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Bisphosphonate Binding Affinity Affects Drug Distribution in Both Intracortical and Trabecular Bone of Rabbits

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Bisphosphonate Binding Affinity Affects Drug Distribution in Both Intracortical and Trabecular Bone of Rabbits
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  ORIGINAL RESEARCH Bisphosphonate Binding Affinity Affects Drug Distributionin Both Intracortical and Trabecular Bone of Rabbits John Turek  • F. Hal Ebetino  • Mark W. Lundy  • Shuting Sun  • Boris A. Kashemirov  • Charles E. McKenna  • Maxime A. Gallant  • Lilian I. Plotkin  • Teresita Bellido  • Xuchen Duan  • James T. Triffitt  • R. Graham G. Russell  • David B. Burr  • Matthew R. Allen Received: 10 June 2011/Accepted: 28 November 2011/Published online: 17 January 2012   Springer Science+Business Media, LLC 2012 Abstract  Differences in the binding affinities of bis-phosphonates for bone mineral have been proposed todetermine their localizations and duration of action withinbone. The main objective of this study was to test thehypothesis that mineral binding affinity affects bis-phosphonate distribution at the basic multicellular unit(BMU) level within both cortical and cancellous bone. Toaccomplish this objective, skeletally mature female rabbits( n  =  8) were injected simultaneously with both low- andhigh-affinity bisphosphonate analogs bound to differentfluorophores. Skeletal distribution was assessed in the rib,tibia, and vertebra using confocal microscopy. The stainingintensity ratio between osteocytes contained within thecement line of newly formed rib osteons or within thereversal line of hemiosteons in vertebral trabeculae com-pared to osteocytes outside the cement/reversal line wasgreater for the high-affinity compared to the low-affinitycompound. This indicates that the low-affinity compounddistributes more equally across the cement/reversal linecompared to a high-affinity compound, which concentratesmostly near surfaces. These data, from an animal modelthat undergoes intracortical remodeling similar to humans,demonstrate that the affinity of bisphosphonates for thebone determines the reach of the drugs in both cortical andcancellous bone. Keywords  Anti-remodeling    Skeletal distribution   Drug accumulation    Fluorescent bisphosphonate Introduction All bisphosphonates have a common P-C-P backbone yetvary with respect to their two side groups. Differences inthese side chains affect both the potency of osteoclastenzyme inhibition and the binding affinity to bone min-eral [1–3]. Binding affinity has been suggested to play a key role in a number of clinically important effectsincluding the speed of onset for fracture reduction, therecovery of bone turnover following treatment with-drawal, and differences in fracture risk reduction acrossskeletal sites [3]. M. Allen has received funding from Warner Chilcott. D. Burr hasreceived remuneration from Eli Lilly and Amgen; serves as consultantfor Eli Lilly, Procter and Gamble, Amgen, and PharmaLegacy; and hasreceived funding from Eli Lilly, Procter and Gamble, Pfizer,NephroGenex, The Alliance for Better Bone Health, and Amgen.C. Mckenna, M. Lundy, and G. Russell have had consultant/advisoryroles to WarnerChilcott. F. Ebetino is an employee of Warner Chilcott.All other authors have stated that they have no conflict of interest.J. Turek Department of Basic Medical Sciences, Purdue University,West Lafayette, IN, USAJ. Turek     M. W. Lundy    M. A. Gallant   L. I. Plotkin    T. Bellido    D. B. Burr    M. R. Allen ( & )Department of Anatomy and Cell Biology,Indiana University School of Medicine,635 Barnhill Dr., MS 5035, Indianapolis, IN 46202, USAe-mail: matallen@iupui.eduF. H. EbetinoWarner-Chilcott, Dundalk, Co Louth, IrelandS. Sun    B. A. Kashemirov    C. E. McKennaDepartment of Chemistry, University of Southern California,Los Angeles, CA, USAX. Duan    J. T. Triffitt    R. G. G. RussellNuffield Department of Orthopaedics, Rheumatology &Musculoskeletal Sciences, Nuffield Orthopaedic Centre, OxfordUniversity Institute of Musculoskeletal Sciences, Headington,Oxford OX3 7LD, UK   1 3 Calcif Tissue Int (2012) 90:202–210DOI 10.1007/s00223-012-9570-0  Binding affinity determines the strength of drugattachment to mineral, yet it also affects where the drug isdistributed throughout the skeleton. Upon exiting the vas-culature, bisphosphonates will move through nonmineral-ized space and become bound to any mineral theyencounter. Using radiolabeled [4] or fluorescent [5–9] bisphosphonates, studies have documented binding of drugto trabecular and endocortical bone surfaces, vascularchannels, and osteocyte lacunae. Compared with radioac-tive labeling, the new fluorescent imaging probes derivedfrom modern clinical nitrogen-containing bisphosphonatessuch as risedronate (RIS) offer important advantages of convenience, safety, resolution, sensitivity, and versatility[10]. Near-infrared fluorescent dyes can be applied in thisapproach to create imaging probes with emission wave-lengths of 600–1,000 nm. These are ideal tools for in vivoimaging because autofluorescence from the tissues isminimized in this optical window [11]. Using such fluo-rescently labeled bisphosphonates that possess differentbinding affinities, it has been demonstrated that drugs withlower affinity penetrate deeper into the matrix at the tra-becular or endocortical surfaces relative to those withhigher affinities [7–9]. These data suggest that agents with lower binding affinity may have greater access to theosteocyte – canalicular network compared to higher-affin-ity compounds. As bisphosphonates attenuate osteocyteapoptosis [12, 13], this differential distribution could influence their pharmacological actions.The main objective of this study was to test thehypothesis that mineral binding affinity affects bis-phosphonate distribution at the basic multicellular unit(BMU) level within either cortical or cancellous bone. Toaccomplish this objective, skeletally mature female rabbitswere injected simultaneously with both low- and high-affinity bisphosphonate analogs and then skeletal distribu-tion at the BMU level was assessed using confocalmicroscopy. Materials and Methods AnimalsSkeletally mature female New Zealand white rabbits(9 months old,  n  =  8) were obtained from Myrtle’s Rab-bitry (Thompson Station, TN). Rabbits were used for thisstudy as they, unlike rodents, have haversian systems and,therefore, a cortical bone vascular pattern similar to that of humans. Animals were housed individually at the IndianaUniversity animal care facility and had ad libitum access tofood and water. Prior to the study, all protocols wereapproved by Indiana University School of Medicine Insti-tutional Animal Care and Use Committee.Synthesis and Purification of Fluorescent ProbesRIS was kindly provided by Warner Chilcott Pharmaceu-ticals (Dundalk, Ireland). 5(6)-X-Rhodamine succinimidylester (SE) and Alexa Fluor 647 SE were purchased fromInvitrogen (Carlsbad, CA). The high-affinity compoundAF647-RIS was synthesized by direct reaction betweenRIS and the linker in aqueous alcohol, followed by linkerdeprotection and conjugation with the activated dye, andthen purified and characterized as described previously [10,14]. The low-affinity compound, a phosphonocarboxylate(ROX-3-PEHPC), was synthesized similarly using a rho-damine-2 fluorophore. Previous work has shown thatphosphonocarboxylates, which substitute one phosphonategroup of a bisphosphonate with a carboxylate group, arefunctional analogs of traditional bisphosphonates yet havelower mineral binding affinity [2]. The specific phospho-nocarboxylate used in this study has a 50% lower mineralbinding affinity compared to the parent RIS compound,determined using a hydroxyapatite column binding assay.The addition of fluorescent tags has been shown to reducebone affinity by about 25% relative to the parent compound[5, 10]. All probes had satisfactory  1 H and  31 P NMRspectra, LC-MS and HRMS, and emission and absorptionspectra and were [ 98% pure by HPLC.Experimental DesignFollowing 1 week of acclimatization at the Indiana Uni-versity School of Medicine, animals were randomlyassigned to two groups ( n  =  4/group). One group was fed alow-calcium diet (Harlan Teklad, Madison, WI; TD.87370,0.1% calcium) in order to stimulate bone turnover [15].The second group was fed a normal-calcium diet (HarlanTeklad Global Rabbit Diet 2030, 0.89% calcium). Asdynamic histomorphometric measures revealed no differ-ence in remodeling rate between these two groups(Table 1), all animals were pooled together for analyses.After 8 weeks on the diet, a duration corresponding toroughly two remodeling cycles in a rabbit [16], animalswere injected intramuscularly with calcein (10 mg/kg doseat a concentration of 20 mg/ml) to label actively formingbone surfaces; a second injection of calcein was adminis-tered 10 days later. One week after the second calceininjection, all animals were given simultaneous intravenousinjections of two different fluorescent bisphosphonateanalogs. Agents were dissolved in phosphate-bufferedsaline and administered at a dose of 3  l g phosphorus/kgbody weight [5]. As a control, two additional rabbits, fed anormal-calcium diet, were treated as described above withcompounds labeled with the opposite fluorophore (AF647on the low-affinity compound and rhodamine-2 on thehigh-affinity compound) to qualitatively determine if the J. Turek et al.: Bisphosphonate Binding Affinity 203  1 3  specific fluorophore had an effect on the imaging propertiesor penetration depth characteristics.Twenty-four hours after dosing of the bisphosphonateanalogs, animals were killed by intravenous injection of alethal dose of sodium pentobarbital. The rib, tibia diaphy-sis, and third lumbar vertebra were dissected free andplaced in 10% neutral buffered formalin for 24 h and thenswitched to 70% ethanol. The rib and tibia were used ascortical bone sites with high and low basal turnover,respectively. The vertebra was used as a trabecular bonesite of clinical relevance.Tissue ProcessingBones were embedded in methyl methacrylate using astandard protocol [17]. The rib and tibia were sectioned inthe transverse plane using a diamond wire saw and thenhand-ground to a final thickness of   * 50  l m. Lumbarvertebrae were sectioned (8  l m thickness) in the sagittalplane using a rotary microtome. All slides were covers-lipped using a nonfluorescent mounting medium (Eukitt;Kindler, Freiburg, Germany).Confocal MicroscopyBone sections were examined using an Olympus (Tokyo,Japan) FV-1000 confocal microscope with a 60 9  oilimmersion lens (Fig. 1). Fluorescence intensity from eachchannel (calcein, rhodamine-2, and AF647) was optimizedfor black level without any oversaturated pixels. Excitationwavelengthsforcalcein,rhodamine-2,andAF647were488,539, and 635 nm, respectively. Emission spectra collectionranges were adjusted around the emission peaks of 520 nm(calcein), 577 nm (rhodamine-2), and 688 nm (AF647) toprovide the maximal signal intensity with minimal channelcross-talk. Image Z stacks (1  l m optical thickness) of 10–12 image planes (rib and tibia) and 5–8 image planes(lumbar vertebrae) were collected sequentially with onlyone laser on per fluorophore. Image stacks from threedouble calcein-labeled osteons in the cortical bone of therib and tibia or hemi osteons in the trabecular bone of lumbar vertebrae were collected from each animal. Hemiosteons in lumbar vertebrae were identified by collagenfiber orientation using brightfield polarized light.Image AnalysisImages were exported as 24-bit composite RGB TIFF filesusing the Olympus FV-10-ASW viewer software. ImageJ(NationalInstitutesofHealth,BethesdaMD)wasusedforallsubsequent image analyses [18]. A maximum intensity Zprojection of each image stack was made, and the red(AF647-HIGH) and blue (RHOD2-LOW) channels wereseparated. The lookup table was inverted so that stainedregions were dark upon a light-colored background. Abrightfieldimagewasusedtoidentifytheosteoncementlineor hemi osteon structure, and this image was synchronizedwith the red and blue channel windows so that tracings onone image were identically traced in other windows.Stained regions around osteocytes were traced (Fig. 2),and the staining intensity was determined using the meangray-scale value of all pixels outlined. All osteocytes were Table 1  Effects of calcium restriction on cortical and trabecularbone remodelingLow-calciumdiet ( n  =  4)Normal-calciumdiet ( n  =  4) P TibiaMAR ( l m) 2.24  ±  0.71 1.58  ±  1.01 0.39BFR (%/year) 8.54  ±  4.99 1.19  ±  1.07 0.08RibMAR ( l m) 1.44  ±  0.32 1.64  ±  0.41 0.15BFR (%/year) 37.55  ±  9.84 50.30  ±  23.5 0.25VertebraMAR ( l m) 2.27  ±  0.34 2.39  ±  0.17 0.77MS/BS (%) 42.6  ±  13.6 31.2  ±  3.33 0.25BFR ( l m 3  /  l m 2  /year) 99.8  ±  41.2 74.6  ±  11.3 0.25Data are presented as mean  ±  standard deviation  MAR  mineral apposition rate,  BFR  bone formation rate,  MS   /   BS  mineralizing surface per unit bone surface P  values from Mann–Whitney nonparametric tests Fig. 1  Maximum-intensity projection of confocal image stack show-ing a double calcein label ( green ) at a remodeling site on theendocortical surface of the rib ( arrow ). A single labeled osteon canalso be seen nearby. The fluorescent agents can be observed near theendocortical surface and the bone adjacent to the Haversian canal.  Blue  (RHOD2 low-affinity compound) staining penetrates farther intothe bone at the endocortical surface.  Magenta  is a combination of thehigh-affinity (AF647-HIGH) and low-affinity (RHOD2-LOW) agentscolocalized at a given site (bar  =  50  l m)204 J. Turek et al.: Bisphosphonate Binding Affinity  1 3  traced in two areas: (1) those within the osteon/hemi osteonand (2) those within a 100  l m distance outside the struc-ture. For both high and low compounds, the average gray-scale value (osteocyte staining) of all osteocytes within thecement line was compared to the same number of osteo-cytes outside the cement line to provide a staining intensityratio. This method of analysis was chosen as it allows eachimage to serve as its own control and removes variation of staining intensity between the different fluorophores as afactor in the analysis. At each skeletal site data from theindividual osteons were summed, and those values wereused to calculate ratios.Dynamic HistomorphometryDynamic histomorphometric measurements were made onthe tibia, rib, and vertebrae using a Nikon (Tokyo, Japan)Optiphot 2 fluorescent microscope and Bioquant image Fig. 2  A typical osteon withinthe rib cortex that was analyzedfor drug distribution. a  Brightfield image of theosteon used to identify thecement line that defines theouter boundary.  b  Brightfieldimage of the osteon depictingthe tracing of the cement linefor analyses.  c  3D imagereconstruction showing calceinlabeling ( green pseudocolor  ). d  3D image reconstruction of image stack showing calceinlabeling and both fluorescentbisphosphonate stains of boneadjacent to the haversian canals,canaliculi, and osteocytes. Green pseudocolor   representscalcein,  blue  representsRHOD2-LOW staining,  red  represents AF647-HIGHstaining, and  magenta represents the combination of RHOD2 and AF647colocalization.  e  Isolation of RHOD2 channel showinglocalization of the low-affinitydrug (no pseudocolor,  dark  pixels  indicate regions of drugbinding) independent of allother fluorophores.  f   Isolation of AF647 channel showinglocalization of the high-affinitydrug (no pseudocolor,  dark  pixels  indicate regions of drugbinding) independent of allother fluorophores(bar  =  50  l m). The greaternumber of   darker pixels  in e  than  f   shows that the low-affinity bisphosphonate isdistributing more deeply intothe tissue than the higher-affinity bisphosphonateJ. Turek et al.: Bisphosphonate Binding Affinity 205  1 3  analysis system (R&M Biometrics, Nashville, TN). Forvertebrae, mineralizing surface/bone surface (MS/BS)—{[double label surface  ?  (0.5* single label surface)]/bonesurface}  9  100—mineral apposition rate (MAR)—distance between calcein labels/days between sets of labels—and bone formation rate (BFR/BS)—MAR *(MS/ BS/100) *365—were collected using standard methods aspreviously described [17]. For rib and tibia diaphysis, in-tracortical measures of MAR and bone formation rate(BFR/B.Ar)—MAR* [(labeled surface/2)/cortical bonearea] *365—were determined [19]. Two histological sec-tions were analyzed for each animal at each region.StatisticsAll statistical tests were run using GraphPad (San Diego,CA) Prism software. The effects of calcium restrictionand between-staining ratios were assessed using Mann-Whitney and Wilcoxon nonparametric tests.  P  B  0.05 wasconsidered statistically significant. Results Effect of Calcium Restriction on Bone Turnover RateFollowing 8 weeks of calcium restriction, bone remodelingwas not significantly altered in any of the skeletal sitesassessed (Table 1). Therefore, the two groups of animalswere pooled for all analyses.Fluorescent Bisphosphonate Localization in CorticalBoneQualitative observations in the rib revealed that the low-and high-affinity compounds labeled the endocortical sur-face and the bone surrounding the haversian canals withhigh intensity, while the canaliculi and osteocyte lacunaewere labeled to a lesser degree (Fig. 2). Endocortical sur-faces and haversian canals were routinely observed to havetwo bands of fluorescence, with the low-affinity drugslightly deeper in the matrix compared to the higher-affinity drug (Fig. 1). This was likely due to the fact thatthe low-affinity drug penetrated deeper into the matrixcompared to the higher-affinity drug, consistent with pre-vious reports using two different-affinity compounds [7, 8]. In control animals that were dosed with agents havingflipped fluorophores, the qualitative banding pattern wasreversed, indicating that the banding was not a function of the fluorophore but rather a function of the binding affinityof the drug (Fig. 3).Quantitatively, the staining intensity ratio betweenosteocytes contained within the cement line of newlyformed rib osteons compared to osteocytes outside thecement line was higher for the high-affinity drug comparedto the low-affinity drug (Fig. 4a). This indicates that thelow-affinity drug distributes more equally across the oste-onal cement line compared to the high-affinity drug, whichconcentrates mostly within the osteonal structure. Stainingintensity ratios were calculated in the ribs of the two ani-mals with the flipped fluorophores, and although the patternwas similar, the data were not significantly different (highaffinity 1.50  ±  0.17, low affinity 1.20  ±  0.06). In tibialosteons the staining of the two fluorophores was almostexclusively within the cement lines of the forming osteons.Therefore, ratios of staining were not possible to calculate.Fluorescent Bisphosphonate Localization in TrabecularBoneQualitative observations on cancellous bone surfacesshowed a similar banding pattern as was noted in corticalsurfaces (Fig. 5). Similar to the rib, there was a signifi-cantly lower ratio of the low-affinity compound comparedto the high-affinity compound (Fig. 4b). Discussion The ability of bisphosphonates to bind to hydroxyapatitehas been long appreciated [20], yet the details of thisphysical–chemical interaction have been elucidated onlyrecently. We know that bisphosphonates have differentbinding affinities that contribute to the adsorption andrelease from mineral [1, 3]. We also have begun to learn how binding affinity affects the global and tissue-leveldistribution of bisphosphonates [4–6]. Previous work on skeletal distribution has predominantly focused on trabec-ular bone as this is where bisphosphonates produce theirmost prominent skeletal effect. Yet bisphosphonates alsosignificantly benefit cortical bone by reducing porositythrough suppression of intracortical remodeling [21–24]. Rodents represent a useful model for studying trabecularbone but have significant limitations in cortical bonestudies due to their lack of intracortical remodeling. Thegoal of the current work was to determine whether differ-ences in bisphosphonate binding affinity affected distribu-tion in cortical and cancellous bone. Our results show thatwithin the cortical bone of the rib compounds with highaffinity are mostly localized within the osteonal units whilethose with lower affinities have a greater ability to diffusebeyond the osteonal cement line. These results differed inthe tibia, where neither the high- nor the low-affinity drugwas detected beyond the osteonal cement line.Fluorescently labeled bisphosphonates with differentbinding affinities provide unique tools to study drug 206 J. Turek et al.: Bisphosphonate Binding Affinity  1 3
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