Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists

Abstract

Substance P (SP) is thought to play a cardinal role in emesis via the activation of central tachykinin NK1 receptors during the delayed phase of vomiting produced by chemotherapeutics. Although the existing supportive evidence is significant, due to lack of an appropriate animal model, the evidence is indirect. As yet, no study has confirmed that emesis produced by SP or a selective NK1 receptor agonist is sensitive to brain penetrating antagonists of either NK1, NK2, or NK3 receptors. The goals of this investigation were to demonstrate: 1) whether intraperitoneal (i.p.) administration of either SP, a brain penetrating (GR73632) or non-penetrating (e.g. SarMet-SP) NK1 receptor agonist, an NK2 receptor agonist (GR64349), or an NK3 receptor agonist (Pro7-NKB), would induce vomiting and/or scratching in the least shrew (Cryptotis parva) in a dose-dependent manner; and whether these effects are sensitive to the above selective receptor antagonists; 2) whether an exogenous emetic dose of SP (50 mg/kg, i.p.) can penetrate into the shrew brain stem and frontal cortex; 3) whether GR73632 (2.5 mg/kg, i.p.)-induced activation of NK1 receptors increases Fos-measured neuronal activity in the neurons of both brain stem emetic nuclei and the enteric nervous system of the gut; and 4) whether selective ablation of peripheral NK1 receptors can affect emesis produced by GR73632. The results clearly demonstrated that while SP produced vomiting only, GR73632 caused both emesis and scratching behavior dose-dependently in shrews, and these effects were sensitive to NK1-, but not NK2- or NK3-receptor antagonists. Neither the selective, non-penetrating NK1 receptor agonists, nor the selective NK2- or NK3-receptor agonists, caused a significant dose-dependent behavioral effect. An emetic dose of SP selectively and rapidly penetrated the brain stem but not the frontal cortex. Systemic GR73632 increased Fos expression in the enteric nerve plexi, the medial subnucleus of nucleus tractus solitarius, and the dorsal motor nucleus of the vagus, but not the area postrema. Ablation of peripheral NK1 receptors attenuated the ability of GR73632 to induce a maximal frequency of emesis and shifted its percent animals vomiting dose-response curve to the right. The NK1-ablated shrews exhibited scratching behavior after systemic GR73632-injection. These results, for the first time, affirm a cardinal role for central NK1 receptors in SP-induced vomiting, and a facilitatory role for gastrointestinal NK1 receptors. In addition, these data support the validation of the least shrew as a specific and rapid behavioral animal model to screen concomitantly both the CNS penetration and the antiemetic potential of tachykinin NK1 receptor antagonists.

Figure 1

The dose-response emetic effects of varying doses of intraperitoneally-administered substance P (Graphs A and B) and the brain penetrating NK₁ receptor selective agonist GR73632 (graphs C and D), during the 30 min post-injection observation period in the least shrew. Graphs A and C depict increases in the frequency of emesis (mean ± S.E.M.), whereas graphs B and D show the percentage of shrews vomiting. Significantly different from corresponding vehicle control (0 mg/kg) at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

Figure 2

The ability of varying doses of intraperitoneally administered GR73632 (a selective NK₁ receptor agonist) to induce dose-dependent increases in the frequency of scratching behavior (mean ± S.E.M.) during a 30 minute observation period in the least shrew (Graph A). Graphs B and C show the ability of varying doses of two structurally diverse but selective NK₁ receptor antagonists (CP99,994 and L733060) in suppressing the scratching behavior produced by a 5 mg/kg intraperitoneal dose of GR73632. Significantly different from corresponding vehicle-pretreated control at P < 0.05 (*) and P < 0.001 (***).

Figure 3

The antiemetic effects of the neurokinin NK₁ receptor selective antagonist CP99,940 against substance P (graphs A and B)- and GR73632 (graphs C and D)-induced emesis in the least shrew. Graphs E and F show the ability of another NK₁ receptor selective antagonist L733060 to suppress emesis produced by GR73632. Different groups of shrews received i.p. vehicle (0 mg/kg), or varying doses of CP99,994 (5, 10 or 20 mg/kg) or L733060 (5, 10 and 20 mg/kg), 30 min prior to an emetic dose of either substance P (50 mg/kg) or GR73632 (5 mg/kg). Emetic parameters were recorded for 30 min post emetic injection. Graphs A, C and E depict attenuations in the frequency (mean ± S.E.M.) of emesis, whereas graphs B, D and F show reductions in the percentage of shrews vomiting. Significantly different from vehicle control at P < 0.01 (**) and P < 0.001 (***).

Figure 4

The lack of effect of either a selective NK₂ receptor antagonist GR159897 (20 mg/kg, i.p.), or a selective NK₃ receptor antagonist SB218795 (20 mg/kg, i.p.), on the ability of a 5 mg/kg intraperitoneal dose of the selective NK₁ receptor agonist GR73632 to produce emesis and scratching behavior. Graph A represents the frequency of emesis (mean ± S.E.M.), graph B depicts the percentage of shrews vomiting, and graph C shows the frequency of scratching (mean ± S.E.M.).

Figure 5

Demonstrates time-dependent distribution of exogenously administered substance P (50 mg/kg, i.p.) in: A) brain stem (▬) and frontal cortex (----), B) duodenum (▬) and jejunum(----), and C) blood serum. Significantly different from corresponding basal level at P < 0.05 (*) and P < 0.01 (**).

Figure 6

Fos Immunoreactivity (Fos-IR) in the DVC of GR73632 injected shrews and controls. A) Coronal hemisection of the dorsal vagal complex (DVC) area of a non-vomiting least shrew (saline control) stained for Fos-IR. B) Fos-IR stained coronal section of the DVC in a shrew which vomited after being given 2.5 mg/kg (i.p.) GR73632. Insets in A and B diagrammatically represent the coronal level studied, and asterisks represent the central canal in the image and corresponding inset. The NTS shows a strong induction of Fos-IR, the DMNX exhibits weaker induction, while the AP is devoid of it following either saline or GR72632 injection. Scale bar for A and B = 100 µm. C) Fos-IR in the myenteric plexus and intestinal wall of a control shrew. Cells in crypts and villi did not produce Fos-IR, but scattered Fos-IR nuclei were found in the myenteric plexus of shrews following either saline (C) or GR73632 (D) injection. D) Fos-IR is greatly enhanced in the myenteric layers (arrowheads) of the GR73632-injected shrew. Scale bar for C and D = 40 µm. Abbreviations: 12 – 12th (hypoglossal) cranial nerve nucleus; AP – area postrema; Cr - intestinal crypts; Cu – cuneate nucleus and fiber tract; DMNX – dorsal motor nucleus of the vagus nerve; Gr – gracile nucleus; MP – intestinal wall layers including myenteric plexus; NTS – nucleus of the solitary tract; V – intestinal villi.

Figure 7

Immunohistochemical analysis of immunotoxin lesion. SSP-SAP (1.2 mg/kg, i.p.) was injected to lesion NK₁ receptor-containing cells in the gut. Immunolabeling for NK₁ receptors and Substance P (SP) was used to assess the lesion. A–B) Labeling in the dorsal vagal complex (DVC) of saline-(A/B) or SSP-SAP-injected (A’/B’) shrews appeared normal for both NK₁ receptor (A/A’) and SP (B/B’). C–D) Relative to saline control (C), labeling in the small intestine showed a distinct and extensive loss of NK₁ receptor (C’) containing cell bodies and fibers in the myenteric plexus, crypts, and villi, although the loss was not complete. SP-containing cell bodies in the intestinal nerve plexi were present in both saline (D) and SSP-SAP (D’) treated shrews, but fibers extending into the intestinal villi and crypts appeared to be reduced in number relative to controls. The inset in B’ shows the presence of Fos-IR within the DVC following i.p. injection of GR73632 and vomiting in a SSP-SAP-injected shrew, demonstrating that the NTS is still functionally responsive to emesis. Scale bars (Except inset) = 50 µm.

Figure 8

The emetic and scratching dose-response effects of intraperitoneally administered doses of the brain penetrating selective NK₁ –receptor agonist GR63762 in normal (O) and in peripherally NK₁-receptor ablated (•) shrews during the 30 min observation period immediately following NK₁ agonist injection. On day 1 groups of shrews were treated i.p. either with saline (control) or 1.2 mg/kg SSP-saporin (peripheral NK₁ receptor-ablated shrews) and on day 4 were challenged with varying doses of GR73632. Graph A shows dose-dependent increases in the frequency of emesis (mean ± S.E.M.), whereas graph B depicts the percentage of shrews vomiting. Graph C presents dose-dependent increases in the frequency of (mean ± S.E.M.) scratching behavior. Significantly different from corresponding vehicle control at P < 0.01 (**) and P < 0.001 (***); or significantly different from corresponding dose in NK₁ receptor -intact shrew control group (normal, O) at P < 0.05†.