RESUMO
Chronic kidney disease (CKD) is characterized by kidney damage and loss of renal function. CKD mineral and bone disorder (CKD-MBD) describes the dysregulation of mineral homeostasis, including hyperphosphatemia and elevated parathyroid hormone (PTH) secretion, skeletal abnormalities, and vascular calcification. CKD-MBD impacts the oral cavity, with effects including salivary gland dysfunction, enamel hypoplasia and damage, increased dentin formation, decreased pulp volume, pulp calcifications, and altered jaw bones, contributing to clinical manifestations of periodontal disease and tooth loss. Underlying mechanisms are not fully understood, and CKD mouse models commonly require invasive procedures with high rates of infection and mortality. We aimed to characterize the dentoalveolar effects of an adenine diet (AD)-induced CKD (AD-CKD) mouse model. Eight-week-old C57BL/6J mice were provided either a normal phosphorus diet control (CTR) or adenine and high-phosphorus diet CKD to induce kidney failure. Mice were euthanized at 15 weeks old, and mandibles were collected for micro-computed tomography and histology. CKD mice exhibited kidney failure, hyperphosphatemia, and hyperparathyroidism in association with porous cortical bone in femurs. CKD mice showed a 30% decrease in molar enamel volume compared to CTR mice. Enamel wear was associated with reduced ductal components, ectopic calcifications, and altered osteopontin (OPN) deposition in submandibular salivary glands of CKD mice. Molar cusps in CKD mice were flattened, exposing dentin. Molar dentin/cementum volume increased 7% in CKD mice and pulp volume decreased. Histology revealed excessive reactionary dentin and altered pulp-dentin extracellular matrix proteins, including increased OPN. Mandibular bone volume fraction decreased 12% and bone mineral density decreased 9% in CKD versus CTR mice. Alveolar bone in CKD mice exhibited increased tissue-nonspecific alkaline phosphatase localization, OPN deposition, and greater osteoclast numbers. AD-CKD recapitulated key aspects reported in CKD patients and revealed new insights into CKD-associated oral defects. This model has potential for studying mechanisms of dentoalveolar defects or therapeutic interventions. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Distúrbio Mineral e Ósseo na Doença Renal Crônica , Hiperfosfatemia , Insuficiência Renal Crônica , Camundongos , Animais , Distúrbio Mineral e Ósseo na Doença Renal Crônica/complicações , Adenina , Microtomografia por Raio-X , Hiperfosfatemia/complicações , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/complicações , FósforoRESUMO
Mineralization of the skeleton occurs by several physicochemical and biochemical processes and mechanisms that facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Two key phosphatases, phosphatase, orphan 1 (PHOSPHO1) and tissue-non-specific alkaline phosphatase (TNAP), play complementary roles in the mineralization process. The actions of PHOSPHO1 on phosphocholine and phosphoethanolamine in matrix vesicles (MVs) produce inorganic phosphate (Pi) for the initiation of HA mineral formation within MVs. TNAP hydrolyzes adenosine triphosphate (ATP) and the mineralization inhibitor, inorganic pyrophosphate (PPi), to generate Pi that is incorporated into MVs. Genetic mutations in the ALPL gene-encoding TNAP lead to hypophosphatasia (HPP), characterized by low circulating TNAP levels (ALP), rickets in children and/or osteomalacia in adults, and a spectrum of dentoalveolar defects, the most prevalent being lack of acellular cementum leading to premature tooth loss. Given that the skeletal manifestations of genetic ablation of the Phospho1 gene in mice resemble many of the manifestations of HPP, we propose that Phospho1 gene mutations may underlie some cases of "pseudo-HPP" where ALP may be normal to subnormal, but ALPL mutation(s) have not been identified. The goal of this perspective article is to compare and contrast the loss-of-function effects of TNAP and PHOSPHO1 on the dentoalveolar complex to predict the likely dental phenotype in humans that may result from PHOSPHO1 mutations. Potential cases of pseudo-HPP associated with PHOSPHO1 mutations may resist diagnosis, and the dental manifestations could be a key criterion for consideration.
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Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.
Assuntos
Distúrbio Mineral e Ósseo na Doença Renal Crônica , Hiperfosfatemia , Monoéster Fosfórico Hidrolases , Insuficiência Renal Crônica , Animais , Densidade Óssea/fisiologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/complicações , Distúrbio Mineral e Ósseo na Doença Renal Crônica/genética , Hiperfosfatemia/complicações , Camundongos , Camundongos Knockout , Fosfatos , Monoéster Fosfórico Hidrolases/metabolismo , Insuficiência Renal Crônica/genéticaRESUMO
Patients with chronic kidney disease (CKD) suffer from arterial media calcification and a disturbed bone metabolism. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the calcification inhibitor pyrophosphate (PPi) into inorganic phosphate (Pi) and thereby stimulates arterial media calcification as well as physiological bone mineralization. This study investigates whether the TNAP inhibitor SBI-425, PPi or the combination of both inhibit arterial media calcification in an 0.75% adenine rat model of CKD. Treatments started with the induction of CKD, including (i) rats with normal renal function (control diet) treated with vehicle and CKD rats treated with either (ii) vehicle, (iii) 10 mg/kg/day SBI-425, (iv) 120 µmol/kg/day PPi and (v) 120 µmol/kg/day PPi and 10 mg/kg/day SBI-425. All CKD groups developed a stable chronic renal failure reflected by hyperphosphatemia, hypocalcemia and high serum creatinine levels. CKD induced arterial media calcification and bone metabolic defects. All treatments, except for SBI-425 alone, blocked CKD-related arterial media calcification. More important, SBI-425 alone and in combination with PPi increased osteoid area pointing to a less efficient bone mineralization. Clearly, potential side effects on bone mineralization will need to be assessed in any clinical trial aimed at modifying the Pi/PPi ratio in CKD patients who already suffer from a compromised bone status.
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Background Homoarginine ( hA rg) has been shown to be cardioprotective in a model of ischemic heart failure; however, the mechanism remains unknown. hA rg can inhibit tissue-nonspecific alkaline phosphatase ( TNAP ), an enzyme that promotes vascular calcification. We hypothesized that hA rg will exert beneficial effects by reducing calcification in a mouse model of coronary artery disease associated with TNAP overexpression and hypercholesterolemia. Methods and Results TNAP was overexpressed in the endothelium in mice homozygous for a low-density lipoprotein receptor mutation (wicked high cholesterol [ WHC ] allele). WHC and WHC -endothelial TNAP mice received placebo or hA rg supplementation (14 mg/L in drinking water) starting at 6 weeks of age simultaneously with an atherogenic diet. Outcomes were compared between the groups after 4 to 5 weeks on treatment. Experiments were performed in males, which presented a study limitation. As expected, WHC -endothelial TNAP mice on the placebo had increased mortality (median survival 27 days, P<0.0001), increased coronary calcium and lipids ( P<0.01), increased left ventricular end-diastolic diameter ( P<0.0001), reduced ejection fraction ( P<0.05), and increased myocardial fibrosis ( P<0.0001) compared with WHC mice. Contrary to our hypothesis, hA rg neither inhibited TNAP activity in vivo nor reduced coronary artery calcification and atherosclerosis in WHC -endothelial TNAP mice; however, compared with the placebo, hA rg prevented left ventricular dilatation ( P<0.01), preserved ejection fraction ( P<0.05), and reduced myocardial fibrosis ( P<0.001). Conclusions The beneficial effect of hA rg supplementation in the setting of calcified coronary artery disease is likely due to its direct protective actions on the myocardial response to the ischemic injury and not to the inhibition of TNAP activity and calcification.
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Fosfatase Alcalina/efeitos dos fármacos , Doença da Artéria Coronariana/fisiopatologia , Endotélio/efeitos dos fármacos , Coração/efeitos dos fármacos , Homoarginina/farmacologia , Calcificação Vascular/patologia , Função Ventricular Esquerda/efeitos dos fármacos , Fosfatase Alcalina/genética , Animais , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Dieta Aterogênica , Dilatação Patológica/diagnóstico por imagem , Dilatação Patológica/genética , Dilatação Patológica/fisiopatologia , Modelos Animais de Doenças , Ecocardiografia , Endotélio/metabolismo , Fibrose , Hipercolesterolemia/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Miocárdio/patologia , Receptores de LDL/genética , Volume Sistólico/efeitos dos fármacos , Volume Sistólico/genética , Taxa de Sobrevida , Sístole , Calcificação Vascular/genética , Função Ventricular Esquerda/genéticaRESUMO
Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.
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Niacinamida/análogos & derivados , Pseudoxantoma Elástico/tratamento farmacológico , Pirofosfatases/antagonistas & inibidores , Sulfonamidas/administração & dosagem , Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Difosfatos/sangue , Difosfatos/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Niacinamida/administração & dosagem , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudoxantoma Elástico/sangue , Pseudoxantoma Elástico/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , Pele/metabolismo , Pele/patologia , Calcificação Vascular/sangue , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/genéticaRESUMO
Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePPi), a potent mineralization inhibitor, to phosphate (Pi). By the controlled hydrolysis of ePPi, TNAP maintains the correct ratio of Pi to ePPi and therefore enables normal skeletal and dental calcification. In other areas of the body low ePPi levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePPi. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.
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Fosfatase Alcalina/antagonistas & inibidores , Inibidores Enzimáticos/química , Niacinamida/análogos & derivados , Niacinamida/química , Sulfonamidas/química , Administração Oral , Fosfatase Alcalina/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Concentração Inibidora 50 , Camundongos , Niacinamida/metabolismo , Niacinamida/farmacocinética , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinéticaRESUMO
Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2 (-/-) ) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2 (-/-) mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP.
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The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Furthermore, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared with wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates.
Assuntos
Fosfatase Alcalina/fisiologia , Intestinos/microbiologia , Trifosfato de Adenosina/farmacologia , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Fosfatase Alcalina/farmacologia , Ampicilina/farmacologia , Animais , Desoxirribonucleotídeos/farmacologia , Farmacorresistência Bacteriana , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fezes/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morganella morganii/efeitos dos fármacos , Fenilalanina/farmacologia , Inanição/fisiopatologia , Estreptomicina/farmacologiaRESUMO
Generalized arterial calcification (AC) of infancy (GACI) is an autosomal recessive disorder that features hydroxyapatite deposition within arterial elastic fibers. Untreated, approximately 85% of GACI patients die by 6 months of age from cardiac ischemia and congestive heart failure. The first-generation bisphosphonate etidronate (EHDP; ethane-1-hydroxy-1,1-diphosphonic acid, also known as 1-hydroxyethylidene-bisphosphonate) inhibits bone resorption and can mimic endogenous inorganic pyrophosphate by blocking mineralization. With EHDP therapy for GACI, AC may resolve without recurrence upon treatment cessation. Skeletal disease is not an early characteristic of GACI, but rickets can appear from acquired hypophosphatemia or prolonged EHDP therapy. We report a 7-year-old boy with GACI referred for profound, acquired, skeletal disease. AC was gone after 5 months of EHDP therapy during infancy, but GACI-related joint calcifications progressed. He was receiving EHDP, 200 mg/day orally, and had odynodysphagia, diffuse opioid-controlled pain, plagiocephaly, facial dysmorphism, joint calcifications, contractures, and was wheelchair bound. Biochemical parameters of mineral homeostasis were essentially normal. Serum osteocalcin was low and the brain isoform of creatine kinase and tartrate-resistant acid phosphatase 5b (TRAP-5b) were elevated as in osteopetrosis. Skeletal radiographic findings resembled pediatric hypophosphatasia with pancranial synostosis, long-bone bowing, widened physes, as well as metaphyseal osteosclerosis, cupping and fraying, and "tongues" of radiolucency. Radiographic features of osteopetrosis included osteosclerosis and femoral Erlenmeyer flask deformity. After stopping EHDP, he improved rapidly, including remarkable skeletal healing and decreased joint calcifications. Profound, but rapidly reversible, inhibition of skeletal mineralization with paradoxical calcifications near joints can occur in GACI from protracted EHDP therapy. Although EHDP treatment is lifesaving in GACI, surveillance for toxicity is crucial.
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Doenças Ósseas/induzido quimicamente , Ácido Etidrônico/efeitos adversos , Ácido Etidrônico/uso terapêutico , Calcificação Vascular/tratamento farmacológico , Adulto , Doenças Ósseas/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Radiografia , Calcificação Vascular/diagnóstico por imagemRESUMO
Bone graft substitutes have become an essential component in a number of orthopedic applications. Autologous bone has long been the gold standard for bone void fillers. However, the limited supply and morbidity associated with using autologous graft material has led to the development of many different bone graft substitutes. Allogeneic demineralized bone matrix (DBM) has been used extensively to supplement autograft bone because of its inherent osteoconductive and osteoinductive properties. Synthetic and natural bone graft substitutes that do not contain growth factors are considered to be osteoconductive only. Bioactive glass has been shown to facilitate graft containment at the operative site as well as activate cellular osteogenesis. In the present study, we present the results of a comprehensive in vitro and in vivo characterization of a combination of allogeneic human bone and bioactive glass bone void filler, NanoFUSE(®) DBM. NanoFUSE(®) DBM is shown to be biocompatible in a number of different assays and has been cleared by the FDA for use in bone filling indications. Data are presented showing the ability of the material to support cell attachment and proliferation on the material thereby demonstrating the osteoconductive nature of the material. NanoFUSE(®) DBM was also shown to be osteoinductive in the mouse thigh muscle model. These data demonstrate that the DBM and bioactive glass combination, NanoFUSE(®) DBM, could be an effective bone graft substitute.
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Materiais Biocompatíveis/farmacologia , Matriz Óssea/química , Substitutos Ósseos/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Osseointegração/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Matriz Óssea/ultraestrutura , Adesão Celular/efeitos dos fármacos , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Gelatina/farmacologia , Cobaias , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteogênese/efeitos dos fármacos , CoelhosRESUMO
BACKGROUND: Hypophosphatasia results from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates extracellularly, leading to rickets or osteomalacia. Severely affected babies often die from respiratory insufficiency due to progressive chest deformity or have persistent bone disease. There is no approved medical therapy. ENB-0040 is a bone-targeted, recombinant human TNSALP that prevents the manifestations of hypophosphatasia in Tnsalp knockout mice. METHODS: We enrolled infants and young children with life-threatening or debilitating perinatal or infantile hypophosphatasia in a multinational, open-label study of treatment with ENB-0040. The primary objective was the healing of rickets, as assessed by means of radiographic scales. Motor and cognitive development, respiratory function, and safety were evaluated, as well as the pharmacokinetics and pharmacodynamics of ENB-0040. RESULTS: Of the 11 patients recruited, 10 completed 6 months of therapy; 9 completed 1 year. Healing of rickets at 6 months in 9 patients was accompanied by improvement in developmental milestones and pulmonary function. Elevated plasma levels of the TNSALP substrates inorganic pyrophosphate and pyridoxal 5'-phosphate diminished. Increases in serum parathyroid hormone accompanied skeletal healing, often necessitating dietary calcium supplementation. There was no evidence of hypocalcemia, ectopic calcification, or definite drug-related serious adverse events. Low titers of anti-ENB-0040 antibodies developed in four patients, with no evident clinical, biochemical, or autoimmune abnormalities at 48 weeks of treatment. CONCLUSIONS: ENB-0040, an enzyme-replacement therapy, was associated with improved findings on skeletal radiographs and improved pulmonary and physical function in infants and young children with life-threatening hypophosphatasia. (Funded by Enobia Pharma and Shriners Hospitals for Children; ClinicalTrials.gov number, NCT00744042.).
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Fosfatase Alcalina/uso terapêutico , Terapia de Reposição de Enzimas , Hipofosfatasia/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Raquitismo/tratamento farmacológico , Fosfatase Alcalina/administração & dosagem , Fosfatase Alcalina/farmacologia , Disponibilidade Biológica , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Pré-Escolar , Terapia de Reposição de Enzimas/efeitos adversos , Feminino , Humanos , Hipofosfatasia/complicações , Imunoglobulina G/administração & dosagem , Imunoglobulina G/farmacologia , Lactente , Recém-Nascido , Infusões Intravenosas , Injeções Subcutâneas/efeitos adversos , Masculino , Radiografia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Raquitismo/diagnóstico por imagem , Raquitismo/etiologia , Resultado do TratamentoRESUMO
The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.
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Fosfatase Alcalina/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoenzimas , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator's laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen.
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Fosfatase Alcalina/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Animais , Biocatálise/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Colorimetria , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Medições Luminescentes , Especificidade de Órgãos/efeitos dos fármacos , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , Sulfanilamida , Sulfanilamidas/químicaRESUMO
Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) for small molecule TNAP inhibitors led to the identification of hits in the sub-micromolar potency range. We report the design, synthesis and in vitro evaluation of a series of pyrazole derivatives of a screening hit which are potent TNAP inhibitors exhibiting IC(50) values as low as 5nM. A representative of the series was characterized in kinetic studies and determined to have a mode of inhibition not previously observed for TNAP inhibitors.
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Fosfatase Alcalina/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pirazóis/síntese química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Cinética , Pirazóis/farmacologiaRESUMO
INTRODUCTION: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. MATERIALS AND METHODS: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. RESULTS: Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. CONCLUSIONS: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.
Assuntos
Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/uso terapêutico , Terapia Biológica , Hipofosfatasia/tratamento farmacológico , Hipofosfatasia/enzimologia , Fosfatase Alcalina/deficiência , Fosfatase Alcalina/farmacocinética , Animais , Humanos , Hipofosfatasia/diagnóstico por imagem , Hipofosfatasia/genética , Camundongos , Camundongos Knockout , Radiografia , Fatores de TempoRESUMO
Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.
Assuntos
Difosfatos/metabolismo , Proteínas de Membrana/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Pirofosfatases/fisiologia , Sialoglicoproteínas/fisiologia , Fosfatase Alcalina/análise , Animais , Sequência de Bases , Osso e Ossos/citologia , Calcificação Fisiológica , Calcinose , Primers do DNA , DNA Complementar , Líquido Extracelular/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Osteoblastos/fisiologia , Osteopontina , Proteínas de Transporte de Fosfato , Diester Fosfórico Hidrolases/deficiência , Diester Fosfórico Hidrolases/genética , Pirofosfatases/deficiência , Pirofosfatases/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genéticaRESUMO
The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.