Best use of a probiotic supplement (Symprove TM )
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1 UCL School of Pharmacy You ve got to be in it to win it Best use of a probiotic supplement (Symprove TM ) Professor Simon Gaisford
2 Probiotics Probiotic market estimated to reach $46.6bn by 2020 *, but probiotic products often misunderstood or viewed with scepticism by healthcare professionals Ineffective probiotic species or strains Reduced viability of probiotics in product Product unable to deliver viable probiotics to the gut Patient use Future changing through Better formulated products and clinical data Next-generation probiotics (NGP) Live biotherapeutic products (LBP) *Nature Microbiol. 2017(2), 17057
3 Effective probiotic products Selection of probiotic species is important, but if it is not formulated correctly product will be ineffectual We started a project to Understand efficacy of a range of products See which formulation strategy was most effective Produce patient recommendations
4 Symprove TM A liquid probiotic suspension containing L. plantarum L. rhamnosus L. acidophilus E. faecium Bacteria suspended in germinated barley extract We are interested in understanding why its effective in clinical studies
5 To assess products, we considered 3 key aspects of performance ARRIVED In a live state SURVIVED Stomach acid transit THRIVED In the target area of the gut
6 1. Enumeration Test Were the claimed number of bacteria recoverable? 2. Tolerance Test Do the bacteria survive exposure to gastric Juices? 3. Real-time Growth Test Are the bacteria able to grow after exposure to gastric juices?
7 Principal findings Enumeration - All products contained viable bacteria, but only liquid probiotic products contained equal to or more than the label-claim Gastric tolerance testing Very simple in-vitro model Varied exposure time to gastric medium
8 Viable cells / log (cfu/ml) Beneficial Microbes 6 (2015) liquid products & 1 solid product Actimel Align Biobalance Biokult Probio7 Symprove VSL3 Liquid product Yakult 0 Solid products Time / min
9 Real-time growth test Difficult in real gut, so we used a novel in-vitro method Isothermal calorimetry Measurement of heat More cells produce more power
10 P (µw) Rate of heat production (or Power) 500 Exponential growth of bacteria Reduction of ph and/or exhaustion of nutrients Inoculate TIME (h)
11 P (µw) Growth of probiotics in products after 30 min exposure to gastric fluid 1000 Key: Symprove 800 Dairy Dairy Dry 600 Big Brand Dry Dry Beneficial Microbes 6 (2015) TIME (h)
12 P (µw) Growth of probiotics in products after 30 min exposure to gastric fluid 1000 Key: Symprove 800 Dairy Dairy Dry 600 Big Brand Dry Dry Beneficial Microbes 6 (2015) TIME (h)
13 Recommendations on patient use These findings give guidance for patients thinking of using a probiotic 1. Choose the product carefully Liquid products seem more resistant to gastric fluid that solids Lactobacilli seem the most tolerant bacteria 2. Take the product in the fasted state to minimise gastric residence time Don t take with meals and/or hot drinks
14 Better IVIVC Calorimetry model is quite simple wanted a more realistic in-vitro model SHIME Simulator of the Human Intestinal Microbial Ecosystem
15 SHIME testing 4 tests carried out Symprove TM (two doses) Symprove TM plus an adherent invasive E. coli strain (AIEC) AIEC plus broad spectrum antibiotic (ciprofloxacin) Aim to show Survival of Symprove TM bacteria in the fasted upper gut Metabolic activity and growth of Symprove TM bacteria in colon Antipathogenic activity against AIEC Effect (stimulatory or not) on colonic microbiota
16 ph Upper GIT survival Test product Stomach conditions Small intestinal conditions Viable bacteria enumerated on agar plates Shaker Temperature and ph-controlled incubation reactor Total (viable and non-viable) bacteria determined with flow cytometry ph-profile fasted conditions 8,5 7,5 6,5 5,5 4,5 3,5 2,5 1,5 Start SI SI2 SI3 SI1 ST0 ST Time (min)
17 Log Total viable bacterial counts Log CFU CFU in UPPER GIT on MRS-plates The bacteria in Symprove TM remained in a viable and culturable and hence metabolic active state upon storage ST0 ST45 SI1 SI2 SI3 Average 10,10 10,22 10,19 10,21 10,12 12 Total viable bacterial counts in UPPER GIT Bacteria did not lose culturability upon upper GIT passage Bacteria remained viable upon upper GIT passage * Bacteria enter the colon in a metabolic active state and at their concentration of dosage 6 ST0 ST45 SI1 SI2 SI3 Average 10,39 10,39 10,34 10,33 10,32
18 Concentration (mm) Log (16S copies/ml) Lactobacillus a b a b No detectable numbers of lactobacilli present in colonic microbiota and during antibiotic treatment 4 T0 T24 T48 neg ctrl < 0,00 LOQ < 0,00 LOQ < 0,00 LOQ pos ctrl < 0,00 LOQ < 0,00 LOQ < 0,00 LOQ dose 1 < LOQ 0,00 8,84 8,76 dose 2 < LOQ 0,00 9,11 9, Lactate a b c d a,b a b d 0 T24 T48 Neg control 1,34 0,83 Pos control 2,00 1,33 Dose 1 8,52 0,45 Dose 2 9,88 0,00 Addition of Symprove TM resulted in an increased concentration of lactobacilli in dose-dependent fashion Addition of Symprove TM resulted in an increased concentration of lactate in dose-dependent fashion Lactate was subjected to crossfeeding interactions
19 Treatment of AIEC Addition of antibiotic resulted in major reduction in AIEC Addition of Symprove TM resulted in significantly lower concentrations of AIEC at the end of the incubations in a dosedependent fashion The highest dose of Symprove TM resulted in a reduction of AIEC up to 87% after 48h of incubation
20 Log (16S copies/ml) Influence on acetate-producing colonic bacteria Bifidobacterium a b a c a b a c Antibiotic treatment prevented outgrowth 6 4 T0 T24 T48 neg ctrl 6,76 8,95 8,53 pos ctrl 6,76 6,54 6,46 dose 1 6,76 9,09 8,63 dose 2 6,76 9,27 8,98 Addition of highest Symprove TM dose resulted in significantly higher numbers at the end of the incubations Antibiotic treatment prevented acetate production Addition of highest Symprove TM dose resulted in significantly higher concentrations at the end of the incubations
21 Log (16S copies/ml) Influence on propionate-producing colonic bacteria Veillonellaceae a 8 a b 6 4 T0 T24 T48 neg ctrl < 0,00 LOQ < 0,00 LOQ < LOQ 0,00 pos ctrl < 0,00 LOQ < 0,00 LOQ < LOQ 0,00 dose 1 < 0,00 LOQ 7,00 8,42 dose 2 < 0,00 LOQ 6,68 8,61 b No detectable numbers of Veillonellaceae present in colonic microbiota and during antibiotic treatment Addition of Symprove TM resulted in an increased concentration of Veillonellaceae in dose-dependent fashion Antibiotic treatment prevented propionate production Addition of Symprove TM resulted in an increased concentration of propionate Conversion of lactate into propionate
22 Log (16S copies/ml) Log (16S copies/ml) Influence on butyrate-producing colonic bacteria Clostridium coccoides/eubacterium rectale Faecalibacterium prausnitzii a b c d a b b 10 8 a a a a a a a a T0 T24 T48 neg ctrl 5,48 7,20 7,60 pos ctrl 5,48 5,34 0,00 dose 1 5,48 7,91 8,06 dose 2 5,48 7,75 8,04 4 T0 T24 T48 neg ctrl 5,72 6,11 5,67 pos ctrl 5,72 5,95 5,86 dose 1 5,72 5,79 5,79 dose 2 5,72 5,81 5,52 Addition of Symprove TM resulted in an increased concentration of Clostridium coccoides but no effect on Faecalibacterium prausnitzii Addition of Symprove TM resulted in an increased concentration of butyrate Conversion of lactate to butyrate
23 Take-home points Survival of Symprove TM bacteria through upper GIT? YES Metabolic activity of Symprove TM bacteria in the colon? YES Anti-pathogenic activity against a number of species? YES Anti-pathogenic activity in the colon? YES Negative effect on colonic microbiota? NO Stimulatory effect on colonic microbiota? YES Stimulatory effect on SCFA production? YES Proposed mechanism of action production of lactic acid reduces ph and retards pathogen. Other bacterial genera then metabolise lactic acid to normalise ph and flourish
24 Thanks to Dr Cornelius Dodoo Dr Mansa Fredua-Agyeman Dr Jawal Said Vivienne Clark Barry and the Symprove Dr Frédéric Moens, Dr Pieter Van den Abbeele and the Prodigest team
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