WO1997031021A1 - Oral calcitonin preparation and method for recovering calcitonin from eggshells - Google Patents

Abstract

The invention relates to calcitonin obtainable from eggshells. In addition, the invention relates to a method for obtaining an orally applicable calcitonin product, wherein calcitonin is released from eggshells, and to an oral preparation which contains calcitonin obtained from eggshells. Finally, the invention relates to a method for administering calcitonin, wherein an eggshell product is administered orally and to the use of eggshells as calcitonin source.

Description

ORAL CALCITONIN PREPARATION AND METHOD FOR RECOVERING CALCITONIN FROM EGGSHELLS

The invention relates to an oral preparation comprising calcitonin or a product having calcitonin-like activity, and to a method for obtaining calcitonin or a product having calcitonin-like activity and products containing calcitonin or said product having calcitonin-like activity.

Calcitonin is a polypeptide hormone which contains 32 amino acids. In mammals it is produced in the parafollicular cells of the thyroid gland (thyrocalcitonin) ; and in birds and fish in the ultimobranchial body. Calcitonin is used therapeutically, inter alia in the treatment of Page ' s disease, in hypercalcemia and (post-menopausal) osteoporosis. It inhibits the action of osteoclasts and promotes the clearance of calcium and phosphate via the urine. In addition, it has been reported that calcitonin reduces pain phenomena associated with these disorders .

Because of the therapeutic application, there is a great need for calcitonin. However, a drawback is that it can only be obtained through complicated and costly processes.

As a natural product, calcitonin is particularly recovered in pure and highly active form from the ultimobranchial glands of salmon, eel or fowl.

According to European patent application 0 606 816, calcitonin is recovered from salmon through specific binding to columns and subsequent purification. The calcitonin thus recovered is 40 times more active than calcitonin of human origin.

In EP-A-0 146 842 it is described that calcitonin is recovered from the ultimobranchial glands of poultry by acid extraction followed by a number of purification steps involving the use of hydrophilic organic solvents, cation exchangers and gel chromatography. The activity of this recovered product was found to be equal to or stronger than that of calcitonin recovered from salmon or eel.

Synthetic analogs and products obtained through recombinant DNA techniques have also been described. Calcitonin is employed both in natural and in modified form.

In European patent application 0 347 105 it is, for instance, described that cysteine sites in the polypeptide are sulfonated, while according to international patent application 95/18152 combinations with (poly) cyclic compounds are to be aimed for.

European patent application 0 261 552 describes the production of calcitonin through recombinant DNA techniques. As is generally known, the simplest and for patients least interfering route for administering a pharmaceutically active agent is via the gastrointestinal tract, with the pharmaceutical being ingested orally. However, this application route is not always so efficient for polypeptides such as calcitonin. Intestinal enzymes, mainly proteases, lead to the decomposition of the polypeptide into smaller peptides and amino acids, which decomposition products are inactive.

For this reason, the known calcitonin preparations, which may or may not be derivatized or modified, have hitherto been chiefly administered parenterally, through inhalation, injection, or vaginal or rectal application.

Owing to the fact that calcitonin has to be administered regularly to achieve a sufficient effect, the last-mentioned application methods have many drawbacks. The most suitable application route for patients is the oral route.

Proposals have been made to improve the absorption efficiency of oral preparations.

For instance, Japanese publication 93-274737 describes special calcitonin derivatives having an improved enteric absorption. To that end, fatty acid groups or lower alkoxy- carbonyl groups are coupled to the polypeptide chain. The derivatives are suitable for the treatment of pain resulting from osteoporosis and hypercalcemia, and improve the growing-on of bone mass.

European patent application 0 535 827 proposes the incorporation of calcitonin into fatty (emulsifier) material and the encapsulation of calcitonin in gelatin. These calcitonin products are better absorbed without necessitating an absorption promoter.

Further, Japanese publication 62-010020 describes a composition which, in addition to calcitonin, contains a fatty ester with an HLB of 11-16. This composition is claimed to have an improved absorption behaviour.

Furthermore, international patent application 87/00750 describes a method for the same purpose, in which calcitonin is enveloped in a thin phospholipid skin, whereby it is brought into a liposome form.

The proposals for orally suitable calcitonin preparations all require relatively expensive modification steps to protect calcitonin per se against rapid inactivation in the gastrointestinal tract. In particular, the basic raw material used as starting material is always pure calcitonin which has been obtained through complicated and/or costly technologies.

It has now been found that calcitonin or a product having calcitonin-like activity is present in eggshells and can be recovered therefrom. Moreover, this product which will be referred to in this description and the appending claims as calcitonin from eggshells, can be efficiently- applied orally without necessitating an additional derivatization or other modification step. Accordingly, in a first aspect the invention relates to calcitonin originating from eggshells.

In addition, the invention relates to a method for obtaining an orally applicable calcitonin product, in which calcitonin is released from eggshells. To that end, for instance, calcitonin can be taken up into an aqueous medium or an aqueous solvent system, but also in other solvents in which calcitonin dissolves, in contrast with the inorganic eggshell matrix. Moreover, calcitonin can be obtained by dissolving the eggshell matrix at least partly.

Eggshells, with or without membrane residues, constitute an amply available waste product. In order to optimize the extraction of calcitonin, the eggshells are first ground, preferably to form an eggshell flour as fine as possible. For this purpose, it is not necessary to separate the membrane residues from the eggshells.

Optionally, before being ground, the eggshells can be dried, which facilitates the grinding treatment. Also, the starting product can be priorly sterilized without the activity of the calcitonin diminishing unacceptably. Apparently, the calcitonin is sufficiently protected by the lime matrix of the eggshell . A major advantage of the method according to the invention is that no extensive steps for purifying the calcitonin product are necessary. It is no problem, indeed an advantage, if not all residues of the eggshell matrix are removed. By administering calcitonin in combination with a calcium product, the effect of calcitonin is considerably enhanced.

It is supposed that one of the reasons why calcitonin from eggshells can be applied orally resides in the fact that this form of calcitonin, by complexing with constituents of eggshells or by embedding in the eggshell matrix, is protected against decomposition under the influence of enzymes or juices in the gastrointestinal tract.

In another aspect of the invention, calcitonin can be administered by administering eggshells, in whatever form, orally. Eggshells contain calcitonin in a form and amount such that its decomposition in the gastrointestinal tract can be avoided or inhibited to a sufficient extent, and that the calcitonin can be absorbed into the system and can work the desired effect there.

Experiments (see infra) have shown that the calcitonin products according to the invention give a physiological pain-reducing effect upon oral administration. Moreover, these products can be employed in all mammals suffering from a disorder that can be treated with calcitonin. Further, the calcitonin product according to the invention can be used prophylactically. The invention further relates to an oral preparation containing calcitonin obtained from eggshells.

It is noted that it is known from international patent application PCT/NL/95/00396 of the present group of inventors, that milk products enriched with ground eggshell lead to an improved calcium resorption. That international patent application further describes that additional advantages are obtained with ground eggshell because of the presence of magnesium compounds therein. It is also pointed out that it can be useful to add vitamin D sources . Further, it is known from Arch. Ger. Geriatr. (1991) 487-490 that administering eggshells can lead to a pain relieving effect.

However, in neither publication is it described or suggested that in fact calcitonin is responsible for the effects.

In the oral preparations according to the invention, preferably a magnesium source is present as well. This magnesium contributes to the pain reducing action of the calcitonin. In a preferred embodiment, the ratio of calcium to magnesium in the preparation according to the invention is between 3:1 and 12:1. Both the calcium and the magnesium can originate from eggshells and/or from external sources . This ratio is so chosen for physiological reasons, namely, to effect an optimum calcium retention in bones. It is noted that in Magnesium Res. 2 (1989) 142 it is described that magnesium upon administration to osteoporosis patients has a pain relieving effect.

Further details about the amounts of calcium and magnesium are mentioned in the aforementioned international patent application PCT/NL/95/00396.

Further, it is of advantage to combine the preparations according to the invention with components which are known to positively influence calcium retention, in particular with vitamin D or with a source rich in vitamin D. Other examples are vitamins A, K and/or C, sources of fluoride ions, vanadium ions, boron ions and/or silicon ions, glycine, and casein phosphopeptides. The oral preparation according to the invention can be used in any known orally applicable form. Preferably, the preparation is in the form of a pill, a tablet to be swallowed or chewed, or a liquid preparation. In this last case, by choosing a suitable liquid medium in the method according to the invention, the product directly obtained can be administered.

Also, the calcitonin obtained from eggshells can be incorporated in a food or be used as a food supplement, for instance for prophylactic reasons. The invention further relates to a method for administering calcitonin, in which an eggshell product is administered orally.

Finally, the invention relates to the use of eggshells as a calcitonin source. The invention is presently explained in and by the following, non-limiting examples.

Example 1

Fine-ground eggshell flour which had been obtained as described in international patent application

PCT/NL/95/00396 contained approximately 38% by weight of calcium, 0.4% by weight of magnesium, 0.1% by weight of phosphorus and 0.6% by weight of nitrogen. This product was suspended in an amount of 10% in 90 ml water, followed by a test for calcitonin. In the suspension the calcitonin content was determined according to the standard method of Nicholson (Nicholson Institute) .

The suspension contained 0.09 μg calcitonin.

Example 2

Prepared were capsules for oral use containing 0.7 g fine calcitonin-containing eggshell flour and 0.05 g of a mixture containing sodium ascorbate, zinc lactate, vitamins E, D3 , B6, K, B2, Bl, B12, silicon dioxide, boric acid and milk protein (casein, whey protein) .

Example 3

The product according to Example 2 was prepared, but 0.7 g calcitonin-containing eggshell flour was replaced with 0.5 g calcitonin-containing eggshell flour and 0.2 g magnesium hydroxycarbonate.

Example 4

A powdery product was prepared by mixing milk powder having a reduced fat content with a mixture of eggshell flour and maltodextrin, jointly dried on a wheel drier, as well as a vitamin-mineral premix. Per 100 grams the product contained 21 g milk protein, 14 g fat, 2.2 g calcium, of which 1.5 g originating from fine eggshell flour, and 0.6 g magnesium. Added as microingredients by way of the vitamin-mineral premix were iron, zinc, copper, iodide, vitamins A, C, D, E, K, Bl, B2, B6, B12, folic acid, niacin, panthothenic acid, biotin. The Ca/Mg ratio was < 4:1.

Example 5 The product according to Example 4 was prepared, but the milk powder of reduced fat content was replaced with a mixture of cereal flour and soybean.

Example 6 The product according to Example 4 was given for a period of 4 months to experimental subjects with proven osteoporosis, who had pain complaints which had hitherto been fought with current pain killers.

None of the participants used hormone preparations (for instance, estrogens) , diphosphonates or other preparations which might be supposed to have an influence on the pain. The subjects daily ingested 3 g calcitonin-containing fine eggshell flour via the product. The pain experience was monitored by specialists who monitored the pain in the back or bones, the increase in pain after strain, and insomnia due to pain, on a rating scale of 1-4 (1 = no complaints; 2 = small complaints; 3 = quite some complaints; 4 = major complaints) .

It was further monitored to what extent the use of pain killers had been stopped or continued.

It was concluded that after 4 months the product had given a significant reduction of the pain complaints in 73% of cases.

A comparable result was achieved through solely administering 6 g of calcitonin-containing eggshell flour to experimental subjects.

Claims

Claims

1. Calcitonin obtainable from eggshells.

2. A method for obtaining an orally applicable calcitonin product, wherein calcitonin is released from eggshells.

3. An oral preparation containing calcitonin obtained from eggshells.

4. A preparation according to claim 3, which has been enriched with a magnesium source.

5. A preparation according to claim 3 or 4, which has been enriched with a vitamin D source.

6. A preparation according to claim 3, 4 or 5, in the form of a pill, a tablet to be swallowed or chewed, or a liquid preparation.

7. A preparation according to claim 3, 4 or 5, incorporated into a food.

8. A method for administering calcitonin, wherein an eggshell product is administered orally.