FI60568C - FOERFARANDE FOER FRAMSTAELLNING AV ETT KJJUGAT FOER BLOCKERING AV ALLERGISKA REAKTIONER - Google Patents

Description

R5R M (H ^ ANNOUNCEMENT PUBLICATION 605 6 8

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C .._ Patent granted 10 02 1932 * 1 ^^ ”Patent aeddelat ^ (51) Kv.lk.3 / lnt.CI.3 C 07 G 7/001 A 61 K 31/35, 37/02 FINLAND-FINLAND (21) Pttenttlhtkemu * - PatunttniBknlnf 800292 (22) H «keml» p * M - Ameknlncsd ·! 31/1/80

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Patent * and Registry Board .... M ... ..,. . ,,,. .

_ _. . (44) Date of dispatch and date of issue. -

Patent · och registrarstyrelsen 'Ansdlun uttagd oeh utUkrlft.n publtcarad 30.10.8l (32) (33) (31) Pyydetty atuolkau * —Bagird priorat 12.10. T6

USA (US) T31U1U

(Tl) Research Corporation, 1 + 05 Lexington Avenue, New York, NY, USA (T2) Arthur Malley, Portland, Oregon, USA (Tl *) Ruska & Co (5l +) Method for preparing a conjugate for the prevention of allergic reactions The present invention relates to a process for the preparation of a conjugate for the prevention of allergic reactions (62) which conjugate has the following structure H ° YTV ^ öfltc · · U ^ A ^^ OH glutathione or the same structure covalently linked to a protein or peptide by a peptide bond via a glutathione moiety.

Methods for treating allergies and allergic reactions have heretofore comprised administering to a subject an antigen that initiates the formation of antibodies that compete with the antibodies whose formation the sensitizer causes, thereby controlling or reducing the allergic reaction.

Hitherto, a common practice in the treatment of allergic patients involves the preparation of a crude extract of an allergenic substance in an aqueous buffer solution. Patients are then treated with the crude extract first in small doses and then in increasing doses over a long period of time. Usually, three or more years of treatment is necessary before a significant lasting improvement is noted in 2,60568. As the treatment progresses, the amount of crude extract administered to the patient is gradually increased. The amount of dose added and the frequency of administration will depend on the patient's sensitivity and degree of response to previous doses.

Injection of the entire crude sensitizer extract over long periods of time results in the formation of inhibitory or competing antibodies. Theories have been suggested that inhibitory antibodies compete with an allergic antibody for antigen and provide protection to the allergic patient.

An expensive and time-consuming method of removing sensitivity from a patient involves many obvious drawbacks. Very often, administration of the crude extract results in a severe allergic reaction if the doses have not been carefully controlled and dosed. Further, the formation of competing antibodies in an allergy patient very often causes side effects that may be more detrimental to the patient than the allergic reaction itself.

What is very significant is that the previously known method of removing sensitivity from a patient used so far has not been entirely successful. In many cases, the time-consuming desensitization method does not result in adequate protection for a patient who is still continuously suffering from an allergy when exposed to the antigen.

♦ The object of the present invention is to provide a product which is suitable for administration to an allergy patient and which neutralizes the existing antibodies and completely prevents the formation of antibodies which either compete with or are characteristic of the allergenic antigen.

In the manufacture of such products which prevent an allergic reaction, a method for treating allergic individuals to prevent allergic reactions has been provided. The product in question can be formulated as an injectable preparation for administration to an allergic patient to prevent allergic reactions.

The process according to the invention is mainly characterized in that quercitine of the formula

Hcy ^ n 6 0 5 6 8 3 is reacted with glutathione in the presence of an oxidizing agent, e.g. silver oxide, followed by activation of the carboxyl groups of the glutathione moiety with, for example, Woodward's reagent or carbodiimide, and then conjugation with the peptide via glutamic acid or glycine moiety. , such as human serum albumin or a d-amino acid copolymer of lysine and glutamic acid, wherein the molar ratio of quercitin-glutathione conjugate to peptide or protein is more suitably 2 to 30 moles of conjugate per mole of peptide or protein.

The term "conjugate" refers to a conventional chemical compound in which the reagents in question are chemically bonded to each other - as opposed to a complex in which the reagents are bound only loosely to each other by hydrogen bonds and the like.

Thus, these conjugates can be administered to an allergy patient to completely prevent an allergic reaction without the need for a long and time-consuming and expensive desensitization method as described above. Because the administration of the conjugates completely prevents the formation of allergic antibodies, the allergic reaction itself is prevented, as are any side effects caused by the formation of competing antibodies formed according to the usual desensitization method.

Results have been obtained with a conjugate having the structure

· OH

ΥΎ ° Ί- <J-0H

s "glutathione or a conjugate containing at least one quercitin-glutathione conjugate covalently linked to a protein or peptide via a peptide bond via a glutathione moiety. .

Thus, the quercitin-glutathione and quercitin-glutathione-protein conjugates described above are effective in inhibiting the allergic reaction.

The quercitin-glutathione ion conjugate is formed by oxidation-addition reaction using AgO or another suitable oxidant: glutamyl-cysteinyl-glycine κ (glutathione)

OH

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OH O glutamyl-cysteine-glycine

The resulting conjugate can then be conjugated to the peptide or protein by activating the carboxyl moieties of the glutathione moiety with, for example, Woodward's reagent, carbodiimides, or the like, followed by conjugation with the protein or peptide molecule. Quercitin-glutathione is bound to the protein by a peptide bond via either the glutamic acid moiety or the glycine moiety.

The peptide or protein conjugated to the quercitin-glutathione conjugate can be any peptide or protein suitable for administration to allergens. Preferred proteins and pent Ids / • / it I - in serum a Lbum L in i or lysir and gLutamine n L acid d-amir.o- 5 60568 acid copolymer (d-GL).

It will be appreciated that the quercityl glutathione molecule may be attached to different sites on the protein or peptide molecule. It is usually preferred to bind about 2.0 to 30 moles, preferably 5 to 20 moles of the quercitin-glutathione compound to 1 mole of the protein or peptide. The molar ratio can be varied by varying the molar ratio of the reactants in the manufacturing process. ·

The antigen-protein complexes described above may be incorporated into any suitable medium, such as buffered saline, for administration to an allergy patient.

Ordinary additives, excipients and other substances may be added to the preparation for injection. However, it is understandable that they are not necessary.

The preparations are usually injected subcutaneously, intramuscularly or intravenously.

Generally, injectable doses of about 0.01 to 0.25 mg of product, preferably 0.025 to 0.1 mg / kg of body weight, will be sufficient to provide an acceptable level of allergic response.

It will be appreciated that the dose will be determined by various factors such as the duration of the complex in the circulation, the antibody titer of the patient being treated, the state of resistance of the patient and the duration of inhibition of the IgE response.

The products prepared by the method of the invention are useful in treating grass-sensitive patients to reduce allergy symptoms. They prevent the formation of allergic antibodies to pollen antigens in the grass and also neutralize the antibodies present in the patient's body. It will be appreciated that the invention is useful in preventing allergic reactions not only to timothy but also to pollen from pollen and other grass pollen which cross-react.

6 60568

The following Examples 1 and 2 illustrate the preparation methods of the invention.

Example 1

Preparation of Quercitin Glutathione 1.5 g of AgCl was stirred with 10 mL of 4N NaOH for 20 minutes. The AgO precipitate was filtered on a Buchner funnel.

0.2 g of glutathione was dissolved in 4 ml of 0.25 N NaOH and placed in an ice bath. 0.2 g of quercitin was dissolved in 8 ml of 0.25N NaOH. 1.2 g of freshly prepared AgO was added to the latter and the mixture was stirred for 15-20 minutes in an ice bath.

The glutathione solution was then added and the mixture was stirred for one hour in an ice bath. Excess AgO was removed by filtration. The resulting solution was adjusted to pH 1-2.5 with HCl and stirred for 30 minutes.

The solution was filtered to remove free quercitin and the pH was adjusted to 7 with NaOH. Free glutathione was determined by the DNTB method (Anal. Biochem. 48, 1557, 1972).

Example 2

Preparation of Quercitin-Glutathione Protein Conjugate 40 mg of quercitin-glutathione was added to 100 mg of Woodward's reagent and stirred for 10 minutes at room temperature. The pH was adjusted to 6.2 with 0.3 M cocodylate buffer (pH 6.8).

100 mg of human serum albumin (HSA) was then added while maintaining the pH above 6.2. The mixture was allowed to stand at room temperature for 10-15 minutes and then overnight at 4 ° C. The solution was concentrated under reduced pressure. Dialysis removed free quercitin-glutathione from its conjugate with the protein.

7 60568

The following experiments illustrate the inhibitory properties of the products prepared by the method of the invention against allergic reactions.

Biological experiments Experiment 1

The release of histamine from monkey lung tissue passively sensitized with the serum of allergy patients was determined according to the method of Malley et al., J. Immunol, ion, 915 (1968). The whey used to sensitize monkey lung tissue was obtained from a group of 20 timothy-sensitive patients with a mean allergic antibody titer of 1: 750. The monkey lung tissue was then incubated for two hours at 57 ° C with serum. Prior to sensitization, the tissue was incubated for 10 minutes at 37 ° C with inhibitor. The antigenic effect was obtained by incubating the crude pollen extract (WST) at the optimum concentration for 15 minutes at 37 ° C.

The results are shown in Table 1, where: WST denotes an aqueous extract of whole timothy pollen that has been dialyzed to remove low molecular weight fragments; HSA stands for human serum albumin.

As can be seen from the results shown in Table 1, the conjugates prepared by the method of the invention are effective inhibitors against allergic reactions.

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The passive transfer reactivity of the products prepared by the method of the invention was compared to those antigens present in timothy pollen.

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Experiment 2 0.1 ml of blood whey containing antibodies (whey from a group of 20 time-sensitive patients with an average P-K titer of 750) was injected intradermally into the skin of a non-allergic volunteer.

48 hours later, each site previously sensitized to that whey, were treated with different conjugates.

Allergic antibodies in a given blood serum react with allergenic antigenic antibodies as a local allergic reaction.

If the antigen did not cause allergic reactions, no local reaction was observed.

The results of various experiments performed on the products prepared by the method of the invention as well as the antigenic substance in timothy pollen are shown in Table 2, where dGL means a d-amino acid copolymer of glutamic acid and lysine with a molecular weight of about 40,000.

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4 »11 60568

The results shown in Table 2 show that the products prepared by the method of the invention do not initiate allergic reactions.

The following experiment illustrates the ability of the products prepared by the method of the invention to prevent the formation of allergic antibodies.

Test 3

Preliminary studies showed that LAF1 mice developed good IgE (allergic antibodies) against timothy pollen.

Animals were injected intraperitoneally with 10 μg WST in alum and after three weeks they received a second intraperitoneal injection of 10 μg WST in alum. IgE titers increased between 1/100 and 1/200 as measured by PCA after initial immunization. IgE titers after the second injection of antigen increased to values between 1/600 and 1/800 as measured by PCA.

Passive skin anaphylaxis (PCA) was measured by injecting 0.025 ml of diluted serum into the flank of a mouse or rat. 72 hours (mouse) or 3 hours (rat), animals were injected intravenously with WST in Evans Blue Dye.

The reaction between the circulating antigen and the tissue-attached IgE elicited a local allergic reaction and this site became visible as a blue color seepage into the reactive area. The IgE titer was determined as the lowest dilution that gives a positive PCA reaction.

The product of the invention selected for experimental purposes was:

Quercitin-glutathione-dGL (Q-G-dGL): prepared by conjugating an average of 12 to 20 Q-G groups / mole of dGL to quercitin-glutathione dGL.

Q-G-dGL was administered intraperitoneally 3 days before the second immunization of WST.

12 60568 days 0 l8 21 28 injection · WST Q-G-dGL WST seepage in alum alum

Results: Control animals receiving saline on day 18 have the following IgE titer-600; average reaction in 18 animals.

Animals treated with Q-G-dGL: PCA titer Group of animals

Low dose (0.24 mg quercitin) 600 6

Medium dose (1.8 mg quercitin) 300 6

High dose (3S6 mg quercitin) 100 6 These values indicate that the conjugate significantly reduces IgE levels when administered 3 days before the second antigen injection, indicating neutralization and disappearance of the antibody. Similar experiments have shown that administration even 7 days before the second antigen injection gives essentially the same results.

Claims (1)

60568 13 A method for preparing a conjugate for inhibiting allergic reactions having the following structure TT 1 -O- glutathione or the same structure covalently linked to a protein or peptide by a peptide bond via a glutathione moiety, characterized in that the quercitin of formula in the presence of an oxidizing agent, e.g. silver oxide, followed, if desired, by activating the carboxyl groups of the glutathione moiety with e.g. wherein the molar ratio of quercitin-glutathione conjugate to peptide or protein is preferably 2-3 moles of conjugate per mole of peptide or protein.