IE57998B1 - Insulin formulations,processes for their preparation and their use - Google Patents

Abstract

1. Pulsation damper (10) for elastically disposed objects (26), including a damper housing which is at least partially filled with 3 viscose damping medium (12) and which is open at the top relative to its installation position ; a hollow plunger pipe (17) which is open at the bottom relative to its installation position and continuously conically formed and which can be moved up and down into the damper medium (12) ; a top plate (16), linked with the plunger pipe (17), for receiving the object (26), in which respect the plunger pipe (17) is connected with the top plate (16) by its smaller diameter (28), and its larger diameter (29) is emersed into the damper medium (12), characterised in that the interior of the plunger pipe (17) is divided into two chambers by a sealing plate (22) ; that a buffer plate (24) including passages (33) is disposed in the larger diameter (29) at the bottom of the plunger pipe (17) which plate carries a plurality of interior pipes (21) which extend into the plunger pipe (17), the upper ends of the interior pipes (21) being at a distance (35) from the sealing plate (22), and the plunger pipe (17) has a plurality of peripheral bores (19) in the casing surface below the sealing plate (22).

Description

The invention relates to the preparation of novel insulin formulations with a delayed action, the preparation of stabilized insulin formulations for use in automatic metering apparatuses, and the use thereof, in particular for the treatment of diabetes mellitus.

It is generally known that particular requirements are imposed on parenteral replacement therapy with insulin. These include, in particular, the question of delayed pharmacokinetics, which enable the diabetic to be stabilized with one or a few injections per day. A few clinically proven principles exist to achieve such depot effects, these principles including the use of zinc or protamine sulfate as depot auxiliaries.

These known depot principles are based on the physical effect of the slow redissolving of an insulin form which is sparingly soluble at the physiological pH, for example the 2-zinc crystal form. If the product already has a neutral pH, which is advantageous with regard to chemical stability and long storage, these products are suspensions which must be very carefully homogenized by shaking before metering, in order to avoid incorrect dosages.

The insulin depot products previously known furthermore have very specific action profiles which can be varied only within 'certain limits by adding dissolved insulin. There are always patients for whom alternative action profiles, for example those with a somewhat less rapid onset of an action of about the same length, are desirable. If the physician has such products available, he can suit the specific habits and characteristics of the diabetic. In contrast, if the patient were to be required to change his habits, this would lead to the problem of patient compliance, which in the end effect substantially influences the therapeutic result.

Kraegen et al. report, in Brit. Med. J. 1975, 3., 464-466, a stabilizing effect of adding up to 3.5% of Haemaccel (sic) to very dilute insulin solutions (0.04 I.U./ml), by which means, inter alia, the adsorption of the insulin in the stock vessel and tube system in infusion systems can be prevented.

It has now been found that an aqueous insulin formulation with an insulin concentration above 1 I.U./ml and a content of stabilizing physiologically acceptable agents surprisingly exhibits a synergistically improved physical stability and other improved properties, for example in respect of the action profile, if it has a viscosity of at least 1.75 mPa.s at 4°C and contains* as stabilizing physiologically acceptable agents, a combination A) of a physiologically acceptable thickener of the polygeline and/or dextran type with B) a physiologically acceptable surface-active substance from the group comprising BI) an ethylene glycol/propylene glycol copolymer or block polymer, at least half of the chain members, however, being units of 1,2-propylene glycol; B2) lecithin and/or B3) polyoxyethylene 23 lauryl ether.

This formulation is particularly highly stable towards mechanical stress, in particular under elevated temperature, for example shaking and pumping movements and is therefore in particular suitable, inter alia, for use in peristaltic pumps.

The question of the stability of insulin formulations has already previously been a serious problem. Thus, it is known that dissolved proteins such as insulin are adsorbed at interfaces (including also the aqueous solution/ air interface) (C.W.N. Cumber and A.E. Alexander Trans. Faraday Soc. 46., 235 (1950)). Various secondary reactions are observed as a result of this adsorption at interfaces, and these are generally referred to by the term denaturing. A change in the shape of the protein molecules adsorbed occurs (change in the tertiary and/or secondary structure). In addition, aggregation of adsorbed molecules to soluble or insoluble polymeric forms may also occur. The turbulence which occurs when insulin solutions pass through narrow channels also appears to promote insulin denaturing.

The tendency of insulin to precipitate out of commercially available solutions and thereby to block mechanical components and supply lines has proved to be the main obstacle in further development and clinical use of continuous infusion devices. There is also the tendency to reduce the size of these devices in order thus to obtain systems which can be implanted, which results in a need for highly concentrated stable insulin solutions, which in turn renders the above problems even more serious.

The question of the physical stability of insulin solutions has been under particular discussion since the development of automatic metering apparatuses. It is generally known that specially stabilized insulins must be used in such apparatuses. In connection with the inadequate physical stability of insulins, not only is the reduced biological activity under discussion, but recently also a process for the formation of amyloid A protein in the serum, which proceeds by stimulation of the macrophages and can lead to amyloidosis in various organs (Brownlee et al., Lancet (1984), 411-413).

A number of proposals have already been made to solve these problems where, however, nowhere has the combination of thickener + surface-active substance been described as a stabilizing agent: principally in order to achieve a sustained action, various thickeners have already been added to insulin formulations (US-A-2,474,729: polyvinylpyrrolidone; US-A-2,574,889: methylated or alkylated gelatins; C.A. Vol.77, 1972, ref. 56944t: dextran).

One component of the solid insulin formulation according to GB-A-2,101,482 is a water-soluble copolymer of n-vinylpyrrolidone, acrylamide and ethyl acrylate; a specific salicylic acid derivative is used as a stabilizer here.

A compilation of different low and also high molecular weight compounds which are suitable for the stabilization of insulin is found in the article by W.D. Lougheed et al. Insulin aggregation in artificial delivery systems in Diabetologia 19, 1-9 (1980).

DE-A-2,917,535 discloses aqueous solutions of insulin which contain, for protection from denaturing, a surfaceactive substance of the general formula I Ra RbG— (-CH--CH-O-) ~RC (1) 2 P in which R" denotes hydrogen, methyl or ethyl, p denotes a number from 2 to 80, preferably 8 to 45, and Rb and Rc are identical or different and denote hydrogen, alkyl alcohol radicals with 1-20 carbon atoms, carboxylic acid radicals with 2 - 20 carbon atoms, alkylphenol radicals with an alkyl chain of 1 - 10 carbon atoms or alkylamine radicals with 1-20 carbon atoms, as a homopolymer, block polymer or copolymer in a concentration of 2 to- 200 mg/1.

EP-A-18,609 describes aqueous solutions of insulin and a large number of other proteins which are stable towards denaturing and contain a surface-active substance with a chain-like basic structure, the members of which contain weakly hydrophobic and weakly hydrophilic regions in alternating arrangement.

WO-A-83/00288 describes stable aqueous insulin formulations which are for use in insulin-metering devices, have a pH of 6.5 to 9 and contain up to 1,000 ppm of a polyoxyethylenealkyl ether of the formula R7-O-[CH2-CH2-O]m-H (II), in which R7 denotes a saturated or unsaturated (C8-C15)-alkyl group and m denotes an integer from 2 to 25.

Finally, physically stabilized insulin solutions are known from DE-Ar-3,240,177, which contain stabilizing amounts of a phospholipid of the formula III (III) in which R4 and R5, which can be identical or different, represent hydrogen, alkylcarbonyl, alkenylcarbony1, alkanedienylcarbonyl, alkanetrienylcarbonyl or alkanetetraenylcarbonyl, with the proviso that R4 and R5 are not simultaneously hydrogen, and in which R6 represents a hydrophilic group.

These surface-active stabilizers are extremely effective, in that they significantly increase the stability of insulin solutions to shaking. Shaking is without doubt a substantial adverse influence on the insulin in metering apparatuses.

It has now -been found, however, that, especially in peristaltic pumps, the squeezing of the elastomer tube of the pump and/or the shearing effects, such as occur in many pump principles, additionally impair insulin stability. Precipitation of insulin in pump tubes or catheters may in this way occur, in spite of addition of the various stabilizers known hitherto.

Here and in the following text, insulins are understood as single products or mixtures of several insulins, and in particular not only human insulin and insulins of animal origin, such as mammalian insulins (for example from cattle or pigs); the term also includes insulins in the broader sense, i.e. modified insulins, such as des-PheB1-insulins (cf., for example, DE-C-2,005,658 and EP-A-46,979) or insulins which are modified by basic substituents on the C-terminus of the Β-chain (such as insulin-B31-Arg-0H or insulin-B31-Arg-Arg-0H, proposed in German Offenlegungsschriften DE-A-3,326,472, -3,327,709, -3,333,640 and -3,334,407), and human proinsulin or other proinsulins or proinsulin analogs (cf., for example, DE-A-3,232,036), and alkali metal and ammonium salts. It is also possible for several of these insulins to be present in a mixture. Depending on the solubility, the insulin concentration can be up to about 1,500 Ι.ϋ./ml, and is preferably between 5 and about 1,000 I.U./ml. In depot forms, any desired proportion of one or more insulins can be present in each case in the dissolved, amorphous and/or crystalline form, independently of one another.

Possible thickeners (also called gelling agents) are in particular polygeline (= gelatin partial hydrolysates, which can also be crosslinked, for example with diisocyanates) and/or dextran(s), and possible surface-active substances are ethylene glycol/propylene glycol copolymers or block polymers, at least half of the chain members being units of 1,2-propylene glycol, as well as lecithin and/or polyoxyethylene 23 lauryl ether.

The result of the thickeners is that the insulin formulation is slightly viscous to viscous or in the form of a hydrogel at low temperatures, for example 4 °C. The formulations according to the invention preferably have a viscosity, measured at 4 °C, of at least 2 and in particular of at least 2.5 mPa.s. The gels already partly liquefy at room temperature or at temperatures close to body temperature.

The formulation according to the invention, which has a high physical stability, preferably contains more than 1, in particular between 2 and 20, % by weight of thickeners.

However, hydrogels can already be formed when smaller amounts of thickener are added. The upper limit of the content of gelling agent can be 30% or more, depending on its nature.

If the content of thickener is sufficiently high, all embodiments of the formulation according to the invention have the common property that they are in solid form as a hydrogel under storage conditions. This is an advantage in respect of the physical stability, since oligomer formation and denaturing are known to be greatly accelerated by movement, that is to say in principle cannot be reliably excluded when liquid insulin formulations are handled. Storage in gel form can also be advantageous because this form remains considerably more homogeneous on storage than a solution or suspension. In the case of sedimented crystal suspensions, for example, it is entirely conceivable that relatively stable crystal associates are formed on prolonged storage, which can then be shaken up less easily to give a homogeneous suspension, so that metering errors may occur. In contrast, if the crystal suspension is homogeneously frozen in a gel, the insulin molecules can diffuse only slowly at the vessel wall or at the liquid/air interface, and such effects are to be excluded. Movements and turbulences within the gels during handling are also considerably reduced. The formulation thus has a particularly good storage stability.

Before use, the gels, as is also usual with conventional insulins, are brought to room temperature to body temperature, whereupon they liquefy, but nevertheless still have a greater viscosity than conventional insulin solutions. Suspensions do not then usually sediment immediately, so that inhomogeneities and metering errors can occur less easily; an inhomogeneity problem does not of course exist with clear gel formulations. The customary injection apparatuses can be used in all cases.

The increased physical stability of the gels according to the invention is also present to a certain degree at body temperature, i.e. in the liquid state. If such solutions are exposed to thermomechanical stress in a rotation experiment at 37"C, a relative stability of about 3 to 5 is to be observed in comparison with conventional insulin solutions containing no thickener.

All the formulations according to the invention can in principle be prepared from dissolved or from amorphous or crystalline insulin (clear or cloudy gels). They have, in general, a pH between 2.5 and 8.5, but in particular between 6 and 8, and preferably contain a suitable isotonicizing agent, a suitable preservative and, if appropriate, a suitable buffer substance, for example those mentioned below. The active substance is preferably present in dissolved form.

The formulation according to the invention has a certain depot effect - probably due to the content of thickener.

It is possible to intensify the depot effect by combina20 tion with customary auxiliaries with a retarding action, such as, for example, by addition of suitable amounts of zinc, Surfen, globin or protamine sulfate. The amount of zinc added can be up to 100 pg of Zn2+/100 insulin units; it is preferably more than 35 and usually less than 50 pg of Zn2+/100 insulin units. The amount of protamine can be, for example, between 0.28 mg and 0.6 mg per 100 units (based on protamine sulfate). Products which were hitherto inaccessible and have a particularly long-lasting action can be prepared in this manner, the use of these products being of interest because recent knowledge from therapy with insulin-metering apparatuses shows that precisely a basal amount of insulin seems to be therapeutically advantageous.

A suitable physiologically acceptable carrier medium which is compatible with the insulins is a sterile aqueous solution which has been rendered isotonic with blood in the customary manner, for example by glycerol, sodium chloride or glucose, and which also contains one or more of the usual preservatives, for example phenol, m-cresol, benyl (sic) alcohol or p-hydroxybenzoic acid esters. The carrier medium can additionally contain a buffer substance, for example sodium acetate, sodium citrate, sodium phosphate or tris-(hydroxymethyl)-aminomethane. Dilute acids (typically HCl) or alkalis (typically NaOH) are used to adjust the pH.

The insulin formulation according to the invention is prepared by adding a physiologically acceptable thickener and a physiologically acceptable surface-active substance of the abovementioned type to an aqueous insulin formula15 tion. The formulations are distinguished by particular stability and - in the case of subcutaneous or intramuscular administration - by a delayed action.

The invention furthermore relates to the use of these insulin formulations in the treatment of diabetes mel20 litus, in particular by means of devices for continuous release of insulin, and to the use of the formulations for avoiding adsorption or denaturing of insulin on surfaces and other phase interfaces, in particular during purification by chromatography or crystallization, storage and therapeutic use.

The insulin formulations according to the invention can be administered parenterally, i.e. intravenously, subcutaneously or intramuscularly, for the treatment of diabetes mellitus. The depot effect of the formulations with or without surface-active substances manifests itself most markedly in the subcutaneous mode of administration, but also manifests itself clearly on intramuscular injection. When administered intravasally, the formulations according to the invention, in clear solu35 tion, have a rapid action, in the same manner as the known dissolved insulins. They are therefore outstandingly suitable for use in automatic metering apparatuses, such as pumps, in which the infused insulin must be immediately effective, since only in this way is rapid control, for example in accordance with the blood glucose level, possible.

With some metering principles, it is necessary, or at least advantageous, to fill the reservoir with degassed insulin solution. As has been frequently demonstrated, air, together with contact with the materials of the equipment, is the most adverse environment for insulin. This is a practical problem with conventional solutions, since solutions which may have been degassed by the manufacturer in the end effect dissolve air again by movement (for example transportation) and diffusion. In contrast, a solidified gel, which has been degassed as a liquid, is far less susceptible to this influence; troublesome degassing (and the associated risk of nonsterility) directly before use in the pump can then possibly be dispensed with.

Another practical advantage of the formulations according to the invention can be that, if the products are gels which have solidified at the storage temperature, direct contact with the stopper is avoided. In particular, the usual stoppers tend, for example, to absorb the stabilizers, which- can present problems in view of the small amounts added in some cases.

The following examples serve for further illustration, without restricting the invention to these. The formulations prepared therein all have a viscosity of more than 1.75 mPa.s at 4°C. The figures given for dextran, for example 60, multiplied by 103, indicate the molecular weight. The distilled water was in each case p.i. of pH 7.3. Polygeline is a product from Behringwerke AG, Marburg.

Examples 1 and 2) - Insulin formulations containing 20% of polyqeline According to Example 1, in each case a sterile solution of a) 250 g of polygeline, lyophilized, in distilled water, made up to 1 1, and b) 4.464 g of human insulin (28 I.U./mg), 21.25 g of glycerol, 7.50 g of tris-(hydroxymethyl)-aminomethane, 3.375 g of phenol, an amount of anhydrous zinc chloride such that the total zinc content was 0.035 g and 0.0125 g of polypropylene glycol, onto which in each case about 5% of polyethylene glycol had been polymerized on both sides (average molecular weight of 1,800), in distilled water, made up to 250 ml, were combined under sterile conditions.

According to Example 2. in each case a sterile solution of a) 200 g of polygeline, in distilled water, made up to 800 ml, and of b) 1.429 g of human insulin (28 I.U./mg), 1.50 g of m-cresol, 1.00 g of phenol, 17.00 g of glycerol, an amount of anhydrous zinc chloride such that the total zinc content was 0.028 g and 0.030 g of lecithin, in distilled water, made up to 200 ml, were combined under sterile conditions.

The solutions prepared according to Examples 1 and 2 are introduced into small glass bottles in the usual manner. At about 15°C, they solidified as clear gels containing 100 I.U./ml.

Examples 3 to 7)? Insulin formulations containing 8 or 16% of dextran According to Examples 3 and 4, in each case a sterile solution of a) 180 g of dextran 60 or 160 g of dextran 60, in distilled water, made up to 800 ml, and of 3 b) 3.571 g of human insulin (28 I.U./mg), 6.00 g of tris(hydroxymethyl)-aminomethane, 2.00 g of m-cresol, 1.00 g of phenol, 17.00 g of glycerol, an amount of anhydrous zinc chloride such that the total zinc content was 0.028 g and 0.010 g of polypropylene glycol (see Example 1), in distilled water, made up to 200 ml, were combined under sterile conditions.

These formulations were introduced into small glass bottles in the usual manner.

According to Examples 5 to 7. insulin formulations were prepared analogously to Example 3, but with 10 and 20% of dextran of different molecular weight (see Table 1). 8) Proinsulin formulation containing 20% of polygeline In each case a sterile solution of a) 20 g of polygeline, in distilled water, made up to 80 ml, and of b) 100 mg of proinsulin from pigs, 0.21 g of NaH2PO*.2Hz0, 0.30 g of m-cresol, an amount of anhydrous zinc chloride such that the total zinc content was 0.0012 g and 0.010 g of polyoxyethylene 23 lauryl ether of molecular weight 1,200, in distilled water, made up to 20 ml, were combined under sterile conditions.

Physical stability of the formulations according to Examples 1 and 3 to 7 The following stability data were obtained in a standardized pump experiment using a peristaltic pump (37°C, movement, pumping rate of 12 I.U./d): 4 Table 1 Solution Time before first Relative clouding occurs stability H-Insulin Hoechst 3 days 1 Formulation b) according to Example 1, made up to 1.25 1 with distilled water 45 days 15 Formulation according to Example 1 >80 days >27 Formulation containing 8% of dextran (Example 3) >60 days >20 Formulation containing 16% of dextran (Example 4) >80 days >27 Formulation containing 10% of dextran 32 (Example 5) >60 days >20 Formulation containing 10% of dextran 100 (Example 6) >60 days >20 Formulation containing 20% of dextran 100 (Example 7) >80 days >27 Physical stability of the formulations according to Examples 2 and 8 The solutions prepared were investigated for their physical stability in a standardized circulatory pumping experiment using a peristaltic pump (37 °C, movement, recycling pumping at a rate of 5 ml/h = 500 I.U./h = 12,000 I.U./d).

Table 2 Solution Time before first clouding occurs Relative stability H-Insulin Hoechst 20 h 1 Formulation containing 20% of polygeline (Example 2) > 7 days >8 Formulation according to Example 2, solution b)* 2 days 2.4 Formulation containing 20% of polygeline (Example 8) > 7 days >8 Formulation according to Example 8, solution b)* 30 h 1.5 * in each case without polygeline, but made up to 1 1 (Example 2) or 100 ml (Example 8) with distilled water.

Examples 9 and 10: Physical stability of formulations which contain thickeners only According to Example 9. a formulation was used which was prepared according to Example 1, but with the difference that no polypropylene glycol was added to the solution b) .

According to Example 10, in each case a sterile solution of a) 1 1 of a 10% strength solution of polygeline and b) 1.786 g of pig insulin (28 I.U./mg) and 4.25 g of glycerol, 1.50 g of tris-(hydroxymethyl)-aminomethane, 2.50 g of phenol and an amount of anhydrous zinc chloride such that the total zinc content was 0.014 g, in distilled water, made up to 250 ml, were combined under sterile conditions .

According to the biological test, the formulation thus prepared contained 40 I.U./ml.

In each case 5 small bottles of a formulation according to Example 9 and 10 were tested in a standardized rotation experiment at 37°C, 1 Hz. A standard insulin was investigated for comparison. Ιβ Table 3 Solution Time before first Relative stability cloudin< j occurs H-Insulin Hoechst 2 days 1 Formulation b) according to Example 10, made up to 1.25 with distilled water 1 4 days 2 Formulation according to Example 9 15 days 7.5 Formulation according to Example 10 22 days 11 Action profile of insulin formulations according to Example 1 i.v. Administration to dogs and rabbits In each case 0.2 I.U./kg of body weight of the formulation according to Example 1 was administered into the ear vein. Human insulin Hoechst was used as the comparison insulin (II). Figures 1 and 2 show the time course of the blood glucose level (x + SEM). The values were determined from measurements on in each case 5 animals. The formulation according to Example 1 (I) had an action which was as rapid or even somewhat more rapid than the comparison unmodified insulin (II) both in dogs (Figure 1) and in rabbits (Figure 2).

In Figures 1 and 2, the length of the strokes for the individual measurement values indicates the standard deviation from the mean value (SEM) in the customary manner.

Claims (8)

1. An aqueous insulin formulation with an insulin concentration above 1 I.U./ml and a content of stabilizing physiologically acceptable agents, which has a viscosity of at least 1.75 mPa.s at 4°C and contains, as stabilizing physiologically acceptable agents, a combination A) of a physiologically acceptable thickener of the polygeline and/or dextran type with B) a physiologically acceptable surface-active substance from the group comprising BI) an ethylene glycol/propylene glycol copolymer or block polymer, at least half of the chain members, however, being units of 1,2-propylene glycol; B2) lecithin and/or B3) polyoxyethylene 23 lauryl ether.

2. An agent as claimed in claim 1, which has at least one of the features that a) the insulin concentration is up to 1,500, preferably between 5 and 1,000, I.U./ml, that b) the insulin is human insulin, a mammalian insulin, a modified insulin or a human proinsulin, that c) it contains more than 1, preferably 2 to 20, % by weight of thickener, that d) it contains polygeline as the thickener, that e) it has a pH of between 2.5 and 8.5, preferably between 6 and 8, and that f) it is in the form of a hydrogel at a temperature of 4 e C.

3. An agent as claimed in either of claims 1 and 2, which contains a) a suitable isotonicizing agent, b) a suitable preservative, c) a suitable buffer substance and/or d) zinc in an amount of up to 100 μg of zinc ions/100 I.U.

4. An agent as claimed in one or more of claims 1 to 3, which additionally contains an auxiliary with a retarding action. 1 8

5. An agent as claimed in one or more of claims 1 to 4, in which the viscosity of the formulation is at least 2, and in particular at least 2.5, mPa.s.

6. An agent as claimed in one or more of claims 1 to 5, in particular in the form of an injection solution, for use in the treatment of diabetes mellitus.

7.The use of the combination A) of a physiologically acceptable thickener of the polygeline and/or dextran type with B) a physiologically acceptable surface-active substance from the group comprising BI) an ethylene glycol/propylene glycol copolymer or block polymer, at least half of the chain members, however, being units of 1,2-propylene glycol, B2) lecithin, and/or B3) polyoxyethylene 23 lauryl ether for the preparation of a stabilized aqueous insulin formulation as claimed in one or more of claims 1 to 5.

8. An aqueous insulin formulation as claimed in claim 1, substantially as hereinbefore described and exemplified.