GB2119248A - Insulin formulations and method of producing them - Google Patents

Abstract

An insulin formulation comprises a solution of insulin in a dipolar aprotic solvent. A method of purifying insulin includes the steps of introducing a solution of impure insulin in a dipolar aprotic solvent into deionised water and removing the insulin precipitated therefrom. The solvents may be dimethyl sulphoxide, dimethyl sulpholane or 5,5-dimethyl-1,3-cyclohexanedione. The insulin formulations are stable and non-corrosive, and are especially suitable for use in implanted pump delivery systems for continuous insulin administration.

Description

SPECIFICATION Insulin formulations and method of producing them In recent years mechanical insulin delivery systems have been employed to gain better control over the metabolic and hormonal disturbances which accompany conventional insulin therapy. The development of these systems has been retarded by the corrosive and unstable nature of available insulin formulations.

Insulin is barely soluble in pure water. To achieve useable concentrations of insulin in aqueous solutions it is necessary to add salts, buffers and/or acids as hydrotropic agents. As these solutions are very corrosive the range of material and the methods used to construct mechanical insulin delivery systems are limited. Furthermore aqueous solutions of insulin are unstable when exposed to heat, electrical potential or mechanical agitation. Thus when implanted at body temperature, or exposed to galvanic corrosion potentials or subjected to mechanical trauma in a pump the insulin molecules in available formulations aggregate to form a sludge which not only impairs the proper function of the delivery system but also affects the pharmokinetics of the hormone.In a severe case the insulin molecule may be disrupted rendering it biologically inactive, and, as the fragments are no longer identifiable as normal body constituents, hypersenstivity reactions occur in the patient.

In the case of corrosion the aggregation of insulin molecules is rapid. It is known, from electrophoretic studies on insulin, that at physiological hydrogen ion concentrations, (circa pH 7.4) the preferred form of aqeuous solution, that insulin carries an overall negative charge. In a galvanic corrosion cell insulin is therefore attracted to the anode where it meets a high concentration of positive metal ions going into solution. These ions attract large numbers of the negatively charged insulin molecules to form large aggregates of insulin/metal complexes which precipitate out of solution.

A further problem facing the designer of implanted systems is that it is difficult to achieve concentrations of insulin much in excess of 500 international units of insulin per millilitre (iu/ml). Clearly if a higher concentration were available then reservoirs could be smaller for a given period between refills.

According toone aspect of the present invention, there is provided an insulin formulation comprising a solution of insulin in a dipolar aprotic solvent.

According to another aspect of the present invention there is provided a method of preparing an insulation formulation including the step of introducing insulin into a dipolar aprotic solvent.

In an advantageous embodiment of the aspects of this invention, the dipolar solvent is selected from dimethyl sulphoxide, or dimethyl sulpholane or 5,5-dimethyl-1,3-cyclohexanedione.

In another advantageous embodiment a non-toxic dipolar solvent is produced by splitting the long chain fatty acid from phsopholipids.

According to a still further aspect of the invention, there is provided a method of purifying insulin contaminated with other proteins including the step of introducing a solution of the contaminated protein in a dipolar aprotic solvent into deonised water and removing the insulin precipitated out.

In order that the invention may be more clearly understood, one embodiment thereof will now be described, by way of example, with reference to the accompanying drawings, in which: Figures 1 and 2 are simplified schematic representations of the electric charges exhibited by a double chain insulin molecule in solution above and below its isoelectric point of pH 5.5 respectively; Figure 3 is a simplified schematic representation illustrating how ions in solution are held by the insulin molecule of Fig. 1 above the isoelectric point; Figure 4 diagrammatically illustrates the effect of mechanical agitation on the structure of Fig. 3; and Figure 5 is a simplfied schematic representation of the solution of the insulin molecule of Fig. 1 above the isoelectric point of pH 5.5 in dimethyl sulphoxide.

Crystalline insulin normally requires hydrotropic agents, for example sodium chloride and hydrochloric acid, to enable it to dissolve in water. Above the isoelectric point of pH 5.5 the solvated molecule carries an overall negative surface charge. Below the isoelectric point an overall positive charge can be demonstrated. The large, complex, double chain insulin molecule carries both charges. The pH of the solution determines whether these charges are internally or externally placed. Figs. 1 and 2 are simplified, schematic representations of both situations. Obviously the real situation is much more complex and the number of charges would be many times greater. The charges are in the configuration of two Helmholtz double layers interleaved with the A and B chains (1 and 2) of the insulin molecule.

Insulin forms a clear solution which has a well defined absorption maximum at 274-279 nanometre (nm) and is therefore totally solvated. A partially solvated particle would be expected to scatter light to give a cloudy solution with a very broad or multiple absorption maximum.

Based on the schematic representations of Figs. 1 and 2 it is possible to describe how the solvation of the insulin molecule occurs as a result of the presence of hydrotropic ions, for example sodium (NA+), chloride (Cl-), hydrogen (H+) and hydroxyl ions (OH-). The schematic representations in Fig. 3 shows how the charges on the insulin molecule attract, and hold these ions. The representation depicts the situation where the solution is at a pH higher than the iso-electric point of insulin.

As the positively charged ions are attracted to the negatively charged areas on the insulin molecule and the negatively charged ions are attracted to the positively charged areas on the insulin molecule, adjacent ions will as they are like charges repel each other until a state of unstable equilibrium exists. Using this model it can be seen that the polarity of the charge on the molecule is that of the hydrotropic ions and therefore opposite to the charge on the insulin molecule. The A and B chains are indicated at 3 an 4 respectively. The solvation shell is shown at 5.

When this relatively loose, unstable situation is disrupted during mechanical agitation cross linking will occur as depicted in Fig. 4 which shows part of two insulin molecules.

Cross linking to form polymers can continue to a stage where aggregates visible to the naked eye form. These large aggregates, can, because of the momentum they gather during agitation, achieve sufficient energy to impact the containing vessel with sufficient force to denature the insulin molecules trapped on the surface.

During the process of corrosion the situation becomes more unstable due to the flow of electrical current between anodic and cathodic areas in the corrosion cell. In addition the high concentration of cations going into solution at the corrosion cell anode causes insulin to agglomerate.

Since the situation in aqueous solutions with conventional hydrotropic agents is unsatisfactory a new approach is necessary. This new approach comprises introducing the insulin into a non-ionic dipolar solvent so that the dipoles align themselves with the cnarges in the insulin molecule to result in total solvation as dipicted schematically in Fig. 5. In the case where all of the solvent molecules 8 are dipoles the insulin crystals would probably be dispersed until the viscosity of the solution was too high to allow mixing to occur. Due to the surface arrangement of the dipoles on the insulin molecules such solutions are stable and monomeric as linking would not occur between adjacent molecules.This is because, as is shown in Fig. 5, each chain 6, 7 of the double chain exhibit a charge which repels rather than attracts the next adjacent molecule, those charges being provided by the dipoles which are held tightly by the chains themselves.

Dimethyl Sulphoxide (DMSO), which has the empirical formula C2H60S, was chosen as the dipolar solvent to demonstrate the above described action.

Insulin crystals, assayed at 25 international units per milligram (iu per mg), were dissolved in DMSO and concentration of 1 5,000 iu/ml was achieved. The solution, now highly viscous, remained optically clear and observations using both a transmission electron microscope and a photoelectric/photomultiplier tube showed that even at this high concentration insulin was dispersed in monomeric form.

To test its stability to mechanical agitation samples were prepared at concentrations of 80 iu/ml and 1000 iu/ml. These were placed in a Stuart flask shaker with commercially available aqueous solutions of insulin which had concentrations of 80 iu/ml. All of the containers were 10 ml plastic tubes and contained 5 ml of the insulin solutions. The flask shaker was set to reciprocate at 1 ,000 cycles per second in an oven at 40"C and the samples were examined at 1 2 hourly intervals. At the end of 36 hours all of the aqueous solutions contained aggregates visible to the naked eye. The DMSO samples remained clear at 90 days. Samples examined under the electron microscope and by the photoelectric/photomultiplier tube at 30 and 60 days revealed that insulin remained dispersed in the monomeric form.

The resistivity of the DMSO/insulin solution was measured in a Siemens Resistivity cell using a Wayne Kerr bridge. All concentrations had a resistivity of 34 mega-ohms per centimetre. Aqueous solutions, measured using the same equipment, had a resistivity which varied little around 5 ohms/cm.

It was also noted during the course of experimentation that the dispersal of crystalline insulin in aqueous solutions required mechanical agitation. When insulin and DMSO were placed in the same container dispersion occurred without mechanical agitation.

The addition.of DMSO/insulin solution to deionised water caused heat to be generated in the resultant solution (indicating ionic rearrangement) and the dispersed insulin rapidly precipitated out of solution in crystalline form.

Solutions of DMSO made with other body proteins added to de-ionised water did not precipitate. When a solution of DMSO/insulin is added to water containing hydrotropic agents or human plasma no precipitation is noted.

Similar results were obtained using other dipolar solvents, for example, dimethyl, sulphane and 5, 5-dimethyl-1 , 3-cyclohexanedi- one.

The above experimental results demonstrated the following: (1) DMSO/insulin solutions are much more stable when exposed to mechanical agitation, even at temperatures above normal body temperature, than aqueous solutions, (2) Because of the high resistivity of DMSO/insulin solutions corrosion is eliminated as corrosion currents will be minute.

(3) DMSO/insulin solutions are monomeric.

(4) DMSO/insulin solutions can be prepared at higher concentrations than aqueous/insulin solutions.

(5) DMSO/insulin solutions mix, without recrystallisation of the dispersed insulin, in plasma or aqueous solutions containing hydro- tropic agents so they can be injected without recrystallisation occurring to block delivery catheters.

(6) As insulin precipitates out of a DMSO/ insulin solution, when added to de-ionised water and this behaviour is different to that of other proteins so it provides a method for extracting and purifying insulin from contaminating proteins during the manufacture of insulin from pancreas, or other processes, for example bacterial culture.

(7) The preparation of DMSO/insulin solutions do not require mechanical agitation thus making the manufacturing process less costly.

It will be appreciated that the above embodiment has been described by way of example only and that many variations are possible without departing from the scope of the invention. For example splitting the long chain fatty acid residue from phospho-lipids would leave a non-toxic dipolar solvent.

Claims (11)

1. An insulin formulation comprising a solution of insulin in a dipolar aprotic solvent.

2. A method of preparing an insulin formulation comprising the step of introducing insulin into a dipolar aprotic solvent.

3. A formulation as claimed in claim 1, wherein the solvent is dimethyl sulphoxide.

4. A formulation as claimed in claim 1, wherein the solvent is dimethyl sulpholane.

5. A formulation as claimed in claim 1, wherein the solvent is 5,5-dimethyl-1,3-cyclohexanedione.

6. A formulation as claimed in claim 1, wherein the solvent is produced by splitting the long chain fatty acid from phospho-lipids.

7. A method of purifying insulin including the steps of introducing a solution of impure insulin in a dipolar aprotic solvent into deionised water and removing the insulin precipitated therefrom.

8. A method as claimed in claim 2, wherein the solvent is dimethyl sulphoxide.

9. A method as claimed in claim 2, wherein the solvent is dimethyl sulpholane.

10. A method as claimed in claim 2, wherein the solvent is 5,5-dimethyl-1,3-cyclohexanedione.

11. A method as claimed in claim 2, wherein the solvent is produced by splitting the long chain fatty acid from phospho-lipids.

1 2. A formulation as claimed in claim 1, substantially as hereinbefore described.

1 3. A method as claimed in Claim 2, substantially as hereinbefore described.