A COMBINATION OF PLASMA IMMUNOGLOBULIN AND ANTIGEN-SPECIFIC IMMUNOGLOBULIN FOR THE MODIFICATION OF THE IMMUNE SYSTEM AND THE TREATMENT OR PREVENTION OF ALLERGIC DISEASES

Abstract

A method of use of plasma immunoglobulin, such as intramuscular immunoglobulin, in combination with polyclonal antigen-specific immunoglobulin in the treatment or prevention of allergic disease is provided. Also provided is a pharmaceutical composition including plasma immunoglobulin in combination with polyclonal antigen-specific immunoglobulin.

Claims

1. The use of a combination of plasma immunoglobulin and antigen-specific immunoglobulin for the treatment or prevention of allergic disease.

2. The use of claim 1 wherein said plasma immunoglobulin is intramuscular immunoglobulin.

3. The use of claim 2 wherein said intramuscular immunoglobulin is human intramuscular immunoglobulin.

4. The use of claim 1 wherein said plasma immunoglobulin is human plasma immunoglobulin.

5. The use of claim 1 wherein said plasma immunoglobulin is from a non-human species.

6. The use of claim 1 wherein said plasma immunoglobulin and said antigen-specific immunoglobulin are from the same species.

7. The use of claim 1 wherein said antigen-specific immunoglobulin is selected from the group consisting of polyclonal anti-tetanus toxoid immunoglobulin, polyclonal anti-Rh immunoglobulin, polyclonal anti-hepatitis B immunoglobulin, polyclonal anti-rabies immunoglobulin, and polyclonal anti-varicella immunoglobulin.

8. The use of claim 1 wherein said plasma immunoglobulin is greater than 50 mg per kg body weight.

9. A pharmaceutical composition for the treatment or prevention of allergic disease, said pharmaceutical composition comprising: plasma immunoglobulin; and antigen-specific immunoglobulin.

10. The pharmaceutical composition of claim 9 wherein said plasma immunoglobulin is intramuscular immunoglobulin.

11. The pharmaceutical composition of claim 10 wherein said intramuscular immunoglobulin is human intramuscular immunoglobulin.

12. The pharmaceutical composition of claim 9 wherein said plasma immunoglobulin is human plasma immunoglobulin.

13. The pharmaceutical composition of claim 9 wherein said plasma immunoglobulin is from a non-human species.

14. The pharmaceutical composition of claim 9 wherein said plasma immunoglobulin and said antigen-specific immunoglobulin are from the same species.

15. The pharmaceutical composition of claim 9 wherein said antigen-specific immunoglobulin is selected from the group consisting of polyclonal anti-tetanus toxoid immunoglobulin, polyclonal anti-Rh immunoglobulin, polyclonal anti-hepatitis B immunoglobulin, polyclonal anti-rabies immunoglobulin, and polyclonal anti-varicella immunoglobulin.

16. The pharmaceutical composition of claim 9 wherein said plasma immunoglobulin is greater than 50 mg per kg body weight.

17. A method of treating or preventing allergic disease comprising administering injections of plasma immunoglobulin and antigen-specific immunoglobulin.

18. A kit comprising aliquots of plasma immunoglobulin, aliquots of antigen-specific immunoglobulin, and instructions associated with said kit directing use of said aliquots of plasma immunoglobulin and said aliquots of antigen-specific immunoglobulin for the treatment or prevention of allergic disease.

19. A kit comprising aliquots of mixtures of plasma immunoglobulin and antigen-specific immunoglobulin, and instructions associated with said kit directing use of said aliquots of mixtures of plasma immunoglobulin and antigen-specific immunoglobulin for the treatment or prevention of allergic disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0019] At least one mode of carrying out the invention in terms of several examples will be described by reference to the drawings below in which:

[0020] FIG. 1A is a graph showing the attenuation of an anti-OVA IgE response by combined injections of IMIG and polyclonal anti-Tetanus toxoid Ig.

[0021] FIG. 1B is a graph showing no change in an anti-OVA IgG response by combined injections of IMIG and polyclonal anti-Tetanus toxoid Ig.

[0022] FIG. 1C is a graph showing OVA-induced IL-4 in splenocytes of OVA immunized mice receiving combined injections of IMIG and polyclonal anti-Tet Ig.

[0023] FIG. 1D is a graph showing OVA-induced IL-2 in splenocytes of OVA immunized mice receiving combined injections of IMIG and polyclonal anti-Tet Ig.

[0024] FIG. 2A is a graph showing OVA-induced IL-2 in splenocytes of OVA immunized mice injected with IMIG and anti-Varicella Ig.

[0025] FIG. 2B is a graph showing OVA-induced IL-4 in splenocytes of OVA immunized mice injected with IMIG and anti-Varicella Ig.

[0026] FIG. 3A is a graph showing the total serum IgG levels in dog blood before sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0027] FIG. 3B is a graph showing the total serum IgG levels in dog blood after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0028] FIG. 3C is a graph showing the total serum IgE levels in dog blood before sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0029] FIG. 3D is a graph showing the total serum IgE levels in dog blood after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0030] FIG. 3E is a graph showing IL-2 production by ConA-stimulated peripheral blood lymphocytes of dogs before sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0031] FIG. 3F is a graph showing IL-2 production by ConA-stimulated peripheral blood lymphocytes of dogs after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0032] FIG. 3G is a graph showing IL-4 production by ConA-stimulated peripheral blood lymphocytes of dogs before sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0033] FIG. 3H is a graph showing IL-4 production by ConA-stimulated peripheral blood lymphocytes of dogs after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0034] FIG. 3I is a graph showing the ratio of IL-2/IL-4 production levels by ConA-stimulated peripheral blood lymphocytes of dogs before and after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0035] FIG. 3J is a graph showing the IL-2 and IL-4 production by peanut butter Ag-stimulated peripheral blood lymphocytes of dogs after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0036] FIG. 3K is a graph showing the ratio of IL-2/IL-4 production levels by peanut butter Ag-stimulated peripheral blood lymphocytes of dogs after sensitization with peanut butter and treatment with dog IMIG and dog anti-Rabies immune Ig or treatment with human IMIG and human anti-Tetanus Ig.

[0037] FIG. 4A is a graph showing OVA-specific IgE serum levels in OVA immunized mice after combined infusion of varying doses of IMIG and anti-Tetanus Ig.

[0038] FIG. 4B is a graph showing OVA-specific IgG serum levels in OVA immunized mice after combined infusion of varying doses of IMIG and anti-Tetanus Ig.

[0039] FIG. 4C is a graph showing IL-4 production by OVA-stimulated splenocytes in OVA immunized mice after combined infusion of varying doses of IMIG and anti-Tetanus Ig.

[0040] FIG. 4D is a graph showing IL-2 production by OVA-stimulated splenocytes in OVA immunized mice after combined infusion of varying doses of IMIG and anti-Tetanus Ig.

[0041] FIG. 4E is a graph showing the ratio of IL-2/IL-4 production levels by OVA-stimulated splenocytes of OVA immunized mice after combined infusion of varying doses of IMIG and anti-Tetanus Ig.

DETAILED DESCRIPTION OF AT LEAST ONE MODE FOR CARRYING OUT THE INVENTION IN TERMS OF EXAMPLE(S)

[0042] The pharmaceutical compositions of the present invention are a combination of pooled plasma immunoglobulin and antigen-specific polyclonal immunoglobulin.

[0043] Plasma immunoglobulin is typically prepared from the serum of at least 1000 donors and has been used to treat patients with primary immunodeficiency diseases. Depending on how the plasma immunoglobulin is formulated and administered, it may be referred to as intramuscular immunoglobulin (IMIG), intravenous immunoglobulin (IVIG), subcutaneous immunoglobulin (SCIG), or intraperitoneal immunoglobulin (IPIG).

[0044] Antigen-specific polyclonal immunoglobulin is immunoglobulin prepared with a high antibody count against a specific pathogen, such as Varicella-zoster virus, or against a specific antigen, such as Tetanus toxoid.

[0045] The inventors have found that the combination of pooled plasma immunoglobulin and antigen-specific polyclonal immunoglobulin is useful for the treatment or prevention of allergic diseases.

[0046] As will be described in detail below, the inventors conducted experiments using various antigen-specific immunoglobulins against various antigens. In view of the significant differences in preparation and chemical composition between the antigen-specific immunoglobulins used (polyclonal anti-Tetanus immunoglobulin, polyclonal anti-Rabies immunoglobulin, and polyclonal anti-Varicella immunoglobulin), the combination of plasma immunoglobulin with any antigen-specific immunoglobulin (including but not limited to polyclonal anti-Rh immunoglobulin and polyclonal anti-hepatitis B immunoglobulin) is predicted to result in a similar modification of the immune system of the treated human or animal and to similarly prevent and/or treat allergic diseases.

[0047] For the treatment and prevention of allergic diseases in humans and pets, the plasma immunoglobulin component of the combination may be plasma immunoglobulin approved for use in humans such as, but not limited to, Gamunex and Hizentra.

[0048] The plasma immunoglobulin and antigen-specific polyclonal immunoglobulin may be administered in any suitable manner. For example, the antibodies may be administered in a non-immunogenic form, that is without an adjuvant, in a non-immunogenic amount, for example intramuscularly, intravenously, subcutaneously, or intraperitoneally. A pharmaceutical composition comprising the combination of plasma immunoglobulin and antigen-specific polyclonal immunoglobulin may include a pharmaceutically acceptable carrier, such as buffered saline, phosphate buffered saline, or phosphate buffered saline at neutral pH.

[0049] Another aspect of the invention is a kit comprising aliquots of plasma immunoglobulin, aliquots of polyclonal antigen-specific immunoglobulin, and instructions directing use of such two different aliquots for the treatment or prevention of allergic disease.

[0050] Another aspect of the invention is a kit comprising aliquots of a mixture of plasma immunoglobulin and polyclonal antigen-specific immunoglobulin and instructions directing use of such aliquots for the treatment or prevention of allergic disease.

[0051] The antibodies may be administered at any suitable site and time. However, the antibodies are preferably administered contemporaneously or substantially contemporaneously. The antibodies may be administered in separate compositions sequentially or contemporaneously or together as a mixture.

Example 1

[0052] Allergy Experiment Protocol with Polyclonal Human IMIG and Polyclonal Human Anti-Tetanus Ig

[0053] Five BALB/c mice (8 weeks of age at initiation of study) were used per group. All mice except a control group (Group 1see below) received ovalbumin (10 g OVA plus Al(OH).sub.3) intraperitoneally in 0.3 ml phosphate buffered saline (PBS) on day 0 and day 14 with a further boost on day 56. All mice received egg white solution (filtered 20% (w/v) EWS) in their drinking water from day 14.

[0054] Mice receiving intramuscular immunoglobulin (Gamunex 25 g: Grifols) were given intramuscular injections in 0.05 ml PBS in the left gluteus muscle at days 2, 7, 14, 21, 28, 35, and 42. Animals receiving Anti-Tetanus immune Ig (HyperTET 25 g: Grifols) were given intramuscular injections in 0.05 ml PBS in the right deltoid muscle at days 2, 7, 14, 21, 28, 35, and 42. In some cases IMIG or Anti-Tetanus was absorbed with Tetanus toxoid (3) before use. The control groups received PBS only in the same sites.

[0055] The following groups were used. [0056] Group 1: OVA.sup./EWS.sup.+ [0057] Group 2: OVA.sup./EWS.sup.+ [0058] Group 3: As for group 2+IMIG [0059] Group 4: As for group 2+Anti-Tet Ig [0060] Group 5: As for group 2+IMIG+anti-Tet Ig [0061] Group 6: As for group 2+IMIG (absorbed3 with Tet)+ant-Tet Ig [0062] Group 7: As for group 2+IMIG+anti-Tet Ig (absorbed with Tet3)

[0063] All mice were sacrificed at day 63 of the study. On sacrifice, serum IgE to OVA obtained by cardiac puncture was measured by ELISA using plates coated with 100 ng/well of OVA and developed with HRP-anti-mouse IgE and appropriate substrate.

[0064] In addition, 510.sup.6 splenocytes from individual animals were challenged in vitro in 2 ml medium with 1 g/ml OVA for 72 hr and IL-2/IL-4 measured in culture supernatants using commercial ELISA Kits (eBIOSciences).

[0065] Results

[0066] FIG. 1A shows that a combination of IMIG and polyclonal anti-Tetanus Ig results in significant suppression of OVA-specific IgE and FIG. 1B shows no significant effect on OVA-specific IgG. Referring now to FIG. 1A, absorption of IMIG or polyclonal anti-Tetanus with Tetanus (groups 6 and 7) made no difference. This suggests that the active pharmaceutical ingredient is not the anti-Tetanus antibodies.

[0067] FIG. 1C shows a decrease in the levels of OVA-induced IL-4 in the splenocytes of mice in groups 5, 6, and 7 that received combined injections of IMIG and anti-Tetanus Ig. When considered together with FIG. 1D showing OVA-induced IL-2 levels, it is apparent that the ratio of IL-4 to IL-2 is smaller for the groups treated with IMIG plus anti-Tetanus Ig (groups 5, 6, and 7) than the control groups 2, 3 and 4. This result is consistent with suppression of allergy and the results shown in FIGS. 1A and 1B.

Example 2

[0068] Allergy Experiment Protocol with Polyclonal Human IMIG and Polyclonal Human Anti-Varicella Ig

[0069] Eight BALB/c mice (8 weeks of age at initiation of study) were used per group. All mice except a control group (Group 1see below) received ovalbumin (10 g OVA plus Al(OH).sub.3) intraperitoneally in 0.3 ml PBS on day 0, day 14, and day 42. All mice received egg white solution (filtered 20% (w/v) EWS) in their drinking water from days 14-42.

[0070] IMIG (Gamunex 25 g: Grifols) and anti-Varicella Ig (25 g: Grifols) was infused weekly intravenously from days 7-35.

[0071] The following groups were used. [0072] Group 1: OVA.sup./EWS.sup.+ [0073] Group 2: OVA.sup./EWS.sup.+ [0074] Group 3: As for group 2+IMIG [0075] Group 4: As for group 2+Anti-Varicella Ig [0076] Group 5: As for group 2+IMIG+anti-Varicella Ig

[0077] All mice were sacrificed at day 49 of the study. On sacrifice, serum IgE to OVA obtained by cardiac puncture was measured by ELISA using plates coated with 100 ng/well of OVA and developed with HRP-anti-mouse IgE and appropriate substrate.

[0078] In addition, 510.sup.6 splenocytes from individual animals were challenged in vitro in 2 ml medium with 1 g/ml OVA for 72 hr and IL-2/IL-4 measured in culture supernatants using commercial ELISA Kits (eBIOSciences).

[0079] Results

[0080] FIG. 2A shows that there was no attenuation of IL-2 production from the OVA-stimulated individual splenocytes of mice. In contrast, FIG. 2B shows a clear attenuation of IL-4 production. These results are consistent with the study done in EXAMPLE 1 using combined injection of IMIG and anti-Tetanus Ig (see FIGS. 1C and 1D).

Example 3

[0081] Allergy Experiment Protocol with Polyclonal Human IMIG and Polyclonal Human Anti-Tetanus Ig, or Polyclonal Dog Ig and Dog Anti-Rabies Ig, Using Beagle Dogs Sensitized to Peanut Butter

[0082] In a modification of the protocol in EXAMPLE 1 and EXAMPLE 2, the inventors considered whether they could attenuate allergic sensitization in large animals (Beagle dogs) where previous literature reports indicate a high prevalence (>80%) of induced allergic sensitization to peanut butter applied topically.

[0083] Animals received weekly topical exposure to peanut butter (abdomen) with the first exposure occurring 1 week before treatment. The next 5 exposures of peanut better were with 5 weekly treatments with combined dog Igs (IMIG and pooled dog anti-Rabies immune Ig) or with combined human Igs (IMIG and Anti-Tet Ig). After 5 weekly treatments all animals received a further 3 treatments given at 14 d intervals of the same Ig mixes. Finally, all dogs received an oral challenge with peanut butter, and serum IgG, serum IgE, and peanut butter induced IL-2/IL-4 production was measured.

[0084] Dogs receiving dog intramuscular immunoglobulin (Innovative Research, USA) were given 1 mg/kg intramuscular injections in 0.5 ml PBS in the gluteus muscle and dogs receiving pooled dog anti-Rabies immune Ig (prepared from pooled dogs re-immunized with rabies vaccine) were given 1 mg/kg intramuscular injections in 0.5 ml PBS in the opposite gluteus muscle.

[0085] Dogs receiving human intramuscular immunoglobulin (Gamunex: Grifols) were given 1 mg/kg intramuscular injections in 0.5 ml PBS in the gluteus muscle and dogs receiving human Anti-Tetanus Ig (HyperTET: Grifols) were given 1 mg/kg intramuscular injections in 0.5 ml PBS in the opposite gluteus muscle.

[0086] Results

[0087] FIGS. 3A and 3B show a comparison of the total serum IgG levels at the start (t0) and end (t1) of the study respectively. FIGS. 3C and 3D show a comparison of the total serum IgE levels at the start (t0) and end (t1) of the study respectively. While there were no significant changes in the IgG levels, a reduction in the IgE levels was observed both for the treatment with dog IMIG and dog anti-Rabies Ig and for the treatment with human IMIG and human anti-Tetanus Ig.

[0088] FIGS. 3E and 3F show a comparison of the IL-2 production by conconavalin A (ConA) in dog peripheral blood lymphocytes (PBL) before (t0) and after (t1) treatment respectively.

[0089] FIGS. 3G and 3H shows a comparison of the IL-4 production by Con-A in dog PBL before (t0) and after (t1) treatment respectively.

[0090] FIG. 3I shows a comparison of the IL-2/IL-4 induction by ConA in dog PBL before/after Ig treatment. Note that even for this polyclonal response, the IL-2/IL-4 ratio is enhanced in dogs after treatment with either dog IgGs or human IgGs (*, p<0.05).

[0091] FIG. 3J shows that IL-2 and IL-4 productions by peanut butter stimulated dog PBL after treatment with dog IMIG and dog anti-Rabies Ig and after treatment with human IMIG and human anti-Tetanus Ig. FIG. 3K shows the IL-2:IL-4 ratios after treatment. Consistent with the other experiments, IL-4 production was attenuated by Ig treatment, but IL-2 production was not. Additionally, the IL-2:IL-4 ratios were elevated after treatment (*, p<0.05).

Example 4

[0092] Dose Response StudyPolyclonal Human IMIG and Polyclonal Human Anti-Tetanus Ig in Pre-Immune Mice

[0093] The inventors also conducted a dose response study using polyclonal human IMIG and polyclonal human anti-Tetanus Ig in pre-immune mice.

[0094] In the dose response study, all groups had five BALB/c mice each. Mice were approximately 25 grams. All mice received OVA (10 g OVA plus Al(OH).sub.3) on day 0 and day 14 with a boost on day 56. All mice received EWS (filtered 20% (w/v) egg white solution in their drinking bottle) from day 14.

[0095] Mice began treatment with immunoglobulin injections on days 56, 63, 70, 77, and 84.

[0096] The following groups were used: [0097] Group 1 (control): OVA immunization and EWS [0098] Group 2: As for Group 1+IMIG (250 g/mouse)+anti-Tet Ig (10 g/mouse) [0099] Group 3: As for Group 1+IMIG (250 g/mouse)+anti-Tet Ig (50 g/mouse) [0100] Group 4: As for Group 1+IMIG (250 g/mouse)+anti-Tet Ig (250 g/mouse) [0101] Group 5: As for Group 1+IMIG (50 g/mouse)+anti-Tet Ig (10 g/mouse) [0102] Group 6: As for Group 1+IMIG (50 g/mouse)+anti-Tet Ig (50 g/mouse) [0103] Group 7: As for Group 1+IMIG (50 g/mouse)+anti-Tet Ig (250 g/mouse) [0104] Group 8: As for Group 1+IMIG (10 g/mouse)+anti-Tet Ig (10 g/mouse) [0105] Group 9: As for Group 1+IMIG (10 g/mouse)+anti-Tet Ig (50 g/mouse) [0106] Group 10: As for Group 1+IMIG (10 g/mouse)+anti-Tet Ig (250 g/mouse) [0107] Group 11: As for Group 1+IMIG (50 g/mouse)+anti-Tet Ig (50 g/mouse)+CoQ10 ip (100 g/mouse) q2d from days 56-90 [0108] Group 12: As for Group 1+IMIG (250 g/mouse) [0109] Group 13: As for Group 1+Anti-Tet Ig (50 g/mouse)

[0110] A final boost with OVA was on day 90 and the mice were sacrificed on day 97.

[0111] Serum OVA-specific IgG/IgE was measured by ELISA. Additionally, 210.sup.6 splenocytes were challenged in vitro in 2 ml medium with 1 g/ml OVA for 72 hr and IL-2/IL-4 was measured in culture supernatants using commercial ELISA Kits (BioLegend).

[0112] Results

[0113] FIGS. 4A and 4B show that optimal suppression of IgE responses occurred with IMIG doses of 250 and 50 g/mouse and across a broad spectrum of anti-Tet dosing (from 10-2504/mouse). At lower IMIG doses (10 g/mouse) suppression was less evident. There was some synergy in suppression at lower doses of IMIG (50) and anti-Tet (50) if animals also received CoQ10 as anti-oxidant every 2 d (1004/mouse). No attenuation of IgG responses occurred (FIG. 4B).

[0114] FIGS. 4C, 4D, and 4E confirm the data of FIGS. 4A and 4B using IL-4 and IL-2 production as readout. Again suppression of IL-4 but not IL-2 production was seen with IMIG doses of 250 and 50 g/mouse and across a broad spectrum of anti-Tet dosing (from 10-2504/mouse). At lower IMIG doses (10.sub.4/mouse) suppression was less evident. There was synergy in suppression at lower doses of IMIG (50) and anti-Tet (50) if animals also received CoQ10 as anti-oxidant every 2 d (1004/mouse). These data are further emphasized by comparison of IL-2:IL-4 ratios as shown in FIG. 4E.

[0115] In the foregoing description, exemplary modes for carrying out the invention in terms of examples have been described. However, the scope of the claims should not be limited by those examples, but should be given the broadest interpretation consistent with the description as a whole. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.