WO2003088997A2

WO2003088997A2 - Reduction of unwanted immune reactions

Info

Publication number: WO2003088997A2
Application number: PCT/NL2003/000297
Authority: WO
Inventors: Jacoba Johanna Heijnen; Anna Maria Agnes Antonius Kavelaars
Original assignee: Universiteit Utrecht Holding B.V.; Umc Utrecht Holding B.V.
Priority date: 2002-04-22
Filing date: 2003-04-22
Publication date: 2003-10-30
Also published as: AU2003228133A1; WO2003088997A3; AU2003228133A8

Abstract

The invention relates to the field of pharmaceutical products and therapy. The invention provides means and methods to improve the induction of tolerance to treat autoimmune diseases, delayed type hypersensitivity reactions and/or transplant rejection, and/or graft versus host reaction and/or allergic reactions. Mucosal or dermal administration of a ß2 -adrenergic agonist greatly improves the induction of tolerance against antigen administration. Pharmaceutical compositions are claimed that comprise a ß2 -adrenergic agonist and an antigen. Use of these compositions is also claimed

Description

Title: Reduction of unwanted immune reactions.

The invention relates to the field of immunology, more in particular to the field of immune therapy, more in particular to the induction of tolerance against a predefined antigen, more specifically to the treatment of autoimmune disease and/or transplant rejection, and/or graft versus host reaction and/or allergic reaction and/or a delayed type hypersensitivity reaction.

Antigen-specific oral tolerance induction has been suggested as a novel approach to treat autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis (1). However, clinical trials using soluble auto-antigens or related antigens have not been very successful so far. In animal models of arthritis and experimental autoimmune encephalomyelitis (EAE) it has been shown to be difficult to achieve tolerance by feeding antigen during the disease process, whereas feeding the antigen before disease induction was shown to be successful in decreasing the disease symptoms (2-8). There are only a few reports describing tolerance induction during disease or in previously primed animals despite the fact that this is the relevant clinical set-up to investigate the efficiency of therapeutic strategies for the treatment of inflammatory autoimmune diseases. Animals can be rendered tolerant only when high doses of antigen are administered orally immediately after immunization, before the onset of clinical signs. Similarly, multiple feedings of low doses of antigen during disease result in only a very modest suppression of immune responses or disease activity (9-12). In summary, we can conclude that feeding soluble antigen alone is not sufficient to suppress an ongoing immune response or disease process.

Oral feeding of antigen in the context of an activated immune system can only induce immune tolerance when an additional therapeutic strategy is applied to modulate the local cytokine milieu in the gut. There is thus a need for a method to improve the induction of immunotolerance and to prevent unwanted immune reactions against antigen, for example to treat patients with autoimmune disease, or allergic reactions, or chronic inflammation. The abovementioned unwanted immune reactions trigger the β₂-adrenergic receptor (AR). Triggering of the β₂-adrenergic receptor (AR) results in accumulation of intracellular cyclic adenosine mono phosphate (camp), which will influence immune functioning (13)(14,15). β₂-AR stimulation of monocytes and macrophages leads to a decrease in IL-12 production and an increase in IL-10 production thereby favoring an anti- inflammatory response (16,17)(19-21). β2-adrenergic agonists are known to inhibit mitogenic responsiveness of lymphocytes among others by decreasing interleukin (IL)-2 production (14,15)(16-18). In addition they have an important effect on the production of regulatory cytokines. The present invention teaches in one embodiment a method for treating unwanted immune reactions by co-administering a 62 —adrenergic agonist, and a predefined antigen, wherein said antigen is correlated with said unwanted immune reaction. A 62 -adrenergic agonist changes the production of regulatory cytokines thus changing the immune response against said antigen, thereby improving the induction of immunotolerance against said antigen. Said unwanted immune reaction comprises any immune reaction having a (prolonged) deleterious effect on the body, such as for example, an auto-immune disease and/or an allergic disease and/or transplant rejection, and/or graft versus host reaction and/or a delayed type hypersensitivity reaction, for example, but not limited to, Crohn's disease, rheumatoid arthritis, Hashimoto's disease, asthma, allergic skin reaction, hypersensitivity to food allergens like celiac disease, multiple sclerosis, atopic dermatitis and contact dermatitis. Said predefined antigen is related with said unwanted reaction, and/ or belongs to the group of allergens such as food allergens like for example but not limited to, ovalbumin or protein from milk or wheat gluten, and/or inhaled allergen like for example but not limited to pollen, and/or autoimmune antigen like for example but not limited to myelin basic protein or sperm antigens or ocular antigens, and or a tissue antigen like a transplantation antigen, and/or heat shock protein, a functional equivalent of any of these, or a combination of any of these, or a mimotope thereof, mimotope being defined as an epitope having a different sequence but eliciting the same kind of immune response. Because local administration of a sufficient amount and form of 62 —adrenergic agonist causes a local decrease in pro- inflammatory cytokines and a local increase in anti-inflammatory cytokines, said method of the invention improves the induction of immune tolerance against said antigen and can also be applied to other parts of the body. This has the advantage of offering different routes of administration like for example dermal or nasal or oral or enteral administration. Dermal administration is useful for antigens that are digested too fast in the digestive tract or have a strong taste or any other adverse characteristic, like for example but not limited to, oral toxicity, that makes them less suitable for oral administration. For dermal administration the antigen and a 62 —adrenergic agonist are suspended in a fluid, or a cream or a fatty substance to facilitate the application. Nasal or oral administration is useful because of its ease of administration. To assure delivery of certain antigens at the intestinal mucosa without digestion in the stomach, the antigen may be given in capsules or tablets with an enteric coating. The antigen and a 62 -adrenergic agonist can also be administered in a suppository to assure delivery at the intestinal mucosa without digestion in the stomach. Thus the invention in one embodiment discloses a method for improved induction of immune tolerance against said predefined antigen comprising nasal, more preferably dermal, and even more preferably oral or enteral co- administration of a β₂-adrenergic receptor and said predefined antigen, said 62 -adrenergic agonist applied in an amount and in a form capable of decreasing the pro-inflammatory cytokine pattern and increasing the anti-inflammatory cytokine pattern (at least) at the site of antigen uptake and/or antigen handling. Many substances have a β₂- adrenergic agonist function. Some examples of β₂-adrenergic agonists are for instance SALBUTAMOL, TERBUTALINE, or CLENBUTEROL, or PROCATEROL, or SALMETEROL, or FORMOTEROL, or FENOTEROL, but other substances are a functional derivative or analogue thereof (that at least in kind if not in amount have the same function as for example SALBUTAMOL as disclosed herein). In a preferred embodiment of the invention, abovementioned methods for treating unwanted immune reactions and improving the induction of immune tolerance by co-administration of a 62 —adrenergic agonist and a predefined antigen, comprises as a 62 -adrenergic agonist for example SAMBUTAMOL, TERBUTALINE, or CLENBUTEROL, or PROCATEROL, or SALMETEROL, or FORMOTEROL, or FENOTEROL, or a functional derivative thereof that at least in kind if not in amount, has the same function as SALBUTAMOL or a combination of any of these. Local administration of a β₂-adrenergic agonist temporarily modulates the local immune response to an antigen, therefore, said co- administration comprises applying β₂-adrenergic agonist, for example SAMBUTAMOL, and said antigen in the same period of time at the same site of the body, said body comprising a mammal. More preferably, said mammal comprises a human. The same or even a better effect can be achieved by administering β2-adrenergic agonist separately from said antigen to the same body. Therefore, co-administration also comprises applying β2-adrenergic agonist and a predefined antigen separately but in such a way that both compounds are bio-available in said body at the same time. The site of antigen uptake may differ from the site of antigen handling, for instance in lymphoid tissue. The cytokine modulating effect of β₂-adrenergic agonist is important for both antigen uptake and/or antigen handling. Thus, co-administration also comprises applying β₂-adrenergic agonist orally, and/or enterally and/or nasally, and/or dermally, at an amount and in a form sufficient to decrease the pro-inflammatory cytokine pattern and to increase its anti-inflammatory cytokine pattern (at least) at the site of antigen uptake and/or antigen handling.

Both during adjuvant arthritis (AA) in the rat as well as in an ovalbumin (OVA)-specifϊc delayed type hypersensitivity (DTH) model in mice, SALBUTAMOL potentiated the efficacy of oral tolerance induction.

Interestingly, SALBUTAMOL feeding also suppressed the OVA-specific humoral response, especially the antibodies of the I G2a subclass. This is an important finding since humoral immune responses have been shown to be difficult to tolerize when low doses of antigen are used for tolerance induction (9,12,18,19)(10,13,22,23). The invention thus discloses among others that oral tolerance induction during an ongoing immune response is significantly improved by oral co-administration of a β₂-adrenergic agonist, for example abovementioned SALBUTAMOL or derivatives thereof. Feeding SALBUTAMOL together with the antigen OVA from day 5-10 after immunization with OVA induces a long-lasting tolerance. Even after 3 boosters with OVA/IFA the mice were still tolerant both at the humoral and the cellular levels. Thus administration of SALBUTAMOL as an additional therapeutic strategy in tolerance protocols using (auto) antigens for the treatment of human pro-inflammatory auto-immune diseases is useful. 62 - adrenergic agonists like for example SALBUTAMOL have never been used or administered in combination with antigen with the express purpose to induce tolerance. This invention in another embodiment provides the use of a 62 - adrenergic agonist, like for example SALBUTAMOL in combination with a predefined antigen, as a medicament to induce tolerance against said predefined antigen. In one embodiment, the invention provides a pharmaceutical composition comprising a 62 -adrenergic agonist like for example SALBUTAMOL and a predefined antigen with a suitable diluent or carrier substance. In the art, many substances are known and used as diluents and carriers for the administration of 62 —adrenergic agonist. Said diluent or carrier substance comprises any liquid, fatty substance or solid substance, suitable for oral, and/or nasal, and/or dermal administration of the pharmaceutical composition. In one embodiment of the invention, said 62 - adrenergic agonist is administered to a patient 9separately from said antigen. In a preferred embodiment of the invention, said 62 -adrenergic agonist, like for example SALBUTAMOL and said predefined antigen are combined before administration to a patient. Now that said pharmaceuticals are disclosed, the invention also teaches the use of said pharmaceutical compositions for treating patients with unwanted immune reactions. Said pharmaceutical compositions can be used for reducing unwanted immune reactions, and/or improving induction of immune tolerance, and/or treating an allergic reaction or autoimmune disease, or delayed type hypersensitivity.

Said pharmaceutical composition can be given orally or administered by the enteral or intranasal or dermal route for the reasons as described above. Thus, the invention also provides the use of said pharmaceutical composition by enteral, and/or intranasal, and/or dermal administration to increase induction of immunotolerance and/or to decrease unwanted immune reactions, said reactions comprising for example an autoimmune disease, and/or an allergic reaction and/or transplant rejection, and/or graft versus host reaction and/or a delayed type hypersensitivity reaction.

Above-mentioned administration of said pharmaceutical composition teaches a method to treat patients suffering of autoimmune disease and/or allergic disease and/or a delayed type hypersensitivity reaction.

One of the mechanisms via which SALBUTAMOL improves oral tolerance induction is by changing the balance between pro- and anti- inflammatory cytokines. As an immediate effect of oral SALBUTAMOL administration in immunized animals, an increase was observed in production of anti-inflammatory cytokines, such as IL-10, TGF-β and IL-lRa, in both the intestine and in the peritoneal macrophages, whereas pro-inflammatory cytokines, like IFN-γ and TNF-α, decrease at both sites. Although it is not known which cells in the intestine are responsible for the observed changes in cytokine production, it is conceivable that the predominant cellular component of the intestine, i.e. enterocytes, plays a major role. First of all, purified enterocytes express β2-adrenergic receptors (unpublished results, Cobelens et al.). Secondly, it is known that enterocytes or enterocytic cell lines can actively produce a number of cytokines, including TNF-α, TGF-β, IL-10, and IL-lRa (20,21)(24-30). Macrophages also express β₂-adrenergic receptors and in vitro exposure to a β₂-agonist results in decreased TNF-α production as well as in increases in TGF-β, IL-lRa and IL-10 presumably via accumulation of intracellular cAMP (19,20,31-34)16,22,23). Therefore, modulation of cytokine production by oral SALBUTAMOL may be the result of a direct effect of the drug on enterocytes as well as macrophages. Another immediate effect of OVA/SALBUTAMOL administration is an increase in antigen-induced IL-10 production by splenocytes. Sanders et al. have shown that only THI cells express β2-adrenergic receptors, whereas mature TH2 cells do not, either at the mRNA or receptor protein level (24). In view of these data, OVA-induced IL-10 increases in cells of mice fed with OVA and SALBUTAMOL probably do not result from a direct effect of SALBUTAMOL on TH2 cells. Probably, SALBUTAMOL will act by inhibiting IL-12 production or increasing IL-10 production by macrophages, thereby favouring IL-10 production by antigen- specific cells. It may well be possible that the IL-10 is produced by Trl-cells, which may express β2-AR (25,27). The invention also shows here that co- administration of OVA/SALBUTAMOL results in long-lasting tolerance for at least 12 weeks. At this time point, however, cytokine patterns in the intestine are no longer different from PBS-treated animals (data not shown). Moreover, antigen-induced IL-10 production has returned to normal levels. In contrast, a marked increase was observed in OVA-induced IFN-γ production after 1 booster (at 5 weeks) and after 3 boosters (at 12 weeks) in OVA/SALBUTAMOL treated animals. Thus IFN-γ plays an important role in the maintenance of tolerance. In line with an important role for IFN-γ in tolerance induction, tolerance could not be achieved in IFN-γ knockout mice (27). In addition, results from studies on animal models of autoimmune diseases suggest that IFN-γ plays a role in down-regulating tissue inflammation (28). This invention discloses that long-lasting tolerance induction is not simply mediated by a decrease in the balance between pro- and anti-inflammatory cytokines, but will involve immediate up regulation of anti-inflammatory cytokines during oral antigen administration as well as long-lasting up regulation of some pro- inflammatory mediators, i.e. IFN-γ, to control the ongoing immune or disease process. The present invention among others discloses that both the cytokine production (intestinal and systemic) and the specific antibody responses are involved in the potentiating effect of β2-adrenergic agonist, for example SALBUTAMOL, on tolerance induction, for example in an ovalbumin (OVA) - specific delayed-type hypersensitivity (DTH) model. The invention further discloses the clinical relevance of oral co-administration of β2-adrenergic agonist, for example SALBUTAMOL, with a predefined antigen for tolerance induction in a rat model of adjuvant arthritis (AA).

Because this invention discloses a method to induce immune tolerance, it is clear to anyone skilled in the art that also a method is disclosed with which antigens can be selected that cause unwanted immune reactions. This can for example be achieved by administering a number of candidate antigens in combination with a 62 -adrenergic agonist, to groups of experimental animals suffering from an unwanted immune reaction, and recording in which group of animals the unwanted immune reaction is diminished of decreased.

Oral co-administration of SALBUTAMOL with HSP65 to rats during adjuvant arthritis has proven to be very effective in down regulating the clinical symptoms of the disease. It is already documented that no significant tolerance can be induced in AA in the rat by oral administration of multiple doses of the (cross-reactive) antigen mycobacterial HSP65 only (29). This invention discloses that effective tolerance can be achieved with oral HSP 65 treatment by co-administration of SALBUTAMOL. Moreover, in the OVA model we showed that a single course of 6 feedings with SALBUTAMOL and antigen induces a long-lasting suppression of the immune response for at least 12 weeks. The latter results underline the clinical importance and thus that SALBUTAMOL, or other β₂-adrenergic agonists, are of great value for improving the therapeutic efficacy of oral tolerance induction protocols using auto-antigens for the treatment of pro-inflammatory auto-immune diseases in man and mammals. The use of SALBUTAMOL, or other β₂-adrenergic agonists, is also of great value for improving the therapeutic efficacy of oral tolerance induction protocols using allergens for the treatment of allergic disease symptoms in man and mammals. The invention is further explained in the detailed description.

Detailed Description

Example 1

Oral administration of SALBUTAMOL potentiates HSP65-induced disease suppression in adjuvant arthritis. We tested the possible clinical use of SALBUTAMOL to potentiate oral tolerance induction during a disease process in the adjuvant arthritis (AA) model in Lewis rats. In an earlier study we have shown that ongoing AA in Lewis rats cannot be suppressed by feeding the cross-reactive antigen mycobacterial heat shock protein (HSP) 65 alone. Therefore we now co- administered SALBUTAMOL together with HSP65. We started treatment at the onset of clinical signs of arthritis (on day 11 after immunization). From this time point, rats received 450 μg of SALBUTAMOL and 30 μg of HSP65 orally every other day for a total of 5 doses. Control rats received SALBUTAMOL or HSP65 alone or 30 μg of superoxide dismutase (SOD) as an irrelevant antigen. Feeding HSP65 in combination with SALBUTAMOL significantly suppressed the clinical signs of arthritis (mean cumulative arthritis score 40,2 + 2,5 in SOD-treated rats vs. mean cumulative arthritis score 18,3 ± 2,4 in HSP65/SALBUTAMOL-treated animals) (Fig. 1). Feeding irrelevant antigen SOD in combination with SALBUTAMOL had no significant effect on the arthritis score. In addition, treatment with SALBUTAMOL alone did not affect disease severity.

Example 2

Oral co-administration of SALBUTAMOL and OVA after immunization with OVA-CFA induces tolerance.

Since AA in rats is a mono-phasic disease, the model cannot be used to determine whether SALBUTAMOL modulates long-term tolerance induction during an ongoing immune process. Therefore we examined the effect of SALBUTAMOL on oral tolerance induction in a murine model of OVA- induced delayed type hypersensitivity (DTH). We immunized mice i.p. with OVA in CFA and 18 days later animals received a booster with OVA in IFA. One week after boosting, we challenged the animals with OVA in the footpad and measured paw swelling 24 hours later. Five days after immunization with OVA in CFA we started oral tolerance induction. From this time point onwards mice received orally 10 μg of SALBUTAMOL and 250 μg of OVA every other day for a total of 5 doses. Control mice received PBS, OVA alone, or SALBUTAMOL alone.

Oral administration of OVA or SALBUTAMOL alone had no significant effect on the DTH-response (Fig. 2d). However, when we co- administered OVA and SALBUTAMOL the DTH-response was reduced by 70% (P < 0.01; Fig. 2d).

From a clinical perspective it is crucial to test the long-term efficacy of oral co-administration of OVA and SALBUTAMOL. To this end mice that had been treated with one regimen of OVA/SALBUTAMOL from day 5-10 after immunization, received a second and a third booster (i.p. injection of OVA in IFA) on day 43 and 68 after immunization. One week after each booster we determined the OVA-specific DTH-response. OVA/SALBUTAMOL-treated animals were still tolerant after the second booster and even after a third booster (Fig. 2b).

We also analysed sera after the first booster (day 35 after immunization) from each group of mice for anti-OVA IgGtotai. The antibody response was lower in both the OVA (123 ± 13 U/ml) and SALBUTAMOL- treated (150 ± 17 U/ml) groups compared to PBS-treated mice (267 ± 31 U/ml). However, the response was suppressed more profoundly in the

OVA/SALBUTAMOL-treated group (69 ± 7 U/ml). To get an impression of the contribution of Tiil-driven and TH2-driven antibody responses, we looked at the ratio of OVA-specific IgG_2a over IgGi. Feeding SALBUTAMOL in combination with OVA resulted in a large decrease in the OVA-specific IgG2a/IgGι ratio compared to PBS-treated mice (Fig. 2c). Feeding OVA or SALBUTAMOL alone did not change the relative contribution of I 2a and

Even after the third booster (day 82 after immunization) the effect of one treatment regimen with OVA and SALBUTAMOL on antibody production was maintained (Fig. 2d).

Example 3

SALBUTAMOL modulates intestinal cytokine production. We next investigated the effect of SALBUTAMOL on the expression of regulatory cytokines in the small intestine. Five days after immunization with OVA in CFA, mice received a single dose of SALBUTAMOL orally. Eight hours later we prepared a homogenate of whole intestinal samples and determined pro and anti-inflammatory cytokine expression on both mRNA and protein level. Oral SALBUTAMOL treatment resulted in down regulation of IFN-γ and up regulation of IL-10 mRNA expression in the small intestine (Fig. 3σ). The expression of IL-12 mRNA was below detection limit. SALBUTAMOL did not affect mRNA expression of the pro-inflammatory cytokine IL-lβ. However, mRNA encoding the anti-inflammatory mediator IL-lRa was significantly augmented in mice given oral SALBUTAMOL compared to those given oral PBS (Fig. 3α).

We confirmed some of our mRNA data by measuring IFN-γ and IL- 10 at the protein level in intestinal samples by ELISA. In addition, we measured the inflammatory cytokine TNF-α and the anti-inflammatory cytokine TGF-β by ELISA. The production of the inflammatory cytokines IFN-γ and TNF-α were down regulated in SALBUTAMOL-fed mice, whereas the anti-inflammatory cytokines IL-10 and TGF-β were unregulated compared to PBS-treated mice (Fig. 36).

Example 4

SALBUTAMOL modulates cytokine production by peritoneal macrophages.

We examined possible systemic effects of feeding SALBUTAMOL on cytokine expression by peritoneal macrophages. Again, we treated mice with a single dose of SALBUTAMOL at day five after immunization with OVA in CFA. Eight hours later we analyzed peritoneal macrophages for cytokines at both mRNA and protein level. The expression of IL-10 and IL-12 mRNA was below detection limit. However, comparable to the intestinal cells, SALBUTAMOL decreased macrophage mRNA expression for IFN-γ and increased mRNA expression for IL-lRa, whereas expression of IL-lβ mRNA was not altered (Fig. Ad). In addition, oral treatment with SALBUTAMOL significantly suppressed the production of TNF-α protein by peritoneal macrophages (Fig. 4b). Example 5

SALBUTAMOL modulates antigen-induced cytokine production by splenocytes.

One of the important questions is how SALBUTAMOL treatment alters regulatory cytokine production of antigen-specific cells. To address this question we collected spleens at day 9 after immunization i.e. after 2 feedings with OVA and SALBUTAMOL. Subsequently, we stimulated splenic cells in vitro for 72 hours with OVA and determined IFN-γ and IL-10 production. Control cultures without OVA did not contain detectable levels of IFN-γ and IL-10. Feeding OVA or SALBUTAMOL alone elevates OVA-induced IFN-γ production by splenocytes in vitro. Co-administration of OVA and SALBUTAMOL did not further enhance IFN-γ production (Fig. 5α). In contrast, feeding OVA alone or SALBUTAMOL alone did not change IL-10 production by splenocytes. Only the co-administration of OVA and SALBUTAMOL resulted in an increase in IL-10 production after antigen- specific stimulation of splenocytes in vitro (Fig. 5b).

Example 6

Feeding OVA/SALBUTAMOL results in long-term changes in antigen- induced cytokine production in vitro by splenocytes.

To address the question whether the long-term tolerance of the DTH response is reflected by long-term changes in the T cell compartment, we investigated antigen-induced cytokine production at day 35 after immunization. We treated mice orally with OVA/SALBUTAMOL from day 5 after immunization with OVA in CFA. Mice received a booster at day 18 after immunization. Two weeks later (day 35) we cultured the splenocytes in vitro in the presence of OVA for 72 hours. At this time point, IFN-γ production was significantly enhanced in OVA SALBUTAMOL-treated animals as compared to PBS-treatment, whereas treatment with OVA or SALBUTAMOL alone did not significantly change IFN-γ production (Fig. 5c). In contrast to the enhanced splenic IL-10 production immediately after feeding OVA/SALBUTAMOL (day 9), there was no longer a significant change in OVA-induced IL-10 production by splenocytes from OVA/SALBUTAMOL-treated animals on day 35 (Fig. 5d). Interestingly, we observed the same effects of OVA/SALBUTAMOL treatment on OVA-induced cytokine production after 3 boosters of OVA in IFA (day 82 after immunization). No significant difference in IL-10 production, but significantly elevated levels of IFN-γ by splenocytes from OVA/SALBUTAMOL-treated mice (Fig. 5e).

Example 7

Salbutamol reduces disease severity in human patients suffering from rheumatoid arthritis.

40 Patients suffering from rheumatoid arthritis are selected from a group of well described patients (UMCU). Inclusion criteria are as follows: established diagnosis of rheumatoid arthritis for more than 2 years with active arthritis. Exclusion criteria are: treatment with more than 10 mg prednisolone per day. The patients are randomly divided in four groups: group 1, 2, 3 and 4.

Patients in group 1 are treated by oral administration every other day for a total of 5 doses of Salbutamol (4.2 mg/person and bovine type II collagen (0.5 mg/person). Patients in group 2 are treated in the same way as the patients in group 1, except that they receive ovalbumin as a control antigen.

Patients in group 3 are treated in the same way as the patients in group 1, except that Salbutamol is replaced by a harmless placebo substance. Patients in group 4 are treated in the same way as the patients in group 1, except that both Salbutamol and the antigen are replaced by a harmless placebo substance.

The observation period is 6 months during which period the patients are examined at regular monthly intervals and a questionnaire is filled out at every examination.

Results of group 1 are significantly different from groups 2 to 4, meaning that in the patients in group 1, disease activity scores are significantly reduced.

Materials & Methods

Animals.

Lewis rats (6-8 weeks) (University of Limburg, Maastricht, The Netherlands) and Balb/c mice (6-8 weeks) (Charles River, Sulzfeld, Germany) were kept at the Utrecht University animal facility, fed a standard diet (Hope Farms, Woerden, The Netherlands) and water ad libitum.

Induction and clinical evaluation of AA.

AA was induced in male Lewis rats by a single intradermal injection of 100 μl of Freund's complete adjuvant (5 mg M tuberculosis (strain

H37Ra)/ml incomplete Freund's adjuvant (IFA) (Difco, Detroit, MI) in the base of the tail. Severity of arthritis was assessed daily by standard methods in a blinded protocol (29)(38). A cumulative score was calculated for each individual animal by summation of the individual disease scores from day 11 until day 20.

Mycobacterial HSP65 from Mycobacterium bovis was expressed in Escherichia coli and isolated as described (28). Rats were treated orally with 450 μg of SALBUTAMOL (Sigma; St. Louis, MO) together with 30 μg of HSP65 or superoxide dismutase (SOD)(Sigma) in 1 ml of PBS using an 18-gauge animal-feeding needle (Popper & Sons, In., New York, NY). Treatment was started when most of the animals lost weight, that is, at the onset of clinical arthritis. Oral administration of HSP65 or SOD and SALBUTAMOL was repeated every other day for an additional 4 doses.

Induction and assessment of DTH.

For the OVA-specific immune response a modified protocol from Chung et al. was used (9). Female Balb/c mice were immunized i.p. with 20 μg of OVA (grade V; Sigma) emulsified in Freund's complete adjuvant (CFA, Difco). Eighteen days after immunization, mice were boosted i.p. with 20 μg of OVA in IFA (Difco). One week later, mice were challenged by a subcutaneous injection into the footpad with 50 μl PBS containing 400 μg/ml OVA and 1 mg/ml AIOH3. After 24 hours we measured thickness of the footpad.

Mice were treated orally with 10 μg of SALBUTAMOL and 250 μg of OVA dissolved in 0,5 ml PBS using a 20-gauge stainless steel animal-feeding needle (Popper & Sons, In.). Treatment was started 5 days after the first immunization. Oral administration of the drugs was repeated five times every other day. To analyse long-term effects of the tolerance protocol, mice were boosted again with OVA in IFA on day 43 and day 68 after immunization, followed by a challenge 7 days later. In the pre-treatment protocol, mice were fed six times 10 μg of

SALBUTAMOL and 250 μg of OVA dissolved in PBS every other day from 13 days prior to immunization with OVA in CFA.

OVA-specific antibody production. Microtiter plates (Maxisorp F96; Nunc, Roskilde, Denmark) were coated with OVA (5 μg/ml) in PBS. After washing the plates with PBS/Tween (0.05%), the plates were blocked with 200 μl/well PBS/1% BSA (Sigma). Diluted serum samples were added to the wells and incubated for 2 hours at room temperature. Biotin-conjugated goat anti-mouse IgG (Zymed; San Francisco, CA) diluted in PBS/1% BSA/0.005% Tween-20 (IgG_totai: 1:10000; IgGi and IgG₂a: 1:1000) was added and incubated. Plates were developed with horseradish peroxidase-labelled streptavidin (1:10000) and 3, 3', 5,5'- tetramethylbenzidine substrate (TMB)(ICN Biomedicals, Zoetermeer, The Netherlands). Antibody concentrations in arbitrary U/ml were determined from standard curves constructed from serial diluted samples. The IgG2a/IgGι- ratio was calculated by dividing the concentrations.

Splenocyte culture.

Splenocytes were cultured serum-free in quadruplicate in 200 μl at 1.5 x 10⁵ cells per well in presence or absence of OVA (125 μg/ml final concentration).

Preparation of intestinal samples and peritoneal macrophages.

Five days after immunization with OVA in CFA, mice were treated orally with 10 μg SALBUTAMOL. Eight hours later mice were sacrificed and peritoneal lavages were obtained as a source of macrophages. In addition, 2 cm of distal ileum was collected, homogenized in PBS and used for determining cytokine production.

Cytokine mRNA analysis.

RNA was isolated using Trizol reagent (Gibco) and cytokine mRNA expression was analysed by RNase Protection kit (PharMingen, San Diego, CA). RNA expression was visualized by phospor-imaging and Molecular Analyst software (Bio-Rad, Richmond, CA). Results are expressed as the percentage of household gene (L32) expression.

Cytokine ELISA.

IFN-γ and TNF-α were determined using ELISA kits from U-Cytech (Utrecht, The Netherlands). IL-10 and TGF-β were determined using OptEIA kits from PharMingen. Oral dose of Salbutamol for rheumatoid arthritis patients:

Extended release tablets:

Each Proventil Repetab extended-release tablet contains a total of 4 mg (2 mg in the coating for immediate release and 2 mg in the core for release after several hours) of salbutamol as 4.8 mg of salbutamol sulphate. The inactive ingredients for salbutamol extended-release tablets include: butylparaben, calcium phosphate, calcium sulphate, lactose, magnesium stearate, oleic acid and titanium dioxide. The slower acting extended release tablets have a duration of action of at least 12 hours after a single dose.

Collagen II tablets are prepared by absorption onto a lactose base.

Clinical assessment

Clinical assessments are performed at weeks -1, 0, 1, 4, 8, 12, 16, 20, and 24. Assessments are based on the ACR and European League Against Rheumatism core data sets and included duration of early morning stiffness, fatigue score, the number offender and swollen joints (28-joint score), visual analogue scale for pain, patient's and physician's global assessment of disease activity, ESR, and modified Health assessment Questionnaire.

Statistical analysis.

Group differences were analysed by one-way ANOVA, followed by Fisher's least significance difference (LSD) test or by Student's t-test for unpaired data. Figure legends

Figure 1

Oral co-administration of mycobacterial HSP65 and SALBUTAMOL reduces AA scores. From day 11 after induction of AA we treated rats (n=8 per group) every other day with oral administration of 30 μg HSP65 with or without 450 μg of SALBUTAMOL. Control rats received SOD with or without SALBUTAMOL. Similar results were seen in 2 separate experiments. **, P < 0.01, HSP65/SALBUTAMOL vs SOD and vs HSP65

Figure 2

Oral OVA/SALBUTAMOL after immunization reduces cellular and humoral immune responses.

At day 0 we immunized the mice and at day +5 we started treatment every other day with oral administration OVA and sal. We boosted the mice with OVA in IFA at day +18 (a), day +43 (6), and day +68 (b). One week later, we determined the DTH response (a and b). At day +35 (c) and day +82 (d) we analyzed sera for OVA-specific antibody production. All data represent mean ± SEM of 8 individual animals in each group and are representative for two independent experiments.

*, P < 0.05, OVA/SALBUTAMOL vs PBS **, P < 0.01 OVA/SALBUTAMOL vs PBS

Figure 3

Oral SALBUTAMOL changes cytokine production in the small intestine mRNA (a) and protein cytokine expression (b) of homogenates from the small intestine from PBS- ( ■ ) and SALBUTAMOL- ( GH ) treated mice. mRNA cytokine expression is expressed as percentage of household gene expression (L32). All data represent mean ± SEM of 8-10 individual animals in each group. *, P < 0.05, SALBUTAMOL vs PBS **, P < 0.01 SALBUTAMOL vs PBS

Figure 4

Oral SALBUTAMOL changes cytokine production in peritoneal macrophages. mRNA (a) and protein (b) cytokine expression of peritoneal macrophages from PBS- ( ■ ) and SALBUTAMOL- ( Q ) treated mice. mRNA cytokine expression is expressed as percentage of household gene expression (L32). All data represent mean ± SEM of 8-10 individual animals in each group. *, P < 0.05, SALBUTAMOL vs PBS **, P < 0.01 SALBUTAMOL vs PBS

Figure 5

Oral OVA SALBUTAMOL alters antigen-induced cytokine production by splenocytes. We stimulated splenocytes with OVA on day +9 after immunization (a,b), at day +35 after immunization (c,d), and at day +82 after immunization (e). We assayed supernatant for IFN-γ and IL-10 production. Data represent mean ± SEM of 6 individual animals in each group. *, P < 0.05, OVA/SALBUTAMOL vs PBS, **, P < 0.01 OVA/SALBUTAMOL vs PBS Reference List

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Claims

1. A method for treating unwanted immune reactions by co- administering a 62 -adrenergic agonist, and a predefined antigen, wherein said predefined antigen is correlated with said unwanted immune reaction.

2. A method for improving the induction of tolerance against a predefined antigen, before and/or during an ongoing unwanted immune reaction, comprising oral, and/or enteral, and/or nasal, and/or dermal co- administering a 62 —adrenergic agonist, and a predefined antigen, wherein said predefined antigen is correlated with said unwanted immune reaction.

3. A method according to claim 1 or 2, wherein said 82 —adrenergic agonist is applied at an amount and in a form capable of decreasing the pro- inflammatory cytokine pattern and increasing the anti-inflammatory cytokine pattern, (at least) at the site of antigen uptake and/or antigen handling.

4. A method according to any one of claims 1 to 3, wherein said 62 - adrenergic agonist is applied at an amount and in a form capable of improving the induction of immunotolerance.

5. A method according to any one of claims 1 to 4, wherein said unwanted immune reaction comprises an autoimmune disease, and/or transplant rejection, and/or graft versus host reaction and/or an allergic reaction, and/or a chronic inflammatory reaction.

6. A method according to any one of claims 1 to 5, wherein said predefined antigen belongs to the group of allergens such as food allergen, and/or inhaled allergen, and/or auto immune antigen, and/or heat shock protein, a functional equivalent of any of these, or a combination of any of these, or a mimotope thereof.

7. A method according to any one of claims 1 to 6, wherein said 62 - adrenergic agonist comprises SALBUTAMOL, and/or TERBUTALINE, and/or CLENBUTEROL, and/or PROCATEROL, and/or SALMETEROL, and/or FORMOTEROL, and/or FENOTEROL, or a functional derivative or analogue thereof.

8. A pharmaceutical composition comprising a 62 -adrenergic agonist and a predefined antigen, further comprising a suitable diluent.

9. A pharmaceutical composition according to claim 8, wherein said predefined antigen is combined with said 62 —adrenergic agonist before administration to a patient.

10. A pharmaceutical composition according to claim 8 or 9 for reducing unwanted immune reactions.

11. A pharmaceutical composition according to anyone of claims 8 - 10 for improving induction of immune tolerance.

12. A pharmaceutical composition according to anyone of claims 8 - 11 for treating an allergic reaction.

13. A pharmaceutical composition according to anyone of claims 8 - 12 for treating an autoimmune disease.

14. A pharmaceutical composition according to anyone of claims 8 - 13 for treating a delayed type hypersensitivity.

15. A pharmaceutical composition according to anyone of claims 8 - 14 for treating a delayed type hypersensitivity and/or transplant rejection, and/or graft versus host reaction.

16. A method to increase induction of immunotolerance, and/or decrease unwanted immune reactions, comprising providing a pharmaceutical composition according to any one of claims 8 —15 by oral, and/or enteral, and/or intranasal, and/or dermal administration.

17. A method according to claim 16 for treating a patient suffering of unwanted immune reactions, said reactions comprising an auto-immune disease, and/or an allergic reaction, and/or transplant rejection, and/or graft versus host reaction and/or a delayed type hypersensitivity reaction.

18. A method for selecting an antigen causing unwanted immune reactions, comprising co-administering an antigen with a 62 —adrenergic agonist according to any one of claims 1-7, further comprising recording the immune response against said antigen.