MXPA97001455A - Pharmaceutical compositions containing a salvage and a regulator to increase the biodisponibility of a compound act - Google Patents

Abstract

Compositions containing a bile salt and a regulator such as a carbonate or bicarbonate salt which is adapted to regulate the intestine at a pH of from 7.5 to 9 are capable of increasing the bioavailability of an active molecule while minimizing toxic side effects. they are usually associated with bile salts

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING A BILIARY SALT AND A REGULATOR TO INCREASE THE BIODISPONIBILITY OF AN ACTIVE COMPOUND The present invention relates to pharmaceutical compositions and in particular to orally administrable compositions of proteins, peptides and other active molecules "which in general are not readily absorbed from the gastrointestinal tract. Medical practice for many years has prescribed or recommended the administration of many biologically active materials for the treatment or prophylaxis of a wide variety of diseases or conditions. One of the best known, but by no means the only, biologically active protein material prescribed is insulin, which is used to control diabetes. Other protein materials include growth factors, interleukins and calcitonin and non-protein biologically active materials include oligonucleotides and polysaccharides. Possibly the easiest method to take any medication is oral ingestion. This route of administration, "which may be by syrup, elixir, tablets, capsules, granules, powders or any other convenient formulation, is generally simple and direct and is frequently the least inconvenient or unpleasant route of administration from the point of view of the patient. It is therefore unfortunate that most of these materials are very poorly absorbed when administered orally. First, the preferred route of administration of protein drugs and other biologically active materials involves passing the material through the stomach, which is a hostile environment for many materials, including proteins, according to the acid, hydrolytic and protein environment of the stomach. has evolved efficiently for. digesting protein materials in amino acids and oligopeptides for later anabolism, it is hard to be surprised that very little or anything from a wide variety of biologically active protein material, if simply taken orally, would survive this passage through the stomach to be absorbed by the body in the small intestine. It is possible to provide coated enteric formulations "that are protected from the acidic environment of the stomach, but even so, relatively large molecules such as peptides and proteins are poorly absorbed in the small intestine. The result, as many diabetics can testify, is "that many proteinaceous drugs have to be taken parenterally, often by subcutaneous, intramuscular or intravenous injection, with all the inconveniences, discomfort and difficulties of patient submission that it entails. This is not an isolated problem, as the diseases that need control by the administration of protein material can be very widespread. Diabetes, for example, claims a large number of patients in many countries of the world and there are many other conditions that require treatment by administering a protein compound or another macromolecule. Osteoporosis is another example of a condition that can be treated using one. protein - in this case calcitonin - and protein growth hormones can be used to treat dwarfism. Clearly, therefore, there is a need for pharmaceutical formulations of proteins and other macromolecules "that can be administered orally and that provide acceptable bioavailability of the active material. It is known that when pharmaceutically active compounds are administered together with. certain bile salts, their bioavailability is increased. This is discussed by Kakemi et al (Chem. Pharm. Bull, 18 (2) 275-280 (1970)) who showed that the bioavailability of several substances would be increased by administration in combination with taurocholate or glucocholate. Other experiments by Kojima et al. (Chem. Pharm. Bull, 25 (6) 1243-1248 (1977)) showed «that sodium cholate is able to increase the absorption of different substances but also accelerates the release of the components of the cell membrane. In addition there are many other references in the prior art with the use of bile salts as absorption enhancers including GB-A-2244918 which shows that the bioavailability of somatosta ina can be improved when it is administered with a derivative of colanic acid such as chenodeoxycholic acid or ursodeoxycholic acid. It is believed that bile salts improve absorption. of biologically active materials due to their action on the cell membranes of epithelial cells. Cell membranes become more permeable, possibly due to "that the detergent action of the bile salts removes the lipids from the cell membranes and makes them become more fluid. One theory is "that the increased permeability of the cell membrane allows active materials to pass through the epithelial cells. Alternatively, it is possible that the cytoskeletal structure of the epithelial cells is modified as a result of changes in the cytoplasmic levels of sodium or calcium arising from the increased permeability of the membrane. This would result in an alteration of the integrity of the hermetic joints so that there would be gaps between the cells through which the active materials can pass. Whatever the mechanism, it seems clear that the increased absorption arises from the increased permeability of cell membranes. However, the inclusion of bile salts in pharmaceutical formulations has always been a problem because they have an unacceptably high toxicity when used in amounts required by them to exert their bioavailability enhancing effect. It has been suggested that the reason for this is that the increase in the permeability of the epithelial cells put in contact with the biliary salts gives as. resulting in an increased flow of ions in and out of the cell. In order to "keep the cells viable, the intracellular concentrations of ions such as sodium, potassium and calcium must remain within relatively narrow ranges and it may be that the increased flow of ions caused by the presence of bile salts causes the concentrations of intracellular ions move out of the ranges within which the cells are viable. Thus, the problem with the use of bile salts as absorption enhancers is "that they have always been considered to have an unacceptably low therapeutic index. Through the present specification the term "therapeutic index" has been used to refer to the toxic dose ratio: absorption enhancing dose. It would thus be extremely useful to be able to reduce the toxicity and therefore effectively increase the therapeutic index of the bile salts to the point where they could be used in pharmaceutical compositions without unacceptable toxic side effects. In a first aspect of the present invention there is provided a pharmaceutical and / or veterinary composition comprising a biologically active material, an acid or bile salt and an agent adapted to adjust the pH of the intestine to the pH value of from 7.5 to 9. WO -A-9012583 shows a pharmaceutical composition comprising an active agent, a bile salt and an additional bile component. However, the only additional components suggested herein as likely to be useful in these compositions are additional bile salts and bile lipids, in particular phospholipids. Even though bicarbonate (a preferred pH adjusting agent useful in the invention) is a bile component, bicarbonate salts are not mentioned in WO-A-9012583 and it is clear that bicarbonate salts are not considered as useful components in a composition containing a bile salt and a pharmaceutically active agent. Thus the effect of bicarbonate or other pH adjusting agents to effectively increase the therapeutic index of bile salts was certainly not described in this prior document. In the present invention it should be understood that the terms bile salt and bile acid are used interchangeably because if the salt or its conjugate acid is present it will depend on the pH of the surrounding environment. In this way, the solid formulations according to the invention can contain either a bile salt or a bile acid. However, compositions as administered frequently will have a pH such that if an acid is used, it will be converted to a salt form when it is in solution. When a bile salt is used in the compositions of the present invention, it is preferred that there be a soluble counter ion present such as sodium or potassium. It is also possible to use, for example, an ammonium ion but is preferred to a lesser degree. Bile salts are surfactants that occur naturally. They are a group of compounds with a common "central" structure based on colanic acid found in all mammals and higher plants. Bile salts can be mono, di, or trihydroxylated; they always contain a 3a-hydroxyl group while the other hydroxyl groups, most commonly found in C6, C7 or C? 2, can be placed either on top (ß) or below (a) of the plane of the molecule. Within the class of compounds described as bile salts include the amphiphilic polyhydric steels bearing carboxyl groups as part of the primary side chain. The most common examples of these in mammals are a result of the metabolism of cholesterol and are found in bile and, in a derivative form, through the intestine. In the context of this specification, the term may also apply to synthetic analogs of bile salts "that occur naturally that exhibit similar biological effects, or to microbially derived molecules such as fusidic acid and its derivatives. The bile salt can be unconjugated or conjugated. The term "unconjugated" refers to a bile salt in which the primary side chain has a single carboxyl group "which is in the terminal position and which is not substituted. Examples of unconjugated bile salts include cholate, ursodeoxycholate, chenodeoxycholate and deoxycholate. A conjugated bile salt is one in which the primary side chain has a carboxyl group that is substituted. Frequently the substituent will be an amino acid derivative which is linked via its nitrogen atom to the carboxyl group of the bile salt. Examples of bile salts include taurocholate, glucocholate, taurodeoxycholate and glucodeoxycholate. The amount of bile acid contained in a single dose of the formulation will vary depending on the bile acid chosen and the rate and degree to which that bile acid dissolves in the aqueous fluid contained in the intestine. For chenodeoxycholic acid, and most other bile acids, this is probably in the range of 10 milligrams to 1 gram, preferably from 20 milligrams to 200 milligrams, and more preferably 30 milligrams to 100 milligrams. For deoxycholic acid, the maximum will generally not exceed 500 milligrams, in view of this slightly greater activity. The intestine of many animals, particularly humans and other mammals) is regulated naturally a. a pH below neutrality. The compositions of the invention comprise an agent adapted to adjust the pH of the intestine to a pH of from 7.5 to 9. The agent is "adapted" to adjust the pH either by its chemical nature or by the amount in which it is present or , usually, both. The optimal pH at which the intestine adjusts is in the range of 7.8 to 8.3. Although simple agents adapted to adjust the pH of the intestine in the range specified above can be used successfully in the invention, it is preferred that the pH adjusting agent also has the ability to regulate the intestine at a pH within the established range. This can give a longer lasting effect, which can be desired in many circumstances. Also, a regulator has a greater capacity to accommodate the patient-to-patient variability of endogenous bowel pH, as well as the viability of the bowel pH seen over time in any individual patient; in particular, a regulator can act as a safety barrier to ensure "that the pH of the patient's intestine does not radically change beyond safe limits during the administration of the formulations of the invention. Two of the most favored pH adjusting agents suitable for use in the invention, either separately or in combination, are carbonate and deionic ions. baking soda. In the discussion "below it refers to bicarbonate as an illustration of the principles involved, but the same principles apply in the same way to other agents capable of exerting a similar effect on intestinal pH. It is difficult to directly determine the amount of agent necessary to adjust the pH within the contemplated range because the intestinal pH can vary between 5 and 7, and its aqueous content can vary, such as its intrinsic regulation capacity, which acts to Keep a low pH. Consequently, the considerations directed to determine the appropriate amount of agent that controls the pH need to take into account a scenario of the "worst of cases" . An indication can be obtained • observing the pH reached after adding a potential pH adjusting agent to a 50 mM solution of MES (morpholino ethanesulfonic acid) adjusted to pH 6.0. The concentrations useful in the invention are those that result in pH adjustment within the range of 7.5 to 9; the preferred concentrations result in an adjustment within the range 7.8 to 8.3. (For reasons to be explained below, the weight amount of the pH adjusting agent is generally that which produces the desired concentration in 10 milliliters of liquid.) The pH values achieved are shown below for the pertaining bicarbonate at different. concentrations, dispersed in distilled water or in MES. Concentration of bicarbonate (molarity) Water MES 0.002 8.86 6.42 0.004 8.67 6.58 0.008 8.63 6.86 0.016 8.57 7.14 0.031 8.55 7.40 0.062 8.49 7.68 0.125 8.41 7.93 0.250 8.30 8.14 0.500 8.16 8.05 1.000 8.02 7.96 Therefore, the minimum amount of bicarbonate useful in the invention in most cases is about 0.045 M, which gives a pH in the gut of about 7.5. This translates to approximately 40 milligrams of sodium bicarbonate, for a dispersion volume of 10 milliliters. A saturated solution of bicarbonate in water (< 2M) has a pH of 8.01. From this it will be seen that, regardless of the concentration of the preferred bicarbonate administered, the pH will not rise above 9.0. Under certain circumstances, it may be that a concentrated solution of a bile acid salt itself has sufficient capacity to raise the pH to the appropriate level, although the effect will not be as strong as for the preferred bicarbonate. Therefore, it is possible, although not always desirable, that the pH adjusting agent be the same bile salt, or a different bile salt. The greater the regulatory capacity of the composition, the pH specified above will last longer in the intestine and therefore the benefits of the invention will prevail for a longer time. Two of the most favored regulating agents suitable for use in the invention, either separately or in combination, are carbonate and bicarbonate ions. As already mentioned, the advantage of the compositions of the present invention is that they are able to effectively increase the therapeutic index of the bile salt present in the composition. The beneficial effect is present for both conjugated and unconjugated bile salts but the action of the carbonate or bicarbonate ions on conjugated bile salts is slightly different from the action on unconjugated bile salts. With conjugated bile salts, the presence of sufficient bicarbonate or carbonate has the effect of increasing the permeability of the cell membrane during exposure of the cells to a given amount of bile salt without affecting the viability of the cell. This means "that the amount of bile salt necessary to obtain a given increase in permeability is reduced and thus the permeability of the cells can be increased without increasing the adverse effects of the bile salt. With unconjugated bile salts in the presence of carbonate or bicarbonate, permeability is not increased during exposure of the cell to the bile salt but, instead, the toxic effect on the cells is reduced after exposure to the bile. biliary salt. Again, this means that the amount of bile salt that can be administered without affecting the viability of the cells is increased in the presence of bicarbonate or carbonate ions. In general, the amount of pH adjusting agent will be such that when a unit dose of the composition is dispersed in the amount of liquid that would be present in the length of the intestine over which the composition will be distributed upon administration to a patient, the concentration of the pH adjusting agent is at least about 0.01 M, although you can have a more accurate estimate referring to the MES regulation test referred to above. Usually, and preferably for the bicarbonate or carbonate, however, the bicarbonate or carbonate concentration will be greater than 0.05 M. and it is more preferred that the concentration be at least about 0.1 M, even though higher concentrations may be used, for example up to 1 M. A typical length of the small intestine on which a composition of. the present invention would be 30 centimeters and the amount of liquid that would be present in that length of intestine would probably be 10 milliliters. However, it should be noted that the choice of 30 centimeters as a convenient length is arbitrary and the invention is not intended to be limited to compositions that are distributed over this distance in the small intestine. There may be reasons why it would be desirable for a composition to be distributed over a shorter or much longer period of time, for example a sustained release composition may disperse much more slowly and therefore be distributed over a much longer length of time. small intestine. These compositions are familiar to those skilled in the art that could easily determine the most convenient type of composition to meet a particular therapeutic requirement. If the composition were to be adapted to disperse over a longer or shorter length of the small intestine then the amount of water in which a unit dose of the composition would be dispersed to give one. particular concentration in accordance with this would become greater or lesser. The unit dose that is dispersed in water to determine the minimum amount of bicarbonate required will be the dose that would be administered to a patient at any time. For liquid formulations, this will be a previously determined amount calculated by a physician or pharmacist. For solid formulations, such as tablets or capsules, the unit dose will generally be a single tablet or capsule. However, there may be circumstances in which a patient would require more than one tablet or capsule, for example if large doses of active substances are needed, and in this case the amount of bicarbonate can be divided between two or more tablets or capsules. It seems that an additional beneficial effect of the preferred carbonate or bicarbonate ions may arise because these can increase the solubility of the bile acids. In general bile salts begin to be converted to their conjugated acid at a pH of about 6.8 or less and the acid form is insoluble in aqueous solutions. Since the regulating agent has the effect of regulating the compositions of the invention at a pH of about 7.5 or more, the solubilized bile salt will be present instead of the insoluble bile acid. A solubilized bile salt will be able to act on the epithelial cells when it is in. solution, while "that this may not be possible in the solid acid form. The higher the concentration of the regulating agent, the faster a satisfactory pH will be reached, resulting in a faster dissolution of the acid or bile salt; this will result in a higher local concentration of bile salt in solution, leading to greater efficiency to increase the permeability of the bioactive materials. It is also possible "that the reason for the particularly beneficial effect of carbonate and bicarbonate ions is associated in some way with the fact that" bicarbonate receptors are expressed on the surfaces of intestinal cells. However, the nature of this link, if it really exists, is not clear in the present and, in any case, it should be emphasized that the correctness or otherwise of this theory does not limit the effectiveness of the present invention. The results obtained using bicarbonate are generally superior. As discussed above, it is thought that one of the functions of bicarbonate or carbonate ions is to ensure that the bile salt is in soluble form. However, the presence of calcium ions increases the pH at which salt becomes the prevalent form and below which insoluble bile acid begins to precipitate out of solution. This pH varies according to the particular bile salt but it is desirable to avoid precipitation of the bile acid. For this reason, it is often advantageous to include in the composition a calcium ion chelator such as a di- or tricarboxylic acid salt (for example a citrate salt), ethylenediaminetetraacetic acid (EDTA), bis- (0-aminoethyl ether) ) N, N, N ', N' -tetraacetic of ethylene glycol (EGTA) or phytate and other polyphosphorus compounds. When a citrate salt is used as the "calcium chelator" ion, it should preferably be in water soluble form. The most convenient salts are therefore sodium and potassium citrate although in some circumstances ammonium citrate can also be used. The term "biologically active material" includes, in particular, pharmaceutically active protein materials. The protein material can be a pure protein, or it can comprise protein, such that a glycoprotein comprises both protein residues and sugar residues.

The material may be useful in human or veterinary medicine, either as a form of treatment or prophylaxis of diseases or their symptoms, or it may be useful cosmetically or diagnostically. Examples of biological protein material that can be provided as orally or rectally administrable formulations in accordance with this invention include hormones from proteins or peptides or hormone releasing factors such as insulin, calcitonin and growth hormone, either from humans or animals or semi- or totally. synthetically prepared, or other bioactive peptides such as interferons including human alpha interferon and interleukins including interleukin 1, interleukin 2, interleukin 3, interleukin 4 and interleukin 5. It can also be. use analogues and active fragments of these and other proteins. It is particularly noteworthy "that the invention not only works, but works well, with insulin, since insulin is usually poorly soluble at the pH values contemplated by the present invention. It seems "that insulin is only able to disperse as a result of some unexpected interaction between insulin and the bile salt that stabilizes it in bicarbonate or another high pH solution. Oligonucleotides such as antisense oligonucleotides and their analogs "which may be useful for interfering with the replication of nucleic acids in virally infected or cancerous cells and for correcting other forms of improper proliferation of cells may also be biologically active materials. Polysaccharides such as heparin are also suitable for use in the present invention as well as combinations of one or more of protein, nucleic acid or polysaccharide. The molecular weight of the biologically active material should preferably not be greater than about 20,000 Da. This is because, even with the increasing permeability of the cells resulting from the use of the compositions of the present invention, it is not an easy matter to achieve effective bioavailability with active molecules of larger size than this one. In general, however, the smaller the size of the active molecule, the easier it will be to administer it and therefore it is preferred that the biologically active molecule have a molecular weight of less than about 10,000 Da and, more conveniently, less than about 5,000. Gives. The amount of naturally occurring biologically active material will depend on its intrinsic potency. All that is needed is that it be present in sufficient quantity to manifest its desired activity when it is swallowed. For pharmaceutical products, the amount administered will be under the guidance of the doctor or clinician. Other excipients that may be present include lactose, used as a filler, to ensure the homogeneity of the composition, and to aid in the handling of the preparation, Ac-di-sol ™ (sodium cross-carmellose), an inflatable agent that aids in disintegration of the tablet, and polyvinyl pyrrolidone, commonly used as a binding agent during granulation processes. The formulation will usually be in a solid or exceptionally liquid form. Solid forms are preferred because the active material can easily be digested in the stomach by an enteric coating, and, it is much easier to ensure that the active ingredient or ingredients and the bile salt reach the intact small intestine and at the same time weather; This is much more difficult to achieve with a liquid formulation. If the composition is formulated as a solid, which must, during a prolonged period of shelf life, be substantially dry, it may be in the form of a tablet, bolus, powder, granules or icrogranules and may also contain appropriate fillers or binders. which are well known to those skilled in the art. The powders, granules or microgranules can be encapsulated. As mentioned above, a bile acid or a bile salt can be used in a solid formulation. The liquids suffer from the disadvantages referred to above. Nevertheless, if for some reason it is particularly desired to formulate a composition of the invention as a liquid, probably an enteric coated capsule is the best way of administration of the content, which may be a syrup or elixir. The compositions can be formulated for rapid or sustained release or a combination of these two release forms can be used. The compositions can also be incorporated in immediate, delayed or pulsed release formulations. The amount of regulatory agent included in the composition will depend on the manner in which the composition. It is formulated and the time taken by it to disperse in the intestine and thus the amount of the regulatory agent included in a sustained release formulation will generally be much greater than the amount needed for a rapid release formulation. In general, the compositions of the invention can be prepared by simply mixing the ingredients using techniques well known to those skilled in the art to prepare pharmaceutical formulations. The invention also relates to a method of improving the bioavailability of an active material, the method comprising co-administering to a patient an active material, a bile salt and an agent adapted to adjust to the intestine at a pH of from 7.5 to 9. therefore, in a second aspect of the invention there is provided the use of a bile salt and an agent adapted to adjust the intestine to a pH of from 7.5 to 9 in the preparation of an agent to increase the bioavailability of a biologically active material for be co-administered with the agent. Preferred features are as detailed for the first aspect of the invention, mutatis utandis. The invention will now be described with reference to the following examples and drawings in which: Figure 1 shows a comparison of the viability of Caco-2 cells after exposure to bile salts in vi tro in balanced salt or in solution bicarbonate, the results are expressed as maximum tolerated concentrations of bile salt in milligrams / milliliters; Figure 2 shows a comparison of neutral red permeability of Caco-2 cells in vi tro during and after exposure to conjugated bile salts in HBSS or bicarbonate solution, the results being expressed as maximum tolerated concentrations of bile salts in milligrams / milliliters; Figure 3 is a comparison of neutral red permeability of Caco-2 cells in vi tro during and after exposure to unconjugated bile salts in either HBSS or bicarbonate solution, the results being expressed as maximum tolerated concentrations of bile salt in milligrams. / milliliters; Figure 4 is a graph showing the percentage release of dye after incubation of cells stained with chenodeoxycholate in different regulators; Figure 5 is a graph showing the percentage reduction of activity of the mitochondria after incubation with chenodeoxycholate in different regulators, - Figure 6 is reference to Example 3 and shows the effect of pH and bile salt concentration in the . absorption of salmon calcitonin (SCT) after intrayeyunal administration in pigs; and Figure 7 is a reference of Example 4 and shows the increase in the efficacy of the oral insulin formulation in solid dose when combined with bicarbonate.

Preparation of Solutions for Use in the Examples A neutral red broth solution was prepared at a concentration of 0.5 milligrams / milliliter in DMEM (Eagle Medium Modified from Dubelcco) (pH 4.5) which was then diluted to 1:10 with TCM (Tissue Culture Medium) or HBSS ( Balanced Salt of Hanks) before use. The Hanks Balanced Salt Solution (HBSS) was prepared containing the following ingredients: g / L mM CaCl2.2H20 0.19 1.26 KCl 0.40 5.37 KH2P04 0.06 0.44 MgCl2.6H20 0.10 0.49 MgSO4.7H20 0.10 0.41 NaCl 8.00 133.33 NaHCO3 0.35 4.17 Na2HP04 0.48 3.38 D-Glucose 1.00 5.56 Red Phenol 0.01 A 0.1 M solution of sodium bicarbonate was prepared in distilled water. Similar 0.1M solutions of sodium acetate, sodium borate and sodium carbonate in distilled water were also prepared. These are also called regulators in the examples. Test solutions were prepared containing different concentrations of a bile salt in HBSS or in one of the 0.1 M buffer solutions. The following bile salts were tested: Non-conjugated bile salts: sodium cholate, sodium ursodeoxycholate, sodium chenodeoxycholate, deoxycholate of sodium, - Conjugated bile salts: sodium taurocholate, sodium glucocholate, sodium taurodeoxycholate, sodium glucodeoxycholate. A solution of MTT broth was prepared at a concentration of 5 milligrams / milliliter in distilled water, which was then diluted 1:10 with TCM before use. The tests described in Examples 1 and 2 are well-known tests and conform to the British Standards (BS 5750). Caco-2 cells are well recognized by those skilled in the art as the best available model in the intestinal cell.

EXAMPLE 1 In vitro culture experiments for the determination of cytotoxicity and permeability of the cell membrane Preparation of the Cells Test plates were fixed by aliquoting 200 μl of suspension of Caco-2 cells containing 1 × 10 5 cells / milliliter in each well. the 96-well microtiter tray. The outer wells were filled with 200μl of saline alone, to counteract the effects of evaporation. The cells were incubated in Dulbecco's modified medium high glucose (DMEM) tissue culture medium (TCM) at 37 ° C, 5 percent C02 in air, for 8 days, with feeding when necessary. The cells were then used to perform an initial assault or a recovery study.

A. Assessment of Toxicity After Initial Assault The neutral red spot was actively absorbed by the viable cells. Cells with more permeable membranes lost dye in the bath medium and thus the permeability of the cell membrane can be directly assessed after exposure to the test material by quantifying the dye. remaining in the cell. In this procedure, therefore, the cells were first stained and then incubated with test materials and measurements were made to determine the degree to which incubation with test materials caused the dye to leave the cells during exposure. An exposure duration of 2 hours was used and the experiment was conducted according to the following protocol. TCM was removed from the cell monolayers in each well and replaced with 200μl of dye. Plates were incubated for 2 hours at 37 ° C, 5 percent of C02, and then it was verified even dyeing and normal morphology. The dye was removed from each well and replaced with 150μl of the appropriate regulator. The regulator was removed from the wells in column 2 and replaced with 150 milliliters of test solutions at the highest concentration. 150μl of test solutions was added to each well in column 3, mixed gently by repeated aspiration, and 150μl was transferred to the wells in column 4. This process was repeated on the tray until serial dilutions were obtained twice from the well material. test along each row. A row of each tray was assigned with HBSS as a control, and a row for a controller control if necessary. The cells were incubated for 2 hours at 37 ° C in 5 percent C02. The dye was then aspirated from the wells and the trays were incubated with shaking for 20 minutes in a desorbent solution (100μl / well) consisting of 1 percent glacial acetic acid in 50 percent ethanol. In order to quantify the amount of dye remaining in the cells, the absorbencies were measured on a plate reader at 550 nm, and the readings were compared with regulator controls to assess the dye spill from the cells. The test was then used as a measure of the permeability of the cell membrane at the time of exposure to bile salts *.

B. Recovery from Toxic Assault The neutral red dye is actively absorbed by viable cells and the non-viable cells do not stain. Cells with enhanced cell membrane permeability will have a reduced dye content. In this variant of the procedure, the cells are first incubated with test materials, then incubated with dye, and measurements are made to determine the degree to which incubation with the test materials has caused "the cells to be unable to absorb." retain neutral red after exposure. An exposure period of 2 hours is used together with a recovery period of 3 hours in optimum growth material during which the dye is absorbed. He followed the. following protocol: TCM was removed from each well and replaced with 150μl of the appropriate regulator. The regulator was removed from the wells in column 2 and replaced with 150 μl of the test solutions at the highest concentration. 150 μl of test solutions were added to each of the wells in column 3, mixed gently by repeated aspiration, and 150 μl was transferred to the wells in column 4. This process was repeated throughout the tray to obtain serial dilutions twice of test material along each row. One row of each tray was assigned with HBSS as control, and one row for regulator control if necessary. The cells were incubated for 2 hours at 37 ° C in 5 percent C02. The supernatant liquid was then removed from the cell monolayers in each well and replaced with 200 μl of dye. The plates were incubated for 3 hours at 37 ° C, 5 percent C02. The dye was then aspirated from the wells and plates were incubated with shaking for 20 minutes in desorbent solution (100 μl / well) consisting of 1 percent glacial acetic acid in 50 percent ethanol. Absorbances were measured in a plate reader at 550 nm, and the readings were compared with controls. regulator to assess the spill of neutral red outside the cells after the active absorption of the dye. The test was thus used as a measure of the permeability of the cell membrane during the recovery period after exposure to bile salts. A comparison of the effects of bicarbonate and HBSS on cells treated with bile salts is presented in Tables 1 and 2 and Figures 2 and 3. The effects of HBSS and bicarbonate, acetate and borate regulators are compared in Figures 4 and 5. It should be noted that in the HBSS the Proportions of the components have been adjusted to reach a pH of 7.0 to 7.2 when dissolved together in distilled water. In the small intestine, the pH will be lower than this. With 0.1M of bicarbonate, on the contrary, as can be seen from the table presented above, the pH will be close to pH 8.0, and it was confirmed to be at the end of each of the experiments described here.

EXAMPLE 2 In vitro culture experiments for the determination of cytotoxicity using MTT staining to see the activity of the mitochondria Preparation of the Cells Test plates were fixed by aliquoting. 200μl of suspension of Caco-2 cells - containing 1 x 10 5 cells / milliliter in each well of the 96-well microtiter tray. The outer wells were left empty, and filled with 200 μl of saline alone, to counteract the effects of evaporation. - Cells were incubated in medium Dulbecco's modified high glucose elas (DMEM) tissue culture medium (TCM) at 37 ° C, 5 percent C02 in air, for 8 days, with feeding when necessary.

Test for mitochondrial activity MTT is a tetrazolium salt "that is converted by dehydrogenases from mitochondria into viable cells to form an insoluble purple crystal. Dead cells do not perform this conversion. The crystal formation can be quantified colorimetrically and is used to assess the activity of the mitochondria and therefore the cell viability. The preparation of the MTT broth solution, test solutions and the regulatory solution described above. In this procedure, the cells are first incubated with test materials and then incubated with MTT and measurements are made to determine the degree to which the incubation with test materials has altered the mitochondrial activity of the cells. A two-hour exposure period is used along with a three-hour recovery period during which the dyeing is carried out. The TCM was removed from each well and replaced with 150 μl of the appropriate regulator. The regulator was removed from the wells in column 2 and replaced with 150 μl of the test solutions at the highest concentration. 150 μl of test solutions were added to each of the wells in column 3, mixed gently by repeated aspiration, and 150 μl was transferred to the wells in column 4. This process was repeated throughout the tray to obtain serial dilutions twice of test material along each row. One row of each tray was assigned with HBSS as control, and one row for controller control if necessary. The cells were incubated for 2 hours at 37 ° C in 5 percent C02.

The cells were incubated for 2 hours at 37 ° C in 5 percent C02. The supernatant was then removed from the cell monolayers in each well and replaced with 200 μl of MTT. The plates were incubated for 3 hours at 37 ° C, 5% C02 and the wells were examined to assess the level of spotting with the naked eye. The dye was then aspirated from the wells and the plates were incubated with shaking for 20 minutes in desorbent solution (100 μl / well) -consisting of 0.1 M HCl in anhydrous isopropanol. Absorbances were measured in a plate reader as the difference between 550 and 650 nm, and the readings were compared with regulator controls to assess the change in mitochondrial activity. A reduction in staining below that of the controls indicates a reduction in the activity of the mitochondria and is highly indicative of toxic action of materials in the incubation medium.

RESULTS The results of Examples 1 and 2 are presented in Tables 1 and 2, and in Figures 1 through 5, which illustrate the effects exerted by conjugated and unconjugated bile acids in the presence of high pH regulators, using bicarbonate as a particular example.

TABLE 1 Maximum concentration of unconjugated bile salt (ma / mL) tolerated by Caco-l cells in the presence of bicarbonate or HBSS TABLE 2 Maximal concentration of conjugated bile salt (mg / mL) tolerated by Caco-l cells in the presence of bicarbonate or HBSS Tables 1 and 2 show the results of the experiments described in examples 1 and 2. The first columns of the tables list the bile salts "which are present in the test medium and the same test means are presented in the second column . The test medium was HBSS or the 0.1 M solution of sodium bicarbonate. The results of the neutral red tests during and after exposure to bile salt give the maximum concentration of bile salt in mg / mL at which the. The permeability of the cell remains unaffected (compared to cells incubated in medium without bile salt). The results in the MTT column represent the maximum concentration of bile salt at which the viability of the cell remained unaffected after incubation. Table 1 gives the results for unconjugated bile salts and shows that, in each case, the figures in neutral red (after exposure) and in the MTT column are higher for bile salts administered with bicarbonate than for bile salts. administered with HBSS although there is no significant difference in the figures in the neutral red column (during exposure). This indicates that the permeability of the cells during incubation with the bile salt solution does not change much in the presence of bicarbonate ions, but "that much more bile salt is needed for a toxic effect to be apparent on the cells. Because it is possible to increase the amount of bile salt, the permeability of the cells after incubation can also be increased without affecting the viability of the cells. Table 2 gives the results for the conjugated bile salts and shows that, in each case, the figures in the neutral red column (during the exposure) increase when the bile salt is administered in the presence of the bicarbonate while the figures in the columns of neutral red (after exposure) and MTT are the same. This indicates that much less bile salt is needed to increase the permeability of the cells when they are incubated with bile salts in the presence of bicarbonate than when the bicarbonate is not present. The results also show «that the permeability and viability of the cells after incubation is not affected by the presence of the bile salt. Thus, in the presence of bicarbonate ions, it is possible to increase the permeability of the cells during incubation with conjugated bile salts without increasing the concentration of bile salt and thus without adversely affecting the viability of the cells. Figures 1 to 3 are graphical representations of the results presented in Tables 1 and 2. Figure 1 shows the effect of bicarbonate on the viability of the incubated cells when measuring bile salt solutions via the MTT viability test. For conjugated bile salts, it can be seen that the presence of bicarbonate does not affect the viability of the cells after incubation with the bile salt but for unconjugated bile salts, the concentration of bile salt with which the cells can be treated without affecting their viability increases considerably in the presence of bicarbonate. Figure 2 shows the results for the cell permeability measured by the neutral red test. both during and after incubation with conjugated biliary salt. It can be seen that during exposure, the amount of bile salt necessary to increase the permeability of the cells is considerably lower in the presence of 0. 1 M bicarbonate than in the presence of HBSS. However, after exposure, the permeability of the cells returns to normal. This means that for a given amount of bile salt, cell permeability can increase significantly during exposure. However, after exposure, the viability of the cells remains unchanged with reference to the control (cells incubated with bile salts and HBSS without bicarbonate). Figure 3 is similar to Figure 2 but in this case the results for unconjugated bile salts are presented. In this case it can be seen «that the bicarbonate does not have a clear cut effect on the permeability of the cells during exposure to the bile salt but that after exposure, the maximum concentration of bile salt that can be administered without causing a Increase in cell permeability is increased and thus cell permeability decreases significantly in the presence of bicarbonate. Figure 4 is a graph showing the percentage release of spot in the neutral red recovery test after exposure of stained cells to bile salt at different pH values, using chenodeoxycholate. as an example. This graph shows quite clearly that much less dye is released from the cells that have been incubated at a pH above 7.5, and this is an indication that the prospects for short and long term recovery of the cells are much better under conditions of high pH Similarly, Figure 5, which is a graph showing the percentage reduction of mitochondrial activity after incubation with chenodeoxycholate in different regulators, indicates that a marked reduction in toxicity is achieved under high pH conditions. The pH 7.0 medium was prepared using HBSS containing 3 percent (volume / volume) 1 M HEPES solution (hydroxyethylpropylidenetene sulfonic acid) adjusted to pH 7.0.

The pH 7.5 medium was prepared using HBSS containing 50 mM TRIZMA ™, and adjusted to pH 7.5. The pH 8.0 medium was prepared using HBSS «containing 50 M TRIZMA ™, and adjusted to pH 8.0.

The pH 8.5 medium was prepared using HBSS containing 50 mM Tricine, and adjusted to pH 8.5. The results also indicate that this phenomenon is observed with 0.1 M bicarbonate at pH 8.0, but not with HBSS only at pH 7.0. The confirmation that the bicarbonate concentrations used actually have the desired effect in the intestinal environment are obtained in the live experiments reported in the following examples.

EXAMPLE 3 In vivo efficacy study - Calcitonin from salmon Pigs (large white x large white / landrace, male, 65 kg) were used for the study and were surgically prepared ten days before starting by implantation, under anesthesia, of two catheters in the dorsal aorta via the middle saphenous arteries. A cannula of approximately 1.6-2 millimeters in internal diameter was implanted in the jejunum. They were given water at will, fasted during the night and fed twice a day in the morning and in the afternoon. On the days of treatment, the morning meal was retained until after the last blood sample was taken. Doses of salmon calcitonin (sCT) were administered by instillation via the cannula lodged in the jejunum, followed by rinses with warm sterile water. Blood samples of 8 milliliters were removed several times before and after dosing, and divided into two portions. A portion was allowed to clot at room temperature, and the serum was stirred and stored at -20 ° C until ready for use. The other portion was transferred to heparinized tubes to which 4000 kiu of aprotinin was added, and the plasma was decanted after centrifugation and stored at -20 ° C. Serum calcium levels were measured colorimetrically after complexing with the calcium-sensitive chelating agent arsenazo III. Nine animals were used in the study, and formulations were administered on three separate days, each separated by at least one day, in a random three-way cross-base, so that all pigs received all three treatments. Each of the treatments consisted of 625 iu of sCT dissolved in 2 milliliters of phosphate buffered saline at pH 7.5, either alone (-o-), or together with 25 milligrams of chenodeoxycholate (-? -), along with 50 milligrams of sodium chenodeoxylate (-B-), or with 50 milligrams of "chenodeoxycholate" where the pH was subsequently lowered to 6.5 with hydrochloric acid (-x-). The results are shown in Figure 6 and are expressed as variations in the concentration of calcium in the plasma over time due to calcitonin, from which we can see «that the effectiveness of bile salts to increase the absorption of calcitonin depends on the concentration of bile salt present, and the pH at which it is administered.

EXAMPLE 4 In Vivo Efficacy Study - Insulin Pigs prepared in an identical manner to that described in Example 3 were employed. The blood samples taken were analyzed for glucose using standard enzyme-based methodology. The animals were dosed with a solution of 200.iu insulin in 2 milliliters (at pH 5 to put the peptide in solution), or with insulin formulated as a solid dose, either with or without sodium bicarbonate. Each dose contained 300 milligrams of each formulation, rapidly suspended in 2 milliliters of phosphate buffered saline immediately before administration. The composition of the two formulations is listed below on a weight-for-weight basis. No bicarbonate (w / w) With bicarbonate (w / w) Insulin bovine 2.4 Insulin bovine 2.4 A Acciiddoo squueennooddeess-- Acid chenodes-oxycolic 16.7 oxycolic 16.7 Lactose 74.9 Lactose 66.6 Ac-di-sol 3.0 Ac-di-sol 3.0 Pyrrolidone Pyrollidone from ppoolliivviinniilloo 3 3..00 polyvinyl 3.0 sodium bicarbonate 8.3 PH < 7 pH > 7.5 The results are shown in Figure 7, and are expressed as variations in the concentration of plasma glucose over time due to insulin, from which the efficacy of binary acid chenodeoxycholic acid can be seen to increase the absorption of the peptide to through the intestine. It is markedly improved by the presence of sodium bicarbonate in the solid dose formulation.

Claims (20)

1. A pharmaceutical or veterinary composition comprising: (i) a biologically active protein material, oligonucleotide or analog thereof or polysaccharide; (ii) an acid or bile salt; and (iii) an agent adapted to adjust the pH of the intestine to a value of from 7.5 to 9.

2. A composition as claimed in claim 1, wherein the agent is adapted to adjust the pH of the intestine to a value of pH from 7.8 to 8.3.

3. A composition as claimed in claim 1 or 2, wherein a soluble counter ion is present.

4. A composition as claimed in claim 1, 2 or 3, wherein the acid or bile salt is unconjugated.

5. A composition as claimed in claim 4, wherein the acid or bile salt is chenodeoxycholic acid or ursodeoxycholic acid or a salt thereof.

6. A composition as claimed in claim 1, 2 or 3, wherein the acid or bile salt is conjugated.

7. A composition as claimed in any of claims 1 to 6, wherein the pH adjusting agent is adapted to regulate the pH of the intestine within the established range.

8. A composition as claimed in claim 7, wherein the regulating agent comprises carbonate and / or bicarbonate ions.

9. A composition as claimed in claim 8, which comprises an amount of carbonate. or bicarbonate such that when the composition is dispersed in the amount of liquid that would be present in the length of the intestine over which the composition is intended to be dispersed, the concentration of the bicarbonate or carbonate is at least 0.1 M.

10. A composition as claimed in claim 8 or claim 9, which contains sufficient bicarbonate or carbonate ions to give a concentration of at least 0.045 M when a unit dose is dispersed in 10 milliliters of water.

11. A composition as claimed in any one of claims 1 to 10, wherein the pH adjusting agent itself comprises an acid or bile salt.

12. A composition as claimed in any one of claims 1 to 11, which includes a calcium ion chelator such as a salt of a di- or tricarboxylic acid, for example a citrate salt, ethylenediaminetetraacetic acid (EDTA). or bis- (6-ether to inoethyl) -N, N, N ', N'-tetraacetic acid of ethylene glycol (EGTA) or polyphosphate as phytate.

13. A composition as claimed in any of the claims of la la 12, wherein the biologically active material is a protein such as insulin, calcitonin, a growth hormone, an interferon, an interleukin or an active fragment of any of these .

14. A composition as claimed in any one of claims 1 to 13, wherein the molecular weight of the biologically active material is less than about 20,000 Da.

15. A composition as claimed in claim 14, wherein the molecular weight of the biologically active material is less than about 10,000 Da.

16. A composition as claimed in any of claims 1 to 15 which is formulated as a dry solid.

17. A composition as claimed in claim 16, which is in the form of a tablet, bolus, powder, granule or microgranule.

18. The use of a bile salt and an adapted agent for adjusting the pH of the intestine to a pH value of from 7.5 to 9 in the preparation of an agent to increase the bioavailability of a biologically active material to be coadministered with the agent.

19. A method for improving the bioavailability of an active material, the method comprising co-administering to a patient an active material, a bile salt and an agent adapted to adjust the pH of the intestine to a pH value of from 7.5 to 9.

20. A method for improving the therapeutic index of a bile salt (with respect to its ability to act as a permeation enhancer for a bioactive material in the intestine), the method comprising co-administering to a patient an active material, a bile salt and a agent adapted to adjust the pH of the intestine to a pH of from 7.5 to 9.