AU656014B2 - Method of preparing starch esters for clinical, in particular parenteral, applications - Google Patents

Abstract

In order to prepare physiologically tolerated water-soluble starch esters, the invention proposes that starch is converted by acid hydrolysis or enzymatic hydrolysis to a partially hydrolysed form with a mean molecular weight in the range from 10,000 to 500,000 Daltons. This partial hydrolysate is then acylated, in aqueous solution, with the anhydride or halide of an aliphatic monocarboxylic acid with 2 to 4 carbon atoms or an aliphatic dicarboxylic acid with 3 to 6 carbon atoms, or mixtures thereof, and an alkalinization agent, to give a product with a substitution ratio in the range from 0.1 to 1.0 mole/mole. Salts are removed from the resulting reaction mixture. The starch esters thus obtained are highly suited for clinical use, in particular parenteral administration and administration in the diet.

Description

02,i DATE 11/02/93 AOJP DATE 08/04/93 APPLN. ID PCT NUMBER AU9222775

)IE

I l (51) Internationale Patentkdlassilfikation 5 C08B 35/02, 31/04 (11) Internationale Veriiffentlichungsnummer: WO 93/0121.7 Al (43) Internationales Veriiffentlichungsdatum: 21, Januar 1993 (21.01.93) (21) Internationales; Aktenzeich en: (22) Internationales; Anineldedatumn: PCT/EP92/0 1553 9. Juli 1992 (09.07.92) Prioritiitsdaten: P 4123 000.0 11. Juli 1991 (11.07.9 1) (71) Anmelder (ir alle Bestininungsswiaen ausser US): LAEVO- SAN-GESELLSCHAFT M BH [AT/AT]; Estermannstrage 17, A-4020 Linz/)Donau (AT).

(72) Eriindcr; und Erfinder/Anmnelder (nurfir US) :FORSTER, Harald [DE/ DEl; Wilhielm-Kobelt-Stralle 67, D-6000 Frankfurt/Main 71 ASSKALI, 'Fatima [MA/DE]; Gerauerstrafle D-6000 Frankfurt/Main 71 NITSCH, Ernst [AT/AT]; Voltastrafle 33, A-4040 Linz (AT).

(74)Aniilte: WEICKMANN, H. usw. Kopernikusstralle 9, D-8000 Mdnchen 80 (DE).

(81) Bestimmungsstaatcn: AT, AU, BB, BG, BR, CA, CR, CS, DE, DK, ES, Fl, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, US, europilisches Patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), OAPI Patent (BF, BJ, CF, CG, Cl, CM, GA, GN, ML, MR, SN, TD, TG).

Verbffentlicht Mit ine, ,:c-.ialen Reclwrclienberichl.

56 014 (54)Title: METHOD OF PREPARING STARCH ESTERS FOR CLINICAL, IN PARTICULAR PARENTERAL, APPLI-

CATIONS

(54) 43ezeichnung: VERFAHREN ZUR HERSTELLUNG VON STARKEESTERN FOR KL1NISCHE, INSBESONDERE PARENTERALE ANWENDUNG (57) Abstract In order to prepare physiologically tolerated water-soluble starch esters, the invention proposes that starch is converted by acid hydrolysis or enzymatic hydrolysis to a partially hydrolysed form with a mean moleiular weight in the range from 10,000 to 500,000 Daltons. This partial hydrolysate is then acylated, in aqueous solution, with the ginhydride or haide of an aliphatic monocarboxylic acid with 2 to 4 carbi atoms or an aliphatic dicarboxylic acid with 3 to 6 carbon atoms, or mixtures thereof', and an alkalinization agent, to give a product with a substitution ratio in the range from 0.1I to 1.0 mole/mole. Salts are removed from the resulting reaction mixture. The starch esters thus obtained are highly suited for clinical use, in particular parenteral administration and administratio~n in the diet.

(57) Zusammenfassung Zur Herstellung von wasserlt~slichien, physiologisch vertrllglichen Stfirkeestern wird Stirke durch Sflurchlydrolyse oder Enzymhydrolyse in ciii Teilbydrolysat nit cinem mittleren Molekulargewicht Mw mm Bereich von 10000 bis 500000 Dalton 0lberfohrt, dieses Teilhydrolsat in wllssriger L~sung mit dem Anhydrid oder H-alogenid ciner aliphatischen Monocarbonsllure mit 2 bis 4 Kohl enstoffatomen oder cuner aliphatischen Dicarbonslure mit 3 bis 6 Kohlenstoffatomen oder Gernischen davon und ciacm Alkalisicrungsmnittel bis zu euner molaren Substitution in Bercich von 0,1 bis 1,0 Mol/Mol acyliert. Aus dcii so erhaltenen Reaktionsgemisch werden die Salze entfernt. Die so erhflitlichen Stllrkeester sind schr gut fOr klinische, insbesondcre parenterale und dilltetische Anwendungen gecignet.

I t i 1 1, Process for the production of starch esters for clinical, in particular parenteral use Description The invention concerns a process for the production of water-soluble, physiologically compatible starch esters for clinical and in particular parenteral uses.

There is a great need for water-soluble biocompatible polymers for infusion solutions, as plasma substitutes, for haemodilution or in solutions for peritoneal dialysis as well as for tube feeding. In particular gelatins, gelatin derivatives, dextran and hydroxyethyl starch have previously been used for such purposes (cf.

e.g. US-A-3937821; DE-A-3313600; "Rbmpp's Chemie Lexikon", Vol. 9, page .9 and 1509).

At present the polysaccharides dextran and hydroxyethyl starch are mainly used for clinical applications, in particular for plasma substitute solutions. The primary disadvantage of dextran is that anaphylactic reactions can occur due to the presence of preformed antibodies, which make the use of this substance dangerous or complicate it by a compulsory pretreatment with a neutralizing low molecular hapten. Although direct sideeffects were only seldom observed in a short-term direct administration of hydroxyethyl starch, the prolonged administration of hydroxyethyl starch is, however, very limited by its storage especially in reticuloendothelial tissue. Hydroxyethyl starch can only be slowly and incompletely degraded by endogenous enzymes because the etherification impedes attack by endogenous T- 2 Lglycosidases. Although it is not yet known with certainty whether this storage impairs the function of the reticuloendothelial system, the occurrence of itching for example as a side-effect which is observed when administering hydroxyethyl starch has been linked to this storage. Recently the use of starch esters with lower aliphatic mono or dicarboxylic acids, in particular of acetyl starch, has also been proposed which compared to the aforementioned substances have a major advantage in that they lack antigenicity and can be completely excreted or metabolized.

A polymer which is intended for clinical and in particular parenteral use must, apart from a good watersolubility, also meet the following requirements: S The polymer solution should have a viscosity at the concentrations used therapeutically which is not higher than that of plasma; S The substance should have a retention time in the organism which is adapted to the respective application and should be completely degradable after parenteral administration. This requirement can be accomodated in the case of acyl starches by means of the degree of esterification because the degradability by endogenous a-amylase slows down with increasing molar substitution.

The substance should not be toxic, pyrogenic or antigenic.

The previously known starch esters do not meet some 6r all of these requirements.

t- 3 L' For application in foods, acetyl starch of high viscosity is used with a maximum acetyl content of 2.5 corresponding to a molar substitution (MS) of 0.064 mol/mol maximum. Such products are not suitable for clinical applications because of the pastiness of aqueous dispersions and because of their rapid degradation due to the low molar substitution. Acetyl starches of low viscosity with a low molecular weight are used to dress textiles and to size paper; such products are also unsuitable for clinical uses because of their low molar substitution, undefined distribution of molecular weight and low purity (for the state of the art cf. e.g. O.B. Wurzburg, Modified Starches, Properties and Uses, CRC Press Inc.; Boca Raton, Florida, 1986, page 55 to 74).

The object of the present invention is therefore to provide suita~l water-soluble, physiologically compatible starch esters for clinical and in particular parenteral uses which avoid the aforementioned disadvantages of the products which have previously been used for such applications and which in particular meet all requirements that are required of polymers for clinical and in particular parenteral use. This object is achieved by the present invention.

The invention concerns a process for the production of water-soluble, physiologically compatible starch esters which is characterized in that a starch is converted by acid hydrolysis or enzyme hydrolysis into a partial hydrolysate with an average molecular weight Mw in the range 10000 to 500000 Daltons, this partial hydrolysate is acylated in aqueous solution with the anhydride or halogenide of an aliphatic monocarboxylic .acid with 2 to 44 carbon atoms or of a aliphatic dicarboxylic acid with 4 carbon atoms or of ai aliphatic dicarboxylic acid with

A

-4- 3 to 6 carbon atoms or mixtures thereof and an alkalisation agent up to a molar substitution in the range 0.1 to 1.0 and the salts are removed from the reaction mixture obtained in this way and low molecular weight components are removed before and/or after the acylation.

A starch which is preferably used according to the invention is a starch which is composed of virtually amylose-free amylopectin that contains no more than 1 by weight amylose. Preferred examples of a starch used according to the invention are wax corn, wax rice or wax sorghum starch.

A process for the production of acetyl starches is known from US-A 2,362,282 with which starch hydrolysates with S.a low.dextrose equivalent value should be Sproduced which are intended as adhesives. In this case a "!depolymerization and acetylation is carried out at the same time by heating in high-percentage acetic acid.

This acid hydrolysis is difficult to control and the final product is not physiologically compatible due inter alia to its molecular weight which is too low.

GB-A-1 476 057 teaches the use of a starch derivative containing mixtures for the production of throat lozenges. In this case an amylopectin esterified with vinyl acetate is set forth as a starch derivative without details about production procedures, degree of substitution and molecular weight. Such a product is unsuitable for parenteral use.

Clear soluble, low substituted starch syrups are known from US-A-3 639 389 which are produced starting with starch hydrolysates with a D.E. value of less than 941 lO,pAopcrdab,22775.spc4 Such products are unsuitable for parenteral uses due to their low molecular weights alone.

The molecular structure of amylopectin corresponds to a large extent to that of endogenous glycogen so that no antigenicity or toxicity would be, expected. After cleavage of the acyl groups that can be readily hydrolysed by endogenous enzymes, the residual molecule is virtually indistinguishable from glycogen and is subjected to the same metabolism.

The partial degradation of the starch which occurs by the process according to the invention enables the viscosity of the solutions which are to be used therapeutically to be brought into the required range.

The starch is preferably hydrolysed until the average molecular weight Mw is in the range 40000 to 250000 Daltons.

The hydrolysis can be carried out in a well-known manner by means of an acid hydrolysis or an enzyme hydrolysis.

Strong mineral acids such as hydrochloric acid or sulphuric acid are preferably used for the acid hydrolysis. a-amylase is preferred as the enzyme for the enzyme hydrolysis. It is also possible to combine an acid hydrolysis with an enzyme hydrolysis and namely in any desired order.

The degree of hydrolysis can be easily monitored by measuring the viscosity of a sample diluted with water in order to thereby determine the desired degradation and end of the hydrolysis reaction.

1- 6 1- In the step in the procedure according to the invention which follows the hydrolytic degradation, the partially hydrolysed starch is acylated in aqueous solution with the anhydride or halogenide of an aliphatic monocarboxylic acid with 2 to 4 carbon atoms or of an aliphatic dicarboxylic acid with 3 to 6 carbon atoms up to a molar substitution in the range 0.1 to 1.0 mol/mol, a range of 0.3 to 0.7 mol/mol being preferred. The anhydride or halogenide which is used for this is derived from an aliphatic monocarboxylic acid with 2 to 4 carbon acids or from an aliphatic dicarboxylic acid with 3 to 6 carbon atoms. Aliphatic monocarboxylic acids are for example butyric acid, isobutyric acid, propionic acid, and in particular acetic acid. The aliphatic dicarboxylio acids with 3 to 6 carbon atoms used according to the invention can be saturated or unsaturated; they are preferably derived from a-dicarboxylic acids and are for example glutaric acid, adipic acid and in particular succininic acid and maleic acid. The chlorides are preferred as halogenides, but bromides and iodides are also well suited. According to the invention it is particularly preferable to use acetyl chloride and in particular acetic anhydride.

It is also possible to use a mixture of two or several of the mono or dicarboxylic acids according to the invention.

The acylation is carried out in the presence of an alkalising agent by which means acids which are formed at the same time are neutralised. Alkaline and alkaline earth hydroxides, carbonates or oxides are preferably used as alkalising agents' and in particular sodium hydroxide, sodium carbonate, calcium hydroxide and/or

I

7 magnesium oxide.

The reaction conditions for the acylation with the acid anhydride or the acid halogenide correspond to the usual conditions for such acylations; the reaction is preferably carried out at room temperature while stirring vigorously.

The salts are preferably removed from the reaction mixture obtained after the acylation by means of dialysis, ultrafiltration (diafiltration) or ion exchange. It is also possible to use two or several consecutive desalting procedures which may be identical or different.

Depending on the intended use of the starch esters according to the invention, it may also be expedient to also remove, at least to a substantial extent other low molecular components apart from the salts, such as low molecular degradation products of the initial starch.

This can be carried out before and/or after the acylation step by e.g. dialysis and in particular by ultrafiltration (diafiltration) in which membranes with an appropriate exclusion limit can be selected depending on the intended use. It is expedient to remove the low molecular cmponents together with the salts after the acylation. Depending on the desired degree of removal, it is possible to carry out two or several consecutive purification steps.

The reaction mixture obtained after the hydrolysis (partial hydrolysate) and/or in particular the reaction mixture obtained after removing the salts and, if desired, other low molecular components is preferably

X

I I 8 L subjected to sterilization by filtration before further processing or use.

The starch ester solution obtained after removing the salts and, if desired other low molecular co 'ponents, can be used5 directly as such; the starch ester solution is preferably converted into a dried product for better handling and shelf-life. This is carried out in particular by gentle concentration of the solution in a vacuum and subsequent drying in a vacuum. It is, however, also possible to convert the starch ester solution into a freeze-dried product by lyophilization, Due to their properties, in particular their good ilStersolubility and physiological compatibility, the starch esters obtainable by the process according to the invention are very well suited for clinical and in particular parenteral uses.

Starch esters according to the invention are in particular used for the production of pharmaceutical compositions for peritoneal dialysis as well as for the production of blood plasma substitutes (cf. the German patent application P 41 22 999.1 "Metabolizable plasma substitute" and the German patent application P 41 23 001.9 "Pharmaceutical composition for peritoneal dialysis" by the same applicant and with the same application date).

The following examples are intended to elucidate the invention in more detail. If not stated otherwise the percentages stated above and below refer to by weight and parts refer to parts by weight. Details of temperatures refer to the celsius scale.

L_ 9 Examples Example 1 Production of a partial hvdrolysate from wax corn starch (Mw ca. 200000) 4000 g wax corn starch with a water content of 10.0 corresponding to 3600 g anhydrous starch, was suspended in 12.5 kg desalted water, then 3.85 g calcium chloride dihydrate and 1 g a-amylase (Termamyl 60 L; NOVO Co.

Copenhagen) was added and quickly heated to 950C while stirring. The starch dissolved without forming a highly viscous phase. It was kept at 95 0 C until a sample diluted to 20 with water had a relative viscosity of at 20 0 C compared to water. The degradation was stopped by addition of hydrochloric acid until the pH value was 3.0; the reaction mixture was then kept at 950C for a further 10 minutes, subsequently cooled to room temperature, adjusted to pH 5.5 with sodium hydroxide solution and filtered over sterile layers. The light-yellow coloured degraded starch solution, 15.6 kg, with a dry solids content of 22.4 was subjected to diafiltration over an ultrafiltration membrane with an exclusion limit of 30000 Daltons in order to remove low molecular components in which 10.2 kg of a 19.6 solution containing 2.0 kg degraded amylopectin was obtained. Aliquots of this solution were used in the following examples 2 and 3.

t Example 2 Production of an acetyl starch (Mw ca. 200000, MS ca. 0.35) 40.8 g (0.40 mol) acetic anhydride was added constantly over a period of one hour to 826.5 g of the degraded starch solution obtained according to example 1 which contained 162 g dry weight (1 mol CH 6

H

10 0 5 while stirring vigorously with a magnetic stirrer and at the same time 2 N sodium hydroxide solution was added by means of pH-stat circuit to maintain a pH value between to 8.5. 225 ml (0.45 mol) was consumed for this.

The solution obtained in this way was diafiltered over an ultrafiltration membrane (exclusion limit 30000 Daltons) until the conductivity was 1 micro S/cm.

After addition of 1.6 g active charcoal, the solution was filtered over sterile layers, concentrated in a vacuum to form a thick syrup and afterwards oven-dried in a vacuum to yield a brittle, white blistered mass.

The yield was 165.0 g after grinding. The molar substitution was determined as being 0.355 mol/mol. The substance proved to be pyrogen-free when tested on rabbits according to Ph.Eur. and was tolerated by the animals without reaction.

Example 3 Poduction of an acetvl starch (Mw ca. 200000, MS ca. 0,50) 64.85 g (0.635 mol) acetic anhydride was added 11 constantly over a period of two hours to 826.5 g of a degraded starch solution obtained according to example 1 containing 162 g dry weight (1 mol CH 6

H

10 0 5 while stirring vigorously with a magnetic stirrer and at the same time 2 N sodium hydroxide solution was added by means of a pH-stat circuit to maintain a pH value between 8.0 to 8.5. 500 ml (1.0 mol) was consumed for this.

The solution obtained in this way was diafiltered over an ultrafiltration membrane (exclusion limit 30000 Daltons) until the conductivity was 1 micro S/cm.

After addition of 1.6 g active charcoal, the solution was filtered over sterile layers, concentrated in a vacuum to form a thick syrup and afterwards oven-dried in a vacuum to yield a brittle, white blistered mass.

The yield was 166.4 g after grinding. The molar substitution was determined as being 0.502 mol/mol. The substance proved to be pyrogen-free when tested on rabbits according to Ph.Eur. and was tolerated by the animals without reaction.

Application example Production of plasma substitute solutions An acetyl starch according to the invention with an average molecular weight (Mw) of ca. 200000 Daltons and a degree of substitution of 0.3 or 0.5 was used as the starch ester.

The following plasma substitute solutions were prepared from this: 12 1. Acetyl starch solution at a concentration of 3, 6 and 10 by weight in physiological saline solution (0.9 by weight).

2. Acetyl starch solution at a concentration of 6 by weight in 2.5 by weight glycerol solution (electrolyte-free plasma substitute solution) Investigations on biodegradability The acetyl starch solutions prepared as described above with a concentration of 3, 6 and 10 by weight acetyl starch (degree of substitution 0.3 or 0.5) in physiological saline solution were administered intraveneously into rats (18 ml in 3 hours).

The infusions were very well tolerated and no direct side-effects were detected. Immediately after completion of the infusions dose-dependent blood levels of acetyl starch were present (6 to 25 mg/ml). These blood levels were comparable to those which were obtained using hydroxyethyl starch solutions (HES 200/0.5; Mw ca.

200000 Daltons, molar substitution 0.5 mol). Even 3 hours after completion of the infusion acetyl starch was still detectable in the blood of the animals (1.0 to mg/ml); these amounts also corresponded to the amounts which we a obtained under comparable conditions with the hydroxyethyl starch solution (HES 200/0.5). However, 24 hours after completion of the infusion no acetyl starch could be detected in the blood of the treated animals.

Fig. 1 shows the serum content (in mg/ml) after a 3 hour infusion which was determined immediately after the infusion was completed (A to E) and 3 hours after 13 completion of the infusion G, H).

Fig. 2 shows the content of hydrocolloid in the kidney (in mg/g) which was determined immediately after the infusion was completed C, D, E and NaC1), 3 hours after completion of the infusion G) and 18 to 24 hours after completion of the infusion J, K, L).

Fig. 3 shows the results corresponding to Fig. 2 for the lung.

The data A to L in Fig. 1 to 3 refer to the following plasma substitute solutions: A 3 acetyl starch; B 6 acetyl Starch; C 10 acetyl starch; D 6 HES 200/0.5; E 10 HES 200/0.5; F 6 acetyl starch; G 10 acetyl starch; H 3 acetyl starch; I 6 acetyl starch; J 10 acetyl starch; K 6 HES 200/0.5; L 10 HES 200/0,5. NaCl sodium chloride.

13a Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

e *0 •a0 o *et 94)104,p-oprdab,22775sp C,13

Claims (1)

14- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Process for the production of water-soluble, physiologically compatible starch esters, wherein a starch is converted by acid hydrolysis or enzyme hydrolysis into a partial hydrolysate with an average molecular weight Mw in the range 10000 to 500000 Daltons, this partial hydrolisate is acylated in aqueous solution with the anhydride or halogenide of an aliphatic monocarboxylic acid with 2 to 4 carbon atoms or the anhydride or mono-halogenide of an aliphatic dicarboxylic acid with 3 to 6 carbon atoms or mixtures thereof and an alkalisation agent up to a molar substitution in the range 0.1 to 1.0 mol/mol and the salts are removed from the reaction mixture obtained in this way 15 rnd low molecular weight components are removed before and/or after the acylation. U S*.i 2. Process as claimed in claim 1, wherein the starch used is composed of virtually amylose-free amylopectin. 3. Process as claimed in claim 1 or 2, wherein the starch is wax corn, wax rice or wax sorghum starch. S4. Process as claimed in any one of the claims 1 to 3, ?5 wherein the starch is hydrolysed until the average molecular weight Mw is in the range 40000 to 250000 Daltons. Process as claimed in any one of the claims 1 to 4, wherein u-amylase is used for enzyme hydrolysis. 6. Process as claimed in any one of the claims 1 to wherein it is acylated until the molar substitution is 0.3 to 0.7 mol/mol, 7. Process as claimed in any one of the claims 1 to 6, wherein an anhydride or halogenide of acetic acid is used as g the anhydride or halogenide of a monocarboxylic acid. 941 104,p'opcrtdab,22775spec14 8. Process as claimed in any one of the claims 1 to 6, wherein an anhydride or halogenide of succinic acid or maleic acid is used as the anhydride or halogenide of a dicarboxylic acid. 9. Process as claimed in any one of the claims 1 to 8, wherein sodium hydroxide, sodium carbonate, calcium hydroxide or magnesium hydroxide is used as the alkalisation agent. 10. Process as claimed in any one of the claims 1 to 9, wherein salts and/or low molecular weight components are removed by dialysis, ultrafiltration and/or ion exchange. 11. Starch esters obtained by a process as claimed in any one of the claims 1 to 12. Use of a starch ester as claimed in claim 11 for the production of pharmaceutical compositions for clinical and in particular parenteral use or of dietary formulations. 13. Processes for the production of water-soluble, physiologically compatible starch esters, starch esters obtained by said processes or uses of said starch esters, substantially as hereinbefore described with reference to the Examples. DATED this 4th day of November, 1994 Laevosan-Gesellschaft mbH By Its Patent Attorneys DAVIES COLLISON CAVE 941 04,p:\opekab,2277,spcI1 Abstract For the production of water-soluble, physiologically compatible starch esters, starch is converted by acid hydrolysis or enzyme hydrolysis into a partial hydrolysate with an average molecular weight Mw in the range 10000 to 500000 Daltons, this partial hydrolysate is acylated in aqueous solution with the anhydride or halogenide of an aliphatic monocarboxylic acid with 2 to 4 carbon atoms or an aliphatic dicarboxylic acid with 3 to 6 carbon atoms or mixtures thereof and an alkalisation agent up to a molar substitution in the range 0.1 to 1.0 mol/mol. The salts are removed from the reaction mixture obtained in this way. The starch esters that can be obtained in this way are very well suited for clinical and especially for parenteral and dietary applications.