Anti-HIV And Anti-Cancer Drug-Drug Interactions CORONA G Experimental and Clinical Pharmacology Unit, NATIONAL CANCER INSTITUTE CRO-IRCCS. Via Franco Gallini 2, 33081 Aviano (PN) ITALY
The challenge in the treatment of human immunodeficiency virus (HIV)-related malignancies is represented by the need to maintain an adequate control of HIV infection while managing the malignant disease with anticancer therapy. Combination therapy with highly active antiretroviral therapy (HAART) and anticancer chemotherapy (CT) has been found to be effective and may contribute in reducing morbidity and extend survival of HIV patients with cancer. However, an increased toxicity of the treatment often limits the clinical benefits of HAART+CT combination leading to the interruption or dose reduction of both the anticancer and antiviral therapies, thus increasing the risk of viral rebound and tumor relapse. It was believed that such unexpected toxicity could be related to pharmacokinetic interactions between antiretroviral and antineoplastic drugs. Cytotoxic anticancer drugs have a narrow therapeutic index, and small variations in drug exposure consequent to metabolic interactions with the antiretroviral drugs may explain the increased toxicity observed during coadministration of HAART with chemotherapy. HAART consists of a combination of nucleoside analogue reverse transcriptase inhibitors (NRTIs), with protease inhibitors (PIs) and non-nuclease analogue reverse transcriptase inhibitors (NNRTIs). PIs, as well as many anticancer drugs are metabolized by the liver cytochrome P450 CYP3A4 isoform. The metabolic clearance of anticancer drugs sharing this common enzymatic pathway of PIs can be inhibited by concomitant administration of PIs. Moreover, PIs could also compete for cellular ABC transporter proteins such as P-glycoprotein (Pgp) or multi-drug resistance proteins (MRP), interfering with both biliary and renal excretions of the anticancer drugs. The current use in HAART of the new boostered formulations containing ritonavir (RTV), which is the most potent CYP3A4 and ABC transporter inhibitor among the PIs, increases the need for formal drug-drug interaction study to establish the extent and the clinical consequence of the pharmacokinetics interaction between PIs and anticancer agents. In this contest we have studied the effect of bostered lopinavir/ritonavir (LPV/RTV) on the pharmacokinetics of irinotecan (CPT11) in HIV patients with Kaposi’s sarcoma. CPT11 is a prodrug that is activated in vivo by the liver carboxylesterase 2 to 7-ethyl-10-hydroxycamptothecin (SN38), a potent topoisomerase inhibitor. SN38 is inactivated to SN38 glucuronide (SN38G) by the UDP-glucuronosyl transferase 1A1 isoform (UGT1A1). In addition, CPT11 is oxidized to the inactive 7-ethyl-10-[4-N-(5-aminopentanoic-acid)-1-piperidino]-carbonyloxycamptothecin (APC) metabolite by CYP3A enzymes. Excretion pathways of CPT11 and metabolites in the bile and urine involve ABCB1 and ABCC2 transporters. This complex in vivo metabolism of CPT11 gives the opportunity to evaluate the effect of LPV/RTV on diverse metabolic pathways demonstrating that the inhibition of marginal oxidative metabolism route of CPT11, mediated by CYP3A4/5, could have dramatic consequence on the pharmacokinetics and pharmacodynamic profile of CPT11.
Docking calculations of heme derivatives on CYP2B4
Sección de Estudios de Posgrado e Investigación y Departamento de Bioquímica, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Casco de Santo Tomás, México, D.F. 11340, México, jcorreab@ipn.mx
Cytochrome P-450 is a group of enzymes involved in the biotransformation of many substances, including drugs. These enzymes possess a heme group (1) that when it is properly modified induces several important physicochemical changes that affect their enzymatic activity. In this work, the five structurally modified heme derivatives 2–6
and the native heme 1 were docked on CYP2B4, (an isoform of P450), in order to determine whether such modifications alter their binding form and binding affinity for CYP2B4 apoprotein. In addition, docking calculations were used to evaluate the affinity of CYP2B4 apoprotein-heme complexesfor aniline (A) and N-methyl-aniline (NMA). Results showing the CYP2B4 heme 4- and heme 6-apoprotein complexes to be most energetically stable indicate that either hindrance effects or electronic properties are the most important factors with respect to the binding of heme derivatives at the heme-binding site. Furthermore, although all heme-apoprotein complexes demonstrated high affinity for both A and NMA, the CYP2B4 apoprotein-5 complex had higher affinity for A, and the heme 6 complex had higher affinity for NMA. Finally, surface electronic properties (SEP) were calculated in order to explain why certain arginine residues of CYP2B4 apoprotein interact with polarizable functionalities, such as ester groups or sp2
carbons, present in some heme derivates. The main physicochemical parameter involved in the recognition process of the heme derivatives, the CYP2B4 apoprotein and A or NMA, are reported.
The Interaction of the Organophosphorous Pesticide Methyl-Parathion with Serum Albumin by Fluorescence Spectroscopy CORTEZ CM, SILVA D, BASTOS JC.
Rio de Janeiro State University, Rio de Janeiro, Brazil.
Background: The aim of this work was to study the interactions of methyl-parathion (MP) with human (HSA), bovine (BSA) and fish (FSA) albumins by using the fluorescence quenching technique. The fish species used was the pacu (Piaractus mesopotamicus), a typical inhabitant of Brazilian rivers. MP is a pesticide still used in agriculture and fish hatcheries in many countries.
Methods: MP was purified from a commercial grade preparation by thin layer chromatography, using dichloromethane as the mobile phase. HSA and BSA were purchased from chemical company. FSA was isolated from the pacu serum by affinity chromatography. We excited the intrinsic fluorescence of the tryptophan in albumins and observed the quenching by titrating the protein solutions with MP. The Stern-Volmer graphs were plotted and the quenching constants were evaluated.
Results: The titration of HSA and BSA at 25ºC produced linear Stern-Volmer plots. At 37oC, the plot is still linear for HSA, but not for BSA. The titration of pacu albumin by the pesticide at 20ºC, 25ºC and 30ºC showed relevant slope deviations by the temperature changes. This behavior of Stern-Volmer plot of pacu albumin is analogous to the BSA. Examining the influence of the temperature at low MP concentrations on the plots, we found the occurrence of static quenching for the three albumins.
Conclusions: (1) The three studied albumins interact with MP to form complexes. (2) The pprimary binding sites for the pesticide on HSA and BSA are close to tryptophan residues 214 and 212, respectively. (3) Estimated association constants for HSA and BSA were, respectively: 3.07x104 (1.2x103) M-1 and 1.96x104 (4.5x102) M-1 at 25ºC; 1.08x104 (2.0x102) M-1 and 8.16x103 (1.9x102) M-1 at 37°C. (4) For pacu albumin, the Stern-Volmer constants were 1,19x104 (3,4x102) M-1 at 20ºC, 9,73x103 (±4,9x102) M-1 at 25ºC, 9,37x103 (4,4x102) M-1 at 30ºC.
Small Peptides Challenge Thymidylate Synthase Dimerization and Inhibit Ovarian Cancer Cells Growth. COSTI MP1, STEFANO M2, WADE R3, MORUZZI MS4, MARVERTI G4 1Dipartimento di Scienze Farmaceutiche, Universita’ di Modena e Reggio Emilia, via Campi 183, 41100 Modena,Italy. 2 Dipartimento di Chimica, Università degli Studi di Siena, Via Moro 1, 53100 Siena, Italy. 3 EML Research gGmbH Villa Bosch Schloss-Wolfsbrunnenweg 33 D-69118 Heidelberg, Germany 4 Dipartimento di Scienze Biologiche, Universita’ di Modena e Reggio Emilia, via Campi 187, 41100 Modena, Italy. Presenting author: mariapaola.costi@unimore.it
Background: Ovarian cancer is the fifth most common cause of death from cancer in women. The standard first-line treatment is a combination of paclitaxel and carboplatin or carboplatin alone. In the case of progressive disease or drug resistance treatment with platinum, either alone or in combination, especially investigational compounds should be used. The mechanisms behind acquired resistance to cDDP and its derivatives are not clear yet, although it is evident that the process is multifactorial including, enhanced DNA repair. In the human ovarian carcinoma cell line A2780, a 3-fold-cDDP-resistance was associated with cross-resistance to the thymidylate synthase (TS) inhibitors 5-fluorouracil and to methotrexate, a 2.5-fold increase in TS level, and an increase in the intracellular pools of the TS cofactor 5, 10-methylentetrahydrofolate and of tetrahydrofolate. Our ultimate goal is to directly halt tumour progression and the development of drug resistance upon treatment with platinum derived drugs by inhibiting the protein regulatory function of monomeric TS through small molecule cellular perturbation.
Methods: To this aim we applied a multidisciplinary approach to identify new molecules that could bind to specific pocket at the protein interface. We included antipeptide design, site directed mutagenesis, tethering of thiol ligands at the protein interface, Mass spectrometry, x-ray crystallography.
The biological profile of the discovered inhibitors was characterized and the mechanism of action described.
Results: Four peptides among the ones identified showed inhibitory activity versus human Thymidylate synthase in the range of 10-400 M. The most active and specific compounds identified showed effective TS inhibitory properties and cell growth inhibition properties against both sensible and resistant cancer cells.
The project is supported by FP6 european grant (LIGHTS), LSH 038752 and MIUR-PRIN 2006030430_004 MPC.
Magic Bullets - the Lantibiotic approach COTTER PD1, FIELD D1, DRAPER LA1, LAWTON EM1, O’CONNOR PM2,3, ROSS RP2,3, HILL C1,3 1 Department of Microbiology, University College Cork, Cork, Ireland; 2 Teagasc Dairy Products Research Centre, Moorepark, Fermoy, County Cork, Ireland; 3Alimentary Pharmabiotic Centre, Cork, Ireland.
Background: Lantibiotics are antimicrobial peptides that frequently function at nanomolar concentrations by targeting the essential cell wall precursor, lipid II. Being gene-encoded, the lantibiotics can serve as biological templates for novel bioengineered antimicrobials. Bioengineering of lantibiotics first occurred in 1992, an event which prompted great optimism with predictions that lantibiotics might ultimately be regarded as the equivalent of primitive antibodies, potentially having constant and variable regions. However, unlike the mammalian immune system, the bacterium has no mechanism for generating a diverse population of peptides. Here we used molecular biology-based techniques to address this issue.
Methods:nisA, the gene encoding the structural peptides of the lantibiotic, nisin, were cloned into plasmid pCI372 and used as a template for error prone PCR-based random mutagenesis. The bioengineered genes were introduced the L. lactis NZ9800, which possesses all other genes required for nisin biosynthesis, to generate a bank of 8,000 strains producing different nisin derivatives. The bioactivity of a number of these lantibiotic producers and the specific activity of the most potent of the peptides produced was quantified.
Results: The approach described led to the identification of a nisin-producing strain with enhanced bioactivity against Streptococcus agalactiae resulting from a residue change in the middle ‘hinge’ region of the peptide (K22T). Further ‘hinge’-specific mutagenesis took place, resulting in the identification of additional derivatives, most notably N20P, M21V and K22S, with enhanced bioactivity and specific activity against gram-positive pathogens including Listeria monocytogenes and/or Staphylococcus aureus. In a number of cases this antimicrobial activity was species specific.
Conclusions: The identification of these derivatives represents a major step forward in the bioengineering of nisin, and lantibiotics in general, and confirms that peptide engineering can deliver derivatives with enhanced antimicrobial activity which could be regarded as being ‘Magic Bullets’ with respect to the targeting of specific problematic spoilage and pathogenic microbes.
Aspirin resistance myth or reality? COX D1, MCCALL1 M, TEDESCO A1, PEACE A1 AND FOLEY D2.
1Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, and 2Department of Cardiology, Beaumont Hospital, Dublin, Ireland
Background. The concept of aspirin resistance is controversial with reports of clinical studies showing an association with outcome in cardiovascular patients and other researchers claiming that it doesn’t exist. This study was designed to determine if the recent trend to use low dose enteric-coated aspirin may play a role in the phenomenon of aspirin resistance
Methods. 71 healthy volunteers were administered a low dose aspirin preparation and in a subsequent study 144 cardiovascular patients on low dose aspirin were recruited. The effect of aspirin was monitored by arachidonic acid-induced platelet aggregation, VerifyNow® and serum thromboxane. The definition of aspirin resistance was assay dependent.
Results. None of the healthy volunteers were considered to be aspirin resistant as they showed complete inhibition to at least one aspirin preparation. However there was variation in the response to different preparations of aspirin as determined by serum thromboxane. In particular the response to enteric-coated aspirin was not as good as that to plain aspirin (P<0.001) and this was exacerbated by weight with a poorer response among heavier subjects (P<0.001). Among the patients the incidence of aspirin resistance was between 13% and 21% depending on the assay (P>0.05) although each assay detected different patients and there was little agreement between the assays, however, when patients were re-tested with a reminder to take their medication only 2% were considered to be resistant.
Conclusions. Aspirin resistance appears to be a rare phenomenon. In most cases it is due to poor compliance. In many of the remaining patients the lower bioavailability of enteric-coated aspirin, especially when combined with higher weight results in a significant reduction in the dose/kg that the patient is exposed to. Thus, the phenomenon is best described as aspirin non-response rather than aspirin resistance. However, it is still clinically relevant as aspirin is only effective if the patient takes it in sufficient quantities.
Mechanisms of anti-CD20 immunotherapy: Why Type II mAb are better Beers SA1, Chan CHT1, James S1, French RR1, Attfield KE1, Brennan CM1, ahuja A2, shlomchik M2, Glennie MJ1andCragg MS1 1Cancer Sciences Division, Southampton University School of Medicine, UK
2Yale University School of Medical, CT, USA
Background: Rituximab is an anti-CD20 monoclonal antibody (mAb) that was the first of its kind to be approved by the FDA. It is now part of the standard treatment for many B cell malignancies and is finding utility in a range of autoimmune diseases where depletion of normal B cells, although not as yet explained mechanistically, appears highly beneficial. Treatment is not always successful however and not surprisingly the pursuit of improved reagents to replace rituximab is intense. Anti-CD20 mAb can be sub-divided into Type I (like rituximab) or Type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions such as complement. Therefore, we wished to address whether other (Type II) anti-CD20 mAb might be more effective than rituximab in deleting B cells.
Methods: To compare Type I and II anti-CD20 mAb directly in vivo and maximize Fc effector function, we selected and engineered mAb with the same mouse IgG2a isotype and assessed their B cell depleting activity in two different strains of human CD20 transgenic mice. The ability to deplete peripheral blood and secondary lymphoid organs was assessed by flow cytometry and immunohistochemistry.
Results: Despite being the same isotype, having similar affinity, opsonising activity for phagocytosis, and in-vivo half-life, the Type II mAb tositumomab provided substantially longer depletion of B cells from the peripheral blood compared with the Type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular the spleen. Failure to engage complement did not explain the efficacy of the Type II reagents, since Type I mAb mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. We have recently determined the likely mechanism through which Type II mAb outperform Type I mAb and will present this data.
Conclusions: These results provide strong support for the development of Type II anti-CD20 mAb for the treatment of B cell diseases and expect that their lack of complement engagement should reduce the toxic side effects often associated with the use of rituximab and other Type I reagents. This work also provides insight into how mAb to other targets might be optimized for better therapeutic efficacy.
Authors’ disclosure statement:
The authors declare no competing financial interests.
Allergy vaccines: dreams and reality
CRAMERI R1 , KÜNDIG T2 1Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland
2Unit for Experimental Immunotherapy, Department of Dermatology, University Hospital, CH-8091 Zurich, Switzerland
Allergy, asthma, and atopic eczema derive from deregulated immune responses against innocuous antigens. The incidence of atopic diseases is affecting around 30% of the population in industrialized countries and has become a relevant socio-economic burden for the modern society. Although much progress has been achieved in the development of efficient symptomatic treatments for allergic diseases, the only curative treatment remains allergen-specific immunotherapy (SIT). In contrast to classical vaccines which elicit strong host immune responses after one of a few injections, SIT require a long treatment time of 3 to 5 years to confer some protection. During the last year, three promising allergy vaccination concepts [RPE 04 (Curalogic); GT-14 (ALK-Abelló); TOLAMBA (Dynavax)] have been put on hold in Phase III due to the lack of efficacy. The reality is that “allergy vaccines” achieve beneficial effects through immunomodulation which takes a long time to establish, the dream would be to develop “true” allergy vaccines able to cure the disease with a few injections. We have engineered modular antigen translocation vaccines (MAT) for direct intracellular targeting of the MHC class-II presentation pathway aimed to increase the efficacy of antigen presentation. MAT vaccines administrated directly into the lymph node in mouse models of allergy were able to completely protect the animals from anaphylactic shock in a very short time. Intranodal administration of low doses of pollen extracts to allergic patients was able to confer a long lasting protection after only three injections administrated over a period of eight weeks only. Now we are combining a MAT-Fel d 1 vaccine with intranodal administration in a Phase I clinical study ready to start at beginning of July 2008. The combination of a new route of administration with a direct targeting of the MHC class-II presentation pathway has to potential to realize the dream of curing allergy in a few weeks.
Naked Models of Compound I of Heme Enzymes CRESTONI ME, FORNARINI S
Università “La Sapienza”, Roma, Italy
Background: High valent iron porphyrins at the formal Fe(V) oxidation level are known as Compound I intermediates, invoked in the oxidation pathways catalyzed by heme proteins and by their synthetic model compounds.1 A dominant feature of these processes is the dramatic change in product patterns and selectivities that Compound I may exhibit under the influence of reaction conditions. In this context, gas phase studies may have a great potential in revealing the intrinsic behaviour of this key intermediate.
Methods: Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance mass spectrometry is used to characterize the gas phase reactivity of high-valent oxoiron intermediates formed by two distinct procedures. In the first one, the oxoiron(IV) porphyrin cation radical intermediate, (TPFPP)∙+FeIV=O+ (TPFPP = 5,10,15,20-tetrakis (pentafluorophenyl)porphinato dianion) ion is prepared in solution by the reaction of the iron(III) porphyrin chloride, [(TPFPP)FeIII]Cl, and H2O2 in methanol and then transferred to the gas phase by ESI.2, 3 Alternatively, the naked core of Compound I is synthesized by the reaction of iron-protoporphyrin-IX (heme) ions, [(PP-IX)FeIII]+, with ozone in the gas-phase.
Results: The formation of the oxo-complex, described as a gaseous iron(IV)-oxo protoporphyrin IX radical cation species, [(PP-IX)·+ FeIV=O]+, allows a viable entry to a species that proves to be elusive in solution where it evolves presumably by activating the rapid growth of degradation products.4
The reactivity properties of the so-obtained high valent oxo iron intermediates with exemplary biologically active molecules and model compounds of naturally occurring substrates (L) of hemeproteins are reported. The reaction efficiencies, which measure the % fraction of reactive collision events, appear to increase with the oxophilic character of the active site of L (C
Conclusions: Gas phase studies may contribute to elucidate complex mechanisms in enzyme chemistry providing highly simplified models.
References
Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev.2004, 104, 3947.
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I
Prognostic value of total AgNOR area/Nucleus area per cell in urinary bladder carcinoma via two-dimensional image analysis CUCER N1, IMAMOGLU N2, TOZAK H3, DEMIRTAS H1, SARAC F2, TATLISEN A4, OZTURK F5 Erciyes University, Kayseri, Turkey 1 Medical Faculty, Medical Biology Department, 2 Halil Bayraktar Health Services Vocational College, Biomed. Tech. Prog., 3 Medical Faculty, Histology 4 Medical Faculty, Urology Department, 5 Medical Faculty, Pathology Department
Background: Traditional criterions are not sufficient to predict accurately the recurrence of transitional cell carcinoma of the urinary bladder. Therefore, we aimed to evaluate comperatively the nucleolar organizer region-associated proteins (AgNORs) via total AgNOR area/nucleus area (TAA/NA) for each cell, and as a prognostic parameter, comparing with total AgNOR number/nucleus (TAN/N), in TCC of urinary bladder.
Methods: Tumor tissues of 20 consecutive cases of male bladder cancer patients were divided into two groups as middle differentiated (LG) and high grade (HG). The extra-tumoral tissue (ETT) samples of 10 males served as control group. A second control group (HC) consisted of five healthy and normal bladder tissue samples. The 3 µm of sections from each paraffin embedded tumoral, extra-tumoral and normal tissue samples served as patient and control groups. After deparaffinization and rehydratation steps, AgNORs silver stained. Instead of Giemsa stain, we used Hematoxylin for contra staining. The images of the 100 analyzable nuclei from each tissue sample transferred by means of a video camera and video capture card from microscope and recorded onto a computer. Software was prepared in Delphi language for analysis.
Results: Mean (E+02) TAA/NA values were significantly different between all groups (p values < 0,001). While the mean TAN/N values of the groups were able to distinguish only malign samples from normals (p values < 0.05). The data is given in the table.
Table . TAN/N and TAA/NA values of control and patient groups
Conclusion: As a new approach the evaluation of mean TAA/ NA per cell has a great potential to be a prognostic parameter and it has been found more sensitive and more accurate than the TAN/N determination. Therefore, further evaluation of big patient series will be useful.
Do we need a smaller Magic Bullet for combating Staphylococcal infections? : Mechanism of vancomycin-resistance in vancomycin-intermediate S. aureus CUI L Department of Bacteriology ＆ Infection Control Science, Juntendo University, 2-1-1 Bunkyo-Ku, Tokyo, Japan 113-8421
Since our reports on the discovery of vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously vancomycin-resistant Staphylococcus aureus (hetero-VISA) in 1997 (JAC, 1997' 40:135-6 and Lancet, 1997' 385:1670-3), we have published a dozen serial papers on the molecular mechanism of vancomycin resistance in VISA, mainly in JAC (98’ 42[2]:199-209 and 42[3]:315-20), AAC (2000’ 44:1176-1185), Lancet (2001’ 357: 1225-40), JCM (2003’41:5-14), Mol Micro (2003’ 49;807-21), Lancet (2004’ 364: 565-6) and AAC (2005’ 49: 3404-13, 2006’ 50’ 428-38 and 2008’ 52: 45-53 ). By performing the above serial studies, we have elucidated the molecular mechanism of vancomycin resistance: VISA resists vancomycin via a thickened cell wall, which in turn results in a novel mechanism of “clogging” of the peptidoglycan mesh, whereby incoming vancomycin molecules are trapped in the thickening cell wall, preventing them from reaching the cell membrane. Quantitative measurement and mathematic analysis revealed that the “clogging” is related with the bigger molecule of vancomycin. Moreover, in a recent study with daptomycin-nonesusceptible S. aureus, we found a strong positive correlation between vancomycin and daptomycin resistance in VISA, and the result suggests the thickened cell wall acts as a common obstacle to daptomycin and vancomycin penetration. Even though daptomycin does not bind peptidoglycan to form subsequent physical barriers within the cell wall, it might be hard for daptomycin, with as big a molecule as over 1620, to penetrate smoothly through the cell wall when the cell wall become as thick as that of VISA. Taking all together, we propose that development of new antibiotics with smaller molecular size than that of vancomycin and daptomycin may be a new potential to overcome the vancomycin- and daptomycin-resistant Staphylococci infections.
Fundamental Understanding on Interactions of Bisphosphonates with Bone (by Use of Different Techniques, Including Computational Modelling). CUKROWSKI I University of Pretoria, Depatment of Chemistry, Pretoria 0002, South Africa.
Background: Computational modelling of BP-bone interactions is rare and hence there is very little to link the molecular structure of BP with its potency. Aims: 1) To determine stability constants of BPs with metal ions;. 2) To investigate interactions between BPs and a bone surface. 3) To model computationaly the interactions of BPs with a bone surface.
Methods: Raman spectroscopy (RS) was used to study the interactions of bovine bone, hydroxyapaptite (HA), and CaHPO4 with HEDP (a reference compound in scale of potency). Generalised AMBER Force Field (GAFF) was used to model over 50 BPs (C(R1)(R2)(PO32-)2) and the interaction of MDP (R1 = R2 = H), HEDP (R1 = OH, R2 = CH3), APD (R1 = OH, R2 = (CH2)2NH3+), ALN (R1 = OH, R2 = (CH2)3NH3+) and NER (R1 = OH, R2 = (CH2)5NH3+) with the (001), (010) and (100) faces of HA. Metal-BP stability constants were established by voltammety and potentiometry; this allowed us to conclud on mode of complexation at blood plasma pH. Linnear Free Energy Relationships generated allowed us to predict stability constants of radioactive elements with BPs; potential use in bone cancer therapy.
Results: The Raman spectra of the products from the reaction of 0.5 M HEDP solution with bone, HA and CaHPO4 could be considered virtually identical meaning that the complexes of HEDP formed were the same. From GAFF, all BPs react with HA exothermicaly (10 and 20 kcal mol-1). With a dielectric constant of 78o and <10o, non-bonded and electrostatic interactions dominate, respectively. The order of increasing interaction with HA, MDP < HEDP < APD < ALN > NER, accords with the observed in vivo order of pharmacologic activity (potency) and parallels the increase in molecular volume up to ALN; the side chain of NER fails to interact fully with HA. There is no significant difference in the structure of the BP-HA complex if BP is a mono- or bis-protonated. Relationships established from metal equilibria allowed prediction of radiisotops’interactions with BPs.
Conclusions: 1) HA can be substituted for bone in fundamental studies of BP-bone interactions. 2) The consonance between experimental and molecular modelling results suggests that GAFF may be a useful tool to aid in the design of novel BP ligands. 3) From stability constants it was concluded that side chain of BPs is not involved in complexation with metals at blood plasma pH.
