Patent Application
20160346320
The present invention relates to the use of a substance for inhibiting tissue calcification and tissue fibrosation, and for delaying the onset of age-related diseases and methods associated therewith.
The aging process of a living being is typically characterized by the increasing onset of diseases and organ disfunctions. Such so-called age-related diseases or aging syndromes ultimately result in the death of the being. Tissue calcification and tissue fibrosation play a decisive role in the aging process.
The tissue calcification plays a decisive role in particular in the accelerated aging of patients with renal failure. The decline of functioning organ tissue when replaced by connective tissue (fibrosation) plays a central role in renal failure, cirrhosis of the liver, Crohn's disease, fibrotic pancreatitis, pulmonary fibrosis, heart failure and scarring. Furthermore, tissue fibrosation leads to an impairment of the effectiveness of peritoneal dialysis.
Tissue calcification and fibrosation are both stimulated by the “transforming growth factor” TGFβ1, which also contributes to the development of Alzheimer's disease. An activation of the alkaline phosphatase and the increased expression of the transcription factor Runx2 also contribute to the signal transduction of tissue calcification.
Excessive tissue calcification, early onset of age-related diseases and shortened lifespans has been observed in mice with a klotho deficiency (kLothm). With an inhibition of the tissue calcification and extension of the lifetime after an agent has been administered to the mouse it can therefore be concluded that this agent delays tissue calcification and aging.
There are numerous references in the prior art to reputed substances for reducing tissue calcification and tissue fibrosation, and for delaying the onset of age-related diseases. In most cases, however, there is no scientific evidence for the effectiveness of these substances.
Based on this, the invention addresses the object of finding a substance for reducing tissue calcification and organ fibrosation, as well as age-related diseases in a living being.
This object is achieved by providing ammonium sulfate, ammonium chloride, the carbonic anhydrase inhibitor acetazolamide, chloroquine, ammonium nitrate, ammonium citrate, or ammonium lactate.
This discovery on the part of the inventor is surprising.
Ammonium sulfate is the salt from ammonia and sulfuric acid. In food technology, ammonium sulfate is used as an additive for regulating acidity, and is generally regarded as safe by the U.S. Food and Drug Administration (generally regarded as safe: [GRAS]). In the European Union it has the number E517.
Ammonium citrate is the salt from ammonia and citric acid, and is approved under the number E380.
Ammonium lactate is the salt from ammonia and lactic acid, and is listed in the European Union under the number E328 as an acidity regulator.
Ammonium nitrate is used as fertilizer and in explosives.
Ammonium chloride having the molecular formula NH4Cl, also referred to as ammonium muriate, ammonia salt, or sal ammoniac, and having the CAS-No. 12125/02/9, is the ammonium salt of hydrochloric acid. It is a colorless, crystalline solid. Ammonium chloride is used in food technology as an additive, and has the number E 510. In medicine, ammonium chloride is used as an expectorant, i.e. as mucus expectorants.
Chloroquine [(RS)—N′-(7-chloroquinoline-4-yl)-N,N-diethyl-pentane-1,4-diamine] alkalizes lysosomes and is used against malaria, for immunosuppression, for treating viral diseases, and to combat tumors.
The carbonic anhydrase inhibitor acetazolamide inhibits the enzymatic conversion of bicarbonate to carbon dioxide, and can therefore act on the local pH. It is used as a diuretic.
It is known that an acidosis, such as can be triggered by ammonium chloride (NH4Cl), can inhibit tissue calcification. It is furthermore known that acetazolamide can lower the phosphate concentration, resulting in a reduction in tissue calcification. In the experiments on the klothohm mouse, the tissue calcification is inhibited by ammonium chloride, but without increasing the acidosis, and by acetazolamide, without lowering the plasma phosphate concentration (see below).
The use of ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate, ammonium chloride (NH4Cl), chloroquine or acetazolamide for inhibiting signal transduction, which leads to tissue calcification and tissue fibrosation, and delays the onset of age-related diseases, is not described in the prior art.
The inventor was able to prove, on an established cell model, that ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate and ammonium chloride (NH4Cl) inhibit the formation of TGFβ1, a key molecule in the regulation of tissue calcification and tissue fibrosation (
More extensive tests provided insight into the participating cellular mechanisms: it could be demonstrated that aortas of klotho-deficient animals exhibit a massive expression of the transcription factor NFAT5 (
In accordance with the invention, “use” is to be understood to mean that at least one of the specified substances induces the claimed effect. In accordance with the invention thereby, the use of the specified substances can occur in the framework of monotherapies, in which ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate, ammonium chloride (NH4Cl), chloroquine and acetazolamide are used as the active substance, or the sole active substance, respectively. Combination therapies may also be implemented, however, in which two or more of these active substances are deployed simultaneously.
The fundamental objective of the invention is fully achieved herewith.
According to a preferred further development of the invention, age-related diseases are selected from the group composed of: arterial sclerosis, pulmonary emphysema, atrophoderma, myasthenia, acquired immune deficiency syndrome, infertility, kyphosis, disruption of the CaPO4 metabolism, osteoporosis, low immunity (thymus degeneration), and neurodegeneration.
According to a preferred further development of the invention, the tissue fibrosation is based on a disease selected from the group composed of cirrhosis of the liver, Crohn's disease, fibrotic pancreatitis, pulmonary fibrosis, heart failure, scarring, fibrosation in peritoneal dialysis, Alzheimer's disease.
The measure has the advantage that substances are provided with which important fibrotic diseases can be treated prophylactically and therapeutically.
Ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate, ammonium chloride (NH4Cl), chloroquine and acetazolamide can be substances in a pharmaceutical compound, which is preferably designed for an oral, rectal, parenteral, intraperitoneal, local or transdermal application. Furthermore, the pharmaceutical composition can preferably be designed as a powder, tablet, juice, drops, dialysis fluid, capsule, suppository, solution, injection solution, aerosol, ointment, rinse, patch, pellet, lozenge, or modified release dosage form.
The absolute quantity of ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate, ammonium chloride (NH4Cl), chloroquine and acetazolamide in a dosage unit of the pharmaceutical composition is determined by the person skilled in the art on a case-by-case basis. Compositions for administration to adult humans may provide for daily doses of ca. 25 g ammonium sulfate, 25 g ammonium citrate, 35 g ammonium lactate, 25 g ammonium nitrate, 20 g ammonium chloride (NH4Cl), 900 mg chloroquine, and 800 mg acetazolamide. The person skilled in the art may, however, provide other absolute quantities deviating therefrom.
This measure has the advantage that the active substance is provided in an absolute quantity that achieves the desired effect.
The pharmaceutical composition can contain a pharmaceutically acceptable carrier, and possibly other additives, which are generally known in the prior art. They are described, by way of example, in the publication by Kibbe, A., Handbook of Pharmaceutical Excipients, third edition, American Pharmaceutical Association and Pharmaceutical Press 2000. Additives comprise any compound or composition that is advantageous for the intended use of the compound according to the invention, which include salts, binders, solvents, dispersants, and other substances typically used in conjunction with the formulation of pharmaceuticals.
Ammonium sulfate, ammonium citrate, ammonium lactate, ammonium nitrate, ammonium chloride (NH4Cl), chloroquine and acetazolamide can/may be used according to the invention as additive(s) in a food product.
This measure takes advantage of the fact that, in part, the substances have already been put to use in food technology, and are distinguished by tolerability and substantial tastelessness. Any arbitrary food product may be considered according to the invention, in particular beverages, but also solid foods.
The preferred concentration of the active substance can be readily determined by means of methods known to the person skilled in the art, e.g. via titration experiments, in which different concentrations are deployed. The effective quantity can be established on an individual basis. In the case of a therapeutic application, the concentration is based on the concrete age-related disease that is to be treated, the course, the severity, the patient that is to be treated, in particular according to his immune response status, sex, age, disease history, etc. When used in beverages, the concentration can amount to ca. 25 g ammonium sulfate, 25 g ammonium citrate, 35 g ammonium lactate, 25 g ammonium nitrate, 20 g ammonium chloride (NH4Cl), 800 mg chloroquine, or 800 mg acetazolamide. The person skilled in the art may, however, also make use of concentrations deviating therefrom.
This measure has the advantage that the active substance, or the additive, is already provided in such a concentration that it ensures the desired effect.
A further subject matter of the invention relates to a method for producing a pharmaceutical composition for reducing tissue calcification and tissue fibrosation, as well as for delaying the onset of age-related diseases, having the following steps:
Moreover, a further subject matter of the present invention relates to a method for producing a food product for reducing tissue calcification and tissue fibrosation, as well as for delaying the onset of age-related diseases, having the following steps:
Lastly, a further subject matter of the present invention relates to a method for reducing tissue calcification and tissue fibrosation, as well as for delaying the onset of age-related diseases, which comprises the administration of a substance to the living being, wherein the substance is selected from the group composed of: ammonium sulfate, ammonium chloride (NH4Cl), acetazolamide, chloroquine, ammonium nitrate, ammonium citrate and ammonium lactate.
The properties, features and advantages of the use according to the invention apply accordingly to the methods according to the invention. As such, the substances can be used individually as sole active substances or additives, or in combinations thereof.
It is to be understood that the features specified above and to be explained below can be used not only in the respective given combinations, but also in other combinations or in and of themselves, without abandoning the scope of the present invention.
The invention shall now be explained in greater detail based on exemplary embodiments from which further properties and advantages can be derived. The exemplary embodiments are purely illustrative, and do not limit the scope of the invention. Reference is made thereby to the attached drawings.
In the attached Figures:
The following experimental studies were executed: Primary human aortic smooth muscle cells (HAoSMCs, Invitrogen) were cultivated in Waymouth's MB 752/1 medium and Ham's F12 nutrient mixture (1:1, Gibco, Life Technologies) with the addition of 10% FBS (Gibco, Life Technologies). In all experiments, confluent HAoSMCs, passages 4 to 11, were used. The cells were treated for 24 hours with 2 mM β-glycerophosphate (Sigma-Aldrich) with or without simultaneous addition of 0.5 mM ammonium salts or 100 μM chloroquine diphosphate (Sigma-Aldrich). Quantitative RT-PCR (real time polymerase chain reaction) were carried out, as described previously (Voelkl, J., Alesutan, I., Leibrock, C. B., Quintanilla-Martinez, L., Kuhn, V., Feger, M., Mia, S., Ahmed, M. S., Rosenblatt, K. P., Kuro, O., Lang, F.: Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice. J. Clin Invest 2013; February 1; 123(2):812-22). For this, HAoSMCs were washed and total RNA were isolated using Trifast reagent (Peqlab) according to the directions from the manufacture. For the in vivo experiments, aortas from klotho+/+ mice and klothohm mice, in each case with and without treatment with NH4Cl, were removed and quick-frozen. The total RNA were likewise isolated with Trifast reagent (Peqlab) in accordance with the directions from the manufacture. In each case, 2 μg RNA of the human as well as the murine samples were used for the reverse transcription of the RNA with oligo(dT)12-18 primers (Invitrogen) and SuperScripIII Reverse Transcriptase (Invitrogen). Quantitative real-time PCR were carried out with an iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories) and iQ™ Sybr Green Supermix (Bio-Rad Laboratories) according to the manufacturer's instructions. The following primers were used (5′ orientation):
The specificity of the PCR products was checked by means of melting curve analysis and agarose gel electrophoresis. All PCRs were each carried out twice, and the multiple mRNA quantities were calculated by means of the 2−ΔAct methods with GAPDH as the internal reference.
All of the animal experiments were carried out in compliance with the German Animal Protection Law, and were approved by the local authorities.
Male and female hypomorphic klotho mice (klothohm) were compared with male and female wild mice (klotho+/+). The source of the mice, the breeding, and the genotyping are described in the prior art; cf. Kuro-o et al. (1997), Mutation of the mouse klotho gene leads to a syndrome resembling ageing, Nature 390: 45-51. Through repeated backcrossings (>9 generations) with animals of the 129/Sv inbreeding strain, congenic strains of the klotho mice were produced and used in this study. The mice had random access to water or an aqueous solution of (NH4)2SO4 (0.14M), NH4Cl (0.28M), NH4NO3 (0.28M), acetazolamide (800 mg/1) and chloroquine diphosphate (0.288 mg/ml) and were fed with a control feed (Sniff, Soest, Germany). The treatment with NH4Cl (0.28M) or acetazolamide (800 mg/1) started with the pairing of the parental generation and was continued throughout the pregnancy, until the descendants were killed.
1.2 Blood Chemistry
For blood withdrawal, the mice were anesthetized with diethyl ether (Roth, Karlsruhe, Germany) and blood samples of 50 to 200 μl were withdrawn in capillaries containing heparin by puncturing the retro-orbital plexus. The phosphate and calcium concentrations in plasma were determined using a photometric method (FUJI FDC 3500i, Sysmex, Nordstedt, Germany). The FGF23 and PTH concentrations in plasma were determined using commercial ELISA kits (FGF23: ImmunDiagnostics, Boston, USA; PTH; Immunotopics, San Clemente, USA, MPG: Cloud-Clone Corporation, Houston, USA). The measurement of the concentration of calcitriol [1.25(OH) vitamin D3] in plasma likewise occurred using a commercial ELISA kit (IDS, Boldon, United Kingdom). The ammonia concentration was enzymatically measured using glutamate dehydrogenase with NADPH as a cofactor. The evaluation likewise occurred with a photometric method (ADVIA 1650 analyzer, Siemens, Fernwald, Germany).
1.3 Histology
For the examination of the trachea, lunges, kidneys, heart, the stomach and the vessels, corresponding tissue was removed from male klotho+/+ mice (age: 8 weeks) and male klothohm mice (age: 8 weeks), without and with an aqueous solution treatment composed of (NH4)2SO4 (0.14 M), NH4Cl (0.28 M), NH4NO3 (0.28 M) and chloroquine (0.288 mg/ml), or in female animals without and with acetazolamide treatment (800 mg/l in drinking water) embedded in paraffin, cut into slices of 2 to 3 μm, and dyed with H&E and von Kossa; cf. Mossbrugger et al. (2007), Standardized morphological phenotyping of mouse models of human diseases within the German Mouse Clinic, Verh. Dtsch. Ges. Pathol. 91; 98-103.
1.4 Statistics
The data were presented as average±SEM, wherein n represents the number of independent experiments. All of the data were tested for significance using ANOVA or the paired or unpaired student t-tests. For the experiments regarding lifespan, SAS Jmp Version 8.0.1 (SAS Institute Inc., Cary, N.C., USA) was used. Only results having a p<0.05 were regarded as statistically significant.
a. Results
klotho is a transmembrane protein, related to the β-glucuronidase. A reduced production of this protein was observed in patients having chronic kidney failure, frequently accompanied by degenerative processes such as arterial sclerosis, osteoporosis, and skin atrophy. Mutations in these proteins were able to be connected to aging processes.
In the examined mouse model, the klotho expression was massively reduced by a defect in the klotho gene. The mice having this defect were referred to as hypomorphic mice. The deficit in klotho resulted in a syndrome that resembles human aging. The (accelerated) onset in these animals of tissue and/or vessel calcification, arterial sclerosis, pulmonary emphysema, skin atrophy, myasthenia, acquired immune deficiency syndrome, infertility, kyphosis, disrupted CaPO4 metabolism, osteoporosis, low immunity (thymus degeneration), hearing loss and neurodegeneration, was observed. The animals having this defect furthermore have a significantly reduced life expectancy and are infertile.
The expression of TGFβ1 mRNA in human aortic smooth muscle cells (HAoSMCs) is depicted in
The expression of Runx2 mRNA in human aortic smooth muscle cells (HAoSMCs) is shown in
Furthermore, the expression of the alkaline phosphatase (ALP) in human aortic smooth muscle cells (HAoSMCs) was studied (
It is shown in
The clear growth deficit of untreated hypomorphic klotho mice (klothohm) in comparison with their wild cousins (klotho+/+) is shown in
As is depicted in the following table, the pH value of the blood from untreated klotho mice was significantly lower than from untreated klotho+/+ mice. In klotho+/+ mice, the administration of NH4Cl tends to lead to a lowering of the pH value in blood, but does not achieve statistical significance. Accordingly, a treatment of klothohm mice with NH4Cl also does not lead to a significant increase in acidosis (Table 1).
The treatment of klothohm mice with acetazolamide likewise led to an increase in weight and size in the animals. As can be seen in
As is depicted in
The plasma concentrations of Ca++, phosphate and 1.25 (OH)2D3 in klotho+/+ mice are depicted, in each case with and without treatment with acetazolamide. Neither the concentrations of 1.25 (OH)2D3 nor phosphate are affected by the treatment thereby. The calcium level in the treated klothohm mice also remained slightly elevated, but displayed a significant different to neither the untreated klothohm mice nor the klotho+/+ mice. It was also possible to fully normalize the concentration of matrix gla protein (MGP) in the animal plasma through treatment with acetazolamide.
As is shown in
As is depicted in
3. Conclusion
The inventors provided substances with ammonium sulfate, ammonium chloride (NH4Cl), acetazolamide, chloroquine, ammonium nitrate, ammonium citrate, and ammonium lactate, which are suitable for preventing tissue calcification and tissue fibrosation, and delaying the onset of age-related diseases, and thus to extend the lifetime of a living being. On one hand, this was demonstrated by the effects on the expression of calcification and fibrosation indicators in the cell culture, and on the other hand, by the impressive effects of ammonium chloride and acetazolamide on an established animal model. The animals treated accordingly display significantly reduced aging syndromes, impressively illustrated by the tissue and vessel calcification, and live significantly longer than untreated animals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2013 110 608.0 | Sep 2013 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/070333 | 9/24/2014 | WO | 00 |
