WO1999048520A1

WO1999048520A1 - Novel preparation of protracted acting human insulin

Info

Publication number: WO1999048520A1
Application number: PCT/DK1999/000157
Authority: WO
Inventors: Klavs Holger JØRGENSEN
Original assignee: Joergensen Klavs Holger
Priority date: 1998-03-24
Filing date: 1999-03-23
Publication date: 1999-09-30
Also published as: AU2827199A

Abstract

An injectable preparation of protracted acting human insulin in suspension form, wherein the concentration of insulin is strongly increased. The preparation is adapted for injection by an insulin pen and exhibits potential therapeutic advantages.

Description

1

NOVEL PREPARATION OF PROTRACTED ACTING HUMAN INSULIN

BACKGROUND OF THE INVENTION

When good blood glucose control of insulin requiring diabetes is aimed at today, intensive treatment is performed by 3-4 subcutaneous injections of rapid-acting insulin at meal times and one injection of protracted acting insulin in the evening. Some patients also receive a morning injection of protracted acting insulin. Another feature of modern insulin therapy is the use of reusable or disposable insulin pens, providing convenience for the patients and increase of life quality. Protracted acting insulin is also, and often as the only insulin, used for control of maturity onset diabetes.

Most of the insulin used to day is human insulin. The standard strength of insulin preparations is presently 100 IU/ml. Only soluble insulin preparations are available in higher strengths for special purposes. One formulation of protracted acting human insulin is human NPH with a concentration of insulin of 100 IU/ml ('TJ 100") . NPH (Neutral Rrotamin Hagedorn) was invented in the era of animal insulin by Krayenbϋhl and Rosenberg (Rep Steno Mem Ηosp Νord Insulinlab 1, 60-73(1946), USP 2,538,018). ΝPΗ contains tetragonal crystals of zinc, insulin and the basic polypeptide protamine in an amount just sufficient for the positive charge of protamine to neutralise the negative charge of the insulin, thereby lowering the solubility of the crystals to produce a protracted acting drug. The formation of the crystals is dependent on the presence of phenolic substances. In fact, only phenol and m-cresol are used in practise. The ratio between the amounts of protamine and insulin (in the order of 10 % by weight on the basis of insulin) is called the isophane ratio and is determined as the ratio giving minimum solubility of both insulin and protamine. The isophane ratio varies with different protamine preparations, pΗ, zinc and auxiliaries. It was found that ΝPΗ preparations could be mixed with neutral soluble insulin without loss of the fast action of the soluble insulin part and of the protracted action of the ΝPΗ part. Such preparations, in a premixed state, with the insulin components in different proportions have found widespread use for twice a day injections. This also goes for mixed preparations of human insulin (examples: Mixtard® 50/50 Νovo Νordisk and Ηumulin® 30/70 Lilly).

.Another protracted acting human insulin is human Ultralente which contains rhombohedral crystals of zinc and insulin. The protracted action of Ultralente is due to a high content of zinc (much higher than in ΝPΗ) and a large crystal size. For use as protracted acting human insulin in pen cartridges only human ΝPΗ is available, although the other and longer acting insulin, human Ultralente, is on the market as well, but 2 only in vials. The reason for that is the fact that the needle shaped protamine insulin crystals of NPH are so small (length less than 15 μm) , that they sediment slowly and are able to pass unfractionated through the finest injection needles of today. However, Ultralente crystals are so big (20-40 μm), that they sediment fast and have a tendency to clog fine needles by alignment across the inside of the needle, vide USP 5,462,535.

This last mentioned patent deals with an insulin pen with a G30 needle and a cartridge containing an insulin type which may flow freely through a G30 needle, including insulin crystals with max. size of 15 μm. NPH is fit for this pen but it is only mentioned indirectly in the patent in the following remark: "..the use of suspensions of insulin types having very short and needle shaped crystals totally eliminates the risk of bridging in a G30 needle when no dimension of the insulin crystals exceeds 15μm."

In the intensive insulin therapy human NPH is often used as the protracted acting insulin injected once per day, which however is not satisfactory for covering the insulin demand through 24 hours in many patients. Another disadvantage is an inappropriately early onset of action. There appears to be a tendency to a faster absorption of human compared with porcine and bovine NPH (Hildebrandt in Subcutaneous absorption of insulin in insulin-dependent diabetic patients, p 6, Lsgeforeningens Forlag, Copenhagen (1991)).

In the Danish Catalogue of Medicaments (Laegemiddelkataloget) from 1997, the information can be found that the onset of action of human NPH (Humulin® NPH Lilly or human Insulatard® NovoNordisk) occurs after /2-2 hours and that maximum effect is obtained after 4-8 hours. This means that NPH taken in the evening have maximum effect during sleep at which time hypoglycemia is very undesirable. For human Ultralente (Humutard Ultra Lilly) the corresponding figures are 4-6 hours and 8-24 hours, respectively. Importantly, this means that the maximum peak of action is less pronounced with human Ultralente. The original Ultralente (Schlichtkrull, in Insulin Crystals, p 92, Ejnar Munksgaard

Publishers, Copenhagen (1958)) was made from bovine insulin and was longer acting than 24 hours. Due to that, and the steady release of insulin, this preparation was considered the ideal preparation for control of the basal blood glucose level by only one injection per day. Unfortunately, the bovine Ultralente proved to be immunogenic. leading to the formation of insulin antibodies. When the originally applied impure bovine insulin was substituted by pure, monocomponent bovine insulin the immunogenicity was reduced. A further reduction was obtained with substitution by monocomponent porcine insulin. With the advent of semisynthetic followed by biosynthetic monocomponent human insulin this insulin was naturally used for an Ultralente preparation being the least or only slightly immunogenic. The prolonged effect of Human Ultralente proved to be less pronounced than that of bovine Ultralente, whereas porcine Ultralente showed an intermediate effect (Owens in Human Insulins, pp 170-177, MTP Press Limited, Lancaster(1986)). A longer lasting effect of human Ultralente has been obtained by adding zinc to this preparation to a total formulation concentration between about 0.5 and about 20 mg per 100 units of insulin, corresponding to 77-3086 zinc atoms/insulin hexamer (USP 5,534,488). However, this type of preparation can not be administered by an insulin pen with a fine needle, since the crystal size is the same as in unmodified human Ultralente. Another attempt of achieving a long-acting human insulin preparation is described in PCT

Application WO 88/02633. The preparation contains protamine, zinc and insulin (human, porcine or bovine). The crystals have a claimed length below 20 μm and a claimed insulin concentration in the range of from 20 to 500 IU/ml. A particularly prolonged action is obtained by ratios between the amounts of protamine and insulin and between the amounts of zinc and insulin being extremely higher than those of therapeutically applied human NPH. The preparation according to Application WO 88/02633 has never been made available for general use and is outside the scope of the present invention.

Together with the original long-acting bovine Ultralente an injectable short-acting suspension of amorphous porcine zinc insulin was introduced under the name of Semilente. At the same time an intermediate-acting 3:7 mixture of Semilente and Ultralente was introduced under the name of Lente. The size of the individual amorphous particles in Semilente is about 1 μm allowing unhindered passage of the particles through the finest injection needles. However, even though Semilente is more protracted acting than soluble insulin, it is less protracted acting than NPH. Human Semilente is a component in a 3:7 mixture with human Ultralente under the name of Monotard® Novo Nordisk (Brange et al., in Galenics of Insulin, pp 36-38, Springer-Verlag, Berlin (1987))

Insulin analogues have been prepared for use as long-acting preparations and may be well suited for administration by insulin pen once per day. Recently, two promising candidates have been described. B-3 l-B32-Di-Arg-human insulin has shown a flat insulin profile between 1 and 24 hours and a more slow absorption than NPH after subcutaneous injection in healthy subjects (Coates et al., Diabetes 44 (Suppl 1) 130A (1995)). Soluble fatty acid acylated insulin, capable of binding to albumin, has shown a slower absorption and a more flat profile of action than NPH after subcutaneous injection in pigs (Markussen et al., Diabetologia 39, 281-288 (1996)). However, no matter which preclinical models have been used, insulin analogs can always be suspected of having unwanted effects such as mitogenesis and immunogenicity, until long term clinical trials have confirmed whether an analog is safe (Johannesen et al., Diabetologia 40, B89-B93 (1997)). Indeed, serious side effects have been found unexpectedly, both with the rapid-acting insulin analog, Asp^B1° (showing in vitro mitogenic and in vivo carcinogenic properties in rats), and with the long-acting insulin analog, Novo Sol Basal (showing a local inflammatory reaction and increasing dosage requrements), as described by Barnett and Owens (Lancet 349, 47-51 (1997)). Another rapid-acting analog, insulin lispro, appears safe and efficient after long term clinical trials, vide the last quoted paper. The rapid-acting analog, insulin aspart, also appears promising so far (Home et al., 4

Diabetes Care, 1904-1909 (1998)). The long-acting insulin analogs, however, await more thorough investigations and long term trials before it can be decided, whether or not they are safe and efficient. Insulin analogs are outside the scope of the present invention. The main technical problem addressed by the present invention is the too fast absorption of current human insulin suspension preparations capable of being injected by means of an insulin pen comprising a fine injection needle without problems of clogging of the needle.

SUMMARY OF THE INVENTION

The object of the invention is to provide an injectable, protracted acting preparation comprising a liquid carrier and suspended therein human insulin containing particles, which regarding their kind and size of less than 15 μm are known for use in conventional insulin therapy, in which preparation the concentration of suspended human insulin is between about 200 and about 1200 IU/ml The above stated main technical problem is solved by the preparation of the present invention according to Claim 4, viz. an injectable, protracted acting preparation comprising a liquid carrier and suspended therein tetragonal crystals containing human insulin, protamine and zinc, which crystals regarding their kind and size of less than 15 μm are known for use in conventional insulin therapy in the form of human NPH , in which preparation the concentration of suspended human insulin is between about 200 and about 1200 IU/ml.

The preparation according to Claim 4 is new and useful, since the invention is based on the discovery that, when the insulin concentration in human NPH is strongly increased, preparations are obtained, which combine the advantages of the two prior art products NPH and Ultralente (both having a strength of maxJOO IU/ml), i.e. a small particle size of lower than 15 μm, and a slow onset of action combined with a long duration of action, respectively. It is surprising that the protraction properties are substantially improved by the increase of insulin concentration.

The preparation preferably exhibits such a slow onset of action, that the risk of hypoglycaemia is reduced, and such a high degree of protraction that appropriate control of the basal blood glucose level can be provided by one injection per day only.

Another technical problem, a too fast final action of the NPH part in premixed conventional preparations (such as Mixtard® 50/50 Novo Nordisk and Humulin® 30/70 Lilly), is met by the preparation of the present invention according to Claim 5. A further technical problem, a too fast action of human Semilente, is met by the preparation according to Claim 6. Human Semilente is known as a component admixed with human

Utralente in Monotard® Novo Nordisk. There is still an interest in Semilente for treatment of adolescents (Holl and Heinze, Exp Clin Endocrinol Diabetes, 104 (1996)). The more concentrated and more slowly acting amorphous human insulin may therefore cover a demand 5 within a certain segment of insulin therapy.

The higher insulin strength (at least twice the normal) in the preparation of the present invention is advantageous per se, in that the smaller volume thus obtained for a given insulin dose might be less painful for the patient to inject. The insulin particles in the preparation of the present invention are small enough to allow free flow without fractionation through the finest needles available today.

By use of human insulin it is ensured that the injected insulin after having been absorbed and delivered to the blood stream is behaving exactly as endogenous insulin, thereby minimising the risk of unwanted effects in patients. This is important, inter alia in light of the serious unwanted effects of the two insulin analogs mentioned earlier in the text..

In a preferred embodiment of the invention the concentration of suspended human insulin is between about 500 and about 1100 IU/ml, more preferred about 1000 IU/ml.

In another preferred embodiment of the invention the particles are tetragonal protamine zinc insulin crystals, as in NPH. In an alternatively preferred embodiment of the invention the suspended crystals are admixed with dissolved human insulin in the concentration range of from about 20 to about 1200 IU/ml.

In a further preferred embodiment of the invention the particles are amorphous, as in Semilente. It is preferred, that the preparation according to the invention is contained in a cartridge for insertion into an insulin pen.

The present invention further relates to a preparation as defined in claim 1 for use as a medicament for treating or preventing diabetes mellitus.

Also, the present invention relates to the use of a preparation as defined in claim 1 for the manufacture of a medicament for treating or preventing diabetes mellitus.

Finally, the present invention relates to a method of treating or preventing diabetes mellitus, which comprises administering subcutaneously to an individual in need of such treatment or intervention the preparation of the invention.

Studies have shown a preventive effect of administering insulin subcutaneously to persons (prediabetics) susceptible for developing diabetes. For these clinically healthy persons the risk of hypoglycemia provoked by the insulin administration is existing and should be minimised.

DETAILED DESCRIPTION OF THE INVENTION

In the preparation according to Claim 4 the zinc content in the crystals, the auxiliaries and their concentrations can be chosen to be similar to those of a current human NPH preparation. However, changes can be made on condition that the kind of the crystals is not changed, and that their size is still less than 15 μm. The ratio between the amounts of protamine and insulin 6 should be close to the isophane ratio determined as known in the art, e.g. as described by Krayenbuhl and Rosenberg (Rep Steno Mem Hosp Nord Insulinlab 1, 60-73(1946)).

The preparation according to Claim 4 can be produced by removing so much of the liquid carrier from a human NPH preparation (by filtration or by suction of supernatant after sedimentation or centrifugation), that the desired degree of concentration is obtained, as illustrated in Example 1. However, the preparation is preferably produced in such a way that the desired strength of insulin is obtained directly, as illustrated in Example 5.

The premixed preparation according to Claim 5 is made in analogy to a method known in the art of producing of a current premixed preparation. The preparation according to Claim 6 can be produced by removing so much of the liquid carrier from a human Semilente U100, (by filtration or by suction of supernatant after sedimentation or centrifugation), that the desired degree of concentration is obtained, as illustrated in Example 2. However, the preparation is preferably produced in such a way that the desired strength of insulin is obtained directly, as illustrated in Example 7. When the insulin concentration is raised, the concentration of total zinc has to be adjusted, as it is exemplified for Lente preparations with strengths from 40 to 100 IU/ml by Brange et al. (in Galenics of Insulin, p 37, Springer- Verlag, Berlin (1987)). Apart from zinc, auxiliaries and their concentrations can be chosen to be similar to those of Semilente. In order to avoid lump formation in the more concentrated Semilente preparations, prepared in the direct way, it is recommended that zinc salt and base solutions are added in a balanced way, as in Example 7. If the preparation, according to the invention, is filled into conventional vials, the syringe size and the scale graduation of the syringe used for injection should be adapted to the new insulin strength, such that the patient is able to inject the intended dose. However, because of the risk of administering the wrong dose by unintentional interchange of these syringes and conventional syringes, and for the sake of accuracy and convenience for the patient, the preparation should preferably be injected by a reusable or rather a disposable insulin pen with a cartridge different from conventional cartridges, in order to avoid interchange, and with a pen adapted for the special use. The cartridge should contain a ball that could easily be moved from one end to the other, when the insulin pen is turned upside down in order to ensure that an even distribution of crystals is obtained at the time of injection.

The invention is further illustrated in the following examples, which however are not to be construed as limiting.

EXAMPLE 1

1.6 ml of human NPH U100, supplied by Novo Nordisk under the name of Protaphan Zn HM with 5 zinc atoms/insulin hexamer, was pipetted into a tube and centrifuged. 1.44 ml of the supernatant was sucked off and the precipitate suspended in the residual supernatant. This suspension was named NPH U1000.

EXAMPLE 2

A solution of 85.5 mg/ml human insulin (2311 IU/ml) with a pH of 7.8 was prepared by dissolving 1.05 g human monocomponent insulin containing 2.2 zinc atoms/insulin hexamer in 9.5 ml water and 0.8 ml 1 mol/1 HCl, followed by pH adjustment with 1.2 ml 1 mol 1 NaOH. 216 μl of this insulin solution (500 IU) was mixed with 4 ml water, 500 μl 1.6 g/1 methyl-p- hydroxybenzoate, 150μl 4 mol 1 NaCl and 12.5 μl 4 mol/1 sodium acetate. 105 μl 0.1 mol/1 zinc acetate was then added under vigorous stirring, resulting in an amorphous suspension. The pH value was adjusted to 7.4 with 1 mol 1 NaOH, and the volume was adjusted to 5 ml with water. 1.6 ml of this amorphous suspension, named AS U 100, was pipetted into a tube and centrifuged. 1.44 ml of the supernatant was sucked off and the precipitate suspended in the residual supernatant. This suspension with an insulin concentration of 1000 IU/ml was named AS U1000.

EXAMPLE 3

250 μl of the preparations, NPH UlOO from Example 1 and AS UlOO from Example 2 were pipetted into tubes and rotated for 3 hours at 37 °C. The tubes were then centrifuged and the insulin contents of the supernatants determined by HPLC on a C4 column. The found percentages of insulin dissolved per se are shown in Table 1. An Artificial Interstitial Liquid, AIL, was prepared containing 20 g/1 human albumin, 111 mmol/1 NaCl, 4 mmol/1 KC1, 31 mmol/1 NaHCO₃, 2.5 mmol/1 CaCl₂, 1 mmol/1 MgCl₂, 1 mmol/1 Na₂HPO₄ and 0.5 mmol/1 Na₂SO₄. The pH of AIL was adjusted to 7.3 with HCl just before use. 125 μl of NPH UlOO and NPH U1000 from Example 1, and 125 μl AS UlOO and AS U1000 from Example 2 were pipetted into 4 tubes. 125 μl AIL was added to each tube that was of such a size, that just a small air bubble was left in the tube after it was stoppered. The tubes were then rotated for 3 hours at 37 °C, the movement of air bubbles up and down allowing the particles to be kept evenly suspended. The tubes were centrifuged and the insulin contents of the supernatants determined by HPLC on a C4 column effecting a complete separation of insulin and albumin. The found percentages of insulin dissolved in the 1:1 mixtures of preparation and AIL are shown in the table below.

Preparation NPH UlOO NPH Ul 000 AS 100 AS 1000 |

% ins. dissolved - AIL < 0.2 _ 0.5 1

% ins. dissolved + AEL 9 1.5 15 < 0.2 8

It appears that % insulin in solution without AIL was very low for both NPH UlOO and AS UlOO (< 0.2% and 0.5 %, respectively), whereas % insulin in solution with 1: 1 AIL was 9% and 15%, respectively. However, NPH U1000 and AS U1000 surprisingly displayed a much reduced solubility (% insulin in solution with 1: 1 AB : 1.5% and < 0.2%, respectively), indicating that they would also be absorbed more slowly after a subcutaneous injection.

EXAMPLE 4

For use in absorption studies in pigs preparations of NPH UlOO and NPH U500 were labelled with trace amounts of mono- 17 ~5J-(Tyr Al 9)-insulin, in the following way.

12 ml of NPH UlOO (Insulatard® Novo Nordisk, 5 zinc atoms/insulin hexamer) was centrifuged in a tube. 11.4 ml of the supernatant was transferred to another tube. 30μl was removed from this tube and 15μl 5 mol/1 NaOH added. 15 μl 5 mol 1 HCl was added to the first tube and mixed with the precipitate and the residual supernatant while stirring.

19 -5J-insulin was added to the acid solution thus obtained. The solution was then transferred by a pump to the stirred content of the other tube, in the course of about half an hour. The mixture was left overnight without stirring. The next day the precipitate was totally crystalline with crystals having a length of 5-10 μm and with an appearance like the crystals of the parent

NPH. The pH value was 7.3, the same as that in the parent NPH. The radioactivity concentration was 13 μCi/ml. The preparation so prepared was named *NPH UlOO and used as a reference in all of the 14 pig experiments. 7 ml of the *NPH UlOO was centrifuged in a tube. 5.6 ml of the supernatant was removed, and the precipitate suspended in the residual supernatant. This preparation was named *NPH

U500 and used in 6 of the 14 pig experiments. *NPH U500 with a ratio between the amounts of protamine and insulin close to the isophane ratio and with 5 zinc atoms/insulin hexamer was also made in such a way that the correct strength was obtained directly, in the following way. A solution of 90.3 mg/ml (2441 IU/ml) human insulin with 2.2 zinc atoms/insulin hexamer and with a pH of 7.8 was prepared in analogy with the insulin solution in Example 1. From this solution 267 μl was pipetted into a tube and mixed with 129 μl water, 24 μl 1 mol 1 HCl,

1

1.8 μl 1 mol/1 zinc chloride and 228 μl 10 mg/ml protamine sulphate. I-insulin was added to the acid solution thus obtained. 600 μl of the solution (600 IU) was then transferred by a pump, in the course of about half an hour, to the stirred solution in another tube, containing a mixture of 120 μl 160 g/1 glycerol, 180 μl 10 g/1 m-cresol, 19.5 μl 40 g/1 phenol, 156 μl 0.1 mol/1 Na₂HPO 20.5 μl 1 mol/1 NaOH and 104 μl water. The pH value (7.2) was adjusted to 7.3 with 1 μl NaOH. The mixture was left overnight at room temperature without stirring. The next day the precipitate was totally crystalline with crystals having a length of 5-10 μm and with an appearance like the crystals of normal NPH. The concentration of radioactivity was 4.5 μCi ml. This preparation was also named *NPH U500 and used in 8 of the 14 pig experiments.

The labelled preparations were filled into 1.5 ml NovoPen cartridges, each containing a glass ball. Each cartridge was then inserted in a NovoPen® 1.5.

In each of 14 experiments in 7 pigs 10 IU of *NPH UlOO (100 μl) and 10 IU of *NPH U500 (20μl) were injected contralaterally by the insulin pen to a depth of 5 mm in the neck of the pig. The absorption was followed by measuring the radioactivity over the injection site after injection and at approx. 2, 4, 24 and 48 hours, and in 4 cases furthermore at approx. 6 and 8 hours after injection. The measured values were transformed to values at exactly 0, 2, 4, (6), (8), 24 and 48 hours, respectively, by calculation based on first order kinetics.

The table below shows the mean values in % of the value at zero time. It appears that *NPH U500 is absorbed more slowly throughout the time course than the same dose of *NPH UlOO.

Hours after N % Residual radioactivity, mean values p-values injection *NPH U500 SEM *NPH U100 SEM paired t-test

0 14 100 0 100 0 -

2 14 89 1.6 87 1.6 not sign.

4 14 79 1.8 73 1.6 0.004

6 4 67 3.5 57 4.9 0.06

8 4 56 4.3 45 5.2 0.08

24 14 26 2Λ 16 JO 0.001

48 13 5 0.7 3 0.4 0.02

The impact of the difference may be better understood from the table below showing calculated mean values of T75%, T50% and T25%, designating the number of hours at which just 75%, 50% and 25% of the original radioactivity, respectively, are still at the injection site. It appears that all three values are significantly higher for *NPH U500 than for *NPH UlOO, being about 5 hours vs. 4 hours for T75%, 12 hours vs. 9 hours for T50% and 24 hours vs. 18 hours for T25%.

T75%. hours T50%. hours T25%. hours

^♦NPH U500 *NPH U100 *NPH U500 *NPH U100 *NPH U500 *NPH U100 j

Mean 5.0 3.8 12.2 9.0 24.2 17.9 |

SEM 0.5 03 0.9 0.6 1.2 1.2 ^'<

N 14 14 14 14 14 14 1 p (paired t-test) 0.007 0.003 0.001

Difference in 1.2 3.2 6.3 relation to

NPH UlOO 32% 35% 35% Interestingly, the T25% values indicate that human NPH U500 has about 6 hours longer maximum duration in patients than the usual maximum duration of 14-18 hours for current 10 human NPH (re the table on page 36 in "1999 Resource Guide", Diabetes Forecast). This prolongation is precisely of the order, that clinicians consider appropriate for a protracted acting preparation given once a day. Furthermore, as the results point to a later onset of action for human NPH U500 , the risk of hypoglycemic attacks could well be reduced in patients by substitution of this preparation for current human NPH UlOO.

EXAMPLE 5

100 ml of an injectable preparation of human NPH U500, for use in insulin therapy, according to the present invention, is prepared in the following way.

An amount of dry, crystallized human insulin (about 1.85 g), corresponding to 50000 IU, is dissolved in 20 ml of water and 1.38 ml 1 mol/1 HCl. An amont of protamine sulphate, determined beforehand to provide the isophane ratio together with 50000 IU human insulin, is added in the form a 10 mg/ml protamine sulphate solution. Then a solution of 1 mol/1 ZnCl is added in a volume that gives a total content of 5 zinc atoms per insulin hexamer (inclusive the zinc content in the applied dry insulin). Finally the volume is adjusted to 50 ml with water.

50 ml of an aqueous solution with a total content of 1.3 mmol Na₂HPO 0.2 g m-cresol, 0.085 g phenol, 1.6 g glycerol and 1,77 mmol NaOH is pressed through a sterilization filter into a sterilized container. The 50 ml acid solution is then pressed, in the course of about half an hour, through a sterilization filter into the container, in which the mixture is stirred. Five minutes after the addition of acid solution has been finished, pH is measured and if outside the range of 7.2-7.4 adjusted hereto. The mixture is left overnight at 15-20 °C without stirring.

The next day pH is controlled again and the preparation inspected in the microscope. If the appearance and the size of crystals and the results of other required examinations are acceptable, the preparation is transferred aseptically to cartridges for insertion in insulin pens.

EXAMPLE 6

For use in absorption studies in pigs human amorphous insulin preparations of Semilente type, HSL UlOO and HSL U500 were labelled with trace amounts of mono-¹²⁵I-(Tyr A19- insulin, in the following way. From the solution of 90.3 mg/ml (2441 IU/ml) human insulin with 2.2 zinc atoms/insulin hexamer and with a pH of 7.8 (described in Example 4) 287 μl (700 IU) was pipetted into a tube and mixed with 4.375 ml 1.6 g/1 methyl-p-hydroxybenzoate, 1.875 ml water and 30 μl 1 mol/1 HCl. I-insulin was then added to the acid solution thus obtained. While stirring 30 μl 1 mol/1 NaOH, 203 μl 4 mmol/1 NaCl, 147 μl 0.1 mol/1 zinc acetate and 17 μl 4 mol/1 sodium acetate were added. The pH value (6.5) was then adjusted to 7.3 with 1 mol/1 NaOH. Finally water was added to 7 ml. The labelled preparation, named *HSL UlOO, contained fine 11 amorphous particles without lumps.

5 ml of the *HSL UlOO was centrifuged in a tube. 4 ml of the supernatant was removed, and the precipitate suspended in the residual supernatant. This preparation was named *HSL U500. The labelled preparations were filled into 1.5 ml NovoPen cartridges, each containing a glass ball. Each cartridge was then inserted in a NovoPen® 1.5.

Absorption studies with *HSL UlOO and *HSL U500 were performed in 4 pigs, in the same way as described in Example 4.

The two tables below show the mean values in % of the value at zero time and the T75%, T50 % and T25 % values, respectively.

Hours after N % Residual radioactivity, mean values p-values t injection *HSL U500 SEM *HSL U100 SEM paired t-test

0 4 100 0 100 0 .

2 4 79 6.9 62 3.0 0.05 |

4 4 59 9.6 34 5.0 0.05 <

24 4 4 1J 2 03 0.05 1

48 4 2 0.5 1 0.1 0.07 !

T75%. hours T50%. hours T25%, hours

*HSL U500 *HSL U100 *HSL U500 *HSL U100 *HSL U500 *HSL U100

Mean 2.7 1.2 5.3 2.8 10J 5.8

SEM 0.7 0J 1.0 0.3 1.5 1.0

N 4 4 4 4 4 4 p (paired t-test) 0J 1 0.07 0.05

Difference in 1.5 2.5 4.3 relation to

NPH UlOO 125 % 89 % 74 %

It appears that *HSL U500 is absorbed more slowly throughout the time course than the same dose of *HSL UlOO, which is surprising. By comparison with the corresponding tables in Example 4, however, it can be seen that *HSL U500 is absorbed faster than *NPH UlOO.

EXAMPLE 7

100 ml of an injectable preparation of human HSL U1000, for use in insulin therapy, according to the present invention, is prepared in the following way.

An amount of dry, crystallized human insulin (about 3.7 g), corresponding to 100000 IU, is dissolved in 50 ml 2 g/1 methyl-p-hydroxybenzoate, 8 ml water and 2.76 ml 1 mol/1 HCl. Then 4.14 ml 1 mol/1 NaOH and 2.85 ml 4 mol/1 NaCl are added while stirring. The solution is pressed through a sterilization filter into a sterilized container. 14.3 ml of 0.1 mol 1 zinc acetate with a content of 0.002 mol/1 HCl and 14.3 ml 0.08 mol/1 NaOH are then pressed at 12 the same rate, and in the course of about 10 minutes, through separate sterilization filters into the container, in which the mixture is stirred. Five minutes after the addition of the two solutions has been finished, pH is measured and if outside the range of 7.2-7.4 adjusted hereto. Finally the volume is adjusted to 100 ml with water and the preparation inspected in the microscope. If the appearance of the amorphous material and the results of other required examinations are acceptable, the preparation is transferred aseptically to cartridges for insertion in insulin pens.

Claims

13 CLAIMS

1. An injectable, protracted acting preparation comprising a liquid carrier and suspended therein human insulin containing particles, which regarding their kind and size of less than 15 ╬╝m are known for use in conventional insulin therapy, in which preparation the concentration of suspended human insulin is between about 200 and about 1200 IU/ml.

2. The preparation according to Claim 1, in which the concentration of suspended human insulin is between about 500 and about 1100 IU/ml.

3. The preparation according to claim 2, in which the concentration of suspended human insulin is about 1000 IU/ml.

4. The preparation according to any of claims 1 to 3, in which the suspended particles are tetragonal protamine zinc insulin crystals, as in human NPH.

5. The preparation according to Claim 4, in which the suspended crystals are admixed with dissolved human insulin in the concentration range of from about 20 to about 1200 IU/ml.

6. The preparation according to any of claims 1 to 3, in which the particles are amorphous, as in human Semilente.

7. The preparation according to any of the preceding claims, in which the preparation is contained in a cartridge for insertion into an insulin pen.

8. An injectable, protracted acting preparation comprising a liquid carrier and suspended therein human insulin containing particles, which regarding their kind and size of less than 15 ╬╝m are known for use in conventional insulin therapy, in which preparation the concentration of suspended human insulin is between about 200 and about 1200 IU/ml, the said preparation being for use as a medicament for treating or preventing diabetes mellitus.

9. Use of an injectable, protracted acting preparation comprising a liquid carrier and suspended therein human insulin containing particles, which regarding their kind and size of less than 15 ╬╝m are known for use in conventional insulin therapy, in which preparation the concentration of suspended human insulin is between about 200 and about 1200 IU/ml, for the manufacture of a medicament for treating or preventing diabetes mellitus.

10. A method of treating or preventing diabetes mellitus which comprises administering subcutaneously to an individual in need of such treatment or intervention the preparation of the invention. AMENDED CLAIMS

[received by the International Bureau on 10 August 1999 (10 08 99), original claims 1,2,7,8 and 9 amended, remaining claims unchanged (1 page)]

1 An injectable, protra╬▒ed acting human insulin preparation compnsing a liquid earner and suspended therein human insulin contaimng particles, which regarding their kind and size of less than 15 ╬╝m are known for use conventional insulin therapy, in which preparation the concentration of suspended human insulin is between 200 and 1200 IU/ml

2 The preparation according to Claim 1, in which the concentration of suspended human insulin is between 500 and 1100 IU/ml

3 The preparation according to claim 2, in which the concentration of suspended human insulin is about 1000 IU/ml

4 The preparation according to any of claims 1 to 3, in which the suspended particles are tetragonal protamine zinc insulin crystals, as in human NPH

5 The preparation according to Claim 4, in which the suspended crystals are admixed with dissolved human insulin in the concentration range of from about 20 to about 1200 IU/ml 6 The preparation according to any of claims 1 to 3, in which the particles are amorphous, as in human Semilente

7 The preparation accordmg to any of the preceding claims, the said preparation bemg contained in a cartndge for insertion into an insulin pen

8 -An injectable, protracted acting preparation accordmg to any of the preceding claims, the said preparation being for use as a medicament for treating or preventing diabetes mellitus

9 Use of an injectable, protracted acting preparation according to Claim 8, for the manufacture of a medicament for treating or preventing diabetes mellitus

10 A method of treatmg or preventing diabetes mellitus which compnses administering subcutaneously to an individual in need of such treatment or intervention the preparation of the invention

AMENDED SHEET (ARTICLE 1S)