SEARCH

SEARCH BY CITATION

Keywords:

  • antagonism;
  • inflammatory bowel disease;
  • mouse colitis;
  • vanilloid receptor 1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Neurogenic mechanisms have been implicated in the induction of inflammatory bowel disease (IBD). Vanilloid receptor type 1 (TRPV1) has been visualized on nerve terminals of intrinsic and extrinsic afferent neurones innervating the gastrointestinal tract and local administration of a TRPV1 antagonist, capsazepine, reduces the severity of dextran sulphate sodium (DSS)-induced colitis in rats (Gut 2003; 52: 713–91). Our aim was to test whether systemically or orally administered TRPV1 antagonists attenuate experimental colitis induced by 5% DSS in Balb/c mice. Intraperitoneal capsazepine (2.5 mg kg−1, bid), significantly reduced the overall macroscopic damage severity compared with vehicle-treated animals (80% inhibition, P < 0.05); however, there was no effect on myeloperoxidase (MPO) levels. An experimental TRPV1 antagonist given orally was tested against DSS-induced colitis, and shown to reverse the macroscopic damage score at doses of 0.5 and 5.0 mg kg−1. Epithelial damage assessed microscopically was significantly reduced. MPO levels were attenuated by approximately 50%, and diarrhoea scores were reduced by as much as 70%. These results suggest that pharmacological modulation of TRPV1 attenuates indices of experimental colitis in mice, and that development of orally active TRPV1 antagonists might have therapeutic potential for the treatment of IBD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Inflammatory bowel diseases (IBD) such as ulcerative colitis, Crohn's disease and celiac disease are characterized by diminished intestinal barrier function, erosive loss of intestinal mucosa, inflammatory infiltrates in the submucosa and mucosa, and dysregulated cytokine and T-helper cell profiles.2 In addition to classical patterns of inflammation, there also exist neuronal or neurogenic inflammatory pathways 3 in IBD.4–11 For example, changes in intrinsic and extrinsic afferents innervating the intestinal tract occur during clinical and experimental IBD.11–15 This has prompted interest in the role of neurotransmitters in colitis, especially those involved in nociceptive pathways.

Among the more recently described nociceptive receptors is vanilloid receptor type 1 (TRPV1).16,17 TRPV1 is sensitive to heat,16 low pH,18,19 endogenous lipids derived from metabolism of arachidonic acid such as anandamide,20,21 and to exogenous substances that possess a vanilloid moiety, as found in the plant substance capsaicin.22–24 The family of vanilloid receptors includes six related subtypes with TRPV1, the only one reported to respond physiologically to capsaicin.25 TRPV1 immunoreactivity has been reported in many somatic nociceptive pathways 21,26–28 but only recently has it been shown to be present in intrinsic and extrinsic neurones innervating the gastrointestinal tract.29,30

The levels of TRPV1 are elevated in colonic tissues from patients with both Crohn's disease and ulcerative pancolitis, and there is a marked increase in TRPV1-immunoreactive fibres in the submucosal plexus of diseased tissue.31 Experimental data suggest a protective role of TRPV1 antagonists in acute experimental colitis. For example, treatment with a TRPV1 antagonist, capsazepine, reduced the inflammatory infiltrate and tissue damage in acute ileitis evoked by either Clostridium difficile toxin A 32 or anandamide33 administration to the ex vivo ileum. However, it is not clear whether this type of acute experimental ileitis mimics the progression of inflammatory changes associated with IBD.

The dextran sulphate sodium (DSS) model of experimental colitis in rodents provides pathology similar to human colitis, which is characterized by a discontinuous pattern of mucosal epithelial damage in the distal colon, shrinkage in colon length and increases in the wet colonic tissue weight, infiltration of inflammatory cells and diarrhoea.34–39 This model was recently shown to be sensitive to daily intraperitoneal dosing with nordihydrocapsiate, a naturally occurring capsaicin analogue, which during a 6-day DSS regimen prevented the disruption of mucosal epithelia and loss of glandular crypts that is associated with DSS treatment.40 However, in that study effects of this treatment on macroscopic damage to the colon or myeloperoxidase (MPO) levels were not reported. A beneficial effect of capsazepine on DSS-induced colitis in rats has been reported 1 in which capsazepine (100 μg) was administered locally as an enema infusion twice daily and shown to improve disease indices, and reduce MPO levels and epithelial damage.1 This effect may have been due to a local effect on the colonic mucosa.

The aims of the present study were to extend the reported findings on the effect of capsaicin analogues on experimental colitis by using systemic capsazepine administration, and to test the proposition that a synthetic, non-vanilloid small molecule TRPV1 antagonist, JNJ 10185734, given orally can manifest anti-colitic activity in a 7-day DSS regimen in mice.

Mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Female Balb/c mice (Charles River Laboratories), 11–14 weeks of age, were used for most studies. All procedures were performed with the approval of the Institutional Animal Care and Use Committee of Johnson and Johnson Pharmaceutical Research and Development, LLC.

Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

The TRPV1 antagonist JNJ 10185734 was synthesized by the Analgesics Research Team, Johnson and Johnson Pharmaceutical Research and Development, LLC. JNJ 10185734, whose structure is shown in figure 1, is a synthetic small molecular weight non-capsacinoid compound that is not derived from a natural substance. It inhibits resiniferotoxin binding to human TRPV1-transfected HEK293 cells with a Ki of 13.1 nmol L−1 (S.-P. Zhang, unpublished results). JNJ 10185734 demonstrated antagonist properties by inhibiting capsaicin-stimulated calcium flux in human TRPV1-transfected cells (A.E. Dubin, unpublished results). DSS (40 kDa MW) was purchased from ICN pharmaceuticals (Newport, CA, USA). Capsazepine (CPZ) was obtained from Sigma-Aldrich chemicals (St Louis, MO, USA), as were phosphate-citrate buffer with sodium perborate, o-dianisidine and hexadecyltrimethylammonium bromide (HTAB).

image

Figure 1. The chemical structure of JNJ 10185734.

Download figure to PowerPoint

Induction of colitis and dosing regimens

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Balb/c female mice (n = 10/treatment group) were provided with a solution of tap water containing 5% DSS (ICN pharmaceuticals) ad libitum over a 7-day period. The DSS solution was replenished daily and the amount consumed measured. During this same time, selected groups of test animals were administered a preparation of an experimental TRPV1 antagonist, JNJ 10185734, which is orally bioavailable. Mice were dosed by oral gavage either with vehicle (10% hydroxypropyl beta-cyclodextrin), or with JNJ 10185734 starting on the day of induction with DSS, and twice daily thereafter in the morning and afternoon for 7 days. Capsazepine was used as a reference standard and was administered intraperitoneally twice daily in the morning and afternoon in 10% Tween 80–10% Ethanol-80% D5W. Capsazepine, which is not water-soluble and which has not been shown to be active via oral administration, has previously been reported to be active in other studies using this vehicle with intraperitoneal administration.22

At the end of this 7-day period, the animals were killed and their colons collected for further analysis. For those analyses, the distal colon segment between the first and the fourth centimetre was dissected into two halves. One half was placed in 10% neutral-buffered formalin for subsequent histological analysis. The other half was cleaned in a bath of physiological buffer, patted dry, weighed and then placed in a solution of 0.5% HTAB (Sigma-Aldrich) dissolved in 50 mmol L−1 phosphate buffer, adjusted to pH 5.4. The tissue sample thus treated was then frozen at −80 °C and reserved for MPO determinations.

Among the macroscopic parameters analysed before tissue samples were collected were length of the colon starting from 1 cm above the anus to the top of the caecum, weight of the colon still containing faecal contents, the consistency of any stools found within the colon, and the gross macroscopic appearance of the colon.

Decreases in filled colon weight are commonly observed in the mouse DSS colitis model and are indicative of colonic hypermotility. Indeed, colons from mice with severe colitis can be seen to be nearly devoid of faecal contents. Colon shrinkage is also a commonly observed parameter in DSS colitis and is indicative of neuromuscular-induced contraction of the colon smooth muscle.36,37

The decreases in the various colitis parameters by experimental compounds in individual mice were compared with those parameters in colitic mice not given those treatments. Baseline levels from normal, non-colitic mice were subtracted from all groups and a percentage inhibition of DSS-induced colitis was ascertained according to the following formula:

  • image

A scoring system was used to describe the changes for each of these parameters and is detailed in Table 1. The four scores for each animal were added together to provide a total macroscopic score.

Table 1.  Total macroscopic score criteria
Stool score
 0 = normal (well-formed faecal pellets)
 1 = loosely shaped moist pellets
 2 = amorphous, moist, sticky pellets
 3 = diarrhoea
 Presence of blood in stool: add 1 to scores
Colon damage score
 0 = no inflammation
 1 = reddening, mild inflammation
 2 = moderate inflammation or more widely distributed
 3 = severe inflammation and/or extensively distributed
Colon weight score
 0 = <5% weight loss
 1 = 5–14% weight loss
 2 = 15–24% weight loss
 3 = 25–35% weight loss
 4 = >35% weight loss
Colon length score
 0 = <5% shortening
 1 = 5–14% shortening
 2 = 15–24% shortening
 3 = 25–35% shortening
 4 = >35% shortening.
Maximum score = 15

Myeloperoxidase measurement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Myeloperoxidase is an intracellular enzyme found in neutrophils and has been considered to be a surrogate marker for neutrophil infiltration into tissues, including those afflicted with colitis.36 The tissue samples previously frozen at −80 °C in 0.5% HTAB were thawed, then re-frozen and thawed three times before being homogenized twice for 15 s using a Brinkman Polytron homogenizer fitted with a TS10 generator (Brinkmann Instruments, Westbury, NY, USA). The crude homogenate was next sonicated at 60 W for two 5-s bursts and then centrifuged for 20 min at 15 000 g. The supernatant of this homogenate was collected for MPO analysis wherein 100 μL of sample diluted in HTAB (0.5% in 50 mmol L−1 phosphate buffer, pH 5.4) was added to a 96-well plate in duplicate. The enzyme reaction was initiated by addition of 100 μL of substrate solution consisting of phosphate-citrate buffer with sodium perborate and 0.10 mg mL−1 o-dianisidine. After 10 min of incubation at room temperature, the reaction was stopped by adding 50 μL of 4 N H2SO4. Absorbance was read at 405 nm minus absorbance at 650 nm on a 96-well microplate reader.

Histology evaluations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Segments from the distal colon of each animal were removed, rinsed in saline and then fixed in 10% neutral-buffered formalin. They were embedded longitudinally in paraffin, sectioned and stained with haematoxylin/eosin. The sections were examined by light microscopy (Olympus Model BX-50 microscope, Olympus Instruments, Melville, NY, USA) and were scored by an investigator blinded to the experimental groups tested. Scoring for extent of epithelial damage was according to the following scale: 0 = intact epithelium, 1 = ≤1/3 loss or disruption of epithelium, 2 = 1/3 to 2/3 loss or disruption of epithelium, 3 = >2/3 loss or disruption of epithelium. Photomicrographs were taken with a Leitz Laborlux S microscope (Wild Leitz, Wetzlar, Germany) using a 4x objective and a SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, MI, USA).

Statistical analyses

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

One-way anova statistical analyses with Bonferroni test were performed using Graphpad Instat to evaluate differences between DSS-treated mice and DSS-treated mice given experimental compounds. Differences of P < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

Our evaluation method included scores that monitored filled colon weight and stool consistency as measures of bowel dysfunction, colon length shrinkage as an indicator of inflammation and functional changes, and a macroscopic damage evaluation that is based on gross observations of inflammation, erythema and hyperemia. This scoring method is routinely used,13,41,42 and has potential to offer mechanistic insights into the efficacy of experimental compounds. In addition to this gross score, we also examined distal colon tissue by histology for epithelial erosions and loss of architecture as a definitive end point for disease, and MPO in a tissue homogenate from a paired tissue section as an indicator of polymorphonuclear (PMN) infiltration. The latter also has mechanistic value as it addresses vascular permeability, immune cell recruitment and the contribution to tissue damage by recruited cells.

Mice receiving the DSS regimen and vehicle daily exhibited the expected macroscopic changes and increased MPO levels compared with untreated animals. Capsazepine (2.5 mg kg−1, bid, i.p.) significantly inhibited colon weight decrease, colon shrinkage (67 ± 14%), colon damage score (89 ± 10%), stool score (89 ± 10%) and total macroscopic score (80 ± 11%). There was no significant effect on MPO levels (Table 2).

Table 2.  Reductions in colitis parameters by subcutaneous administration of capsazepine (2.5 mg kg−1, sc, bid, for 7 days), in Balb/c mice on a concurrent regimen of 5% DSS (n = 10/group)
ParameterUntreated miceDSS-treated DSS + CPZ
  1. * = P < 0.05 vs DSS-treated mice by 1-way anova using Bonferroni test. %Inhibition = (1−[(DSS+drug)−untreated]/[(DSS−drug)−untreated])×100.

Colon weight (gm)0.85 ± 0.03*0.65 ± 0.070.91 ± 0.07*
Colon length (cm)9.01 ± 0.19*6.9 ± 0.288.3 ± .31*
Colon damage score0*1.1 ± 0.310.13 ± 0.11*
Stool score0*1.1 ± 0.310.13 ± 0.11*
Total macroscopic score0*6.88 ± 1.21.88 ± 0.68*
Net MPO (OD/gm wet wt.)0 ± 1.6*19.7 ± 2.014.7 ± 2.1

The experimental TRPV1 receptor antagonist, JNJ 10185734, is a synthetic small molecular weight non-capsacinoid compound. This compound was tested in vivo over a range of 0.05–10 mg kg−1 (Fig. 2) against mice given DSS to induce experimental colitis. Colon weight decrease and colon shortening were inhibited in a dose-related fashion at doses of 0.05–5.0 mg kg−1 with statistical significance obtained at 0.5 and 5.0 mg kg−1. Overall, the total macroscopic score and the epithelial damage score were significantly improved compared with vehicle treatment. Stool scores were strongly inhibited, attaining as high as 70% inhibition (P > 0.05) with 0.5 mg kg−1. Although MPO was inhibited by as much as 50% (P > 0.05), there was no observable dose-related effect regarding its inhibition. The highest dose of compound (10 mg kg−1) did not improve any of the macroscopic damage scores and in fact trended towards being less effective than lower doses.

image

Figure 2. Examination of JNJ 10185734 tested at 0.05, 0.5, 5 or 10 mg kg−1, po, bid on DSS colitis in Balb/c mice. n = 8–10 mice/group. Results are mean of results from individual animals pooled from three separate experiments in which parameters for each animal were compared against those parameters from control animals. * = P < 0.05 vs DSS – vehicle treatment by 1-way anova using Bonferroni test for significance.

Download figure to PowerPoint

Sections of the distal colon from mice dosed with this compound were examined by histology for extent of epithelial damage. The data shown in Fig. 2 for the two most active doses reveal significant inhibition of histological damage, with scores decreasing by 50 and 70% for 0.5 and 5 mg kg−1, respectively. Histology shown in Fig. 3 reveals the vehicle-treated animal had severe loss of epithelium, the compound-treated animal had a markedly intact epithelium and normalised smooth muscle architecture. The incidence of colitis, as determined by the number of mice exhibiting histological damage, was decreased by 45% after dosing with JNJ 10185734 at 0.5 mg kg−1, bid, and by 75% after dosing with 5 mg kg−1, bid (Table 3).

image

Figure 3. Prevention of epithelial damage in the distal colon from Balb/c mice administered DSS and treated with JNJ 10185734 at 5 mg kg−1, po, bid. Haematoxylin/eosin staining of fixed tissue sections embedded in paraffin. Photomicrographs were taken using a 4X objective and are each shown at identical magnifications. DSS-treated tissue demonstrates destruction of epithelium and smooth muscle changes, while this is reversed and normalized in tissue taken after treatment with JNJ 10185734.

Download figure to PowerPoint

Table 3.  Incidence of colitis after dosing with JNJ 10185734
 Number of Mice Positive for Colitis
Expt 1Expt 2Total
  1. Colitis was determined as a function of the presence or absence of epithelial damage in distal colon sections examined histologically (H&E). Within each respective experiment or for combined data, *P < 0.05 vs untreated, **P < 0.05 vs DSS, anova Student's t-test.

Untreated0/10**0/10**0/20**
DSS9/10*8/10*17/20*
DSS + 0.5 mpk JNJ 101857344/106/10*10/20*
DSS + 5 mpk JNJ 101857344/101/10**5/20**

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

These data demonstrate that the TRPV1 antagonist, capsazepine, when given systemically, can reduce the macroscopic and microscopic disease severity of DSS-induced colitis in mice. Oral administration of our experimental TRPV1 antagonist gave a similar improvement in macroscopic disease severity as capsazepine. Oral administration of this compound also resulted in dose-related improvements in the macroscopic disease score as well as the epithelial damage in the dose range 0.5–5.0 mg kg−1, but was ineffective at 10 mg kg−1. These data show for the first time that systemically and orally administered TRPV1 antagonists can ameliorate the symptoms of experimental colitis and will be discussed in the light of recent evidence that TRPV1 is involved in experimental colitis.

Subcutaneous administration of capsazepine and oral dosing of the experimental vanilloid receptor antagonist JNJ 10185734 corrected the total macroscopic score that is one measure of disease severity. Specific parameters that these agents markedly reversed were colon shrinkage and colon weight loss. Colon weight loss and shrinkage commonly occur in DSS colitis. The former is indicative of bowel dysfunction, while colon shrinkage is indicative of inflammation-related neuromuscular stimulation resulting in longitudinal muscle contractions. The reversal of these parameters by JNJ 10185734 exhibited a bell-shaped dose response. This may have been due to the compound interacting with other as yet unidentified receptors. Alternatively, the inhibition of TRPV1 activation at higher doses may result in intracellular increases in endogenous endocannabinoids such as anandamide that were prevented from binding at TRPV1 and which now are able to bind to cannabinoid receptors or other unidentified sites. This may allow actions by these higher concentrations to occur at other receptors 43 that may act in opposition to TRPV1 antagonism.

The experimental TRPV1 antagonist JNJ 10185734 reduced MPO by as much as 50%, but without statistical significance due to high variability in measurements of this parameter, and with no obvious dose relationship. Similarly, systemic capsazepine had a minimal effect on MPO levels. The latter is unlike results reported in other studies where CPZ was reported to prevent PMN cell accumulation in ileal loops in response to C. difficile toxin A32,33 or in experimental colitis 1 when administered rectally. Our data are compatible with those of Kihara et al.1 that show amelioration of colitis severity, but are different with respect to decreases in MPO expression. This discrepancy may be due to a number of factors. The MPO inhibition observed by Kihara et al.1 may reflect higher local concentrations of CPZ due to its intrarectal administration by those investigators. Similarly, toxin A-induced ileitis is a short-term response that responded to CPZ only if the agent was administered 1 h before toxin A administration.32 Therefore, the differences in the acute (toxin A) and chronic DSS-induced colitis could contribute to the different effects of capsazepine in these models. In addition, there are differences in the underlying mechanisms for each model. For example, toxin-A-induced ileitis is predominantly a substance P (SP)-dependent response,44,45 and two significant SP-mediated responses can occur at the endothelial lining; increased permeability and upregulation of ICAM-1 and VCAM-1 adhesion molecules for transendothelial PMN migration.4,46–48 However, PMN recruitment in DSS-induced colitis is not wholly dependent on SP-mediated mechanisms. Thus, recruited monocytic and lymphoid cells can secrete tumour necrosis factor (TNF) that subsequently promotes epithelial production of IL-8 49–51 to promote PMN recruitment.

Oral administration of JNJ 10185734 markedly improved the histological appearance of the epithelium, and epithelial damage scores were significantly better in distal colon tissues from treated vs control animals. Epithelial architecture was maintained and cellular infiltration was dramatically reduced. Careful examination of those tissues revealed small dense pockets of recruited cells below areas of damaged epithelium in the small numbers of tissue samples that were not completely healed, and occasionally sparse numbers of recruited cells in some healed tissues. The latter cells may not have been activated, but would nonetheless contain MPO and could explain the observation that JNJ 10185734 non-significantly attenuated MPO levels. The prevention of epithelial damage is consistent with the observation that intraperitoneal administration of the naturally occurring capsiate, nor-dihydrocapsiate, preserved epithelial integrity in DSS-induced colitis in mice.40 Together, these data suggest that a striking beneficial therapeutic effect of TRPV1 antagonists would be to prevent epithelial damage in IBD.

The expression of TRPV1 and its messenger RNA are increased in dorsal root ganglia of animal models of paw inflammation.31,43,52–54 Because TRPV1 is found in dorsal root ganglia innervating the gastrointestinal tract via sensory extrinsic neurones,30 and on neurones in the enteric nervous system (ENS),29,30,55 it is highly likely that TRPV1 antagonists reduce the disease severity of DSS-induced colitis by inhibiting these neuronal pathways. However, other sites of action may also be possible. For example, TRPV1 antagonism may result in increased availability of anandamide or other endocannabinoids that can act as cannabinoid receptor 1 (CB1) ligands.43 This possibly increased CB1 activation could lead to downstream effects on nuclear factor kappa B and TNF. In addition, increased CB1 activation in the ENS may affect bowel motility. Furthermore, increased anandamide levels may engender a feedback response on intracellular fatty acid amide hydrolase activity that may affect the balance of intracellular levels of endogenous lipids. This phenomenon has not yet been examined in the context of TRPV1 blockade. JNJ 10185734 has not been tested against other vanilloid receptors.

Diminished epithelial damage in mice dosed with TRPV1 antagonists may not only be caused by decreased PMN recruitment or activation, but may also be caused by decreases in signalling downstream of TRPV1-dependent pathways that would culminate in the release of tissue-destructive mediators. It is worth noting that an experimental SP antagonist was reported to ameliorate tissue damage in DSS colitis and other putative secretomotor aspects of bowel dysfunction.56 In that context, colon weight loss as measured in these present studies is an indirect measure of bowel dysfunction as the weight of the intact colon included faecal contents. This weight is decreased when diarrhoea occurs, is restored towards normalcy by TRPV1 antagonist treatment, and thus is potentially an indirect measure of secretomotor function.

The present body of results highlight and reinforce the role of neuronal control in inflammation by demonstrating that antagonists of a neuronal receptor, TRPV1, implicated in sensory perception were capable of moderating the symptoms and tissue damage associated with a model of experimental colitis. Preliminary data reported that TRPV1 gene knockout animals on a DSS regimen showed significantly lower disease activity scores than wild type controls as early as the second day of treatment with DSS and this improvement was maintained for the course of treatment until day 7.57 These findings are supportive of the results reported herein which indicate the potential for oral, systemically active TRPV1 antagonists in the treatment of IBD and possibly in functional bowel disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References

The authors would like to acknowledge Dr Ellen Codd, Dr Scott Dax, Dr Christopher Flores, Dr Ray Colbourn, James McNally and Michelle Jetter (Analgesics Research Team), Dr Adrienne Dubin (Neuroscience Team) and Norah Gumula (MorphoMetrics Team, Johnson and Johnson Pharmaceutical Research and Development, LLC) for their assistance, advice and support offered during the conduct of these studies, and to Dr Jeffrey Palmer (Enterology Research Team, Johnson and Johnson Pharmaceutical Research and Development, LLC) for his critical review of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Materials
  7. Induction of colitis and dosing regimens
  8. Myeloperoxidase measurement
  9. Histology evaluations
  10. Statistical analyses
  11. Results
  12. Discussion
  13. Acknowledgments
  14. References
  • 1
    Kihara N, de la Fuente SG, Fujino K, Takahashi T, Pappas TN, Mantyh CR. Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats. Gut 2003; 52: 7139.
  • 2
    Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Ann Rev Immunol 2002; 20: 495549.
  • 3
    Herbert MK, Holzer P. Neurogenic inflammation. I. Basic mechanisms, physiology and pharmacology. Anasthesiologie, Intensivmedizin, Notfallmedizin, Schmerztherapie 2002; 37: 31425.
  • 4
    Mantyh CR, Gates TS, Zimmerman RP et al. Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease. Proc Natl Acad Sci U S A 1988; 85: 32359.
  • 5
    Di Sebastiano P, Grossi L, Di Mola FF et al. SR140333, a substance P receptor antagonist, influences morphological and motor changes in rat experimental colitis. Dig Dis Sci 1999; 44: 43944.
  • 6
    Vrees MD, Pricolo VE, Potenti FM, Cao W. Abnormal motility in patients with ulcerative colitis: the role of inflammatory cytokines. Arch Surg 2002; 137: 43945; discussion 445–6.
  • 7
    Perdue MH, McKay DM. Integrative immunophysiology in the intestinal mucosa. Am J Physiol 1994; 267: G15165.
  • 8
    Al-Saffar A, Hellstrom PM. Contractile responses to natural tachykinins and selective tachykinin analogs in normal and inflamed ileal and colonic muscle. Scand J Gastroenterol 2001; 36: 48593.
  • 9
    Li M, Johnson CP, Adams MB, Sarna SK. Cholinergic and nitrergic regulation of in vivo giant migrating contractions in rat colon. Am J Physiol Gastrointest Liver Physiol 2002; 283: G54452.
  • 10
    Mayer EA, Raybould H, Koelbel C. Neuropeptides, inflammation, and motility. Dig Dis Sci 1988; 33: 71S7S.
  • 11
    Kachur JF, Keshavarzian A, Sundaresan R et al. Colitis reduces short-circuit current response to inflammatory mediators in rat colonic mucosa. Inflammation 1995; 19: 24559.
  • 12
    Blandizzi C, Fornai M, Colucci R et al. Altered prejunctional modulation of intestinal cholinergic and noradrenergic pathways by alpha2-adrenoceptors in the presence of experimental colitis. Br J Pharmacol 2003; 139: 30920.
  • 13
    Linden DR, Sharkey KA, Mawe GM. Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon. J Physiol 2003; 547: 589601.
  • 14
    Lecci A, Carini F, Tramontana M et al. Nepadutant pharmacokinetics and dose-effect relationships as tachykinin NK2 receptor antagonist are altered by intestinal inflammation in rodent models. J Pharmacol Exp Ther 2001; 299: 24754.
  • 15
    Kimura M, Masuda T, Hiwatashi N, Toyota T, Nagura H. Changes in neuropeptide-containing nerves in human colonic mucosa with inflammatory bowel disease. Pathol Int 1994; 44: 62434.
  • 16
    Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 1999; 398: 43641.
  • 17
    Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Ann Rev Neurosci 2001; 24: 487517.
  • 18
    Olah Z, Karai L, Iadarola MJ. Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1. J Biol Chem 2001; 276: 3116370.
  • 19
    Kollarik M, Undem BJ. Mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig. J Physiol 2002; 543: 591600.
  • 20
    Smart D, Gunthorpe MJ, Jerman JC et al. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 2000; 129: 22730.
  • 21
    Zygmunt PM, Petersson J, Andersson DA et al. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 1999; 400: 4527.
  • 22
    Jaggar SI, Scott HC, James IF, Rice AS. The capsaicin analogue SDZ249-665 attenuates the hyper-reflexia and referred hyperalgesia associated with inflammation of the rat urinary bladder. Pain 2001; 89: 22935.
  • 23
    Schicho R, Schemann M, Pabst MA, Holzer P, Lippe IT. Capsaicin-sensitive extrinsic afferents are involved in acid-induced activation of distinct myenteric neurons in the rat stomach. Neurogastroenterol Motil 2003; 15: 3344.
  • 24
    Szallasi A. Vanilloid (capsaicin) receptors in health and disease. Am J Clin Pathol 2002; 118: 11021.
  • 25
    Szallasi A, Szabo T, Biro T et al. Resiniferatoxin-type phorboid vanilloids display capsaicin-like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1. Br J Pharmacol 1999; 128: 42834.
  • 26
    Cortright DN, Crandall M, Sanchez JF, Zou T, Krause JE, White G. The tissue distribution and functional characterization of human VR1. Biochem Biophys Res Commun 2001; 281: 11839.
  • 27
    Stenholm E, Bongenhielm U, Ahlquist M, Fried K. VRl- and VRL-l-like immunoreactivity in normal and injured trigeminal dental primary sensory neurons of the rat. Acta Odontol Scand 2002; 60: 729.
  • 28
    Tohda C, Sasaki M, Konemura T, Sasamura T, Itoh M, Kuraishi Y. Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation-induced increase in capsaicin sensitivity. J Neurochem 2001; 76: 162835.
  • 29
    Anavi-Goffer S, McKay NG, Ashford ML, Coutts AA. Vanilloid receptor type 1-immunoreactivity is expressed by intrinsic afferent neurones in the guinea-pig myenteric plexus. Neurosci Lett 2002; 319: 537.
  • 30
    Patterson LM, Zheng H, Ward SM, Berthoud HR. Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract. Cell Tissue Res 2003; 311: 27787.
  • 31
    Yiangou Y, Facer P, Dyer NH et al. Vanilloid receptor 1 immunoreactivity in inflamed human bowel. Lancet 2001; 357: 13389.
  • 32
    McVey DC, Vigna SR. The capsaicin VR1 receptor mediates substance P release in toxin A-induced enteritis in rats. Peptides 2001; 22: 143946.
  • 33
    McVey DC, Schmid PC, Schmid HH, Vigna SR. Endocannabinoids induce ileitis in rats via the capsaicin receptor (VR1). J Pharmacol Exp Ther 2003; 304: 71322.
  • 34
    Blumberg RS, Saubermann LJ, Strober W. Animal models of mucosal inflammation and their relation to human inflammatory bowel disease [erratum appears in Curr Opin Immunol 2000; 12: 226]. Curr Opin Immunol 1999; 11: 64856.
  • 35
    Cooper HS, Murthy SNS, Shah RS, Sedergran DJ. Lab Invest. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993; 69: 23849.
  • 36
    Diaz-Granados HK, Lu J, McKay DM. Dextran sulfate sodium-induced colonic histopathology, but not altered epithelial ion transport, is reduced by inhibition of phosphodiesterase activity. Am J Pathol 2000; 156: 216977.
  • 37
    Egger B, Bajaj-Elliott M, MacDonald TT, Inglin R, Eysselein VE, Buchler MW. Characterization of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency. Digestion 2000; 62: 2408.
  • 38
    Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method of induction of reliable experimental acute and chronic colitis in mice. Gastroenterology 1990; 98: 694702.
  • 39
    Stevceva L, Pavli P, Husband AJ, Doe WF. The inflammatory infiltrate in the acute stage of the dextran sulphate sodium-induced colitis: B cell response differs depending on the percentage of DSS used to induce it. BMC Clin Pathol 2001; 1: 313.
  • 40
    Sancho R, Lucena C, Macho A et al. Immunosuppressive activity of capsaicinoids: capsiate derived from sweet peppers inhibits NF-kappaB activation and is a potent antiinflammatory compound in vivo. Eur J Immunol 2002; 32: 175363.
  • 41
    Wallace JL, McCafferty DM, Sharkey KA. Lack of beneficial effect of a tachykinin receptor antagonist in experimental colitis. Regul Pept 1998; 73: 95101.
  • 42
    Linden DR, Chen JX, Gershon MD, Sharkey KA, Mawe GM. Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol 2003; 285: G20716.
  • 43
    Mang CF, Erbelding D, Kilbinger H. Differential effects of anandamide on acetylcholine release in the guinea-pig ileum mediated via vanilloid and non-CB1 cannabinoid receptors. Br J Pharmacol 2001; 134: 1617.
  • 44
    Mantyh CR, Maggio JE, Mantyh PW, Vigna SR, Pappas TN. Increased substance P receptor expression by blood vessels and lymphoid aggregates in Clostridium difficile-induced pseudomembranous colitis. Dig Dis Sci 1996; 41: 61420.
  • 45
    Castagliuolo I, Keates AC, Qiu B et al. Increased substance P responses in dorsal root ganglia and intestinal macrophages during Clostridium difficile toxin A enteritis in rats. Proc Natl Acad Sci USA 1997; 94: 478893.
  • 46
    Evangelista S, Paoli S, Giachetti A, Manzini S. Involvement of tachykinin NK1 receptors in plasma protein extravasation induced by tachykinins in the guinea pig upper airways. Neuropeptides 1997; 31: 6570.
  • 47
    Quinlan KL, Song IS, Naik SM et al. VCAM-1 expression on human dermal microvascular endothelial cells is directly and specifically up-regulated by substance P. J Immunol 1999; 162: 165661.
  • 48
    Nakagawa N, Sano H, Iwamoto I. Substance P induces the expression of intercellular adhesion molecule-1 on vascular endothelial cells and enhances neutrophil transendothelial migration. Peptides 1995; 16: 7215.
  • 49
    Kunkel SL, Strieter RM, Chensue SW et al. Tumor necrosis factor-alpha, interleukin-8 and chemotactic cytokines. Prog Clin Biol Res 1990; 349: 43344.
  • 50
    Goh J, Baird AW, O'Keane C et al. Lipoxin A(4) and aspirin-triggered 15-epi-lipoxin A(4) antagonize TNF-alpha-stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF-alpha-induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo. J Immunol 2001; 167: 277280.
  • 51
    Beales IL, Calam J. Stimulation of IL-8 production in human gastric epithelial cells by Helicobacter pylori, IL-1beta and TNF-alpha requires tyrosine kinase activity, but not protein kinase C. Cytokine 1997; 9: 51420.
  • 52
    Amaya F, Oh-hashi K, Naruse Y et al. Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons. Brain Res 2003; 963: 1906.
  • 53
    Maggi CA, Borsini F, Santicioli P et al. Cutaneous lesions in capsaicin-pretreated rats. A trophic role of capsaicin-sensitive afferents? Naunyn Schmiedebergs Arch Pharmacol 1987; 336: 53845.
  • 54
    Renzi D, Evangelista S, Mantellini P et al. Capsaicin-induced release of neurokinin A from muscle and mucosa of gastric corpus: correlation with capsaicin-evoked release of calcitonin gene-related peptide. Neuropeptides 1991; 19: 13745.
  • 55
    Nozawa Y, Nishihara K, Yamamoto A, Nakano M, Ajioka H, Matsuura N. Distribution and characterization of vanilloid receptors in the rat stomach. Neurosci Lett 2001; 309: 336.
  • 56
    Stucchi AF, Shofer S, Leeman S et al. NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats. Am J Physiol - Gastrointest Liver Physiol 2000; 279: G1298306.
  • 57
    Fujino K, de la Fuente SG, Pappas TN, Mantyh CR. Dextran sulfate sodium-induced enterocolitis is attenuated in vanilloid receptor-1 knock out mice. Gastroenterology 2003; 124: A300.