1. vitamin K2 A scientific overview of by Trygve Bergeland, phd
Vice President Science & Product Development
Kappa Bioscience AS

2. Overview Vitamin K2 Biochemistry Biomarkers Physiology & metabolism
Health effects
As will 1/5 Men!

3. Timeline overview – Discovery and rediscovery of vitamin K2
History
Introduction
Timeline overview – Discovery and rediscovery of vitamin K2
Japanese warriors consumed K2-rich fermented soya beans – natto
Dam an Doisy shared the Nobel Prize in Medicine for discovery of vitamin K family
K2 as MK4 became an approved drug in Japan
Motohara et. al. studied the transfer of K2 between mothers and newborns
Knapen et. al and Kurna-towska et. al. demonstra-ted K2 effect on reducing cardiovascular risk
1600s
1943
1960s
1990s
2015
1930s
1950s
1970s
2000s
2016
Dam discovered a fat-soluble substance that reduced bleeding; Doisy determined the chemical structure of vitamin K & succeeded in synthesizing it
K2 forms were first identified. Price’s ‘Activator X’ (K2), the missing ingredient for Teeth and bone health
Vermeer started scientific work on enzymes an coagulation
Studies investigation K2 and bone health as well as K2 biomarkers
Møller et. al. demonstrated that synthetic K2 (K2VITAL®), and natural-fermented K2 are biologically equivalent

4. PubMed results showing a spike in K2 publications in the last decade.
Scientific attention
Introduction
PubMed results showing a spike in K2 publications in the last decade.
For internal use ONLY
Vitamin K2 as MK-4
approved as a drug in
Japan.
Vermeer started scientific
work on enzymes
and coagulation.
Vitamin K2-PubMed Results by Year

5. Chemistry Vitamin K Introduction Vitamin K1 Vitamin K2 MK-4
Phylloquinone
Vitamin K2 MK-4
Menaquinone-4
Vitamin K2 MK-7
Menaquinone-7

6. Dietary sources Western diets are K2 deficient Introduction Meat Fish
Milk
Cheese
Butter
Margarine
Green Vegetables
Natto Dish
Fruit

7. Function - biochemistry
Carboxylation (protein activation)
Vitamin K cycle
Coagulation factors
Osteocalcin
Matrix gla-protein (MGP)
Vitamin K
epoxide
Vitamin K
Active protein
Inactive protein

8. Carboxylation Coagulation factors Osteocalcin MGP
Booth et al. 2013, Nature Review
Vitamin K dependent modifications of glutamic acid residues in prothrombin (proton magnetic resonance spectroscopy/ mass spectrometry)
Proc. Nat. Acad. Sci. USA – Vol. 71, No. 7, pp. 2730–2733, July Johan Stenflo*, Per Fernlund*, William Egan † and Peter Roepstorff ‡

9. The Vitamin K Cycle
Takes place in different tissues, in particular the liver and bone
Involves two membrane bound enzymes:
VKOR – vitamin K epoxide reductase
GGCX - ɣ-glutamyl carboxylase
Sensitive to warfarin
Tie et al. 2011, Blood 117(10);

10. Osteocalcin Endocrine functions Only synthesized in osteoblasts
Insulin
Pancreas Insulin synthesis β-cell proliferation
Brain Neurotransmitter Production
Muscle Insulin sensitivity
Adipose Insulin sensitivity Adiponectin Fat mass
Testes Testosterone
Osteocalcin
Booth et al. 2013, Nature Review
Zoch et al., Bone 2016; 82;42-9
Only synthesized in osteoblasts
Carboxylation allows calcium binding and mineralization
Mechanical function by binding hydroxyapatite and collagen

11. Association between OC and fractures
Design
Observational trial
Population
Age of 70 and older
Sample size
792
Follow-Up Period
5 years
Main Endpoints
Fractures, carboxylated and total osteocalcin
Fracture cases
Controls
Sex and OC form n
mean (SD) p*
Men
TOC 21
9.45 (4.76)
280
9.30 (4.69)
0.786
COC
6.66 (4.22)
279
8.54 (4.03)
0.022
COC/TOC
0.74 (0.36)
0.96 (0.30)
0.002
Women 85
13.45 (7.56)
406
11.42 (6.63)
0.006 84
9.17 (5.32)
403
9.82 (4.72)
0.073
0.77 (0.46)
0.96 (0.44)
0.001
* t-Test
Luukinen, J Bone Miner Res 2000; 15(12);

12. Matrix gla protein (MGP)
Secreted by smooth vascular muscle cells & chondrocytes
Human vascular smooth muscle cell Anti cMGP
Calcified artery, diabetic patient
Anti ucMGP
Green: Anti cMGP, Blue: DNA
A: adventitia, Ca: calcification, I: intima, M: media
Cranenburg et al., Thromb Haemost 2007; 98:

13. Association between MGP and mortality
Population
Severe aortic stenosis
Sample size
147
All cause mortality
dp-ucMGP (pM)
Ueland et al., J Intern Med 2010; 268(5);483-92

14. Physiology & Metabolism
Intake
Clinical trials
Uptake
Distribution

15. Overview: Clinical documentation on MK-7
Number
of trials
Range of
participants
Tot
participants
Bioequivalence 2
17-48 65
Bone 13
4167
Cardiovascular 5
26-244
422
ucOC
cOC
Children 2
20-55 75
ucMGP
cMGP
Biomarkers/other 25
4-214
1382

16. 19 RTC trials, 6759 participants, MK-7 and MK-4
Does vitamin K2 play a role in the prevention and treatment of osteoporosis for postmenopausal women: a meta-analysis of randomized controlled trials
Z.-B. Huang, S.-L. Wan, Y.-J. Lu, L. Ning, C. Liu, S.-W. Fan Osteoporos Int. 2015; 26(3);
Included trials:
Study ID
Study design
Country
Number
OS?
Age
Follow-up
Intervention
Comparison intervention
Shiraki et al.
RCT without details
Japan
121/120 Y
67.2±0.8
24 M
45 mg/day menatetrenone + C
150 mg/day Ca
Iwamoto et al.
29/21
55–81
0.75 μg/day VD3
24/23
53–78
45 mg/day menatetrenone
2 g/day Ca
Ishida et al.
RCT with details
66/66
50–75
Menatetrenone 45 mg/dμ
Placebo
Orimo H et al.
41/39
∼72
24 W
90 mg/day menatetrenone
Emaus et al.
Norway
167/167 N
50–60
12 M
360 μg MK-7
Binkley et al.
America
129/126
∼62
Koitaya et al.
24/24
50–65
1.5 mg/day MK-4
Kasukawa et al.
50/51
>60
17.5 mg/week risedronate
Kanellakis et al.
Greece
38/39
54–73
100 μg MK-7 + C
800 mg Ca 10 μg VD3
Moschonis et al.
26/24
55–65
Je et al.
Korea
40/38
6 M
400 IU VD3qd 315 mg Ca bid
60/62
68.6±7.6
133.8 mg/day Ca
Inoue et al.
2193/2185
>50
48 M
Oral Ca
Hirao et al.
26/22
∼68
45 mg/day vitamin K2 + C
5 mg/day alendroate
Knapen et al.
Netherlands
164/161
55–75
36 M
Purwosunu et al.
Indonesia
30/33
60–75
48 W
1500 mg Ca
Ushiroyama et al.
43/43
∼53.4
1 μg/day VD3
124/120
180 μg MK-7/day
19 RTC trials, 6759 participants, MK-7 and MK-4

17. Bone health Conclusion
Vitamin K2
Control
Mean Difference
Study or Subgroup
Mean SD
Total
Weight
IV, Random, 95% CI
Year
1.1.1 patients without osteoporosis
Binkley et al.
0.04
2.92
126
0.4
2.84
129
19.2%
-0.36 [-1.07, 0.35]
2009
Je et al.
1.32
5.34 18
-1.04
5.09 27
12.4%
2.36 [-0.77, 5.49]
2011
Subtotal (95% CI)
144
156
31.6%
0.56 [-1.96, 3.08]
Heterogeneity: Tau2 = 2.36; Chi2 = 2.77, df = 1 (P = 0.10); I2 = 64%
Test for overall effect: Z = 0.43 (P = 0.67)
1.1.2 patients with osteoporosis
Shiraki et al.
1.4
0.7
105
-1.8
0.6
100
19.8%
3.20 [3.02, 3.38]
2000
Iwamoto et al.
1.49
1.45 21
-0.44
3.86 29
17.3%
1.93 [0.39, 3.47]
Ushiroyama et al.
4.1
5.88 31
0.115
3.07 32
14.9%
3.98 [1.66, 6.31]
2002
Purwosunu et al. 33 1 30
16.4%
1.40 [-0.46, 3.26]
2006
190
191
68.4%
2.70 [1.72, 3.69]
Heterogeneity: Tau2 = 0.53; Chi2 = 6.58, df = 3 (P = 0.09); I2 = 54%
Test for overall effect: Z = 5.38 (P < )
Total (95% CI)
334
347
100.0%
2.01 [0.21, 3.81]
Heterogeneity: Tau2 = 4.26; Chi2 = 96.95, df = 5 (P < ); I2 = 95%
Test for overall effect: Z = 2.19 (P = 0.03)
Test for subgroup differences: Chi2 = 2.42, df = 1 (P = 0.12); I2 = 58.6%
-10 -5 10 5
Favours Control
Favours Vitamin K2
Huang et al. 2015
Conclusion
Vitamin K2 plays a role in the maintenance and improvement of vertebral BMD and the prevention of fractures in postmenopausal women with osteoporosis

18. Bone Health
Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women
M. H. J. Knapen, N. E. Drummen, E. Smit, C. Vermeer, E. Theuwissen Osteoporos Int. 2013; 24(9);
Response to advertisements
(n=441)
Questionnaires returned
(n=343)
Assessed for eligibility
(n=244)
Did not meet inclusion criteria (n=99)
Randomized to non MK-7 treatment (n=124)
Randomized to MK-7 treatment (n=120)
Lost to follow-up within year 1 (n=5)
Not motivated n=2
Complaints capsules n=2
Soybean allergy n=1
Lost to follow-up within year 2 (n=5)
Interfering medication n= 2
Without reason n=1
Lost to follow-up within year 3 (n=2)
Completed study
(n=112)
(n=111)
Lost to follow-up within year 1 (n=6)
Deceased of concer n=1
Complaints capsules n=1
Interfering medication n=1
Lost to follow-up within year 2 (n=1)
Tscore < -2.5 (start med) n=1
Not motivated n=1
Design
Double-blinded, randomized, placebo controlled
Intervention
180µg/d MK-7 for 3 years
Participants
244 healthy postmenopausal women
Endpoints
Carboxylated osteocalcin
Uncarboxylated osteocalcin
Bone mineral content
Bone mineral density

19. Bone health Osteocalcin Bone mineral content & Bone mineral density
(uncarboxylated osteocalcin)
(carboxylated osteocalcin)
Bone mineral content
& Bone mineral density
Knapen et al. 2013
Placebo
MK-7

20. Cardiovascular Health
Data on MK-7
Publications
Participants
Population
Dose
Duration
Results
Knapen et al. 2015
244
Healthy postmenopausal women
180µg/d
3 years
Significant reduction in arterial stiffness
Kurnatowska et al. 2015 42
Kidney patients
90µg/d
9 months
Slower progression of calcification by MK-7
Mc Farlin
et al. 2017 26
Healthy aerobically trained athletes
µg/d
8 weeks
Increased maximal cardiac output during exercise
Mansour 60
Renal transplant patients
360µg/d
Improved arterial stiffness

21. Reversion of calcification in animal model
Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats Schurgers et al. 2007
6 weeks on Warfarin + low dose Vit. K1
6 weeks on high dose K2
Thoracic aorta
12 weeks on Warfarin + low dose Vit. K1
A Calcium (by Von Kossa),
B Total MGP,
C ucMGP,
D cMGP
High intake of vitamin K:
Higher levels cMGP in thoracic aorta
Lower levels of ucMGP in thoracic aorta
Reversed arterial calcification
Blood 109(7):

22. Heart Health
Dietary Intake of Menaquinone is associated with a Reduced Risk of Coronary Heart Disease: The Rotterdam Study
Men (n = 1836)
Women (n = 2971)
Age, y
66.9 ± 7.4
67.7 ± 8.0
BMI, kg/m2
25.7 ± 2.9
26.6 ± 4.0
Smoking status, %
Current
29.8
19.1
Former
61.5
28.8
Never
8.7
52.0
Alcohol use, %
88.1
74.4
Educational level, %
Low
23.3
41.1
Intermediate
57.1
52.1
High
19.7
6.8
Blood pressure, mm Hg
Systolic
± 21.9
± 22.2
Diastolic
74.8 ± 11.4
73.1 ± 11.0
Serum cholesterol level, mmol/L
Total
6.3 ± 1.1
6.9 ± 1.2
HDL
1.2 ± 0.3
1.5 ± 0.4
Diabetes mellitus, %
3.1
3.6
Johanna M. Geleijnse†, Cees Vermeer, Diederick E. Grobbee‡, Leon J. Schurgers, Marjo H. J. Knapen, Irene M. van der Meer, Albert Hofman and Jacqueline C. M. Witteman
Design
Observational trial
Participants
4807 men and women (55yrs and above)
Follow-up period
7.2 yrs ± 1.9 (mean ± SD)
Endpoints
Dietary intake of vitamin K1 and K2
incidences of CHD
all-cause mortality
J Nutr. 2004; 134(11);

23. The Rotterdam study
… showing the benefits of vitamin K2 on cardiovascular health
Geleijnse et al. 2004

24. Heart Health
Menaquinone-7 supplementation improves arterial stiffness in healthy postmenopausal women: double-blind randomised clinical trial
Design
Double-blinded, randomized, placebo controlled
Participants
244 healthy postmenopausal women
Intervention
180µg/d or placebo in 3 years
Main Endpoints
Arterial stiffness by pulse wave velocity and echotracking
ucMGP
Marjo H. J. Knapen, Lavienja A. J. L. M. Braam, Nadja E. Drummen, Otto Bekers, Arnold P. G. Hoeks, Cees Vermeer
VitaK & Cardiovascular Research Institute (CARIM), Maastricht University, The Netherlands; Central Diagnostic Laboratory, University Hospital Maastricht, The Netherlands; Biomedical Engineering, Maastricht University, The Netherlands
Thromb Haemost. 2015; 113(5);

25. Elevated stiffness at baseline
Arterial stiffness
Whole population
Elevated stiffness at baseline
Change in serum ucMGP
(mean±SD)
MK-7
Placebo
MK-7
Placebo
Knapen et al. 2015
Conclusion
Long-term use of MK-7 supplements improves arterial stiffness in healthy postmenopausal women, especially in women high arterial stiffness

26. Summary Vitamin K2 Biochemistry Biomarkers Physiology & metabolism
Health effects

27. Thank you Trygve.Bergeland@kappabio.com Trygve Bergeland, PhD
Vice President Science & Product Development
Headquarter Kappa Bioscience AS Silurveien 2 | Building B | 0380 Oslo Norway