Summary of a field trial investigating UTP08 as a treatment for EGUS in racing thoroughbreds

Robinson RS¹, Moore A², Pearson W³

¹ Selected Bioproducts Ltd., Guelph ON; ² VitaTech Laboratory Services, Mississauga ON; ³ Dept Plant Agriculture, University of Guelph, Guelph ON

 

Introduction

EGUS is recognized as a significant health concern in mature and immature horses.  It is most common among performance horses, with an incidence as high as 100% (Bell et al., 2007).  Clinical presentation of EGUS varies and is dependent on location and severity of ulcers. Signs may include poor body condition, retained hair-coat, reduced appetite, mild colic and compromised athletic performance (Bell et al., 2007). Clinical signs in foals may also include diarrhea, suppressed growth rate, grinding of teeth, a pot-bellied appearance, interrupted nursing and excessive salivation (Borrow 1993).

Bioflavonoids are increasingly recognized as potential preventives or treatments for ulcers in laboratory animals (Jung et al., 2007; Baggio et al., 2007) and humans (Ares and Outt 1998). However, the effectiveness of bioflavonoids in preventing or treating EGUS is not documented. Information on mode of action is substantively incomplete (Campos et al., 2008), and no data are available in the equine model. Information on the utility, safety and mode of action of bioflavonoids on EGUS will allow for evidence-based application of bioflavonoid-rich treatments and effectively reduce the economic and welfare impact of EGUS in performance horses. Furthermore, information on mechanism of action will allow practitioners to make educated predictions about interactions between bioflavonoids and other medications which may be co-administered.

UTP08 is an aqueous extract of 7 plants (marshmallow, meadowsweet, licorice, fenugreek, yarrow, chamomile, and slippery elm) which was developed as a bioflavonoid-rich supplement to treat EGUS in performance horses. The objectives of the current study were 1) to detect bioflavonoid species which are present in UTP08, and 2) to conduct a pilot investigation on the efficacy of UTP08 in performance horses diagnosed with EGUS. It was hypothesized that UTP08 would reduce ulcer score in horses with spontaneous EGUS.

Methods

The bioflavonoid profile of UTP08 was determined by LC/MS at the University of Guelph. A sample of UTP08 was extracted using CHCl₃ / ACN and a total ion chromatograph was detected by electrospray ionization mass spectroscopy with negative ions measured over the range of 100 – 1000 m/z. Peaks were compared to retention times and molecular mass of pure standards of bioflavonoids known to be present in the constituent herbs.

Twelve thoroughbreds in active race training with endoscopic evidence of EGUS (score ³ 1; see Table 1), as determined by a qualified veterinary practitioner (AM), received 0.4 mL (0.23 g; n=6) per kg BW UTP08 in their feed twice daily for 28 days in an open-label trial. All horses were campaigning at Woodbine Racetrack in Toronto Ontario Canada. Horses were removed from any/all ulcer medication for a minimum of 7 days prior to entry into the study, and no concurrent anti-ulcer medication was permitted for the 28 days of the trial. At the end of 28 days, all horses were fasted for 12 hours, and EGUS was scored endoscopically by the same veterinarian as conducted the baseline examinations. Ulcer score at the end of the trial was compared to that from the beginning using a paired Student t-test.

Table 1: Scoring method utilized for quantifying EGUS
Score
0	No visible ulceration
0.5	Mild gastritis
1	Mild ulceration – few small ulcers
2	Moderate ulceration - medium size ulcers on a number of sites on squamous epithelium with signs of inflammation; may be bleeding ulcers
3	Severe ulceration - large ulcers covering a large area of the squamous epithelium with severe inflammation +/- bleeding ulcers

 

Results

The bioflavonoids definitively identified in UTP08 were genistein/apigenin, kaempferol, rutin, and isoquercitin.

Upon recruitment into the field trial, the mean ulcer score of all horses was 1.8 ± 0.29. By the end of the supplementation period this fell to 0.8 ± 0.10 (p<0.001) (Figure 1).

 

 

 

 

 

 

Figure 1: Ulcer score (0 – 3) of 12 racing thoroughbreds which received 0.4 mL/kg BW (0.23 g/kg BW) UTP08 in their feed twice daily for 28 days in an open-label trial. Change in ulcer score from the beginning of the trial (Day 0) to the end (Day 28) was statistically significant (p<0.001).

 

Discussion

This study demonstrates significant benefit of feeding UTP08 to racing thoroughbreds with pre-existing gastric ulceration. In this pilot field study we utilized horses that were actively racing, as these horses are at higher risk for EGUS than their sedentary counterparts due to the influence of exercise on gastric pH coupled with gastric compression (Lorenzo-Figueras and Merritt, 2002). Gastric compression forces gastric contents to the proximal portion of the stomach, where the squamous mucosa is more susceptible to injury.  This may explain the increase in gastric ulceration of the non-glandular stomach that is associated with continual and strenuous exercise.

Conventional veterinary treatment of EGUS usually involves antacids, histamine H₂ receptor antagonists and proton pump inhibitors. Antacids are basic compounds that act to neutralize the acid within the stomach.  Their actions are short-lived, and in many cases it is difficult to administer sufficient quantity of antacid to achieve a satisfactory clinical effect (Murray and Grodinsky 1992).  These drugs may be more effective as a prophylactic rather than as a treatment.  Histamine H₂ receptor antagonists work by inhibiting the secretion of gastric acid by the parietal cells of the GI tract.  This action is effected by blocking the interaction of histamine with H₂ receptor on the parietal cells (Bruneton 1996).  Drugs in this class that have shown effectiveness in increasing gastric pH in horses include cimetidine and ranitidine (Sangiah et al., 1988).  However, there is currently a lack of research demonstrating efficacy in healing ulcers that are already present (MacAllister et al., 1994), and it is likely that these drugs are also better suited to a prophylactic role. Arguably the most effective and most widely utilized drugs for the treatment and healing of gastric ulceration in horses are proton pump inhibitors.  In particular, omeprazole is effective for healing ulcers (Murray et al., 1997), and is generally considered the gold standard against which other drugs are compared.  However, research has shown that when treatment with omeprazole is discontinued without concurrent removal of the stimulus for ulceration, horses quickly re-develop the ulceration (Andrews et al., 1999), and up to 25% of horses with EGUS do not respond to treatment with omeprazole at all (White et al. 2007).  At approximately $1200 per month per horse, this is a very expensive treatment regimen, and may be cost prohibitive for many horse owners. 

How UTP08 produced the effect seen in the current pilot study is not known, and requires further research. However, a growing body of evidence implicates plant bioflavonoids in the prevention/treatment of gastric ulceration in mammals. A specific flavan (3-methoxy-5,7,3’,4’-tetrahydroxyflavan) is as effective as omeprazole in reducing total gastric secretion, reducing the free and total acids in the gastric secretions, and reducing gastric mucosal histamine (Parmar and Hennings 1984). A related flavan, 5-methoxyflavone, reduced indomethacin-induced gastric damage in rats by up to 99% (Blank et al.1997).  Another naturally-occurring flavone, narigenin, was found to effectively prevent gastric ulceration initiated by absolute ethanol. There were fewer ulcers formed, together with increased production of gastric mucous and increased concentration of total proteins and hexosamines in gastric tissue (Motilva et al. 1994). In addition, the flavonic fraction of Bidens aurea (Martin Calero et al. 1996), Erica andevalensis (Reyes et al. 1996) and Silybum marianum (Alarcon de la Lastra et al. 1995) prevented the development of and/or augmented the healing of rat gastric mucosa after gastric challenge.

The mechanism of action of bioflavonoids in gastroprotection is not well understood.  Hypotheses include an ability of flavonoids to reduce leukocyte activation (evidenced by reduced myeloperoxidase activity) which reduces their adherence to the endothelial surface (Blank et al. 1997; Kahraman et al. 2003). Increased leukocyte adherence is associated with reduced perfusion of the mucosa, which predisposes to injury. Activation of these cells (by inflammatory mediators such as LTB₄) leads to release of oxygen-derived free radicals, which damage the endothelium and mucosa (Martin Calero et al. 1996). Myeloperoxidase is a leukocyte-specific enzyme which is used as a marker for leukocyte infiltration (Berenguer et al., 2007). Additionally, bioflavonoids inhibit the enzyme histidine decarboxylase (Middleton 1988).  Histamine, as liberated from histidine decarboxylase, stimulates gastric acid secretion (Parmar et al. 1984), and is involved with the pathogenesis of ulcers. The suppressive effect of flavonoids on tissue histamine levels has been demonstrated in several studies. Twelve flavonoids were tested for their ability to inhibit the activity of histidine decarboxylase in vitro, and several are reported effective (Wendt et al. 1980). It is also shown that 3-methoxy-5,7,3’,4’-tetrahydroxyflavan is a specific histidine decarboxylase inhibitor and effectively reduces tissue histamine in pylorus-ligated rats (Parmar et al. 1984).

Another possible mechanism of anti-ulcer action is stimulation of gastric PGE₂ formation (Choi et al. 2007; Brzozowski et al. 1998). PGE₂ stimulates production of mucous by goblet cells to protect cells from acidic injury (Akiba et al. 2000) and by promoting healing of existing ulcers through increased angiogenesis (Hatazawa et al. 2007).

The current study provides rationale for further study into UTP08 as a gastroprotective agent in horses and other performance animals. Future studies should evaluate the longterm safety of UTP08 in the target animal species, mechanistic studies of UTP08 interactions with gastric mucosa, prophylactic effect on occurrence of EGUS, and dose-optimization studies.

 

Conclusions

This open field study on the efficacy of UTP08 in treating existing EGUS in racehorses demonstrates that the product is effective in reducing gastric ulcer score to a clinically relevant degree. The data provide rationale for further investigating this product in controlled trials to determine whether it can prevent occurrence and/or severity of EGUS in exercising horses.

Acknowledgements

Financial support for this study provided by Selected Bioproducts Ltd.

 

References

Akiba Y, Guth PH, Engel E, Nastaskin I, Kaunitz JD. Dynamic regulation of mucus gel thickness in rat duodenum. Am J Physiol Gastrointest Liver Physiol. 2000 Aug;279(2):G437-47.

Alarcón de la Lastra AC, Martín MJ, Motilva V, Jiménez M, La Casa C, López A. Gastroprotection induced by silymarin, the hepatoprotective principle of Silybum marianum in ischemia-reperfusion mucosal injury: role of neutrophils. Planta Med. 1995 Apr;61(2):116-9.

Andrews FM, Sifferman RL, Bernard W, Hughes FE, Holste JE, Daurio CP, Alva R, Cox JL. Efficacy of omeprazole paste in the treatment and prevention of gastric ulcers in horses. Equine Vet J Suppl. 1999 Apr;(29):81-6.

Ares JJ, Outt PE. Gastroprotective agents for the prevention of NSAID-induced gastropathy. Curr Pharm Des. 1998 Feb;4(1):17-36.

Baggio CH, Freitas CS, Otofuji Gde M, Cipriani TR, Souza LM, Sassaki GL, Iacomini M, Marques MC, Mesia-Vela S. Flavonoid-rich fraction of Maytenus ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both H+,K+ -ATPase activity and formation of nitric oxide. J Ethnopharmacol. 2007 Sep 25;113(3):433-40.

Bell RJ, Mogg TD, Kingston JK. Equine gastric ulcer syndrome in adult horses: a review. N Z Vet J. 2007a Feb;55(1):1-12.

Berenguer B, Trabadela C, Sánchez-Fidalgo S, Quílez A, Miño P, De la Puerta R, Martín-Calero MJ. The aerial parts of Guazuma ulmifolia Lam. protect against NSAID-induced gastric lesions. J Ethnopharmacol. 2007 Nov 1;114(2):153-60.

Blank MA, Ems BL, O'Brien LM, Weisshaar PS, Ares JJ, Abel PW, McCafferty DM, Wallace JL. Flavonoid-induced gastroprotection in rats: role of blood flow and leukocyte adherence. Digestion. 1997;58(2):147-54.

Borrow HA. Duodenal perforations and gastric ulcers in foals. Vet Rec. 1993 Mar 20;132(12):297-9.

Brzozowski T, Konturek SJ, Kwiecien S, Pajdo R, Drozdowicz D, Sliwowski Z, Muramatsu M. SU-840, a novel synthetic flavonoid derivative of sophoradin, with potent gastroprotective and ulcer healing activity. J Physiol Pharmacol. 1998 Mar;49(1):83-98.

Campos DA, de Lima AF, Ribeiro SR, Silveira ER, Pessoa OD, Rao VS, Santos FA. Gastroprotective effect of a flavone from Lonchocarpus araripensis Benth. (Leguminosae) and the possible mechanism. J Pharm Pharmacol. 2008 Mar;60(3):391-7.

Choi SM, Shin JH, Kang KK, Ahn BO, Yoo M. Gastroprotective effects of DA-6034, a new flavonoid derivative, in various gastric mucosal damage models. Dig Dis Sci. 2007 Nov;52(11):3075-80.

Hatazawa R, Tanigami M, Izumi N, Kamei K, Tanaka A, Takeuchi K. Prostaglandin E2 stimulates VEGF expression in primary rat gastric fibroblasts through EP4 receptors. Inflammopharmacology. 2007 Oct;15(5):214-7.

Jung HJ, Choi J, Nam JH, Park HJ. Anti-ulcerogenic effects of the flavonoid-rich fraction from the extract of Orostachys japonicus in mice. J Med Food. 2007 Dec;10(4):702-6.

Kahraman A, Erkasap N, Köken T, Serteser M, Aktepe F, Erkasap S. The antioxidative and antihistaminic properties of quercetin in ethanol-induced gastric lesions. Toxicology. 2003 Feb 1;183(1-3):133-42.

Lorenzo-Figueras M, Merritt AM. Effects of exercise on gastric volume and pH in the proximal portion of the stomach of horses. Am J Vet Res. 2002 Nov;63(11):1481-7.

MacAllister CG, Lowrey F, Stebbins M, Newman MS, Young B. Transendoscopic electrocautery-induced gastric ulcers as a model for gastric healing studies in ponies. Equine Vet J. 1994 Mar;26(2):100-3.

Martín Calero M, La Casa C, Motilva V, López A, Alarcón de la Lastra C. Healing process induced by a flavonic fraction of Bidens aurea on chronic gastric lesion in rat. Role of angiogenesis and neutrophil inhibition. Z Naturforsch [C]. 1996 Jul-Aug;51(7-8):570-7.

Motilva V, Alarcón de la Lastra C, Martín MJ. Ulcer-protecting effects of naringenin on gastric lesions induced by ethanol in rat: role of endogenous prostaglandins. J Pharm Pharmacol. 1994 Feb;46(2):91-4.

Murray MJ, Haven ML, Eichorn ES, Zhang D, Eagleson J, Hickey GJ. Effects of omeprazole on healing of naturally-occurring gastric ulcers in thoroughbred racehorses. Equine Vet J. 1997 Nov;29(6):425-9.

Murray MJ, Grodinsky C. The effects of famotidine, ranitidine and magnesium hydroxide/aluminium hydroxide on gastric fluid pH in adult horses. Equine Vet J Suppl. 1992 Feb;(11):52-5.

Parmar NS, Hennings G, Gulati OP. Histidine decarboxylase inhibition: a novel approach towards the development of an effective and safe gastric anti-ulcer drug. Agents Actions. 1984 Dec;15(5-6):494-9.

Reyes M, Martín C, Alarcón de la Lastra C, Trujillo J, Toro MV, Ayuso MJ. Antiulcerogenicity of the flavonoid fraction from Erica andevalensis Cabezudo-Rivera. Z Naturforsch [C]. 1996 Jul-Aug;51(7-8):563-9.

Sangiah S, McAllister CC, Amouzadeh HR. Effects of cimetidine and ranitidine on basal gastric pH, free and total acid contents in horses. Res Vet Sci. 1988 Nov;45(3):291-5.

White G, McClure SR, Sifferman R, Holste JE, Fleishman C, Murray MJ, Cramer LG. Effects of short-term light to heavy exercise on gastric ulcer development in horses and efficacy of omeprazole paste in preventing gastric ulceration. J Am Vet Med Assoc. 2007 Jun 1;230(11):1680-2.