1. Vaginal dysbiosis and its correction with vaginal probiotic Neoprobio
XXXII Kongres Polskiego Towarzystwa Ginekologicznego
Łodz, September 3, 2015
Vaginal dysbiosis and its correction with vaginal probiotic Neoprobio
Taras Lyaskovsky
PhD, Laboratory of Lactic Acid Bacteria
Zabolotny Institute of Microbiology and Virology
National Academy of Sciences, Kiev, Ukraine

2. Zabolotny Institute of Microbiology and Virology of National
Academy of Sciences of Ukraine
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

3. Zabolotny Institute of Microbiology and Virology of National
Academy of Sciences of Ukraine
Institute is the main scientific organization in Ukraine in the field of microbiology and virology research.
17 departments and labs
State depositary of non-pathogenic microorganism
Accredited laboratory in the sphere of control of biological products in Ukraine
Publish “Microbiological Journal”
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

4. Zabolotny Institute of Microbiology and Virology of National
Academy of Sciences of Ukraine
Department of Physiology of Industrial Microorganisms
Department of antibiotics
Department of General and soil microbiology
Department of Biochemistry microorganisms
Department of phytopathogenic bacteria
Department of Biology extremal microorganisms
Department of Physiology and Systematics of micromycetes
Department of Genetics of microorganisms
Department of phytopathogenic viruses
Department of microbiological processes on solid surfaces
Department mycoplasmology
Department of Molecular Genetics of Bacteriophages
Division problems interferon and immunomodulators
Department of Innovation and Technology Transfer
Laboratory of secondary metabolites micromycetes
Laboratory reproduction of viruses
Laboratory biological polymer compounds
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

5. Bacterial Vaginosis is the most common vaginal infection in women ages 15-44.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

6. Most women found to have BV (84%) reported no symptoms.
Bacterial Vaginosis is the most common cause of vaginal symptoms among women.
The prevalence in USA is estimated to be 21.2 million (29.2%) among women ages 14–49.
Most women found to have BV (84%) reported no symptoms.
Women who have not had sex can still be affected by BV (18.8%), as can pregnant women (25%), and women who have ever been pregnant (31.7%).
Prevalence of BV increases based on lifetime number of sexual partners.
Source: Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J, Sutton M, Markowitz LE. The prevalence of bacterial vaginosis in the United States, ; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis Nov;34(11):864-9.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

7. What is Bacterial Vaginosis?
BV is a polymicrobial clinical syndrome resulting from replacement of the normal hydrogen peroxide producing Lactobacillus sp. in the vagina with high concentrations of anaerobic bacteria (e.g., Prevotella sp. and Mobiluncus sp.), G. vaginalis, Ureaplasma, Mycoplasma, and numerous fastidious or uncultivated anaerobes.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

8. Factors promoting Bacterial Vaginosis
uncotrollable drugs administration – mainly antibiotic
frequent use of contraceptive products
changes of hormonal status at menstrual cycle
stress
travelling
suppression of immune system
inflammation processes
other
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

9. Composition of the vaginal microbiota in women of reproductive age
Genetic analysis with the use of 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays for:
• healthy women
• intermediate
• Bacterial Vaginosis
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

10. Healthy women
Microorganism %
Lactobacillus iners
38.1
Lactobacillus crispatus
43.2
Other Lactobacillus species
5.4
Gardnerella vaginalis
7.2
Atopobium vaginae
0.6
Eggerthella
0.001
Prevotella
0.2
BVAB 1
0.02
BVAB 2
0.01
Finegoldia magna
0.1
Megasphaera type 1
Megasphaera type 2
Sneathia sanguegens
Leptotrichia amnionii
BVAB-NV7
0.002
Mobiluncus curtisii/mulieris
Other non-Lactobacillus species
4.8
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

11. Intermediate
Microorganism %
Lactobacillus iners
43.9
Lactobacillus crispatus
1.2
Other Lactobacillus species
6.1
Gardnerella vaginalis
30.5
Atopobium vaginae
1.5
Eggerthella
0.2
Prevotella
0.9
BVAB 1
BVAB 2
0.5
Finegoldia magna
0.1
Megasphaera type 1
4.2
Megasphaera type 2
0.001
Sneathia sanguegens
Leptotrichia amnionii
2.4
BVAB-NV7
Mobiluncus curtisii/mulieris
0.01
Other non-Lactobacillus species
8.3
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

12. Bacterial Vaginosis (BV)
Microorganism %
Lactobacillus iners
6.6
Lactobacillus crispatus
0.3
Other Lactobacillus species
Gardnerella vaginalis
29.1
Atopobium vaginae
7.1
Eggerthella
1.2
Prevotella
13.2
BVAB 1
6.1
BVAB 2
6.3
Finegoldia magna
0.1
Megasphaera type 1
9.8
Megasphaera type 2
Sneathia sanguegens
4.3
Leptotrichia amnionii
6.8
BVAB-NV7
0.8
Mobiluncus curtisii/mulieris
0.2
Other non-Lactobacillus species
7.9
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

13. Probiotic strain screening
Report of a Joint FAO/WHO Working Group on
Drafting Guidelines for the Evaluation
of Probiotics in Food London Ontario, Canada
April 30 and May 1, 2002
Food and Agriculture Organization of the United Nations & World Health Organization
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

14. EVALUATION OF PROBIOTICS
Strain identification
Strain identification by phenotype (API test systems)
Strain identification by genotype (sequencing)
Functional characterization
In vitro tests
Animal studies
Safety assessment
In vitro and/or animal
Phase 1 human study
Clinical trials (Phase II (DBPC), Phase III (effectiveness in comparing with standard treatment)
Deposit strain in IMV culture collection
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

15. PROBIOTIC SCREENING STRAIN IDENTIFICATION • Study of phenotype characteristics of LAB • Study of genotype characteristics of LAB
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

16. PHENOTYPE CHARACTERISTICS
Colony: size, surface, color
Cells: form, size, positioning,
Motion
Fermentation of 49 carbohydrate substrates (API 50 CH)
Gas production from glucose, NH3 from arginine
Higrolitic activity: gelatin, eskuline
Acidification and the growth
in milk
Growth on different media (substrates)
6,5% NaCl; pH 9,6; 40% bile
Growth at temperatures 150С, 300С, 370С, 450С, 500С
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

17. GENOTYPE CHARACTERISTICS
Electrophoregram of amplification products with the use of genus specific primers
The result of the sequence of amplification products of DNA fragment of the strain L. plantarum 200 Д with primers Lp3_sp and Lp2 ( 543 b.p. fregment)
[GGC TGC TGG CAC GTA GTT AGC CGT GGC TTT CTG GTT AAA TAC CGT CAA TAC CTG ААС AGT TAC TCT CAG ATA TGT TCT TCT TTA АСА АСА GAG TTT TAC GAG CCG AAA CCC TTC TTC ACT CAC GCG GCG TTG CTC CAT CAG ACT TTC GTC CAT TGT GGA AGA TTC CCT ACT GCT GCC TCC CGT AGG AGT TTG GGC CGT GTC TCA GTC CCA ATG TGG CCG ATT ACC CTC TCA GGT CGG СТА CGT ATC ATT GCC ATG GTG AGC CGT TAC CCC ACC ATC TAG СТА ATA CGC CGC GGG ACC ATC CAA AAG TGA TAG CCG AAG CCA TCT TTC AAG CTC GGA CCA TGC GGT CCA AGT TGT TAC GCG GTA TTA GCA TCT GTT TCC AGG TGT TAT CCC CCG CTT CTG GGC AGG TTT CCC ACG TGT TAC TCA CCA GTT CGC CAC TCA CTC AAA TGT AAA TCA TGA TGC AAG CAC CAA TCA ATA CCA GAG TTC GTT CGA CTT GCA TGT ATT AGG CAC GCC GCC AGC GTT CGT CCT GAG CCA GGA TCA ААС TCT ]
99% identical to gene 16S рРНК of L. plantarum, presented in GenBank.
Gel picture showing the PCR amplified product of probiotic strains of lactic acid bacteria :
М- marker, 1- E.durans Sm, 2-E.faecium 77Д, 3- Str.thermophilus S8, 4- L.plantarum 200 Д
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

18. PROBIOTIC STRAIN SCREENING FUNCTIONAL CHARACTERIZATION • Adhesion Activity • Antagonistic Activity • Lysozyme Synthesis • Vitamin Synthesis • Interferon Induction
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

19. ADHESION ACTIVITY
Adhesion activity is studied in vitro on the model of pig enterocytes
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

20. LACTIC ACID BACTERIA ADHESION TO EPITHELIUM OF DIFFERENT BIOTOPES OF HUMAN ORGANISM
BUCCAL
VAGINAL
INTESTINAL
x 4000
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

21. ANTAGONISTIC ACTIVITY
Bacillus cereus ATCC 11778
Escherichia coli ATCC 25922
Staph. epidermitis ATCC 12228
Pseudomonas aeruginosa ATCC 9027
Pribiotic strains
of LAB
Candida albicans ATCC
Proteus vulgaris ATCC 6896
Klebsiella pneumonia ATCC 10031
Staphylococcus aureus
ATCC 12228
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

22. ANTAGONISTIC ACTIVITY SYNERGISM OF PROBIOTIC LAB+ 1 2 3 4 5 6 7 8 9 10
L. plantarum 200D 1 2 3 4 5 6 7 8 9 10
Pathogens growth inhibition zones, mm
E.durance Sm
Bacillus cereus
Escherichia coli
Proteus vulgaris
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

23. ANTAGONISTIC ACTIVITY
Growth inhibition of gas producing putrefactive microflora by LAB probiotic strains
TEST (Growth inhibition by LAB )
CONTROL
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

24. ANTAGONISTIC ACTIVITY
Growth inhibition of gas producing putrefactive microflora by LAB probiotic strains
Number of gas producing putrefactive microorganisms, %
Control
Physiologic solution
L.plantarum 200D
E.durance Sm
Blend of
strains
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

25. Micrococcus lysodeikticus lysozyme assay
LYSOZYME SYNTHESIS
Lyses of micrococcus zone diameter, mm
Micrococcus lysodeikticus lysozyme assay
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

26. VITAMIN SYNTHESIS L. plantartum 3 L. plantartum 45 L. plantartum 200D
B5 (NICOTINAMIDE)
B2 (RIBOFLAVIN)
H (BIOTIN)
B6 (PYRIDOXINE)
μg/ml
B1 (THIAMINE)
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

27. INTERFERON INDUCTION BY LACTIC ACID BACTERIA IN EXPERIMENTS IN VITRO
Cultures
Interferon activity INF AU/ml on media
№5 (bean broth)
№7 (enzyme lysate cornmeal)
Streptococcus thermophilus Sn
320
E.durance Sm
1280
Str. thermophilus S24
640
Str. thermophilus S8
160
E. faecium 77D 80
Lactobacillus plantarum 195D
L plantarum 200D
L. acidophilus 4356
Суміш: Sn +Sm + 200D
Полі {І}: полі {Ц}
2560
Контроль середовища
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

28. OPTIMISED BLEND OF LAB Lactobacillus plantarum 200D
Bacterial vaginoses
Enterococcus faecium 77D
Neoprobio
Enterococcus durance Sm
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

29. OPTIMISED BLEND OF LAB
High adhesion activity (provide fast colonization of vaginal canal)
High antagonistic activity (provide inhibition of growth of pathogenic microflora)
Bacterial vaginoses
Neoprobio
Synthesis of biologically active substances (H2O2, lysozyme, vitamins
Interferon induction
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

30. Lactic acid bacteria blend Lactose Ascorbic acid Folic acid Hard fat
UNIQUE COMPOSITION
Lactic acid bacteria blend
Lactose
Ascorbic acid
Folic acid
Hard fat
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

31. FUNCTION OF EACH INGREDIENT
LACTIC ACID BACTERIA (LAB) BLEND
LACTOSE
ASCORBIC ACID
FOLIC ACID
HARD FAT
Probiotic properties of the product
Starting and protective ingredient for growth of lactic acid bacteria
Provide acidic conditions which create more favorable condition for the growth of LAB then other BV microflora
Provide acidic conditions and makes positive effect on vaginal epithelium regeneration.
Base of the suppository and protective media for LAB. Slow melting of the suppository provide controlled release of LAB and effectively colonies the vaginal canal by LAB
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

32. CLINICAL TRIALS OF VAGINAL OVULES WITH PROBIOTIC LAB BLEND
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

33. CLINICAL TRIALS
Name of the study Clinical studies of efficacy and tolerability of XXX (vaginal suppositories with probiotic lactic acid bacteria) in treatment of patients with vaginal disbacteriosis (Bacterial Vaginosis). 
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

34. CLINICAL TRIALS
Clinical base:
Ministry of Health of Ukraine, Shupik National Medical Academy of post-graduate education Department of Obstetrics, Gynecology and Reproduction, Hospital No 4
Investigation Product:
XXX (vaginal suppositories with probiotic lactic acid bacteria)
Type of study:
Under restricted program
Study design:
Open, comparative, controlled, randomized study
Study methods:
Clinical study. General laboratory analysis, cytological, bacterioscopic, bacteriological analysis of vagina discharge
Patients:
Vaginal disbacteriosis in patients years of age
Number of patients:
Investigation group – 40 (Metronidazole+probiotic); Control group – 30 (Metronidazole);
Efficacy criteria:
- clinical symptoms normalization dynamics;
- dynamics of bacteriological, bacterioscopic, cytological analysis of vaginal discharge for disbacteriosis.
Objectives:
- study the efficacy of the study medication in vaginal disbacteriosis patients.
- study the tolerability of the product and possible adverse product-related events.
- compare the findings of treatment between the investigating and control groups.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

35. Selection criteria for the patients in the trial
CLINICAL TRIALS
Selection criteria for the patients in the trial
Clinical symptoms (vaginal discharge that is usually thin and grayish white, vaginal odor (foul-smelling or unpleasant fishy odor), itching irritation.
Microbiological examination (dominative microflora is Gardnerella vaginalis, Prevotella and other, Lactobacilli in low quantities).
pH greater than 4.5.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

36. Treatment CLINICAL TRIALS
Metronidazole 500 mg orally twice a day for 7 days
Vaginal ovules with probiotic LAB blend twice a day for 10 days
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

37. Day 7 after start of the treatment
CLINICAL TRIALS
Day 7 after start of the treatment
Microorganism
Investigation group (Combined treatment (Metronidazole + vaginal ovules with probiotic LAB blend)
Control group (Metronidazole) %
Lactobacillus iners 22
3.6
Lactobacillus crispatus
29.4
0.3
Other Lactobacillus species
5.4
2.4
Gardnerella vaginalis
16.3
17.3
Atopobium vaginae
4.1
3.7
Prevotella
6.4 7
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

38. Day 20 after start of the treatment
CLINICAL TRIALS
Day 20 after start of the treatment
Microorganism
Investigation group (Combined treatment (Metronidazole + vaginal ovules with probiotic LAB blend)
Control group (Metronidazole) %
Lactobacillus iners
25.4
7.9
Lactobacillus crispatus 36
3.5
Other Lactobacillus species
7.2
8.4
Gardnerella vaginalis
14.3
16.4
Atopobium vaginae
5.1
3.9
Prevotella
5.8
6.2
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

39. Conclusions CLINICAL TRIALS
Combined treatment (Metronidazole + vaginal suppositories with probiotic LAB blend) was effective in achieving therapeutic cure or in restoring healthy vaginal lactobacilli
Fast colonization with beneficial microflora
Decrease the risk of BV relapse
Decrease the risk of local adverse events of standard treatment
Treatment with Metronidazole reduced colonization with BV-associated bacteria, but was not effective in achieving therapeutic cure or in restoring healthy vaginal lactobacilli
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

40. PRODUCTION OF LAB BLEND
LAB blend is produced by Nutraceutix Inc, USA at a GMP certified facility.
Guaranteed required quality parameters.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

41. STRAINS INDENTIFICATION
As probiotic features of the lactic acid bacteria are strain dependent the strains for each batch of production are re-identified before and after the production to validate that the LAB blend conceits of the same probiotic strains. This identification is done by DNA sequencing technique.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

42. MANUFACTURE OF FINAL PRODUCT
Neoprobio ovules are manufactured in Poland by FARMINA SP. Z O.O., KRAKÓW. A unique technology of low temperature production has been specially developed to allow safe processing of the LAB, keeping a therapeutic concentration of 109 to 107 of viable LAB cells per ovule.
The facility is regularly audited according to EU GMP standards.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

43. NEOPROBIO IS NOT ANOTHER VAGINAL PROBIOTIC!
NEOPROBIO IS UNIQUE!
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

44. UNIQUE OVULE FORM
Ovules provide controlled release of LAB and promote fast colonization of healthy microflora.
Capsules are not the correct vaginal form (may cause individual reactions to the compound of the capsule which contains unfavourable biological material and the rest of cultivation media).
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

45. UNIQUE OVULE FORM
Ovules provide direct therapeutic concentrations of LAB to vaginal canal.
The use of oral tablets or capsules decreases concentration of LAB which pass through the aggressive environment of the stomach and then depend on an unproven mechanism of „finding their way” from from anal canal into the vaginal canal.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

46. UNIQUE LAB BLEND
LAB of the blend have been tested in vitro as well as in vivo and possess main characteristics:
High adhesion activity (provide fast colonization of vaginal canal)
High antagonistic activity (provide inhibition of growth of pathogenic microflora)
Synthesis of biologically active substances (H2O2, lysozyme, vitamins
Interferon induction
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

47. UNIQUE LAB BLEND
Unique composition helps create conditions for fast microflora balance change.
API of Neoprobio consists of tested and scientifically proved probiotic LAB according to WHO requirements. Validated technology of production and testing ensure that exactly specific probiotic strains are incorporated in the ovule.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

48. Indications are supported by positive clinical trials.
UNIQUE MEDICAL EFFECTS
Neoprobio is indicated for the prevention and treatment of vaginal dysbiosis of different etiologies.
Neoprobio is indicated following therapy with antibiotics, antifungal and antiviral drugs to maintain and restore the balance of vaginal flora.
Neoprobio is indicated for the prevention and elimination of dry erosion of women of different age groups, as an aid to epithelialization due to the components included in the product.
Indications are supported by positive clinical trials.
dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

49. Thank you! www.neoprobio.pl FOR MORE INFORMATION PLEASE CONTACT:
Taras Lyaskovsky, PhD or
Michał Szczęsny, MPharm