BONE TREK: Next-Gen Drugs for Bone Diseases

BONE TREK
The “Next Generation”of Drugs for
Osteoporosis, Osteoarthritis &
Bone Cancer
Michael R. Doschak, MSc, PhD
Associate Professor
Faculty of Pharmacy & Pharmaceutical Sciences
University of Alberta

Outline of Talk

• Bone Metabolism
– Bone Cells and Remodeling
• Calcium Physiology
– Key Hormones Involved in Bone Health
• Bone-Targeting Drug Delivery
– Case study: Calcitonin delivery to Bone
• Imaging Dynamic Bone Turnover
– Synchrotron Imaging of Strontium Drugs

‘Bone’ is often
perceived as
being ‘dead’ - but
in reality, bone is
very dynamic
and very much
‘alive’
- constant remodeling
- maintains Calcium levels
- balanced turnover:
- acromegaly
- osteoporosis

•‘Bone’ is often
perceived as
being ‘dead’ - but
in reality, bone is
very dynamic
and very much
‘alive’
•Normal Bone •Osteoporotic Bone
- constant remodeling
- maintains Calcium levels
- balanced turnover:
- acromegaly
- osteoporosis

Bone: Matrix and Mineral
• Matrix
– 90% Type I collagen
– Non-collagenous proteins
(growth / differentiation / mineralization)
– Bone cells (Osteoclast, Osteoblast, Osteocyte)
and other cells (marrow, blood vessels,
nerves)
• Mineral
– Calcium Phosphate – Hydroxyapatite
Ca10(PO4)6(OH)2
– Deposited on (and within) matrix

Adult Human Skeleton
– Comprised of 206 bones
– Classified primarily by
• Location: axial vs. appendicular
• Shape: long, short, flat, irregular
– Two structural forms of bone
• Cortical (dense)
• Trabecular (honeycomb-like)
– Bones function as
• sites of attachment
• for protection
• locomotion
• haematopoietic function
• mineral storage and homeostasis

- Trabecular bone - compact bone
remodels according to - spongy bone
lines of stress
(mechanical loading) •compact bone:
- formed (active)
- Cortical bone is the - osteon (Haversian
thickest system)
- concentric lamellae
- central canal
- Volkmann canals
- interstitial lamellae
- circumferential
lamellae
•spongy bone:
- missing?
- trabeculae

Cortical bone
 Compact (dense) bone
 Osteon - Concentric layers (lamellar)
 Central Haversian Canal
 (blood vessels, nerves, fluid)

 Volkmann canal (Transverse)
 Microscopic canaliculi
 Channels linking osteocytes
 (osteocyte cellular processes,
extracellular fluid)

Osteocyte

Osteocytes:
- mature bone cells
- lacunae
- canaliculi

Osteocytes: Osteoblasts: Osteoprogenitors:
- mature bone cells - surface - surface
- lacunae - secrete bone matrix - daughter cells
- canaliculi - osteoid - osteoblasts

Osteocytes: Osteoblasts: Osteoprogenitors: Osteoclasts:
- mature bone cells - surface - surface - giant cells
- lacunae - secrete bone matrix - daughter cells - multinucleated
- canaliculi - osteoid - osteoblasts - erode bone

Bone Remodeling (metabolism)
• Resorption of existing bone
• Formation of new “osteoid” bone matrix
• Mineralization

Dynamic process that allows
• Bone renewal and microcrack repair
• Liberation of bone calcium stores
• Fracture healing

Calcium Physiology – The Major Players

• Calcium
• Phosphate
• Calcium Sensing Receptor
• Parathyroid Hormone (PTH)
• Vitamin D (pro-hormone!)
• Kidneys / Small Bowel / Bone
• Other hormones (calcitonin, estrogen)

Calcium

• Ca/PO4 - the major mineral of bone
• 99% of total body Ca is bone
• 1% is extracellular
– Co-factor for enzymatic rxns
– Coagulation
– Muscle contraction
– Neurotransmitter release
– Endocrine/exocrine secretion

Phosphate

• 85% of total body PO4 is bone-
resident
• 15% as organic form
– Part of biological molecules
– Nucleic Acids, phospholipids, CHO,
Enzymes, cofactors, ATP

Parathyroid Hormone (PTH)

• 84 aa peptide hormone that controls
minute to minute ionized [Ca]
• Tight control of serum calcium
(2.10 – 2.55 mmol/L)
• Synthesized and secreted by “chief
cells” of the parathyroid glands
• Primary function to maintain (raise)
serum calcium levels

Actions of Parathyroid Hormone (PTH)
• Acts on kidney (within minutes)
– Increases renal calcium reabsorption
– Increases production of active Vitamin D
(intestinal Ca++ absorption)
• Increases bone resorption
– Mobilizes calcium from bone by stimulating
cellular matrix dissolution
• Subsequently, PTH stimulates bone formation
(as bone resorption-and-formation are coupled!!)

Factors that influence PTH secretion

• Low serum Ca++ - primary stimulus
• High serum PO4 – stimulates PTH
– Direct effect to increase PTH mRNA
– Indirect effect by decr. Serum Ca
• High Vitamin D (active form)
– Direct effect on gland that decreases PTH
synthesis

Low serum
Parathyroid
calcium
Gland

Parathyroid
calcium
Gland

PTH

phosphate
Parathyroid
calcium
Gland

PTH

phosphate
Parathyroid
calcium
Gland

PTH

Vitamin D (active)

phosphate
Parathyroid ?
calcium
Gland

PTH

Vitamin D (active)

Summary: Actions of PTH

Parathyroid
Gland
PTH

PTH
Synthesis
Release Conserves of active Absorb
Ca++ in Vitamin D
Ca++ from Kidneys Ca++ and PO4
Bone But Promotes In small intestine
Phosphaturia

Maintain (raise) Serum Ca++ Levels

Calcitonin
• Produced by Thyroid C cells
• 32 aa polypeptide hormone
• blocks osteoclast activity (Calcitonin Rc)
• role in calcium regulation
(will restore resting plasma calcium levels)
• Synthetic human and salmon
• s.c., i.m. (Intranasally for Osteoporosis)

Therapeutic dosage forms:
– Salmon calcitonin [Miacalcin; Calcimar]

Vitamin D

This term describes 2 molecules:
• Ergocalciferol (Vitamin D2)
– Produced by UV irradiation of plant steroid ergosterol
– Often the major form of supplemental Vitamin D
• Cholecalciferol (Vitamin D3)
– Formed in skin (epidermis) under action of ultra-violet
(UV) light on 7-dehydrocholesterol

Vitamin D conversion to active form

• Vitamin D2 & D3, ingested or made in the
skin, metabolized in liver to
25-hydroxyvitamin D (25-OH-VitD [calcidiol]),
the major circulating form!

• Further hydroxylation in kidney to form the
highly biologically active:
1,25-Dihydroxyvitamin D (1,25-[OH]2-VitD [calcitriol])

1,25-[OH]2-VitD (calcitriol)

Promotes:
• Absorption of Calcium and PO4 from
small intestine
• Extracellular Calcium homeostasis,
directly & also indirectly through ↓PTH
• Mineralization of the skeleton

Childhood “Rickets”

•ASBMR Bone Curriculum

Adult Osteomalacia

• Pseudofracture
• Pathognomonic for
Osteomalacia

•ASBMR Bone Curriculum

Vitamin D Deficiency
• Lack of sun exposure
• Dietary deficiency
• Malabsorption pathology
• Liver or Renal disease (conversion & loss)
• Increased liver metabolism (drug-induced)
• Genetic causes:
– Renal 1-α-hydroxylase deficiency
(Vit D dependant rickets, VDDR)
– Vit D receptor defect (Vit D “resistance”)

• Incidence reduced in North America due to
supplemented foods (milk, orange juice)

• Still identified in young, elderly, new immigrants
– Dietary insufficiency
– Countries with little sun exposure
– Sunscreen overuse
– Traditional clothing that covers skin
– Dark skin requires more sunlight exposure

Produces Osteomalacia in stages
• Initially ↓Calcium absorption
Results in ↑⁭ PTH (2ary Hyperparathyroidism)
• PTH prevents hypocalcemia
At expense of phosphaturia and osteopenia
• Bone mineralization impaired
But Osteoblasts still actively form osteoid
Leads to “hungry bone” effect with treatment
(hypocalcemia, despite supplementation)

Bone Health and Bone Disease
• Excessive bone turnover associated with:
– Osteoporosis
– Rheumatoid Arthritis
– Paget’s Disease
– Bone cancers
– Post-traumatic Osteoarthritis
– Fracture pain (particularly vertebral bone
fractures, secondary to Osteoporosis)

Therapeutic agents

Four main types of therapeutics currently
indicated:
• Selective estrogen receptor modulators
(SERM’s)
• Potent Nitrogen Bisphosphonates (BP)
• Calcitonin (CT)
• Parathyroid Hormone (PTH)

Problems with Current Bone Therapeutics

• Selective estrogen receptor modulators (SERMs) have a variety of
hormonal effects on the body and are associated with increased
number of blood clots*
• Nitrogen-containing Bisphosphonates (BP) are highly selective for
bone but have been associated with osteonecrosis of the jaw and
other severe side effects**
• Native peptide hormones (e.g., Calcitonin, PTH) lack potency and
have very short half-lives (~43 mins), which requires more
frequent and supra-physiological dosing of the drug

*SERMs for the treatment and prevention of breast cancer. Rev Endocr Metab Disord 2007
**Systematic review: bisphosphonates and osteonecrosis of the jaw. Ann Int Med 2006;
Spontaneous femoral shaft fracture after long term bisphosphonate therapy

Next Generation Drugs –
Targeted Drug Delivery
Site-specific Drug Delivery:
• Enhances therapeutic potency
• Diminishes off-site drug side-effects
• Can be achieved by
– Identifying cellular metabolic process as a
means of uptake
– Synthesizing a “pro-drug” by chemical
conjugation of the active ligand to a carrier
that is uptaken or incorporated

Targeting Bone with BP-conjugates
This image cannot currently be displayed.

BISPHOSPHONATES (BPs)
– First used to treat Paget’s disease in 1971
– Initially termed “di-phosphonates”)
– Analogues of inorganic pyrophosphate
– Non-hydrolyzable (stable P-C-P bonds)

Targeting Bone with BP-conjugates
This image cannot currently be displayed.

BISPHOSPHONATES (BPs)
– High affinity for bone mineral
– Adsorb to bone (large avail. surface area)
– Uptake with osteoclast resorption, interfere
with cell function
– Many different bisphosphonates
constructed, with differing affinity for
hydroxyapatite

General Hypothesis

The targeted delivery of Calcitonin to
hydroxyapatite bone surfaces will
increase drug interaction with bone cell
Calcitonin-receptors, and will preserve
bone mass in Osteoporosis

Radiolabeled
Bisphosphonate
Distribution
(Bone scan)
of Knee
OsteoArthritis (OA)

Image from: Addison S. et al. Arthritis Rheum. 2009 Nov;60(11):3366-73.

Bone-Targeting Peptide Hormones:
BP-conjugated Calcitonin (BP-CT)
and
BP-conjugated Parathyroid Hormone (BP-PTH)

• Derivatize peptide hormones with bone-specific
bisphosphonate (BP) moieties
• Our lead compounds BP-Calcitonin and BP-PTH
significantly increase bone specificity of CT or
PTH
• Bone targeting lowers dose levels (and dosing
frequency) and decreases side effects relative to
the competing therapeutics in the market

Salmon Calcitonin-Bisphosphonate Conjugation Scheme

1. 0.06 µMol Calcitonin in DMF + 0.3 µMol Sulfo-SMCC in DMF + 0.1 %
TEA…………..Reacted at room temperature for 1 hr.
2. 3 µMol Thiol-BP in 10 mM PBS pH 6.7……..Reacted for 2 hr at room
temperature then overnight at 4°C

(Bhandari and Doschak, Int J Pharmaceut, 2010)

Scientific (BP-CT)
• Chemical Coupling of
Bisphosphonate (BP) moiety to
salmon Calcitonin (sCT)
• Di-conjugate utilizes amine
groups of Lysine11 and Lysine18
• Bone-targeting delivery
system for sCT whilst retaining
bioactivity
•PEGylated formulations of
BP-CT have also been
developed to increased
terminal half-life and decrease
dosing interval
• sCT-Tri (SMCC-BP)

Characterization of Salmon Calcitonin (sCT): MALDI-TOF

x104
Intens. [a.u.]

3414.808
3430.449
3436.956
1.25

1.00

0.75

0.50
3654.569
3636.524
1830.709
1321.509

1578.592

2069.884
2112.864
2301.009

2816.187

0.25
Salmon Calcitonin:
Peak at 3430.449
0.00
000 1500 2000 2500 3000 3500 4000 4500 5000 5500
m/z

Derivatization of sCT with crosslinker: MALDI-TOF
Intens. [a.u.]

3872.108
3868.030
6000

5000

Salmon Calcitonin:
4000 Peak at 3430.449 is
lost (i.e., converted)
3000
Mono-substituted
crosslinker:
Peak at 3648.999
4088.172

2000
3648.999
1935.555

3690.977

Di-substituted
4313.169
2045.553
2046.077

1000

crosslinker:
Peak at 3868.03
0
000 1500 2000 2500 3000 3500 4000 4500 5000 5500
m/z Tri-substituted
crosslinker:
Peak at 4088.172

Conjugate Characterization: MALDI-TOF:
sCT-crosslinker-bone targeting moiety
(Proof-of-principle compound)
Intens. [a.u.]

4113.367
5000

4000 Salmon Calcitonin: Peak at
3430.449 is absent (i.e.,
conjugates are stable)
3000

sCT-Mono substituted bone
targeting conjugate:
3650.261

2000
Peak at 3770.219
4453.437
2096.811

3991.436
1404.516

2212.872
2057.734
2126.989
1267.510

3814.270
1170.493

2244.846
1890.881
1897.616

4319.479
3770.219
1630.678
1766.711
1827.173

1000
sCT-Di substituted bone
0
Peak at 4113.367
000 1500 2000 2500 3000 3500 4000 4500 5000 5500
m/z
sCT-Tri substituted bone
Peak at 4453.437

Hence we hypothesized targeting sCT to bone by
conjugation to Bisphosphonate (BP)
Osteoclast
CTR CTR CTR
sCT sCT sCT

Localization & retention
of sCT to bone BP BP
BP

Bone resorption site

[sCT] at its site of
BP binds to antiresorptive action
hydroxyapatite

Improved
Bone bioavailability
& efficacy of sCT

(Pierce, 1987; Kasugai, 2000; Yokogawa, 2001; Orme, 1994; Fujisaki, 1997).

Scientific (BP-PTH)

•Chemical Coupling of Bisphosphonate (BP)
moiety to synthetic human 1-34 PTH
• Mono-conjugate utilizes Lysine13 amine
group

Mechanism: Intermittently administered PTH
(in low doses) presents an anabolic effect on
bones; stimulates osteoblasts

Localization of •PTH
PTH to bone
•BP
after single dose

PTH-mediated
BP binds to
Hydroxyapatite osteoblast activity
surfaces

Improved
Bone bioavailability
& efficacy of PTH
therapy

Intens. [a.u.]

4119.990

Intens. [a.u.]

4119.990
6000

6000

4000

8195.185
8107.682
8019.068
7973.798
8063.079
8153.478
8239.548
8283.341
7929.963

8327.843
7886.641

8371.998
3970.689
4015.333

8416.423
7844.018
3926.007

7753.375
3882.898

7799.245

8504.527
3795.031
4000 2000

3750.510
0

8107.682
4000 5000 6000 7000 8000 9000
m/z
3882.898

2000

0
000 4000 6000 8000 10000 12000 14000 16000 18000
m/z

Qualitative MALDI-ToF mass spectrum of PTH-PEG-BP after HPLC purification.
PTH MW 4119 & PTH-PEG-BP (mono-substituted species) MW 8107;
a, 0 to 20,000 Da scan of sample; b, Zoom-in view of mass spectra.

•*
•*

•*
•* •*
•* * SIG DIF P<0.05

PEGylated Bone-targeting Calcitonin shows
significantly reduced kidney accumulation,
and increased retention in bone after 24 hours “in vivo”

Calcitonin Receptor Binding
and In Vitro Bioactivity
Intracellular cAMP stimulation in human T47D cells.

120

100
cAMP (% maximal)

80

s CT
60 s CT-SMCC
s CT-SMCC-BP
40

20

0
0 20 40 60 80 100
Dose equivalent to [sCT] (nM)

Ability to trigger calcitonin receptor was retained
by sCT-SMCC and sCT-BP

Procedures
• Six week old female OVX rats were injected
sub cu with 2.5 IU/kg/day sCT or equivalent
analogues
• Analyzed using “in vivo” micro-CT (0, 4, 8,
12, 16 wks) to measure bone mass (%
bone volume) and volumetric bone mineral
density (BMD, after calibration against
known HA “phantoms”)

Micro-Computed Tomography

Skyscan 1076 “in vivo”
Micro-CT (9 µm, 100kV)

Pharmacy Micro-CT Imaging and
Pharmaceutical Research Labs

(Jones and Doschak, Arth Rheum, 2010)

Untreated
OVX rat

baseline 4 weeks 8 weeks 12 week 16 week

Risedronate
treated
OVX rat

STR (Protos)
treated
OVX rat
baseline 4 weeks 8 weeks 12 week 16 week

RIS+STR
treated
OVX rat

(Doschak et al, ISCD, 2008)

Initial BP-CT Data – Rat Osteoporosis
25
Bone Volume / Tissue Volume %

20

Baseline 4 week
15
* *
8 week 12 week
10

5

0
OVX untreated OVX + Calcimar OVX + BP-CT(v2)

* BP-CT significantly outperforms regular Calcitonin “in vivo”

How? Evidence of New Bone Formation in Rat OVX Model
[Dynamic Bone Labeling (light blue colour)]

Light blue colour shows new BP-CONJUGATE RISEDRONATE
bone formation DOSED DOSED

Osteometabolix Pharmaceuticals Inc.

Bone-targeting biologics
The “Next Generation” of Bone Drugs

•79

Osteometabolix (OMX)

• Start-up company based on a new
bone-targeting drug delivery platform
developed at the University of Alberta, Canada
• OMX has expertise in development of bone-
targeting peptide hormone therapeutics, and
expertise in high-resolution Micro-CT bone imaging
• OMX’s Two Lead Products are transformational
drug compounds that offer the promise of safer
and more effective treatments for Bone disease

Summary
BP-conjugated Calcitonin and
BP-conjugated Parathyroid Hormone
• We have developed a novel bone-targeting drug
platform and several lead products (BP-CT and BP-PTH)
that link peptide hormones with bone-specific BP
molecules
• Preclinical studies show significantly increased bone
specificity
• BP-conjugation lowers dose levels and decreases the
side effects relative to unmodified competing
therapeutics in the market
• Patented bone-targeting methodology
•81

Summary 2:
BP-conjugated Calcitonin and
BP-conjugated Parathyroid Hormone
• OMX has engaged local Alberta government innovation
entities (AITF), matching funds, and in-kind research
contributions which are continuing to move our drug
program towards Investigational New Drug (IND) status
• Total Budget = $494,200 with AITF contributing
$248,700. Approved Dec 2012. Expected completion is
January 2014
• The Joint Development Initiative (JDI) has 2 parts:
 chemical synthesis, scale-up and purification = $93k (Duration:
4 months)
 Pre-clinical toxicology studies and supporting analytical work =
$401,200 (Duration: 1 year) •82

3M Microneedle Technology
hollow Microstructured Transdermal System
(hMTS)

• 18 hollow microneedles / cm2
• 900 µm tall (0.9 mm)
• Needles allow for fluid flow
from device into skin
• Wear time 3 to 40 mins

Next Steps
• Projected Costs for FY13-FY15
– $1M to bring compound to Investigational New Drug (IND)
– $1M for Phase I Clinical Trial (administered through the
Alberta Osteoarthritis Team – Doschak is a key PI)
– http://www.oarthritis.com/
• Projected Revenue
– $494K from provincial innovation entities (Approved Dec 2012)
– (Alberta Innovates – Technology Futures Joint Development
Initiative)
– $300K from federal government Grants (Industry partnered)
• Required
– $1.2M from Pharma Partner (or Shareholder Round of
Investment)
– (Current discussions with Merck, Grunenthal, Asahi Kasei)

Evidence of New Bone Formation in Rat OVX Model
[Dynamic Bone Labeling (light blue colour)]

Light blue colour shows new BP-CONJUGATE RISEDRONATE
bone formation DOSED DOSED

Quantitative Imaging Data
(% Vertebral Bone Volume)

(Bhandari and Doschak; Wu and Doschak, ASBMR 2010)

Principle of K-Edge Subtraction (KES)
imaging of Strontium in Bone

Strontianite
Below Sr K-edge (16.020 keV)

Above Sr K-edge (16.180 keV)

Subtraction Image

Strontium Solution Standards
(3-D Synchrotron KES micro-CT Imaging)

Dynamic Strontium Label in Remodeling
Rat Bone (3-D Synchrotron KES Render)

Acknowledgements
• Pharmaceutical Orthopaedic Research Lab
(PORL) Staff & Students
– Dr. Krishna Bhandari (Senior Scientist, Pharmasciences)
– Dr. Yang Yang (Postdoctoral Fellow)
– Madhuri Newa (PhD/ Pharmaceutical Sciences)
– Arash Panahifar (PhD/ Pharmaceutical Sciences)
– Kathy Tang (PhD/ Pharmaceutical Sciences)
– Yuchin Wu (PhD/Biomedical Engineering)
– Imran Khan (Micro-CT Imaging Technician)

BONE TREK: Next-Gen Drugs for Bone Diseases

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Andere mochten auch

Andere mochten auch (8)

Ähnlich wie BONE TREK: Next-Gen Drugs for Bone Diseases

Ähnlich wie BONE TREK: Next-Gen Drugs for Bone Diseases (20)

Kürzlich hochgeladen

Kürzlich hochgeladen (20)

BONE TREK: Next-Gen Drugs for Bone Diseases