WO1990002559A1

WO1990002559A1 - Pharmaceutical compositions for eliciting an immunostimulant effect

Info

Publication number: WO1990002559A1
Application number: PCT/GB1989/001081
Authority: WO
Inventors: Jack Melling; John Robert Stephenson
Original assignee: Public Health Laboratory Service Board
Priority date: 1988-09-15
Filing date: 1989-09-14
Publication date: 1990-03-22
Also published as: GB8821656D0; DE68908414D1; CA1337113C; ZA897005B; AU4324389A; JPH04500675A; JP3072489B2; DE68908414T2; EP0434749A1; AU633851B2; EP0434749B1

Abstract

Pituitary hormones, particularly growth hormones have been found to be capable of eliciting an immunostimulant effect. Co-administration of such hormones with vaccine antigens enhances the effectiveness of the vaccines.

Description

PHARMACEUTICAL COMPOSITIONS FOR ELICITING AN IMMUNOSTIMULANT EFFECT

This invention relates to pharmaceutical compositions for eliciting an immunostimulant effect in humans or non-human animals. Particularly, but not exclusively the invention relates to vaccine preparations and to pharmaceutical compositions for use in conjunction with vaccine preparations in order to enhance the effectiveness of such preparations.

Many circumstances exist in which it is desirable to produce an immunostimulant effect in an animal. Thus, for example, pathological conditions occur in which an animal's immune response is diminished, as, for example, in the case of chronic infections which HIV (Human Immunodeficiency Virus). Further, it would be desirable to produce an immunostimulant effect when administering antigens in the form of. vaccines intended to produce a protective immunological effect. Thus, for example, in order to produce an effective degree of immunisation against many pathological microorganisms (including bacteria, fungae, protozoa, viruses, viroids and prions) it is necessary to inoculate on more than one occasion. Similar considerations apply to so-called "contraceptive vaccines", i.e. vaccines, the use of which produce antibodies against hormones involved in reproduction. The use of such "multi-shot" inoculation regimes is a particular drawback in the veterinary field where the inoculation of large numbers of animals is time-consuming and it is inconvenient to record whether or not a particular animal has received the necessary number of inoculations. Achieving an enhanced immunological response is a particularly desirable objective, particularly in Third World countries, where "multishot" inoculation procedures are impractical and where effective contraceptive vaccines would be desirable.

Pituitary hormones, including pituitary growth hormone, luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone and prolactin, have widespread use in medicine. For example, pituitary growth hormone is used in the treatment of pituitary dwarfism whereas luteinising hormone, follicle stimulating hormone and prolactin are.used in treatment, therapy and diagnosis of conditions (including pregnancy) related to reproduction. Thyroid stimulating hormone is, as its name implies, used in therapy of conditions associated with^* thyroid deficiency.

Conventionally, such pituitary hormones have been extracted from pituitary tissue obtained from cadavers and from slaughter-house by-products, but recently, pituitary hormones have been produced synthetically using, for example, recombinant DNA techniques and synthetic chemistry techniques. The term "pituitary hormone" as used herein includes such hormones which have been extracted from tissues in which they normally occur as well as synthetic forms, such as those produced by recombinant DNA techniques.

Using recombinant DNA techniques and synthetic chemistry techniques it is possible to produce polypeptides which can either be identical to native polypeptides or differ from native polypeptides by deletions, substitutions and insertions of one or more amino acid residues. In the context of the present invention, the term "pituitary hormone" is intended to include not only such hormones produced by recombinant DNA techniques which are identical to native polypeptides but also such hormones which differ from native polypeptides by up to 60 percent of the native sequence. Preferably they should not differ by more than 5 percent, most preferably 30 percent of the native sequence. The term "pituitary hormone" as used herein is also intended to include pituitary hormones having different patterns of glycosylation from the native hormones, including polypeptides which are totally unglycosylated.

A further class of compounds intended to be covered by the term "pituitary hormone" consists of fragments of pituitary hormones possessing at least one of the characteristic biological activities of the native hormone. In addition, "pituitary hormone" is also intended to include pituitary hormones having different patterns of acylation, phosphorylation, sulphation and other forms of post-translational modification. A given polypeptide may be identified as being a pituitary hormone in the context of the present invention by reference to the relationship of its structure to a native sequence (as indicated above) or by reference to its possession of at least one of the biological properties of the native hormone in a standard assay.

The present invention finds basis in the surprising discovery that pituitary hormones are capable of producing an immunostimulant effect.

Thus according to one aspect of the invention there is provided the use of a pituitary hormone in the manufacture of a pharmaceutical composition for eliciting an immunostimulant effect in a human or non-human animal. Preferably the pituitary hormone is a pituitary glycoprotein hormone, most preferably pituitary growth hormone. Other examples of pituitary hormones include luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone and prolactin.

Pituitary growth hormone used in accordance with the invention may be characterised in terms of biological activity. Thus, for example, pituitary growth hormone may be defined as a polypeptide capable of stimulating long ,bone growth and releasing somato edin. Such activity may be detected by the methods described in Marx et al. (1942), Endocrinology, 30, 1. and Greenspan et al. (1949), Endocrinology, 5, 4 5- Alternatively, pituitary growth hormone may be defined in terms of its antigenic reaction with anti-sera as described, for example in Rabin (1959) » Recent Progress In Hormone Research, 15, 71-105- A further characterisation of pituitary growth hormone is by reference to a ino acid sequence. Amino acid sequences of known pituitary growth hormones are given in Li (197 ) . Hormonal Proteins and Peptides, 3. 1-40, Academic Press. A description of the biological activities of fragments of pituitary growth hormone is also provided in "Structure and Function in Growth Hormones", Wilhelmi, A.E. Proc of the USP Workshop on Drugs and Reference Standards for Insulin Somatotropin and Thyroid Axis Hormones (1982) Bethesda, Maryland.

According to a preferred aspect of the invention, the immuno¬ stimulant effect is produced in a human or non-human animal in which it is desired to raise antibodies against a selected antigen. The selected antigen may, for example, be a component of a vaccine. Examples of typical antigens include proteins, glycoproteins, peptides, glycopeptides and polysaccarides and include such antigens derived from viruses, viroids, prions, bacteria, fungae, protozoa and hormones.

The pituitary hormone may be administered prior to, simultaneously with or subsequent to the administration of the antigen. Preferably however the hormone and vaccine comprise a single composition adapted for parenteral administration.

The pharmaceutical preparations according to the invention may comprise a single composition containing both the antigen and the pituitary hormone or these two components may be present in the form of a kit comprising separate aliquots thereof.

As indicated above, in carrying out the invention, the pituitary hormone is advantageously administered in a form which encourages localised retention. Pharmaceutical conditions embodying such forms (whether they additionally comprise one or more of the aforementioned antigens or not) form a further aspect of the invention.

Thus according to a further aspect of the invention there is provided an injectable pharmaceutical composition for use in conjunction with an antigenic component of a vaccine in order to produce an immunostimulant effect, said pharmaceutical composition comprising a pituitary hormone and a pharmaceutically acceptable carrier, said carrier (or an optional additional component of the composition) being selected so as to promote localised retention of the pituitary hormone at or adjacent to the injection sites. Preferably, said composition comprises an adjuvant such as, for example, aluminium hydroxide.

In carrying out the method of the invention, it has been found that the desired immunological effect (for example a protective effect in the case of immunisation),'is obtainable with only a single injection of antigen.

As indicated below, it is believed that the mode of action of pituitary hormones according to the invention is via the cell-mediated immune response (rather than by a humoral or antibody response) . A mode of administration of pituitary hormones which maximises such a response is preferred, i.e. one in which the pituitary hormone is administered in a form which encourages localised retention of the hormone after injection. Such forms include injectable compositions which include known adjuvants such as, for example, aluminium hydroxide.

Pharmaceutical preparations may be produced in accordance with the invention comprising both the pituitary hormone and the antigen in a single composition. The antigen may be an antigenic component of a vaccine, for example an antigen capable of producing a protective immunological effect against the causative organisms of herpes, rubella, Japanese encephalitis. Rift Valley Fever, yellow fever, measles, mumps, poliomyelitis, louping 111, dengue fever, lassa fever, Central European Tick - borne encephalitis, Russian Spring-Summer encephalitis, Omsk Haemorrhagic Fever, Kyanasur Forest Disease or Wesselsbron disease. Others include antigens capable of producing a protective immunological effect against such organisms as HIV, influenza virus, parainfluenza virus, Epstein-Barr virus and parvo viruses (e.g. the causative organism of so-called B19 otherwise known as slapped-cheek syndrome or Sixth disease) . Further examples include the organisms responsible for foot and mouth disease and canine distemper, respiratory syncitial virus, feline leukemia virus, canine parvo viruses, Neisseria sp., Bordatella sp. , Hae aphilus sp. and Mycobacterium sp.

Included amongst the causative organisms referred to specifically above are the so-called "flaviviruses".

Flaviviruses have one, single stranded RNA molecule, approximately 11,000 bases long as a genome. The RNA is positive stranded and has a 5' cap but does not have a 3' polyA tail. This genome codes for 3 virus structural proteins and 7 non-structural proteins and in addition has substantial non-coding regions at both the 5' and 3' ends. The genomic RNA is encapsulated in an icosohedral core of about 30 nanometers in diameter, made up entirely of the C protein. This core is surrounded by the E protein as well as a few molecules of M protein. The virus envelope also contains several molecules of lipid incorporated from the membranes of the infected cell.

The C protein is the only structural component of the core of the virus particle. No other functions are known and it does not appear to stimulate the immune system.

The E protein is glycosylated in most flaviviruses and contains all the biological activities associated with the virus particle, and is a major stimulator of the immune system.

The M protein has no known function at present but is present in low numbers in the virus particle. The NS1 protein is glycosylated, is a major stimulator of the immune system, and has a limited protective function in some systems, but its function is unknown.

The NS3 and NS5 proteins are probably constituents of the viral replicase, but the functions of NS2a, NS2b, NS4a and NS4b are unknown at present.

The flavivirus group of viruses is responsible for some of the most dangerous and widespread diseases of man and his domestic animals including Yellow fever, Dengue fever, Japanese encephalitis and Louping 111. In spite of good vaccine being available, Yellow fever is still a major public health problem in S.E. Asia, Africa and South America. Dengue fever is of growing concern in S.E. Asia and Central America and has been targeted by WHO as one of the most important viral diseases against which there is no current acceptable vaccine. Although this infection is normally 'flu-like in symptomology, there are increasing incidents of Dengue haemorrhagic fever and Dengue Shock Syndrome being reported; these conditions are frequently life-threatening if not treated immediately. Japanese encephalitis is the most rapidly expanding viral disease in the world at present and again has been targetted by the WHO. Tick-borne encephalitis is a major public health problem in the USSR with mortality rates of up to 40% being reported. This disease is also of growing public concern in most Eastern European countries as well as Austria and West Germany. TBE has been reported in most other European countries although it is only recognised as a veterinary problem (Louping 111) in the United Kingdom. However four or five cases of Louping 111 infection of humans are reported in Scotland every year and these can result in hospitalisation for several months.

The present 17D vaccine against Yellow Fever is one of the safest and most efficacious vaccines developed, but as it is a live, attenuated vaccine it requires a cold chain, which is a problem in tropical third world countries where it is most needed. Development of a non-living vaccine would almost certainly require a multi-shot regime. The only recognised vaccine against Japanese encephalitis is made from an inactivated, partially purified mouse-brain preparation and is effective, but requires three doses for good protection. A live attenuated vaccine has been developed in China and is widely used there as Japanese Encephalitis is a major public health problem, but this vaccine has yet to be tested in the West. Both the Russian and Austrian vaccines against TBE are also inactivated preparations but are made in avian cells and are purified; these also require 2 to 7 doses for complete protection. The veterinary vaccine against Louping 111 is made in the United Kingdom by Wellcome and is manufactured from infected tissue culture cells, inactivated with formalin and has been subjected to only the most rudimentary purification. This vaccine requires at least three of even more doses for a good protection. At present there is no good vaccine against Dengue fever, although the US army has been working on a vaccine for many years. However a group of attenuated vaccines have been developed in Thailand and these are now under trial by the WHO. The inactivated vaccines against Japanese encephalitis, TBE and Louping 111 could all benefit from a one-shot inoculation regime and even the live attenuated vaccines against Yellow fever, Dengue and Japanese encephalitis may derive a beneficial effect from co-injection with an immunostimulant.

The areas where single-shot vaccination regimens are almost needed are in the military, veterinary vaccines and rural populations in developing countries; it could also be argued that these groups are the most at risk.

The stimulating effect of human growth hormone (also known as somatotrophin or HGH) in mice is demonstrated in the following Examples.

Example 1 - Demonstration of Immunostimulant Effect The effect of administering HGH on the effectiveness of a vaccine against Tick-Borne Encephalitis (TBE) vaccins was tested in a murine model. The murine model had been developed by the Molecular Virology Group of PHLS Centre for Applied Microbiology & Research to assess the protective ability of various vaccine preparations (both commercial and experimental) against TBE virus (Neudorfl isolate) . From previous experiments it was known that a commercially available TBE virus vaccine required a two dose regimen of at least ^'•_. g of vaccine in order to give 100 percent protection, whereas a single dose was known to give only 10 to 30 percent protection. In view of this established bank of data it was not necessary to include simultaneous controls in which the efficacy of vaccine alone was tested. In order to assess the effect of HGH on the efficacy of a single vaccine dose, groups of 15 Balb/C mice were injected intra-peritoneally with various combinations of TBE virus vaccine, HGH and PBS (phosphate buffered saline) .

Stock solutions were prepared as follows:

1. PBS - NaCl, 0.139M; KC1, 0.0269M; Na₂-HPO4 (2H₂0) ,

0.0075M, KH₂P0i , 0.00147M.

2. HGH - One vial of Batch Number TPL 29 (four units) was dissolved in 1 ml of sterile glass-distilled, water. The HGH was a polypeptide obtained from extraction from pituitaris.

3. Vaccine - One vial of "FSME-Immun" (lot no: 37028601) was diluted 1/20 in PBS.

Combined vaccine/HGH preparations where produced by mixing 6 μl of stock solution (2) with 0.2 ml of stocl: solution (3).

On 10th February 1988 fifteen Balb/C mice were divided into three groups A, B and C and injected intra-peritoneally (i.p.) as follows:

Group A - 0.2 ml of vaccine + HGH

Group B - 0.2 ml of PBS + HGH

Group C - 0.2 ml of vaccine + HGH. On 24th February I988 three mice from each of groups A, B and C were bled and the remainder of group A were vaccinated with vaccine and HGH as on 10th February 1988. On 9th March 1988 three mice from each of groups A, B and C were bled and each of the remaining mice in groups A, B and C were challenged subcutaneously (s.c.) with 0.2 ml PBS containing lθ3 plaque forming units of the Neudorfl isolate of TBEV.

The results were as follows:- .^•

Example 2- Demonstration of Immunostimulant Effect The effect of administering HGH on the effectiveness of a vaccine against Tick-Borne Encephalitis at sub-optimal doses was carried out using the protocol used in Example 1 except that the TBE vaccine was diluted 1:100 before use.

The results were as follows:-

Assuming that each mouse weighs, on average 20 g and an average 5 year old child weighs 40 kg; each mouse received the equivalent dose per kilogram of ten times the normal human dose of HGH when used to treat pituitary dwarfism. During childhood and on into adult life, the pituitary continuously excretes HGH, but at levels below that required to obtain the enhanced immunostimulant activity according to the invention. The invention therefore requires the administration of quantities of hormone sufficient to result in a plasma level of hormone above normal basal levels.

In the case of HGH, it is thus desirable to administer at least 10~2 units of HGH per kilogram of body weight, preferably from 10~² to 10^-1 units/kg.

Example 3 - Demonstration of Immunostimulant Effect

The experiment of Example 1 was repeated, except that the HGH used was a commercially available polypeptide produced by recombinant DNA technology.

The following results were obtained:

Animal Grou Vaccine No. of Survivors % Survivors

9/9 100 0/9 0

9/9 100

Example 4 - Analysis of Mouse Sera For TBVE-Specific Antibodies The experiment of Example 1 was repeated and blood samples were analysed for TBVE-specific antibodies. The results obtained were as follows: Animal Grou f ELISA ^"titre at various times after vaccination

a. Blood sample taken when second shot of vaccine was given to group A. b. Blood sample taken when virus challenge was given. c. Blood sample taken at the end of the experiment. d. Although all mice in this group are normally dead at this time, a sample from an occasional survivor was analysed. e. No sample available as all mice died. f. Groups are designated as in the tables of the survival data. g. In this assay a titre of 300 or less would be considered a negative; therefore it appears that vaccine alone stimulates very little antibody production and even in the presence of HGH antibody titres are low. It is only after challenge with live virus that significant levels of antibody are detected. Although there is a stimulation in antibody production with HGH it is probably insufficient to account for the increased levels of protection that are observed.

Example 5 - Dose Response Of The Ability Of HGH To Stimulate Protection

The dose response of the HGH preparation was tested by performing a series of protection experiments, identical in design to those described in Example 1, except that the HGH solution was diluted before addition to the vaccine preparation. A total of six experiments were performed and the data for two of them is presented below. Experiment A was performed with a 1:4 dilution of HGH, i.e. the highest dilution at which no reduction in the immunostimulant effect of HGH was observed. Experiment B was performed with a l:4θ dilution of HGH, i.e. the lowest dilution at which the immunostimulant effect of HGH was not observed..

Experiment A

HGH, i.e. a concentration of 0.2 international units m/1.

From the results of Examples 1 and 2 it can be seen that HGH induces a marked immunostimulant action and enhances the immune response against TBE vaccine both using a less than optimal number of inoculations and using a less than optimal vaccine concentration.

Thus for TBE vaccine in the mouse protection model described above, HGH is capable of improving the protective efficacy of a single optimal dose of vaccine from 33 to 100% and is capable of improving the protective efficacy of two sub-optimal doses from 22% to 55%. The results of Example 3 demonstrates that recombinant HGH is capable of exerting an immunostimulant effect. This establishes that HGH itself (as opposed to an incidental impurity in cadaver-derived material) is capable of exerting an immunostimulant effect.

The mechanism of this potentiation of the immune system is at present unknown, but experiments show only a slight increase in overall specific antibody levels. This .would suggest that HGH might act directly on the stimulation of the cell mediated immune system. Thus as shown in the results of Example 4, the administration of HGH resulted in no significant elevation of ELISA titre at 14 days, a marginal elevation of ELISA titre at 28 days and a marked elevation at 42 days after vaccination (i.e. 14 days after challenge with live virus) .

It can be concluded that the effect of coadministering vaccine plus HGH does not result in an increase in antibody levels (compared to controls which receive only vaccine) and that consequently the cell mediated immune system is involved.

The method of the invention has considerable potential in human and animal therapy in enhancing the effectiveness of standard vaccines and enabling a significant degree of protection to be obtained using one-shot inocculation regimes.

Claims

1. The use of a pituitary hormone in the manufacture of a pharmaceutical composition for eliciting an immunostimulant effect in a human or non-human animal.

2. The use as claimed in Claim 1 wherein the pituitary hormone is a pituitary glycoprotein hormone.

3. The use as claimed in Claim 1 wherein the pituitary hormone is selected from pituitary growth hormone, luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone and prolactin.

4. The use as claimed in Claim 1 wherein the pituitary hormone is pituitary growth hormone.

5. The use as claimed in Claim 4 wherein the pituitary hormone is human growth hormone.

6. The use as claimed in any preceding claim wherein the pituitary hormone is produced by recombinant DNA techniques.

7. The use as claimed in Claim 6 wherein the pituitary hormone comprises a polypeptide which is identical to a native polypeptide or differs from a native polypeptide by up to sixty percent of the native sequence.

8. The use according to any preceding claim wherein the immuno¬ stimulant effect is produced in a human or non-human animal in which it is desired to elicit or stimulate an immune responseagainst a selected antigen and includes the administration of antigen in order to produce a protective effect.

9- The use according to Claim 8 wherein the selected antigen is a component of a vaccine.

10. The use according to Claim 8 or Claim 9 wherein the pituitary hormone is administered prior to, simultaneously with or subsequent to the administration of the antigen.

11. The use according to Claim 10 wherein the pituitary hormone is administered within 2 _ hours of the administration of the antigen.

12. The use according to any of Claims 7 to 11 wherein the selected antigen is capable of producing a protective immunological effect against the causative organisms of herpes, rubella, Japanese encephalitis, Rift Valley Fever, yellow fever, measles, mumps, poliomyelitis, TBEV, louping 111, dengue fever, lassa fever, Central European Tick - borne encephalitis, Russian Spring-Summer encephalitis, Omsk Haemorrhagic Fever or Kyanasur Forest Disease.

13. The use according to any of Claims 7 to 11 wherein the selected antigen is capable of producing a protective immunological effect against the causative organism, of Wesselbron disease, or against HIV, influenza virus, para influenza virus, Epstein-Barr virus and parvovirus B19.

14. The use claimed in any .preceding claim wherein the pituitary hormone is derived from an animal species other than the one in which it is desired to produce an immunostimulant effect.

15. The use claimed in any of Claims 1 to 13 wherein the pituitary hormone is derived from the same animal species as the one in which it is desired to produce an immunostimulant effect.

16. A pharmaceutical preparation for use in producing an immune response in a human or non-human animal comprising an antigen and at least one pituitary hormone.

17. An injectable pharmaceutical preparation for use in producing an immune response in a human or non-human animal, comprising at least one pituitary hormone and a pharmaceutically acceptable carrier or added ingredient for encouraging localised retention of the pituitary hormone at or adjacent the injection site.

18. A pharmaceutical composition according to Claim 16 or Claim 17 wherein the pituitary hormone is a pituitary glycoprotein hormone.

19. A pharmaceutical composition according to any of Claims 16 to 18 wherein the pituitary hormone is selected from pituitary growth hormone, luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone and prolactin.

20. A pharmaceutical composition according to Claim 19 wherein the pituitary hormone is pituitary growth hormone.

21. A pharmaceutical composition according to Claim 20 wherein the pituitary hormone is human growth hormone.

22. A pharmaceutical composition according to any of Claims 16 to

21 wherein the pituitary.hormone is produced by recombinant DNA techniques and comprises a polypeptide which is identical to a native polypeptide or differs from a native polypeptide by not more than sixty percent of the native sequence.

23. A pharmaceutical composition according to any of Claims 16 to

22 wherein the pituitary hormone is derived from an animal species other than the one in which it is desired to produce an immunostimulant effect.

24. A pharmaceutical preparation according to any of Claims 16 to

23 comprising at least one antigen which is an antigenic component of a vaccine.

25. A pharmaceutical preparation according to Claim 24 wherein the antigen is an antigenic component as defined in any of Claims 12 to 14.

26. A pharmaceutical preparation according to any of Claims 16 to

2 wherein the pituitary hormone is derived from an animal species other than the one in which is is desired to produce an immune response.

27. A pharmaceutical preparation according to any of Claims 16 to

26 in the form of an injectable composition containing both the antigen and the pituitary hormone.

28. A kit for use in producing an immune response in a human or non-human animal comprising separate aliquots of an antigen and at least one pituitary hormone.

29. A kit according to Claim 28 wherein the pituitary hormone is as defined in any one of Claims 18 to 23.

30. A kit according to Claim 28 or Claim 29 wherein the antigen is as defined in Claim 24 or 28.

31. A method of eliciting an immunostimulant effect in a human or non-human animal which comprises administering a a pituitary hormone.

32. A method according to Claim 31 wherein the pituitary hormone is as defined in any one of Claims 18 to 23.

33. A method according to Claim 31 or Claim 3 wherein the immunostimulant effect is produced in a human or non-human animal in which it is desired to raise antibodies against a selected antigen.

34. A method according to any of Claims 31 to 33 wherein the pituitary hormone is administered prior to, simultaneously with or subsequent to the administration of the antigen.

35- A method according to any of Claims 31 to 3 wherein the antigen is as defined in Claim 24 or 25.