Pharmaceutical composition for oral delivery of a physiologically active peptide agent and method of strengthening its bioavailability

 

(57) Abstract:

The composition comprises a therapeutically effective amount of a peptide, a pharmaceutically acceptable pH-lowering agent, power absorption, effective to increase the bioavailability of the indicated peptide, and acid-resistant protective filler. The specified filler contributes to the transport of the pharmaceutical composition through the stomach of the patient and prevents the contacting of the peptide with gastric proteases. The use of a new pharmaceutical composition provides oral administration of the peptide to the patient and selective target the release of the above peptide in the intestine. Reduced proteolytic degradation of the peptide under the action of gastric proteases, as well as intestinal and pancreatic proteases. Improved bioavailability of the peptide allows you to maintain its concentration in pharmaceutical drug at a relatively low level. 2 C. and 18 h.p. f-crystals, 9 PL.

The technical field to which the invention relates

The present invention relates to a peptide pharmaceutical compositions for oral administration in which the active compounds include a variety of amino acids and at least one of pepti the different active compounds when they are orally administered.

Description of the prior art

Numerous human hormones, neurotransmitters and other important biological compounds contain peptides as a significant part of their molecular structures. Many diseases respond positively to the increase in the content of such peptide compounds in the patient's body. A therapeutically effective amount of such biologically relevant peptides can be administered to patients in a variety of ways. However, as discussed in detail below, the preferred oral administration, the active compounds of this type is very difficult.

So, for example, calcitonin salmon is a peptide hormone that reduces the leaching of calcium from the bones. When used for the treatment of bone diseases and disorders associated with calcium (such as osteoporosis, Paget's disease, malignant hypercalcemia, and so on), this substance assists in maintaining bone density. Were allocated calcitonin different types (human calcitonin, salmon calcitonin, eel calcitonin, elcatonin, porcine calcitonin, chicken calcitonin). Among calcitonin different types of observed znacili human calcitonin, and amino acids, components of salmon calcitonin. Despite the difference in molecular structure of salmon calcitonin may be used to treat people suffering from the above-mentioned calcitonin-reactive diseases.

The peptide of the pharmaceutical composition used in the known methods, often administered by injection or nasal. An example of a peptide of the pharmaceutical agent, which is often introduced in the form of injections, can serve as insulin. Preferred oral administration is problematic because the peptide active compounds are susceptible to degradation in the stomach and intestines. For example, in the earlier literature there are no reports on the ability to achieve reproducible levels of salmon calcitonin in the blood when administered orally. Apparently, this is due to the fact that salmon calcitonin does not have sufficient stability in the gastrointestinal tract and has a tendency to bad transport through the intestinal wall into the blood. However, injection and nasal introduction of significantly less convenient and more uncomfortable for the patient than oral administration. Often such inconvenience or discomfort result in resultspopulation oral administration of peptide pharmaceutical agents similar to insulin, salmon calcitonin and other substances discussed in detail in the description.

Proteolytic enzymes as stomach and intestines can break down peptides, making them inactive before they can be absorbed by the blood stream. Any amount of a peptide which resists proteolytic degradation under the action of gastric protease (having, as a rule, acid pH value), then exposed to proteases small intestines and enzymes secreted by the pancreas (usually having an optimum pH value in the range from neutral to basic). Specific difficulties encountered by oral administration of such a peptide as salmon calcitonin, include the relatively large size of the molecule and the charge distribution in the media. These circumstances may impede the penetration of salmon calcitonin through the mucus along the walls of the intestine or the intersection of membrane and brush the edges of the intestine and subsequent admission into the blood. These additional problems can contribute to limited biocosmology drugs.

Summary of the invention

In accordance with the above purpose in which notizie for reliable delivery of pharmaceutical peptides, for example, a physiologically active peptide agent as insulin, salmon calcitonin, vasopressin, and other substances discussed in the present description.

Another objective of the present invention is to provide therapeutic ways of improving the bioavailability of these peptides.

Another objective of the present invention is to provide methods of treatment of bone diseases and disorders associated with calcium, by oral administration of salmon calcitonin.

In accordance with one aspect of the present invention provides a pharmaceutical composition for oral delivery of a physiologically active peptide agent comprising:

(A) a therapeutically effective amount of the specified active peptide agent;

(B) at least one pharmaceutically acceptable pH-lowering agent;

(C) at least one power absorption, effectively increasing biotechonomy specified active agent; and

(D) acid-proof protective filler, efficient in terms of transport specified in the pharmaceutical composition through the stomach of the patient and thus preventing contact between the specified active peptide is cautiously composition, if it is complemented with 10 ml of 0.1 M aqueous solution of sodium bicarbonate in a quantity sufficient to lower the pH value of the specified fluid to the values above 5.5.

Preferred peptide active agents include, but are not limited to, insulin, vasopressin, salmon calcitonin and other substances discussed below, especially salmon calcitonin.

In accordance with another aspect of the invention provides a method of increasing the bioavailability of therapeutic peptide active agent is delivered orally, and this method is selective release of the peptide active agent together with at least one pH-lowering agent and at least one amplifier absorption in the intestine of the patient after completion of the peptide active agent, a pH-lowering agent and amplifier absorption through the oral cavity and the stomach of the patient under the protection acid-resistant protective filler, which substantially prevents contact between gastric proteases and the specified peptide agent,

wherein said pH-lowering agent and others entered with him the compounds are released into the specified intestine in number, kotoroe specified solution to a value not higher than a 5.5.

In accordance with another aspect of the present invention provides a method of increasing the bioavailability of salmon calcitonin delivered by oral method, and this method is selective release of salmon calcitonin in conjunction with at least one pH-lowering agent and at least one amplifier absorption in the intestine of the patient after passing the specified salmon calcitonin specified pH-lowering agent and amplifier absorption through the oral cavity and the stomach of the patient when protected by an enteric coating that substantially prevents contact between gastric proteases and salmon calcitonin;

in which the specified pH-lowering connection is released by the specified media in the intestine in number, which, being completed to 10 ml of 0.1 M aqueous solution of sodium bicarbonate, should be sufficient to lower the pH of the specified fluid to the values above 5.5.

The present invention involves the reduction of the probability of proteolytic degradation of the peptide active compounds by simultaneous protect the peptide from proteolytic attack gastric protease, usually the most the e active at pH values in the range from basic to neutral values).

Further, the present invention contributes to the process by which the peptide crosses the membrane of the intestinal brush borders and into the blood stream while continuing to protect the peptide from proteolytic degradation.

Acid-resistant protective filler protects the peptide active agent from the acid-existing gastric proteases. Significant amounts of acid (which is mixed peptide active agent) then reduce the activity of intestinal proteases acting in conditions of neutral to basic (for example, luminally or digestive proteases and protease membrane brush edges) by lowering the pH to values below the optimal interval of intestinal activity of such proteases. Amplifiers absorption of the present invention can be used to enhance transport of the peptide agent in the blood through the mucous layers of the intestine and through the membrane of the brush edges.

In accordance with the present invention the simultaneous use of amplifiers absorption and connections, reducing pH, provides an unexpected synergistic effect on the bioavailability compared with the action of the amplifier, absorption or connection, lowering the pH, separately. You can see osely calcitonin and pH-lowering compound) and the data of table 4 for recipe 2 (salmon calcitonin and power absorption) with the data of table 4 for recipe 3 (salmon calcitonin, pH-lowering agent and power absorption).

Other distinctive features and advantages of the present invention will become clear from the following detailed description of the invention.

This description of the invention

In accordance with the present invention, patients requiring treatment of the peptide active ingredients, provided their pharmaceutical composition for oral administration (in the appropriate dosage) is preferably, but not necessarily, in the form of tablets or capsules, which have a traditional size for the pharmaceutical industry. Dosage and frequency of introduction of the products of the invention are discussed in detail below. This treatment may benefit those patients who suffer from disorders that favorably responds to elevated levels peptidases connection. For example, in accordance with the invention for oral drug on the basis of salmon calcitonin can be used to treat patients suffering from disorders associated with calcium or bone diseases. The present invention can be applied, for example, for the treatment of osteoporosis, Paget's disease, hypercalcemia in malignant socoety calcitonin for a number of reasons is the preferred active ingredient for use in accordance with the present invention. For example, he has a number of advantages in comparison with human calcitonin, even when used as a pharmaceutical agent in humans. Among the benefits of salmon calcitonin instead of a human calcitonin for treatment of osteoporosis person can be noted increased efficiency, analgesia and increased value of half-life. Salmon calcitonin is more effective in treatment than natural human calcitonin, because it requires lower dosage compared to human calcitonin. Between salmon and human calcitonin is no significant homology and these two calcitonine there is only 50% identity in their amino acid sequences.

It was found that salmon calcitonin has unexpectedly high bioavailability when administered orally in accordance with the present invention in comparison with what one would expect on the basis of its molecular weight. It was found that the use of oral formulations of the invention in an internal comparative tests bioavailability of salmon calcitonin much higher than the one which had >With no regard to any particular theory, it is assumed that the pharmaceutical composition of the invention overcomes a number of different and unrelated natural barriers to bioavailability. The various components of the pharmaceutical compositions are such that overcome various barriers through mechanisms corresponding to each of them, and this provides a synergistic effect on the bioavailability of the peptide active ingredient. As discussed below, specific physical and chemical properties of salmon calcitonin and other peptides do some amplifiers absorption more efficient than others in the course of forcing their bioavailability.

Peptide active compound is administered orally. In accordance with the present invention decreases the proteolytic degradation of the peptide under the action of gastric proteases (most of which are active in the range of acidic pH values), as well as intestinal and pancreatic proteases (most of whom are active in the range of pH from neutral to basic). Amplifiers solubility facilitate the passage of the peptide active agent through the intestinal epithelium is a worthwhile invention the peptide is transported through the stomach under the protection of the appropriate acid-resistant protective vehicle with the aim of preventing contact between salmon calcitonin or other active peptide, and any of gastric proteases, able to degrade. After the pharmaceutical composition of the invention passes through the stomach and enters the intestinal region, dominated by pH values in the range from basic to neutral and where protease tend to purchase pH values in this interval, intersolubility floor or other media release the peptide and acid (in close proximity to each other).

It is assumed that the acid lowers the local pH value (at the place where stands the active agent) to values below the optimum interval for a large number of intestinal proteases. This decrease in pH reduces the proteolytic activity of the intestinal proteases, which protects the peptide from potential degradation. The activity of these proteases is attenuated temporary acidic environment provided by the present invention. It is preferable to provide a sufficient amount of acid, so that local intestinal pH value temporarily decreased to 5.5 or below, preferably to 4.7 or below and most preferably up to 3.5 or below. To determine the required amount of acid is conducted as described below (in the section titled "pH-lowering agent") test application existed during the time, sufficient to protect the peptide agent from proteolytic degradation, as long as at least part of the peptide agent will be able to pass through the intestinal wall into the bloodstream. The experiments allowed to establish Tmaxin the range of 5-15 minutes for levels of salmon calcitonin in the blood, when the active components are entered directly into the duodenum, ileum or the colon. Amplifiers absorption invention synergistically activate the absorbance of the peptide in blood at the prevalence of the condition of reduced proteolytic activity.

It is assumed that the mechanism for achieving the objectives of the present invention, consisting in enhanced bioavailability contributes to the implementation of almost simultaneous joint release of the active components of the pharmaceutical composition. To this end it is preferable to keep as low as possible volume Intercollege cover provided protection from the action of gastric proteases. Thus, intersolubility floor probably isn't a hindrance to the release of the peptide or other components that are released almost simultaneously with him. Typically, n is the number of components, excluding intersolubility floor). Preferably it amounts to less than 20% and more preferably this coating is 10-20% by weight of uncoated ingredients.

Power absorption, which may be an amplifier solubility and/or power transport (as described in more detail below), promotes the transport of the peptide agent from the intestines into the blood and can accelerate this process in the case where it operates during the existence of low intestinal pH and reduced intestinal proteolytic activity. Many surface-active agents have the potential to act as amplifiers of solubility and amplifiers transport (absorption). Again, without limiting to a specific theory, we can assume that the increased solubility provides (1) more simultaneous release of the active components of the invention in the aqueous part of the contents of the intestine, (2) better solubility of the peptide in the mucous layer along the walls of the intestine and improved transport through it. After the peptide active ingredient reaches the colon wall, power absorption provides improved transport through the membrane wenige, many preferred compounds are able to perform both functions. In such situations, the preferred embodiments of using both of these functions can be realized by introducing the pharmaceutical composition of only one additional connection. In other embodiments, the implementation of the various amplifiers absorption can provide these two functions separately.

Below, separately discuss each of the preferred ingredients of the pharmaceutical compositions of the invention. Can be used as a combination of many pH-lowering agents or multiple amplifiers, and only one pH-lowering agent and/or a single amplifier. Some preferred combinations are also discussed below.

Peptide active ingredients

In accordance with the present invention the peptide active ingredients, which are beneficial to deliver orally, include any therapeutic agent which is physiologically active, the molecular structure of which contains many amino acids and at least one peptide bond. The present invention through several mechanisms involves the suppression of degradation of the asset is x links active ingredient. This molecular structure may additionally include other substituents or modification. For example, salmon calcitonin, which is the preferred peptide active agent, lidiruyut on it-the end. In accordance with the invention orally delivered can both artificial and natural peptides.

Peptide active compounds of the invention include, but are not limited to, insulin, vasopressin, calcitonin (including not only the preferred salmon calcitonin, and other calcitonin). Other examples include a peptide related gene calcitonin, paratertiary hormone, factor in the release of luteinizing hormone, erythropoietin, activators of plasma tissue, human growth hormone, adrenocorticotropin, various interleukins, enkephalin, etc. In the art known, and many other substances. It is expected that any pharmaceutical compound with the peptide bond, which can be decomposed in the digestive tract, it would be useful to deliver oral in accordance with the present invention, since the present invention provides a reduction in the extent of cleavage.

When Ispolin composition (excluding intersolubility floor). Salmon calcitonin is a commercially available product (manufactured, for example, the firm VASHEM, Torrence, CA). On the other hand, it can be synthesized by known methods, some of which are briefly discussed below. Other peptide active agents must be present in greater or lesser concentrations, depending on the desired target concentration of active compound in the blood and its bioavailability in the system oral delivery of the present invention (some of these compounds noted in table 8).

Predecessors of salmon calcitonin can be obtained by chemical or recombinant synthesis known in this field. Similar methods can be obtained from precursors other emitirovannykh peptide active agents. It is assumed that the recombinant production will be significantly more efficient in terms of cost. The precursor is transformed into an active salmon calcitonin by amidation reactions, which are also known in the art. So, for example, enzymatic amidation described in U.S. patent 4708934 and in published European patent 0308067 and 0382403. Recombinant production of avseenko in the salmon calcitonin. Such recombinant getting discussed in Biotechnology, so 11 (1993), pp. 64-70, where it is additionally describes the transformation of the precursor in aminirovanie product. Described in this work, recombinant product is identical to natural salmon calcitonin and salmon calcitonin produced using chemical peptide synthesis in solution and in the solid phase.

The preferred production of recombinant salmon calcitonin (rsCT) can be carried out, for example, by obtaining the glycine-incarcerating predecessor of salmon calcitonin in E. coli as soluble fused protein in the presence of glutathione-S-transferase. Extended glycine precursor has a molecular structure that is identical to the structure of active salmon calcitonin, with the exception of the fragment at the C-end (where salmon calcitonin ends with a fragment of pro-NH2while the predecessor ends of the fragment pro-gly). -Aminirovanie the enzyme described in the above publications, catalyzes the conversion of precursors in the salmon calcitonin. This enzyme preferentially receive recombinant method, for example, in the ovary cells of Chinese Hoki different emitirovannykh peptides can be obtained in a similar way. The same method can also be derived peptides, which do not require amidation or other additional functionalization. Other peptide active agents are commercially available products or can be obtained by methods known in the art.

pH-Lowering agent

Preferably, the total amount of pH-lowering connection, subject to the introduction of each application salmon calcitonin, was sufficient to ensure that when it is released into the intestine to lower local intestinal pH below the optimal pH for being there proteases. The quantity required will necessarily vary depending on several factors including the type of pH-lowering agent (discussed below) and the number of proton equivalents, provide data pH-lowering agent. In practice, the quantity required to ensure good bioavailability is the amount that when added to 10 ml of 0.1 M sodium bicarbonate solution lowers the pH of this solution to a value not above 5.5, preferably not higher than the 4.7, most preferably no higher than a 3.5. In some embodiments, the implementation can be used on edocfile in the composition of the invention is used, at least 300 ml, and more preferably at least 400 ml of pH-lowering agent. The above preferred values apply to the total combined weight of all pH-lowering agents, when two or more of such agent are used in combination. Oral formulation should not contain any ground, which in a joint release with a pH-lowering compound is able to prevent the lowering of pH in the above-mentioned test with sodium bicarbonate to 5.5 or below.

pH-Lowering agent of the invention can be any pharmaceutically acceptable compound, non-toxicity in the gastrointestinal tract and is able to deliver hydrogen ions (traditional acid) or to induce an increased content of hydrogen ions from the local environment. Can also be used in combination with such compounds. Preferably, at least one pH-lowering agent of the invention had a pKa value not exceeding 4.2 and preferably not higher than 3.0. Also preferably, the agent that lowers the pH, had a solubility in water at room temperature at least 30 g per 100 ml of water.

Examples of compounds indusiry acceptable traditional acid include, but not limited to, acid salts of amino acids (for example, hydrochloride amino acids) or derivatives thereof. Examples of such compounds can serve as sour salt acetylglutamine acid, alanine, arginine, asparagine, aspartic acid, betaine, carnitine, carnosine, citrulline, creatine, glutamine acid, glycine, histidine, hydroxylysine, hydroxyproline, hepatorenal, isoleucine, leucine, lysine, methylhistidine, norleucine, ornithine, phenylalanine, Proline, sarcosine, serine, taurine, threonine, tryptophan, tyrosine and valine.

Other examples of applicable pH-lowering compounds include carboxylic acid, acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glycerin, glycosuria, dixileta, solimena, salariesa, lactic, maleic, salewoman, pyruvic, succinic, propionic, salelevitra, wine, Valerian, etc.

Other useful pH-lowering agents, which in the literature is related to the traditional "acids" and which, in spite of this, can be used in the present invention, constitute a complex phosphate esters (for example, 1,6-bisphosphate fructose 1,6-diphosphate change CARBOROL(trade name: BF Goodrich) and such polymers as polycarbophil.

Can be used in any combination on the basis of pH-lowering agent that provides the desired level of pH not above 5.5, in the above-mentioned test with sodium bicarbonate. In one of the preferred embodiments as at least one pH-lowering agents, pharmaceutical compositions used acid selected from the group consisting of citric acid, tartaric acid, and acidic salts of amino acids.

When using salmon calcitonin as the active peptide agent has been shown that some correlation between the pH-lowering agent and salmon calcitonin are particularly effective. Preferably, the ratio by weight between the pH-lowering agent and salmon calcitonin exceeded 200:1, preferably 800:1 and most preferably 2000:1.

Power absorption

Amplifiers absorption is preferably present in amount of 0.1-20.0 wt.% of the total weight of the pharmaceutical composition (excluding intersolubility floor). Preferred enhancers of absorption are surface-act the tea amplifiers solubility improves the ability of the components of the invention to solubilize in any aquatic environment, they were originally released, or in the lipophilic environment of the mucous layer lining the intestinal wall, or in both. "Amplifiers transport (absorption)" (which often represent the same surface-active agents that are used as amplifiers solubility) are substances that facilitate the penetration of the peptide agent through the intestinal wall.

In the framework of the invention provides that one or more amplifiers, absorption can only perform one function (e.g. solubility) or one or more amplifiers, absorption can only perform a different function (e.g. absorption). It is also possible to use a mixture of several compounds, some of which provide improved solubility, others provide superior absorption and/or some of which perform both functions. Not limited to any theory, it can be assumed that the absorption enhancers may act by (1) increasing violations of the hydrophobic region of the outer membrane of intestinal cells, thereby providing a reinforced transcellularly transport; or (2) leaching of membrane proteins that resulting paracellular transport.

It is assumed that the surface-active agents can be used as amplifiers solubility and as absorption enhancers. So, for example, the beneficial effect of detergents is to (1) rapid solubilization of all active components in the aquatic environment, where they were initially released, (2) enhancing solid components of the invention, particularly a peptide active agent, which contributes to its passage into and through the intestinal mucus, (3) strengthen the capacity usually polar active agent to pass through the epithelial barrier membrane brush edges and (4) strengthening transcellular or paracellular transport, as described above.

When using surface-active agents as enhancers of absorption it is preferable that they were free flowing powders to facilitate mixing and loading of the capsules during the manufacturing process. In connection with the specific characteristics of salmon calcitonin and other peptides (e.g., in terms of their isoelectric point, molecular weight, amino acid composition, etc.,) some surface-active agents interact with some peptides are better than others. Resumesession and prevent its absorption, that undesirable way, can lead to reduced bioavailability. When trying to increase the bioavailability of salmon calcitonin or other peptides preferably, any surface-active agent used as the amplifier absorption, selected from the group consisting of (i) anionic surface-active agents which are derivatives of cholesterol (e.g., bile acids), (ii) cationic surface-active agents (for example, acylcarnitines, phospholipids, etc.,), (iii) non-ionic surface-active agents, and (iv) mixtures of anionic surface-active agents (especially those that have a linear hydrocarbon plots) with negatively charged neutralizers. Negatively charged neutralizers include, but are not limited to, acylcarnitine, chloride, pyridinium, etc., it is Also preferable to power absorption was soluble at acid pH values, especially in the range of 3.0 to 5.0.

One of the particularly preferred combinations that work well with salmon calcitonin, is a mixture of cationic surface-active agents, anionic surface-active agents which are derivatives of cholesterol, and obese mixture of soluble bile acids with cationic surface-active agent. A good combination is a mixture of acylcarnitine with a complex ester of sucrose. In the case where the specific power absorption is used by itself, it is preferable that he was a cationic surface-active agent. Acylcarnitine (for example, lauroylsarcosine), phospholipids and bile acids are especially good amplifiers absorption, especially acylcarnitine. In some embodiments, the implementation also used anionic surface-active agents which are derivatives of cholesterol. The application of the above preferred substances to exclude interactions with the peptide agent that prevent absorption of the peptide agent in the blood.

To reduce the likelihood of side effects preferred detergents for use in the amplifier of the absorbance of the invention are either biodegradable or able to reabsorption (for example, such compounds are capable of biological recycling, as bile acids, phospholipids and/or acylcarnitine), preferably biodegradable. It is assumed that acylcarnitine especially useful in strengthening paracellular transport. In that case, is combinatii with cational detergent, salmon calcitonin better is transported to and through the wall of the intestine.

Preferred enhancers of absorption include: (a) salicylates such as sodium salicylate, 3-methoxysilyl, 5-methoxysilyl, homovanillic; (b) such bile acids, as human beings need it, taurodeoxycholate, desoxycholate, cholic, Glagoleva, lithocholate, chenodeoxycholic, ursodeoxycholic, orseolia, dehydrocholic, positiva and so on; (C) such non-ionic surface-active agents, as polyoxyethylene esters (e.g., Brij 36T, Brij 52, Brij 56, Brij 76, Brij 96, Texaphor A6, Texaphor A14, Texaphor A60, etc.,), p-tert-octylphenoxypolyethoxyethanol (Triton X-45, Triton X-100, Triton X-114, Triion X-305, and so on), nonylphenoxyacetic (for example, Igepal CO series), polyoxyethylene sorbitol complex esters (such as Tween-20, Tween-80, etc); (d) such anionic surface-active agents, as sulfosuccinate dioctyl sodium; (e) such lysophospholipid as lysolecithin and lysophosphatidylethanolamine; (f) such acylcarnitine, acylcholine and acylaminoalkyl as lauroylsarcosine, myristoylation, palmitoylcarnitine, maureilhan, myristoylation, palmitoylation, hexadecimalisan, N-allenylidenes, N-acylpyrin etc.; (g) such water-soluble phospholipids, as diheptanoate what it is, di - and triglycerides containing fatty acids with medium chain length (Caprylic, capric and lauric acid); (i) ethylenediaminetetraacetic acid; (j) such cationic surface-active agents, as chloride, pyridinium; (k) such fatty acid derivatives of polyethylene glycol, as Labrasol, Labrafac, and so on; and (l) such alkalihalide as lauryl maltoside, lauroyl sucrose, myristoyl sucrose, Palmitoyl sucrose, etc.

In some preferred embodiments, the implementation without any theoretical restrictions are cationic ion-exchange agents (e.g. detergents) to ensure enhance the solubility of other possible mechanism. Such agents, mainly, can prevent the binding of salmon calcitonin or other peptide active agents with mucus. Preferred cationic ion-exchange agents include Protamine chloride or any other polycation.

Other optional ingredients

Preferably, a water-soluble barrier separated the pH-lowering agent from the acid-resistant protective filler. In some of the following examples to create such a barrier is used usual pharmaceutical to the droxyprogesterone and traditional pharmaceutical gelatin.

Preferred embodiments of include another peptide (e.g., albumin, casein, soy protein, other proteins of animal and vegetable origin, etc.,) to reduce nonspecific adsorption (for example, binding of the peptide with the intestinal mucous barrier and, therefore, may decrease the concentration of expensive peptide active agent. The peptide is preferably added in the amount of 1.0-10.0 wt.% based on the weight of the pharmaceutical composition (excluding protective filler). Preferably this second peptide has no physiological activity, and most preferably is a food peptide as soy or so on, are Not limited to the special theory, it should be noted that this second peptide may also increase the bioavailability, acting as acceptor protease, which competes with the peptide active agent in interaction with the protease. The second peptide may also contribute to the passage of active compounds through the liver.

All the pharmaceutical compositions of the invention can also

optional to include conventional pharmaceutical diluents, polysaccharides, lubricants, relate the Ah and quantities.

Protective filler

Any carrier or filler, which protects salmon calcitonin from the action of gastric proteases and then dissolved, in consequence of which the other ingredients of the invention can be released in the intestine, is a suitable substance. Many of these intersolubility coatings known in the art and they can be used in accordance with the invention. Examples of such agents can serve acetate cellulose phthalate, succinate of hydroxypropylmethylcellulose, phthalate of hydroxypropylmethylcellulose, carboxymethylcellulose and copolymer of methacrylic acid and methyl methacrylate. In some embodiments, the active peptide, such amplifiers absorption, as the amplifier(s) solubility and/or absorption, and pH-lowering compound is introduced in a sufficiently viscous protective syrup order to secure passage of components of the invention through the stomach.

Suitable intersolubility coatings for protection of the peptide agent from the action of gastric proteases can be used, for example, the capsules after the remaining components of the invention loaded in such capsules. In other embodiments, implementation of enterosorbent who were then pressed into tablets, or loaded into the capsule, which is preferably covered intersolubility the floor.

It is highly desirable that all components of the invention would be released from the carrier or excipient and, almost simultaneously, were dissolved in the intestinal environment. Preferably, the filler or carrier released the active components in the small intestine, where absorption enhancers that increase transcellularly or paracellular transport, less likely to cause unwanted side effects than in the case when the same amplifiers absorption later released in the colon. However, it should be emphasized that the present invention provides for efficient use of both the colon and the small intestine. In the art there are many fillers or carriers, in addition to those mentioned above. It is desirable to use a small amount Intercollege coverage (especially in terms of optimization of the simultaneous release of components of the invention). Preferably intersolubility floor add no more than 30% by weight of residue pharmaceutical compositions (and the term "residue" refers to a pharmaceutical composition, except for the mass of the uncoated composition. Intersolubility coating preferably should be sufficient to prevent the destruction of the pharmaceutical compositions of the invention in HCl 0.1 N. for at least two hours, it must be able to provide a full release of all components of the pharmaceutical composition within 30 minutes after raising the pH to 6.3 in the bath for dissolution, where the specified composition rotate with a speed of 100 revolutions per minute.

Other preferences

Preferably, the ratio by weight between the pH-lowering agent and amplifier absorption had a value in the range 3:1 to 20:1, preferably 4:1 to 12:1 and most preferably 5:1 to 10:1. The total mass of all pH-lowering agents, and the total mass of all amplifiers absorption in this pharmaceutical composition are covered above, the preferred ratios. For example, if the pharmaceutical composition comprises two pH-lowering agent and three amplifier absorption, the above ratio should be calculated on the total combined weight of the pH-lowering agents, and the total combined weight of all three amplifiers absorption.

Preferably, the pH-lowering agent, a peptide active agent and the amplifier and the us in the pharmaceutical composition. According to one embodiments the pharmaceutical composition comprises granules, including pharmaceutical binder, which is distributed homogeneously peptide active agent, a pH-lowering agent and power absorption. Preferred granules can also be composed of acid core surrounded by a uniform layer of an organic acid, a layer of an amplifier and a layer of a peptide which is surrounded by an outer layer of organic acids. Granules can be obtained from an aqueous mixture consisting of such pharmaceutical binder agents, as polyvinylpyrrolidone or hypromellose, together with pH lower agents, enhancers of absorption and peptide active agents of the present invention.

The method of obtaining

Preferred pharmaceutical composition of the invention includes gelatin capsule size 00, filled with 0.25 mg of salmon calcitonin, 400 mg of granulated citric acid (supplied, for example, Archer Daniels Midland Corp. ), 50 mg taurodeoxycholic acid (supplied, for example, SIGMA), 50 mg of lauroylsarcosine (SIGMA).

All these ingredients are designed for ultimate introduction into a gelatin capsule and predpochtitel is given approximately five minutes before thorough mixing of powders. Then, the mixed powders are loaded into the large ends of the gelatin capsules. Next add the other end of the capsule and the capsule is closed with a snap. 500 or more of these capsules can be loaded into the device for coating (for example, in the Vector LDCS 20/30 Development Laboratory Coating System supplied Vector Corp., Marion, Iowa).

The solution for applying Intercollege coating is prepared as follows. Weighed 500 g of EUDRAGIT L30 D-55 (methacrylic acid copolymer with methyl ether metallinou acid, intersolubility coating, manufactured by ROHM Tech Inc., Maidan Mass.). Add 411 grams of distilled water, 15 g of triethylcitrate and 38 g of talc. Such a number of cover should be enough to cover about 500 capsules size 00.

Capsules are weighed and placed in a dryer device for coating. The device is switched to the mode of rotation of the drum (containing now capsules) with a speed of about 24-28/min Temperature input atomizer is preferably 45oC. the outlet Temperature is preferably 30oC. the temperature of the uncoated capsule preferably is about 25oC., the Air supplied at a rate of about 38 cubic feet (0.75 m3in minutaie includes a pump for supplying the solution in the device for applying the coating. The coating process is carried out in automatic mode. The device can be stopped at any time for weighing capsules with a view to establishing the fact of applying a sufficient amount of coverage. Typically, the coating process lasts for 60 minutes. The pump is then shut off for five minutes, leaving the device in the status of the work to facilitate the drying of coated capsules. The device will then turn off. The coating on the capsule ends, although it is recommended to dry the capsules on the air for two days.

Because the present invention provides improved bioavailability, concentration of salmon calcitonin in the pharmaceutical preparation of the invention can be maintained at a relatively low level. Below is a special prescription examples.

Patients

In the case when the salmon calcitonin is chosen as the active ingredient for the treatment of osteoporosis, it is recommended that periodic introduction. After subcutaneous administration to the person of salmon calcitonin rapidly undergoes metabolism with a half-life of only 20 to 40 minutes. However, its beneficial effects on osteoclasts lasts much bollyone after approximately two hours after the injection of salmon calcitonin in conventional doses, its level in blood is not detected. In accordance with this prefer to carry out periodic introduction of one dose per five-day period during the week. Subcutaneous administration of salmon calcitonin (100 International units) often creates its maximum concentration in the serum of about 250 picograms per milliliter. Nasal introduction of salmon calcitonin (200 International units) proved effective against osteoporosis at the maximum dosage of only 10 picograms per milliliter. Some patients reported gastrointestinal disorders at high maximum levels of use (for example, 200 picograms per milliliter or higher). Accordingly, it is preferable that the minimum concentration of salmon calcitonin in serum was 10-150 picograms per milliliter, more preferably 10 to 50 picograms per milliliter. Levels in serum can be measured by radioimmunoassay methods known in this field. The attending physician can monitor the patient's response, the levels of salmon calcitonin in the blood or equivalent markers of bone diseases (such as urinary pyridinoline or deoxypyridinoline), especially during the initial treatment phase (1-6 months). He may then h is asthema with the present invention, allows you to deliver oral salmon calcitonin in the blood with the above, the preferred concentration values using only 100-1000 micrograms of salmon calcitonin per capsule, preferably 100-400 micrograms, especially 100-200 micrograms.

Preferably, each introduction has used only the capsule, as in this case, the best way is provided by the simultaneous release of the polypeptide, the pH-lowering agent and amplifiers, absorption. This is a very desirable circumstance, because the acid is best able to reduce unwanted proteolytic effect on the polypeptide in the case when the acid is released almost simultaneously with the release of the polypeptide. Close to simultaneous release is best accomplished by the introduction of all components of the invention in the form of a single pill or capsule. However, the present invention also provides, for example, the distribution of the required amount of acid and amplifiers for two or more capsules that can be entered together in such a way that they together provide the necessary number of all ingred the particular introduction of the person, regardless of the separation, if it involves the simultaneous introduction.

Below is a series of tables illustrating the effects on bioavailability to the variation of some parameters. Except with regard to human studies, as mentioned, the number of ingredients may vary from those stated in the text, taking into account differences between humans and animals used in animal models.

Way

Female rats varieties Wistar (250-275 g) (n=3 for each formulation) were anestesiologi ketamine and xylazine before the introduction of the cannula in the carotid artery. The cannula has been implementing the three-way valve through which samples were taken and blood was replaced them with saline solution. Did the median abdominal incision and 0.5 ml of the formulation was injected directly into the Nude the duodenum. the pH of the formulation was adjusted by the adulteration of various amounts equimolar concentrations of citric acid and sodium citrate. Blood samples (0.5 ml) were collected before administration and after 5, 15, 30, 60 and 120 minutes after administration of the formulation. The blood samples were centrifuged for 10 minutes at a load of 2600 g and the resulting supernatant plasma was stored at -20oC. Concentric (i.e., relative to an intravenous dose of calcitonin) was calculated from the area under the curve graph of the dependence of the concentration of calcitonin in plasma from time to time.

Results and discussion

At low pH buffer 5 (formulation 1) to 4 (formulation 2) the value of the absolute bioavailability was increased five times from 0.02% to 0.1%. Upon lowering the pH to 3 (formula 3) absolute bioavailability was increased 6.4 times. Noted only a slight increase in the bioavailability of calcitonin in reducing the pH to 2. The total bioavailability of calcitonin increased 32 times with decreasing pH of the buffer from 5 to 3 (see tab.1).

Way

Formulations consisting of a constant number of taurodeoxycholic acid and 2 different amounts of citric acid in a total volume of 0.5 ml was injected into the duodenum shot rats as indicated in the explanation to table 1. As a marker for measuring paracellular transport in the formulation was administered mannitol. Blood samples were taken after various time intervals and analyzed for calcitonin as described above.

Results and discussion

The bioavailability of salmon calcitonin, applied in the presence of 9.6 g lemanstv fixed number taurodeoxycholic acid bioavailability of salmon calcitonin increased about 10 times with increasing content in the formulation of citric acid only 5 times (see table.2).

Way

Formulations consisting of citric acid, calcitonin and amplifiers of various classes with a total volume of 0.5 ml was injected into the duodenum shot rats as described in the explanation to table 1. Mannitol was included in the formula 5 as a marker for measuring paracellular transport. Blood samples were taken after different time intervals and analyzed for the content of calcitonin by the method described above.

Results and discussion

In the absence of amplifier absolute bioavailability of calcitonin was 0.69%. The inclusion of water-soluble phospholipid (formulation 7) increased bioavailability 4.3 times before 2,97%. The most effective power belongs to the class of esters of sugars (formula 5), in the presence of bioavailability of calcitonin was 5.83%. The application of a mixture of bile acids and cationic detergent (recipe 3), non-ionic detergent (recipe 4) and acylcarnitine (formulation 6) resulted in intermediate values of bioavailability, lying in the interval 3,03-4,53%. Differences in the values of bioavailability of calcitonin in the presence of different classes of amplifiers are small compared with the values of policeacademy, containing lauroylsarcosine, calcitonin, and various other compounds with a total volume of 0.5 ml was injected into the duodenum shot rats in accordance with the methodology described for the results of the table 1. Blood samples were taken after various time intervals and analyzed for calcitonin in accordance with the previously described method.

Results and discussion

In the absence of citric acid and of any amplifier (formulation 1) the absolute bioavailability of calcitonin was 0,096%. In the presence of 5 mg of chloride lauroylsarcosine (formulation 2) bioavailability was increased 1.8-fold and amounted to 0.17%. Adding citric acid with Eurointernational (formulation 3) bioavailability was increased in 27 times and was a 4.53%. A five-fold decrease in the number of lauroylsarcosine, but not citric acid (formula 4) was slightly reduced the bioavailability of salmon calcitonin. Introduction 5 mg digitalofficepro in formula 3 to obtain recipe 5 slightly increased the value of bioavailability (1.4 times). Replacement of citric acid 25 mg albumin calf serum (formulation 6) reduced bioavailability with a 4.53% (formulation 3) to 0.42% (see tab.4). A joint is considered the traditional acid, and this amplifier, as lauroylsarcosine.

Way

Modified muscle inlet surgically implanted into the duodenum, pouzdanog intestine and the colon of male greyhounds. Bulkhead/tank body "gate" implanted under the skin and used as sites for the introduction of calcitonin recipes. Before and after the introduction calcitonin recipes in conscious dogs openings (gates) was washed with 2 ml of the formulation without calcitonin. Blood samples (2 ml) was collected through angiocatheter tube of foot vein at time t= 30, 15 and 0 prior to the introduction of calcitonin and through 5, 10, 20, 30, 40, 50, 60 and every 15 minutes during the following 2 hours after application. Blood samples were centrifuged for 10 minutes at a load of 2600 g and the resulting supernatant plasma was stored at -20oC. the Concentration of calcitonin in plasma were determined using a competitive radioimmunoassay. The average absolute bioavailability (i.e., the values obtained in comparison with the effect of intravenous doses of calcitonin) was calculated from the area under the curve obtained from the graphs of the dependence of the concentration of calcitonin in plasma from time to time.

Rezultatai 192 mg of citric acid (2) bioavailability of calcitonin increased 25 times. Introduction 20 mg taurodeoxycholic acid in the formula (3) led to an additional increase in the absolute bioavailability of 2.2 times, to the value 0,81% (see tab.5). Using a combination of pH-lowering compounds, citric acid and amplifier, taurodeoxycholic acid, provided the total to 54-fold increase in the absolute bioavailability of salmon calcitonin.

Ways

Starch and gelatin capsules were filled with these recipes and covered for 60 minutes in a hinge device for coating or phthalate of hydroxypropylmethylcellulose 50 (1, 2, 3) (gain 1 wt.%), or Eudragit L30-D55 (4) (gain 10 wt.%). The stability of the capsules in HCl 0.1 N. was determined in the bath for dissolution using a "basket (basket) method". At least two dogs orally applied each of said capsules and took blood samples, which were analyzed by the method described above on the content of calcitonin.

Results

Bioavailability of 10 mg of calcitonin, mixed with 100 mg of citric acid and 100 mg taurodeoxycholic acid and delivered in a starch capsule (1), was 0.07%. When using the same recipes for dogs in the form of gelatin capsules (2) biodone amount of calcitonin (3) has resulted in an almost 3-fold increase in the bioavailability of calcitonin (see table.6).

When replacing Intercollege coverage of the phthalate of hydroxypropylmethylcellulose 50 on the floor of the methacrylate polymer Eudragit L30-D55 while maintaining the same formulation (4) the bioavailability of salmon calcitonin increased from 0.62% to 1.48%. Replacement Intercollege coverage of the phthalate of hydroxypropylmethylcellulose 50 on the coating of Eudragit L30-D55 resulted in improved stability of the capsules in 0.1 G. of HCl. This improved stability provided manifestation of maximum levels of calcitonin in the blood of dogs in later periods of time. Instability capsules 1, 2 and 3 in Hcl suggests that such capsules may be opened in the stomach of dogs, while the improved stability of the capsules 4 assumes that it is completely stable in the stomachs of dogs and opens in the intestines of dogs. The obtained result indicates that the preferred is a minimum number of Intercollege coverage. At the same time too much coverage may delay the release of calcitonin after the release of other important components (e.g., acid and detergent). Preferably intersolubility floor add 5-15% by weight of uncoated who you are, 105 mg taurodeoxycholic acid and 10.5 mg of salmon calcitonin. Capsules for 20 minutes covered with a phthalate of hydroxypropylmethylcellulose 50 in the hinge device for coating and kept at 4oC. in the Morning in the beginning of the study patients on an empty stomach took 1 capsule which washed down with a glass of orange juice. Blood samples were taken 15 minutes before taking the capsules and at specified time intervals after administration of calcitonin capsules. The concentration of calcitonin in the blood was determined by the method of competitive radioimmunoassay. Absolute bioavailability (i.e., a value relative to an intravenous dose of calcitonin) was calculated from the area under the curve obtained from the plot of concentration of calcitonin in plasma from time to time.

Results

Only the use of salmon calcitonin in the amount of 10 milligrams did not, resulting in the appearance of detectable levels of salmon calcitonin in the blood plasma. However, when the objects of treatment received composition of the invention, described in table 7, the maximum levels of calcitonin were detected in the blood within 30-60 minutes after taking the capsule patients. The maximum concentrat was 173 PG/ml at t=30 minutes The absolute value of bitestrenos had a value of 0.02-0.06% of the average total value of 0.03%.

Way

Formulations consisting of either [arg8]-vasopressin, recombinant salmon calcitonin or insulin and specified additives in a total volume of 0.5 ml was injected into the duodenum shot rats according to the method described in the explanation to table 1. Blood samples were collected in different periods of time and analyzed for the contents of the specified peptide in accordance with the described methodology.

Results and discussion

In the absence of any additives, the absolute bioavailability intraduodenal used [AGD8]-vasopressin was 0.38%. When injected into the formulation of citric acid and lauroylsarcosine bioavailability of vasopressin increased to 8.1%. Bioavailability of calcitonin in the absence of acid and amplifier was 0,096%, which is lower than the values for the formulation of vasopressin without additives. However, the introduction to the recipe citric acid and lauroylsarcosine absolute bioavailability was increased 50-fold and reached a 4.53%. In the absence of citric acid human insulin may not even be dissolved in water. In the presence of citric acid all p the amounts of 0.07%. With the introduction of the formulation of lauroylsarcosine absolute bioavailability of insulin is increased 10 times. The results show that the bioavailability of peptides without prescription additives is not more than 0.38% and that the introduction of organic acids, such as citric acid, and amplifier, such as lauroylsarcosine, increases the bioavailability of the peptide to 8.1% (see tab. 8).

Ways

Gelatin capsules filled 473 mg of citric acid, 75 mg taurodeoxycholic acid, 75 mg of lauroylsarcosine and 0.82 mg of salmon calcitonin. Capsules within 60 minutes covered Eudragit L30-D55 in Cup device for coating and kept at 4oC. On the morning of the beginning of the studies, patients on an empty stomach took 1 capsule, drinking her glass of orange juice. Blood samples were taken 15 minutes before taking the capsules and at specified time intervals after administration of the capsules with calcitonin. The concentration of calcitonin in the blood were determined using a competitive radioimmunoassay. The average absolute bioavailability (i.e., a value relative to an intravenous dose of calcitonin) was calculated from the area under the curve obtained from the graphs of the dependence of the concentration of calcitonin in plasma from time to time.

apsule patients. The maximum concentration of calcitonin in the blood was 211-623 PG/ml Average maximum concentration (Cmax) calcitonin for 5 patients was 411 PG/ml, which is approximately 5-10 times higher than the target therapeutic level in the plasma. Absolute bioavailability had a value of 0.14 to 0.68% at a cumulative average of 0.38% (see tab. 9). These results show that even with the reduction in the content of the peptide is about 10 times the bioavailability of sCT in comparison with bioavailability obtained in table 7, were increased 10 times in the replacement of starch capsules gelatin capsules, use as Intercollege coating Eudragit L30-D55 instead of phthalate hydroxymethylcellulose, increasing the amount of citric acid and the introduction to the recipe lauroylsarcosine.

Although the present invention is described with reference to its specific embodiments of the specialists in this field will be able to find many other variations and modifications and other uses. In this regard, the present invention is not limited to the above description but only by the claims.

1. Pharmaceutical composition for oral delivery of a physiologically active peptide agent, the enta; (C) at least one pharmaceutically acceptable pH-lowering agent; (C) at least one power absorption, effective to increase the bioavailability of the specified active peptide agent, and (D) acid-resistant protective filler, contributing to the transport specified in the pharmaceutical composition through the stomach of the patient, but prevent the contacting of the specified active peptide agent and stomach proteases, where the specified pH-lowering agent is present in the specified pharmaceutical composition in an amount which, when you add specified tracks to 10 ml of 0.1 M aqueous sodium bicarbonate solution, would be sufficient to lower the pH of the specified solution until no values above 5.5.

2. The pharmaceutical composition under item 1, characterized in that said pH-lowering agent is present in an amount which when added to the specified composition to 10 ml of 0.1 M aqueous sodium bicarbonate solution, would be sufficient to lower the pH of the specified solution to a value not higher than a 3.5.

3. The pharmaceutical composition under item 1, characterized in that the mass of the specified protective filler is not more than 30% of the mass of the rest of specified pharmaceuti is a surface-active agent, selected from the group consisting of acylcarnitines, phospholipids and bile acids.

5. The pharmaceutical composition according to p. 4, characterized in that it further comprises an ester of sucrose.

6. The pharmaceutical composition under item 1, characterized in that it contains at least two amplifiers absorption, one of which is a cationic surface-active agent, and the other anionic surface-active agent, a derivative of cholesterol.

7. The pharmaceutical composition under item 1, characterized in that it further comprises a second peptide in an amount effective to increase the bioavailability of the specified active peptide agent.

8. The pharmaceutical composition under item 1, characterized in that it further contains a water-soluble barrier that separates the specified pH-lowering agent from the specified protective filler.

9. The pharmaceutical composition under item 1, characterized in that it is a solid dosage form in which the ratio by weight between the specified pH-lowering agent and the specified power absorption has a value in the range 3:1 to 20:1.

10. The pharmaceutical composition under item 1, the best of viteska composition on p. 1, characterized in that said active peptide agent is a vasopressin.

12. The pharmaceutical composition under item 1, characterized in that the said active peptide agent is a salmon calcitonin.

13. The pharmaceutical composition under item 1, characterized in that the said active peptide agent is an insulin.

14. The pharmaceutical composition under item 1, characterized in that it's intended for oral delivery of salmon calcitonin and contains (A) a therapeutically effective amount specified salmon calcitonin; (C) at least one pharmaceutically acceptable pH-lowering agent; (C) at least one power absorption, effective in increasing the bioavailability of the specified salmon calcitonin, and (D) acid-resistant protective filler, representing intersolubility floor, where the specified pH-lowering agent is present in the specified pharmaceutical composition in a quantity which when you add specified tracks to 10 ml of 0.1 M aqueous sodium bicarbonate solution, would be sufficient to lower the pH of the specified solution until no values above 5.5.

15. Farmatsevticeski the major salmon calcitonin is, at least 200:1.

16. The pharmaceutical composition under item 1, characterized in that it's intended for oral delivery of salmon calcitonin and contains (A) a therapeutically effective amount specified salmon calcitonin; (C) at least one pharmaceutically acceptable pH-lowering agent having a pKa value not exceeding 4.2 and the solubility in water of not lower than 30 g per 100 ml of water at room temperature; (C) at least one power absorption, enhancing bioavailability specified salmon calcitonin; (D) acid-resistant protective filler, representing intersolubility floor, with a mass component 10-20% of the mass of the rest of specified pharmaceutical composition; and further comprises (E) a water-soluble barrier that separates the specified pH-lowering agent from the specified Intercollege coating, where the specified pH-lowering agent is present in the composition in an amount which when added to the specified composition to 10 ml of 0.1 M aqueous sodium bicarbonate solution, would be sufficient to lower the pH of the specified solution to a value not above 5.5, and where specified in the pharmaceutical composition in the form of pellets, is shown pH-lowering agent and power absorption.

17. Method of enhancing the bioavailability of therapeutic active peptide agent is delivered orally, provided a pharmaceutical composition which is capable of selectively release the specified active peptide agent, together with at least one pH-lowering agent and at least one amplifier absorption in the intestine of the patient after passing the specified active peptide agent, a pH-lowering agent and amplifier absorption through the oral cavity and the stomach of the patient under the protection acid-resistant protective filler, which substantially prevents contact between gastric proteases and the specified active peptide agent, wherein said pH-lowering agent and other compounds entered with him, are released into the specified intestine in an amount which when added to 10 ml of 0.1 M aqueous sodium bicarbonate solution, would be sufficient to lower the pH of the specified solution until no values above 5.5.

18. The method according to p. 17, characterized in that said pH-lowering agent is present in an amount which when added to the specified pharmaceutical compositions to 10 ml of 0.1 M new to 3.5.

19. The method according to p. 17, characterized in that the ratio by weight between the specified pH-lowering agent and the specified power absorption has a value in the range 3:1 to 20:1.

20. The method according to p. 17, characterized in that the said active peptide agent is a salmon calcitonin.

 

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