PharmacologyPharmacology ofof bisphosphonatesbisphosphonates Dr.Dr. FraserFraser CoxonCoxon Bone & Musculoskeletal Research Programme Institute of Medical Sciences University of Aberdeen OutlineOutline

•• IntroductionIntroduction toto osteoclastsosteoclasts •• CultureCulture andand analysisanalysis ofof osteoclastsosteoclasts inin vitrovitro •• MechanismMechanism ofof actionaction ofof bisphosphonatesbisphosphonates •• MechanismMechanism ofof actionaction ofof phosphonocarboxylatephosphonocarboxylate analoguesanalogues ofof bisphosphonatesbisphosphonates •• LocalisationLocalisation ofof thesethese compoundscompounds inin bonebone FormationFormation ofof osteoclastsosteoclasts

M-CSF Osteoclast precursor 1,25(OH)2D3 osteoblastosteoblast PTH Oestrogen NFκB

RANKLRANKL Pre-Pre- OsteoclastOsteoclast c-fos RANKRANK

MatureMature OsteoclastOsteoclast

RANKRANK == receptorreceptor activatoractivator ofof NFNFκκBB RANKLRANKL == RANKRANK ligandligand IdentifyingIdentifying featuresfeatures ofof osteoclastsosteoclasts

• Multinucleated Phase contrast • Have high levels of TRAP (tartrate-resistant acid phosphatase) • Abundance of acidic vesicles

• αvβ3 integrin (vitronectin receptor) on the cell surface • Expression of cathepsin K

Staining for TRAP vitronectin receptor (green) Cath K activity (red) activity acidic vesicles (red) (live osteoclast) IdentifyingIdentifying featuresfeatures ofof resorbingresorbing osteoclastsosteoclasts

• Formation of distinct membrane domains (e.g. ruffled border) • Polarisation of cytoskeleton into an F-actin ring • Resorption of bone! Resorption pits on Actin rings visualised by bone surface staining with fluorescent visualised reflected phalloidin conjugates light microscopy OsteoclastsOsteoclasts areare specialisedspecialised bonebone--resorbingresorbing cellscells

F-actin ‘ring’ sealing zone

H+ CK H+ CK H+ BONE

ruffled border

Bone is resorbed by the secretion of acid and cathepsin K (CK) at the ruffled border of the osteoclast Analysis of resorbing osteoclasts

• Osteoclasts need to be cultured on a mineralised substrate –Dentine – Cortical bone – Hydroxyapatite discs

axial views: hOsteoclasts on dentine 30µm

10µm hOsteoclast on glass UnusualUnusual membranemembrane domainsdomains inin osteoclastsosteoclasts

basolateral Vesicular - basolateral domain stomatitis virus in epithelial cells

apical basolateral Influenza - apical domain in virus epithelial cells

apical Salo et al. 1996 J Cell Sci 109, 391

LAMP-2

Ruffled border: Properties of an endosomal/lysosomal Vitronectin membrane receptor

Palokangas et al 1997 J Cell Sci 110, 1767 Unique membrane domains in osteoclasts

FSD (apicalFSD (apicaldomain) domain)

basolateralBL BLbasolateral domain domain

sealing zone SZSZ SZ

ruffled border Bone

FSD- Functional secretory domain SZ- sealing zone (F-actin ring) Vesicular trafficking routes in osteoclasts

FSD (apicalFSD (apicaldomain) domain)

basolateralBL BLbasolateral domain domain

sealing zone SZSZ SZ

ruffled border Bone

FSD- Functional secretory domain SZ- sealing zone (F-actin ring) SourcesSources ofof osteoclastosteoclast culturescultures IsolationIsolation ofof maturemature osteoclastsosteoclasts

• relatively low numbers • impure cultures • useful for resorption assays or Mince and single cell studies scrape the bones to release osteoclasts washing or digestion

Mixed culture of Pure culture of TRAP osteoclasts/other +ve osteoclasts bone cells GenerationGeneration ofof osteoclastsosteoclasts inin vitrovitro

• high numbers • fairly pure cultures • useful for formation assays or biochemical analyses flush bone marrow from long bones Isolate PBMCs from human blood

M-CSF M-CSF + M-CSF + M-CSF + M-CSF Seed cells in RANKL RANKL RANKL petri-dishes

Day 3 Day 6 Day 9 Day 12 Day 15 Re-seed on to Day 0 resorbable substrate Resorbing osteoclasts e.g. dentine cortical bone GenerationGeneration ofof osteoclastsosteoclasts inin vitro-vitro-beforebefore RANKL!RANKL!

flush bone Isolate marrow from marrow from osteoblasts Osteoclast long bones from calvaria

Co-culture cells in the presence of 1,25(OH)2D3 ~ 8 Days

Useful for assessing osteoblast-osteoclast interactions in osteoclast formation e.g. whether a defect is in the osteoblast or osteoclast lineage IsolationIsolationIsolation ofofof osteoclastsosteoclastsosteoclasts exexex vivovivovivo

Isolate osteoclasts using antibody to surface marker (e.g. VNR) and magnetic bead separation

Pure culture of VNR+ve osteoclasts

Useful for assessing the effect of a drug on osteoclasts in vivo BisphosphonatesBisphosphonates Bisphosphonates bind calcium ions and target bone mineral

OO - = Side chain = OO PP OHOH

CC Ca2+ OHOH 1 PP = OHOHRR = OO- OO Localisation of a fluorescent BP (RIS) in mouse vertebra

cartilage

trabecular bone Bone marrow Bone

Green = FAM-RIS Blue = nuclei 0.1 mm OsteoclastsOsteoclastsOsteoclasts engulfengulfengulf bisphosphonatebisphosphonatebisphosphonate intointointo intracellularintracellularintracellular vesiclesvesiclesvesicles Coxon et al 2008, Bone 42:848-60

osteoclast

resorption pit

dentine surface

zx green = bisphosphonate Green- F-ALN red = actin cytoskeleton Red- F-actin Blue-VNR Bisphosphonates are selectively internalised from mineralised surfaces by osteoclasts Rabbit osteoclasts cultured on dentine labelled with fluorescent alendronate Bisphosphonate dextran * (endocytic marker) * * * * * *

Green- F-ALN Green- dextran Red- F-actin Red- F-actin Blue-VNR Blue-VNR EffectsEffects ofof BPsBPs onon osteoclastosteoclast morphologymorphology

Ruffled border Ruffled border cell morphology in vivo cytoskeleton in vivo in vitro (F-actin ring) resorbing osteoclast

retraction apoptosis

+ RIS

Sato et al 1991, J Clin Invest 88, 2095 TwoTwo groupsgroups ofof bisphosphonatesbisphosphonates withwith differentdifferent modesmodes ofof actionaction

Simple Bisphosphonates Nitrogen-containing Bisphosphonates

O O O =

OH = Cl = OH OH P P P OH H N OH OH 2 N H2N OH OH OH HO P HO P P =

Cl = = OH OH OH O O O pamidronate alendronate clodronate O

CH3 = OH P OH N ibandronate O OH = OH HO P

H C = 3 P OH O OH = O O OH

= OH P OH OH HO P P = OH OH N N OH O HO P OH = OH HO P N etidronate = OH O zoledronate O risedronate BisphosphonatesBisphosphonates inhiinhibitbit farnesylfarnesyl diphosphatediphosphate synthasesynthase inin thethe mevalonatemevalonate pathwaypathway

HMG-CoA Luckman et al, 1998 Fisher et al, 1999 Van Beek et al, 1999 mevalonate Keller et al, 1999 Bergstrom et al, 2000 phosphomevalonate Grove et al, 2000 Dunford et al, 2001 mevalonate diphosphate

dimethylallyl diphosphate isopentenyl diphosphate N-BPs e.g. pamidronate alendronate geranyl diphosphate geranyl diphosphateFPP synthase Ibandronate x risedronate zoledronate cholesterol squalene farnesyl diphosphate

geranylgeranyl diphosphate MostMost smallsmall GTPasesGTPases areare modifimodifieded byby prenylationprenylation

O O O -P - O - P Ras O O farnesyl diphosphate farnesyl C S A transferase A Ras proteins X

O O O -P - O - P Rho Rab O Ogeranylgeranyl diphosphate Rab proteins Rab diphosphate geranylgeranyl C S geranylgeranyl Transferase I A A C S transferase X Rho proteins X Rap1 C S SmallSmall GTPasesGTPases actact asas molecularmolecular switchesswitches

Guanine nucleotide exchange factor (GEF)

GTP GEF GDP GDP Ras ““OffOff”” ““OnOn””

GDI GTP GDP Rho GDPGTPase GTPase Effector Translocation to membrane

GDP Rab Pi GAP downstream REP signalling GTPase activating protein (GAP) BisphosphonatesBisphosphonates inhibitinhibit proteinprotein prenylationprenylation HMGCoA Ctl

A- Rab GTPases A 14 B B- 21 kDa GTPases [ C]mevalonate

dimethylallyl diphosphate isopentenyl diphosphate

FPP geranyl diphosphate FPP synthase

farnesyl diphosphate [Ras14C] Ras proteins FTase Ras S

[14C] Rab Geranylgeranyl Rho[14 familyC] Rho proteins Rab GGTase diphosphate GGTase I Rap1proteins Rap1 S Rab Rho S S BisphosphonatesBisphosphonates inhibitinhibit proteinprotein prenylationprenylation HMGCoA Ctl RIS

A- Rab GTPases A 14 B B- 21 kDa GTPases [ C]mevalonate

dimethylallyl diphosphate isopentenyl diphosphate

BPs FPP e.g. RIS synthase

FTase Ras

Rab GGTase GGTase I

Rab Rho RIS disrupts the localisation of Rab6 in rabbit osteoclasts

Rab6 WGA (Golgi) merge Ctrl

RIS Loss of prenylated small GTPases disrupts osteoclast function

cytoskeleton disruption Induction of apoptosis lossloss ofof prenylatedprenylated smallsmall GTPasesGTPases

Rho, Rac, Cdc42 Rho, Rac (regulate the actin (regulate cell survival) cytoskeleton)

Rabs Loss of the ruffled border (regulate vesicular Transport) FPPFPP synthase synthase catalyses catalyses the the condensation condensation of of isoprenoid isoprenoid lipid lipid chainschains

5-carbon 5-carbon 10-carbon + IPP 15-carbon IPP + DMAPP GPP FPP O O O O - - - = = =

= - = = - = O O O = O P P OH P OH P OH P OH O O O O P OH P OH P OH P OH ======

- = O- O- O- = O- O O O O

FPP synthase DMAPPGPP substratesubstrate IPP DMAPP/GPP substrate RisedronateRisedronate co-ordinates co-ordinates withwith 33 magnesiummagnesium ionsions inin thethe GGPGGP binding binding site site of of FPPS FPPS Kavanagh et al 2006, Proc. Natl. Acad. Sci 103, 7829-7834 Courtesy of Jean-Michel Rondeau, Novartis CorrelationCorrelation between between inhibition inhibition of of FPP FPP synthase synthaseinin vitro vitro andand anti anti-resorptive-resorptive potencypotency in in vivo vivo Dunford et al, J Pharm Exp Ther 2001

10000 IC50 (nM) for inhibition r=0.95, p<0.0001 of rhFPP synthase 1000

100

10

1 0.0001 0.001 0.01 0.1 1 Lowest effective dose (mgP/kg) in vivo TheThe nitrogennitrogen inin thethe sideside chainchain of of risedronate risedronate or or zoledronate zoledronate interactsinteracts withwith aa conservedconserved threoninethreonine and and lysinelysine residueresidue inin FPPFPP synthasesynthase

ideal distance is ~3Å

Zoledronate 2.96

Risedronate 2.97

Alendronate 3.84

Ibandronate 4.03

Zoledronate Risedronate

This interaction enables ‘slow tight binding’ of the Kavanagh et al 2006 Rondeau JM et al 2006 enzyme Proc. Natl. Acad. Sci Chem Med Chem PPhosphonocarboxylatehosphonocarboxylate analogueanalogue ofof RISRIS

Bisphosphonate Phosphonocarboxylate

O O OH OH P OH P OH

OH C O HO P HO N OH N O OH Risedronate (RIS) NE10790 (3-PEHPC) 50x lower affinity for bone 5,000x lower anti-resorptive potency NE10790NE10790 isis aa novelnovel inhibitorinhibitor ofof RabRab GGTaseGGTase

[14C]mevalonate Coxon et al 2001 J Biol Chem 276, 48213 Ctl RIS

A B DMAPP/IPP

A- Rab GTPases FPP B- 21 kDa GTPases synthase

farnesyl diphosphate [14C] Ras (FPP) FTase Ras S

Ras S Geranylgeranyl [14C] Rab [14C] Rho proteins diphosphate proteins Rap1 (GGPP) GGTase I Rab GGTase

Rho S Rab S S Rho S

Rab S S NE10790NE10790 isis aa novelnovel inhibitorinhibitor ofof RabRab GGTaseGGTase

[14C]mevalonate Coxon et al 2001 J Biol Chem 276, 48213 Ctl RIS NE10790 [ C]mevalonate

A B DMAPP/IPP

A- Rab GTPases FPP B- 21 kDa GTPases synthase

14 farnesyl diphosphate [ C] Ras NE10790 (FPP) FTase Ras S

Ras S Rab Geranylgeranyl [14C] Rho proteins diphosphate Rab Rap1 (GGPP) GGTase I Rab Rab GGTase

Rho S

Rho S NE10790 disrupts localisation of Rab6 in rabbit osteoclasts

Rab6 WGA (Golgi) merge Ctrl

NE10790 TheThe RabRab familyfamily ofof smallsmall GTPasesGTPases

Family of small GTPases (21-28kDa) with more than 70 human isoforms

Rab GTPases are crucial regulators of vesicle trafficking: •Vesicle formation •Vesicle motility •Vesicle docking •Membrane fusion Try Schwartz?

Schwartz et al (2007) J Cell Sci 120: 3905-3910 NE10790NE10790 inhibitsinhibits bonebone resorptionresorption inin vitrovitro withoutwithout affectingaffecting thethe cytoskeletoncytoskeleton 140 140 RIS NE10790 120 120 100 100 Resorption area 80 80 actin rings % of ctrl % of ctrl % of ctrl % of ctrl 60 60 40 40 20 20 0 0 0.1 1 10 100 0.1 0.5 1 Concentration (µM) Concentration (mM) RIS NE10790 Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Coxon et al 2001, J Biol Chem 276, 48213-48222

Control

1mM NE10790 Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Coxon et al 2001, J Biol Chem 276, 48213-48222 Control 1mM NE10790 SEM

confocal * (axial views) DoDo thesethese drugsdrugs inhibitinhibit proteinprotein prenylationprenylation inin osteoclastsosteoclasts inin vivovivo?? DoDo thesethese compoundscompounds inhibitinhibit proteinprotein prenylationprenylation inin vivovivo??

2mgP/kg 23c6 & anti- NE10790 mouse IgG- or RIS coated 24 hrs Dynal beads

VNR+ve osteoclasts Purified osteoclast pellet

In vitro prenylation assay using rh Rab GGTase Assay for unprenylated Rap1A BPs and PCs inhibit protein prenylation in osteoclasts in vivo

+ve: osteoclast fraction Unprenylated Rabs detected -ve: non-osteoclast bone marrow 3 by prenylating with [ H]GGPP cells in vitro: J774 cells 0.5mg/kg 45mg/kg in vitro CTL RIS PC Ctl NE10790 +ve -ve +ve -ve +ve -ve Rab proteins prenylated in vitro Rab S Rab S S S

Rab S Rab S Rab western blotting for unprenylated Rap1A: in vitro prenylation assay CTL RIS PC +ve -ve +ve -ve +ve -ve Rab S +ve -ve Rab S S S unprenylated Rab S 3H S Rap1A Rab S S Rab S 3H S actin AreAre thethe effectseffects ofof BPsBPs reallyreally duedue toto inhibitioninhibition ofof FPPS?FPPS? Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Fisher et al 1999, Proc Natl Acad Sci, 96:133-138; Coxon et al 2000, J Bone Miner Res 15:1467-1476

120

mevalonate 100

Bone resorption 80 ALN (% of control) 60

40

farnesyl 20 farnesylated diphosphate proteins 0 Ctrl 60µM ALN+ ALN+ Farnesol ALN GGOH FOH (FOH)

Risedronate (µM) geranylgeranyl geranylgeranyl Geranylgeranylated + GGOH diphosphate diphosphate Rho proteins 0 12.5 25 50 12.5 25 50 β-actin unprenylated geranylgeraniol Geranylgeranylated Rab 6 (GGOH) Rab proteins Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Coxon et al 2000, J Bone Miner Res 15:1467-1476 Coxon et al 2003, Calcif Tissue Int 72:80-84

mevalonate Osteoclast polarisation:

BPs

FTI-277 farnesyl farnesylated diphosphate proteins Ctrl 10µM FTI 10µM Osteoclast resorption: GGTI Resorption GGTI-298 area (mm2) 0.1 geranylgeranyl 0.08 Geranylgeranylated diphosphate Rho proteins 0.06 0.04 0.02 Geranylgeranylated 0 Rab proteins Ctrl 5µM10µM 5µM 10µM FTI FTI GGTIGGTI Summary

• Assays using osteoclasts in vitro and ex vivo have enabled us to determine the mechanism of action of bisphosphonates and the related phosphonocarboxylates

– Inhibition of prenylation of small GTPases

• Morphological analysis of osteoclasts treated with these compounds has also shed light on the importance of prenylated small GTPases for osteoclast function LocalisationLocalisation ofof BPsBPs andand PCsPCs inin bonebone BisphosphonatesBisphosphonates havehave differentdifferent affinitiesaffinities forfor hydroxyapatite hydroxyapatite Nancollas et al 2006, Bone 38: 617-627

The R2 side-chain of BPs also contributes to bone affinity…

HAP Adsorption Affinity Constants at pH 7.4

4

3 ZOL 1 1 - - L mol L mol 6 6 2 /10 /10 ALN L L K K

1 RIS

0 CLO ETD RIS IBN ALN ZOL

Lawson, Triffitt, Ebetino & Russell ASBMR & Davos 2005 & 2006 AreAre osteocytesosteocytes exposedexposed toto bisphosphonatesbisphosphonates inin vivovivo??

Photo by Lilian Plotkin and Lynda Bonewald

Lower affinity BPs should be able to gain access to more sites in bone than higher affinity BPs which will get ‘stuck’ at sites of first contact Anti-apoptotic effect of BPs on osteocytes (MLO-Y4 cells)

BPs can open Connexin 43 hemichannels and activate anti-apoptotic signalling pathways via ERKS risedronate etidronate 150 150

100 100 * 50 induced apoptosis 50

- * * * * * * * * * 0 0 % etoposide %

-50 -50 0100 10-11 10-10 10-9 10-8 10-7 10-6 10-5 0100 10-11 10-10 10-9 10-8 10-7 10-6 10-5

concentration (M) concentration (M)

IC50 values in the nM range!

Plotkin, Manolagas, Bellido, Bone 39 (2006) 443-452 Methods

• Mice or rats injected subcutaneously with fluorescently labelled conjugates (fluorescein, rhodamine or AlexaFluor 647) of RIS and 3-PEHPC • Animals sacrificed 24h later and tibiae fixed and embedded in MMA resin • Resin blocks cut transversely and bone examined directly by confocal microscopy FF--RISRIS localiseslocalises toto osteocyteosteocyte lacunaelacunae inin mousemouse corticalcortical bonebone

osteocyte

Vascular channel

Green- F-RIS Blue-nuclei Red- wheat germ agglutinin AcknowledgementsAcknowledgements

Bone Research Group, Procter & Gamble Pharmaceuticals Aberdeen Hal Ebetino Mike Rogers Mark Lundy Miep Helfrich Adam Taylor University of Southern California Anke Roelofs Charles McKenna Debbie Scott Imperial College, London Aysha Khalid Miguel Seabra University of Oxford Mimi Mules Graham Russell Queen Mary, University of London Jim Dunford Alan Boyde