The Pharmacological Profile of a Novel Highly Potent Bisphosphonate,OX14 (1‐Fluoro‐2‐(Imidazo‐[1,2‐α]Pyridin‐3‐yl)‐Ethyl‐Bisphosphonate)

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen‐containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1‐fluoro‐2‐(imidazo‐[1,2 alpha]pyridin‐3‐yl)‐ethyl‐bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short‐term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3‐NSG murine model of myeloma‐induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Effects of 4‐chloro‐2,6‐bis‐(2‐hydroxyl‐benzyl)‐phenol on Healing of Skin Wounds and Growth of Bacteria

In this investigation, the effects of synthesized 4‐chloro‐2,6‐bis‐(2‐hydroxyl‐benzyl)‐phenol (CBHBP) on cutaneous wound healing and growth of some of the wound contaminating microorganisms were studied. The antibacterial effects of this compound were then evaluated on Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Klebsiella spp., using solid dilution method. It was demonstrated that CBHBP has a significant antimicrobial activity against S. aureus but it is not effective in the case of other microorganisms studied in this experiment. The effect of local administration of CBHBP on healing of a standard full‐thickness 2 cm skin incision of skeletally mature rats was evaluated. Histological changes together with mechanical properties and dry weight content of the healing tissues at the site of the lesions were assessed in treated and untreated animals. It was observed that the injured area of the treated animals was more organized and showed more fibroblasts and less inflammatory cells. Much better maturation criteria in treated tissues were observed in comparison with those of the untreated ones which contained numerous polymorphonuclear inflammatory cells after 14 days post‐injury. Many infiltrated macrophages and lymphocytes were present even 28 days after injury induction in the haphazardly organized dermis and also in subcutaneous tissues of the untreated animals. The percentage dry weight content of the treated lesions at 14 days post‐injury was remarkably higher than those of the untreated animals. The results of biomechanical tensile testing showed that the ultimate tensile strength and stress of the injured skin of the treated animals were higher than those of the untreated ones. From these results, it could be concluded that CBHBP can be effective on wound healing and may be considered as a treatment regimen after evaluating its mechanism of action as well as testing its contraindications.     

Magnesium Metabolism in 4‐Year‐Old to 8‐Year‐Old Children

Magnesium (Mg) is a key factor in bone health, but few studies have evaluated Mg intake or absorption and their relationship with bone mineral content (BMC) or bone mineral density (BMD) in children. We measured Mg intake, absorption, and urinary excretion in a group of children 4 to 8 years of age. Mg absorption was determined using a dual‐tracer stable isotope technique, with ²⁵Mg given intravenously and ²⁶Mg given orally. We found a small, but significantly greater Mg absorption efficiency (percentage absorption) in males than females (67% ± 12% versus 60% ± 8%, p = 0.02) but no difference in estimated net Mg retention (average of 37 mg/d in both males and females). Relating dietary Mg intake to estimated Mg retention showed that an intake of 133 mg/d, slightly above the current estimated average requirement (EAR) of 110 mg/d, led to a net average retention of 10 mg/d, the likely minimum growth‐related need for this age group. Covariate analysis showed that Mg intake and total Mg absorption, but not calcium intake or total absorption were significantly associated with both total body BMC and BMD. These results suggest that usual Mg intakes in small children in the United States meet dietary requirements in most but not all children. Within the usual range of children's diets in the United States, dietary Mg intake and absorption may be important, relatively unrecognized factors in bone health. © 2014 American Society for Bone and Mineral Research.     

HLA‐A, ‐B, ‐C, ‐DR,and ‐DQ Matching in Pancreas Transplantation: Effect on Graft Rejection and Survival

To enhance selection of appropriate deceased donors for pancreas transplants, we sought to determine whether HLA matching improved posttransplantation outcomes. In this single‐center study of 1219 pancreas transplants, we correlated posttransplantation outcomes with HLA‐A, ‐B, ‐C, ‐DR, and ‐DQ matches and mismatches. Rejection was linearly correlated with the number of mismatches. The individual number of HLA mismatches reached significance at four or more with a 2.3‐ to 2.9‐fold increase in rejection. The effect was most predominant with HLA‐B (1.8‐fold with one mismatch and 2.0‐fold with two mismatches) and ‐DR (1.9‐fold with two mismatches) loci, whereas HLA‐A, ‐C, and ‐DQ matches or mismatches did not independently predict acute rejection. The affect was strongest in solitary pancreas transplants, with little impact for simultaneous pancreas and kidney (SPK). In contrast, HLA matching did not affect graft or patient survival rates but was associated with a reduced risk of opportunistic infection. Avoidance of acute rejection saved an estimated $32 000 for solitary pancreas recipients and $52 000 for SPK recipients in hospital costs. Our data do not support the use of HLA matching for predicting pancreas graft survival but do support its significance for the reduction of acute rejection, particularly for solitary pancreas recipients.     

“Inside‐Out” PEGylation of Bovine β‐Cross‐Linked Hemoglobin

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside‐out PEGylation, has been used here to conjugate a multiarm maleimide‐PEG (Mal‐PEG) to β‐cross‐linked (βXL‐Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL‐Hb to one Mal‐PEG. Thermal denaturation studies showed that the cross‐linked and PEGylated species were more stable than native Hb. Cross‐linking under oxy‐conditions produced a high oxygen affinity Hb species (P₅₀ = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P₅₀ = 9.67 Torr). Inside‐out PEGylation can be used to produce a hemoglobin‐based oxygen carrier and potentially for other multiprotein complexes.     

Hemorrhage‐Induced Interleukin‐1 Receptor Pathway in Lung Is Suppressed by 3,5‐Bis(2‐Fluorobenzylidene)‐4‐Piperidone in a Rat Model of Hypovolemic Shock

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa‐B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin‐1 receptor (IL‐1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 μL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll‐like receptor 4 (TLR4), IL‐1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL‐1R‐related (SIGIRR). The tissue‐associated pro‐inflammatory cytokines, tumor necrosis factor alpha (TNF‐α) and IL‐6, were measured by enzyme‐linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL‐1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down‐regulators of the pro‐inflammatory IL‐1R pathway) was increased in EF24‐treated hemorrhaged rats. The concentration of hemorrhage‐induced TNF‐α and IL‐6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide‐stimulated dendritic cells that EF24 beneficially modulates the IL‐1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.     

Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs

Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.