Selective catalytic oxidation of glycerol: perspectives for high value chemicals

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

https://jmg.bmj.com/content/jmedgenet/early/2005/08/23/jmg.2005.036673.full.pdf

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment

The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro.

c-Src is a proto-oncogene, belonging to the nonreceptor protein kinases family, which plays a prominent role in carcinogenesis. In this study, we tested the hypothesis that c-Src could promote breast cancer metastasis acting on several cell types and that pharmacological disruption of its kinase activity could be beneficial for the treatment of metastases. Female BALB/c-nu/nu mice were subjected to intracardiac injection of the human breast cancer cells MDA-MB-231 (MDA-231), which induced prominent bone and visceral metastases. These were pharmacologically reduced by treatment with the c-Src inhibitor [7-{4-[2-(2-methoxy-ethylamino-ethoxy]-phenyl}-5-(3-methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine] CGP76030 (100 mg/kg/day p.o.), resulting in decreased morbidity and lethality. Metastases were more severe in mice injected with MDA-231 cells stably transfected with wild-type c-Src (MDA-231-SrcWT), whereas transfection in injected cells of a c-Src kinase-dead dominant-negative construct (MDA-231-SrcDN) resulted in reduced morbidity, lethality, and incidence of metastases similar to the mice treated with the inhibitor. An analogous beneficial effect of c-Src inhibition was observed in subcutaneous and intratibial implanted tumors. In vitro, c-Src suppression reduced MDA-231 cell aggressiveness. It also impaired osteoclast bone resorption both directly and by reducing expression by osteoblasts of the osteoclastogenic cytokines interleukin-1beta and interleukin-6, whereas parathyroid hormone-related peptide was not implicated. c-Src was also modestly but consistently involved in the enhancement of endothelial cell proliferation in vitro and angiogenesis in vivo. In conclusion, we propose that c-Src disruption affects the metastatic process and thus is a therapeutic target for the treatment of breast cancer.

Inhibition of Protein Kinase c-Src Reduces the Incidence of Breast Cancer Metastases and Increases Survival in Mice: Implications for Therapy

Celiac disease is an autoimmune disorder characterized by atrophy of the intestine villi triggered by ingestion of gluten in genetically susceptible individuals. The association between celiac disease and low BMD has been recognized, but the mechanisms of disturbance are poorly understood. We show imbalance of cytokines relevant to bone metabolism in celiac patients' sera and the direct effect of these sera on in vitro bone cell activity. Introduction: Celiac disease is associated with mineral metabolism derangement and low BMD. We investigated whether imbalance of serum factors in celiac patients could affect human bone cell activity in vitro. Materials and Methods: We studied two groups of celiac patients— one on a gluten-free diet and another before the diet— both with decreased bone mass. Patients were investigated for bone turnover markers, and their sera were used for culturing bone cells from healthy donors and evaluate changes in cell activity. Results: The N-terminal telopeptide of procollagen type I and interleukin (IL)-6 were higher than normal in patients not on the gluten-free diet. IL-1 and TNF-/ were normal in all patients. IL-12 was reduced in all patients, whereas IL-18 was reduced only in patients on the diet. The RANKL/Osteoprotegerin (OPG) ratio was increased in patients not on the gluten-free diet. Persistently increased osteoclast numbers were obtained from peripheral blood mono- nuclear cells of healthy donors on incubation with sera of patients not on the gluten-free diet versus control sera and sera from patients on the diet. In human osteoblasts from healthy individuals, IL-18 was reduced on incubation with sera from all patients, whereas OPG expression was lower when sera from patients not on the diet were used. Proliferation, alkaline phosphatase, and nodule mineralization were increased in osteoblast cultures containing sera from all celiac patients, either on or not on the gluten-free diet. Conclusions: We conclude that bone loss in celiac disease might also be caused by a cytokine imbalance directly affecting osteoclastogenesis and osteoblast activity. J Bone Miner Res 2004;19:1112-1121. Published online on March 22, 2004; doi: 10.1359/JBMR.040319

https://asbmr.onlinelibrary.wiley.com/doi/pdf/10.1359/JBMR.040319

Imbalance of Osteoclastogenesis-Regulating Factors in Patients With Celiac Disease

Two decades of investigations have failed to unequivocally clarify the functions and the molecular nature of imidazoline-2 receptors (I2R). However, there is robust pharmacological evidence for the functional modulation of monoamino oxidase (MAO) and other important enzyme activities by I2 site ligands. Some compounds of this class proved to be active experimental tools in preventing both experimental pain and opioid tolerance and dependence. Unfortunately, even though these compounds bind with high potency to central I2 sites, they fail to represent a valid clinical opportunity due to their pharmacokinetic, selectivity or side-effects profile. This paper presents the preclinical profile of a novel I2 ligand (2-phenyl-6-(1H-imidazol-1yl) quinazoline; [CR4056]) that selectively inhibits the activity of human recombinant MAO-A in a concentration-dependent manner. A sub-chronic four day oral treatment of CR4056 increased norepinephrine (NE) tissue levels both in the rat cerebral cortex (63.1% ±4.2%; P < 0.05) and lumbar spinal cord (51.3% ± 6.7%; P < 0.05). In the complete Freund’s adjuvant (CFA) rat model of inflammatory pain, CR4056 was found to be orally active (ED50 = 5.8 mg/kg, by mouth [p.o.]). In the acute capsaicin model, CR4056 completely blocked mechanical hyperalgesia in the injured hind paw (ED50 = 4.1 mg/kg, p.o.; ED100 = 17.9 mg/kg, p.o.). This effect was dose-dependently antagonized by the non-selective imidazoline I2/α2 antagonist idazoxan. In rat models of neuropathic pain, oral administration of CR4056 significantly attenuated mechanical hyperalgesia and allodynia. In summary, the present study suggests a novel pharmacological opportunity for inflammatory and/or neuropathic pain treatment based on selective interaction with central imidazoline-2 receptors.

/pdf/analgesic-efficacy-of-cr4056-a-novel-imidazoline-2-receptor-29ica0hw50.pdf

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain.

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM-nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.

Tartronates: A New Generation of Drugs Affecting Bone Metabolism

Ketoprofen lysine salt inhibits disuse-induced osteopenia in a new non-traumatic immobilization model in the rat.

Colchicine, a known inhibitor of microtubule polymerization and a suppressor of granulocyte development and motility, has been shown, by the group of Tokyo Medical and Dental University, to induce the formation of new bone in the marrow cavities of rat long bones [1–3]. A recent paper by Wlodarski and Wlodarski [4], however, states that in their hands, this model could not be reproduced, either in WAG rats, or in mice. On the basis of the data obtained in mice and in 10 WAG rats (six treated and four controls), these authors conclude that the colchicine model “seems to be doubtful also in Wistar rats.” Moreover, they suggest that the effect observed by Arai et al., erroneously interpreted as a medullary ossification, was actually the result of a serendipitous selection of osteopetrotic (op/op?) rats. Over the past 2 years, we have also been working on the colchicine model as, like Wlodarski, we consider this model a “most dynamic and efficient” model of bone induction. More important, it appears to be an authentic model of medullary bone formation in mammals, not related to the mechanical disruption (and subsequent repair) of the vascular and stromal network of the bone marrow, which is instead the case in many current models of myeloablation. As such, the colchicine model appears to date as a unique model of pharmacological ablation of hematopoiesis (not ablation of marrow, because the stroma is not ablated) followed by de novo medullary bone formation, and a unique way to probe interactions of hematopoiesis and osteogenesis in the postnatal animal in vivo, a topic of major current interest in the field. Hence our own interest, which led us to verify and reproduce, first, the results of Arai et al. using essentially the experimental protocol of Arai et al. (a single (1 mg/kg) iv. dose of colchicine in 6-week-old animals) [2, 3], from which ours only diverges in the use of SpragueDawley instead of Wistar rats. We were therefore surprised to read the report of Wlodarski and Wlodarski. We believe, of course, that different strains of rats may exhibit different responses to colchicine administration, and we plan to look at the behavior of WAG rats in the near future. We cannot see why this should be construed as a denial of the value of others’ data, obtained using different protocols, and fully documented. Microradiographs and histology (Fig. 1) leave no doubt about the formation of new medullary bone in colchicine-treated rats. The formation of the new bone is preceded by hyperplasia of osteogenic marrow stromal cells (Western-Bainton, ALP+ reticular cells), occurs at the abluminal side of marrow sinusoids [5] Caselli et al. (manuscript in preparation), and is followed by resorption of the new bone and restoration of the marrow cavity. We have reproducibly obtained these results in more than 80 rats/ group (controls and colchicine-treated animals) on nine different occasions, and we are currently using this model to investigate the complex interplay of influences of age, bone growth, hormonal status, and hematopoietic activity on the osteogenic potential of the bone marrow stroma in vivo.We have determined the dose dependency of the phenomenon and the impact of animal age, site, and gonadal status on its production. Others have reproduced the same results in mice (K Satomura, et al., personal communication). Furthermore, by using serial dual x-ray absorptiometry (DXA) assessments on individual colchicine-treated animals, we determined that the increase in bone mass observed (and verified by histology) is indeed induced by the treatment, is transient, and is reversible. Therefore, the interpretation offered by Wlodarski and Wlodarski [4] of the data obtained by Arai et al. [2] (that osteopetrotic rats were being individually studied as representative of the effects of colchicine at certain time points) is, at best unwarranted. The mechanisms of colchicine effects on bone and marrow remain poorly understood. An inordinate proliferation of osteogenic cells is hard to reconcile with the negative effect that colchicine exerts on cell division. Tissue repair is also not in order, as the stromal scaffold and the vascular network of the bone marrow remain intact upon colchicine administration. It is our working hypothesis that the selective ablation of hematopoietic cells caused by colchicine may release marrow osteogenic cells associated with sites of bone growth from an inhibitory influence. This, of course, remains to be proven. For the time being, the need to understand the mechanism underlying the de novo medullary osteogenesis induced by high doses of colchicine in young rats adds, we believe, further appeal to the model. Which is subject to question, of course, as long as one remains critical about his/her own data (more so if negative), is fair about others’ data, and is objective about both.

Does Colchicine Really Induce Bone Formation in the Rodent Bone Marrow? Yes, it Does

Compounds of formula I: (see fig. I) wherein Ra, Rb, .PHI., B and R are as defined in the disclosure, have antagonistic activity on osteoclast hyper-reactivity.

Simonetta Fiorentino

Papers

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain.

Tartronates: A New Generation of Drugs Affecting Bone Metabolism

Ketoprofen lysine salt inhibits disuse-induced osteopenia in a new non-traumatic immobilization model in the rat.

Does Colchicine Really Induce Bone Formation in the Rodent Bone Marrow? Yes, it Does

Geminal carboxylic acids and esters thereof pharmaceutical formulations containing them useful in the treatment of bone dysmetabolism