In Vivo Characterization of the Novel Neuropeptide Y Y₁ Receptor Antagonist H 409/22

Abstract

We studied the effects of the novel neuropeptide Y (NPY) Y₁ receptor antagonist H 409/22, and its inactive enantiomer H 510/45, on vascular responses evoked by endogenous and exogenous NPY in the pig in vivo. H 409/22 and H 510/45 were given as 30-min infusions, and the antagonistic effects and circulating plasma concentrations were measured. The initial and terminal half-lives of H 409/22 in plasma were ∼3 and 30 min, respectively. In pigs pretreated with reserpine and transection of sympathetic nerves (depletion of noradrenaline), sympathetic nerve stimulation evoked nonadrenergic vasoconstrictor responses in kidney and hindlimb, mediated by neuronally released NPY. Significant inhibition of these vasoconstrictor responses, as well as of vascular responses to injections of exogenous NPY, were seen during a low-dose infusion of H 409/22 (1.8 nmol/kg/min), when plasma levels of the antagonist reached 77 ± 8 nM. Greatest inhibitory effects were seen at the highest dose of H 409/22 (180 nmol/kg/min, giving plasma levels of 7.4 ± 0.6 μM) when all vascular responses evoked by NPY were strongly attenuated or largely abolished. H 510/45 did not affect any of the vascular responses studied. It is concluded that H 409/22 potently and dose-dependently antagonizes vascular responses to exogenous and endogenous NPY in the pig, and thus represents an interesting tool for studies on NPY Y₁ receptor-mediated effects in vivo.

Neuropeptide Y (NPY), a peptide of 36 amino-acid residues ⁽¹⁾, is abundantly present in the sympathetic nervous system. Stored with noradrenaline in sympathetic nerve terminals ⁽²⁾, NPY is released with noradrenaline, especially on stronger nerve activation ⁽³⁾. The NPY Y₁-receptor subtype is the main postjunctional receptor involved in vasoconstriction (see 4), but postjunctional NPY Y₂ receptors also may occur (e.g., in pig spleen) ^(5,6). In recent years, nonpeptide NPY Y₁-receptor antagonists such as BIBP 3226 and SR 120107A ^(7,8) have been introduced, and, with them, final pharmacologic evidence for the involvement of endogenous NPY in sympathetic vasoconstriction both in vitro and in vivo has been presented ⁽⁴⁾. Thus, neurogenically released NPY evokes contractions of guineapig caval vein in vitro ^(9,10) and vasoconstriction in several vascular beds in the reserpine-treated pig in vivo ^(11,12). The involvement of NPY in sympathetic vascular responses is less obvious in pigs with normal noradrenaline levels ⁽¹³⁾, presumably due to an effective prejunctional α-adrenergic regulation of NPY release ⁽¹⁴⁾. Reserpine, in combination with transection of sympathetic nerves, creates a situation of depleted noradrenaline levels with maintained levels of NPY within the sympathetic nerve terminals ⁽¹⁴⁾. In the absence of noradrenaline, the vascular actions of neuronally released NPY are unmasked. This model was used in our study to investigate the in vivo potency of the novel NPY Y₁-receptor antagonist H 409/22, (2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(1R)-1-(4-hydroxyphenyl)ethyl]-pentanamide (Fig. 1)^(15,16). H 409/22 is a new nonpeptide antagonist structurally similar to the previously described NPY Y₁-receptor antagonist BIBP 3226 ⁽⁷⁾, and differs from this latter compound by the presence of an additional (R)-methyl-group in the alpha position of the N-terminal benzamide moiety (Fig. 1). H 409/22 possesses high affinity for rat and human NPY Y₁ receptors ^(15,16). Thus, receptor binding experiments in vitro have revealed that H 409/22 displaces [³H]NPY with median inhibitory concentration (IC₅₀) values in the low nanomolar range in rat brain cortex (16 ± 3 nM), human SK-N-MC cells expressing the NPY Y₁ receptor (13 ± 1 nM), and CHO cells transfected with the human NPY Y₁ receptor (4.8 ± 1.1 nM). In SK-N-MC cells, H 409/22 displaced [¹²⁵I]-peptide YY (PYY) with an IC₅₀ value (2.3 ± 0.4 nM) in a range similar to that of BIBP 3226 (1.6 ± 0.3 nM). In addition, H 409/22 is devoid of affinity for NPY Y₂, Y₄, and Y₅ receptors ^(15,16). Thus, H 409/22 showed no effect on [¹²⁵I]-PYY binding to the porcine NPY Y₂ receptor (pig splenic membranes; IC₅₀ value, >10 μM), to the human NPY Y₅ receptor (baculovirus-infected insect cells; IC₅₀ value, >10 μM), or on [¹²⁵I]-pancreatic polypeptide (PP) binding to the human NPY Y₄ receptor (transfected CHO cells; IC₅₀ value, >10 μM). Furthermore, the enantiomer to H 409/22, H 510/45, (2S)-5-([amino(imino)methyl] amino)-2-[(2,2-diphenylacetyl)amino]-N-[(1S)-1-(4-hydroxyphenyl)ethyl]-pentanamide, lacks affinity for NPY Y₁ receptors ⁽¹⁵⁾. Thus, H 510/45 showed no effect on either [¹²⁵I]-PYY or [³H]H 409/22-binding to the NPY Y₁ receptor in human SK-N-MC cells (IC₅₀ values, >10 μM each). H 510/45 may therefore be suitable to serve as a control.

FIG. 1: Chemical structure of the NPY Y₁-receptor antagonist H 409/22.

In this study the ability of H 409/22 to antagonize vascular responses to endogenous and exogenous NPY was investigated in the reserpine-treated pig in vivo. Two vascular beds were studied in detail: kidney and hindlimb. The kidney responds with a rapid vasoconstrictor response to both exogenous NPY and sympathetic nerve stimulation, which, to a major extent, is evoked by NPY after reserpine treatment ⁽¹⁷⁾. In contrast, a long-lasting vasoconstrictor response is seen in skeletal muscle in response to both exogenous and endogenous NPY, released from sympathetic nerves ⁽¹⁷⁾. The aim of this study was to investigate the antagonistic activity of different doses of H 409/22 on these vascular responses, and to compare the actions with its circulating plasma levels. Furthermore, the effects of H 510/45, the inactive enantiomer of H 409/22, were studied on the same vascular responses.

METHODS

In vivo study

This study was approved by the local ethics committee for animal research.

Surgical preparation

Pigs of either sex (16-22 kg), premedicated with ketamine (20 mg/kg i.m.) and atropine (0.02 mg/kg i.m.), were anesthetized with sodium pentobarbitone (20 mg/kg i.v.). The animals were intubated and ventilated by a respirator (Servo ventilator 900; Siemens-Elema, Sweden), and skeletal muscle relaxation was induced (pancuronium, 0.5 mg/kg i.v.). The anesthesia depth was checked by pinching the interdigital skin before administration of pancuronium. The retroperitoneal space was reached via a flank incision below the left costal margin, where the postganglionic sympathetic nerves to the left kidney and the sympathetic lumbar chains of both sides (level L3-L4) were exposed and sectioned. The incision was closed, and reserpine (1 mg/kg i.v.) was administered before extubation. After 24 h, the pigs were reanesthetized (see earlier) and ventilated by the respirator via a tracheal tube. A catheter was inserted into the right brachial vein for infusion of drugs to maintain anesthesia (sodium pentobarbitone, 8 mg/kg/h), skeletal muscle relaxation (pancuronium, 0.5 mg/kg/h), fluid balance (sodium chloride, 154 mM, and glucose, 28 mM, 2 ml/min), and to prevent intravascular coagulation (heparin, 250 IU/kg/h). A catheter also was placed into the left brachial artery, for collection of systemic arterial blood. Another catheter, connected to a Statham P23 AC pressure transducer, was inserted into the right brachial artery for measurement of mean arterial pressure (MAP). Heart rate was recorded by a tachograph unit triggered by the blood pressure. Ultrasonic flow probes (2RB), connected to Transonic flowmeters (T202; Transonic Instruments, Ithaca, NY, U.S.A.), were placed around the splenic artery, the left renal artery, and the femoral arteries of both sides to measure local blood flows. Electrodes were placed on the distal ends of the cut left and right lumbar sympathetic chain (supplying hindlimbs) and the left renal periarterial nerves, for electrical stimulation. The saphenous arteries of left and right hindlimbs were cannulated with a catheter in a retrograde direction, for local intraarterial (i.a.) injection of NPY. The abdomen was then closed, after which the pigs were allowed to stabilize for 1 h before the experiments were commenced.

Experimental procedures

Atropine (0.5 mg/kg i.v.) was administered every fourth hour to prevent any cholinergic vasodilatory response evoked by lumbar sympathetic stimulation in hindlimb ⁽¹⁸⁾. Electrical stimulation [two high-frequency bursts of 20 Hz for 1 s at 10-s intervals (5 ms, 25 V)] of sympathetic nerves was performed with a Grass stimulator. Five minutes later, these nerve stimulations were followed by an i.v. bolus injection of NPY (230 pmol/kg) and i.a. bolus injections of NPY (1.2 nmol) into the right and left saphenous artery. The doses of NPY were chosen to evoke largely similar vasoconstrictor responses to sympathetic nerve stimulation of the corresponding vascular beds. Consecutive infusions of H 510/45 and H 409/22 were given, either as H 510/45 (18 nmol/kg/min) and three following infusions of H 409/22 (1.8, 18, and 180 nmol/kg/min), or as four consecutive infusions of H 409/22 at increasing doses between 180 pmol/kg/min and 180 nmol/kg/min (equal to between 0.1 and 100 μg/kg/min). The infusions were given during 30 min, each at 1-h intervals. The set of nerve stimulations and NPY injections was then repeated between minutes 20 and 30 of each infusion. Thereafter a recovery period of 90 min followed, after which the set of nerve stimulations and exogenous NPY injections was performed once again. Systemic arterial blood samples were collected before and at minutes 20 and 30 of each infusion for determination of plasma levels of H 510/45 and H 409/22. Systemic arterial blood samples also were collected 1, 2, 5, 10, 15, 30, 60, and 90 min after cessation of the last infusion of H 409/22 (180 nmol/kg/min during 30 min), to determine the half-life in plasma of the antagonist.

The vascular responses studied are reproducible and not susceptible to any spontaneous decline, as was demonstrated in a previous study ⁽¹⁷⁾. In one separate series (n = 4), i.v. bolus injections of the α₁-adrenoceptor agonist phenylephrine (15 nmol/kg), angiotensin II (10 pmol/kg), α,β-methylene adenosine triphosphate (ATP) (mATP, 20 nmol/kg), the NPY Y₁-receptor agonist [Leu³¹Pro³⁴]NPY (470 pmol/kg), and the NPY Y₂ receptor agonist N-acetyl[Leu²⁸Leu³¹]NPY(24-36) (1.1 nmol/kg), were given before and during infusion of H 409/22 (60 nmol/kg/min during 30 min), to investigate the specificity and selectivity of the antagonist in vivo.

Determination of H 409/22 and H 510/45 in plasma

Blood samples were collected in prechilled tubes containing EDTA (final concentration of 10 mM), centrifuged for 10 min (+4°C), after which the plasma was pipetted off and stored at −20°C before being analyzed for H 409/22 by high-performance liquid chromatography. H 510/45 was determined in the same assay as H 409/22, as this method makes no distinction between the two enantiomers. The proteins in the plasma sample, 100 μl, were precipitated with acetonitrile, mixed, centrifuged, and H 409/22 in the supernatant was extracted as an ion pair in a liquid-solid extraction on an extraction disk (3M Empore C18 SD, High Performance Extraction Disk Cartridges). After elution with methanol, the pH-adjusted eluate was injected onto the reverse phase liquid chromatographic system, consisting of a Supelcosil ABZ+ column (3-μm particles), a mobile phase with acetonitrile/perchloric acid (6.5 mM) in water/acetic acid (37.8:62:0.2% vol/vol), and monitored with a fluorescence detector at excitation wavelength of 272 nm, emission wavelength of 305 nm. With use of an adequate internal standard, the SD of repeatability (within-day) was 2.9%, and the SD of reproducibility (between-day) was 1.7%. H 409/22 could be measured at levels down to 25 nM (limit of quantitation, SD <20%) in a 100-μl sample.

Calculations

All vascular responses were calculated as minimal remaining vascular conductance, calculated as blood flow divided by MAP ⁽¹⁹⁾, in percentage of basal vascular conductance (before vascular response), and expressed in percentage of the control response (seen before H 510/45 and H 409/22 were given). The calculations of half-lives of H 409/22 in plasma were performed using the WinNonlin Pro, version 1.5 (Scientific Consulting, Inc., Apex, NC, U.S.A.). Data in the text are given as mean ± SEM, and statistical significance was calculated with the multiple analysis of variance (ANOVA) followed by the post test of Tukey.

Drugs

Ketamine (Parke-Davis, CA, U.S.A.), sodium pentobarbitone (NordVacc, Sweden), atropine and sodium heparin (KabiVitrum, Sweden), pancuronium bromide (Organon, The Netherlands), reserpine, angiotensin II, phenylephrine hydrochloride, α,β-methylene ATP and atropine chloride (Sigma, St. Louis, MO, U.S.A.), NPY, [Leu³¹Pro³⁴]NPY and N-acetyl[Leu²⁸Leu³¹]NPY(24-36) (Auspep, Australia). H 409/22, (2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(1R)-1-(4-hydroxyphenyl)ethyl]-pentanamide, and H 510/45, (2S)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(1S)-1-(4-hydroxyphenyl)ethyl]-pentanamide (AstraZeneca R&D Mölndal, Mölndal, Sweden).

RESULTS

Effects of H 510/45 and H 409/22 per se

H 510/45 (18 nmol/kg/min) and the three lower doses of H 409/22 (0.18-18 nmol/kg/min) did not have any cardiovascular effects per se. The last infusion of H 409/22 (180 nmol/kg/min) was accompanied by a slight increase in splenic vascular conductance (to 111 ± 3% of basal, p < 0.05) without any effect on MAP.

Vascular responses to exogenous NPY

Intravenous administration of NPY (230 pmol/kg) evoked elevation of MAP (by 25 ± 3 mm Hg) and vasoconstriction in spleen and kidney (Fig. 2). Hence, vascular conductance in spleen and kidney was reduced to 27 ± 3% of basal and 51 ± 3% of basal, respectively. These vascular responses to exogenous NPY were all gradually attenuated in the presence of increasing doses of H 409/22 (Figs. 2-4). Significant inhibition of the NPY-evoked effects on MAP and renal vascular conductance was seen at 1.8 nmol/kg/min (Figs. 3 and 4), and the greatest inhibition was seen at the highest dose of H 409/22 (180 nmol/kg/min), when 21 ± 6% of the MAP elevation and 8 ± 2% of the renal vasoconstrictor effect to exogenous NPY remained (Figs. 3 and 4). The ID₅₀ value for the latter response was 5 ± 1 nmol/kg/min (n = 11). Significant inhibition of the NPY-evoked effect on splenic vascular conductance was seen at 18 nmol/kg/min (Fig. 3), and the greatest inhibition was seen at the highest dose of H 409/22 (180 nmol/kg/min), when 65 ± 5% of the splenic vasoconstrictor effect to exogenous NPY remained (Fig. 3).

FIG. 2: Original recording of the renal arterial blood flow in the reserpine-treated pig in vivo. Vascular responses on high-frequency stimulation of renal sympathetic nerves (two 1-s bursts at 20 Hz at 10-s intervals) and to exogenous NPY (230 pmol/kg), given i.v., are shown before (control) and at the end of four consecutive 30-min infusions of H 409/22 (0.18-180 nmol/kg/min) and 90 min later (recovery).

FIG. 3: Elevation of MAP (a) and splenic vasoconstrictor response (b) to i.v. NPY administration (230 pmol/kg) in the reserpine-treated pig in vivo. The vascular responses are compared with the control response to NPY and shown during consecutive 30-min infusions of H 510/45 (18 nmol/kg/min) and H 409/22 (0.18-180 nmol/kg/min) and after a recovery period of 90 min. Data are given as mean ± SEM, n = 11. Significant differences compared with control, ***p < 0.001. Significant differences between H 409/22 (180 nmol/kg/min) and recovery, ^###p < 0.001.

FIG. 4: Renal vasoconstrictor responses to i.v. NPY administration (230 pmol/kg) (a) and sympathetic nerve stimulation (b) in the reserpine-treated pig in vivo. The vascular responses are compared with the control responses and shown during consecutive 30-min infusions of H 510/45 (18 nmol/kg/min) and H 409/22 (0.18-180 nmol/kg/min) and after a recovery period of 90 min. Data are given as mean ± SEM, n = 8-11. Significant differences compared with control, **p < 0.01, ***p < 0.001. Significant differences between H 409/22 (180 nmol/kg/min) and recovery, ^##p < 0.01, ^###p < 0.001.

Local i.a. injection of NPY (1.2 nmol) into the saphenous artery elicited vasoconstriction in the hindlimb. Thus, vascular conductance in the femoral artery was reduced to 47 ± 3% of basal on local NPY administration. In the presence of increasing doses of H 409/22, the vascular response in hindlimb to exogenous NPY was gradually attenuated (Figs. 5 and 6A). Significant attenuation of NPY-evoked responses in hindlimb was seen during infusion of H 409/22 at 1.8 nmol/kg/min, and the greatest inhibition of these responses was seen during infusion of the highest dose of H 409/22 (180 nmol/kg/min) when 3 ± 1% of this vascular response remained (Fig. 5).

FIG. 5: Vasoconstrictor responses evoked in hindlimb by i.a. NPY administration (1.2 nmol) (a) and sympathetic nerve stimulation (b) in the reserpine-treated pig in vivo. The vascular responses are compared with the control responses and shown during consecutive 30-min infusions of H 510/45 (18 nmol/kg/min) and H 409/22 (0.18-180 nmol/kg/min) and after a recovery period of 90 min. Data are given as mean ± SEM, n = 16-20. Significant differences compared with control, *p < 0.05, **p < 0.01, ***p < 0.001. Significant differences between H 409/22 (180 nmol/kg/min) and recovery, ^##p < 0.01, ^###p < 0.001.

FIG. 6: A: Original recording of the arterial blood flow of hindlimb in the reserpine-treated pig in vivo. Vascular responses to exogenous NPY (1.2 nmol), given i.a., are shown before (control) and at the end of four consecutive 30-min infusions of H 409/22 (0.18-180 nmol/kg/min) and 90 min later (recovery). B: Original recording of the arterial blood flow of hindlimb in the reserpine-treated pig in vivo. Vascular responses on high-frequency stimulation of lumbar sympathetic nerves (two 1-s bursts at 20 Hz at 10-s intervals) are shown before (control) and at the end of four consecutive 30-min infusions of H 409/22 (0.18-180 nmol/kg/min) and 90 min later (recovery).

Ninety minutes after the last infusion of H 409/22 (180 nmol/kg/min), the vascular responses to exogenous NPY had partially recovered (Figs. 2-6A). Thus, at this time, the NPY-evoked elevation of MAP was back to 52 ± 10% of control (Fig. 3), whereas the renal and splenic vasoconstrictor responses had returned to 43 ± 5% (Fig. 4) and 88 ± 5% (Fig. 3) of control, respectively. The response to NPY given i.a. in the hindlimb also had returned to 72 ± 5% of control (Fig. 5).

H 510/45 (18 nmol/kg/min) did not affect any of the vascular responses to exogenous NPY (Figs. 3-5).

Vascular responses to various exogenous agonists

Intravenous injection of [Leu³¹Pro³⁴]NPY (470 pmol/kg) evoked vasoconstrictor responses in spleen and kidney (vascular conductance reduced to 47 ± 7% of basal and 29 ± 8% of basal, respectively) and elevation of MAP (by 15 ± 3 mm Hg). These responses were strongly inhibited during H 409/22 (60 nmol/kg/min), when only 11 ± 3% (p < 0.001) of the renal vasoconstriction, 31 ± 9% (p < 0.01) of the splenic vasoconstriction, and 15 ± 6% (p < 0.001) of the MAP elevation evoked by [Leu³¹Pro³⁴]NPY remained. N-acetyl[Leu²⁸Leu³¹]NPY(24-36) (1.1 nmol/kg, i.v.) evoked splenic vasoconstriction (vascular conductance reduced to 51 ± 4% of basal) only, accompanied by a modest elevation of MAP (by 3 ± 1 mm Hg). This response was not affected by H 409/22 (60 nmol/kg/min). Phenylephrine (15 nmol/kg, i.v.), angiotensin II (10 pmol/kg, i.v.), and mATP (20 nmol/kg, i.v.) evoked elevation of MAP (by 38 ± 5, 15 ± 2, and 14 ± 3 mm Hg, respectively), renal vasoconstriction (vascular conductance reduced to 27 ± 2% of basal, 27 ± 3% of basal, and 58 ± 6% of basal, respectively), and splenic vasoconstriction (vascular conductance reduced to 51 ± 4% of basal, 25 ± 3% of basal, and 20 ± 2% of basal, respectively). These vascular responses were not affected by H 409/22 (60 nmol/kg/min).

Vascular responses to sympathetic nerve stimulation

High-frequency stimulation (two 1-s bursts at 20 Hz) of the renal and lumbar sympathetic nerves evoked rapid vasoconstriction in the renal and femoral vascular beds, respectively (Figs. 2 and 6B). In the hindlimb, the initial effect was followed by a slowly declining long-lasting vasoconstriction, peaking about a minute after the initial rapid phase (Fig. 6B). Vascular conductance was maximally reduced to 60 ± 5% of basal in the kidney and 55 ± 4% of basal in the hindlimb on this nerve stimulation. The peak of the long-lasting phase of nerve-evoked vasoconstriction in hindlimb was at 75 ± 3% of basal vascular conductance.

The vascular response in kidney to sympathetic nerve stimulation was gradually attenuated in the presence of increasing doses of H 409/22 (Fig. 2), and the effect reached significance at 1.8 nmol/kg/min (Fig. 4). The greatest inhibitory effects were seen at the highest dose of H 409/22 (180 nmol/kg/min) when 29 ± 8% of the control vasoconstrictor response remained (Fig. 4).

The rapid phase of the vascular response in hindlimb was not affected by H 409/22, whereas the long-lasting phase of the sympathetic vasoconstriction was gradually attenuated (Fig. 6B). Significant inhibition of the long-lasting phase of sympathetic vasoconstriction in hindlimb was seen at 1.8 nmol/kg/min, and greatest inhibition was seen at the highest dose of H 409/22 when 12 ± 4% remained of the control response (Fig. 5).

Ninety minutes after the last infusion of H 409/22 (180 nmol/kg/min) these vascular responses had partially recovered (Figs. 2 and 4-6B). Thus, the renal response to high-frequency sympathetic nerve stimulation had returned to 60 ± 13% of control (Fig. 4), and the peak of the long-lasting phase of sympathetic vasoconstriction in the hindlimb was back to 43 ± 10% of control (Fig. 5).

H 510/45 (18 nmol/kg/min) did not affect any of the vascular responses to sympathetic nerve stimulation (Figs. 4 and 5).

Plasma levels of H 409/22 and H 510/45

H 510/45 (18 nmol/kg/min) reached plasma levels of 1,100 ± 100 nM. During the lowest-dose infusion of H 409/22 (0.18 nmol/kg/min), all plasma levels were below limit of detection (25 nM). During the intermediate infusions of H 409/22 (1.8 and 18 nmol/kg/min), when significant effects were observed in both vascular responses to exogenous NPY and sympathetic nerve stimulation, plasma levels reached 77 ± 8 nM (n = 9) and 910 ± 90 nM (n = 9), respectively, and remained stable from minutes 20 to 30 of infusion. Because the present assay does not distinguish between the two enantiomers, the plasma levels of the second infusion of H 409/22 (1.8 nmol/kg/min) may have been marginally exaggerated because of the preceding (1.5 h earlier) infusion of H 510/45. During the last infusion of H 409/22 (180 nmol/kg/min), plasma levels were steady from minutes 20 to 30 of infusion at 7,400 ± 600 nM. The elimination of H 409/22 from plasma follows a biexponential function (Fig. 7). The half-lives of the initial and terminal phases were ∼3 and 30 min, respectively.

FIG. 7: Plasma levels of H 409/22 in the pig in vivo, plotted against time. H 409/22 was given as consecutive 30-min infusions. The plasma concentrations during and after the last infusion (180 nmol/kg/min) are shown. Data are given as mean ± SEM, n = 9.

DISCUSSION

In this study H 409/22 was demonstrated to dose-dependently and potently antagonize NPY Y₁ receptor-mediated vasoconstrictor responses evoked by both neuronally released and exogenous NPY in the pig in vivo. H 409/22 is a newly developed nonpeptide antagonist for the NPY Y₁ receptor. High affinity (in the low nanomolar range) for rat and human NPY Y₁ receptors has been demonstrated in binding assays in vitro, and selectivity for NPY Y₁ receptors has been shown as H 409/22 is devoid of affinity for NPY Y₂, Y₄, and Y₅ receptors in vitro ⁽¹⁵⁾. That H 409/22 exerts competitive antagonism has been shown in functional assays in vitro. Thus, H 409/22 antagonized NPY-evoked inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) production in SK-N-MC cells (expressing NPY Y₁ receptors) and extracellular acidification in response to NPY-induced metabolic stimulation of human NPY Y₁ receptor-transfected CHO-cells with pA₂ values of 8.0 ⁽¹⁵⁾. In addition, H 510/45, the enantiomer of H 409/22, lacks affinity for the NPY Y₁ receptor in binding assays in vitro and can therefore serve as control ⁽¹⁵⁾.

To investigate the actions of H 409/22 in vivo, two vascular beds of the reserpine-treated pig were studied in detail. Previous studies have shown that NPY-evoked vasoconstriction in the kidney and hindlimb of the pig in vivo can be entirely abolished in the presence of NPY Y₁-receptor antagonists ⁽⁴⁾. In this respect, the NPY Y₁ receptor seems to be the sole receptor mediating vasoconstrictor responses in these two vascular beds in the pig. Furthermore, previous studies using NPY Y₁-receptor antagonists in the pig in vivo also showed that after pretreatment with reserpine and preganglionic transection of sympathetic nerves (depletion of noradrenaline), high-frequency stimulation of renal and lumbar (supplying hindlimb) sympathetic nerves evokes vasoconstrictor responses in kidney and hindlimb, which, to a major extent, are mediated by NPY acting on NPY Y₁ receptors ⁽⁴⁾. The kidney responds with short-lasting vasoconstriction to both neuronally released NPY and circulating NPY, given i.v. In hindlimb, moderate circulating levels of NPY do not evoke any vascular responses. Instead, NPY has to be injected locally and then exerts long-lasting vasoconstrictor responses, similar to those seen in response to neuronally released NPY. The cause of these different characteristics concerning NPY-evoked vascular responses is not known, but the difference may, at least in part, be due to a difference in the endothelial permeability. Hence, the renal vascular bed possesses fenestrated endothelium in contrast to the skeletal muscle ⁽²⁰⁾, which in turn may be less permeable to a large molecule like NPY.

In our study H 409/22 dose-dependently inhibited the vascular responses evoked in kidney and hindlimb by both endogenous, neuronally released NPY and exogenous NPY, given either i.v. or i.a. The vascular responses to endogenous and exogenous NPY were of roughly the same magnitude in both vascular beds. Significant inhibition was seen simultaneously of vasoconstrictor responses to endogenous and exogenous NPY both in kidney and hindlimb, indicating that H 409/22 antagonizes vascular responses to exogenous and endogenous NPY with equal potency. Furthermore, significant inhibitory effects were seen simultaneously in kidney and hindlimb, indicating that H 409/22 exerts similar antagonistic effects in these two vascular beds, independent of their different characteristics concerning ability to respond to, and pattern of response to, NPY.

H 409/22 was found to be a potent NPY Y₁-receptor antagonist in vivo. Thus, significant inhibition of all NPY Y₁ receptor-mediated events was seen during a low-dose infusion of the compound, giving rise to moderate plasma levels. For a feasible comparison with the previously described NPY Y₁-receptor antagonist BIBP 3226, the antagonistic effects exerted on the NPY-evoked renal vasoconstriction are discussed later. An ID₅₀ value of 5 ± 1 nmol/kg/min was calculated for the NPY-evoked renal vasoconstrictor response in this study. Thus, the potency of H 409/22 seems similar to that of BIBP 3226 [ID₅₀ value of 7 ± 1 nmol/kg/min ⁽¹⁷⁾ for the same response] in vivo. Furthermore, at the highest dose of H 409/22, all NPY Y₁ receptor-mediated vascular responses were strongly attenuated or even nearly abolished. At the highest dose of H 409/22 (180 nmol/kg/min), 92 ± 2% of the renal vasoconstrictor response to NPY was inhibited. Again, the potency of H 409/22 seems similar to that of BIBP 3226, exerting 88 ± 2% inhibition ⁽¹⁷⁾ of this response at its highest dose (190 nmol/kg/min). H 409/22 was less potent in antagonizing the NPY-evoked vasoconstrictor response in pig spleen than in, for example, the kidney. Thus, (a) a large portion of the vascular response to NPY evoked in spleen remained, even at the highest dose of the antagonist; and (b) a higher dose of the antagonist was required to inhibit significantly the splenic vascular response compared with the other responses studied. This is explained by (a) the fact that NPY mediates vascular responses predominantly through the NPY Y₂ receptor in the pig spleen ^(5,6). However, part of the vasoconstrictor effect evoked by NPY in pig spleen is NPY Y₁ receptor mediated, as shown by the partial inhibition of this response exerted by H 409/22 and other NPY Y₁-receptor antagonists ^(4,17). Furthermore, (b) augmented splenic vascular responses have been demonstrated on repeated NPY administration (in the absence of antagonist) in the pig in vivo ⁽⁴⁾; the cause of this phenomenon is unknown. In our study, it also was observed that a small portion of the renal nonadrenergic sympathetic vasoconstrictor response as well as all of the initial rapid phase of the nonadrenergic sympathetic vasoconstrictor response in hindlimb remain after NPY Y₁-receptor blockade with H 409/22. Similar findings have been seen with other NPY Y₁ receptor antagonists as well ^(12,17). If not explained by an incomplete NPY Y₁-receptor blockade, these rapid nonadrenergic sympathetic vasoconstrictor responses, resistant to NPY Y₁-receptor blockade, may very well be mediated by purinergic mechanisms, as this and previous studies ⁽¹²⁾ have shown that mATP potently evokes vasoconstriction in both these vascular beds.

Specificity for NPY receptor-mediated events was demonstrated in this study, as H 409/22 did not affect vascular responses evoked either by mATP, angiotensin II, or the α₁-adrenoceptor agonist phenylephrine. In accord with these results, H 409/22, at doses that inhibit the renal vasoconstrictor response to NPY ⁽²¹⁾, does not inhibit adrenergic or angiotensin II-mediated renal vasoconstriction in the rat and dog in vivo (Nordlander, personal communication). Selectivity for the NPY Y₁ receptor in vivo was shown in our study. Thus, the vascular responses evoked by the NPY Y₁-receptor agonist [Leu³¹Pro³⁴]NPY were strongly antagonized by H 409/22. In contrast, the vascular response mediated by the other known vascular NPY receptor subtype (Y₂) was not affected, as H 409/22 exerted no inhibitory effect on the splenic vasoconstrictor response to the NPY Y₂-receptor agonist N-acetyl[Leu²⁸Leu³¹]NPY (24-36).

The results of this study are strengthened by the fact that a partial recovery of all vascular responses was seen after a recovery period of 90 min (after completion of the last H 409/22 infusion). Most of the vascular responses seen after this recovery period corresponded to those seen during the second or third infusion of H 409/22 (1.8 and 18 nmol/kg/min). Accordingly, 90 min after completion of the last infusion of H 409/22 (180 nmol/kg/min), some of the antagonistic actions remained. The plasma level of H 409/22 was 270 ± 70 nM at 90 min after the last infusion, and this level is between what was observed at the second and third H 409/22 infusions, which would also correspond to the moderate antagonistic actions evoked by H 409/22 at this moment. It was demonstrated that 57 ± 5% of the inhibitory effects exerted by H 409/22 on the NPY-evoked renal vasoconstrictor response remained after the recovery period, indicating that H 409/22 may possess a slightly longer duration of action in vivo than does BIBP 3226 (42 ± 6% inhibition remaining after a 90-min recovery) ⁽¹⁷⁾. H 409/22 was found to possess a rather short half-life in plasma. Hence it is suggested that in vivo studies with H 409/22 are preferably performed during infusions of the compound, at least when the antagonistic actions are to be correlated to the dose.

The highest dose of H 409/22 was accompanied by a slight splenic vasodilatation, without causing any decrease in blood pressure. Hypotensive effects have been observed when using other NPY Y₁-receptor antagonists in vivo ^(11,12,22). It was postulated that these were nonspecific effects, possibly due to histamine release ⁽⁴⁾. High doses of NPY, and C-terminal fragments of NPY, may release histamine from mast cells in both rats and humans ^(23,24), and this effect was attributed to the basic structures within the peptide. Because H 409/22 shares some of its structural characteristics with the C-terminal part of NPY, such nonspecific effects may be shared as well. As with any newly developed compound, it is of great importance to use a proper control to ascertain that the results obtained are specific. H 510/45, the enantiomer of H 409/22 devoid of affinity for the NPY Y₁ receptor, was used as control in this study. It was demonstrated that H 510/45, at a dose at which H 409/22 exerted strong antagonistic effects, had no effects on any of the vascular responses studied, which in turn strengthens the results obtained with H 409/22.

It is concluded that H 409/22 is a potent and selective NPY Y₁-receptor antagonist, that dose-dependently, and with similar potency, inhibits vascular responses to exogenous and endogenous, neuronally released NPY in the pig in vivo. In contrast, the enantiomer of H 409/22, H 510/45, which is devoid of NPY Y₁-receptor affinity, exerted no such inhibitory effects. Thus, especially because enantioselective actions can be confirmed using H 510/45, the antagonist H 409/22 represents an interesting tool for studies on NPY Y₁ receptor-mediated transmission in vivo.

Acknowledgment: This study was supported by grants from AstraZeneca R&D Mölndal, and the Queen Victoria and Gustav V Foundation. We thank Ms. Margareta Stensdotter for expert technical assistance and Mr. Thomas Antonsson for supplying the chemical structure of H 409/22.