Recombinant activated protein C attenuates endothelial injury and inhibits procoagulant microparticles release in baboon heatstroke.

OBJECTIVE: We tested the hypothesis that the antithrombotic and cytoprotective effects of recombinant human activated protein C (rhAPC) protect baboons against the lethal effects of heatstroke. METHODS AND RESULTS: Fourteen anesthetized baboons assigned randomly to rhAPC (n=7) or control group (n=7) were heat-stressed in a prewarmed incubator at 44 to 47 degrees C until systolic blood pressure fell below 90 mm Hg, which signaled severe heatstroke. rhAPC was administered intravenously (24 microg/kg/h) for 12 hours at onset of heatstroke. Heat stress induced coagulation and fibrinolysis activation as evidenced by a significant increase from baseline levels in plasma levels of thrombin-antithrombin (TAT) complexes, tissue plasminogen activator, and D-dimer. Heat stress elicited cell activation/injury as assessed by the release of interleukin (IL)-6, soluble thrombomodulin, and procoagulant microparticles (MPs). rhAPC did not significantly reduce heatstroke-induced thrombin generation, and D-dimer and had no effect on fibrinolytic activity. In contrast, rhAPC infusion attenuated significantly the plasma rise of IL-6 and inhibited the release of soluble thrombomodulin and MPs as compared with control group. No difference in survival was observed between rhAPC-treated and control group. CONCLUSIONS: rhAPC given to heatstroke baboons provided cytoprotection, but had no effect on heatstroke-induced coagulation activation and fibrin formation. Inhibition of MPs by rhAPC suggested a novel mechanism of action for this protein.

Structure-activity relationships among the nitrogen containing bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl pyrophosphate synthase.

The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.

Glucocorticoids do not protect against the lethal effects of experimental heatstroke in baboons.

The mortality and neurological morbidity in heatstroke have been attributed to the host's inflammatory responses to heat stress, suggesting that anti-inflammatory therapy may improve outcome. We tested the hypothesis that a high dose of dexamethasone protects baboons against the lethal effects of heatstroke. Ten anesthetized baboons (Papio hamadryas) were assigned randomly to dexamethasone (n = 5) or control group (n = 5). Dexamethasone (2 mg/kg i.v.) was administered in four divided doses every 6 h starting immediately before heat stress and continuing during cooling. All animals were heat-stressed in a prewarmed neonatal incubator at 44 degrees C to 47 degrees C until systolic blood pressure fell less than 90 mmHg and then cooled passively at the ambient temperature. Mortality and neurological morbidity were noted, and biochemical markers of tissue injury/organ dysfunction were determined. Circulating interleukin (IL) 6 and complement components (C3 and C4) were measured sequentially. All heat-stressed animals had systemic inflammation indicated by increased plasma IL-6 and decreased C3 and C4 levels. Dexamethasone attenuated the complement system activation and maintained a higher plasma concentration of IL-6, with a significant augmentation of arterial blood pressure. Dexamethasone did not prevent the occurrence of severe heatstroke but unexpectedly aggravated significantly the tissue injury and multiorgan system dysfunction. Two animals (40%) in the control group and one in the steroid group survived (P > 0.05). Dexamethasone failed to protect the baboons from the lethal effects of heatstroke. These results do not support clinical testing of corticosteroids as beneficial in preventive or therapeutic strategies for the treatment of heatstroke in humans.

Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy.

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

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 American Society for Bone and Mineral Research.

RAGE-mediated neutrophil dysfunction is evoked by advanced glycation end products (AGEs).

The accumulation of advanced glycation end products (AGEs) in the tissue and serum of subjects with diabetes has been linked to the pathogenesis of vascular complications. Because diabetes may be also complicated by increased susceptibility to recurrent infection, we investigated the effects of AGEs on human neutrophils, because their burst of activity immediately upon engagement of pathogens or other inflammatory triggers is critical to host response. We demonstrate the presence of receptor for advanced glycation end products (RAGE) at the message and protein levels. We also demonstrate that AGE albumin (but not control albumin) binds with high affinity to human neutrophils (K(d) of 3.7 +/- 0.4 nM). The binding was blocked almost completely by excess soluble RAGE, anti-RAGE antibodies, or antibodies to CML-modified albumin. AGE albumin induced a dose-dependent increase in intracellular-free calcium as well as actin polymerization. Further, AGE albumin inhibited transendothelial migration and Staphylococcus aureus-induced but not fMLP-induced production of reactive oxygen metabolite. Moreover, although AGE albumin enhanced neutrophil phagocytosis of S. aureus, it inhibited bacterial killing. We conclude that functional RAGE is present on the plasma membrane of human neutrophils and is linked to Ca(2)(+) and actin polymerization, and engagement of RAGE impairs neutrophil functions.

The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

The relationship between the chemistry and biological activity of the bisphosphonates.

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.

Hsp-72, a candidate prognostic indicator of heatstroke.

Exposure of rats to environmental heat enhances the expression of heat shock protein-72 (Hsp-72) in most of their organs proportionally to heat stress severity. Pre-induction or over-expression of Hsp-72 prevents organ damage and lethality, suggesting that heat shock proteins (Hsps) may have a pathogenic role in this condition. We investigated the expression profile of Hsps in baboons subjected to environmental heat stress until the core temperature attained 42.5 degrees C (moderate heatstroke) or occurrence of hypotension associated with core temperature > or = 43.5 degrees C (severe heatstroke). Western blot analysis demonstrated a differential induction of Hsp-72 among organs of heat-stressed animals with the highest induction in the liver and the lowest in lung. Hsp-60 and Hsc-70 expression was similar between control and heat-stressed animals. ELISA studies indicated a marked release of Hsp-72 into the circulation of baboons with severe heatstroke with a peak at 24 h post-heatstroke onset and remained sustained up to 72 h. Hsp-72 release was not associated with core temperature or systolic blood pressure, but correlated with markers of liver, myocardium, and skeletal muscle tissue necrosis. Non-survivors displayed significantly higher Hsp-72 levels than survivors. No Hsp-60 was detected in the circulation. These findings add further evidence that increased expression of Hsp-72 may be an important component of the host response to severe heatstroke. They also suggest that extracellular Hsp-72 is a marker of multiple organs tissue damage. Whether extracellular Hsp-72 plays a role in the host immune response to heat stress merits further studies.

Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a gammadelta-T lymphocytes-mediated activation mechanism.

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human gammadelta T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate gammadelta T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of gammadelta T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.