Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Alendronate sodium.

This chapter is a review on physical and chemical properties, methods of preparation, analysis, as well as pharmacodynamics and pharmacokinetics of Alendronate sodium (4-amino-1-hydroxybutane-1,1-diphosphonic acid sodium salt), a bone metabolism regulator, indicated for the treatment of excessive bone resorption and osteoporosis.

Design and synthesis of novel bone-targeting dual-action pro-drugs for the treatment and reversal of osteoporosis.

There is an important medical need for effective therapies to redress the general bone loss associated with advanced osteoporosis. Prostaglandin E(2) and related EP4 receptor agonists have been shown to stimulate bone regrowth but their use has been limited by systemic side effects. Herein is described the design and synthesis of novel dual-action bone-targeting conjugate pro-drugs where two classes of active agents, a bone growth stimulating prostaglandin E(2) EP4 receptor subtype agonist (5 or 6) and a bone resorption inhibitor bisphosphonate, alendronic acid (1), are coupled using metabolically labile carbamate or 4-hydroxyphenylacetic acid based linkers. Radiolabelled conjugates 9, 11a/b and 25 were synthesized and evaluated in vivo in rats for uptake of the conjugate into bone and subsequent release of the EP4 agonists over time. While conjugate 11a/b was taken up (9.0% of initial dose) but not released over two weeks, conjugates 9 and 25 were absorbed at 9.4% and 5.9% uptake of the initial dose and slowly released with half-lives of approximately 2 weeks and 5 days respectively. These conjugates were well tolerated and offer potential for sustained release and dual synergistic activity through their selective bone targeting and local release of the complimentary active components.

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass.

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4ß1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.

Fluorescence imaging of osteoclasts using confocal microscopy.

In order to understand osteoclast cell biology, it is necessary to culture these cells on a physiological -substrate that they can resorb in vitro, such as bone or dentine. However, this creates problems for analysis by fluorescence microscopy, due to the depth of the sample under investigation. By virtue of its optical sectioning capabilities, confocal microscopy is ideal for analysis of such samples, enabling precise intracellular localisation of proteins in resorbing osteoclasts to be determined. Moreover, by taking a series of images in the axial dimension, it is possible to create axial section views and to reconstruct 3D images of the osteoclasts, enabling the spatial organisation of the structures of interest to be more easily discerned.

N4-[Alkyl-(hydroxyphosphono)phosphonate]-cytidine-new drugs covalently linking antimetabolites (5-FdU, araU or AZT) with bone-targeting bisphosphonates (alendronate or pamidronate).

Amino-bisphosphonates (alendronate, pamidronate) were covalently linked in a three step synthesis, with protected and triazolylated derivatives of therapeutically used nucleoside analogs (5-FdU, araC, AZT) by substitution of their triazolyl residue. From the deprotected and chromatographically purified reaction mixtures N4-[alkyl-(hydroxyphosphono) phosphonate]-cytidine combining two differently cytotoxic functions were obtained. This new family of bisphosphonates (BPs) contains as novelty an alkyl side chain with a cytotoxic nucleoside. The BPs moiety allows for a high binding to hydroxyapatite which is a prerequisite for bone targeting of the drugs. In vitro binding of 5-FdU-alendronate (5-FdU-ale) to hydroxyapatite showed a sixfold increased binding of these BPs as compared to 5-FdU. Exploratory cytotoxic properties of 5-FdU-ale were tested on a panel of human tumor cell lines resulting in growth inhibition ranging between 5% and 38%. The determination of IC50-concentrations of the conjugate in Lewis lung carcinoma and murine macrophages showed an incubation time dependent growth inhibition with higher sensitivity towards the tumor cells. We assume that the antimetabolite-BPs can be cleaved into different active metabolites that may exert cytotoxic and other therapeutic effects. However, the underlying mechanisms of these promising new antimetabolite-BPs conjugates remain to be evaluated in future experiments.

Synthesis and in vivo bioactivity of lipophilic alendronate derivatives against osteoporosis.

New lipophilic alendronate amidated derivatives anchored alkyl chains (C(n)H(2n+1), n = 12, 14, 16, 18) had been obtained through the reaction of alendronate with carboxylic acid under anhydrous condition. The physicochemical parameters, such as the solubility and partition coefficient P(o/w) in n-octanol/water, were determined through calculation by performing reversion phase high-performance liquid chromatography (RP-HPLC). The results showed that the derivatives had improved lipophilicity compared with alendronate. The in vivo bioactivities of the derivatives were investigated using the hindlimb unloading growing rats' model. The results showed that the derivatives had in vivo bioactivity against hindlimb unloading growing rats' bone loss, which indicated that the lipophilic derivative would be a promising new potent bisphosphates for treatment of the osteoporosis.

188Re(CO)3-dipicolylamine-alendronate: a new bisphosphonate conjugate for the radiotherapy of bone metastases.

The palliation of pain due to bone metastases using targeted compounds containing beta-emitters such as rhenium-188 ((188)Re) is an accepted and effective form of treatment. Here, we describe the efficient synthesis and preclinical evaluation of (188)Re(CO)(3)-dipicolylamine(DPA)-alendronate, a novel bifunctional bisphosphonate for the palliative treatment of bone metastases. (188)Re(CO)(3)-DPA-alendronate can be easily synthesized with high specific activities and yields (18.8 GBq/mg, radiochemical yield > or =96%) in two steps using kit-based methodology, and in contrast with the clinically approved bisphosphonate (186/188)Re-HEDP, it forms inert, single species that have been well-characterized. In vivo imaging and biodistribution studies demonstrate that (188)Re(CO)(3)-DPA-alendronate is superior to (188)Re-HEDP in targeting and accumulating in areas of high metabolic bone activity while having low soft-tissue uptake. In addition to these studies, a simple and convenient new method for purifying its precursor, fac-[(188)Re(CO)(3)(H(2)O)(3)](+), is described.

Microencapsulation of sodium alendronate reduces drug mucosal damage in rats.

Sodium alendronate is an effective treatment for osteoporosis, but its oral administration is associated with adverse gastrointestinal effects. The aim of this work was to evaluate gastroresistant sodium alendronate-loaded microparticles prepared by spray-drying using Eudragit S100 or a blend of Eudragit S100/Methocel E4M. Both formulations presented high encapsulation efficiencies, mean diameters below 17 microm, and similar collapsed shape. Dissolution experiments showed good gastro-resistance for the microparticles at pH 1.2. At pH 6.8, the blended microparticles retarded the drug release. In vivo studies showed that the formulations were able to protect the rat stomachs against ulcer formation by sodium alendronate. In conclusion, the microparticles seems to be promising oral carriers for sodium alendronate.

Synthesis and assessment of 99mTc chelate-conjugated alendronate for development of specific radiopharmaceuticals.

Indole-based alendronate (AI) was derived from the condensation reaction of indole 3-carboxaldehyde with sodium alendronate (ALN) and was characterized by various spectroscopic methods (e.g., ultraviolet, fourier-transform-infrared, and liquid chromatography mass spectrometry). The AI was labeled with (99m)Tc and radiochemical purity was above 97%, which was ascertained by instant thin-layer chromatography, using different solvent conditions, with a specific activity 2-5 mCi/mg. The receptor ligand assay on human bone cell line Soas-2 showed K(D) = 0.55 nM. The derivative (AI) was stable, which was determined under physiologic conditions up to 24 hours The blood kinetic study showed a biexponential pattern as well as quick wash-out from the circulation with varying biologic t(1/2)(F) and t(1/2)(S). Excellent-quality radio images were recorded of bone, showing a rapid clearance of background activity, at an early visualization at 1.5 hours. The excretory pathway of the derivative was through the kidneys, which was evidenced by biodistribution studies. Thus, the newly synthesized derivative can be considered as a specific bone-seeking agent.

Ab initio structure determination of anhydrous sodium alendronate from laboratory powder X-ray diffraction data.

Sodium alendronate, a member of bisphosphonate class of compounds commonly used for treatment of generalized bone disorders, exists in various hydrated forms. Dehydration of sodium alendronate trihydrate has been studied using variable temperature X-ray powder diffraction technique. The crystal structure of anhydrous sodium alendronate, prepared by heating the trihydrate sodium alendronate at 150 degrees C, has been determined from X-ray powder data using direct space global optimization technique for structure solution, followed by the Rietveld refinement. The structure of the anhydrous form of sodium alendronate is compared with that of the trihydrate form, which was determined previously from single crystal X-ray diffraction data. Both anhydrous and trihydrate sodium alendronate crystallize in monoclinic system with space group P2(1)/n. The crystal structure of the anhydrous sodium alendronate is built by edge-sharing of NaO(6) octahedra into a two-dimensional molecular sheet in the (011) plane, whereas in the trihydrate compound, one-dimensional chain along the (010) direction is generated by corner sharing of NaO(6) octahedra.

Electrospray tandem mass spectrometry of alendronate analogues: fingerprints for characterization of new potential prodrugs.

1-hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are important drugs for the treatment of a variety of bone diseases. Since these compounds have no chromophore, their detection is challenging and mass spectrometry (MS) appears to be an appropriate sensitive tool. Our work deals with the analysis by electrospray ionization tandem mass spectrometry (ESI-MSn) of the well-known nitrogen-containing HMBP alendronate and of three analogues, considered as potential prodrugs. These four molecules share a common structure with different protecting groups on the phosphonic acid and on the amine functions. We describe the dissociation mechanisms of nitrogen-containing HMBPs in positive ion mode and we compare, in negative ion mode, our results with literature data. In both modes, the dissociations are essentially losses of ROH, and of phosphorus-containing species (HPO2, ROP(OH)2 and ROPO(OH)2), where R=H, C6H5, or CH3OC6H5. These fingerprints will be of great value for differentiating alendronate from its potential prodrugs in complex biological mixtures.

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates.

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.

Conjugation of poorly absorptive drugs with mucoadhesive polymers for the improvement of oral absorption of drugs.

Mucoadhesive poly(vinylamine) conjugates for improving oral absorption of alendronic acid were designed and prepared. Alendronic acid was conjugated via spacers containing brush border peptidase-susceptible amino acid residues. Alanylalendronic acid and alanylprolylalendronic acid were synthesized as expected substrates against brush border aminopeptidase N and dipeptidyl peptidase IV, respectively. In vitro release profiles of alendronic acid from them during incubation with luminal contents and brush border membrane vehicles of the rat's intestine were examined. The studies indicated that alanylproline was a useful peptide spacer for local release of alendronic acid in brush border membranes. We subsequently designed and prepared poly(vinylamine)-alendronic acid conjugates with succinoylglycylglycylphenylalanylalanylproline spacers, in consideration of steric hindrance of polymer chains on cleavability of the spacers and the substrate specificity of dipeptidyl peptidase IV. Oral absorption of alendronic acid after administration of the conjugates was compared with that of free alendronic acid in rats. Conjugation successfully resulted in a 2.5-fold increase in the oral absorption with statistical significance. This novel approach has a potential to improve oral absorption of drugs with poorly absorptive properties caused by low membrane permeability.

Synthesis and study of alendronate derivatives as potential prodrugs of alendronate sodium for the treatment of low bone density and osteoporosis.

Alendronate derivatives were evaluated as potential prodrugs for the osteoporosis drug alendronate sodium in an attempt to enhance the systemic exposure after oral administration. An investigation of the chemical behavior of alendronate derivatives led to development of practical synthetic strategies and prediction of each structural class's prodrug potential. Pharmacokinetic studies of N-myristoylalendronic acid revealed that 25% have been converted in vivo after i.v. administration in rat, providing an important proof-of-concept for this strategy.

Effects of weekly administrations of alendronate+clodronate on young mouse tibia: localized action at the proximal growth plate.

Alendronate (A), a typical aminobisphosphonate (aminoBP), has a strong bone-resorption-inhibitory activity (BRIA). However, like other aminoBPs it has inflammatory side effects. In contrast, the BRIA of clodronate (C), a non-aminoBP, is much weaker, and in animal experiments it suppresses aminoBP-induced inflammatory reactions. In the present study, we examined the effects of weekly administrations of A (1.6 micro mol/kg) + C (160 micro mol/kg) on the tibias in young mice and compared them to those induced by A or C alone. Radiophotography showed that A increased bone density at a selective site in the tibia. Indeed, one week after the final injection of A (given alone), clear sclerotic lines (tentatively called BP-lines) were visible at sites corresponding to the location of the growth plate at the time of the each injection. C also produced BP-lines, although they were weaker than those produced by A. Combined administration of A and C produced similar BP-lines as seen in mice given A alone. These results together with other physicochemical effects of A on the tibia suggest that (1) each injection of A and C inhibits bone resorption selectively and transiently at the tibial growth plate in young mice, although minor effects on other sites cannot be excluded, and (2) the combination of A and C keeps still a strong BRIA. Our findings may suggest a strategy for the prevention or reduction of some inflammatory side effects of A or other aminoBPs.

Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems.

Four polymeric bone-targeting conjugates were synthesized based on poly(ethylene glycol) (PEG, two conjugates) and poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA, two conjugates). The well-known bone-targeting compounds, alendronate and aspartic acid peptide, were used as bone-targeting moieties. Fluorescein isothiocyanate (FITC) was attached to the conjugates as a model drug for detection purposes. The bone-targeting potential of these conjugates was tested in vitro with hydroxyapatite (HA) and in mice. The data obtained indicated that these novel delivery systems could specifically accumulate in the bone tissue.

Synthesis, characterization and biodistribution of bisphosphonates Sm-153 complexes: correlation with molecular modeling interaction studies.

Bisphosphonates (BPs) are characterized by a P-C-P backbone structure and two phosphonic acid groups bonded to the same carbon, and are established as osteoclast-mediated bone resorption inhibitors. The nature of the groups attached to the central carbon atom are responsible in determining the potency of bisphosphonates as anti-resorption drugs. However, it is not yet clear the exact relationship between their molecular structure and pharmacologic activities. In this study, molecular geometries of pamidronate, alendronate and neridronate, differing only in the length of the aliphatic chains, were predicted by molecular mechanics and their interactions with hydroxyapatite, the main bone mineral component, were examined. We report the synthesis and radiochemical characterization of 153Sm complexes with pamidronate, alendronate and neridronate. Hydroxyapatite binding and biodistribution studies of these complexes have shown a good correlation with the theoretical molecular modeling interaction studies. So, it is possible to conclude that computational chemistry techniques are a good approach to evaluate specific interactions and may play a relevant role in determining the relative ability of BPs to mineral bone, and open new perspectives to the design of new BPs with increased pharmacological activity. These techniques could be extended to BPs as ligands to carrier radioactive metals, aiming for new bone therapeutic radiopharmaceuticals.

Labelling of Re-ABP with 188Re for bone pain palliation.

Etidronate and medronate have been labelled with technetium-99m (99mTc-HEDP, 99mTc-MDP) for bone scanning and, with rhenium-188 (188Re-HEDP) to palliate the pain resulting from bone metastases. The objective of this study was to label alendronate, ABP, a new bisphosphonate, with SnF2-reduced-188Re. The reagents for the 5 mg ABP kit were SnF2, KReO4 and gentisic acid at acid pH. The chemical, spectroscopic and microscopic characteristics, quality control, rat bone uptake of [188Re]Re-ABP and similarities with 99mTc-ABP are presented. We conclude that this is a promising new radiopharmaceutical for bone metastases pain palliation.

A peptide prodrug approach for improving bisphosphonate oral absorption.

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.