WO1990001328A1

WO1990001328A1 - Tnf and formulations thereof in the treatment or prophylaxis of hepatitis b

Info

Publication number: WO1990001328A1
Application number: PCT/GB1989/000897
Authority: WO
Inventors: Roger Samuel Williams; Graeme James Mackay Alexander; Christopher R. Franks
Original assignee: Roger Samuel Williams; Graeme James Mackay Alexander; Franks Christopher R
Priority date: 1988-08-04
Filing date: 1989-08-04
Publication date: 1990-02-22
Also published as: GB2221619A; GB8818567D0

Abstract

A medicament for the treatment of hepatitis B in mammals comprising TNF (Tumor necrosis factor) together with methods for administering such a medicament.

Description

- ι -

TNF and formulations thereof in the treatment or prophylaxis of hepatitis B

This invention relates to TNF and formulations thereof in the treatment or prophylaxis of hepatitis B. In particular, the invention also includes a method of making a medicament and methods for the treatment or prophylaxis of hepatitis B in mammalian hosts. Preferably, the TNF is recombinant TNF (rTNF) purified to homogeneity.

It has been estimated that perhaps 200 million people in the world today are chronically infected with hepatitis B virus (Morbidity and Mortality Report 31/24) : 317) . Hepatitis B virus (HBV) infection often leads to chronic persistent hepatitis, chronic active hepatitis or cirrhosis, potential hepatic failure, or hepatocellular carcinoma (HCC) (Annals of Intern Med 101: 613-616) . The latter two conditions are associated with a significant 5 year mortality (15% and 45%, respectively) .

The signs and symptoms experienced by patients, with these hepatic disorders, vary from none to mild degrees of fatigue and malaise, to more severe maifestations of liver dysfunction (such as bleeding disorders, ascites, and hepatic encephalopathy) which may be present in very advanced cases of chronic active hepatitis.

The biochemical abnormalities usually include an elevation of liver enzymes in the serum (serum glutamic oxaloacetate transaminase and serum glutamic pyruvic transaminase) . These tend to be more marked in patients with chronic active hepatitis. Reduction in serum albumin, elevation of serum bilirubin and a prolongation of prothrombin time occur as late manifestations of chronic active hepatitis or due to the development of cirrhosis.

The high number of chronic HBsAg carriers in the world suggests that elimination of this virus by vaccination programs is unlikely in the near future. Some carriers represent a great hazard for the spread of infection, e.g. dentists and other health care personnel with HBsAg and Hepatitis Be antigen (HBeAg) or viral DNA in the blood. The spread of the infection to multiple patient contacts, has been documented.

To date, there is no proven treatment for this infection and its associated diseases. Effective therapy for this chronic viral infection is important for three reasons:

(a) it has been shown that loss of hepatitis B viral particles whether or not accompanying treatment with antivirals has been associated with improvement in histologic liver disease and decrease in levels of serum markers of liver inflammation (Harrison-s Principles of Internal Medicine (1983));

(b) it has been shown that loss of DNAp and HBeAg from sera is accompanied by loss of infectivity of patient sera for susceptible chimpanzees fHepatoloσy l: 228-232); and

(c) there is a possibility of decreased incidence of hepatitis B induced Hepatocellular Carcinoma. Tumour necrosis factor is a macrophage derived cytokine which has an antitumour cytotoxic activity and a regulatory activity on the cell-interactions (Proc. Natl. Acad. Sci, USA, 72: 3666-3670; BR. J. Cancer 38: 310-315; Science 230: 630-632). Production of oxygen radicals by activated PMN's could be one of the tumoricidal effector mechanisms. TNF exerts immunoregulatory activity by enhancing the proliferative capacity of activated T-lymphoσytes via enhancement of high affinity IL-2 receptor expression (Cell. Immunol. 100: 260-267) and stimulates, together with IL-2, IFN-gam a production. IFN-gamma in turn, activates neutrophils, eosinophils and macrophage functions including TNF production (New

Engl. J. Med. 316: 379-385). Further TNF, can exert either direct or indirect antiviral activity for both RNA and DNA viruses (Cancer research 47, June 1, 2793-2798; Nature 323: 816-822), and acts as a growth stimulating factor for diploid fibroblats (Br. J. Cancer 38: 310-315). In contrast, TNF interferes with normal hematopoiesis as revealed by strong inhibition of in vitro colony forming units (Proc. Natl. Acad. Sci. USA 83: 446-450). In addition, TNF may mediate endotoxin shock (J. Immunol. 136: 4487-4495) .

Tumour necrosis factor, produced by activated cells of the monocyte/macrophage lineage, was first demonstrated to cause hemorrhagic necrosis of murine, simian, and human tumours and tumour cell lines (Hepatoloσy 2: 39-49; Proc. Natl. Acad. Sci, USA, 72: 3666-3670; Br. J. Cancer 38: 310-315; Science 230: 630-632) . Evidence now shows that in addition to cytotoxic activity against virally infected or tumour cells, the monokine has, in concert with interleukin-2 and gamma-interferon important autocrine regulatory effects on normal cells as well.

TNF was shown to affect T-lymphocyte differentiation via enhancement of high affinity IL-2 receptor expression (Cell. Immunol. 100: 260-267), acts as a growth stimulating factor for diploid fibroblasts, enhances functional activity of neutrophils and eosinophils (J. Immunol. 138 (in press); J. Immunol., 135: 2069-2073), and adherence to endothelial cells (Proc. Natl. Acad. Sci. USA, 82: 1055-1059) , induces granulocyte/monocyte colony stimulating factor (GM-CSF) production by various cell types (Proc. Nat. Acad. Sci. USA 82: 8667-8671) and enhances HLA expression in various tissues

(Nature 323: 79-82; J. Immunol. 138: 975-980). In contrast, TNF interferes with normal hematopoiesis (Proc. Natl. Acad. Sci. USA 83: 446-450) and may be a mediator in cachexia and entotoxin shock (J. Immunol. 136: 4487-4495).

There appears to be synergy between IL-2 and gamma-interferon in the induction of TNF release by macrophages. In addition, IFN-gamma acts synergistically with TNF to increase target cell susceptibility, and to activate macrophage functions including TNF production (New Engl. J. Med. 316: 379-385) . TNF synergy with interferons was also demonstrated in direct or indirect activity against both DNA and RNA viruses (Cancer research 47, June 1, 2793-2798; Nature 323: 816-822; Nature 323: 819-822) via inhibition of viral replication and/or stimulation of T-lymphocytes to produce IL-2 and TNF, and/or induction of superoxide production and cytotoxicity of neutrophils. There is no knowledge in the art of the use of TNF as such in the treatment of hepatitis B.

Surprisingly, it has also now been found that levels of tumour necrosis factor are markedly reduced in hepatitis B carrier patients compared to age-matched normal controls. Also, in a series of patients treasted with lymphoblastoid interferon, only those patients responding clinically to lymphoblastoid interferon by clearing transiently or permanently markers of hepatitis B virus replication, produced tumour necrosis factor in vitro. In these cases, tumour necrosis factor production was extensive. The treatment of hepatitis B by TNF is clearly, and surprisingly, predicated.

Accordingly, the present invention is directed to a method for the treatment or prophylaxis of hepatitis B in mammals comprising administering, before or after infection of the host, an effective amount of tumour necrosis factor (TNF) from a mammalian species. Recombinant TNF is preferred, and the mammal may be a human.

Other aspects of the invention are:-

(a) the use of TNF in the manufacture of a human or veterinary medicament for the treatment or prophylaxis of hepatitis B;

(b) a pharmaceutical or veterinary formulation comprising an effective amount of TNF formulated for use in the treatment or prophylaxis of hepatitis B;

(c) a pharmaceutical or veterinary formulation comprising TNF which has passed the USP sterility, rabbit pyrogen and general safety tests and has an activity greater than 5 x 10^β units/ g and is formulated for use in the treatment or prophylaxis of hepatitis B;

(d) TNF for use in the treatment or prophylaxis of hepatitis B.

As used herein, the term "recombinant TNF" refers to TNF produced by recombinant DNA techniques wherein generally the gene coding for the TNF is cloned by known recombinant DNA technology. For example, by using human TNF cDNA as a template, the gene showing complementarity to the human TNF cDNA is inserted into a suitable DNA vector such as a bacterial plasmid, preferably an . coli plasmid, to obtain a recombinant plasmid, and the plasmid is used to transform a suitable host. The gene is expressed in the host to produce the recombinant TNF protein. Examples of suitable recombinant plasmids for this purpose include pBR322, pCRl, pMB9 and pSCl. All of these are publicly available. The transformed host may be eucaryotic or procaryotiσ, preferably a proσaryotic host.

As used herein, the term "pharmaceutically or veterinarily acceptable" refers to absence of interference with the effectiveness of the biological activity of the active ingredient and absence of toxicity to the patient.

The method of this invention involves administering to a mammal, preferably a human, an effective amount of TNF. The administration(s) may take place by any suitable technique, preferably parenteral administration. Examples of parenteral administration include intravenous, intraarterial, intramuscular, and intraperitoneal, with intravenous being preferred.

The dose and dosage regimen will depend mainly on whether the TNF is being administered for therapeutic or prophylactic purposes, the stage and degree of infection, the patient, and the patient's history. The amount should ideally be effective to achieve a protection level of at least 50%, preferably at least 70%; dosages which do not achieve this minimal level of effectiveness should not usually be employed. The doses may be single doses, multiple doses over a period of several days or repeated cycles of multiple doses over a period of several days followed by several days of no administration. For purposes herein, a protection level of at least 50% means that at least 50% of the treated hosts exhibit improvement against the infection, including but not limited to improved survival rate, more rapid recovery, or improvements or elimination of symptoms including inter alia clearing transiently, or permanently, markers of hepatitis B virus replication.

For parenteral administration the TNF will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion) , preferably in a pharmaceutically or veterinarily acceptable carrier medium that is inherently non-toxic and non-therapeutic or non-prophylactic. Examples of such vehicles include saline. Ringer's solution, dextrose solution, mannitol, and normal serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. The carrier medium may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The TNF will typically be formulated in such carriers at a concentration of about 0.1 mg/ml to 100 mg/ml.

Ordinary pharmacological techniques and practices known in the art can be used for formulation purposes.

As mentioned above, the TNF employed herein may be obtained from tissue cultures or by recombinant techniques, and from any mammalian source such as, e.g., mouse, rat, rabbit, primate, pig, and humans. Preferably the TNF is derived from a human source, and more preferably is a human recombinant TNF.

Recombinant human TNF may be obtained as described by Pennica et al., Nature, 312:724-729 (1984); Yamada et al. , J. Biotechnology. (1985) 2:141-153; Wang et al.. Science (1985), 228.:149-154; EP 155,549 published September 29, 1985, EP 158,286 published October 16, 1985; EP 168,214 published January 15, 1986; and PCT US85/01921 published April, 1986. The recombinant rabbit TNF may be obtained as described in EP 146,026 published June 26, 1985 and EP 148,311 published July 17, 1985. Preferably the TNF is a human TNF mutein that is N-terminally deleted, preferably wherein the first eight amino acid residues have been deleted, using the procedure described in U.S. Patent No. 4,677,063 issued June 30, 1987 or the TNF is a cysteine-depleted mutein described in U.S. Serial No. 698,939 filed February 7, 1985 and in U.S. Patent No. 4,518,584, supra (for IL-2 but applicable to TNF) .

The TNF should normally be of sufficient purity that it passes the USP sterility test, the rabbit pyrogen test and general safety tests and in addition should preferably have an activity of greater than 5 x 10⁶ units/mg TNF.

Examples

The following Examples do not limit the scope of the invention. Other modifications, techniques and alternatives will be readily apparent to those skilled in the art and these are covered by the present invention.

Example 1 - Storage of TNF

TNF for use in the present methods can be provided in vials kept at a temperature of at least -20°C. The TNF should be allowed to thaw at room temperature prior to use. The vials should not be shaken. Thawed solutions may be refrozen once but it is preferable that the solution once thawed be used within three hours due to sterility considerations.

Each vial can contain, for example approximately 0.3 g TNF in 1.2 ml of solution giving an approximate TNF concentration of 0.25 mg/ml. The solution may also contain suitable pharmaceutically or veterinarily acceptable carriers and/or stabilisers, e.g. 1% annitol, the whole being dissolved in a suitable buffer, e.g. sodium phosphate buffer having a pH between 7.2 and 7.8.

Example 2 - Preparation of TNF for use

The TNF is preferably adminstered in a solution containing serum albumin, e.g. 0.25% serum albumin. This prevents adherence of TNF to the walls of bottles and tubing etc. One method of producing the solution is to add a specified dose of TNF to 100 ml of sodium chloride injection which also contains the specified percentage of normal serum albumin (Human, USP) .

Example 3 - Treatment cvcle (Intramuscular)

Each patient is subject to a treatment cycle consisting of the administration of a specified dose of TNF on a daily basis for five days followed by 3 doses/week for a further three weeks. For example an intramuscular injection of TNF daily, at a dose of 10-100 g/m²/day followed by 3 doses of 10-100 μg/m²/^day f°^{r a} further three weeks.

After a 28 day cycle the patient's response, measured as the presence or absence of the infection, can be evaluated.

If it is desired and, if the patient does not exhibit unacceptable toxicity or disease progression the treatment cycle can be repeated. Up to 4 cycles of treatment, can be given. Continuation of the treatment beyond 4 cycles for a subject showing a complete or partial response to the treatment should be at the discretion of- the treatment supervisor.

Suitable daily doses of TNF are 10, 15, 30, 60, 80 or 100 μg/m²/day.

Example 4 - Treatment cvcle (Intravenous)

Each patient is subject to a treatment cycle consisting of the administration of a specified dose of TNF on a daily basis for five days. For example a 15 minute i.v. infusion of TNF daily, at a dose of 30-250 _/_.g/m²/day followed by 10 days of rest, i.e. no administration of TNF. At the end of each rest period the patient's response, measured as the presence or absence of the infection, can be evaluated.

If it is desired and,, if the patient does not exhibit unacceptable toxicity or disease progression the treatment cycle can be repeated. Up to 10 cycles of treatment, can be given. Continuation of the treatment beyond 10 cycles for a subject showing a complete or partial response to the treatment should be at the discretion of the treatment supervisor.

Suitable daily dosages of TNF are 30, 40, 70, 110, 180 or 250 μg/m²/day.

Claims

CLAIMS :

1. A method for the treatment or prophylaxis of hepatitis B in a mammal which comprises administering tumour necrosis factor (TNF) to the mammal•

2. A method as claimed in claim 1 comprising parenteral administration of TNF.

3. A method as claimed in claim 1 or claim 2 wherein the TNF is made up for administration by dilution into sodium chloride solution containing about 0.25% normal serum albumin.

4. A method as claimed in any one of claims 1 to 3 comprising either

(a) an intramuscular cycle of administration of a daily dose of TNF for a period of five days followed by 3 daily doses per week for a further three weeks, preferably up to 4 such cycles or

(b) an intravenous cycle of administration of a daily dose of TNF for a period of five days, preferably up to 10 such cycles.

5. A method as claimed in claim 4 wherein in

(a) the TNF is administered at a dosage of from about 10μg/m²/day to about 100_/_.g/m²/day and in

(b) the TNF is administered at a dosage of from about 30/_g/m²/day to about 250 g/m²/day.

6. A method as claimed in claim 4 wherein in (a) the TNF daily dosage is from 10 to

80 g/m²/day and in (b) the TNF daily dosage is from 40 to 180μg/m²/day.

7. A method as claimed in any one of claims 1 to 6 wherein the mammal is human.

8. A method as claimed in any one of claims 1 to 7 wherein the TNF is human TNF.

9. A method as claimed in any one of claims 1 to 8 wherein the TNF is recombinant TNF (rTNF) .

10. The use of TNF in the manufacture of a human or veterinary medicament for the treatment or prophylaxis of hepatitis B.

11. The use of TNF as claimed in claim 10 wherein the tnf is human TNF.

12. The use of TNF as claimed in claim 10 or claim 11 wherein the TNF is recombinant TNF (rTNF) .

13. A pharmaceutical or veterinary formulation comprising an effective amount of TNF formulated for use in the treatment or prophylaxis of hepatitis B.

14. A formulation as claimed in claim 13 comprising TNF which has passed the USP sterility, rabbit pyrogen and general safety tests and has an activity greater than 5 x 10^β units/mg.

15. A formulation as claimed in claim 13 or claim 14 also comprising a pharmaceutically or veterinarily acceptable diluent, adjuvant or excipient.

16. A formulation as claimed in any one of claims 13 to 15 which includes a stabiliser.

17. A formulation as claimed in claim 16 wherein the stabiliser is mannitol.

18. A formulation as claimed in any one of claims 13 to 17 comprising an effective dose of TNF in 1% mannitol and sodium phosphate buffer having a pH from about 7.2 to about 7.8.

19. A formulation as claimed in any one of claims 13 to 18 wherein the TNF is human TNF.

20. A formulation as claimed in any one of claims 13 to 19 wherein the TNF is recombinant TNF

(rTNF) .

21. TNF for use in the treatment or prophylaxis of hepatitis B.

22. TNF as claimed in claim 21 which has passed the USP sterility, rabbit pyrogen and general safety tests and has an activity greater than 5 x 10* units/mg, mixed with a pharmaceutically or veterinarily acceptable adjuvant or stabiliser.

23. TNF as claimed in claim 22 which contains mannitol.

24. TNF as claimed in any one of claims 21 to 23 which is in liquid form at a pH of about 7.2 to about 7.8.

25. TNF as claimed in any one of claims 21 to 26 which is human TNF. - 15 -

26. TNF as claimed in any one of claims 21 to 25 which is recombinant TNF (rTNF) ,

10

15

20

25

30

35