Biomarkers of Bone Physiology & Pathology

Christopher Jerome, B.Vet.Med., Ph.D.

SkeleTech, Inc., Bothell, Washington, USA

ILSI Seminar: Biomarkers of Pathologic

Processes and Organ-Specific Injury

Banff, November 15, 2003

Goals and Objectives

• Provide theoretical framework for interpretation of

bone biomarkers

– Sources of bone biomarkers

– Review normal bone biology

– Discuss alterations in bone biology during life phases and in

disease conditions

• Review some experimental data from animal models

Bone RemodelingBone remodels throughout life,

at rates that vary with age and

site. The same cells and

processes participate in

growth, fracture repair and

pathological conditions. Most

biomarkers are byproducts of

bone remodeling.

Cancellous Bone Remodeling

Osteoclasts resorb

bone, leaving a

scalloped surface

Osteoid

mineralizes to

become bone

Osteoblasts

form osteoid

(unminerlized

bone matrix)

Histomorphology of bone remodeling

Double labeling of bone mineralization

Double labeling at sites of

bone formation

Osteoid stained

red

Cortical Bone Remodeling

Like human

beings, monkeys

have osteonal

remodeling of

cortical bone

Haversian osteons in

cynomolgus monkey

femur shaft

Sources of Bone Biomarkers

• Bone cells

– Osteocytes

– Osteoclasts

(bone resorbing

cells)

– Osteoblasts

(bone forming

cells)

• Bone Matrix

• Hormones and

other metabolic

markers

Sources of Bone Biomarkers

• Cell type specific enzymes involved in cell function– TRAP from osteoclasts

– Alkaline phosphatase from osteoblasts

• Secretory products of cells– Osteocalcin from osteoblasts

• Matrix processing or degradation products– Collagen propeptides

– Collagen telopeptides

• Regulatory/Metabolic– PTH, calcitonin

– Ca, P

Distal Radius

Bone Structure

Bone structure varies from site

to site. Most bone in the body is

cortical bone, but vertebrae and

long bone metaphyses also

contain cancellous bone.

Metabolic activity that produces bone biomarkers takes

place on bone surfaces, and cancellous bone has a

much higher surface/volume than cortical bone

Femur Neck

Vertebra

Mid-Radius

Mid-Femur

Dynamic histomorphometry in normal

monkeys

•Circulating biomarkers represent a weighted global average of

biomarker production throughout the body

•Levels of biomarkers in circulation are most highly correlated with

tissue volume (TV) referent data

•Surface-based parameters are comparable across sites, but

BFR/TV is 10x lower in cortical vs. cancellous sites

•Cancellous bone and localized pathological phenomena may be

over-represented in biomarker indices

MR MF FN LV DR

BV/TV 75.2 65.2 43.8 26.0 17.1

BS/BV 0.2 0.1 13.5 22.0 27.2

BS/TV 14.7 8.5 591.3 572.0 465.1

BFR/BS 24.0 21.6 53.1 41.0 44.0

BFR/BV 4.7 2.8 70.6 89.0 110.0

BFR/TV 3.5 1.8 29.6 23.0 18.0

Functions of Bone

• Structural Role– The skeleton adapts its mass and structure to maintain its

integrity during growth and normal usage, using the bone remodeling system

– After fracture, the skeleton restores a functional structure

– In the aged, adaptation to loading may fail

– Adaptive mechanisms may sometimes be activated inappropriately

• Metabolic Role – Calcium Reservoir– Skeleton contains 99% of total body Ca, but regulation of Ca

is primarily at kidney and gut

– Mechanisms by which bone participates in calcium regulation are poorly understood

– Bone remodeling system participates, but role is unclear

Uses and Limitations of Bone Biomarkers

• Bone resorption and formation are coupled, so bone remodeling

markers usually reflect overall bone turnover, which is modified

by age, sex hormones, calcitropic hormones, and bone-active

therapeutic agents.

• Biomarkers reflect global average bone turnover, and are of

limited value for site-specific agents.

• Change in bone mass is related to changes in bone balance,

which cannot be accurately assessed using bone biomarkers.

• Calcium, phosphorus and their regulatory factors (PTH,

calcitonin, vitamin D and its metabolites) are only indirectly

indicative of bone status due to the complexity of mineral

metabolism.

Using Bone Biomarkers in Animals

• Enzymatic assays (alkaline phosphatase, TRAP) and single amino acid-based assays (hydroxyproline, pyridinoline, deoxypyridinoline) are widely applicable, but not specific for bone.

• Clinical immunoassays are usually applicable in primates, and may be applicable in other species.

• When adapting clinical assays, cross-reactivity and matrix interference are the main issues, and can be usually be assessed by dilutional linearity.

• A few assays have been developed specifically for non-primate species.

Effect of Age on Biomarkers of Bone Metabolism

in Rats

Bone biomarker levels decrease with age, and the decline is more marked for the

more specific markers

Female, SD Rats

0

10

20

30

40

50

60

70

0 5 10 15 20

Age (months)

OS

CL

, C

TX

0

50

100

150

200

250

Dp

D

OSCL CTX DpD/CRT

Female, SD Rats

0

5

10

15

20

25

30

0 5 10 15 20

Age (months)

TR

AP

, D

pD

, O

SC

L

140

150

160

170

180

AL

P

TRAP DpD/CRT OSCL ALP

Effect of Age on Bone Dynamics and Biomarkers in

Cynomolgus Monkeys

Both bone biomarker levels and bone formation rate measured

histomorphometrically decrease with age.

Data from Lees CJ, Ramsay H (1999) Bone 24: 25-28.

Female cynomolgus monkeys

0

200

400

600

800

0 5 10 15 20 25

Age (years)

AL

P,

CT

X

0

2

4

6

8

10

12

14

OS

CL

, T

RA

P

ALP CTX OSCL TRAP

Female cynomolgus monkeys

0

50

100

150

200

250

300

350

400

0 5 10 15 20 25

Age (years)

BF

R/B

V

0

5

10

15

20

25

30

BV

/TV

Cn.BFR/BV Ec.BFR/BV Cn.BV/TV

Effect of Pregnancy and Lactation on Bone

Biomarkers in Monkeys

Osteocalcin

0

1

2

3

4

5

0 T3 1MPP 4MPP 10MPP

Seru

m O

ste

ocalc

in (

ng

/ml)

Non-pregnant Pregnancy & Lactation

Deoxypyridinoline

10

15

20

25

30

35

0 T3 1MPP 4MPP 10MPP

Deo

xyp

yri

din

oli

ne/C

reati

nin

eNon-pregnant Pregnancy & Lactation

Bone biomarker levels decrease during pregnancy and rise during lactation.

Data from Lees CJ et al (1998) Bone 22:545-549 and Lees CJ et al (1998) JCEM

83:4298-2302.

Effect of Pregnancy and Lactation on Bone

Mass in Monkeys

Whole Body BMC Change Serum Estradiol

-140

-120

-100

-80

-60

-40

-20

0

20

40

60

BM

C C

han

ge

(m

g/d

ay)

Non-pregnant

Pregnant

Lactation

Weaning

*

Bone mass decreases during lactation and recovers only slowly.

Data from Lees CJ et al (1998) JCEM 83:4298-2302.

Estradiol

10

100

1000

0 T3 1MPP 4MPP 10MPP

Estr

ad

iol (p

g/m

l)

Non-pregnant Pregnancy & Lactation

Development of Osteopenia in Ovariectomized

Cynomolgus Monkeys

Female cynomolgus monkeys become osteopenic when

estrogen-deficient. Spine may show bone loss or failure to gain.

Femur more consistently shows bone loss.

Experiment 2 Distal Femur BMD

-15

-10

-5

0

5

10

15

0 3 6 9 12 15 18

Time (m)

%C

han

ge f

rom

Baselin

e

O S 0%

Experiment 2 LV 1-4 BMD

-8

-6

-4

-2

0

2

4

6

8

10

12

14

0 3 6 9 12 15 18

Time (m)

%C

han

ge f

rom

Baselin

e

O S 0%

NTx

0

50

100

150

200

250

300

350

400

450

0 6 12 18

Time (months)U

rin

ary

NT

x/C

rea

tin

ine

Intact Ovx

Bone Biomarkers are Increased in Ovariectomized

Cynomolgus Monkeys

Osteocalcin

0

5

10

15

20

25

30

35

40

45

0 6 12 18

Time (months)

Se

rum

Os

teo

ca

lcin

(n

g/m

l)

Intact Ovx

Osteocalcin

0

5

10

15

20

25

30

35

40

45

0 6 12 18

Time (months)

Se

rum

Os

teo

ca

lcin

(n

g/m

l)

Intact Ovx L M H

NTx

0

50

100

150

200

250

300

350

400

450

0 6 12 18

Time (months)

Uri

na

ry N

Tx

/Cre

ati

nin

e

Intact Ovx L M H

Bone biomarkers and histomorphometric indices of bone

turnover are increased by estrogen deficiency.

Short-term Bone Efficacy Screening Model Using

Bone Biomarkers in Intact Cynomolgus Monkeys

• Short-term studies are needed for proof-of-concept and for

dose-ranging for long-term studies.

• The GnRH (chemical) ovariectomy model can be used, but this

model is difficult, slow, and expensive.

• Biomarkers respond quickly to bone-active drugs in intact

female monkeys, and can be used for rapid screening in

relatively small numbers of animals.

• Circadian variations need to be considered in experimental

design.

• Short-term models are being developed for additional

indications, such as glucocorticoid osteoporosis

• Biomarker evaluations can also be added to tox studies.

Circadian Rhythms and Sampling Effects on

Biomarkers of Bone Metabolism

0

50

100

150

200

250

300

0:00 4:00 8:00 12:00 16:00 20:00

Time of day

PIC

P (

ng

/ml)

or

CT

x (

(ug

/mM

Cr)

PICP

CTx/CR

Hotchkiss & Jerome 1998

Circadian variations and sampling effects on biomarkers must be considered in

the design of biomarker studies

Bone Biomarkers in Intact Cynomolgus Monkeys

(Short-term Model)

Bone Resorption Inhibitor Bone Formation Stimulator

Serum CTx

0

4

8

12

16

0 1 2 3 4 5

Days

Seru

m C

Tx (

mg

/L)

Placebo

Treatment

Serum Bone-Specific Alkaline Phosphatase

-60

-40

-20

0

20

40

60

80

0 3 6 9 12 15 18 21

Days

BS

AL

K (

% o

f d

ay 0

)

Placebo

Treatment

0%

Bone resorption inhibitor response occurs within 24-48 hours, bone formation

stimulator response within 1-2 weeks

Bone Biomarkers in Glucocorticoid-treated Cynomolgus

Monkeys

Bone Resorption Marker Bone Formation Marker

Serum NTx after Dexamethasone 1

mg/kg

-40

-20

0

20

40

60

80

100

120

140

160

0 5 10 15

Time (days)

%C

ha

ng

e f

rom

Ba

se

lin

e

Placebo

Dexamethasone

0%

Serum Osteocalcin after

Dexamethasone 1 mg/kg

-60

-40

-20

0

20

40

60

80

100

0 5 10 15

Time (days)

%C

ha

ng

e f

rom

Ba

se

lin

e

Placebo

Dexamethasone

0%

Conventional glucocorticoids stimulate bone resorption and depress bone

formation, and may negatively impact bone growth and/or mass

Conclusions

– Most bone biomarkers are byproducts of bone remodeling.

– Calcium regulatory indicators have less direct relevance to bone.

– Circulating and excreted bone biomarkers reflect global rates of bone

turnover, and may over- or under- represent significant focal or site-

specific bone changes.

– Bone resorption and formation are usually coupled, and bone

biomarkers lack the precision necessary to assess bone balance.

– Bone turnover and biomarkers generally decrease with age.

– Bone is responsive to estrogen deficiency, causing increased bone

turnover and osteopenia in lactation and in the post-menopause.

– Bone biomarkers respond rapidly and robustly to bone active agents,

whereas bone mass has a slower and less dramatic response.

– Biomarkers can be useful for proof-of-concept, preliminary efficacy

and dose-ranging in dedicated, short studies or as an adjunct to

toxicology studies, as well as for monitoring long-term response to

therapy.