 min time bins occurring in the 4 h observation period following cisplatin 10 mg/kg i.v. (n=4–6 per group).

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Fig. 3. Enhanced activity of MK–0869 (0.1 mg/kg i.v.) when combined with either dexamethasone (20 mg/kg i.v.) or ondansetron (0.1 mg/kg i.v.). Values shown are the mean (±SEM) numbers of retches for each treatment group occurring in the 4 h observation period following cisplatin 10 mg/kg i.v. (n=6 per group). Significant differences from the vehicle-pretreatment group are shown as *P⬍0.05 (one-way ANOVA followed by Dunnett’s test (BMDP statistical analysis package).

resting or asleep. Although the time spent in this state was not quantified it appeared that there was no marked difference between any of the groups of animals in the three experiments or differences between these animals and untreated ferrets in the time spent inactive (excluding the time spent retching and vomiting). Ferrets dosed with vehicle followed by cisplatin had two distinct periods of retching and vomiting. There was an initial acute phase (which typically was most intense by 16 h after dosing with cisplatin) followed by a period when retching and vomiting was less intense. After this there was a further (delayed) phase which typically peaked by 48 h after injection with cisplatin. 3.3.2. Experiment 1: single dosing with MK–0869 (4, 8 or 16 mg) MK–0869 4, 8 and 16 mg/kg p.o. administered once, 2 h before the cisplatin, dose-dependently decreased the retching and vomiting response during the 72 h observation period (Fig. 4) with significant decreases observed on each of the three test days (P⬍0.05). At 16 mg/kg two out of the four animals tested and at 8 mg/kg one out of four animals tested, were completely protected from retching and vomiting for the entire 72 h following cisplatin injection. 3.3.3. Experiment 2: daily dosing with MK–0869 Following oral dosing with MK–0869 at 2 or 4 mg/kg/day none of the ferrets either retched or vomited to cisplatin during the entire 72 h observation period. Animals dosed with the lower dose of 1 mg/kg/day had a marked and statistically significant decrease in the retching and vomiting response for each of the three days (P⬍0.05: Fig. 5).

3.3.4. Experiment 3 treatment of an established emetic response This experiment examined whether it was possible to inhibit the delayed phase of emesis after the acute phase had become established in all animals and it examined if inhibition of the delayed phase in the previous emesis experiments described here was a consequence of the prevention of the acute phase. MK–0869 (4 mg/kg p.o.) at 24 h and 48 h after cisplatin administration markedly inhibited retching and vomiting with three out of four animals being completely prevented from retching and vomiting (Fig. 5). The one animal which remained unprotected vomited within 1 min of dosing on both occasions (24 and 48 h after cisplatin) suggesting that this may be an artefact produced by the gavage dosing procedure and not a true component of the emetic response to cisplatin.

4. Discussion The novel morpholine NK1 receptor antagonist MK– 0869 and its prodrug L–758,298 have excellent selectivity for the hNK1 receptor and potent in vivo activity in ferrets against cisplatin-induced retching and vomiting. The water soluble N–phosphoryl (phosphoramidate) prodrug L–758,298 was active after intravenous administration suggesting that it is rapidly converted to MK– 0869 in vivo. Given the high degree of binding selectivity and the similarity of anti-emetic activity seen following intravenous dosing with MK–0869 and L– 758,298, it is likely that the anti-emetic activity is mediated by MK–0869 acting at NK1 receptors and is not due to direct actions of L–758,298 at the NK1 recep-

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Fig. 4. Anti-emetic activity of MK–0869 (4, 8 or 16 mg/kg) following a single dose to ferrets administered 2 h before cisplatin 5 mg/kg i.p.. Values are the mean (±SEM) numbers of retches+vomits occurring in 6 h time bins for the 72 h observation period following cisplatin 5 mg/kg i.p.: n=4–8.

tor at which it has lower affinity than MK–0869. We have not measured the rate of conversion of L–758,298 to MK–0869 in ferrets but following i.v. dosing to rats, L–758,298 is converted to MK–0869 (90–100%) and under in vitro conditions, the conversion has been shown to be very rapid in rat blood and in hepatic microsomes prepared from dogs and humans (Huskey et al., 1999). In comparison with ondansetron, both MK–0869 and L–758,298 appeared to have different temporal profiles of activity against cisplatin-induced emesis. At doses of MK–0869 and L–758,298 which did not provide complete protection from emesis these compounds suppressed the emetic response with a flattening of the cumulative numbers of retches and vomits occurring with time, whereas the 5–HT3 receptor antagonist ondansetron appeared to produce a delay in onset of retching and vomiting but there was then breakthrough of the emetic response such that the numbers of retches and vomits gradually increased with time towards those seen in vehicle pretreated ferrets. It is unclear why there is this difference in the profile of emesis between groups of ferrets treated with ondansetron and either MK–0869 or its prodrug L–758,298, but Gonsalves et al. (1996)

have also reported that the NK1 receptor antagonist CP– 122,721 did not significantly increase the latency to the first retch when administered at doses that did not give complete protection against retching and vomiting. It seems unlikely that this difference is related to the short biological half life of ondansetron (Butler et al., 1988; Cohen et al., 1989) since it is also apparent with the longer acting 5–HT3 receptor antagonist granisetron (unpublished data). It may be due to the differing sites of action of these two classes of agents and/or the kinetics of receptor binding observed with NK1 receptor antagonists (see below). Ondansetron and other 5–HT3 receptor antagonists are believed to act predominantly by blocking the stimulation of abdominal vagal afferent fibres by 5–HT released from the enterochromaffin cells of the gut by cytotoxic agents (Sanger, 1992; VeyratFollet at al., 1997) whereas the NK1 receptor antagonists are considered to act centrally. In the ferret the site is probably in the vicinity of the nucleus tractus solitarius in the brain stem (Gardner et al., 1994; Tattersall et al., 1996). Fukuda et al. (1999a, 1999b) have described that in the dog an area which they term “the central pattern generator for vomiting” and have suggested that the anti-

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ity of the commissural subdivision of the nucleus tractus solitarius since it has been shown that activation of cardiorespiratory function in the cat induces an increase in the extraneuronal concentration of substance P in this brain region (Potts et al., 1999). However Fukuda et al. (1998) have shown using electrophysiological techniques in the decerebrate dog that although GR205171 abolished vomiting induced by stimulation of the vagus, firing of neurones in medial nucleus tractus solitarius was not affected. Nonetheless, a central site of action may explain why the NK1 receptor antagonists are effective against not only agents that induce emesis via vagal afferent activation but also drugs which act at the chemoreceptor trigger zone in the area postrema and against motion-induced emesis (Lucot et al., 1997). If the NK1 receptor antagonists exert their anti-emetic actions at or near the final neuronal synapse responsible for the induction of emesis this would allow prevention of emesis irrespective of the locus of simulation by the emetogen. The previously reported NK1 receptor antagonists GR205171 and CP–122,721 have been shown to have good acute anti-emetic activity in ferrets similar to that shown here for MK–0869 (Gardner et al., 1996; Gonsalves et al., 1996). GR205171 0.3 mg/kg s.c. almost completely prevented emesis induced by cisplatin 10 mg/kg i.p. during a 24 h observation period suggesting that this compound has good duration of activity. Both compounds also prevented emesis in dogs and ferrets after oral administration suggesting that they have good oral bioavailability in both species. The anti-emetic activity of MK–0869 was enhanced by combination with either dexamethasone or ondansetron. This supports the use of these established antiemetic agents in combination with an NK1 receptor antagonist in the clinic. Hawthorn and Cunningham (1990) have shown that the anti-emetic activity of ondansetron can be enhanced by combination treatment with dexamethasone in ferrets dosed with the chemotherapeutic agent cyclophosphamide. Rudd and Naylor (1996) have reported a similar interaction between ondansetron and dexamethasone against cisplatin induced acute and delayed emesis. However Marr et al. (1992) reported that combined treatment of dexamethasone with granisetron produced additive anti-emetic effects on cisplatininduced emesis although the dose of dexamethasone

emetic site of action for NK1 receptor antagonists is in the medullary area adjacent to the semi-compact part of the nucleus ambiguus. It is possible that the site of action of NK1 receptor antagonists is located at or in the vicin-

Fig. 5. (a) Anti-emetic activity of daily dosing with MK–0869 (1 mg/kg/day) to ferrets. Ferrets were dosed with MK–0869 (1 mg/kg p.o.) 2 h before cisplatin (5 mg/kg i.p.). Further doses of MK–0869 (1 mg/kg p.o.) were given at 24 and 48 h after the initial administration. (b) Anti-emetic activity of MK–0869 (4 mg/kg p.o.) in ferrets when treatment was started at 24 h and 48 h after cisplatin 5 mg/kg i.p. when retching and vomiting were already established. Values shown are the mean (±SEM) numbers of retches+vomits occurring in 6 h time bins for the 72 h observation period following cisplatin 5 mg/kg i.p.; n=4 per treatment group.

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used was lower than in our studies. In the clinic combined treatment of a 5–HT3 receptor antagonist with dexamethasone has also been shown to be beneficial (Smith et al., 1990) although de Wit et al. (1998) have reported that the initial level of anti-emetic protection afforded by the combination of granisetron with dexamethasone is not maintained over subsequent courses of chemotherapy in humans. Nevertheless it is still encouraging that the combined treatment of dexamethasone with MK–0869 produced a potentiation of the inhibition of cisplatin-induced emesis in our studies, albeit that the dose of dexamethasone used was higher than the dose which could be used in humans. The mechanism by which dexamethasone enhances anti-emetic activity remains unclear (see Sanger, 1993) although it may relate to the inhibition of prostaglandin synthesis or effects on maintaining the blood brain barrier preventing emetogenic substances from entering the central nervous system (Hawthorn and Cunningham, 1990). In the delayed emesis experiments, the profile of the emetic response in the ferrets following cisplatin 5 mg/kg i.p. was similar to that which has been described previously (Rudd et al., 1994; Rudd and Naylor, 1996; Singh et al., 1997). There was an initial (acute) phase which typically was most intense by 10–16 h after dosing with cisplatin. There was then a lag phase when vomiting behaviour was less pronounced followed by a further (delayed) emesis phase which was typically maximal by 48 h after injection with cisplatin. The emetic response in vehicle+cisplatin treated animals was consistent in all studies with only a small degree of variation in the intensity of response being observed. In the experiments presented we have shown that MK–0869 is able to totally prevent retching and vomiting in all animals tested when given as a daily oral treatment at 2 or 4 mg/kg. We have also shown it is possible to prevent the delayed phase of retching and vomiting even after the acute phase has occurred since three out of four ferrets were protected from retching and vomiting on days 2 to 3 by MK–0869 (4 mg/kg p.o.) even after the acute phase had occurred in all animals on day 1. The ferret which remained unprotected retched and vomited within 1 min of dosing on each day, probably indicating that at least some of the drug had been expelled from the stomach on each occasion. The rapid emesis seen with this ferret was probably induced by the gavage oral dosing technique used leading to a gagging behaviour. Interestingly Watson et al. (1995) have reported that the NK1 receptor antagonist CP–99,994 does not block gagging in ferrets and Andrews et al. (1996) have shown that CP–99,994 does not prevent the emetic response to mechanical stimulation of the upper gastrointestinal tract. This anti-emetic activity seen with MK–0869 is a marked improvement over that seen with ondansetron which has only poor anti-emetic activity in this assay

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(Rudd and Naylor, 1994; Singh et al., 1997) and the clinic, (De Mulder et al., 1990). This lack of effect of ondansetron suggests that during the delayed phase of emesis mechanisms come into play other than the stimulation of 5–HT3 receptors on vagal afferent fibres by 5–HT released from enterochromafin cells of the gut. Orally administered MK–0869 appeared more active in the delayed emesis experiments than in the acute emesis experiments. This may be because in the delayed emesis paradigm a lower dose of cisplatin is used and also because, following oral administration to ferrets the maximum plasma concentration of MK– 0869 is reached approximately 2 h after dosing (unpublished observation). MK–0869 4, 8 or 16 mg/kg when given as a single oral treatment 2 h before cisplatin markedly inhibited retching and vomiting on all three days. We believe that the long duration of action of MK–0869 is a result of not only its central site of action but also its kinetics at the NK1 receptor where saturation binding studies with 3 [H]–MK–0869 has shown that it has a slow off rate (rate of dissociation k1=0.0054±0.003 min⫺1 and t 1/2=154±75 min) (Hale et al., 1998). Radioligand binding studies have shown a profile consistent with the idea that MK–0869 behaves as a competitive antagonist with a slow off rate (Hale et al., 1998). A non competitive antagonist profile has been reported for CP–122,721 (McLean et al., 1996) and GR203040 has been shown to exhibit insurmountable antagonism in functional in vitro studies (Beattie et al., 1995). These receptor kinetics may contribute to the good anti-emetic activity seen with these agents. Palmer et al. (1998) have reported that in humans in chemotherapy-induced emesis trials the half life of GR205171 is in the range 4.4 to 11.8 h. CP– 122,721 has been tested for its anti-emetic activity in humans and at doses of 50–200 mg anti-emetic activity was seen which was potentiated by combination with dexamethasone and a 5–HT3 receptor antagonist (Kris et al., 1997). In the delayed emesis experiments MK–0869 provided excellent anti-emetic activity with all animals protected from both retching and vomiting following daily oral treatment with doses of 2 or 4 mg/kg. However, when the NK1 receptor antagonists CP–99,994 and PD 154075 were tested in this delayed emesis protocol in ferrets, they did not completely prevent emesis even at a dose of 10 mg/kg i.p. given three times daily for three days (Rudd et al., 1996b; Singh et al., 1997). In contrast to MK–0869, it has been shown that CP–99,994 (10 mg/kg i.p.) given as a single treatment also failed to inhibit the total retching and vomiting response observed over a three day observation period (Rudd et al., 1996b). In summary, the present studies have demonstrated the excellent anti-emetic activity of the novel morpholine MK–0869 in ferrets for the control of both acute and delayed cisplatin-induced emesis. They provided a basis

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for the testing of MK–0869 and L–758,298 in the clinic for the treatment of emesis associated with anti-neoplastic treatment using cisplatin (Navari et al. 1998, 1999; Van Belle et al., 1998). These studies have shown that L–758,298 and MK–0869 have good anti-emetic activity in humans suggesting that NK1 receptor antagonists have a role in the clinic for the control of emesis to improve the welfare of patients undergoing chemotherapy and that the ferret is an appropriate species in which to examine the anti-emetic activity of this class of compounds.

Acknowledgements The authors thank Dr J.A. Rudd and Professor R.J. Naylor for their advice when establishing the delayed emesis technique.

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