Oral pyrophosphate for use in reducing tissue calcification

Abstract

The invention is concerned with use of oral inorganic pyrophosphate for preventing and/or reducing tissue calcification, particularly soft tissue calcification, and/or diseases or disorders characterized by low plasma PPi levels, as, e.g., occurs in chronic kidney disease (CKD), end-stage renal disease (ESRD), generalized arterial calcification of infancy (GACI), Pseudoxanthoma elasticum (PXE), Arterial Calcification Due to Deficiency of CD73 (ACDC), Ehlers-Danlos syndrome, arteriosclerosis obliterans, venous calcifications, crystal deposition disorders, calcification resulting from neurological disorders, calcinosis universalis, calcinosis circumscripta, scleroderma, dermatomyositis, systemic lupus erythematosus, hyperparathyroidism, neoplasms, milk-alkali syndrome, hypervitaminosis D, tumoral calcinosis, hypophosphatemic rickets, ossification of the posterior longitudinal ligament of the spine, myocardial ischemia, joint calcification, heterotropic ossification of traumatized muscle, angioid streaks, diabetes mellitus type II, cardiovascular disorder, or atherosclerosis.

Claims

1. A method for treating and/or ameliorating generalized arterial calcification of infancy (GACI) or pseudoxanthoma elasticum (PXE), the method comprising the step of: administering to a subject in need thereof a therapeutically effective amount of inorganic pyrophosphate (PPi), wherein said inorganic pyrophosphate is administered in oral form and wherein the inorganic pyrophosphate is sufficient to achieve a transient increase in plasma PPi level in the subject.

2. The method according to claim 1, wherein the transient increase in plasma PPi level is characterized by a PPi level that is at least about 40% of the plasma PPi level in a healthy subject.

3. The method according to claim 1, wherein the transient increase in plasma PPi level is maintained for at least about 15 minutes.

4. The method according to claim 1, wherein the treating is slows the progression of the GACI or PXE.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows that oral PPi attenuates induced cardiac calcification in Abcc6.sup.−/− mice. PPi was provided in various concentrations (0.1-50 mM) in the drinking water to Abcc6.sup.−/− (A) or to C3H/He (B) mice (in which an alternative splice variant in Abcc6 leads to protein deficiency) a day before the cryo injury and continued for four days when the animals were sacrificed. The calcium content of heart tissue was determined. The insert shows calcification of the heart of Abcc6.sup.−/− mice four days after cryo-injury.

(2) FIG. 2 shows that oral PPi attenuates the spontaneous hair capsule calcification of vibrissae in Abcc6.sup.−/− mice. Abcc6.sup.−/− mice were kept on 10 mM PPi (in drinking water) starting 3 weeks after weaning until they were 22 weeks old. The control group was drinking tap water. Tissue blocks with the hair capsules were removed, paraffin-embedded, sectioned and stained with alizarin red for calcium deposits. The extent of calcification was expressed as the ratio of the number of calcifying capsules per total number of capsules (A). Panels B and C show typical alizarin red stained sections of an animal of the control group and that of the 10 mM PPi group, respectively.

(3) FIG. 3 shows that prenatal PPi treatment of the Enpp1.sup.−/− mice attenuates the spontaneous hair capsule calcification of the vibrissae and delays the tiptoe walking phenotype. The mother and the pups were kept on tap water until the pups were 30 days old (“control”); Group 1: as the control group, but for 9 days on 10 mM PPi solution after weaning at 21 days. Group 2: the mothers and the pups were kept on 10 mM PPi during pregnancy, breast feeding and for 9 days after weaning. Group 3: the mothers and the pups were kept on 10 mM PPi during breast feeding and for 9 days after weaning. Group 4: the mothers were kept on 10 mM PPi during pregnancy. Wild type controls are also shown.

(4) The calcium content of the tissue blocks of the hair capsules was determined (panel A). Panels B, C and D show typical alizarin red stained sections of an animal of each group. Panel E: delay of the day of onset of the tiptoe walking phenotype in pups kept on water or on 10 mM PPi either during pregnancy, breast feeding and after weaning or only during the pregnancy, or during breast feeding and after weaning, but not during the pregnancy.

(5) FIG. 4 shows uptake of PPi from drinking water in mice.

(6) A: Uptake from the oral cavity and from the stomach by continuous delivery of 50 mM PPi to Abcc6.sup.−/− mice without ligation. Mice were anesthetized and paper-pads placed into the mouth; 30 ul aliquots of 50 mM PPi were given by 10 minute-intervals for one hour. Blood was collected for PPi assay at the end of PPi administration, at 60 min. 50 mM PPi was provided in 30 ul aliquots directly into the stomach by a gavage into anesthetized Abcc6.sup.−/− mice for 60 minutes in the intervals described above and blood was collected for PPi assay at 60 minutes.

(7) B: Uptake from the oral cavity, from the stomach and from the intestine of C57/Bl6 mice after ligating the esophagus and applied oral delivery of 100 ul 50 mM PPi or the pylorus followed by stomach delivery of 200 ul 50 mM PPi and then ligation the esophagus, or injecting 200 ul 50 mM PPi into the intestine after ligation of the pylorus. In each experiment blood was collected after 15 min and PPi concentrations were determined.

(8) C: Plasma PPi concentrations after delivery of 200 ul of 50 mM PPi by gavage into the stomach of C57/Bl6 mice.

(9) FIG. 5 shows uptake of PPi from drinking water in a human volunteer.

EXAMPLES

(10) Animals and Animal Studies

(11) C57BL/6J mice, designated as wild type were derived from mice purchased from The Jacksons Laboratories. Abcc6.sup.−/− mice were generated on 129/Ola background and backcrossed into a C57BL/6J>10 time.sup.1. Ttw mice were obtained from Ttw.sup.+/− mating and each offspring was genotyped. C3H/He mice were purchased from Harlan (The Netherlands). Both male and female, age-matched Abcc6.sup.−/−, Ttw, C3H/He and wild type mice were used.

(12) All animals were housed in approved animal facilities at the Research Center for Natural Sciences, Hungarian Academy of Sciences. Mice were kept under routine laboratory conditions with 12 hour light-dark cycle with ad libitum access to water and chow. The RCNS, Hungarian Academy of Sciences Institutional Animal Care and Use Committees approved this study. Experiments have been conducted according to national guidelines. Cryo injury was performed and tissue calcium content was determined as described by Brampton, et al. ((2014) Am J Pathol. 184, 159-70). Calcification of the vibrissae was studied by histochemistry following the method described Klement, et al. ((2005) Mol Cell Biol. 25, 8299-310. The number of calcified vibrissae (hair capsules) was counted independently and the onset of tiptoe walking phenotype was determined by two investigators in a blinded fashion.

(13) Pyrophosphate Measurements

(14) Determination of PPi concentration in plasma was performed as described by Jansen et al ((2014) Arterioscler Thromb Vasc Biol. 34, 1985-9). Sodium pyrophosphate tetrabasic decahydrate (BioXtra quality) was purchased from Sigma.

(15) Statistical Analysis

(16) Data were compared by the Student t test. Values are expressed as mean+/−standard error of the mean (SEM). A p value<0.05 was considered statistically significant. Animal numbers used for individual set of data varied and is shown on the figures.

(17) Results

(18) Abcc6.sup.−/− mice faithfully recapitulate human PXE, with calcifying lesions found in skin, eyes and blood vessels. A drawback of the Abcc6.sup.−/− mice is the relatively late onset of the first lesions, making this model less convenient for rapid screening of new treatments. However, cryo-injury applied to the heart results in a calcified lesion within 3-6 days, a phenomenon fully dependent on the absence of Abcc6. As daily intraperitoneal (IP) PPi injections prevented calcification in several animal disease models, IP delivery (6 mg PPi/kg body weight for 7 days) in Abcc6.sup.−/− mice was tested. Indeed, PPi injections reduced the cardiac calcification (ICC) induced by cryo-injury (data not shown).

(19) It was then tested whether orally administered PPi also inhibited the calcification seen in the ICC model. It was found that PPi provided via the drinking water potently inhibited ICC. The extent of calcification inhibition was dose-dependent, with maximal inhibition seen at a PPi concentration of 10 mM (FIG. 1). Next, it was tested whether the spontaneous ectopic calcification seen in Abcc6.sup.−/− mice could also be attenuated by PPi administration via the drinking water. A peculiar phenotype seen in Abcc6.sup.−/− mice is the gradual calcification of the tissue surrounding the vibrissae. Therefore, the effect of oral PPi on the calcification of the dermal sheet surrounding the vibrissae was determined. Just after weaning Abcc6.sup.−/− mice were put on PPi-containing drinking water (10 mM) for a period of 19 weeks. Then the effect of the PPi-containing drinking water on the number of calcified vibrissae was determined. As can be seen in FIG. 2, PPi provided via the drinking water also potently inhibited the spontaneous calcification seen in Abcc6.sup.−/− mice.

(20) Tiptoe walking (ttw) mice have an inactivating mutation in Enpp1 and represent a model for GACI. Due to the complete absence of Enpp1, these mice have extremely low plasma PPi levels and develop extensive calcifications in the skin, blood vessels and joints shortly after birth. Just like Abcc6.sup.−/− mice, the ttw mice show extensive calcification of the dermal sheet surrounding the vibrissae, although in these animals this phenotype shows up much earlier than in mice lacking Abcc6. It was tested whether supplementation of PPi via the drinking water also effectively attenuated this ectopic calcification in the ttw mice.

(21) When PPi was given orally during pregnancy and lactation and afterwards the PPi treatment of the pups was continued, the calcification of the dermal sheet surrounding the vibrissae was greatly reduced (see FIG. 3). To determine when PPi treatment was most effective, the effect of PPi was analyzed separately during pregnancy, lactation and after weaning. When we only treated the ttw pups after weaning, the amount of calcification was no different from the control-treated group. PPi provided during lactation had no significant impact on calcification either. However, the most remarkable finding of these experiments was that the effect of PPi was maximal if administered during pregnancy. The same was true when the tiptoe walking phenotype (due to joint calcification) was determined (FIG. 3), which was delayed by four days in the “PPi during pregnancy” group. An explanation for these results is that microcrystals have already been formed in the control-treated group before birth. Without wishing to be bound by theory, it is hypothesized that these microcrystals might be mostly absent in ttw pups from mothers on PPi during pregnancy and may therefore not be available to accelerate the calcification process after weaning. Our data clearly demonstrate that orally administered PPi attenuates ectopic calcification.

(22) Uptake of PPi from the Oral Cavity and from the Stomach in Mice

(23) Mice were anesthetized and paper-pads placed into the mouth; 30 ul aliquots of 50 mM PPi were given by 10 minute-intervals for one hour. Blood was collected for PPi assay at the end of PPi administration, at 60 min. Alternatively, 50 mM PPi was provided in 30 ul aliquots directly into the stomach by a gavage into anesthetized Abcc6−/− mice for 60 minutes in the intervals described above and blood was collected for PPi assay at 60 minutes. It was found that PPi was taken up from both the oral cavity and the stomach (FIG. 4).

(24) Uptake of PPi into Plasma in Human

(25) A human volunteer drank 300 ml 100 mM Na.sub.4PPi pH8, and plasma PPi concentrations were measured in time. It was shown that orally administered PPi was taken up to provide increase plasma PPi levels (FIG. 5).