WO2008101923A1

WO2008101923A1 - Use of pantothenic acid derivatives for the treatment of hypophosphatemia

Info

Publication number: WO2008101923A1
Application number: PCT/EP2008/051999
Authority: WO
Inventors: Peter Kalisch; Ursula DÜME
Original assignee: Medice Arzneimittel Puetter Gmbh & Co. Kg
Priority date: 2007-02-19
Filing date: 2008-02-19
Publication date: 2008-08-28

Abstract

The present invention is directed to the use of compounds of formula I and/or salts thereof for the treatment of hypophosphatemia. The present invention in particular is directed to the use of those compounds in different enantiomeric forms and in form of different earth alkali or alkali salts. Additionally, the present invention is directed to the use of those compounds in a medicament which is suitable for oral or parenteral administration.

Description

Use of pantothenic acid derivatives for the treatment of hypophosphatemia

Pantothenic acid, also called vitamin B5, is a water soluble vitamin which is needed to form coenzyme A (CoA) and which is critical in the metabolism and synthesis of carbohydrates, proteins and fats. Its systemic name is 3[(2R,4-dihydroxy-3,3-dimethyl-butanoyl)amino] propanoic acid. Since pantothenic acid participates in a wide array of key biological roles, it is considered essential to all forms of life. As such, deficiencies in pantothenic acid may have numerous wide-ranging effects.

Small quantities of pantothenic acid are found in most foods with high quantities found for example in whole grain and eggs. Presently, pantothenic acid experiences a wide-spread use and can be found in many dietary supplements and pharmaceutical compositions (systemic as well as topical).

A deficiency of pantothenic acid in humans is exceptionally rare and has not been thoroughly studied up to now. In the few cases where a deficiency has been observed, nearly all symptoms can be reversed by supplementing pantothenic acid. The symptoms of deficiency are similar to other vitamin B deficiencies. Among others, those symptoms include fatigue, allergies, nausea and abdominal pain. Painful burning sensations of the feet were reported in malnourished prisoners of war ("burning feet syndrome").

Many alternative uses of pantothenic acid have been proposed during the last decades, including hair care and the treatment of acne vulgaris. In particular, however, one derivative of pantothenic acid named panthenol has experienced a wide-spread use in pharmaceutical compositions. Panthenol is the alcohol analogue of pantothenic acid and is the provitamin of B 5. In vivo, it is quickly oxidized to pantothenate. Panthenol is a viscous transparent liquid at room temperature, but salts of pantothenic acid are powders. Panthenol is well soluble in water, alcohol and propylene glycol and many other solvents. Only D-panthenol is said to be biologically active (termed dexpanthenol), however, also its racemic mixture (DL-panthenol) has been used in the preparation of cosmetics. In particular in cosmetics, panthenol is used as a humectant, emollient and moisturizer. It is noted, however, that panthenol may not be absorbed through the skin and does not show systemic effects by topical administration.

There are several recommendations regarding the recommended daily intake of pantothenic acid. Those recommendations usually vary between 4 mg/person/day to 10 mg/person/day depending on the age of the person and certain physiological parameters (pregnancy etc.). See, in this connection, Friedrich Wilhelm: Handbuch der Vitomine, Urban & Schwarzenberg Verlag, 1987, for further information. In the above reference, further indications for pantothenic acid are indicated as, for example, diseases of the skin (wounds, dermatitis, burns), diseases of the mucosa, of the liver and of the central nervous system. Furthermore, it is indicated that calcium pantothenate might be used for the treatment of rheumatic arthritis in a dosage of 500 mg/day.

Karl Heinz Bessler et al. disclose in Vitaminlexikon, 3^rd edition, Urban & Fischer Verlag, 2002, that pantothenic acid might be used for treating the following diseases/conditions:

Substitution of vitamins in the context of parenteral alimentation, supplementation of chronic dialytic patients; burning feet syndrome and for the adjuvant treatment of skin and mucosal lesions.

Hypophosphatemia is an electrolyte disturbance in which there is an abnormally depleted level of phosphate in blood. This can be caused by different circumstances, for example nutrition, alcohol abuse, burns, hepatic failure etc. From the view point of pathophysiology, hypophosphatemia is caused by the following three mechanisms: at first inadequate intake (which is quite uncommon), increase excretion (for example in the case of hyperparathyroidism), and by shifting from extracellular to intracellular space (which can be observed in the treatment of diabetic ketoacidosis, short- term increases in cellular demand and acute respiratory alkalosis).

Basically, phosphate is the most abundant intracellular anion and is essential for membrane structure, energy storage and transport in all cells. Obviously, phosphate is necessary to produce ATP₁ which is the energy carrier for nearly all cell functions. Reducing available phosphate may compromise any organ system. The critical role that phosphate plays in every cell, tissue and organ explains the systemic nature of injury caused by phosphate deficiency.

Serum phosphate or phosphorus normally range from 2,5 to 4,5 mg/dl in adults. Hypophosphatemia is defined as mild (2-2,5 mg/dl), moderate (1-2 mg/dl) or severe (> 1 mg/dl).

The frequency of hypophosphatemia, in particular severe hypophosphatemia is low and occurs in about 2-3% of hospitalised patients in general and in the case of severe hypophosphatemia, of about 0,5% of hospitalised patients.

Up to now, the only available therapy for treating hypophosphatemia is replacement therapy, i. e. administration of usually oral phosphate dosages.

However, although phosphorus/phosphate preparations, for example with sodium and potassium, are available, they have disadvantages including causing osmotic diarrhoea, volume overload or hypercalemia. A further drawback of the substitution of phosphate is that care must be taken by the physician to avoid hyperphosphatemia when administering phosphorous intravenously as this can lead to hypocalcemia (leading to tetany) and calcium- phosphate deposition in tissues (eye, heart, kidney and lung).

Therefore, there is an urgent need to provide further pharmaceutical agents which are capable of being used in the treatment of hypophosphatemia. It is a further object underlying the invention to provide a therapeutical approach for the treatment of hypophosphatemia, wherein the known unwanted side effects of replacement therapy with sodium or potassium phosphate are avoided.

These objects are solved by the subject matter of the independent claim. Preferred embodiments are set forth in the dependent claims.

The present invention is based on the surprising discovery that pantothenic acid and derivatives thereof are capable of dramatically increasing the phosphate uptake in vivo and thus might be used as a well explored, well known and comparably harmless agent for treating hypophosphatemia independent from the underlying causes.

Thus, according to a first embodiment, the invention provides for the use of a compound represented by formula I

or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a medicament for the treatment of hypophosphatemia. Formula I reflects the structural formula of pantothenic acid in the case, R¹ being a carboxyl group. Further derivatives, which are also included in the scope of the present invention, are R¹ being an aldehyde group (= panthenal) or an alcohol group (= panthenol). All of these compounds can be used for treating hypophosphatemia in mammals, in particular in human patients.

The use of the present invention does not only comprise the compounds of formula I per se but also the compounds in specific enantiomeric forms, for example R- enantiomeric, S- enantiomeric, racemic form or mixtures thereof. It is noted that the R- enantiomeric form of pantothenic acid usually is regarded as the biologically active form of pantothenic acid as a vitamin, however, there is evidence existing that also the racemic form (including the S- enantiomeiϊc form) may be used for the treatment of hypophosphatemia. Additionally, the compound of formula I can be used in its salt form, in particular as earth alkali or alkali salt. Here, in particular sodium, magnesium or calcium salts are preferred.

As a further embodiment, mixtures between the compound of formula I as it is and formula I in its salt form can be used in varying amounts. It is noted that, when Rl is being a carboxyl group, than it might form a lactone-compound by reacting with the terminal hydroxy group of formula I. This lactone compound can serve as a prodrug and will be cleaved in vivo after it has been administered to a patient.

As mentioned above, the hypophosphatemia which can be treated by the compounds of the present invention may be caused by alcoholic abuse, burns, hepatic failure, metabolic acidosis, osteomalakia, respiratory alkalosis or hyperparathyroidism.

The compound generally may take the form of a pharmaceutical composition or medicament. Such a pharmaceutical composition contains the compound of formula I itself and a pharmaceutically acceptable carrier. The amounts of the compound of formula I which have to be provided by the pharmaceutical composition is 10 mg to 1000 mg, preferably 50-500 mg, most preferably 100 to 300 mg of the compound per human patient per day.

Based on decades of experience in the administration of pantothenic acid, it is clear that the medicament of the present invention may be administered orally or parenterally. For parenteral administration, the medicament preferably is suitable for i.m or i.v. applications.

The compounds of formula I of the present invention are preferably used in such a pharmaceutical composition or medicament, in the above doses mixed with an pharmaceutically acceptable carrier or carrier material, that the disease can be treated or at least alleviated.

The term "pharmaceutically acceptable" defines a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve a synergistic effects or to minimize adverse or unwanted effects.

Techniques for the formulation or preparation and application/medication of compounds of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. A therapeutically effective dose relates to the amount of a compound which is sufficient to improve the symptoms, for example a treatment, healing, prevention or improvement of such conditions. An appropriate application can include oral and parenteral application, including intramuscular, subcutaneous, as well as intravenous or intranasal injections. The oral route of administration is the preferred treatment of a patient.

The pharmaceutical preparations which can be used orally include push-fit capsules made of gelatine, as well as soft, sealed capsules make of gelatine and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in liquid form that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as in buffered salt solution or in form of an emulsion or suspension (for example in an hydrophobic solvent). In addition, stabilizers may be added.

Furthermore, the pharmaceutical composition may be in the form of granules or spheres containing the pharmaceutical agent represented by above formula I. For example, those granules may be contained in sachets or the like in order to provide a predefined and exact dosage for the patient.

The present invention now is described by means of the enclosed examples and figure. However, it is noted that the present invention is not restricted to their contents and the scope of protection only is determined by the enclosed patent claims.

Figure 1 shows the influence of three different concentrations of D-calcium pantothenate (CP) on the phosphate uptake by colon epithelial cells (Caco-2). CP leads to a remarkable and dosage dependent increase of the phosphate uptake with increasing concentration. Even at lowest concentration of 100 μg/ml, the increase is statistically significant (p < 0,01 ; student's t-test). The mean ± standard deviation is indicated (n=3).

Examples

1. Substances used in the experiments

D-calcium pantothenate

Acros organics, product No. 12258-0000, CAS-No. 1453-82-3, Lot A0214246 Stock solution: 10 mg/ml in culture medium (MEM) without additives; The substance can be solved without problems and has a neutral pH.

2. Cell lines and routine culture

Human colon epithelial cells, cell line Caco-2 (Human Caucasian colon adenocarcinoma, ECACC 86010202, DSMZ ACC 169), passage 67 internal. The Caco-2-ceIls were incubated as a mass culture in minimum essential medium along with L-glutamine (MEM) under addition of 5% fetal bovine serum as well as 100 U/ml penicillin and 100 μg/ml streptomycin and 1% NEAA (non essential amino acid solution, lOOfold).

3. Phosphate stock solution for exposing to the cells

A 100 fold concentrated phosphate stock solution for incubating the cells is containing the following ingredients:

• KH₂PO₄ dry 10,0 g/1 = 100 μg/ml in culture medium for incubation

• Na₂HPO₄ x 2 H₂O 57,5 g/1 = 575 μg/ml in culture medium for incubation. 4. Experimental setup

Caco-2-cells were sown in a density of 750.000 cells/culture dish (growth area per culture dish = 55 cm²) and were incubated for 3 days until a confluence of 90% was reached in minimum essential medium (MEM) under addition of 5% fetal calf serum as well as 100 U/ml penicillin and 100 μm/ml streptomycin and 1% NEAA (non essential amino acid solution, 100 fold).

The Caco-2-cells were preincubated subsequently with culture medium (minimum essential medium; MEM) without any further additives (containing 140 mg/1 sodium dihydrogen phosphate x H₂O) ± 100, 500 and 1000 μg/ml test substance respectively, for 15 minutes in the incubator. The culture medium was aspirated and fresh MEM containing 100 μg/ml potassium dihydrogen phosphate and 575 μg/ml disodium hydrogen phosphate-dihydrate from the 100 fold concentrated phosphate-stock solution ± 100, 500 and 1000 μg/ml test substance, respectively, was added and incubated for 60 minutes at 37°C in the incubator.

The incubation medium was aspirated, washed once with 10 ml per dish each (0,9% saline), the cells were detached by short term treatment with trypsin/EDTA (5 ml trypsin/EDTA for 5 minutes at 37°C) and suspended (+ 5 ml saline). The cell suspension was centrifuged (7 minutes at 240 x g), the supernatant was aspirated with a Pasteur pipette and the cellular sediment was taken up in 1 ml cell line.

The samples were frozen in Eppendorf cups at -20°C until the phosphate-determination. After thawing at the same day of the phosphate determination, Eppendorf cups were shock frozen along with the cellular samples in order to destroy the cellular membranes (in liquid nitrogen) (approximately 30 seconds) and the samples were rethawed subsequently at room temperature. The respective volume (dilution 1:10) for the determination of phosphate was added. A measurement was performed at a wavelength of 820 nm using a ATI unicam UV/Vis spectrometer UV4. All determinations have been performed in triplicate. 5. Results

It turned out that D-calcium pantothenate resulted in an increase of the phosphate uptake in a range of about 70-99%. Already at the lowest test concentrations of 100 μg/ml the increase is already statistically significant (p < 0,01; student t-test).

Table 1

Representation of all measured values of the phosphate uptake under the influence of three different concentrations of the test substance calcium pantothenate.

Phosphate uptake under calcium pantothenate

Figure 1 :

Influence of three different concentrations of D-calcium pantothenate (CP) on the phosphate uptake by colon epithelial cells (Caco-2). CP leads to a remarkable and dosage dependent increase of the phosphate uptake with increasing concentration. Even at lowest concentration of 100 μg/ml, the increase is statistically significant (p < 0,01; student's t-test). The mean ± standard deviation is indicated (n=3).

Claims

Patent Claims:

1. Use of a compound represented by formula I:

Formula I

or a pharmaceutically acceptable salt or derivative thereof, for the manufacture of a medicament for the treatment of hypophosphatemia, wherein R¹ is selected from a carboxyl group, an aldehyde group or an alkohol group.

2. The use of claim 1, wherein the compound of formula I is in its R-enantiomeric, S- enantiomeric, racemic form or mixtures thereof.

3. The use of claim 1 or 2, wherein the compound is used as its earth alkali or alkali salt.

4. The use of claim 3, wherein the compound is in the form of its sodium, magnesium or calcium salt.

5. The use of one or more of claims 1-4, wherein the compound is present as a mixture of the compound according to formula I and its salt form.

6. The use of one or more of claims 1-5, wherein Rl is a carboxyl group, and wherein R¹ is forming a lacton with the terminal hydroxy group of formula I.

7. The use of one or more of the preceding claims, wherein the hypophosphatemia is caused by alcoholic abuse, burns, hepatic failure, metabolic acidosis, osteomalacia, respiratory alkalosis, or hyperparathyroidism.

8. The use of one or more of the preceding claims, wherein the medicament is suitable to provide the compound of formula I in an amount of 10 mg to 1000 mg per human patient per day.

9. The use of claim 8, wherein the amount is 50-500 mg per human patient per day.

10. The use of claim 9, wherein the medicament is suitable for oral or parenteral administration.

11. The use of claim 10, wherein the medicament is suitable for i.m. or i.v. application.