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Zemplar novageneracija VDRA

(specifi nostdelovanja,

selektivnost)

VDRA-Aktivator vitamin Dreceptora (Vitamin D Receptor

Activator)

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Zemplar is Selective

Zemplar stimulates lesscalcium resorption fromthe bone and absorptionfrom the gut

Serum calcium doesnt

tell the complete story Necessary to look

beyond serum calciumto calcium load

Small differences incalcium load can lead tobig differences incalcification over time

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Introduction

Zemplar is the only selective VDRA in our markets

Zemplar is the only selective VDRA therapy among the 3 classes of SHPTtherapies: non-selective VDRA, selective VDRA, and Calcimimetics.

For VDRAs, selectivity means differential VDR binding, transcription, andcomodulator recruitment.Reference: Issa, et al., J Bone Miner Res. 2002

Zemplars selectivity can be illustrated in both gene -specific and cell/tissue-specific ways.Reference: 1. Wu-Wong et al., Atherosclerosis 2006;

2. Slatopolsky et al., Am J Kidney Dis 1998

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Overview of the SHPT Treatment Options

OHHO

OH

OHHO OHHO OHHO

OH

Non-Selective VDRA Selective

VDRA1st

Generation 2nd

Generation

Calcitriol1 ,25-dihydroxyvitamin D 3

Mimics endogenousVDR hormone

Calcijex (IV)Rocaltrol (Oral)Generics (IV & Oral)

SHPT in CKD (pre-dialysis)Osteoporosis,Hypocalcemia

Alfacalcidol/Doxercalciferol1 -hydroxyvitamin D 3 /D 2

Molecular modificationsat the side-chain

One AlphaHectorol

SHPT in CKDOsteoporosis,Hypocalcemia

Paricalcitol19-nor-1 ,25-dihydroxyvitamin D 2

Molecular modificationsat the side-chain and

A-ringZemplar

SHPT in CKD(Stage 3, 4, 5)

Calcimimetics

F3C

HCl

Cinacalcet

CaR activator

Sensipar Mimpara

SHPT in CKD(Stage 5 only)

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TherapeuticTarget

VDR

VDR

RXR

Ligand binding

Heterodimerization

DNA binding

RNA Pol II

CoReg

Transactivation / Transrepression

RNA Pol II

Nucleus

mRNA

VDR

VDR

VDR

RXR

RXR

B

VDRE

Parikalcitol

Interactions of the Ligand-activated VDR-RXR Heterodimer Complex with the NuclearProteins Facilitate the Assembly of the Transcription Preinitiation Complex andRegulate the Rate of Transcription of the Target Gene by RNA Polymerase II

RXR = retinoid x receptor; VDRE = vitamin Dresponsive element.Dusso et al., Semin Nephrol. 2004;24:10-16.

OH

H3C

CH 3

CH 3

HO

CH 3

OH

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RNA Pol II

VDR Bound to Selective VDRAs and Non-selective VDRAs Recruits Different Coactivators

Takeyama et al., Mol Cell Biol 1999; 19:1049-55

RXR VDRRNA Pol II

RXR VDR

SRC1 SRC1

TIF2 TIF2

VDRAIB-1 AIB-1

Non-selectiveVDRA*

SelectiveVDRA**

*1,25(OH)2D3; ** OCT = 22-Oxa-calcitriol

RXR = retinoid x receptor

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Gene-Specific Selectivity: Zemplar Activates andInactivates Different Genes in Vascular Smooth Muscle

Cells from those Regulated by Calcitriol

Wu-Wong et al ., Atherosclerosis 2006; 186:20-28

Zemplar and calcitriolactivate and inactivatedifferent genes

As such, clinical differencesobserved with these productscannot be explained simply asa dosing effect

Red: Increased activation

Blue: Increased inactivation

*Human coronary artery smooth muscle cells treated with0.1 M calcitriol or Zemplar for 30 h

Zemplar Calcitriol

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Selectivity: Differential VDR Binding, Transcription andComodulator Recruitment of Different VDRAs

Issa, et al., J Bone Miner Res . 2002; 17:879-90.

Rat Osteosarcoma (ROS) cells Yeast 2-hybrid Assay

Inhibitory constant (IC 50) indicates therelative binding affinity of VDRAs forVDR compared to calcitriolTranscriptional activities of VDRAs wereassessed in ROS 17/2.8 cells transfectedwith osteocalcin reporter

Zemplar induction of VDRhomodimerization VDR-RXRheterodimerisation and VDR coactivatorinteractions

0

25

50

75

100

125

150

175

% C

a l c i t r i o

l

VDR Binding VDR:VDR VDR:RXR VDR:GRIP1 VDR:RAC3

VDR binding(IC50 nM)

Genetransactivatio

n (ED 50 nM)Calcitriol 0.4 0.3 0.7 0.1

Zemplar 3.7 0.9 0.11 0.03

VDR Binding Affinities and TranscriptionalActivities of Zemplar and Calcitriol

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Cell/Tissue-Specific Selectivity: Zemplar Therapy Associated withSignificantly Less Intestine VDR Expression Compared to Calcitriol

Slatopolsky et al., Am J Kidney Dis 1998;32:S40-47.

0

50

100

150

200

UREMIC25

3 H - C a l c i

t r i o l s p e c i

f i c b i n d

i n g

( f m o l / m

g p r o t e i n )

Calcitriol Zemplar

100 ng620Normal

*

***

*P

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Introduction

Pre-clinical Studies:

Less calcemic and phosphatemic than non-selective VDRAsReference: 1. Brown et al., J Am Soc Nephrol 2000; 2. Slatopolsky et al., Am J Kidney Dis 1998; 3.

Slatopolsky et al., Kidney Int 2002; 4. Nakane et al., J Steroid Biochem. Mol. Biol 2007.

Clinical Studies:

18% less intestine Ca absorption and soft tissue Ca loading than calcitriol.Reference: Lund et al., Nephrol Dial Transplant 2006

Fewer episodes of hypercalcemia or elevated CaxP than calcitriol.Reference: Sprague et al., Kidney Int. 2003

.

Zemplar is the selective VDRA with minimal impact on Ca and P, andeffective PTH suppression

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At Equivalent Doses, Zemplar DemonstratedSignificantly Lower Calcemic and Phosphatemic Effects

than Calcitriol in Normal Rats

n = 4*p

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Doxercalciferol Induced Progressive Increases in SerumCa and P in Uremic Rats, While those of the Zemplar

Groups Remained Unchanged

50 100 250Dose (ngs)

8

9

10

11

12

13

C a ( m g /

d l )

5

6

7

8

9

10

P i ( m g /

d l )

50 100 250

Dose (ngs)

Slatopolsky et al. Kidney Int 2002;62:1277 84

Zemplar

Doxercalciferol

l h d ff b f

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Vehicle Calcitriol Zemplar Doxercalciferol10 ng 100 ng 100 ng

Zemplar had Less Effect on Intestine Absorption of Caand P Than the Non-selective Calcitriol and

Doxercalciferol in Normal Rats

n=21; *p< 0.01 vs. control rats; p< 0.01 vs. Zemplar-treated rats

P h o s p h o r u s a b s o r

b e d / 2 4 h ( % )

*

30

35

40

45

50

25

23%4%

*

C a l c i u m a b s o r b e d

/ 2 4 h ( % )

10

15

20

25

30

35

40

Vehicle Calcitriol Zemplar Doxercalciferol

39%

10 ng

9%

100 ng 100 ng

* *

Slatopolsky et al. Kidney Int 2002;62:1277 84

1xdosing

10xdosing

10xdosing

1xdosing

10xdosing

10xdosing

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Nakane et al, J Steroid Biochem. Mol. Biol 2007;103: 84-89

Zemplar does not Increase Intestine Ca Transport whileNon-selective VDRAs Do in a Dose-dependent Manner

1

8

7

6

5

43

2

0 S e r o s a

l / M u c o s a

l R a t

i o*P < 0.05 vs Nave***P < 0.001 vs Naven = 7-17/grp

Veh calcitriol Doxer-

calciferol Vehicle Zemplar

Treatment ( g/kg, i.p.)

***

***

*

20

10

C a 2 +

T r a n s p o r

t ( n m o l

/ m i n )

30

0

Zemplar

**

*

calcitriol Doxer-calciferol

Ctr -9 -8 -7 -9 -8 -7 -9 -8 -7

*

Test Agent [log M]

In 5/6 Nephrectomized Rats In Caco-2 Cell Culture

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Zemplar Showed a 18% Overall Decrease in CaAbsorption Relative to Calcitriol in Hemodialysis

Patients

F x A b s o n

Z e m p l a r

t h e r a p y

M e a n C a a b s o r p

t i o n

( % )

12.0 12.5 13.0

13.5 14.0 14.5 15.0 15.5 16.0

Zemplar Calcitriol

*

Lund et al. Nephrol Dial Transplant 2006;21(Suppl 7):EDTA 2006 Poster

Due to the intestinal absorption difference in Ca, compared to Zemplar, calcitriol couldresult in up to 46 mg more Ca accumulation in bone and soft tissue everyday (assuming 2g of daily average elemental Ca from diet), which could lead to the doubling of extra-skeletal Ca in 6 months a severe risk for soft tissue calcification.

p=0.022 for Zemplar vs calcitriol Shows combined results of 2 different regimens (calcitriol 2 g, Zemplar 6 g)

0.00 0.05

0.10 0.15 0.20 0.25 0.30 0.35 0.40

0.00 0.05

0.10 0.15 0.20 0.25 0.30 0.35 0.40

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 15% 31%

26%

13%

Regression Equation:Zemplar FxAbs = 0.007+0.815* Calcitriol FxAbsR-Square = 0.813

FxAbs on calcitriol therapyZemplar dosed 3x to 1xcalcitriol

13.5%

15.8%

IV Z l P i H d Si ifi l F S i d

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IV Zemplar Patients Had Significantly Fewer SustainedEpisodes of Hypercalcemia and/or Increased Ca x P Than

Calcitriol

Calcitriol (n=133)

Zemplar (n=130)

0

5

10

15

20

25

30

35

P a t i e n

t s ( % ) w i

t h e v e n

t s

Sprague et al. Kidney Int 2003;63:1483-1490

* p=0.008 for patients with hypercalcemia for at least 2 consecutive blood draws and/or a Ca P product >75 on at least 1 of 4 consecutive blood draws

33%

18%*

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Introduction

Zemplar is better at improving bone health relative to all SHPTtherapies

Pre-clinical Studies:

Less Ca release from the bone and greater collagen synthesis for bone formationReference: Nakane et al, J. Steroid Biochem. Mol. Biol 2006

Clinical Studies:

Reduction in bone-specific alkaline phosphatase and osteocalcin.Reference: 1. Ross et al., ASN 2006;

2. Coyne et al., Am J Kidney Dis. 2006.

Cinacalcet as a mono-therapy has unproven impact on bone biomarkers.Reference: Block et al., N Engl J Med 2004

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Rats were treated with 19-nor-1, 25-(OH)2D2 (19-nor-1,25-D2), 1-(OH)D2, or 1, 25-(OH)2D3 (1,25-D3). Delta serum calciumwas calculated by subtracting the mean of serum calcium in the vehicle treated group from serum calcium in each of the drugtreated animals. Values shown are the mean S.E.M. ( n = 12 16 per treatment group). (*) Different statistically from vehiclecontrol animals; P < 0.001. Statistical comparisons were performed by ANOVA analysis.

Adapted from Nakane et al, J. SteroidBiochem. Mol. Biol 2006, 98, 72-77

Compared to Non-selective VDRAs, Zemplar has the LeastImpact on Serum Ca Mobilized from the Bone

0

D e l

t a S e r u m

C a l c i u m v s . C

o n t r o l

( m g / d L )

1

2

3

4

5

6

0.001 0.01 0.1 1 10* p

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Compared to Non-selective VDRAs, Zemplar is Associatedwith Less Ca Release from the Bone and Greater Collagen

Synthesis for Bone Formation

Zemplar caused less Carelease from mousecalvariae bone primaryorgan culture compared tocalcitriol and 25-OH-

doxercalciferol

Zemplar stimulatedgreater collagensynthesis from mousecalvariae than calcitriol and25-OH-doxercalciferol

0

10

20

30

40

50

60

70

Zemplar Calcitriol Active doxercalciferol

* *** **

***

** **

***

C a r e

l e a s e

( n g

/ L m e

d i u m

)

[log M]

0

1

2

3

4

5

6

7

Zemplar Calcitriol Active doxercalciferol

***

*** ***

***

**

***

** **

*** ***

***

C o

l l a g e n

S y n

t h e s

i s

( % o

f t o t a l p r o

t e i n )

[log M]

Adapted from Nakane et al, J. SteroidBiochem. Mol. Biol 2006, 98, 72-77

ZEMPLAR C l Sig ifi tl D B T

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ZEMPLAR Capsule Significantly Decreases Bone Turnover,as Indicated by a Reduction in Corresponding Bone Activity

Biomarker Levels (BSAP and Serum Osteocalcin)

Similarly, Zemplar also significantly reduces the level of two other bone activity biomarkers, CTx (C-terminal Telopeptide ofCollagen, P

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Paricalcitol Caused Less Calcium Release From MouseCalvariae Compared With Calcitriol and Doxercalciferol

*p

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1

Male 5/6 nephrectomized ratswere placed on a high P dietfor 4 weeks to induceadvanced secondaryhyperparathyroidism

Treatment was initiated withvehicle, paricalcitol,doxercalciferol or calcitriol, IP,three times a week, for 12days

24 hours after last dose, small

intestine was collected formeasurement of Ca absorption

Ca transport was determined inthe mucosal-to-serosaldirection

Nakane M, et al . Steroid Biochem Mol Biol 2007;103:84 9

Effects of Selective and Non-selective VDRAs on IntestinalCalcium Transport in 5/6 Nx Rats

8

7

6

5

4

3

2

0

m e a n s

S E M S e r o s a l

/ M u c o s a l

R a t i o

n=7 17/grp

Veh Calcitriol Doxer-calciferol

Veh Pari-calcitol

Treatment ( g/kg, IP)

***

***

*

# Separate experiment. Different from vehicle control*p

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20

10

C a 2 +

T r a n s p o r t

( n m o l

/ m i n )

Cells grown on permeablefilter supports were treatedwith increasingconcentrations ofparicalcitol, calcitriol, ofdoxercalciferol for 48 hstarting on the 13 th day inculture

Ca transport wasdetermined in the apical tobasolateral direction

Values represent means SD from four determinations

Calcium Transport in Caco-2 Cells

30

0

Paricalcitol

**

*

Calcitriol Doxercalciferol Ctr -9 -8 -7 -9 -8 -7 -9 -8 -7

*

Concentrations (log M)

*p

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Effect of Paricalcitol and Doxercalciferol on SerumPhosphorus in Uremic Rats

5

6

7

8

9

10

P i ( m g /

d L )

50 100 250Dose (ngs)

Doxercalciferol

Paricalcitol

Slatopolsky E, et al. Kidney Int 2002;62:1277 84

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Effect of Paricalcitol and Doxercalciferol on SerumCa in Uremic Rats

50 100 250Dose (ngs)

8

9

10

11

12

13

C a ( m g /

d L )

Slatopolsky E, et al. Kidney Int 2002;62:1277 84

Doxercalciferol

Paricalcitol

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T o t a l C a

( m g /

d L )

Treatment (d)

Paricalcitol 1000 ng

Paricalcitol 100 ng

Calcitriol 10 ng

Paricalcitol 10 ngVehicle

3 6 1008

10

12

14

16

Dosing daily IP for 10 daysSlatopolsky E, et al. Am J Kidney Dis 1995;26:852 60

Paricalcitol Produced Similar Increases in Ca at Doses 10Times Higher Than Calcitriol in Uremic Rats

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Paricalcitol did not Increase P in any Dosing Group;However, Calcitriol was Associated With Significant

Increases in Uremic Rats

*p

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Doxercalciferol Induced Progressive Increases inCa x P in Uremic Rats, While Paricalcitol Groups Remained

Unchanged

IP 3 x week for 2 weeks*p

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Paricalcitol Showed a 17% Overall Decrease in Ca AbsorptionRelative to Calcitriol in Hemodialysis Patients in a Double-blind

Crossover Study

p=0.022 for paricalcitol vs calcitriol

Shows combined results of 2 different regimens (calcitriol 2 g, paricalcitol 6 g)

M e a n

C a a b s o r p

t i o n

( % )

F x A b s o n p a r i c a

l c i t o l

t h e r a p y

0.0

0.1

0.2

0.3

0.4

12

13

14

15

16

Paricalcitol Calcitriol

0.0

0.1

0.2

0.3

0.4

0.0 0.1 0.2 0.3 0.4

15% 31%

26%

13%

Regression Equation:Paricalcitol FxAbs = 0.007+0.815* Calcitriol FxAbsR-Square = 0.813

FxAbs on calcitrioltherapy

*

Paricalcitol dosed 3 xto 1 x calcitriol; equipotent PTH

suppression in both groups

Lund R et al Nephrol Dial Transplant 2006;21(Suppl 4):219A