The present invention relates to novel amino glycoside salts with sucrose-octa-O-sulfonic acid.

These novel salts may be used for the treatment of ul- cerations in the stomach and/or the duodenum, preferably caused by Helicobacter pylori.

Helicobacter pylori (previously known as Campylobacter pylori) is a helical Gram-negative organism which is present in the stomach mucosa. Many recent tests have shown a clear correlation between the presence of H. pylori in the stomach mucosa and histologically shown gastritis. On the basis of this, much seems to indicate that this organism is wholly or partially responsible for the development of gastritis with ensuing ulcerations (Scand. J. Gastroenterol. 1988 (23) suppl. 142, pp. 93-100).

H. pylori is sensitive to a number of known antimicrobial substances in vitro. Furthermore, several publications disclose that the treatment of gastritis with antimicrobi¬ al agents, such as β-lactames (e.g. amoxicillin) or bismuth salts can result in the eradication of H. pylori in vivo (Antimicrobial Agents and Chemother. 1993, pp. 1184-86).

The traditional treatment of ulcerations in the human stomach or the duodenum consists in administering acid neutralising agents or preparations of the anti-histamin H2-inhibitor type which reduces the production of acid, (e.g. ranitidine, ci etidine, etc.) and acid pump-in¬ hibitors, such as omeprazole. This treatment as such is efficient, but it has a short-term effect only as, in almost every case, there is a relapse due to the very cause of the ulcer ailment, H. pylori infection, still being present.

Today the optimum treatment of ulcerations caused by H.

pylori comprise administration of bismuth subcitrate, amoxicillin and metronidazole. This treatment cures 60-90% of the patients (Ann. Rev. Med. 1992 (43) p. 142) .

However, there are certain side effects associated with this therapy:

Bismuth subcitrate may cause constipation and in large doses it may be neurotoxic.

Amoxicillin and metronidazole are systemically acting antibiotics which may cause development of allergy or resistance and influence the icroflora in the colon.

Therefore, there is much need for a preparation for local treatment of H. pylori infections in the stomach and the duodenu .

Amino glycosides form a group of antibiotics which have a good in vitro effect on H. pylori. The following MIC- values are known from the literature:

TABLE 1

Average MIC 90 range AMIKACIN 0.5

GENTAMYCIN 0.04 - 1

KANAMYCIN 0.04 - 2

STREPTOMYCIN 0.04 - 1.28

TOBRAMYCIN 0.04 - 0.64

(Antimicrobial Agents and Chemother. , 1986, pp. 510-511, J. Antimicrobial Chemother. 1986, 17, pp. 309-314, Scand. J. Gastroenterol. , 1988, 23 (suppl. 142), pp. 93-100).

So far amino glycosides .have not been found to be suitable for use in the treatment of H. pylori infections. The reason is that they are not absorbed in case of peroral administration in therapeutic doses. Amino glycosides may

be administered parenterally but this is impractical in the present case as these substances have known oto- and nefro-toxic properties. When known readily soluble salts of amino glycosides are administered orally, they do not influence the stomach and duodenum mucosae due to their poor tissue penetration and thus they will not be capable of eradicating H. pylori. On the contrary, they will have a considerable influence on the colon microflora and may cause diarrhoea.

β-D-fructofuranosyl-α-D-glucopyranoside octakis (hydrogen sulfate) (in the following referred to as sucrose-octa-O- sulfonic acid) is produced by sulphating sucrose with sulphur trioxide in pyridine. In this process eight hydroxy groups in sucrose are esterified with eight molecules of sulphuric acid under formation of semiesters. Sucrose-oc- ta-O-sulfonic acid forms well crystallized salts with Na, K, Cs, Rb and ammonium. (Chem. Pharm. Bull., 1980, 28 (2), pp. 638-341) .

None of these salts have been found suitable for practical use. The only salt of the sucrose-octa-O-sulfonic acid which is used for medical therapy is the aluminium salt produced by treatment of sucrose-octa-O-sulfonic acid with aluminium hydroxide (US 3.432.489 to Chugai, 1969) and it is known under the name of sucralfate having the gross formula C^H^Al^O-^S _g . (Merck Index 11th Edition, pp. 1400-1401) .

Sucralfate is widely used for the treatment of gastric ulcers. Peroral administration of tablets or a suspension causes sucralfate to react with the acidic gastric juice under formation of a sticky gel which adheres to the mucosa and forms a protective layer, especially on the ulcerated areas.

The effect of sucralfate is largely ascribed to the high content of aluminium hydroxide ions which act as acid

neutralizers and absorb pepsin and bile salts. (Clin. Gastroenterol. 1981, 3 (suppl. 2), pp. 117-127).

No other salts of the sucrose-octa-O-sulfonic acid has hitherto been found suitable for use in the treatment of ulcer ailments.

EP 403.048 to Warner-Lambert Co. discloses mixtures of sucralfate with anti-ulcer medicaments which per se either have low solubility or, where readily soluble, are made available in a controlled-release form by means of suitable adjuncts. Among said anti-ulcer agents are antimicrobial substances, such as bismuth salts and antibiotics which may be genta-, strepto-, kana- and neomycins. Mixtures of sucralfate with these amino glycosides, however, are not mentioned in any of the examples. Only mechanically produced mixtures of sucralfate and medicaments are disclosed. No other sucrose-octa-O-sulfonic acid composi¬ tions are disclosed and a chemical interaction between the sucralfate, which as a basic aluminium salt is not capable of further salt formation, and the basic medicaments described will not be possible. Presently, there is no known method of rendering the readily soluble amino glycosides available in controlled-release form in the stomach or the duodenum. Thus, it is not possible to obtain a mechanical mixture of sucralfate and aminoglycosides having the properties necessary to fulfill the object of the invention disclosed in EP 403.048.

WO 92/18143 to Smith-Kline Beecham PLC also discloses a physical mixture of sucralfate and antibiotics of the bacteriocins-type, nisin, gramicidin and tyrothricin are particularly preferred. Nor does this publication disclose or render probable a chemical interaction between sucrose- octa-O-sulfonic acid and the basic antibiotic.

It is an object of the invention to provide novel salts of amino glycosides which are well suitable for local treat-

ent of gastritis and/or ulcerations caused by H. pylori in the stomach and/or the duodenum of mammals including human beings.

It has surprisingly been found that sucrose-octa-O-sulfonic acid and amino glycosides form salts in stoichiometric ratios and that said salts have valuable pharmaceutical properties.

Amino glycosides belong to the group of sugars having a certain number of amino groups, preferably 4, 5 or 6. They are obtained by fermentation of various Streptomyces- or Micromonospora-species. Due to the many hydrophilic hydroxy or amino groups in the amino glycosides said substances and the salts thereof are readily soluble in water. The known salts, except from the aluminium salt, of sucrose-octa-O- sulfonic acid are also readily soluble.

As the amino glycosides have 4, 5 or 6 cation-forming amino groups and sucrose-octa-O-sulfonic acid have 8 anion-for- ming sulfate groups, the skilled person will not readily expect that the amino glycosides will form well defined salts with sucrose-octa-O-sulfonic acid. It should namely be taken into consideration that both amino glycosides and sucrose-octa-O-sulfonic acid have complex spatial construc¬ tions which means that the establishment of so strong ion bondings between e.g. the 4 amino groups in kanamycin A and the 8 sulfate groups in sucrose-octa-O-sulfonic acid that the resulting substance has low solubility in water is not immediately predictable.

It has now been found that most known amino glycosides having an antimicrobial activity with a content of 4 or 5 or 6 amino groups and sucrose-octa-Osulfonic acid form salts with the following general formula I:

([sucrose-octa-O-sulfonic acid^-CR-fNH _j *]..] -M _j "*) (I) wherein (x • y) + (z • n) = 8 x • y e N| [4<x-y<8 ] , N is the set of natural numbers, x e Z|[l<x<6], Z is the set of integers, z e N|[0<z<4], n e Z|[l<n<3],

R is the oligosaccharide sugar moiety, preferably a mono-, di- or trisaccharide, of an amino glycoside, and M is an element capable of forming a pharmaceutically acceptable cation, preferably an element selected from the group consisting of alkaline metals, alkaline earth metals and aluminium, more preferably Na, K, Mg, Ca, Al, or M ^ designates NH ₄ ⁺ .

It has also been found that it is possible to form well defined compounds between sucrose-octa-O-sulfonic acid and an amino glycoside and then to produce salts by introducing cations. The following pharmaceutically acceptable cations are preferred:

- Alkaline metal ions, such as Na ⁺ and K ⁺ , or NH ₄ ⁺ ,

- Alkaline earth metal ions, such as Ca ²⁺ and Mg ²⁺ . Another possibility is to use aluminium as an additional cation, but in this case it is more difficult to describe the resulting compounds with simple sum formulas.

The reasons are:

Normally, the Al cation is written in the following way: Al(H ₂ 0) _χ ³⁺ , however, this ion can exist only in low con¬ centrations. At pH values above 3, the ion is hydrolyzed to more or less soluble polynuclear forms called hydroxo complexes, polycations or hydroxo polymers.

This means, that in the cases where M in the general formula mentioned above is Al, this ion can possibly exist

in more polynuclear forms as :

A1 (H ₂ 0) ₆ ³⁺ , Al (OH) (H ₂ 0) ₅ ²⁺ , Al (OH) ₂ (H ₂ 0) ₄ ⁺ , Al ₂ (OH) _χ (H ₂ 0) ₁₀ . _χ ^6"x+ , Al ₂ (OH) ₂ (H ₂ 0) ₈ ⁴⁺ , or Al ₁₃ (OH) ₂₄ 0 ₄ (H ₂ 0) ₁₂ ⁷⁺ .

It is obvious that it is not possible to describe the double salts formed by sucrose-octa-O-sulfonic acid, an amino glycoside and Al in a form as exact as with cations, such as NH ₄ ⁺ , the alkaline and alkaline earth metals listed above. Therefore the present invention is directed to all possible salts in which sucrose-octa-O-sulfonic acid, amino glycoside and Al are connected to each other by means of ion bonds.

However, in special cases it is possible to describe the new salts with Al-ions more exactly. E.g. it is well known ( J. Chem. Soc. Faraday Trans. JL, 1981, 22, p. 629-639 ) that the structural formula of sucralfate can be described as follows:

C ₁₂ H ₁₄ 0 ₁₁ [S0 ₃ -A1 ₂ (0H) ₅ H ₂ 0] ₈

This means that when sucralfate dissociates the leaving Al ion is

Al ₂ (OH) ₅ ⁺

If some of the novel salts according to the invention are derived from the formula of sucralfate the novel salts can be described as follows:

C ₁₂ H ₁₄ 0 ₁₁ [S0 ₃ -(R-NH ₃ ⁺ ) _s (Al ₂ OH ₅ ) _t ] wherein 1< s <7 , 1< t <7, and s + t = 8, R is oligosaccharide sugar moiety, pre¬ ferably a mono-, di- or trisaccharide, of an antimicrobial- ly active amino glycoside.

It is also possible to describe some of the novel salts in the following way:

([sucrose-octa-O-sulfonic acid ^8" ]-[R -(NH ₃ ⁺ ) _χ ] _y - [Al(OH) _3n . ₁ ^* ] _z

wherein (x • y) + (z • n) = 8 x • y e N| [l≤x-y<6] , N is the set of natural numbers, x e Z|[4≤x<6], Z is the set of integers, z e N|[2≤z<7], n e Z|[l<n<5],

R is the oligosaccharide sugar moiety, preferably a mono-, di- or trisaccharide, of an amino glycoside.

However, it should be pointed out, that it is not possible to describe all the novel double salts consisting of sucrose-octa-O-sulfonic acid, an antimicrobially active amino glycoside and Al according to the novel invention with the formulas mentioned above.

The novel salts produced according to the invention have comparatively low solubility in water. When stirred into water the novel salts form oily or sticky gels with a high degree of affinity to e.g. the inside surface of the glass container wherein the precipitation is effected. Such precipitation will also occur on biological surfaces such as the mucosae of the stomach or the duodenum.

Furthermore, the novel salts will decompose and release the amino glycoside moiety in agueous solutions at a low pH. Therefore, the novel salts will be suitable for the treatment of ulcerations caused by H. pylori in the stomach and the duodenum.

By peroral administration in a suitable pharmaceutical formulation the novel salts will precipitate in the form of a gel which covers the mucosae of the stomach and/or the duodenum when the stomach pH is neutral or slightly below neutral.

Upon consumption of food, the stomach will secernate hydrochloric acid. H. pylori is sensitive to acid, such as gastric acid, but it has developed a protective measure. This protective measure consists in H. pylori producing an enzyme, urease, which will split urea into ammonia and C02. The ammonia thus formed neutralizes the gastric acid.

When the areas of the stomach which are infected with H. pylori come into contact with the salts produced according to the invention, the ammonia formed will cause ion exchange with the amino glycoside portion of said salts and thus release the amino glycoside which subsequently kills the H. pylori.

If the ammonia thus formed is not sufficient to neutralize the gastric acid, a corresponding ion exchange effect will occur as the gastric acid will release the amino glycoside from the novel salts.

The novel salts of amino glycosides and sucrose-octa-O- sulfonic acid are produced in a manner known per se. They are particularly advantageously produced by allowing the aqueous solution of the amino glycoside bases to react with an aqueous solution of sucrose-octa-O-sulfonic acid by titration to obtain the desired stoichiometric ratio.

If it is desired to produce salts in a 1:1 ratio it is necessary to neutralise the excess sulfate groups in the sucrose-octa-O-sulfonic acid with suitable cations, such as Na ⁺ , Ca ⁺ , Mg ²⁺ or Al ³⁺ . In many instances, the salts thus formed will crystallise spontaneously. If this is not the case the salt may be obtained by evaporation, optionally to dryness, or by freeze drying, or by addition of a solvent which is miscible with water, such as MeOH or EtOH.

The novel salts may also be produced by direct reaction of an aqueous solution of a salt of sucrose-octa-O-sulfonic acid with an acid addition salt of an amino glycoside.

In the special cases, where the neutralizing metal ion is Al, it is possible to operate with Al reagents, the constitution of which as mentioned before is dependent upon the chosen pH-value for the precipitation of the novel salts according to the invention. For instance, if the pH is in the range 3.5 - 4, A1C1 ₃ , 6aq is dissolved in water, pH is ajusted to 3.5 - 4 with a base like NaOH or a basic ion exchange resin. In this way, A1(0H ₂ )C1 is formed and used as mentioned in example 11 of the present application.

It is also possible to use sucralfate as the starting material. In this case, the chosen amino glycoside is dissolved in water and pH is ajusted to 4.0. To this solution, an equivalent amount of sucralfate is added with vigorous stirring. A ion exchange reaction takes place whereby the amino glycoside forms ion bonds with one or more of the sulfonic acid groups in sucrose-octa-O-sulfonic acid, and a corresponding amount of Al(OH) ₅ ⁺ dissolves in the aqueous phase. When the reaction is completed the novel salts are filtered off and washed with water.

This reaction will solely take place in a very narrow pH range, i.e. around pH 4. If pH is higher than 4, the Al-ion is not dissolved and, therefore, it cannot be separated from the reaction product by filtration. If pH is lower than 4, a gel formation takes place which also makes a separation impossible. Furthermore, at too low a pH, the equilibrium is moved in such a manner that the amino glycoside will remain dissolved with the Cl ^" as the anion.

The amino glycosides used are preferably selected from among the following substances having a desired anti¬ microbial activity: Amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin, gentamicin, isepa icin, kanamycin, micronimi- cin, neomycin, netilmicin, paromycin, ribostamycin, siso icin, streptomycin, tobramycin.

Amikacin, gentamicin, kanamycin, neomycin, streptomycin and tobramycin are particularly preferred.

The novel salts according to the invention may be for- mulated in a known manner, e.g. in the form of tablets which may optionally be dispersible in water, capsules, powders, aqueous suspensions, syrups, etc.

It is preferred to administer the novel salts of the invention in the form of tablets or capsules which desinte- grate in the stomach or duodenum and each containing from 50 to 250 mg, preferably about 100 g of the active amino glycoside together with conventional adjuvants and/or carriers.

The invention further relates to methods of producing the compounds having the formula I as disclosed in claims 12, 13 and 14, a pharmaceutical composition as described in claims 15 and 16, and the use of the compound having formula I as a medicament and for the production of a medicament as disclosed in claims 17-23.

Example 1

Sucrose-octa-O-sulfonic acid: 60 g (50 mmol) of sodium sucrose-octa-O-sulfonic acid (produced in accordance with J. Chem. Soc. Faraday Trans., 1981, 77, pp. 629-639) is dissolved in 200 ml of water and cation exchanged on Amberlite ^® IR 120 (H ⁺ ) . The combined eluates are diluted to 1 liter corresponding to an 0.05 M solution.

The resulting solution is used in Examples 2 through 7.

Example 2

Kanamycin A salt of sucrose-octa-O-sulfonic acid:

17 g (25 mmol) of kanamycin A sulfate (Maiji Seika Kaisha

Ltd., Tokyo, JP) is dissolved in 100 ml of ion exchanged water. The solution is eluated through an anionic ex¬ changer, A berlyst A26 ^® (OH ^" ). The combined eluates are evaporated on a rotary evaporator to 100 ml and are titrated with an 0.05M solution af sucrose-octa-O-sulfonic acid (from Example 1) to a pH of 5.0. The product precipi¬ tates as a syrup. The above liquid is decanted. To the syrup is added 250 ml of EtOH and stirring is effected for 3 hours thereby effecting crystal precipitation.

The crystals are filtered off, washed with 2 x 25 ml of EtOH and vacuum dried over Sicapent at 45°C.

Yield: 20 g - 82%.

DSC (Differential Scanning Calorimetry) : The substance pyrolyses within the interval ranging from 180 to 195°C and exhibits no melting point.

FTIR (Fourier Transformation Infra Red Spectroscopy) : The spectre is almost identical to that of the substance of Example 6, cf. Figure 2.

HPLC shows that the stoichiometric composition is a kanamycin:sucrose-octa-O-sulfonic acid ratio of 2:1.

Example 2a: Small-scale dissolution test

250 mg of kanamycin salt of sucrose-octa-O-sulfonic acid in 25 ml of ion-exchanged water, 5% NaH ₂ P0 ₄ puffer with a pH = 3 and 0.01 M HC1, respectively, at 37±0.5°C show that the substance has low solubility in water and only dissolves by ion exchange. The solubility is thus proportional with the ion activity.

Example 3

The gentamycin salt of sucrose-octa-O-sulfonic acid:

735 mg (1 mmol) of Gentamycin sulfate (Sigma, G-3632, 648 μg/mg) is dissolved in 25 ml of ion exchanged water. The solution is eluated through an anion exchanger such as in Example 2. The combined basic eluates are titrated with an 0.05M solution af sucrose-octa-O-sulfonic acid (from Example 1) to a pH of 6.5. Evaporation to about 5 ml which are triturated with 75 ml of EtOH and stirring is maintai¬ ned for three hours which results in crystal precipitation.

The crystals are separated off, washed with 2 x 10 ml of EtOH and vacuum dried over Sicapent at 45°C.

Yield 950 mg ~ 98%.

DSC: The substance pyrolyses in the interval of 210-215°C and shows no melting point.

FTIR: Almost identical with that of the substance of Example 6, cf. Figure 2.

Example 4

The neomycin salt of sucrose-octa-O-sulfonic acid:

910 mg (1 mmol) of neomycin sulfate (Sigma, N-1876, 657 μg/mg) is dissolved in 25 ml of ion exchanged water. The solution is eluated through an anion exchanger, such as in Example 2. The combined eluates are titrated with an 0.05M solution of sucrose-octa-O-sulfonic acid (from Example 1) to a pH of 6.5. Evaporation to about 5 ml which are triturated with 75 ml of EtOH and stirring is maintained for three hours which leads to crystal precipitation.

The crystals are filtered off, washed with 2 x 10 ml of

EtOH and vacuum dried over Sicapent at 45°C.

Yield 1 g ~ 90%.

DSC: The substance pyrolyses in the interval of 190-210"C and shows no melting point.

FTIR: Almost identical with that of the substance of Example 6, cf. Figure 2.

Example 5

The streptomycin salt of sucrose-octa-O-sulfonic acid:

1550 mg (1 mmol) of streptomycin sulfate (Sigma, S-6501, 766 units/mg) is dissolved in 25 ml of ion exchanged water. The solution is eluated through an anion exchanger, such as in Example 2. The recovered eluates are titrated with an 0.05 M solution of sucrose-octa-O-sulfonic acid (from Example 1) to a pH of 6.5. Evaporation to about 5 ml which are triturated with 75 ml of EtOH and stirring is maintai¬ ned for three hours which leads to crystal precipitation.

The crystals are filtered off, washed with 2 x 10 ml of EtOH and vacuum dried over Sicapent at 45°C.

Yield 1,5 g - 91%.

DSC: The substance pyrolyses in the interval of 200-210°C and shows no melting point.

FTIR: Cf. Figure 5.

Example 6

The tobra ycin salt of sucrose-octa-O-sulfonic acid:

500 mg (1 mmol) of tobramycin base (Sigma, T-4014, 941

μg/mg) are dissolved in 25 ml of ion exchanged water and are titrated with an 0.05 M solution of sucrose-octa-O- sulfonic acid (from Example 1) to a pH of approximately 6.5.

The emulsion thus formed is evaporated to about 5 ml and is triturated with 75 ml of EtOH and stirring is maintained for three hours which results in crystal precipitation. The crystals are filtered off, washed with 2 x 5 ml of EtOH and vacuum dried over Sicapent at 45°C.

Yield 650 mg ~ 68%.

DSC: The substance pyrolyses in the interval of 215-230°C and shows no melting point, cf. Figure 2

DSC of tobramycin base: Cf. Figure 1.

FTIR: Cf. Figure 4.

FTIR of tobramycin base: Cf. Figure 3.

HPLC shows that the stoichiometric composition is a tobramycin:sucrose-octa-O-sulfonic acid ratio of 1.6:1.

Example 7

Kanamycin/aluminium salt of sucrose-octa-O-sulfonic acid.

Is produced by admixing equimolar amounts of sucrose-oc¬ ta-O-sulfonic acid with kanamycin A-base. pH is subsequent¬ ly adjusted to 5.2 with a slurry of A1(0H) ₃ in water. The resulting gel is evaporated in a rotation evaporator to form an amorphous powder.

The production of corresponding salts with NaOH, Ca(0H) ₂ and Mg(OH ₂ ) is carried out analogously with Example 7.

The following applies to all of the substances of Examples 2 through 7:

The produced reaction with water is a swelling reaction whereby a sticky substance or syrup is formed. The absorp¬ tion of water occurs to a certain point where the particles have become an oily, viscuous fluid which has comparatively low solubility in water. When applying such a drop on smooth skin a membrane is formed which has low solubility in water and which may only be removed by intensive scrub¬ bing with water. By rinsing with ion-containing water the membrane is gradually dissolved until it disappears completely.

Example 8

The production of kanamycin salt of sucrose-octa-O-sulfonic acid with Na-sucrose-octa-sulfate as starting substance.

7.75 g (10 mmol) of kanamycin A-sulfate dissolved in 50 ml of water is admixed with 6.5 g (5 mmol) of sodium sucrose- octa-sulfate, C ₁₂ H ₁₄ S ₈ 0 ₃₅ Na ₈ , 8H ₂ 0, produced in accordance with Example 1.

The mixture is dialysed until the ion strength of the washing water is constant, at this point kanamycin-octa- sulfate is dissolved, and the solution becomes milky.

The emulsion is evaporated to about 25 ml and triturated with 250 ml of ethanol whereby crystallisation occurs. Stirring is maintained for 3 hours and crystals are filtered off and dried as described under Example 2.

Yield 9,55 g ~ 98%.

Analysis of the substance gives the same results as under Example 2.

Example 9

Sucrose-octa-O-sulfonic acid-Al-tobramycin 5.6% salt.

IJ 5 mmol sucrose-octa-0-sulfonic-acid-tobramycin ₁₆ (prepared in ex. 6) and 7 mmol Na-sucrose-octa-O-sulfate is dissolved in 150 ml water.

2) 112 mmol A1C1 ₃ in 100 ml water is mixed with 224 mmol NaOH in 100 ml water.

Solution 1 and 2 are mixed with stirring. pH is ajusted to and maintained at 5.5 with 0.2 M NaOH, whereby a precipita¬ te is formed.

The precipitate is filtered off, washed with 4 x 25 ml of water and dried in vacuum oven at 25°C/P ₄ 0 _1fJ .

Yield: 75%.

The content of tobramycin is analyzed by HPLC to be 5,6%.

Example 10

Test for bioadhesion on mucosal surfaces.

The bioadhesion of the novel salts according to the invention is demonstrated in a test system shown on Fig. 6. wherein (1) is thermostatic water flow at 40°C, (2) is a reservoir containing the washing solution at 37°C, (3) is a peristaltic pump (4) is a stainless steel support, (5) is a model membrane, and (6) is a receiver for collecting the washings.

A segment of jejunum from rabbit, is placed on a support (4) with the mucosa facing upwards, spread and held in position on the support by the adhesive effect of the jejunum itself. Support and jejunum is placed at an angle

of -7° in a cylindrical cell thermostated at 37°C. The sample to be tested is placed on the jejunum, 1 ml buffer is dropped thereon. The buffers used is either a 0.01 M HC1 solution, pH 2 (buffer a) or borate buffer solution 0.05 M, pH 7.4 (buffer b) . Now the segments are left for 10 minutes in the cell to allow the test substance to interact with the glycoproteins of the mucosa. After 10 minutes the jejunum is flushed with either buffer a or b for 30 minutes.

The buffer is collected in a receiver (6) . The amount of test substance remaining on the jejunum is calculated by measuring the amount in the receiver by means of HPLC.

In a first experiment, the tobramycin-SOS salt from example 6 containing 44% tobramycin, was tested. As a reference, a physical mixture of sucralfate 56%/tobramycin 44% was used.

The following results were obtained:

Test substance Buffer a. pH 2.0 Buffer b. pH 7.4

SOS-tobramycin salt from ex. 6 59% 31%

SOS/tobramycin physical mixture 56:44 8^

The percentages indicates the amount of test substance re- maining on the mucosa.

In a second experiment, the following results were obtai¬ ned:

Test substance Buffer a. PH 2.0 Buffer b, PH 7.4

S0S-A1-tobramycin salt from ex. 9 6V, 65%

Sucralfate 94.4%- tobramycin 5.6% physical mixture 40% 50%

The percentages indicates the amount of test substance re¬ maining on the mucosa.

These experiments clearly demonstrates, that the novel salts according to the invention are superior to simple physical mixtures of sucralfate and tobramycin.

Example 11.

Preparation of salts of sucrose-octa-O-sulfonic acid- tobramycin-Al.

I: Preparation of Al(OH) ₂ C1-reagent .1. :

0.2 mol A1C1 ₃ ,6 aq, - 48.2 g is dissolved in 200 ml demineralized water and is ajusted to pH 3.5-4 with 0.4 mol NaOH ~ 200 ml 2 M NaOH. The solution is metastable and is kept at +5°C.

II: Preparation of Al.OHKCl reagent (II ) :

0.2 mol A1C1 ₃ ,6 aq, ~ 48.2 g is dissolved in 300 ml demineralized water and ajusted to pH = 3.9 with a strong basic ion exchange reagent, eg. Amberlite ^® A-26 in hydroxy- form. The ajustment is done over 5 minutes with moderate stirring. The anion exchange reagent is filtered off and eluated with 200 ml of water for 30 minutes. The combined filtrate and eluate are filtered through celite.

The solution is metastable and is kept at + 5°C.

Ill:

100 ml, 0.1 M ~ 10 mmol SOSH ₈ , prepared in example 1 is mixed with 8.5 g tobramycin base, dissolved in 50 ml water. The mixture is stirred for 10 minutes and 400 ml 0.5 M Al- (OH) ₂ -reagent (I) is added - 200 mmol Al(OH) ₂ ⁺ . Stirring is continued for 10 minutes, 100 ml 2 M NaOH - 200 mmol is added which results in the formation of a precipitate. Stirring is continued for 2 minutes and the crystals are filtered off on a glass filter, washed with 50 ml water, and dried in vacuum oven at 80°C/1 Torr/20 hours.

Yield - 35 g, 73%.

IV:

Same procedure as III, but by use of reagent II.

Yield: 36 g - 75%.