PHARMACEUTICAL COMPOSITIONS COMPRISING A CALCITONIN, A GLYCYRRHIZINATE AS ABSORPTION ENHANCER AND BENZYL

Abstract: Pharmaceutical compositions comprising a calcitonin, an effective amount of an absorption enhancer which is a glycyrrhizinate, an effective amount of benzyl alcohol and a pharmaceutically acceptable carrier are useful in the treatment of conditions such as osteoporosis.

Description: PHARMACEUTICAL COMPOSITIONS COMPRISING A CALCITONIN. A GLYCYR ¬ RHIZINATE AS ABSORPTION ENHANCER AND BENZYL The present invention relates to novel pharmaceutical compositions containing calcitonins, and to a novel method of enhancing the absorption of a calcitonin across a mucosal membrane. The calcitonins are a class of pharmacologically active peptides, of both natural and synthetic origin, which contain approximately thirty two amino acids, and which have the ability to regulate serum calcium levels. Various calcitonins, including e.g. natural human, salmon and eel calcitonins and the synthetic eel calcitonin analogue elcatonin are now commercially available and commonly employed, e.g. in the treatment of Paget's disease, Sudeck's disease and osteoporosis. A considerable and well known problem with the administration of peptides is that they are susceptible to rapid acid and enzyme-induced degradation when administered orally. For this reason, parenteral administration has been, hitherto, the most widely used means of administration and, in the case of peptides of higher molecular weight, such as the calcitonins, has been the only significant effective means of administration. It is widely recognised that administration by injection can be both inconvenient and unpleasant for the patient, particularly when the administration has to be repeated at regular intervals for long periods, e.g. in the treatment of post-menopausal osteoporosis with calcitonins. Thus, there has been growing interest in the administration of peptides by more acceptable non-invasive alternative routes, for example in the form of sublingual tablets, suppositories, intrapul onary powders, intranasal drops, sprays, powders, gels, ointments and inserts. A significant problem with many peptides, particularly those of higher molecular weights, is that they are only poorly absorbed across biological membranes, e.g. mucosal membranes and thus the bioavailability of the peptide is often very low. Considerable research has therefore been carried out in order to find methods of improving the trans-epithelial absorption of peptides. One approach is to use an adjuvant o absorption enhancer and there are numerous published reports of compounds which are claimed to have peptide absorption- enhancing properties. Thus, for example, choline esters (EP 214898) , acyl carnitines (EP 215697) , aldoses and glucosamines (Japanese Pat. Appl. No. 61 126034), ascorbates and salicylates (EP 37943) , alpha-cyclodextrin (EP 0094157) , pyroglutamate esters (EP 173990), chelating agents (US 4,476,116) ethanol, benzyl alcohol and polyethylene glycol 400 (EP 371010) have been proposed as absorption enhancers. There are many published reports that surfactants can enhance the absorption of polypeptides, see for example EP 115627 (Armour), GB 2,127,689 (Sandoz) , US 4,548,922 (Carey ≤£ .al) and Hirai ≤ al.. , τ n . .T. Pharm. f 165-184, 1981. However, a recognised problem with surfactant absorption promoters is that they can cause irritation and histolesion at the site of administration. These problems become of great importance when the peptide is administered regularly over a prolonged period. The present applicants have previously found that glycyrrhizinic acid and its salts are excellent absorption promoters for calcitonins and do not give rise to the above- mentioned problems of local toxicity and irritation. Compositions comprising a calcitonin and a glycyrrhizinate are described in our EPA 327756, which includes both liquid and solid formulations. Liquid formulations conventionally contain a preservative and EPA 327756 refers to the use of alkyl p-hydroxybenzoates (parabens) such as methyl and propy p-hydroxybenzoate as suitable preservatives. However, it has subsequently been shown that the antibacterial and preservative actions of the parabens are reduced by the glycyrrhizinate component of the formulation. In addition it would be desirable to increase the absorption of calcitonins still further. We have now surprisingly found that the inclusion of benzyl alcohol in a composition comprising a calcitonin and a glycyrrhizinate not only gives rise to a preservative action which is not diminished by the glycyrrhizinate, but also enhances the absorption of the calcitonin in a synergistic manner. Thus the use of a glycyrrhizinate in combination with benzyl alcohol increases the transmucosal absorption of a calcitonin by more than the sum of the respective effects of benzyl alcohol and glycyrrhizinate alone. In a first aspect, therefore, the present invention provides pharmaceutical compositions comprising a calcitonin; an effective amount of an absorption enhancer which is a glycyrrhizinate; an effective amount of benzyl alcohol and a pharmaceutically acceptable carrier. The present invention also provides a method of enhancing th absorption of a calcitonin across a mucosal membrane, which method comprises co-administering with the calcitonin an effective amount of an absorption enhancer which is a glycyrrhizinate, and an effective amount of benzyl alcohol. Whilst preservatives are generally only used in liquid formulations, absorption enhancers are required in both liquid and solid formulations of calcitonins, and hence the present invention includes within its scope both solid and liquid compositions. The term glycyrrhizinate as used herein is intended to mean both glycyrrhizinic acid and its carboxylate salts. Particular glycyrrhizinate salts are ammonium glycyrrhizinat and the alkali metal salts e.g. sodium glycyrrhizinate and potassium glycyrrhizinate. A preferred salt is ammonium glycyrrhizinate. The term calcitonin as used herein is intended to refer to that class of pharmacologically active polypeptides includin not only naturally occurring calcitonins but also various derivatives and analogues thereof, e.g. in which one or more of the amino acid residues or sequences naturally present is omitted, replaced, reversed or otherwise derivatised or in which the N- or C-terminal is modified. The general term calcitonin, as used hereinafter, is intende to mean all such calcitonins whether naturally occurring or synthetic. Examples of naturally occurring calcitonins include: human calcitonin. Chemical Abstract Service Registry Number (CAS RN) = 21215-62-3, which has the structure: H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly- -Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His- -Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly- -Ala-Pro-NH2; rat calcitonin (CAS RN - 11118-25-5) which has the structur : / \ H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly- -Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His- -Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly- -Ala-Pro-NH 2 ; salmon calcitonin (CAS RN - 47931-85-1) which has the structure: H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly- -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly- -Thr-Pro-NH2/ eel calcitonin (CAS RN = 57014-02-5) which has the structure H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly- -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- -Thr-Pro-NH2; reduced chicken calcitonin I (CAS RN = 96157-98-1) which has the structure: H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly- -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- -Thr-Pro-NH2; chicken calcitonin II (CAS RN = 103468-65-1) which has the structure: H-gamma-Glu-Cys-Gly-OH H-gamma-Glu-Cys-Gly-OH H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly- -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- -Thr-Pro-NH2; ox calcitonin (CAS RN = 26112-29-8) which has the structure: H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser- -Ala-Tyr-Trp-Lys-Asp-Leu-Asn-Asn-Tyr-His- -Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu- pig calcitonin (CAS RN =- 12321-44-7) which has the structure H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser- -Ala-Tyr-Trp-Arg-Asn-Leu-Asn-Asn-Phe-His- -Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu- -Thr-Pro-NH2; and sheep calcitonin (CAS RN = .40988-57-6) which has the structure: H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser- -Ala-Tyr-Trp-Lys-Asp-Leu-Asn-Asn-Tyr-His- -Arg-Tyr-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu- -Thr-Pro-NH 2 • Examples of calcitonins wherein one or more amino acids have been omitted are the des-[Ser 2 , Tyr-^2]—Gly^-calcitonins described in US 4,597,900 and the des-[Tyr 22 ]-salmon calcitonin described in US 4,304,692. Examples of calcitonins wherein the naturally occurring sequence has been modified include the 1,7-dicarba- calcitonins such as eel 1,7-dicarbacalcitonin (elcatonin CAS RN = 60731-46-6) which has the structure: -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- -Thr-Pro-NH 2 ; salmon 1,7-dicarbacalcitonin (CAS RN 60864-37-1) which ha the structure: CO-Ser-Asn-Leu-Ser-Thr-NH-CH-CO-Val-Leu-Gly- -Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln- -Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly- -Thr-Pro-NH2; and human 1,7-dicarbacalcitonin (CAS RN = 66811-56-1) which has the structure: -Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His- -Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly- -Ala-Pro-NH2. In the context of the present invention, a particularly preferred calcitonin is elcatonin (CAS RN = 60731-46- 6) . Th preparation and properties of elcatonin and related 1,7- dicarbacalcitonins are described in British Patent Number 1,516,947 (Toyo Jozo) . Another preferred calcitonin is naturally occurring eel calcitonin (CAS RN = 57014-02-5) . The preparation and properties of eel calcitonin are described in US 3,988,309 (Matsuda ≤£. _al) . The compositions of the present invention suitably can be administered by methods known in the art for transmucosal an transdermal delivery of pharmacologically active substances. The compositions can be administered to, for example, the nasal, sublingual, buccal, rectal, vaginal and colonic mucos and to the skin. They can take the form of drops, aerosols, tablets, capsules, powders, gels, ointments, inserts, suppositories, pessaries, patches and membranes. The compositions can also take the form of enterically coated solid oral compositions as described in, for example, EP 127535 (Hadassah Medical Organisation) . The compositions for sublingual and buccal administration can also take the form of wafers as described in PCT/GB91/00651. Such wafers are formed substantially from starch, and suitably have a thickness of from 0.3 to 1.0 mm. Particular compositions are those intended for administratio to the nasal, buccal, sublingual, rectal and vaginal mucosa. When the composition is intended for delivery to the nasal mucosa, particular dosage forms are solutions, aerosols, drops, gels and powders. Particular dosage forms for buccal and sublingual administration are gels, suspensions, tablets, patches, powders, ointments, solutions, aerosols and wafers. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in a sealed container. The sealed container can take the form of a cartridge or refill for use with an atomising device, or it can take the form of a unitary dispensing device such as a single dose nasal inhaler (see French Patent Application FR 2578426) or an aerosol dispenser fitted with a metering valve and which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as a fluorochlorohydrocarbon. Such aerosol dispensers are well known in the art. The aerosol dosage forms can also take the form of a pump-atomiser and such forms are also well known in the art. The atomising or dispensing devices for dispensing aerosol sprays typically are designed to dispense particles of a size greater than 1 micrometres. In order to ensure that significant quantities of the composition remain in contact with the oral or nasal mucosa, and are not inhaled, the particles suitably are approximately 10-160 micrometres in size. When the composition is intended to be administered as a liquid spray, the viscosity of the liquid composition can b adjusted as necessary according to known methods to ensure that the composition is sprayable. When the composition is intended for application to the rectal and vaginal mucosa particular dosage forms include pessaries, suppositiories, solutions, foams, suspensions, gels, ointments, tablets and soft gelatin capsules. Compositions for rectal or vaginal administration are generally presented as a solid suppository or a semisolid or liquid formulation filled into a soft gelatin capsule. It will be appreciated therefore that the excipients for use in such suppository or capsule formulations will be selected an if necessary admixed to give a formulation of the desired consistency at room and body temperatures. Thus, the suppository base or carrier may for example comprise one or more components selected from an oil, a fat, a polyglycolyse glyceride and a polyethylene glycol. The oil and/or fat preferably comprises one or more triglycerides as the main component, such as coconut oil, fractionated coconut oil (e.g. Miglyol) palm kernel oil, palm fat, cocoa butter or lard. Examples of hard fat suppository bases include Witepsol and Suppocire. A saturated or unsaturated polyglycolysed glyceride may be for example a saturated polyglycolysed glyceride consisting of Cβ-iβ glycerides and polyethylene glycol esters such as are available under the trade name Gelucire e.g. Gelucire 35/10, 37/02 or 44/14; a saturated polyglycolysed CQ-CI Q glyceride such as that available under the trade name Labrasol; or an unsaturated polyglycolysed glyceride consisting of C16-C20 glycerides a polyethylene glycol esters such as those available under th trade name Labrafils e.g. Labrafil WL 2609 BS or M 2125 CS. For use in a capsule formulation the polyethylene glycol component is preferably liquid at room temperature such as polyethylene glycol-200, 300, 400 or 600, whereas for a soli suppository a polyethylene glycol of higher molecular weight is preferred. The relative proportions of the excipients will of course depend inter alia on the consistency of the formulation required. Compositions containing a polyglycolysed glyceride optionall with a polethylene glycol are preferred. Such compositions can also be adapted for oral administration e.g. in hard or soft gelatin capsules, which are preferably enterically coated. When the composition is enterically coated and is intended for oral administration, typically it can take the form of a tablet or capsule coated with a coating agent which ensures passage of the calcitonin through the stomach and its subsequent release preferably in the colon. Suitable coating agents include anionic polymers such as acrylic acid/methacrylic acid ester copolymers (e.g. Eudragit S) . The solvents or liquid carriers used in the present formulations are preferably aqueous but can also be chosen from the physiologically acceptable non-aqueous solvents. Examples of non-aqueous solvents or carriers are alcohols, particularly polyhydroxy alcohols such as propylene glycol and glycerol, and vegetable and mineral oils. Such non- aqueous solvents or carriers can be added in various concentrations to water to form solutions, oil-in-water emulsions and water-in-oil emulsions. The solvent preferably is water. In addition to a solvent or carrier, the liquid formulations of the present invention can contain excipients such as antioxidants, stabilisers, preservatives, agents for adjusting viscosity (e.g. Carbapol, Keltrol or cellulose derivatives), agents for adjusting tonicity (e.g. sodium chloride, glycine or mannitol) , and buffering agents. If desired a further preservative eg. parabens may be used in addition to benzyl alcohol, but in general this is not necessary. The compositions can also contain a protease inhibitor, preferably a non-surfactant protease inhibitor, for example as described in EP 127535. In general, the above-mentioned compositions can be made according to well known pharmaceutical procedures, see for example Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, 1985. Soft gelatin capsules may be prepared for example as described in WO 84/03417 or EPA 122463. Wafer formulations may be prepared for example as described in PCT/GB91/00651. Thus for example the active ingredient may be incorporated into the wafer mix prior to forming the wafer, or applied to the wafer in the form of a layer or a spray. The compositions of the present invention can be used in the treatment of diseases such as Paget's disease (osteitis deformans) , osteoporosis, including post- enopausal osteoporosis; Sudeck's disease and various hypercalcaemic conditions (see, for example, the Physician's Desk Reference 42nd Edition, 1988, pages 1796 and 1797) . The compositions will be administered to the patient in dosages which contain an amount of calcitonin effective to treat the disease in question. The quantity of pharmacologically active substance in a unit dose of the compositions of the present invention will vary according to the potency of the calcitonin and the nature of the composition. However, in general, a unit dose of a composition intended for human use typically contains betwee 1 and 400 International Units (I.ϋ.) of a calcitonin. For elcatonin, a unit dose preferably contains from 5 to 200 I.ϋ. A typical dosage regimen for elcatonin is from 5 to 200 I.ϋ. per day which may be administered in a single dose or in divided doses, for example on consecutive or on alternate days. The term "International Unit" refers to the appropriate International Reference Preparation (I.R.P.) of human, salmon or porcine calcitonins, or elcatonin, established by the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom. When the formulation is a liquid formulation, particularly a spray, the volume of a unit dose typically is in the range 50 to 130 mcl. The pH of the compositions of the present invention can vary within a broad range according to the chemicophysical properties of the different ingredients in the compositions. However, suitably the pH of the composition is in the range from pH 3 to 8, preferably from approximately pH 4 to approximately pH 7. In order to regulate the pH and maintain a suitable value, a buffering agent may be included in the composition. Examples of buffering agents which may be used include citrates, for example a mixture of citric acid and sodiu citrate, acetates and phosphates. In addition to a buffering agent such as those described hereinabove, an alkali metal hydroxide e.g. sodium hydroxide may be incorporated to regulate the pH. The concentration of the benzyl alcohol is between 0.1 and 5.0% (w/w) of the total weight, of the composition. In a liquid or gel composition the benzyl alcohol is suitably present in an amount corresponding to between 0.5 g and 4 g per 100 ml of composition. Preferably the benzyl alcohol is present in an amount corresponding to approximately 2 g/100 ml. In suppositories, tablets and soft gelatin capsules for rectal or vaginal administration the benzyl alcohol is suitably present in an amount corresponding to between 0.1 and 1 g per 100 g of composition. Preferably the benzyl alcohol is present in an amount corresponding to between 0. g and 0.5 g per 100 g. The concentration of the glycyrrhizinate absorption enhance typically is at least 0.1% (w/w), suitably 0.1 to 10% (w/w), and preferably 0.2 to 5% (w/w) of the total weight of the composition. Where the composition is a liquid or gel composition, the glycyrrhizinate suitably is present in an amount corresponding to between 0.5g and 5g per 100 ml of composition. Preferably the glycyrrhizinate is present in a amount corresponding to approximately 2g/100 ml. In suppositories, tablets and soft gelatin capsules for rectal or vaginal administration the glycyrrhizinate is suitably present in an amount corresponding to between 0.1 g and 2 g per 100 g of composition. Preferably the glycyrrhizinate is present in an amount corresponding to between 0.2 g and 1 g per 100 g. For aqueous compositions, the final pharmaceutical form, i.e liquid solution or gel, can also depend upon the pH, the ionic strength of the solution and the concentration of glycyrrhizinate. In general, compositions having a pH of about 5.5 and above will exist as liquids whilst composition having a lower pH value will tend to be more viscous and, at around pH 4.5, will exist in a gel form. The invention will now be illustrated in greater detail by the following examples. Formulations for nasal, sublingual, buccal, rectal or vaginal administration Examples 1-3 Table 1 Elcatonin (meg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate Sodium chloride (mg) Benzyl alcohol (g) Distilled water IN NaOH The formulations of Examples 1 to 3 were prepared by mixing together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and elcatonin were then added. Examples 4-10 The following compositions were prepared according to the method described in Examples 1 to 3. Table 2 Example No. 6 7 8 9 10 Elcatonin (meg) 3690 3690 3690 7380 7380 7380 7380 (6500 I.U./mg potency) Ammonium 0.5 2 5 0.5 1 2 5 glycyrrhizinate ( g ) Citric acid (mg) 37 37 37 37 37 37 37 Sodium citrate 463 463 463 463 463 463 463 dihydrate (mg) Sodium chloride 600 600 600 600 600 600 600 (mg) Benzyl alcohol (g) 2 2 2 2 2 2 2 Distilled water q.s. to 100 ml IN NaOH q.s. to pH 6 Examples 11-15 The following compositions were prepared according to the method described in Examples 1 to 3. Table 3 Elcatonin (meg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Sodium chloride (mg) Benzyl alcohol (g) Distilled water IN NaOH Examples 16-19 Table 4 Elcatonin (meg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Sodium chloride (mg) Benzyl alcohol (g) Methyl p-hydroxybenzoate (mg) Propyl p-hydroxybenzoate (mg) Distilled water IN NaOH The formulations of Examples 16 to 19 were prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, distilled water and sodium hydroxide in a water bath regulated at a temperature of abou 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and elcatonin were then added. Examples 20-21 The following compositions were prepared according to the method described in Examples 1 to 3 Table 5 Elcatonin (meg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Sodium chloride (mg) Benzyl alcohol (g) Distilled water q.s. to 100 ml 0.1N NaOH q.S. to pH 4.5 The formulations of Examples 20 and 21 are gels Example 22 Elcatonin (meg) 7380 (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) 2 Acetic acid (mg) 200 Sodium acetate trihydrate (mg) 200 Sodium chloride (mg) 600 Benzyl alcohol (g) 2 Distilled water q.s. to ml 100 IN NaOH q.S. to pH 5.3 The formulation of Example 22 was prepared by mixing togethe the ammonium glycyrrhizinate, acetic acid, sodium acetate trihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of abou 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and elcatonin were then added. TRIAL A The preparation reported in Example 3 containing ammonium glycyrrhizinate 2% and benzyl alcohol 2% as absorption enhancers, was compared, in a test of pharmacodynamic activity in rats i.e. lowering of calcium concentration in the serum, with the following preparations: a formulation with the same composition as Example 3 with th exception of benzyl alcohol (reference preparation A) ; a formulation with the same composition as Example 3 with the exception of ammonium glycyrrhizinate (reference preparation B) ; a formulation with the same composition as Example 3 wit the exception of both benzyl alcohol and ammonium glycyrrhizinate (reference preparation C) . The preparations were administered intranasally with a small catheter, in the volume of 100 mcl/kg body weight (corresponding to 2 I.U./kg), to groups of 10 Sprague Dawley rats weighing 160±20 g. The animals, fasted overnight, were anaesthetized with tribromoethanol (TBE) 2% (9 ml/kg body weight, given i.p.) 15 min before receiving elcatonin. Serum calcium concentration was measured (with an atomic absorption spectrophotometer VARIAN 30/40) on blood samples obtained in each animal, from the caudal vein, 0, 30, 60, 120 and 180 min after administration of the products. The results, expressed as residual percentage of serum calcium concentration as compared with baseline values (0 time), are reported in Table 6. In order to evaluate the relative efficacy of the preparations, the AUC (0-180 min) values were calculated; being AUC calculated on the residual serum calcium, a lower AUC is indicative of a greater pharmacodynamic effect. The AUC (0-180 min) values and the differences (Δ AUC) between the test preparations and reference preparations A and B in comparison with that of the reference preparation C, which does not contain either ammonium glycyrrhizinate or benzyl alcohol, are reported in Table 7. The results obtained show a clear effect of synergism due to the combination of ammonium glycyrrhizinate and benzyl alcohol. Table 6 Residual percentage of serum calcium as compared with baseline values Table 7 AUC (0-180 min) calculate on the residual serum calcium Preparation of this invention reported in Example 3 Reference preparation A Reference preparation B Reference preparation C TRIAL B The preparation reported in Example 2 containing ammonium glycyrrhizinate 2% and benzyl alcohol 1% as absorption enhancers, was compared, in a test of pharmacodynamic activity in rats i.e. lowering of calcium concentration in the serum, with the following preparations: a formulation with the same composition as Example 2 with t exception of benzyl alcohol (reference preparation A) ; a formulation with the same composition as Example 2 with the exception of ammonium glycyrrhizinate (reference preparation B) ; a formulation with the same composition as Example 2 wit the exception of both benzyl alcohol and ammonium glycyrrhizinate (reference preparation C) . The testing methodologies were the same described for Trial A. The results of residual percentage of serum calcium are reported in Table 8. The AUC and ΔAUC values are reported in Table 9. The results obtained show a clear effect of synergism due to the combination of ammonium glycyrrhizinate and benzyl alcohol. Table 8 Residual percentage of serum calcium as compared with baseline values Table 9 AUC (0-180 min) calculated on the residual serum calcium AUC ΔAUC Preparation of this 14,631 1,959 invention reported in Example 2 Reference preparation A Reference preparation B Reference preparation C Examples 23 and 24 Table 10 Salmon calcitonin (meg) (5500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Benzyl alcohol (g) Sodium chloride (mg) Distilled water IN NaOH q.s. to pH 6 The formulations of Examples 23 and 24 are prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and salmon calcitonin are then added. Examples 25 and 26 Table 11 Example No. 25 26 Eel calcitonin (meg) (5000 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Benzyl alcohol (g) Sodium chloride (mg) Distilled water IN NaOH The formulations of Examples 25 and 26 are prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and eel calcitonin are then added. Examples 27 and 28 Table 12 Chicken calcitonin II (meg) (5000 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Benzyl alcohol (g) Sodium chloride (mg) Distilled water IN NaOH The formulations of Examples 27 and 28 are prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and chicken calcitonin II are then added. Examples 29 and 30 Table 13 Human calcitonin (mg) (200 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Benzyl alcohol (g) Sodium chloride (mg) Distilled water IN NaOH The formulations of Examples 29 and 30 are prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and human calcitonin are then added. Examples 31 and 32 Table 14 Pig calcitonin (mg) (60 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Benzyl alcohol (g) Sodium chloride (mg) Distilled water IN NaOH The formulations of Examples 31 and 32 are prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and pig calcitonin are then added. Powder for nasal administration Examples 33 and 34 Table 15 Elcatonin (mg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Lactose q.s. to (g) The formulations of Examples 33 and 34 are prepared by wetting the lactose with, benzyl alcohol and with an aqueous solution of elcatonin and drying under vacuum. The dried powder is mixed with ammonium glycyrrhizinate and the final mixture is placed into hard gelatine capsules (25 mg each capsule) . The powder is administered, after having pierced the capsules, using a nasal insufflator. Sublingual tablets Examples 35 and 36 Table 16 Example No. 35 36 Elcatonin (mg) 7.7 15.4 (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Sucrose (g) Mannitol (g) Polyethylene glycol 6000 (g) Lactose q.s. to (g) The formulations of Examples 35 and 36 are prepared by mixing together the sucrose, the mannitol and the lactose. The resulting mixture is wetted with benzyl alcohol and with an aqueous solution of elcatonin, granulated through a stainless steel screen and dried under vacuum. The dried granules are mixed with polyethylene glycol and ammonium glycyrrhizinate and then compressed into tablets of 120 mg each. Buccal tablets Examples 37 and 38 Table 17 Elcatonin (mg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Sucrose (g) Mannitol (g) Polyethylene glycol 6000 (g) Carbopol 934 (g) Lactose q.s. to (g) The formulations of Examples 37 and 38 are prepared by mixing together the sucrose, the mannitol and the lactose. The resulting mixture is wetted with benzyl alcohol and with an aqueous solution of elcatonin, granulated through a stainless steel screen and dried under vacuum. The dried granules are mixed with ammonium glycyrrhizinate, Carbopol and polyethylene glycol and then compressed into tablets of 150 mg each. Oral tablets for colonic delivery Examples 39 and 40 Table 18 Elcatonin (mg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Pregelatinized starch (g) Magnesium stearate (g) Lactose q.s. to (g) Eudragit S (g) Polyethylene glycol 6000 (g) The formulations of Examples 39 and 40 are prepared by mixin together the pregelatinized starch and the lactose. The resulting mixture is wetted with benzyl alcohol and with an aqueous solution of elcatonin, granulated through a stainles steel screen and dried under vacuum. The dried granules ar mixed with ammonium glycyrrhizinate and magnesium stearate and then compressed into tablets of 210 mg each. The tablets are coated with an aqueous suspension of polyethylene glycol and Eudragit, to a final weight of 232 mg/tablet. Dosage form for vaginal administration Examples 41 and 42 Table 19 Elcatonin (mg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Corn starch (g) Adipic acid (g) Sodium bicarbonate (g) Magnesium stearate (g) Lactose q.s. to (g) The formulations of Examples 41 and 42 are prepared by mixin together the ammonium glycyrrhizinate, the corn starch, the adipic acid and the lactose. The resulting mixture is wetted with benzyl alcohol and with an aqueous solution of elcatonin, granulated through a stainless steel screen and dried under vacuum. The dried granules are mixed with sodium bicarbonate and magnesium stearate and then compresse into tablets of 1.6 g each. Dosage form for rectal administration Example 43 Elcatonin (mg) (6500 I.U./mg 15.4 potency) Ammonium glycyrrhizinate (g) 6.0 Benzyl alcohol (g) 4.0 Distilled water (g) 100.0 Hard fat q.s. to (g) 1500.0 The formulation of Example 43 is prepared by mixing together the ammonium glycyrrihizinate, benzyl alcohol and distilled water in a water bath regulated at a temperature of about 70°C. The solution is cooled to about 40°C, elcatonin is dissolved and then the resulting solution is incorporated into hard fat melted at about 40°C. The final mixture is poured into suppository moulds and cooled to room temperature, thus obtaining suppositories of 1.5 g each. Formulations for vaginal or rectal administration Examples 44-46 Table 20 Elcatonin (mg) (6500 I.U./mg potency) Polyethylene glycol 600 (g) Gelucire 44/14 (g) Distilled water (g) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Sodium chloride (mg) Sodium citrate dihydrate (mg) Sodium hydroxide (mg) Citric acid (mg) The formulations of Examples 44 to 46 were prepared by mixin together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, sodium hydroxide, distilled water and elcatonin in a water bath regulated at a temperature of about 70°C. The resulting solution, cooled to about 55°C, was incorpor¬ ated into a mixture of Gelucire, polyethylene glycol and benzyl alcohol heated to about 55°C. The final mixture, cooled to about 30°C, was filled into soft gelatin capsules (1 g each capsule) . Examples 47-49 Table 21 Example No. 47 48 49 Elcatonin (mg) (6500 I.U./mg potency) Polyethylene glycol 600 (g) Witepsol S55 (g) Miglyol 812 (g) Distilled water (g) Ammonium glycyrrhizinate (g) Benzyl alcohol (g) Sodium chloride (mg) Sodium citrate dihydrate (mg) Sodium hydroxide (mg) Citric acid (mg) The formulations of Examples 47 to 49 were prepared by mixing together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, sodium hydroxide, distilled water and elcatonin in a water bath regulated at a temperature of about 70°C. The resulting solution, cooled to about 55°C, was incorporated into a mixture of Witepsol, Miglyol, polyethylene glycol and benzyl alcohol heated to about 55°C. The final mixture, cooled to about 30°C, was filled into soft gelatin capsules (1 g each capsule) . Dosage form for transdermal administration Example 50 Elcatonin (mg) 6 (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) 2 Benzyl alcohol (g) 2 Carbopol 934 (g) 2 Citric acid (mg) 37 Sodium citrate dihydrate (mg) 463 Sodium chloride (mg) 600 Distilled water q.s.to 100 g IN NaOH q.S. to pH 6 The formulation of Example 50 is prepared by mixing together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, sodium chloride, Carbopol 934, sodium hydroxide and part of distilled water in a water bath regulated at a temperature of about 70°C. To the resulting gel, cooled to room temperature, a solution of elcatonin and benzyl alcohol in the remaining part of distilled water, is then added. The final gel is filled into patches of 500 mg each. Formulations for nasal, sublingual, buccal, rectal or vaginal administration Examples 51-52 Table 22 Example No. 51 52 Elcatonin (meg) (6500 I.U./mg potency) Ammonium glycyrrhizinate (g) Citric acid (mg) Sodium citrate dihydrate (mg) Mannitol (g) Benzyl alcohol (g) Distilled water IN NaOH The formulations of Examples 51 and 52 were prepared by mixing together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, mannitol, distilled water and sodium hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and elcatonin were then added. Examples 53-54 Table 23 The formulations of Examples 53 and 54 were prepared by mixing together the ammonium glycyrrhizinate, citric acid, sodium citrate dihydrate, glycine, distilled water and sodiu hydroxide in a water bath regulated at a temperature of about 70°C. To the resulting solution cooled to room temperature, benzyl alcohol and elcatonin were then added. Examples 55 - 60 Table 23 Example No. 55 56 57 58 59 60 The compositions are prepared in an analogous manner to Example 1.

Claims: CLAIMS : 1. A pharmaceutical composition comprising a calcitonin, an effective amount of an absorption enhancer which is a glycyrrhizinate, an effective amount of benzyl alcohol and a pharmaceutically acceptable carrier. 2. A composition according to claim 1 wherein the glycyrrhizinate is ammonium glycyrrhizinate. 3. A composition according to either of claims 1 or 2 wherein the glycyrrhizinate is present in a concentration corresponding to at least 0.1% (w/w) of the total weight of the composition. . A composition according to any of claims 1 to 3 wherein the concentration of benzyl alcohol is between 0.1 and 5.0% (w/w) . 5. A composition according to any one of claims 1 to 4 wherein the calcitonin is elcatonin. 6. A composition according to any one of claims 1 to 5 which additionally contains a polyglycolysed glyceride. 7. A composition according to any one of claims 1 to 6 in the form of a liquid, gel or semisolid suitable for application to the nasal, buccal, sublingual, rectal or vaginal mucosa. 8. A composition according to any one of claims 1 to 7 wherein the glycyrrhizinate is present in an amount corresponding to between 0.5 g and 5 g per 100 ml of composition and the benzyl alcohol is present in an amount corresponding to between 0.5 g and 4 g per 100 ml of composition. 9. A liquid pharmaceutical composition comprising, as a carrier, an aqueous solution buffered to approximately pH 6; a non-toxic effective amount of a preservative; and, per 100ml of composition, 20,000-200,000 International Units of elcatonin, approximately 2g of ammonium glycyrrhizinate and approximately 2g of benzyl alcohol. 10. A composition according to any one of claims 1 t 9 which is packaged for administration as a spray. 11. A composition according to any one of claims 1 to 6 adapted for rectal or vaginal administration. 12. A composition according to claim 11 wherein the glycyrrhizinate is present in an amount corresponding to between 0.1 g and 2 g per 100 g of composition and the benzyl alcohol is present in an amount corresponding to between 0.1 g and 1 g per 100 g of composition. 13. A composition according to any one of claims 1 to 12 which has a pH in the range from approximately 4 to approximately 7. 14. A pharmaceutical composition containing a calcitonin, benzyl alcohol and glycyrrhizinate substantially as described herein with reference to Examples 1 to 60. 15. A process for preparing a pharmaceutical composition containing a calcitonin, benzyl alcohol and glycyrrhizinate substantially as described herein with reference to Examples 1 to 60.

Inventors: CHIODINI LAURA, FONIO TEODORO, PINZA MARIO, CHIODINI, LAURA, FONIO, TEODORO, PINZA, MARIO
Applicants: SMITHKLINE BEECHAM FARMA, SMITHKLINE BEECHAM FARMACEUTICI S.P.A.
Application year: 1992
Main classification: A61K47
IPC codes: A61K9, A61K38, A61K47