The NPY Y1 receptor antagonist BIBP 3226 blocks NPY induced feeding via a non-specific mechanism

Regulatory Peptides 75–76 (1998) 377–382

The NPY Y1 receptor antagonist BIBP 3226 blocks NPY induced feeding via a non-specific mechanism D.G.A. Morgan, C.J. Small, S. Abusnana, M. Turton, I. Gunn, M. Heath, M. Rossi, A.P. Goldstone, D. O’Shea, K. Meeran, M. Ghatei, D.M. Smith, S. Bloom* ICSM Endocrine Unit at the Hammersmith Hospital, Francis Fraser Labs., Du Cane Road, London W12 0 NN, UK Received 20 January 1998; received in revised form 4 March 1998; accepted 5 March 1998

Abstract We have previously shown that intracerebroventricular BIBP 3226 inhibits NPY induced feeding in rats. However, this was associated with abnormal behaviour, likely to be due to interaction with Y1 receptors involved in mechanisms other than the control of food intake. In order to minimise such interactions we investigated the effects of paraventricular nucleus (PVN) injections of BIBP 3226 and its inactive enantiomer BIBP 3435. Intra-PVN injection of NPY (0.1–2.5 nmol / animal) increased food intake, with an EC 50 of approximately 0.15 nmol / animal. Injections of BIBP 3226 and BIBP 3435 (0.25–25 nmol) reduced NPY-induced food intake in a dose responsive manner, with BIBP 3226 reducing food intake by 95%, and BIBP 3435 by 65% at the highest dose tested. The reversibility of the effect of BIBP 3226 was investigated by measuring the feeding response to NPY (0.5 nmol) in animals 1 week after BIBP 3226 injection. The response to NPY was less in animals which had received high doses of BIBP 3226. Animals previously injected with saline vehicle alone showed a normal NPY feeding response. These results suggest that BIBP 3226 may be inhibiting NPY-induced food intake in a non-specific manner, not secondary to inhibition of the Y1 receptor. This does not, however rule out a role for the Y1 receptor in the control of food intake by NPY.  1998 Elsevier Science B.V. All rights reserved. Keywords: Neuropeptide Y; Y1 receptor; Food intake; Hypothalamic control

1. Introduction Neuropeptide Y is the most potent orexigenic agent known, and a number of studies support a physiological role for the peptide in the control of food intake. When injected into the cerebral ventricles or into nuclei of the hypothalamus [1,2] it induces a potent increase in food intake [3], and repeated injection leads to hyperphagia and obesity [4]. Injection into a number of different central Abbreviations: NPY, neuropeptide Y; [Pro 34 ]-NPY, NPY with isoleucine – proline substitution at position 34; PVN, paraventricular nucleus of the hypothalamus; ICV, intracerebroventricular; Y1-Y5 NPY receptor subtypes; HPLC, high performance liquid chromatography *Corresponding author. Tel.: 1 44-181-383-3242; fax: 1 44-181-3833142

nervous system (CNS) sites leads to increased food intake. However, either the paraventricular nucleus (PVN) [5], or the neighbouring perifornical area [2] appears to form the nexus of the system. Additionally, hypothalamic NPY immunoreactivity [6] and mRNA [7] are increased by fasting, as is NPY release from the hypothalami of fasted rats, both in vitro [8] and in vivo [9,10]. Immunoneutralisation of endogenous NPY has also been shown to reduce food intake [11,12]. Recently, much attention has been paid to the receptor subtype mediating the orexigenic effect of NPY [13–15]. The Y1 receptor was originally proposed as the receptor involved in this effect, since the Y1 agonist [Pro 34 ]-NPY is a potent orexigenic agent. However, the potency of the N-terminally truncated NPY fragment, NPY 2-36, which has low potency at the Y1 receptor, led to the idea of a

0167-0115 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00091-3

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novel ‘Y1-like’ receptor mediating the effect [16]. We have recently shown that [Pro 34 ]-NPY is only able to stimulate approximately 50% of the food intake seen following the injection of NPY, further suggesting that the Y1 receptor may not be involved, or may be responsible for only a proportion of the effect of NPY [14]. More recently, a novel receptor, Y5, has been cloned from rat hypothalamus [13,15], and this has been nominated as a putative ‘feeding receptor’. This role is supported by antisense [17] and gene deletion (D. Marsh, Abstract OC12 NPY conference, London, 1997) studies. However, whilst the agonist profile of the stimulation of feeding matches that of the Y5 receptor more closely than the Y1 receptor, there are still a number of inconsistencies. For instance, while [D-Trp 32 ]-NPY has been shown to be an agonist at the Y5 receptor [13], two reports suggest that it is an antagonist of the orexigenic effects of NPY [14,18]. Additionally, NPY 13-36 is reported to be a low affinity agonist at the Y5 receptor [13], it has been shown to be unable to stimulate food intake at concentrations up to 70 times the threshold dose of NPY [14], whereas rat PP, which has similar or lower potency at the Y5 receptor [13,15], is able to stimulate food intake [14]. The definitive assignment of a role of any NPY receptors in controlling food intake has been hampered by the lack of specific receptor antagonists. To date no Y5 receptor antagonists are widely available, and few for any other receptors. The only widely available antagonists are for the Y1 receptor. These include the Wellcome peptide compound 1229 U91 [19], and the Thomae non-peptide compound BIBP 3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininamide) [20]. The structure of BIBP 3226 is derived from the two C-terminal amino acids of NPY. It benefits from the existence of an inactive form, BIBP 3435, which is the S-enantiomer of BIBP 3226 [20]. The peptide compound 1229 U91 has been shown to inhibit both NPY-induced, and fast-induced feeding [21]. We have previously reported that BIBP 3226 is able to inhibit NPY induced food intake when injected into the third cerebral ventricle [14]. However, these injections were associated with considerable behavioural side effects. A likely cause of these side effects is inhibition of Y1 receptors, which are widely distributed throughout the brain [22], involved in mechanisms other than the control of food intake. In an attempt to reduce effects caused by activation of such receptors, by giving a lower dose aimed at the region in which the feeding response is thought to be mediated, we have now studied the effects of intra-PVN doses of BIBP 3226 and BIBP 3435 on NPY induced feeding.

fmoc chemistry on an Advanced ChemTech 396MPS peptide synthesiser (Advanced Chemtech, Cambridge, UK). The products comprised one major peak, which was purified to homogeneity by reversed phase HPLC on a C8 column (Phenomenex, Macclesfield, UK). Mass determination by electrospray mass spectrometry was used to confirm the identity of the peptides. BIBP 3226 and BIBP 3435 were supplied by Thomae GmbH (Biberach, Germany). Other reagents were of the highest purity available, and were purchased from Sigma or Merck (both Poole, Dorset, UK) unless otherwise stated.

2.2. In vivo feeding experiments Feeding experiments were carried out as previously described [22]. Briefly, adult male Wistar rats (250–300 g) were maintained in individual cages under controlled temperature (21–238C) and light (11 h light / 13 h dark), with ad libitum access to food (RM1 diet, SDS UK Ltd.) and water. Rats were anaesthetized by intraperitoneal injection of a mixture of Ketalar (ketamine HCl 60 mg / kg, Parke–Davis, Pontypool, UK) and Rompun (xylazine 12 mg / kg, Bayer UK Ltd., Bury St. Edmunds, UK). Permanent 26-gauge stainless steel cannulae were implanted 1.8 mm posterior to bregma, 0.5 mm lateral to the midline and 7.0 mm below the outer surface of the skull using a Kopf stereotactic frame with the incisor bar set at 3 mm below the interaural line. After surgery, a wire stylet was inserted into the cannula to prevent blockage. All animals were allowed a period of 7 days to recover before being used in the study. The animals were handled daily for the 5 days prior to the study to minimise non-specific stress. The placement of the cannulae was verified at the end of the study by the injection of 0.5 ml ink, removal of the brain, and examination of coronal brain slices. Substances were administered by a stainless steel injector, projecting 1.0 mm below the tip of the cannulae. The injector was connected by polyethylene tubing (id, 0.5 mm; od, 1 mm) to a Hamilton syringe (Reno, NV) in a syringe pump set to dispense 0.5 ml solution / min. NPY was dissolved in 0.9% saline and both BIBP compounds in 70% ethanol. Each study involved an injection of 0.5 ml of peptide or saline, preceded 5 min before by a 0.5 ml injection of BIBP 3226, BIBP 3435 or 70% ethanol vehicle. Immediately after the PVN injections rats were placed into their home cage with a preweighed amount of chow and free access to water. After a 2 h period the remaining food was reweighed, and the food intake for each rat calculated. All studies were performed at least 48 h apart, between 08:00 and 11:00 h.

2.3. Study 1 – NPY dose-response relationship 2. Materials and methods

2.1. Materials NPY (porcine sequence) was synthesised in house using

In order to ascertain a suitable dose of NPY for further experiments, the dose-response relationship to intra-PVN NPY was investigated. Six groups of animals were each given an intra-PVN injection of either saline or NPY (0.1–2.5 nmoles / animal). Two hour food intake was

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measured. The doses chosen were approximately 1 / 10th those used previously for ICV studies [14].

2.4. Study 2 – Blockade of NPY-induced feeding with BIBP 3226 and 3435 Five separate groups of rats (n 5 14 to 18 per group) were studied simultaneously at the beginning of the light phase. Groups received two injections separated by 5 min. The first injection contained either 70% ethanol vehicle, or BIBP 3226 or BIBP 3435. Two doses of antagonist (6 and 12.5 nmol) were used. These doses were chosen to give the molar ratio at which BIBP 3226 would be predicted from previous data to inhibit activation of the Y1 receptor by NPY [20]. The second injection contained either saline vehicle or NPY (0.5 nmol). Two hour food intake was measured.

2.4.1. Study 3 – BIBP 3226 dose-response relationship Seven groups (n 5 6–9 per group) were studied. Animals received two injections separated by 5 min. The first injection contained either 70% ethanol vehicle as a control or BIBP 3226 (0.25–25 nmol, 0.5:1–50:1 molar ratio). The second injection contained either saline vehicle or NPY (0.5 nmol). Two hour food intake was measured. 2.5. Study 4 – Reversibility of action of BIBP 3226 on feeding One week after the previous experiment, the same animals were all given a further injection of NPY (0.1 nmol) or saline, in order to test whether the effect of BIBP 3226 was still present. Rats given BIBP 3226 were all given an injection of NPY, except for a control group, in which animals randomly selected from those which had received BIBP 3226 were given an injection of saline. As a further control, rats previously given an injection of the 70% ethanol vehicle were split into two groups, receiving either NPY or saline. Two hour food intake was measured.

Fig. 1. Dose response to intra-PVN injections of NPY. NPY was administered in doses from 0.1–2.5 nmoles / animal in a volume of 0.5 ml of 0.9% saline, and food intake in the subsequent 2 h period was measured. A maximum food intake of 6.460.8 g / 2 h was achieved, and the half maximal dose of NPY was calculated to be 0.15 nmoles. Groups were compared by ANOVA and post hoc Tukey’s HSD test. Results were considered significantly different when P , 0.05, and all doses of NPY given produced a statistically significant increase in food intake. The results shown are the mean6SEM for groups of 6–9 animals.

was found to be approximately 0.15 nmol. For subsequent studies a dose of 0.5 nmol was chosen as being a dose which would give a robust feeding response, but which would still allow inhibition.

3.2. Study 2 – Blockade of NPY-induced feeding with BIBP 3226 and 3435 Both BIBP 3226 and BIBP 3435 were injected at two doses (12:1 and 30:1 molar ratios). At both concentrations, both enantiomers produced approximately a 70% reduction in NPY induced feeding (Fig. 2). There did not appear to be any effect of dose and so the dose responsiveness of BIBP 3226 was investigated further.

2.6. Statistical analysis Differences in food intake between groups were analysed by one way analysis of variance (ANOVA) using the SYSTAT program (Jandel Scientific, Erkrath, Germany). Post hoc comparison of groups was carried out using Tukey’s test, again using SYSTAT.

3. Results

3.1. Study 1 – NPY dose-response relationship NPY induced a significant, dose-dependent increase in food intake when injected into the PVN (Fig. 1). A maximal food intake of 6.460.8 g / 2 h feeding period was achieved with a 0.75 nmol injection of NPY, and the EC 50

3.3. Study 3 – BIBP 3226 and BIBP 3435 doseresponse relationships In the previous experiment, the effects of BIBP 3226 and BIBP 3435 did not appear to be dose responsive. To address this, the inhibition by the compounds of NPYstimulated food intake was measured over a larger range of doses. NPY (0.5 nmol) was administered intra-PVN 5 min after antagonists at doses of 0.25, 1.25, 2.5, 12.5 and 25 nmol (0.5–50:1 molar ratio) and 2 h food intake were measured. In these experiments, both BIBP 3226 and BIBP 3435 inhibited NPY induced food intake (Fig. 3). However, BIBP 3226 appeared more potent, inhibiting . 90% of NPY induced food intake at the highest dose tested, compared to a 65% reduction seen with BIBP 3435.

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Fig. 2. Inhibition of NPY induced food intake by BIBP 3226 and BIBP 3435. NPY (0.5 nmol) was administered intra-PVN 5 min after an injection of BIBP 3226 or BIBP 3435 (6 or 12.5 nmol / animal) or 70% ethanol vehicle. Food intake during the subsequent 2 h period was measured. Groups were compared by ANOVA and post hoc Tukey’s HSD test. Results are considered significantly different when P , 0.05. The results shown are the mean6SEM for groups of 6–9 animals.

3.4. Study 4 – Reversibility of action of BIBP 3226 on feeding Seven days after injection of BIBP 3226 in the previous study, animals were given a further injection of NPY (0.1 nmol) (Fig. 4). Food intake in the 2 h following injection was measured. Animals which had previously been given an injection of 70% ethanol vehicle demonstrated a robust feeding response to NPY (NPY 5 2.960.3 g / 2 h, saline 5 1.460.3 g / 2 h). This feeding response was found to be significantly decreased in animals which had previously received an injection of BIBP 3226 (F(4,29) 5 3.5, P , 0.05). This effect appeared to be dose-dependent, since only two groups of animals that had received the high doses of BIBP 3226 showed a significant difference from those which had received no BIBP 3226 when compared by Tukey’s post hoc test. Animals which had previously been given BIBP 3226 appeared to have normal basal food intake, since those injected with saline in this experiment ate normally (1.460.2 g / 2 h). There was no difference in normal daily food intake between those animals which had previously received BIBP 3226 and those which had received ethanol vehicle, and neither of these were significantly different from non-cannulated animals (results not shown)

4. Discussion We have previously shown that ICV injection of BIBP 3226 causes a blockade of the food intake response to

Fig. 3. Dose-response relationship for the inhibition of NPY induced food intake by BIBP 3226 (A) and BIBP 3435 (B). NPY (0.5 nmol) was administered intra-PVN 5 min after an injection of BIBP 3226 or BIBP 3435 (0.25–25 nmol / animal) or 70% ethanol vehicle. Food intake during the subsequent 2 h period was measured. Groups are compared by ANOVA and post hoc Tukey’s HSD test. Results are considered significantly different when P , 0.05. The results shown are the mean6SEM for groups of 6–9 animals.

NPY [14]. However this was associated by marked behavioural side effects at higher doses, including barrel rolling and induction of a catatonic state. BIBP 3226 was also shown to block noradrenaline and galanin induced feeding. Hence it was felt that the reduction in food intake seen might not be due to blockade of specific NPY/ food intake receptors, but secondary to the blockade of receptors more generally altering behaviour. A large number of Y1-like receptors (i.e. receptors which bind [Leu 31 Pro 34 ]NPY but not human PP) are present throughout the CNS, as shown by receptor autoradiography [22,23], and some of these may be involved in control of mechanisms, the disruption of which might lead to the peculiar behaviour seen following ICV injection of BIBP 3226. The aim of this study was to investigate further the effects of BIBP 3226 in a more controlled system. In order to do this we have investigated the effects of small doses of BIBP 3226 administered directly into the PVN, thought to be the site

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Fig. 4. Reversibility of the inhibition of NPY induced food intake by BIBP 3226. NPY (0.5 nmol) was administered intra-PVN 7 days after an injection of BIBP 3226 (0.25–25 nmol / animal) or 70% ethanol vehicle. Food intake during the subsequent 2 h period was measured. Food intake by animals previously given a 70% ethanol injection was increased by intra-PVN injection of NPY compared to injection of saline. The feeding response to NPY of animals given BIBP 3226 was decreased in proportion to the amount of BIBP 3226 which they had previously received. Both BIBP 3226 and 70% ethanol control animals ate similar amounts following a control injection of saline. Groups are compared by ANOVA and post hoc Tukey’s HSD test. Results are considered significantly different when P , 0.05. The results shown are the mean6SEM for groups of 6–9 animals.

of action of NPY in the control of food intake. It is possible that there might be a spread of the compounds beyond this site of injection, however this would be expected to be less so than for ICV injections, and indeed the side effects seen following ICV injection are not seen with intra-PVN injection. The studies presented in this paper show that BIBP 3226 is able to inhibit NPY induced food intake when injected into the paraventricular nucleus of the hypothalamus in normal Wistar rats. However, the data also suggest that this effect may not be due to specific competitive antagonism of the NPY Y1 receptor, since the supposedly inactive form of BIBP 3226 (BIBP 3435) is as active as the active form, and since the effects of the antagonist appear to last for more than 7 days. It would be interesting to investigate the effects of BIBP 3226 and BIBP 3435 on noradrenaline or galanin stimulated food intake when given intra-PVN. However, this was beyond the scope of this paper. BIBP 3226 ((R)-N2-(diphenylacetyl)-N- [(4-hydroxyphenyl)methyl]-argininamide) was the product of a rational design process, and is modelled on the two C-terminal amino acids of NPY. Hence it contains an argininamide which mimics the arginine at position 35 of NPY. The chirality of this also mimics that of the amino acids in the peptide, and so it might be expected that a change to this chirality would affect their ability to bind to the receptor. This is indeed the case at the Y1 receptor, where the

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S-enantiomer of BIBP 3226, BIBP 3435 is unable to bind. Since neither BIBP 3226 nor BIBP 3435 have been shown to bind to NPY receptors other than the Y1 receptor, we were extremely surprised to find that both enantiomers were able to inhibit NPY induced food intake. This result suggested either that a novel receptor subtype was involved in this process, or that the BIBP compounds were acting via a non-receptor mechanism to inhibit NPY induced feeding. In order to investigate whether the compounds were acting over a short period of time, as would be expected for a competitive antagonist, we investigated the feeding response to NPY in rats which had been given BIBP 3226 7 days previously. These animals exhibited a reduced feeding response to NPY very similar to that seen 5 min after administration of the antagonist. There appeared to be a dose dependent reduction in the response to NPY, with 12.5:1 and 50:1 molar excesses of BIBP 3226 giving food intake very close to that seen in animals not treated with NPY (1.660.3 and 1.560.3 g, respectively, cf. 1.460.3 g for saline group). It is important to note that animals which had previously been treated with 70% ethanol, the vehicle for BIBP 3226 injection, showed a normal response to intra-PVN NPY 1 week later. This suggests that the effects of BIBP 3226 are not simply effects of the vehicle, or of injection. This is also suggested by the dose-responsive effects of BIBP 3226, since all doses were administered in the same volume. It is also interesting to note that the BIBP 3226-treated saline group behaved in a similar manner to the ethanol vehicle-treated saline group (1.460.3 vs 1.460.2 g). Although this group contains animals previously given each of the doses of BIBP, and is therefore not homogeneous, this suggests that, under these experimental conditions at least, normal basal feeding is not affected by previous BIBP 3226 injection. These result suggests that BIBP 3226 is acting not at the Y1 receptor, but via some alternative mechanism. One possible explanation might be that BIBP 3226 is acting as a local toxin. In this manner it may be killing cells near the site of injection which are responsive to NPY, and that this is manifested as a reduced response to NPY, both immediately after injection of the antagonist, and at a time point 1 week later. It is possible that the results observed in this study might be due to a specific action at a receptor. For instance, if BIBP 3226 were to bind to receptors and cause them to become downregulated, either by loss of responsiveness of the receptors to NPY, or by internalisation and destruction, a similar effect might be seen. However two factors suggest that this might not be the case here. Firstly, over the 7 day period between the first and second NPY feeding response tests, it might be expected that there might be some production of new receptors, due to normal protein turnover. If this were the case, a rightshift in the BIBP 3226 dose response curve would be expected as new receptors replaced the old ones blocked

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by the antagonist. This is not the case here, and the two BIBP 3226 dose response curves are remarkably similar. Secondly, as previously mentioned BIBP 3435 has not been shown to bind to any identified NPY receptor subtype. However, it is able to mimic the effects of BIBP 3226, albeit with slightly reduced potency. This suggests that neither antagonist is acting at an NPY receptor. It is possible that this might not be the case, and that either or both are acting at a receptor. It would be difficult to see an effect of any such interaction if it were masked by toxic effects, and so the participation of the Y1 receptor, or indeed of an other unidentified receptor to which BIBP 3226 or BIBP 3435 are able to bind, cannot be ruled out. In this study we have examined the actions of BIBP 3226 and BIBP 3435 in the PVN. These studies demonstrate that both compounds are able to inhibit NPY induced feeding, and that the effects of BIBP 3226 last for at least 7 days. This suggests that the inhibition of NPY induced food intake by BIBP 3226 is not by specific blockade of NPY Y1 receptors. This does not rule out a role for this receptor in the control of food intake, however, since a reduction of food intake caused by blockade of this receptor would be masked by the non-specific effects of BIBP 3226 described above.

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