Mechanism of action of SNF472, a novel calcification inhibitor to treat vascular calcification and calciphylaxis.

BACKGROUND AND PURPOSE: No therapy is approved for vascular calcification or calcific uraemic arteriolopathy (calciphylaxis), which increases mortality and morbidity in patients undergoing dialysis. Deposition of hydroxyapatite (HAP) crystals in arterial walls is the common pathophysiologic mechanism. The mechanism of action of SNF472 to reduce HAP deposition in arterial walls was investigated. EXPERIMENTAL APPROACH: We examined SNF472 binding features (affinity, release kinetics and antagonism type) for HAP crystals in vitro, inhibition of calcification in excised vascular smooth muscle cells from rats and bone parameters in osteoblasts from dogs and rats. KEY RESULTS: SNF472 bound to HAP with affinity (KD ) of 1-10 µM and saturated HAP at 7.6 µM. SNF472 binding was fast (80% within 5 min) and insurmountable. SNF472 inhibited HAP crystal formation from 3.8 µM, with complete inhibition at 30.4 µM. SNF472 chelated free calcium with an EC50 of 539 µM. Chelation of free calcium was imperceptible for SNF472 1-10 µM in physiological calcium concentrations. The lowest concentration tested in vascular smooth muscle cells, 1 µM inhibited calcification by 67%. SNF472 showed no deleterious effects on bone mineralization in dogs or in rat osteoblasts. CONCLUSION AND IMPLICATIONS: These experiments show that SNF472 binds to HAP and inhibits further HAP crystallization. The EC50 for chelation of free calcium is 50-fold greater than a maximally effective SNF472 dose, supporting the selectivity of SNF472 for HAP. These findings indicate that SNF472 may have a future role in the treatment of vascular calcification and calcific uraemic arteriolopathy in patients undergoing dialysis.

Characterization of SNF472 pharmacokinetics and efficacy in uremic and non-uremic rats models of cardiovascular calcification.

End-stage renal disease is strongly associated with progressive cardiovascular calcification (CVC) and there is currently no therapy targeted to treat CVC. SNF472 is an experimental formulation under development for treatment of soft tissue calcification. We have investigated the pharmacokinetics of SNF472 administration in rats and its inhibitory effects on CVC. SNF472 was studied in three rat models: (1) prevention of vitamin D3-induced CVC with an intravenous SNF472 bolus of 1 mg/kg SNF472, (2) inhibition of progression of vitamin D3-induced CVC with a subcutaneous SNF472 bolus of 10 or 60 mg/kg SNF472, starting after calcification induction, (3) CVC in adenine-induced uremic rats treated with 50 mg/kg SNF472 via i.v. 4h -infusion. Uremic rats presented lower plasma levels of SNF472 than control animals after i.v. infusion. CVC in non-uremic rats was inhibited by 60-70% after treatment with SNF472 and progression of cardiac calcification completely blocked. Development of CVC in uremic rats was inhibited by up to 80% following i.v. infusion of SNF472. SNF472 inhibits the development and progression of CVC in uremic and non-uremic rats in the same range of SNF472 plasma levels but using in each case the required dose to obtain those levels. These results collectively support the development of SNF472 as a novel therapeutic option for treatment of CVC in humans.

Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti-O-P Bonding Shows Bone Stimulating Surface Properties and Decreased Bacterial Adhesion.

Myo-inositol hexaphosphate, also called phytic acid or phytate (IP6), is a natural molecule abundant in vegetable seeds and legumes. Among other functions, IP6 inhibits bone resorption. It is adsorbed on the surface of hydroxyapatite, inhibiting its dissolution and decreasing the progressive loss of bone mass. We present here a method to directly functionalize Ti surfaces covalently with IP6, without using a cross-linker molecule, through the reaction of the phosphate groups of IP6 with the TiO2 layer of Ti substrates. The grafting reaction consisted of an immersion in an IP6 solution to allow the physisorption of the molecules onto the substrate, followed by a heating step to obtain its chemisorption, in an adaptation of the T-Bag method. The reaction was highly dependent on the IP6 solution pH, only achieving a covalent Ti-O-P bond at pH 0. We evaluated two acidic pretreatments of the Ti surface, to increase its hydroxylic content, HNO3 30% and HF 0.2%. The structure of the coated surfaces was characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and ellipsometry. The stability of the IP6 coating after three months of storage and after sterilization with γ-irradiation was also determined. Then, we evaluated the biological effect of Ti-IP6 surfaces in vitro on MC3T3-E1 osteoblastic cells, showing an osteogenic effect. Finally, the effect of the surfaces on the adhesion and biofilm viability of oral microorganisms S. mutans and S. sanguinis was also studied, and we found that Ti-IP6 surfaces decreased the adhesion of S. sanguinis. A surface that actively improves osseointegration while decreasing the bacterial adhesion could be suitable for use in bone implants.

Relationship between Urinary Level of Phytate and Valvular Calcification in an Elderly Population: A Cross-Sectional Study.

Pathological calcification generally consists of the formation of solid deposits of hydroxyapatite (calcium phosphate) in soft tissues. Supersaturation is the thermodynamic driving force for crystallization, so it is believed that higher blood levels of calcium and phosphate increase the risk of cardiovascular calcification. However several factors can promote or inhibit the natural process of pathological calcification. This cross-sectional study evaluated the relationship between physiological levels of urinary phytate and heart valve calcification in a population of elderly out subjects. A population of 188 elderly subjects (mean age: 68 years) was studied. Valve calcification was measured by echocardiography. Phytate determination was performed from a urine sample and data on blood chemistry, end-systolic volume, concomitant diseases, cardiovascular risk factors, medication usage and food were obtained. The study population was classified in three tertiles according to level of urinary phytate: low (<0.610 µM), intermediate (0.61-1.21 µM), and high (>1.21 µM). Subjects with higher levels of urinary phytate had less mitral annulus calcification and were less likely to have diabetes and hypercholesterolemia. In the multivariate analysis, age, serum phosphorous, leukocytes total count and urinary phytate excretion appeared as independent factors predictive of presence of mitral annulus calcification. There was an inverse correlation between urinary phytate content and mitral annulus calcification in our population of elderly out subjects. These results suggest that consumption of phytate-rich foods may help to prevent cardiovascular calcification evolution.

Validation of an LC-MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma.

Myo-inositol hexakisphosphate (phytate, IP6) is a naturally occuring compound whose determination in biological matrices is chanllenging. Several benefitial properties have been attributed to IP6 in parallel with the development of suitable analytical methodologies for its analytical determination in urine and some tissues. However, there is a lack of appropriate tools for its determination in plasma samples. In this paper, a direct, sensitive and selective bioanalytical method for the determination of IP6 based on LC-MS is presented. It is the first method published to quantify IP6 in plasma matrices directly through its molecular weight, being consequently a highly specific methodology. The method has been validated in rat, dog and human plasma, according to the acceptance criteria laid down in the FDA guidance Bioanalytical Method Validation. Accuracy and precision were not greater than 15% at medium and high concentrations and not greater than 20% at the LLOQ concentration. The mean absolute recovery obtained ranged from 78.74 to 102.44%, 62.10 to 87.21% and 61.61 to 86.99% for rat, dog and human plasma respectively. The LLOQ was 500ngmL(-1) due to the presence of endogenous IP6 in blank plasma samples and the limit of detection was within the range 30-80ngmL(-1).

Urinary lithogenesis risk tests: comparison of a commercial kit and a laboratory prototype test.

OBJECTIVE: Renal stone formation is a multifactorial process depending in part on urine composition. Other parameters relate to structural or pathological features of the kidney. To date, routine laboratory estimation of urolithiasis risk has been based on determination of urinary composition. This process requires collection of at least two 24 h urine samples, which is tedious for patients. The most important feature of urinary lithogenic risk is the balance between various urinary parameters, although unknown factors may be involved. The objective of this study was to compare data obtained using a commercial kit with those of a laboratory prototype, using a multicentre approach, to validate the utility of these methods in routine clinical practice. MATERIAL AND METHODS: A simple new commercial test (NefroPlus®; Sarstedt AG & Co., Nümbrecht, Germany) evaluating the capacity of urine to crystallize calcium salts, and thus permitting detection of patients at risk for stone development, was compared with a prototype test previously described by this group. Urine of 64 volunteers produced during the night was used in these comparisons. The commercial test was also used to evaluate urine samples of 83 subjects in one of three hospitals. RESULTS: Both methods were essentially in complete agreement (98%) with respect to test results. The multicentre data were: sensitivity 94.7%; specificity 76.9%; positive predictive value (lithogenic urine) 90.0%; negative predictive value (non-lithogenic urine) 87.0%; test efficacy 89.2%. CONCLUSION: The new commercial NefroPlus test offers fast and cheap evaluation of the overall risk of development of urinary calcium-containing calculi.

Evolution of post-ESWL residual lithiasis depending on the type of calculus and urine composition.

OBJECTIVES: Extracorporeal shock wave lithotripsy (ESWL) is one of the most commonly used procedures for removal of renal calculi from the upper urinary tract, but complete expulsion of the fragments generated is not always achieved. This can lead to new lithiasic episodes, and it is considered that 10-26% of fragmented calculi can undergo regrowth. This in vitro study investigated the influence of fragment and urinary composition on post-ESWL growth of fragments, with the aims of establishing the effect and importance of these parameters, and identifying effective prophylactic measures. METHODS: Fragments collected from patients immediately following expulsion after ESWL treatment were selected for regrowth experiments. The particles included 24 calcium oxalate monohydrate (COM) fragments, 48 calcium oxalate dihydrate (COD), 24 hydroxyapatite (HAP), and 16 uric acid. RESULTS: In all treatments, calculi fragments showed a considerable capacity to induce growth of calcium oxalate and calcium phosphate. Under normocalciuria conditions, new COM crystals formed; both COM and COD crystals developed under hypercalciuria conditions at a urinary pH < 6.0; and in hypercalciuric conditions and urinary pH > 6.0 both HAP and brushite (BRU) crystals were formed. The highest growth rates were observed for COD calculi fragments under hypercalciuria conditions and at a urinary pH of 6.5, followed by growth on COM and HAP fragments under the same conditions; growth rates under other conditions tested were similar but 10-fold lower. With regard to the role of crystallization inhibitors, phytate exhibited inhibitory effects under all assay conditions. However, citrate had little effect, even at the highest concentration tested (1,000 mg/L). CONCLUSIONS: This study demonstrates the importance of avoiding heterogeneous nucleant retention (pre-existing solid microparticles) in renal cavities, as these can act as very efficient inducers of the formation of new calculi, the composition of which is mainly dependant on the urine composition.

Phytate reduces age-related cardiovascular calcification.

The aim of this research was to evaluate the effect of dietary phytate on cardiovascular calcification in rats during aging. Male Wistar rats (10 weeks old) were randomly assigned to four diet groups. The control group was fed with a balanced diet (UAR-A04) containing phytate. The AIN group was fed a purified diet (AIN-76A) with an undetectable level of phytate. The PHY group was fed with a purified diet (AIN-76A) enriched with phytate (phytin, as the calcium magnesium salt). The MOD group was fed with the AIN-76A diet (phytate undetectable) enriched with MgO, inositol and CaHPO4. At 76 weeks of age all rats were sacrificed, and the aortas, hearts, kidneys, livers and femurs were removed for chemical analysis. The most significant differences were found in the aorta calcium content. Phytate-treated rats (the control and PHY groups) had significantly lower levels of calcium in the aorta compared to nonphytate-treated rats (AIN and MOD groups). The present study demonstrated that dietary phytate treatment significantly reduced age-related aorta calcification.

Phytate inhibits bovine pericardium calcification in vitro.

OBJECTIVE: The present study examined the inhibitory effects of pyrophosphate, etidronate, and phytate on bovine pericardium calcification in vitro. METHODS: Bovine pericardium was glutaraldehyde fixed and then placed in a flow chamber in the presence of a synthetic physiological fluid alone (control) or the fluid plus various concentrations of pyrophosphate, etidronate, or phytate. Following a 96-h incubation, fragments were removed and assayed for calcification by measuring calcium and phosphorus levels. RESULTS: The data indicated that both pyrophosphate and etidronate at 1 mg/l (5.75 and 4.95 microM, respectively) inhibited bovine pericardium calcification, whereas neither agent had an effect at 0.5 mg/l (2.87 and 2.47 microM, respectively). Phytate was the most potent inhibitor of calcification, and the effects of this agent were apparent at levels as low as 0.25 mg/l (0.39 microM). CONCLUSIONS: While pyrophosphate, etidronate, and phytate were all able to inhibit bovine pericardium calcification in vitro, phytate was found to be the most effective.

Effect of crystallization inhibitors on vascular calcifications induced by vitamin D: a pilot study in Sprague-Dawley rats.

BACKGROUND: Pathological calcification in soft tissues (ie, ectopic calcification) can have severe consequences. Hydroxyapatite is the common mineral phase present in all tissue calcifications. In general, the development of tissue calcifications requires a pre-existing injury as an inducer (heterogeneous nucleant), whereas further progression requires the presence of other promoter factors (such as hypercalcemia and/or hyperphosphatemia) and/or a deficiency in calcification repressor factors (crystallization inhibitors and cellular defense mechanisms). The present study investigated the capacity of etidronate (a bisphosphonate used in osteoporosis treatment) and phytate (a natural product) to inhibit vascular calcification in rats. METHODS AND RESULTS: Six male Sprague-Dawley rats in each of the 3 treatment groups were subcutaneously injected with either a placebo (physiological serum solution), etidronate (0.825 micromol x kg(-1) x day (-1)) or phytate (0.825 micromol x kg (-1) x day(-1)) for 8 days. Four days into this regimen, calcinosis was induced by subcutaneous injections of 500,000 IU/kg vitamin D at 0 h, 24 h and 48 h. Ninety-six hours after the final vitamin D injection, the rats were killed and aortas and their hearts were removed for histological and calcium analyses. The data showed that phytate-treated rats had lower levels of aortic calcium than placebo-treated rats. All groups had similar heart calcium levels. CONCLUSIONS: The present study found that phytate acted as a vascular calcification inhibitor. Thus, the action of polyphosphates could be important in protecting against vascular calcification.

Uric acid as inducer of calcium oxalate crystal development.

OBJECTIVE: This paper deals with the mechanism by which uric acid affects calcium oxalate crystallization and the role of crystallization inhibitors in this process. MATERIAL AND METHODS: Pure uric acid crystals and fragments of uric acid renal calculi were used to induce calcium oxalate crystal formation and development. These studies were performed in flow systems, using synthetic urine and similar conditions to those found in real renal situations. The type and size of the developed crystals were evaluated by scanning electron microscopy and the amount of calcium oxalate crystallized was quantitated by means of inductively coupled plasma atomic emission spectroscopy. RESULTS: The presence of uric acid crystals in a flow system provoked calcium oxalate monohydrate (COM) crystallization at a rate of 3.3 microg/h/mg uric acid. When uric acid renal calculi fragments were used, the amount of COM crystallized varied between 0.048 and 0.161 microg/h/mg of renal calculi depending on the porosity of the calculus. At particular concentrations (3.03 microM phytate, 28.75 microM pyrophosphate, 40 mg/l chondroitin sulphate) the crystallization inhibitors assayed produced a maximum decrease of approximately 50% in the amount of COM crystallized on uric acid crystals. Mucin (a glycoprotein) caused only slight effects. CONCLUSION: Uric acid crystals can clearly induce the development of COM crystals on them through a heterogeneous nucleation process and some crystallization inhibitors can notably delay such a process.

Phytate acts as an inhibitor in formation of renal calculi.

The aim of this study was to assess the inhibitory action of phytate in formation of renal calculi. Hypertension (induced by nicotine) combined with hypercalcemia (induced by D vitamin) was used to induce calcification in renal tissue in male Wistar rats that were fed a purified phytate free diet. Phytate non-treated rats developed significant calcium deposits in kidneys and papillae, as well as in kidney tubules and vessels, whereas calcium deposits were absent in control and phytate treated rats. Fragments of hydroxyapatite (HAP) calculi exhibited the capacity to induce the growth of calcium salts on their surfaces. Presence of 1.5 mg/L of phytate in the synthetic urine inhibited the formation of calcium oxalate monohydrate on HAP renal calculi in normocalciuric conditions. The findings show that the action of phytate as a crystallization inhibitor takes place both in the intrapapillary tissue and urine.

Phytate (Myo-inositol hexakisphosphate) inhibits cardiovascular calcifications in rats.

Calcification is an undesirable disorder, which frequently occurs in the heart vessels. In general, the formation of calcific vascular lesions involves complex physicochemical and molecular events. Calcification (hydroxyapatite) is initiated by injury and is progressed by promoter factors and/or the deficit of inhibitory signals. Myo-inositol hexakisphosphate (phytate, InsP6) is found in organs, tissues and fluids of all mammals and exhibits an important capacity as a crystallization inhibitor of calcium salts in urine and soft tissues. The levels found clearly depend on the dietary intake but it can also be absorbed topically. In this paper, the capacity of InsP6 as a potential inhibitor of cardiovascular calcifications was assessed in Wistar rats. Three groups were included, a control group, an InsP6 treated group (subjected to calcinosis induction by Vitamin D and nicotine and treated with standard cream with a 2% of InsP6 as potassium salt) and an InsP6 non-treated group (only subjected to calcinosis induction). All rats were fed AIN 76-A diet (a purified diet in which InsP6 is undetectable). Animals were monitorized every 12 hours. After 60 hours of calcinosis treatment, all rats of the InsP6 non-treated group died and the rest were sacrificed. Aortas and hearts were removed. A highly significant increase in the calcium content of aorta and heart tissue was observed in the InsP6 non-treated rats (21 +/- 1 mg calcium/g dry aorta tissue, 10 +/- 1 mg calcium/g dry heart tissue) when compared with controls (1.3 +/- 0.1 mg calcium/g dry aorta tissue, 0.023 +/- 0.004 mg calcium/g heart dry tissue) and InsP6 treated (0.9 +/- 0.2 mg calcium/g dry aorta tissue, 0.30 +/- 0.03 mg calcium/g dry heart tissue) animals. Only InsP6 non-treated rats displayed important mineral deposits in aorta and heart. These findings are consistent with the action of InsP6, as an inhibitor of calcification of cardiovascular system.

Factors affecting the regrowth of renal stones in vitro: a contribution to the understanding of renal stone development.

OBJECTIVE: The exact mechanism of renal stone formation is still not totally understood. Thus, the role of crystallization inhibitors at different stages of stone development, the influence of preexisting solid particles and the effects of variations in urine composition require further clarification. The aim of this paper is to clarify some of these questions by studying the regrowth achieved by real spontaneously passed post-extracorporeal shock wave lithotripsy (post-ESWL) fragments of calcium oxalate monohydrate (COM) renal calculi. MATERIAL AND METHODS: An in vitro system was used to study the regrowth of post-ESWL fragments of COM calculi, which was defined as the relative increase in weight of the fragments. RESULTS: It was found that new columnar zones of COM crystals were formed under normal calcium and oxalate urinary conditions and no calcium phosphates were observed, in spite of the urinary pH being >6. The presence of 3.03 microM phytate totally blocked these crystal growth processes. When hypercalciuric urine was used at a pH of 6.5, large brushite crystals and zones totally covered by hydroxyapatite were observed for short periods, and zones containing calcium oxalate dihydrate crystals could be observed for longer periods. In such cases, 9.09 microM phytate totally blocked the growth processes, 69.0 microM pyrophosphate caused a reduction in calculi growth of 93% and 5.35 mM citrate caused no inhibitory effects. CONCLUSION: The results show that when crystallization inhibitors were absent, the growth of calcium oxalate calculi fragments took place even under normal urine conditions, clearly demonstrating the importance of crystallization inhibitors in avoiding or delaying calculi development.

Study of the absorption of myo-inositol hexakisphosphate (InsP6) through the skin.

Recently, some properties of myo-inositol hexakisphosphate (InsP(6)) are related to its dermatological use as discolouring agent, on preventing calcinosis cutis or due to its important role on premature aging. Some studies also seem to demonstrate a capacity of InsP(6) to inhibit skin cancer. In this paper, a first study of the absorption of InsP(6) through the skin is developed. Due to the correlation between InsP(6) absorption and its urinary excretion, these last values were used to evaluate this process. It was found that using a moisturizing cream as vehicle, the InsP(6) sodium salt was absorbed at significantly higher amounts than the InsP(6) calcium-magnesium salt. Maximum InsP(6) urinary concentrations were observed approximately at 14 d of 2% InsP(6) topical cream application, and gave 66.35+/-5.49 mg/l urinary InsP(6) when the sodium salt was used and 16.02+/-2.61 mg/l urinary InsP(6) when the calcium-magnesium salt was applied. When the InsP(6) topical cream administration ceased, the InsP(6) urinary excretion fell dramatically approximately during a period of 10 d. From these results, it can be deduced that by topical administration InsP(6) can achieve important concentrations in tissues and biological fluids, this demonstrating that it is possible to propose the topic use as a new InsP(6) administration route.

Role of the organic matter in calcium oxalate lithiasis.

Urine contains variable amounts of organic matter derived from cell degradation. The cellular detritus is composed by membranous and cytosolic glycoproteins, etc. The aim of this paper was to study the role of organic matter in calcium oxalate crystal development and to evaluate the action of some crystallization inhibitors on this process. Crystallization studies were carried out on urine in stagnant urine as well as under flow conditions, in presence and absence of cellular debris. Low amounts of cellular debris (when batch conditions were used), exhibited some inhibitory activity on heterogeneous nucleation of calcium oxalate monohydrate crystals, probably due to glycoproteins. Increasing amounts of cellular debris, however, promoted the nucleation of calcium oxalate monohydrate crystals. When cellular debris was retained in a cavity with a urine flow, this organic matter effectively induced the development of primary aggregates of calcium oxalate monohydrate crystals (crystallization range 2.8 mg/h per mg of organic matter). Presence of crystallization inhibitors prevented or minimized crystal development. These findings show that cell debris provides the necessary elements for the development of oxalate crystals and that this process can be effectively inhibited by presence of crystallization inhibitors.

Absorption of myo-inositol hexakisphosphate (InsP6) through the skin: study of the matrix effects. mechanism of phytate topical absorption.

Myo-inositol hexakisphosphate (InsP6, phytate) is a molecule to which diverse beneficial properties have been attributed. Some of these properties are related to its dermatological use as discolouring agent, on preventing calcinosis cutis or due to its important role on premature aging. Other studies also seem to demonstrate a capacity of InsP6 to inhibit skin cancer. In this paper, the effect of the vehicle of topical administration of phytate is studied, using four groups of male Wistar rats (n = 6) fed with an InsP6 defficient diet and treated with a hydrophyl gel or an O/W moisturizing cream with two different concentrations of InsP6. Due to the correlation between InsP6 absorption and its urinary excretion, these last values were used to evaluate this process. It was found that phytate was absorbed through the skin using both a gel or a cream, demonstrating that its absorption is independent on the matrix used for topical treatment. However, urinary InsP6 values were slightly higher when using the gel, but in all cases values were much higher than those found with oral InsP6 treatment, due to the formation of insoluble species in the gastrointestinal tract when InsP6 is administered orally.

The role of glycoproteins in calcium oxalate crystal development.

OBJECTIVE: To assess the effects of a glycoprotein (mucine) on calcium oxalate crystal development in different conditions and situations, to clarify some of its possible effects. MATERIALS AND METHODS: Crystallization was assessed using a batch system in presence of mucine suspensions, by kinetic-turbidimetric measurements, and using a flow system in the presence of retained agglomerates of mucine, evaluating the precipitated calcium oxalate. RESULTS: In batch conditions low mucine concentrations (<15 mg/L) inhibited calcium oxalate nucleation and higher concentrations (<250 mg/L) inhibited calcium phosphate nucleation, whereas at high concentrations there was also promotion. The presence of an aggregate of mucine in the flow system provoked calcium oxalate monohydrate crystallization at 0.691 microg/h per mg of mucine. In flow conditions pyrophosphate at 11.5 micromol/L caused a decrease of 84% in the calcium oxalate crystallized on mucine, 1.32 mmol/L of citrate a decrease of a 83%, 20 mg/L of pentosan polysulphate a decrease by 80%, and 7.58 micromol/L phytate totally prevented the crystallization of calcium oxalate on mucine. CONCLUSION: All substances inhibiting calcium oxalate crystallization with the capacity to interact with calcium ions also have crystallization promoting properties when they are at sufficiently high concentrations, because of their capacity to form agglomerates or the insolubility of their calcium salts.