WO1993007890A1 - Treatment of haemophilia - Google Patents

Abstract

Factor IX is used to treat patients suffering from haemophilia B by subcutaneous injection.

Description

TREATMENT OF HAEMOPHILIA

This invention relates to the treatment of haemophilia and is particularly concerned with the administration of factor IX.

Factor IX is a component of the blood-clotting mechanism of warm-blooded animals : it is Involved together with many other blood clotting factors in the blood coagulation cascade as reviewed by Furie B. and Furle B. C, Cell, 53, 1988, pages 505-518. Haemophilia B, or Christmas disease, is a serious inherited bleeding disorder affecting males and caused by a defect in clotting factor IX. Control of the disease is effected by the need for repeated injections of factor IX concentrate, conventionally obtained from blood donors, although more recent technology (as exemplified by EP-A-195592) allows its preparation by recomblnant DNA technology.

Factor IX is known to be a single-chain glycoprotein with a molecular weight of approximately 60,000. It is therefore a sufficiently large molecule to point to the need for direct introduction into the bloodstream. Furthermore quite large doses of factor IX (e.g. of the order of 5,000 international, units) may be required for an adult, for example during surgery. Consequently, factor IX preparations have conventionally been delivered intravenously to patients, either prophylactlcally or in response to bleeding episodes in order to control haemophilia B. Indeed, as early as 1929, it was recognised by Payne and Steen (The British Medical Journal, June 19, 1929, pages 1150-1152) in their article "Haemostatic Therapy in Haemophilia", that treatment of haemophiliacs with citrated human plasma was only effective when given intravenously and was ineffective when given intraperitoneally or intramuscularly. Such repeated use of intravenous injections, while necessary to control the disease, has side effects. Repeated injection leads to the vein at the site of injection becoming fibrosed or occluded, a problem especially acute when treating the elderly. Also, when veins are small, as in babies, it may be difficult for the doctor to insert a needle into the vein to inject the required therapeutic dose.

More recent research has directed attention to the possibility of haemophilia B treatment by gene therapy. Thus Anson et al.,(Mol. Biol. Med., 1987, 4, pages 11-20), St. Louis and Verma (Proc. Natl. Acad. Sci., USA, 85, 1988, pages 3150-3154 and Palmer et al. (Blood, 73 No. 2, 1989, pages 438-445) have shown that factor IX is secreted by mouse skin fibroblasts after infection with retroviral vectors encoding human factor IX, and this human factor IX can be detected in the serum of mice into which the skin fibroblasts have been transplanted. However promising such gene therapy experiments may be, there is still for the forseeable future a clear need for methods of controlling haemophilia B which rely on the introduction of sufficient externally produced factor IX speedily into the blood stream of the patient.

Surprisingly, it has now been found that factor IX can be delivered by subcutaneous injection with sufficiently rapid transport into the bloodstream in biologically active form and in adequate concentrations for control of haemophilia B.

Accordingly, the present invention provides a kit for use in subcutaneous injection comprising factor IX and a pharmaceutically acceptable carrier adapted for delivery of an effective dose of factor IX to a haemophilia B patient by such subcutaneous injection.

The invention also provides factor IX for use in the preparation of a composition for subcutaneous injection of factor IX to a haemophilia B patient.

The invention further provides a method of treatment of a haemophilia B patient comprising subcutaneous injection of the patient with a composition comprising factor IX.

The factor IX employed in the invention may be in the form of a concentrate obtained from blood donors or may have been obtained by recombinant DNA technology. The factor IX is preferably human factor IX for the treatment of human haemophilia B patients, although in principle the invention can be applied to the introduction of human or animal factor IX to other warm-blooded animals with an analogous blood clotting mechanism. The factor IX may be initially supplied as, or made up as, a composition in a pharmaceutically acceptable carrier such as deionised water or physiological saline. Alternatively, solid factor IX in suspension may be employed for administration subcutaneously, allowing slow release into the bloodstream from the site of injection. Other comparatively low molecular weight additives and excipients may be present. High molecular weight protein carriers such as serum albumin may also be used. However, the factor IX is preferably substantially free of other blood clotting factors such as flbrinogen, prothrombin, factors VII, VIII, X and XI which may be present in some commercially available sources of factor IX. For an initially impure source of factor IX, other blood clotting factors may be removed by standard purification procedures such as passing over an immuno-afflnity column loaded with a monoclonal antibody specific for factor IX as described by Rees et al., EMBO J., 7, 1988, pages 2053-2061.

The factor IX, conveniently in lyophilised form, and any carrier may be provided separately in a kit with the intention of combining the factor IX and carrier Immediately prior to use. Instructions for calculating the dosage to be delivered dependent on the patient's body weight and the circumstances of injection and appropriate for subcutaneous injection will conventionally be included in such a kit.

As in the case of the presently used intravenous method of introducing factor IX to the patient, the factor IX can be delivered subcutaneously either prophylactically or in response to bleeding episodes. It will be apparent that the use of subcutaneous delivery is particularly valuable for prophylaxis where there is a lower dose requirement.

It is desirable for subcutaneous injection to minimise the volume of liquid to be introduced to the patient and therefore to employ as little carrier as possible. It is therefore preferred to use sources of factor IX of comparatively high purity, especially those having at least 50%, preferably at least 757. factor IX with respect to the overall protein content of the composition.

The invention will now be further described by way of example and with reference to the accompanying Figures 1 to 5 which illustrate graphically the results of the examples.

EXAMPLE 1

Lyophilised human factor IX (available as "Mononine" from Armour Pharmaceutical Company, Kankakee, II., USA) was dissolved in deionised water at concentrations ranging from 10 to 500 international units (i.u.) per ml. The preparation, containing predominantly factor IX and little other protein, was formulated for human use, and contained low molecular weight additives.

250μg (50 i.u.) of lyophilised factor IX in a volume of 100μl were injected into each of six nude mice, strain MF1. In three control mice, injections were intravenous into the tail veins and in the other three experimental mice, injections were subcutaneous into four sites (25μl per site) over the mouse back. 0.1-0.2 ml blood samples were taken from the tail veins at intervals over one week and the concentration of human factor IX in the plasma assayed at the time points by ELISA (as described below). Fig. 1 shows the results and indicates that factor IX is transported into the bloodstream via subcutaneous injection and reaches a concentration which is approximately 40% of the concentration reached in plasma after intravenous injection of an equivalent amount of factor IX.

In a separate experiment, 5μg (l i.u.) of factor IX in a volume of 100μl was injected subcutaneously into each of two mice. 0.1-0.2 ml blood samples were taken from the tail veins at intervals over two days and assayed for factor IX by ELISA as described below. The results are shown in Fig. 2. It will be seen that factor IX can be detected in the bloodstream as quickly as 10 minutes after subcutaneous injection, reaches a maximum concentration 3-8 hours after injection (depending on the mouse) and then decays.

EXAMPLE 2

Two experiments were carried out using the source of factor IX described in Example 1 and six nude mice, strain MFl (weight approximately 25g). In the first experiment, Img (200 i.u.) factor IX dissolved in 1ml deionlsed water was injected subcutaneously into one mouse and 50μg (10 i.u.) factor IX dissolved in 100μl delonised water was injected intravenously into a second mouse. In the second experiment, 500μg (100 i.u.) of factor IX dissolved in 200μl delonised water was injected subcutaneously into one mouse and 100μg (20 i.u.) factor IX dissolved in 100μl deionised water was injected intravenously into a second mouse. In each experiment a control was conducted with one mouse using intravenous injection of 100μl physiological saline. Blood samples of about 1ml were taken from each of the six mice by means of heart puncture. Blood was collected when the human factor IX concentrations were presumed to be at their maximum from Example 1, i.e. about 4 hours after subcutaneous injection and as soon as possible (less than 10 minutes) after intravenous injection. The results are given in Table 1 below, which gives the concentration of human factor IX in the plasma (given as antigen % of that found normally in human plasma and determined by ELISA), the clotting activity determined as described below, the corrected clotting activity (determined by subtracting the clotting activity of plasma from the control mouse - i.e. the clotting activity of mouse factor IX) and the specific activity (defined in the Table and expressed as %). The results show that subcutaneously injected factor IX reaches the bloodstream in biologically active form with specific activities of 74% and 51% as opposed to specific activities of 63% and 93% (for intravenously injected factor IX). Table 1

Analysis of plasma from mice in which human factor IX

has been Injected either Intravenously or subcutaneously

Experiment 1 Experimeni : 2

Control subcutaneous intravenous control subcutaneous Intravenous

injection Injection Injection Injection

Antigen (%) 186 300 106 700

Clotting activity (%) 63 200 250 66 120 720

Corrected clotting 137 187 54 654 activity (%)

Specific activity (%) 74 63 51 93

(corrected clotting

activity/antigen)

91OCT

ID22

Assay of Factor IX

To determine the concentration of human factor IX in plasma, blood was collected into one-tenth volume of 3.8% sodium citrate in aqueous solution. Cells were removed by centrlfugation and the plasma samples were frozen at -20°C for later assay.

Factor IX in the purified samples was quantified by ELISA for antigen essentially as described by Anson et al., Nature, 315, 1985, pages 683-685. Factor IX clotting activity was quantified by the one-step clotting assay of Austen and Rhymes, A Laboratory Manual of Blood Coagulation, 1975, Blackwell Scientific Oxford, page 59. In both cases, pooled normal human plasma was used as the standard.

EXAMPLE 3

Lyophilised human factor IX as used in Examples 1 and 2 was treated to remove any low molecular weight additives as follows:

Step 1: 20 i.u. factor IX were dissolved in 2ml delonised water. The recommended concentration of factor IX for human use 1s 196 i.u. in 2ml, so this reduces the concentration of additives by 9.8 times.

Step 2: The solution was mlcroconcentrated by centrlfugation for 1 hour and the resulting solution, after an estimated 37% loss (estimated by ELISA), contained 12.6 i.u. in a volume of 85μl. Although the concentration of additives in this factor IX solution remains unchanged from that in the initial solution, they were reduced in amounts by a calculated 23.5 fold (i.e. 2000μl ÷ 85μl).

Step 3: The factor IX solution (12.6 i.u. in 85μl) was then diluted to 1ml with physiological saline, to give a solution containing 12.6 i.u. factor IX/ml. Thus the molarity of the additives was reduced by a further calculated 11.8 fold (i.e. 1000μl ÷ 85μl). The overall dilution of additives is, therefore 9.8 (step 1) × 11.8 (step 3) = 116 fold. So, the concentration of additives in this solution is 116 times lower than it would be if the factor IX preparation had been reconstituted as recommended. 1.26 international units in 100μl of the factor IX treated as above was injected into each of four nude mice (MF1 strain). Two mice were injected intravenously into the tail vein and two mice were injected subcutaneously into the back. In each case the total volume of 100μl was injected into a single site. 0.1-0.2 ml blood samples were taken from the tail vein at intervals over two days and the human factor IX in the plasma quantified by ELISA as described above. The results are shown in Fig. 3 and are comparable with those obtained in Figs. 1 and 2 showing that the presence of low molecular weight additives is not a significant contributory factor to the ability of the factor IX to reach the bloodstream.

EXAMPLE 4

An alternative source of factor IX was employed in this example. This was factor IX "IXMC" (available from Bio Products Laboratory, Elstree, Herts, UK) and dissolved in delonised water to a concentration of 81 international units/ml (as determined by ELISA) which is the recommended concentration for human use of this product. The preparation was believed to further contain low molecular weight additives.

8.1 international units factor IX in 100μl was injected into each of four mice. Two mice were injected intravenously into the tail vein and two mice, were injected subcutaneously into the back. In each case the total volume was injected into a single site. 0.1-0.2 ml blood samples were taken from the tail vein at intervals over two days and the human factor IX in the plasma was quantified by ELISA as described above. The results are shown in Fig. 4 and show that factor IX is transported to the bloodstream from a subcutaneous injection site with an efficiency of transport of 25% and 43% depending on the mouse (comparing the results of the subcutaneously injected mice with the average of the two intravenously injected mice).

EXAMPLE 5

A less pure source of factor IX ("Factor IXA" available from Bio Products Laboratory) was obtained. Information available about this product was as follows: Factor IXA (BPL) 585 i.u./bottle.

Reconstituted solution when dissolved in 20ml H₂O contains per litre not more than: 20g protein, 300 mMoles sodium, 200 mMoles chloride, 50 mMoles phosphate, 60 mMoles citrate, 500 i.u. antithrombin III, 5000 u. heparin; nominal content of factor II 800 u.; factor X 400 u.; factor VII negligible for therapeutic purposes. The recommended concentration for human use (i.v.) was 585 i.u. in 20ml.

Factor IX was dissolved to a concentration of 585 i.u. in 10ml of deionised water. An ELISA assay showed that this product in fact contained 89.4 international units/ml (447 μg/ml).

26.8 i.u. of factor IX in a volume of 0.3ml was injected intravenously into one mouse (in detail, 0.1 ml was injected into each of 3 sites in the tail veins). 89.4 i.u. factor IX in a volume of 1ml was injected subcutaneously into each of 2 mice (in detail, 0.5ml was injected into each of 2 sites on each side of the mouse back). Blood samples of about 1ml were taken by means of a heart puncture 4 hours later (a time at which concentrations of human factor IX were previously estimated to be maximum) from the two mice that received the subcutaneous injection of factor

IX. Similarly, blood was collected after 4 hours from the mouse that had received the intravenous injection.

The results are given in Table 2 below.

For the subcutaneous injections, after 4 hours large clots had formed under the skin in each site where the factor IX had been injected resulting in severe bruising.

Table 2

Mouse Type of Amount of Time after Amount of factor IX injection factor IX injection detected in blood injected

(i.u.)

1 i.v. 26.8 4 hours 2.18 μg/ml

2 s.c. 89.4 4 hours 1.65 μg/ml

3 s.c. 89.4 4 hours 0.80 μg/ml In a yet further experiment, a portion of the "Factor IXA" (BPL) preparation described above was purified by passing it over an A7 monoclonal antibody column, substantially as described by Rees et al., EMBO J., 7, 1988, pages 2053-2061, and then concentrated using a Centrlcon 10 microconcentrator. The final solution contained 2.1 international units factor IX/ml and, as additives, 14.7 mg/ml bovine serum albumin, 150mM NaCl, 20mM Tris-chloride buffer pH 7.5 and trace amounts of sodium thiocyanate.

0.23 international units of this preparation in a volume of 110μl was injected into each of two mice, one of which was injected intravenously into the tail vein and one mouse subcutaneously into the back. In both cases the total volume (110μl) was injected into a single site. 0.1-0.2 ml blood samples were taken from the tail vein at intervals over two days. Factor IX in the plasma was assayed by ELISA as described above. The results are shown in Fig. 5.

Claims

1. A kit for use in subcutaneous injection comprising factor IX and a pharmaceutically acceptable carrier adapted for delivery of an effective dose of factor IX to a haemophilia B patient by such subcutaneous injection.

2. A kit according to claim 1 comprising human factor IX.

3. A kit according to claim 1 or 2 substantially free of other blood clotting factors.

4. A kit according to any one of the preceding claims wherein the factor IX is present at a purity of at least 50% with respect to overall protein content.

5. A kit according to any one of the preceding claims including instructions for use of the kit for subcutaneous injection.

6. A kit according to any one of the preceding claims adapted for delivery of a prophylactlcally effective dose of factor IX.

7. Factor IX for use in the preparation of a composition for subcutaneous injection of factor IX to a haemophi l i a B patient.

8. Factor IX according to claim 7 which is human factor IX for use in the treatment of human haemophilia B patients.

9. Factor IX according to claim 7 or 8 substantially free of other blood clotting factors.

10. Factor IX according to claim 7, 8 or 9 wherein the factor IX is of a purity of at least 50% with respect to overall protein content.

11. Factor IX according to any one of claims 7 to 10 for use in the preparation of a composition for subcutaneous injection of factor IX prophylactlcally to a haemophilia B patient.

12. A method of treatment of a haemophilia B patient comprising subcutaneous injection of the patient with a composition comprising factor IX.

13. A method according to claim 12 wherein the subcutaneous injection employs a kit according to any one of claims 1 to 6.

14. A method according to claim 12 or 13 for the prophylactic treatment of a haemophilia B patient.