Protein hydrolysates: Do they really work ? Vincent C Biourge, DVM PhD Dipl ACVN & ECVCN Royal Canin, Centre de Recherche, Aimargues

Adverse Reactions to Food Dermatologic: pruritus, alopecia,otitis, .. Gastrointestinal: vomiting, abd. discomfort Respiratory: asthma, rhinitis, ... General: headache, arthritis, ... Food Allergy Food intolerance Immune mediated (IgE, cell mediated) Non immune mediated (Lactose, chocolate, bioamines, additives, …) Probably the main cause (Olivry T, J Vet derm 2010)

Adverse Reactions to Food Diagnosis Challenge Dr Thierry Ximenes (France) Double blinded Clin. signs Elimination diet Clinical signs 4 to 12 Weeks Novel ingredients + Serum IgE + Skin test Improvement of the clinical signs

Elimination diets Definition Gold standard Diets composed of ingredients to which the dog has not been exposed. Dietary history Home made Starch sources: Rice, Potato, Tapioca Protein sources: Lamb, Horse, Fish, Turkey, Venison, .. Essential fatty acids, Minerals and Vitamins Commercial Lamb & Rice, Fish & Potatoes, Venison & Potatoes Over the counter Not hypoallergenic ! Intact protein sources ! Sustainability Fish, Rabbit, Duck, Venison, … Raditic et al, JAPAN 2010

A new strategy to treat adverse reaction to food Protein hydrolysates 2001 A new strategy to treat adverse reaction to food Enzymatic Hydrolysis protein polypeptides

Benefits of hydrolysate Easier to digest Short half-life in the lumen Smaller peptides Molecular weight < 16 Kd Cave, 2006

Protein hydrolysates Do they really work ? What is the background ? Should molecular weight be as low as possible ? Are they trully hypoallergenic ? Can they be qualified as allergen free ? Are their efficacies substantiated by clinical studies ?

Elisa immunoreactivity of soy protein depending of its source Background Elisa immunoreactivity of soy protein depending of its source LaMartin.com Adverse reaction to soy in milk replacer Poor appetite and growth Malabsorption/maldigestion – Diarrhea Soy antibodies Villus atrophy Lallès, 1995 Soy flour Soy concentrate Soy hydrolyzate

Protein hydrolysate based milk replacer. Background Protein hydrolysate based milk replacer. http://www.eecs.harvard.edu/~eli/pictures/ Baby allergy to cow’s milk Vomiting diarrhea Atopic dermatitis/ Urticaria Asthma/Rhinitis Babies at risk for allergy  Risk of cow milk allergy

Molecular weight Dalton Arbitrary unit of atomic mass Sir John Dalton, Founder of the atomic theory 1 Dalton (d) = Weight of 1/12 nuclide of 12C 1.657 10-24 gramme Glycin = 75 d - Tryptophan = 204 d Casein = 19 000 - 25 000 d or 19-25 Kd Measurement: electrophoresis -chromatography Human serum albumin = 69 Kd

Molecular weight Mean MW Mean Max Ingredients Vs diets Cave N, 2006

Molecular weight Most common food allergens Glycoproteins Protein with glucide moiete ? Lipids (Bacterial glycolipids) ? Carbohydrates Water - soluble Heat and acid resistants Molecular weight 10 to 40 Kd Small enough to pass the intestinal wall Large enough to induce immune reaction Antigens in selected food in man Cave N, 2006 Looringh van Beeck FA, 2009 Sampson HA, 1993

Highly reduced allergy Molecular weight Molecular weight Less common allergy > 40 kDa Most common 10- 40 kDa Reduced allergy 3 – 10 kDa Guilford GW ,1996 Serra et al, 2006 Highly reduced allergy 1– 3 kDa ER: But we are targeting no allergies. Studies of children have established that only amino-acid-based and extensively hydrolysed formulas might be considered truly hypoallergenic (Hill, 2007) and Diets lower than 1kDa will ensure the greatest chance of reducing allergic reaction (Cave 2006) Olivry T, 2010 Cave N, 2006 No allergy < 1 kDa

Molecular weight Antigenicity Epitope Few Many Close in 3D Hidden Gastroscopic score diameter Olson ME et al 2000 Epitope

Molecular weight Antigenicity AJVR 2006;67:1895-1900

Molecular weight Digestibility Soy source and in vitro N digestibility Soy source and aa ileal digestibility in calfs Lallès, 1995 Royal Canin, 2004 Cave NJ, Marks SL. Evaluation of the immunogenicity of dietary proteins in cats and the influence of the canning process. AJVR 2004; 10,1427-1433

Molecular weight Cut-off « The number of IgE binding sites on the allergen, their location, and the tertiary shape of the protein are probably more important than the molecular weight » S.F. Hefle, 1996 Source of protein (Casein Vs Soy) Process - type of hydrolysis -exposure of epitopes Problem measuring MW on finished product

In vitro testing - ELISA Immunogenicity In vitro testing - ELISA Hannah, 1997

Immunogenicity Western blot native hydrolysed AJVR 2006; 67:484-488

Immunogenicity Skin test Puigdemont et al, 2006   % of reduction of the wheal areas for soy hydrolysed versus native soy protein Soy protein concentration Dog 1 g/ml 10 g/ml 100 g/ml 1T2 53,8 45,7 42,0 1T3 53,5 54,6 52.6 2T2 95,6 18,4 44,6 2T6 81,9 61 86,4 3T2 59,7 58,4 37,4 3T4 47 80,1 57,6 Mean  SD 65,3  19,1 53,0  20,4 53,4  17,7 Puigdemont et al, 2006 Control showed no wheal on both challenges

Immunogenicity Clinical reactions Dogs Native soy protein Hydrolysed specific IgE 8204 Vomits Diarrhoea (1) Pruritus NR +++ 8212 Diarrhoea (2) 8216 ++ Control and 3 other sensitised dogs showed no adverse reactions Puigdemont et al ,2006

Immunogenicity Clinical reactions Jackson et al, 2003 Cutaneous Score (Max 35*3*3) Score max = 315: 3 types de lésions (erythema, excoriation, infection) gradé de 0 à 3. Pas de réaction avec le corn starch No corn and starch diet Corn starch Corn Soy Soy hydrolysate diet 200 mg/kg bw 14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn Jackson et al, 2003

Immunogenicity Stomach reactivity Gastroscopic score diameter Olson ME et al 2000

Immunogenicity Conclusion Hydrolyzed proteins are less antigenic than intact proteins. Hydrolyzed proteins are not anallergenic except if hydrolyzed to single or few amino acids. High digestibility is an important factor in reducing antigenicity.

Clinical trials Shown efficacy Managing adverse reaction to food. Diagnosis of adverse reaction to food. Inflammatory bowel disease. Exocrine pancreatic insufficiency.

Adverse reaction to food Clinical reactions Cutaneous Clinical Score No corn and starch diet Corn starch Corn Soy Soy hydrolysate diet 200 mg/kg bw 14 maltese x Beagle dogs with known clinical hypersensitivity to soy and corn 3/14 dogs increased scores on hydrolyzed diet. Jackson et al, 2003

Adverse reaction to food Evaluation on dogs with demonstrated adverse reactions to food 12 dogs ( breeds, sex, food allergy ) Controlled allergy (no clinical signs) Clinical exam @ 0 and 2 months (CADESI) Owners requested to report: Pruritus, abnormal behavior of the dog Digestive tolerance Palatibility None of the 12 dogs relapsed Perfect tolerance J. Fontaine, CNVSPA 2001

Diagnosis of ARF Challenge Clin. signs Soy hydrolysate diet 2 dermatology specialty pratices J Fontaine (Brussels, B), M Vroom (Oisterwijk, NL) Inclusion in the study: Suspicion of skin hypersensitivity J Nutr2002;134:2062S-2064S Challenge 8 Weeks Clin. signs Soy hydrolysate diet Clinical signs Recovery  challenge +  Adverse Reaction to Food (ARF) 2. Marked improvement  challenge +  ARF + atopy 3. Little or no improvement  other elimination diet  Recovery 4. Little or no improvement other elimination diet  no improvement  ? atopy  No corticotherapy VC Biourge, J Fontaine, MW Vroom, 2004

20 16 22 Diagnosis of ARF Atopy Adverse Reaction to Food 60 dogs included 31 M-3 MC - 13 F - 13FS Age 4.5±0.4 yrs (3 mo – 11 yrs) 26 breeds German Sheperd (10), Bouledogue Français (2), Bouvier des Flandres (2), Boxer(5), English Cocker Spaniel (2), Golden Retriever (4), Jack Russel (2), Labrador (4), Shar Pei (3), Shi Tsu (2), WHWT (5), … Duration of the clinical signs 2.6±0.4 yrs (3 weeks – 10 yrs) 20 16 22 Atopy Adverse Reaction to Food + 2 cases excluded

Adverse reaction to food Diagnosis of ARF Adverse reaction to food 20 dogs 9 M-1 MC - 6 F – 4 FS Age 3.8±0.6 yrs (6 mo – 9yrs) No more pruritus No or very mild clinical signs left. Challenge + 18/20 responded to soy hydrolysate diet Rabbit and Rice Homemade soy diet ***

Diagnosis of ARF Before After Golden Retriever Male, 4.5 yrs, 29.6 kg Golden retriver, mâle, 29,6 kg :   J 0 : Prurit chronique intense et généralisé (initialement à l’abdomen), avec une évolution en alopécie, lichénification-hyperpigmentation et corticosensible seulement à l’apparition des symptômes de l’allergie. J 30 : Reduction du prurit et des lésions cutanées spécialement au niveau de l’abdomen et des yeux. J 60 : Prurit résiduel. Les lésions cutanées sont acceptables. Confirmation de l’allergie alimentaire : réapparition des symptômes suite à la réintroduction de l’ancienne alimentation. Golden Retriever Male, 4.5 yrs, 29.6 kg Generalized intense prurit with lichenification, hyperpigmentation Before After

Adverse reaction to food & Atopy Diagnosis of ARF Adverse reaction to food & Atopy 16 dogs 8 M-1 MC - 2 F – 5 FS Age 5.7±0.7 yrs (3 mo – 11yrs) Pruritus marketly improved Mild to moderate clinical signs left. Challenge + All dogs responded to the soy hydrolysate diet. ***

Diagnosis of ARF Atopy 22 dogs 13 M-1 MC - 5 F – 4 FS Age 4.5±0.6 yrs (1.2 – 11yrs) No or little improvement of pruritus, clinical signs. No response to other elimination diets and + to skin test

ARF: response to the hydrolysate Diagnosis of ARF ARF: response to the hydrolysate 94.4 % of ARF dogs responded to the soy hydrolysate based diet

Diagnosis of ARF 15 20 47 10 11 38 Other study Loeffler et al., Vet Derm 2006;17:273-279 Chicken hydrolysate diet Vs homade diets 181 dogs 17 dogs excluded 35 allergic skin disease Poultry hydrolyzate (109) Homade (72) Excluded b/c did not receive exclusively the food PH 13 – HD 4, did not eat the food: PH 5 – HD 4, needed glucocorticoïds PH 1 – HD 3, Diarrhea PH 1 – HD 1, unrelated reason = 2 15 20 47 10 No significant difference 11 38 ARF Atopy ARF Atopy Excluded: 27 Excluded: 13

1 dog showed severe signs when fed hydrolyzed chicken Veterinary Dermatology,2010,21:358-366 12 dogs selected and divided In 2 groups During PH1, 25 of 26 dogs showed an increased CADESI score after chicken meat once daily for 2 weeks. Twelve subjects, falling into classes 3 and 4, were selected for PH2 of the study. Prior to starting PH2, one dog devel- oped unrelated problems and was substituted with a class 2 dog. Gastrointestinal disturbances were not recor- ded during PH1 or PH2. The average CADESI score (LSMEANS) calculated during the entire study was not influenced by the group. Dogs displayed the most prominent clinical response while eating whole chicken, whereas no significant differences were detected between PH2.1 and PH2.2 (PH1:10.32 versus PH2.1:4.30 versus PH2.2:2.69, P < 0.001; Figure 2). 1 dog showed severe signs when fed hydrolyzed chicken

Diagnosis of ARF Cats After Before Dr Aranda Golden retriver, mâle, 29,6 kg :   J 0 : Prurit chronique intense et généralisé (initialement à l’abdomen), avec une évolution en alopécie, lichénification-hyperpigmentation et corticosensible seulement à l’apparition des symptômes de l’allergie. J 30 : Reduction du prurit et des lésions cutanées spécialement au niveau de l’abdomen et des yeux. J 60 : Prurit résiduel. Les lésions cutanées sont acceptables. Confirmation de l’allergie alimentaire : réapparition des symptômes suite à la réintroduction de l’ancienne alimentation. After Before Dr Aranda

ARF: response to the hydrolysate Diagnosis of ARF ARF: response to the hydrolysate 87.5 % of ARF cats responded to the soy hydrolysate based diet MW Vroom, C. Swinnen, A clinical study of a soy protein isolate hydrolysate diet, in dogs and cats with adverse reactions to food. Proc. of Voorjaarsdagen 2002. 252

Food responsive chronic diarrhea

Food responsive chronic diarrhea Dogs: 26 dogs over a 2 yr-period. 24/26 dogs with IBD 4 IF, 10 SF, 8 IM, 4 CM. Age: 4.3 ± 3.3 yrs (0.6- 11 yrs) Weight 23 ± 12 kg (4.7- 40 kg) Duration of the clinical signs: 1 to 36 months before presentation. Treatments before inclusion: Antibiotics (7), Metaclopramide (6), cimitidine (6) , Prednisolone (4), sulfazalazine (3). Diets before inclusion: Low residue intestinal diets (7), novel protein diets including homemade (12), other diets (7). Mandigers et al., 2010 Objectives To compare the response of dogs with chronic diarrhea on soy hydrolysate Vs intestinal diet.

Food responsive chronic diarrhea Clinical signs after 2 months Weight gain * 11% 12.5% No more clinical signs 23/26 dogs 3 last dogs improved but vomiting and diarrhea persisted

Food responsive chronic diarrhea Follow-up after median 6 months, range 3-15 mo. 15/16 test dogs – 6/7 control dogs 67% 13% 87 % of dogs on hydrolyzate diet remained free of clinical signs on follow-up ! The signs were minor in the remaining 2 dogs.

Food responsive chronic diarrhea Similar findings by other authors. Marks SL, Laflamme D, McCandlish A. Dietary trial using a commercial hypoallergenic diet containing hydrolyzed protein for dogs with IBD. Vet Ther 2002; 3:109-18. Similar finding in cats 8 cats Chronic diarrhea (4-36 Mo) 1 Colitis, 2 gastritis 6 IBD Soy hydrolyzate based diet Resolution of clinical signs within 4-8 d Median weigh gain 0.75 kg within 2 Mo 11Mo follow-up 6/8 cats free of clinical signs

Exocrine pancreatic Insufficiency J Nutr2002;134:2166S-2068S German Shepherds EPI and skin disease GI signs controlled within 7 d Weight gain with 2 months Skin within 3 months

Protein hydrolysates Conclusion Hydrolyzed proteins are less antigenic than intact proteins. Molecular weight (except if extremly low (<1Kd) is a poor predictor of protein immunogenicity. Hydrolyzed protein based diet are not anallergenic. Hydrolyzed proteins are sustainable. Clinical studies to support benefits in: Diagnosis and management of ARF Idiopathic chronic diarrhea - IBD Exocrine pancreatic insufficiency ? Perianal fistula.

If you want to know more … www.ivis.org Obrigado …