RU2711727C1 - Pharmaceutical product containing mite allergen extract(s) and method for production thereof - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention refers to pharmaceutical industry, namely to a pharmaceutical composition for treating allergy caused by house dust mites. Disclosed is a pharmaceutical composition for treating allergy caused by home dust mites, comprising an extract of allergens Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) as an active ingredient, which contains allergen extract: group 1 ticks allergens - Der f 1 and Der p 1 and Group 2 allergens - Der f 2 and Der p 2 and where allergens Der p 1, Der f 1, Der p 2 and Der f 2 are present in predetermined controlled amounts, so that the weight of group 1 allergens - Der f 1 and Der p 1, to allergens of group 2 - Der f 2 and Der p 2, is in ratio from 1: 0.7 to 1:1.5; and where the pharmaceutical composition is a rapidly dissolving sublingual tablet; using a pharmaceutical composition for preparing a medical product for treating allergy caused by home dust mites; a medical product for treating allergy caused by house dust mites; using a pharmaceutical composition for treating allergy caused by home dust mites; method of treating allergy caused by home dust mites, wherein said method involves administering to the patient an effective amount of the pharmaceutical composition.EFFECT: solution described above enables to obtain and use a pharmaceutical preparation containing mite extract with controlled ratio of group 1 allergen to allergen of group 2.12 cl, 1 dwg, 3 tbl, 7 ex

Description

The invention relates to a pharmaceutical product suitable for treating and / or preventing allergies to house dust mites and allergic asthma caused by house dust mites containing a tick allergen, and a method for producing it.

State of the art

In countries with a western lifestyle, allergies are a major public health concern. Moreover, the incidence of allergic diseases is increasing in these countries. Although allergies generally cannot be considered life-threatening diseases, asthma causes a significant number of deaths every year. The exceptionally high prevalence of allergies in approximately 30% of adolescents leads to a significant loss in the quality of life, working days and finances, and serves as the basis for classifying it as a major health problem in Western countries.

Clinically, an allergy and its symptoms appear in several ways and can vary depending on the particular sensitized individual and the causes of the allergy. Common symptoms are swelling, itching, redness, and outflows from the eyes and nose (rhinitis and conjunctivitis) and symptoms from the upper and lower respiratory tract, such as shortness of breath, cough, shortness of breath, skin manifestations, such as eczema, hives, and itching. Other symptoms, such as fatigue or insomnia, also occur. Symptomatic treatment is aimed at reducing or affecting the severity of symptoms or reducing the need to use other drugs prescribed in parallel. Medicines for symptomatic treatment include antihistamines, such as H ₁ and H ₂ receptor antagonists, corticosteroids for intranasal and systemic administration, non-steroidal anti-inflammatory drugs, decongestants for relieving nasal congestion, such as adrenoreceptor agonists. Treating and alleviating one or more allergy symptoms and / or reducing the need for another treatment is an additional objective of the present invention.

Allergen-specific immunotherapy (also related to allergen immunotherapy, allergy immunotherapy, allergy specific vaccination, hyposensitization or desensitization) is a treatment for an allergic disease that addresses its cause. It affects the basic immunological mechanisms, leading to a persistent or long-term improvement in the patient's immune status. Thus, the protective effect of specific vaccination against allergies lasts longer than only during the treatment period, as opposed to symptomatic treatment with drugs. Some patients receiving such treatment are cured and, in addition, most patients have a weakening of the severity and symptoms of the disease, or at least a halt of exacerbation of the disease. Allergen immunotherapy also has prophylactic effects that reduce the risk of hay fever, leading to asthma, and reduce the risk of developing new sensitivity.

Routine allergen immunotherapy is carried out using repeated administration of an allergen that has been used for a long period of time.

Long-term use of immunotherapy with allergens is carried out by injection (also called SCIT, SIT or subcutaneous immunotherapy). Typically, an allergen is administered subcutaneously in increasing doses until a maintenance dose is reached, but treatment regimens are also used with or without group administration. The allergen can be administered for a period of up to 3 years or more until the completion or termination of treatment, depending on the treatment regimen used.

An alternative route of administration is sublingual administration (SLIT), which is more convenient for patients because it can be done at home.

Immunotherapy products for use in immunotherapy typically contain as an active ingredient an allergen that causes an allergy in a patient or its derivatives, which can be formulated in various ways according to the chosen route of administration, such as aqueous compositions, suspensions of aluminum hydroxide or allergoids. The active ingredient may be, for example, an aqueous pollen extract containing pollen allergens and / or an aqueous tick extract containing mite allergens.

The most common house dust mites belonging to the Pyroglyphidae family include Euroglyphus maynei, Dermatophagoides pteronyssinus, and Dermatophagoides farinae. They can be found in most countries of the world, including Europe, the USA, China and many other countries, and allergies and allergic asthma caused by house dust mites are common diseases affecting 25% of the population.

Some patients are sensitized to Euroglyphus maynei, Dermatophagoides pteronyssinus, some others to Dermatophagoides farinae, and others to both species.

Typically, patients are sensitized (although to varying degrees) to the two main house dust mite allergens, that is, group 1 allergen and / or group 2 allergen.

Household dust mites contain over 20 different allergens. However, the two main allergens of group 1 and group 2 are the most important allergens, due to the large number of patients sensitized to these two allergens, the amount of IgE produced in response to these allergens and their content in natural extracts.

During immunotherapy of patients with allergens, they are treated with allergens to which they are sensitized. The ability of allergens to elicit IgE-mediated immune responses makes it important to control the content of allergens, in particular group 1 and group 2 allergens in allergen extracts administered to patients with allergies to avoid side effects.

Group 1 and group 2 allergens from Der f and Der p are closely related but not identical, see "Allergens and Allergen Immunotherapy, Clinical Allergy and Immunology Series 21", Lockey and Ledfors, 4 ^th ed., Page 162-164 .

Currently, all products on the market for immunotherapy for patients with house dust mites are allergic to water extracts of allergens containing mite allergens of group 1 and group 2. Many products contain extracts of both ticks Dermatophagoides pteronyssinus and Dermatophagoides farinae.

Tick allergen extracts are usually prepared by cultivating the tick in a suitable tick environment, followed by extraction of the whole tick culture or extraction of its tick body fraction, Allergens and Allergen Immunotherapy, Fourth Edition, Clinical Allergy and immunotherapy, series 21, Lockey and Ledfors, 4 ^th ed ., page 286.

In the production of an allergen extract, it is important to control variability and achieve consistency and reproducibility to ensure optimal safety and efficacy in subsequent clinical applications. Standardizing an allergen extract is a complex problem and, in principle, involves a complete production chain of processes, including establishing reliable and reproducible production procedures, see Allergy Methods and Protocols, Jones and Lympany (2008), chapter 12 and 13. Since the ability of an allergen extract to bind to Since IgE is associated with the content of one or more major allergens, a general standardization strategy usually involves standardizing the extract with respect to the content of selected data of the main allergens in addition to the general STI IgE binding and composition. Appropriate methods for evaluating allergen extracts are well known in the art and include immunoelectrophoresis, such as CIE, CRIE, FRIE, CLIE, quantitative methods using mono-or multispecific antibodies, such as SRID, RIS, QIE, as well as methods for preparing standards. for standardization. International standards are limited, one example is the standards of the Food and Drug Administration; CBER standard provided for the production of extracts for standardization and labeling of their product with biological labels AI or BAU (allergy units and bioequivalent allergy units).

There is significant batch to batch variability in the weight amount of group 1 and group 2 allergens in allergen extracts, and the weight amount of group 1 allergen in these allergen extracts is generally higher compared to the weight amount of group 2 allergen.

It is well known that group 1 and group 2 house dust mite allergens are found in the bodies and fecal particles of mites produced by mites during cultivation. It is also well known that a group 1 allergen is associated with fecal particles, and a group 2 allergen is associated with tick bodies; see Batard et al., Int. Arch. Allergy Immunol., 2006, 140, p. 295-305.

As mentioned above, there is significant batch to batch variability in the content of group 1 and group 2 allergens in batches of extracts of whole tick mites, including tick body extracts, which makes it difficult to prepare tick extracts with a constant and predetermined allergen content of group 1 and group 2 Of course, this is a serious problem for the industrial or large-scale production of a pharmaceutical product that will contain as much as possible the same amount of active ingredient (c) in each batch.

Therefore, improved methods and product (s) are important in order to obtain maximum production efficiency, as well as to provide high quality products in terms of efficiency and safety for the end user.

SUMMARY OF THE INVENTION

Therefore, the present invention relates to a pharmaceutical product containing an extract of an allergen or allergoid thereof, which contains at least one tick body extract selected from the following groups (a) to (b):

(a) the extract of the bodies of ticks Der p,

(b) Der f tick body extract,

and at least one tick culture extract selected from the following groups (c) to (g):

(c) an extract of fecal particles Der p,

(d) extract of fecal particles Der f,

(f) an extract of a whole Der mite tick culture,

(f) an extract of an entire Der p tick culture,

(g) a combination of extracts (c) - (f).

The present invention also relates to a pharmaceutical product containing an extract of an allergen or allergoid thereof, which contains at least one tick body extract selected from the following groups (a) to (b):

(a) the extract of the bodies of ticks Der p,

(b) Der f tick body extract,

and at least one extract of fecal mite particles selected from the following groups (c) to (d):

(c) an extract of fecal particles Der p,

(d) extract of fecal particles Der f.

The invention also relates to a tick body fraction containing more than 70% v / v. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. bodies of ticks.

The invention also relates to a fraction of fecal mite particles containing more than 70% v / v. fecal particles, preferably more than 75% v / v fecal particles, preferably more than 80% v / v. fecal particles, preferably more than 85% v / v fecal particles, preferably more than 90% v / v. fecal particles, more preferably more than 95% v / v. fecal particles and most preferably more than 98% v / v. fecal particles.

The invention also relates to a method for producing a tick allergen extract containing an allergen Der p 1 and Der p 2, where the method comprises the following steps:

(a) isolating fraction (s) of Der p mite bodies from Der p mite cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p;

(b) optionally combining several of these fractions of the bodies of ticks Der p; and

(c) extracting allergens from said fraction (s) of Der p mite bodies obtained in step (a) or step (b), as described above, to obtain said tick mite allergen extract.

The invention also relates to a method for producing a tick allergen extract containing allergens Der f 1 and Der f 2, where the method comprises the following steps:

(a) isolating fraction (s) of Der f mite bodies from Der f mite cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies Der f, more preferably greater than 75% v / v. mite bodies Der f, more preferably greater than 80% v / v. mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mites of Der f mites, preferably greater than 95% v / v mite bodies Der f and most preferably more than 98% v / v. mite bodies Der f;

(b) optionally combining several of said fractions of Der f mite bodies; and

(c) extracting allergens from said fraction (s) of Der f mite bodies obtained in step (a) or step (b), as described above, to obtain said tick mite allergen extract.

The invention also relates to a method for producing a pharmaceutical composition for treating and / or preventing allergies or allergic asthma caused by house dust mites, which contains an allergen extract or allergoids thereof having a predetermined and controlled content of allergens selected from allergens Der f 1, Der f 2 Der p 1 and Der p 2, and comprising the following steps:

(a) isolating Der pit mite body fractions and / or Der p mite fecal particle fractions from Der p mite cultures and / or isolating Der f mite body fractions and / or Der f mite fecal particle fractions from Der f mite cultures from Der f mite cultures; not necessary

(b) combining several fractions of bodies of mites Der p, optionally combining several fractions of fecal particles of Der p, optionally combining several fractions of bodies of mites Der p, optionally combining several fractions of fecal particles of Der f;

(c) extracting allergens from the tick body fraction (s) obtained in step (a) or step (b), as described above, and / or extracting allergens from the tick fraction (s) fecal particles obtained in step (a) or stage (b) as described above, and then

(d) determining the concentration (w / v) of the group 1 and group 2 allergen in the tick allergen extracts obtained in step (c) as described above; and optional

(e) mixing one or more extracts of tick bodies with one or more extracts of fecal particles obtained in step (c), as described above, to obtain a mixture of allergen extracts with a predetermined concentration (w / v) of group 1 allergen to the allergen groups 2 and optional

(f) converting the extract into allergoids of the extract.

The invention also relates to an allergen extract from a series of allergen extracts for use in a pharmaceutical allergen product for treating and / or preventing allergies and allergic asthma caused by house dust mites, where each allergen extract contains at least one tick body extract, and, at least one extract of fecal mite particles, or an extract of mite bodies from a body fraction containing less than 70% v / v. mite bodies, or a whole mite culture, wherein said allergen extract has a predetermined ratio of group 1 to group 2 allergen, and where the variability coefficient of said ratio is not more than 25, 20, 15, 10, or 7.5% for a series of extracts.

The invention also relates to an allergen extract from a series of allergen extracts for use in a pharmaceutical allergen product for diagnosing allergies caused by house dust mites, where each allergen extract contains at least one tick body extract and at least one fecal extract mite particles, or mite body extract from a body fraction containing less than 70% v / v. mite bodies, or a whole mite culture, wherein said allergen extract has a predetermined ratio of group 1 to group 2 allergen, and where the variability coefficient of said ratio is not more than 25, 20, 15, 10, or 7.5% for a series of extracts.

Brief Description of the Figures

Figure 1 shows the installation, additionally described in example 2.

Definitions

In the sense that the term “tick” is used in this document, it refers to house dust mites. Examples of such ticks are Dermatophagoides farinae and / or Dermatophagoides pteronyssinus, and / or Euroglyphus maynel or other common species belonging to the Pyroglyphidae family.

In the sense that the term “Der p” is used in this document, it means Dermatophagoides pteronyssinus, “Der p 1” means “Der p group 1 allergen”, and “Der p 2” means “Der p group 2 allergen”.

In the sense that the term “Der f” is used in this document, it means Dermatophagoides farinae, “Der f 1” means “Der f allergen of group 1”, and “Der f 2” means “allergen of Der f of group 2”.

Group 1 allergens and group 2 allergens of two tick species Der f and Der p exist in nature as a series of isoforms, see, for example, www.allergen.org from allergen sources (Animalia Anthropoda / Astigmata / Dermatophagoides farinae or Dermatophagoides farinae). In the sense that the terms “group 1 allergen”, “group 1”, “group 2 allergen” and “group 2” are used in this document, they include all naturally occurring isoforms of each of the group 1 and group 2 allergens, including mentioned at www.allergen.org .

As used herein, the terms “tick culture” or “tick culture”, “whole tick culture” and “whole culture”, “Der p tick culture” and “Der f tick culture,” and “tick culture Der p ”and“ Der f mite cultures ”, they mean material obtained after culturing Der p or Der f on a suitable medium, and it includes whole cultures, including ticks, tick fragments, fecal particles, eggs, and components of the medium for ticks etc.

In the sense in which the terms “culture medium or mite medium” or “medium or mite medium” are used in this document, they mean an environment suitable for the cultivation of house dust mites.

In the sense in which the terms “tick body extract”, “tick body extract Der p” and “tick body extract Der f” are used in this document, they mean, respectively, “extract of one fraction or more combined fractions of tick bodies”, “Extract of one fraction or more of the combined fractions of bodies of mites Der p” and “extract of one fraction or more of the combined fractions of bodies of mites of Der f”.

In the sense in which the terms “fecal mite particle extract”, “fecal particle extract”, “fecal mite extract Der p” and “fecal mite extract Der f” are used in this document, they mean respectively “extract of one or more combined fractions of fecal mite particles "," extract of one fraction or more of a combined fraction (s) of fecal mites "," extract of one fraction or more of a combined fraction (s) of fecal mite particles Der p "and" extract of one fraction or in more penalties for the combined fraction (s) of fecal mite particles Der f ".

In the sense that the terms “allergen extract” and “allergen (I) extract” are used in this document, they are used interchangeably and mean an extract of at least one “tick body extract” or “fecal particle extract” optionally in combinations with additional tick extracts and in any combination.

In the sense that the term “tick body fraction (s)” is used in this document, it means one or more combined fractions of tick cultures that contain more than 50% v / v. mite bodies, “Der p mite body fraction (s)” means one or more combined fractions of Der p mite cultures that contain more than 50% v / v. mites of Der p mites, and “Der f mite bodies fraction (s)” means one or more combined fractions of Der f mite cultures that contain more than 50% v / v. mite bodies Der f.

In the sense that the terms “tick fecal particles fraction (s)” and “fecal particle fraction (s)” are used herein, they mean one or more combined tick culture fractions that contain more than 50% v / v . fecal mite particles, “Der p faecal particle fraction (s)” means one or more combined fractions of Der p mite cultures that contain more than 50% v / v. fecal particles Der p, and “fraction (s) of fecal particles Der f” means one or more pooled fractions of Der f cultures that contain more than 50% v / v. fecal particles Der f.

In the sense in which the term “s” is used at the end of a word, it means a word in singular or plural form.

In the sense that the term “treatment” is used in this document, it means “curing or alleviating the symptoms of an allergy”.

In the sense in which the term “rapidly disintegrating sublingual tablet” is used herein, it refers to compressed and unpressed dosage forms that disintegrate in less than about 90 seconds, preferably less than about 60 seconds, preferably less than about 30 seconds , more preferably less than about 20 seconds, even more preferably less than about 10 seconds in the oral cavity, even more preferably less than about 5 seconds and most preferably less than about 2 seconds Ek after placement under the tongue in the oral cavity. Such rapidly disintegrating sublingual tablets are described in international application WO 04/77994.

In the sense that the term “CV” is used in this document, it means the coefficient of variability expressed as a percentage.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical products of the invention are suitable for treating and / or preventing allergies to house dust mites and allergic asthma caused by house dust mites.

As already mentioned above, there is a high variability from batch to batch in the content of tick group 1 allergens and group 2 allergens in batches of extracts of whole tick mites, including tick body extracts, which makes it very difficult to prepare tick extracts with a constant and predetermined allergen content of group 1 and groups 2.

It has now been found that by obtaining several, for example, two extracts for each mite culture batch, for example, isolating a mite culture fraction containing mainly mite bodies and a mite culture fraction containing mainly fecal mite particles, followed by separate extraction of the fraction mite bodies and fecal mite particles fraction, it is possible to control the ratio of group 1 allergen to group 2 allergen in the final allergen extract by mixing the extract of body cells another with an extract of fecal mite particles until the desired weight ratio is reached.

It was also found that if the tick culture obtained by cultivating ticks is cleaned to obtain a fraction of tick bodies containing more than 70% v / v. or more preferably more than 80% v / v. mite bodies, then extraction of this purified mite body fraction will usually produce a mite body extract that contains an equal weight amount of group 1 allergen and group 2 or more group 2 allergen than group 1 allergen.

The tick body extract with a higher weight content of group 2 allergen than the group 1 allergen can be enriched with an extract of fecal tick mites, which usually contains more group 1 allergen than group 2 by weight, to obtain a mixture of extracts with a predetermined and, if required, more equal weight amounts of allergen of group 1 and group 2.

An example of extracts that usually contain more mass of a group 1 allergen than a group 2 allergen and which can be used for enrichment is an extract of a whole tick culture or body fraction (s) containing less than 70% v / v. bodies of ticks.

An extract of tick bodies with a higher content of a group 2 allergen than a group 1 allergen can also be enriched with an extract of a whole tick culture or a fraction of tick bodies containing less than 70% v / v. mite bodies, which usually both contain more mass of group 1 allergen than group 2 allergen, to obtain a mixture of extracts with predetermined and, if required, more equal weight amounts of group 1 and group 2 allergen.

By mixing the extracts of tick bodies and another fraction, preferably a fecal particle fraction, it is possible to obtain a mixture of extracts that has a weight ratio of group 1 to group 2 allergen in a wider range, such as from 1: 0.6 to 1: 1.6; from 1: 0.7 to 1: 1.5; from 1: 0.8 to 1: 1.4; from 1: 0.9 to 1: 1.3; from 1: 0.95 to 1: 1.2; from 1: 0.98 to 1: 1.15, including a mixture that contains equal weight amounts of group 1 and group 2 allergens, i.e. a mixture of Der p 1, Der p 2, Der f 1 and Der f 2 in a ratio of 1: 1: 1: 1.

In one aspect, the invention relates to a method for producing a mixture of tick allergen extracts for a pharmaceutical product having a predetermined, controlled weight amount of allergens Der f 1, Der f 2, Der p 1 and Der p 2.

The invention also relates to a pharmaceutical product containing allergen extracts selected from at least one tick body extract and optionally at least one tick tick fecal particle extract selected from: (a) Der p tick body extract; (b) an extract of fecal particles Der p; (c) Der mite body extract Der and (d) fecal particle extract Der f, where the main allergens Der p 1, Der p 2, Der f 1 and / or Der f 2 are in predetermined, controlled amounts.

In some embodiments, the extracts of the invention are characterized by a higher weight content of group 2 allergen compared to products available on the market. The authors of this application have provided an industrial or large-scale method for producing allergen extracts with a modified weight ratio of group 1 to group 2 allergen.

It was found that if separate extracts of mite bodies and fecal mite particles are prepared for each of two types of mites, and a sufficiently pure fraction of mite bodies (and a fraction of fecal mite particles) is used for extraction, it is possible to obtain mite extracts with an allergen weight ratio of 1 to group 2 allergen, which ranges from 1: 0.6 to 1: 1.6 for each type of tick.

By combining extracts of both types, it is possible to achieve a weight ratio of Der p 1, Der p 2, Der f 1 and Der f 2, which is 1: 1: 1: 1.

Such products are advantageous since the allergen content of pharmaceutical products is well controlled.

Previously, tick allergen extracts, as a rule, contained more group 1 allergen than group 2 allergen by weight, which could be the reason for ineffective treatment of patients with allergies, which, in particular, were sensitized by group 2 allergen.

According to the invention, there is provided an industrial method for producing extracts with a higher weight content of a group 2 allergen than a group 1 allergen, which is usually found in the extracts.

Accordingly, the pharmaceutical product described above contains at least one extract of tick bodies and at least one extract of fecal mite particles selected from groups (a) to (d) above.

Preferably, the tick allergen extract contains Der p mite body extract, Der p fecal particle extract, Der f mite body extract and Der f fecal particle extract.

According to further embodiments of the invention, a tick allergen extract comprises:

- Der p tick body extract and Der p fecal particle extract, and optionally without Der f tick body extracts and Der f fecal particle extracts; or

- Der f tick body extract and Der f fecal particle extract, and optionally without Der p tick body extracts and Der p fecal particle extracts; or

- Der p tick body extract and Der f tick body extract, and optionally without fecal particle extract; or

- extract of tick mites Der p and without extract of tick mites Der f and optionally without extract of fecal mite particles; or

- Der f tick body extract and without Der p tick body extract and optionally without fecal mite particle extract.

In a preferred embodiment, the Der p tick body extract is prepared from one or more Der p tick body fractions, where the extraction of the Der p tick body fraction (s) causes the extract to contain equal weight amounts of an allergen of group 1 and group 2 or more allergen group 2 than the allergen of group 1 by weight, and / or the Der f tick body extract is prepared from one or more Der f tick body fractions, and where the extraction of the Der f tick body fraction (s), the extract contains equal weight amounts allergen of group 1 and group 2, or more he longer allergen Group 2 than in Group 1 allergen by weight.

In a preferred embodiment:

- Der p mite body extract, respectively, is prepared from one fraction or pooled fractions of Der p mite cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p, and / or

- Der f mite body extract, respectively, is prepared from a single fraction or pooled fraction (s) of Der f mite cultures, wherein said fractions contain more than 70% v / v. mites of Der f mites, preferably greater than 75% v / v mites of Der f mites, preferably greater than 80% v / v mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mite bodies Der f, more preferably greater than 95% v / v. mite bodies Der f and preferably more than 98% v / v. mite bodies Der f; and / or

- Der p faecal particle extract is suitably prepared from a single fraction or pooled fraction (s) of tick culture Der p, wherein said fraction (s) contains more than 70% v / v. fecal Der p particles, preferably greater than 75% v / v. fecal Der p particles, preferably greater than 80% v / v. fecal Der p particles, preferably greater than 85% v / v. fecal Der p particles, preferably greater than 90% v / v. fecal Der p particles, more preferably greater than 95% v / v. fecal Der p particles, most preferably greater than 98% v / v. fecal particles Der p; and / or

- Der f faecal particles extract, respectively, is prepared from one fraction or the combined fraction (s) of Der f tick cultures, wherein said fraction (s) contains more than 70% v / v. fecal Der f particles, preferably greater than 75% v / v. fecal Der f particles, preferably greater than 80% v / v. fecal Der f particles, preferably greater than 85% v / v. fecal Der f particles, preferably greater than 90% v / v. fecal Der f particles, preferably greater than 95% v / v. fecal particles Der f and more preferably more than 98% vol./about. fecal particles Der f.

In one embodiment, the fecal particle fraction is not as pure as the body fraction.

In a preferred embodiment, each of the fractions or combined fractions described above contains less than 10% v / v. particles of culture medium for ticks.

The tick body fraction or the combined tick body fractions may contain up to 20% v / v. fragments of mites (limbs, eggs, etc.), the environment and fecal particles (i.e. any that are not mite bodies or are undesirable impurities), provided that the number of particles of the medium is less than 10% vol./about .

The fecal mite particles fraction or the combined fecal mite particles fractions contain up to 20% v / v. fragments of ticks (limbs, eggs, etc.) and particles of the medium (i.e. any that are not fecal particles of ticks or are undesirable impurities), provided that the number of particles of the medium is less than 10% vol./about.

In a further embodiment, each of the fractions or combined fractions as a whole contains no more than 1.0% w / w. detectable foreign substances. Fecal mite particles, mite fragments, mite bodies or mite media are not considered foreign substances.

The volume percentages of tick bodies and fecal tick particles in fractions of tick bodies and fecal particles are determined by microscopy. Count the number of particles (for example, the number of bodies or fecal mite particles) and expressed as% v / v. multiplying by a number that represents the average volume of a given particle (for example, a body or fecal mite particle).

Particle counting by microscopy in tick body fractions

The sample is weighed and suspended in glycerol containing a solution (25% glycerol, 5% Igepal). The sample is poured onto a mesh-applied filter under vacuum to remove liquid and immobilize particles on the filter. The filter is then dried to increase visibility by microscopy. Randomly select grids for counting and count all particles in the selected grid. Grids are selected until at least 800 particles are counted. The procedure described above can be carried out on several samples, for example, two, three, four or five independent samples. When calculating the results, the variability between the samples should satisfy the required criteria for the acceptability of the analysis (for example, more than 80% vol./about. Whole bodies of ticks and / or less than 10% vol./about. Of particles of the medium) to ensure that the data between the samples match. If the results for the samples are the same, then the average number that gives the overall analysis result is displayed.

Voluminous numbers are converted into volumes using predetermined volumes for tick bodies, fecal particles, and the environment.

Particle counting by microscopy in fecal particle fractions

The sample is weighed, placed in a counting chamber with a grid on it and mixed with paraffin oil to obtain a uniform layer. Randomly select grids for counting and count all particles in the selected grid. Grids are selected until at least 800 particles are counted. The procedure described above can be carried out on several samples, for example, two, three, four or five independent samples. When calculating the results, the variability between the samples should satisfy the required criteria for the acceptability of the analysis (for example, more than 80% vol./about. Whole bodies of ticks and / or less than 10% vol./about. Of particles of the medium) to ensure that the data between the samples match. If the results for the samples are the same, then the average number that gives the overall analysis result is displayed. Non-voluminous numbers are converted into volumes using predetermined volumes of tick bodies, fecal particles and the environment.

The volumes established for the bodies of ticks, fecal particles, particles of the medium for ticks are as follows:

mite bodies Der f: 21132000 cubic micrometers

body mites Der p: 12,926,000 cubic micrometers

mite particles in body fractions: 6,721,000 cubic micrometers

fecal mite particles: 19,000 cubic micrometers

fecal particles: 19,000 cubic micrometers

As indicated above, the present invention is based on the finding that by cleaning fraction (s) of tick bodies so that they contain more than 70% v / v. mite bodies or more preferably more than 80% v / v, it is possible to prepare an extract of body particles from such a mite body fraction or combined fractions in which the weight amount of group 1 allergen in the extract is lower than the weight amount of group 2 allergen or the weight concentration of group 1 allergen and group 2 is equal.

According to a preferred embodiment, the weight amount of group 1 allergen is lower than the weight amount of group 2 allergen in the tick body extract (x) and the weight amount of group 1 allergen is significantly higher than the group 2 allergen in tick extracts of fecal particles.

Accordingly, the weight ratio of group 1 allergen to group 2 allergen is closer to the weight ratio of group 1 allergen to group 2 allergen in tick extracts than the weight ratio of group 1 allergen to group 2 allergen in tick extract of fecal particles used for its preparation.

According to the invention, it is possible to change the weight ratio of the group 1 allergen to the group 2 allergen by more equal weight amounts in the extract than are present in the extract of tick bodies and fecal tick particles from which they are prepared. This was not possible earlier when typical batches of mite bodies and fecal particles contained more group 2 allergen than group 2 allergen by weight.

A pharmaceutical product of the invention is provided containing an allergen extract that contains allergens selected from Der f 1, Der f 2, Der p 1 and Der p 1, and where the allergen is in the lowest concentration (w / v) in the extract allergen, is in a concentration that is higher than 50% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 60% concentration allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more than Significantly above 70% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 80% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 85%, of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 located at the highest concentration (w / v), more preferably above 90%, of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 at the highest concentration and (w / v), more preferably above a 95% concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), preferably higher 98% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2 at the highest concentration (w / v) or most preferably above 99% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v).

One daily dose of the product according to the invention accordingly contains from 2 to 10 μg or more preferably from 3 to 8 μg Der p 1, from 3 to 8 μg Der p 2, from 3 to 8 μg Der f 1 and from 3 to 8 μg Der f 2.

In a preferred embodiment, the daily dose of group 1 and group 2 allergens in the sublingual product of the invention is suitably in the range of 3 to 4 μg, more preferably about 3.6 μg Der p 1, Der p 2, Der f 1 and / or Der f 2.

In yet another embodiment, the daily dose of group 1 and group 2 allergens in the sublingual product of the invention is suitably in the range of 6 to 8 μg, more preferably about 7.2 μg, Der p 1, Der p 2, Der f 1 and / or Der f 2.

The pharmaceutical product described above can be formulated as a compressed or unpressed sublingual tablet (for example, described in international application WO 04/77994), a liquid sublingual product (for example, described in international application WO 07/051476) or a liquid product for injection, optional with adjuvant aluminum hydroxide (for example, described in international application WO 10/043675). Preferably, the pharmaceutical product is a rapidly disintegrating sublingual tablet. Preferably, the pharmaceutical product is in the form of tablets, capsules, troches, or capsules.

The allergen extract may be in the form of allergoids in the pharmaceutical products described above.

The present invention also relates to a method for producing a tick allergen extract for a pharmaceutical product, wherein said extract contains a predetermined, controlled and, if desired, more equal weight amount of allergen Der f 1, Der f 2, Der p 1 and / or Der p 2.

The invention also relates to a method for producing a tick allergen extract or allergoids thereof for a pharmaceutical product as described above, wherein said allergen extract has a predetermined and controlled weight amount of allergens selected from allergens Der f 1, Der f 2, Der p 1 and / or Der p 2, and includes the following steps (a) and (c) - (d) and optionally (b), (e) and / or (f):

(a) isolating Der pit mite body fractions and / or Der p mite fecal particle fractions from Der p mite cultures and / or isolating Der f mite body fractions and / or Der f mite fecal particle fractions from Der f mite cultures from Der f mite cultures;

(b) optionally combining several fractions of Der p mite bodies, optional combining several fractions of Der p mite fecal particles, optional combining several fractions of Der f mite bodies, optional combining several fractions of Der f mite fecal particles;

(c) extracting allergens from the tick body fraction (s) obtained in step (a) or step (b) as described above, and / or extracting allergens from the fecal particle fraction (s) obtained in step (a) or step (b) as described above; and then

(d) determining the concentration (w / v) of the group 1 and group 2 allergen in the tick allergen extracts obtained in step (c) as described above; and optional

(e) mixing one or more tick body extracts with one or more tick fecal particle extracts obtained in step (c) as described above, to obtain a mixture of allergen extracts with a predetermined amount and weight ratio of group 1 allergen to group 2 allergen, and optional

(f) the conversion of the allergen extract to its allergoid.

According to the invention, extracts of tick body fractions and extracts of tick fecal particle fractions can be combined or mixed, however, tick body fraction (s) are never combined with fecal particle fraction (s).

Accordingly, one or more extracts of tick bodies and one or more extracts of fecal particles are mixed to form a mixture of allergen extracts.

Preferably, the tick mite allergen extract mixture comprises Der p body extract (s), Der p fecal particle extract (s), Der f tick body extract (s) and Der f 2 fecal particle extract (s).

To prepare the extract, more than one tick body fraction can be combined in step (b), followed by extraction in step (c).

Also, more than one fraction of fecal mite particles can be combined in step (b), followed by extraction in step (c).

Alternatively, the tick body fraction obtained in step (a) is subjected to extraction and / or the fecal tick fraction obtained in step (a) is subjected to extraction.

Accordingly, one or more tick body extracts obtained in step (c) are mixed in step (e) with one or more fecal tick extracts obtained in step (c).

Accordingly, one tick body extract obtained in step (c) is mixed in step (e) with one fecal tick extract obtained in step (c).

In the method described above, one or more tick body extracts obtained in step (c) can be mixed in step (e) with one fecal tick extract obtained in step (c).

Alternatively, one tick body extract obtained in step (c) can be mixed in step (e) with one or more fecal tick extracts obtained in step (c).

In the sense in which the term “blending of extracts” is used in this document, it includes blending of both whole batches of extracts, as well as parts of whole batches.

According to a preferred embodiment of the invention, the tick body extract (s) are enriched with an amount of fecal particle extract that is sufficient to achieve the desired weight ratio of group 1 allergen to group 2 allergen in the mixture of allergen extracts.

In yet another preferred embodiment of the invention, the tick body extract contains an almost equal weight amount of group 1 and group 2 allergen, and it is not necessary to mix it with the extract of fecal tick particles.

The coefficient of variability (CV) is calculated as the ratio of the standard deviation σ to the average value of μ:

In some cases, it is expressed as a percentage, and in this case, the CV value is multiplied by 100. CV is used to describe the improved constancy of the extract and pharmaceutical products (therapeutic or diagnostic) of the invention from batch to batch.

Confidence Intervals (CI), such as 95%, can be used.

Tick cultivation

A number of nutritious materials are known that are suitable for the cultivation of mites, and there are numerous studies on the effectiveness for this purpose of various ingredients of culture media, such as yeast, albumin, animal feed, fish food (Tetramin), exfoliated flakes of human skin, pig skin, eggs shrimp, wheat germ, rat food, dog food, animal liver powder, milk protein hydrolyzate, vitamins, minerals, protein hydrolyzate or soy flour.

Tick nutrition materials are described, for example, in the International Journal of Acarology, Vol. 8, Issue 3 (1982), p. 189-192, Rodriques, Recent Advances in Acarology, Vol. 11 (1979), 211-216, Batard et al., Int. Arch. Immunol., 140 (2006), p. 295-305, Yi et al .; Asian Pacific Journal of Allergy Clin. Immunol., No. 3 (1987), p. 457-466 and European Patent 1236394.

One example of a tick culture medium containing a mixture of yeast and dried daphnia in a 1: 1 ratio is described in Dust Mites by Matthew Colloff (2009), p. 270. Another example of a tick growth medium is described in European Patent 1236394.

It is preferable to use a protein hydrolyzate, as well as other ingredients that have a low risk of allergization to reduce the possibility of anaphylactic reactions in allergen-sensitized patients. In addition, it is preferable to use a medium for ticks that does not contain animal and human proteins.

Accordingly, the tick medium is used in the form of particles that are of sufficient size and integrity to withstand the effects of subsequent processing and can be easily removed during initial screening.

The two types of ticks Der p and Der f are always cultivated in separate containers, but they can be cultivated on the same tick medium. Examples of containers include glass or plastic containers of suitable size and shape, such as an insect container. Disposable mite culture containers are described, for example, in WO 2008/119762.

Ticks are cultivated in containers with culture medium and placed in a controlled environment with control of temperature and humidity of the environment surrounding the growing tick culture. Suitable conditions for Der p and Der f represent the conditions described, for example, in a number of articles, such as Fain et al. (ISBN 90-71868-12-5) and Crowther: Exp. Appl. Acarol., 2007; 41 (1-2): 61-86.

One suitable set of parameters for the cultivation conditions of each tick species is given in table 1a below:

Table 1a Der f Der p Relative humidity 65% ± 5% RH 70% ± 5% RH Temperature 25ºC ± 5ºC such as 27.5ºC ± 2ºC 25ºC ± 7ºC such as 30ºC ± 2ºC Cultivation time 42-70, such as 42-56 days 42-70, such as 42-56 days

Microscopic analysis is carried out with respect to growth and viability during cultivation or to identify the optimal time for collecting ticks.

Killing of ticks can be carried out by freezing. Also, the death of ticks can be caused by "suffocation", for example, treatment with acetone. Storage containers with dead ticks are stored at a temperature of -20ºС until further processing.

Cleaning tick culture

Mite cultures are thawed, dried and sieved to obtain mite culture fractions containing components of various sizes. Drying can be carried out in an air stream to a moisture content below 20%, more preferably at a level of 15%.

Then the culture can be further purified and fractions of tick bodies or fecal particles can be isolated using separation methods including sieving (for example, using sieves with 1 mm, 500 μm, 250 μm, 100 μm sieves on a shaker with a sieve), saturated with sodium chloride suspension and ethanol suspensions, see, for example, Dust Mites, author Matthew Coloff (2009), p. 271.

In one embodiment, the tick body fraction (s) and the tick fecal particles fraction (s) can be prepared by sieving the tick culture to produce four fractions:

a) large particles of the medium> 350 μm

b) tick body fraction 350-90 μm

c) fecal agglomerates and tick fragments 90-50 microns

d) fecal particles <50 μm

Within the indicated particle size limits, there are usually various mite culture components, such as mite bodies, fecal particles and environmental particles, etc. Mite culture fractions isolated within sizes that differ from the size limits indicated above may also be suitable according to the invention, provided that fractions of mite bodies and fecal particles can be prepared which are sufficiently clean and in sufficient quantity.

Fractions (a) and (c) are discarded. Fraction (d) can be used directly in the extraction process, and fraction (b) is further purified before extraction.

Cleaning the tick body fraction

A method for cleaning tick bodies from particles of the medium by flotation in a suitable liquid is described previously, UK patent 1318560 on page 3. In J. Med. Entomol. (1979), Vol. 16, # 2, p. 128-132 describes flotation purification in alcohol (ethanol) to separate tick bodies from tick medium particles.

It has now been found that repeated centrifugation in a density gradient of the tick body fraction in a suitable liquid gives a tick fraction of high purity ticks, which upon extraction provides an extract with a higher weight content of group 2 allergen than group 1 allergen or an equal weight amount of group 1 allergen and groups 2.

Thus, the present invention also relates to a method for isolating and purifying tick bodies, comprising the steps of:

(i) isolating the tick body fraction (s) from the tick culture (p) of Der p and / or Der f mites; and

(ii) centrifuging one or more Der p1 mite body fractions or one or more Der f mite body fractions in a density gradient in a first liquid medium having (iia) higher density than mite bodies and lower density than culture medium at temperature used during centrifugation; and

(iii) collecting mite bodies from the surface of said first liquid medium and repeating steps (ii) and (iii) until the desired mite bodies are clean, then optional

(iv) washing the tick bodies to remove said first liquid with a second liquid that does not extract the group 1 and group 2 allergen from the tick bodies at the temperature used during the washing step.

Preferably, the first and second liquids in the method described above do not contain water (are anhydrous) to limit the extraction of allergens from the source material in the purification step of the soluble source material. In another embodiment of the method for isolating and purifying tick bodies in step (ii), centrifuging one or more fractions of tick body Der p or one or more fractions of tick body Der f in a density gradient in a first liquid medium is carried out with a first liquid having (iia) more higher density than mite bodies and lower density than particles of the culture medium, and (iib) which does not extract the group 1 and group 2 allergen from mite bodies at the temperature used during centrifugation.

Suitable liquids include metrizoic acid derivatives (such as yonohexol, Nycodenz ^® (N-2,3-dihydroxypropylacetamido-2,4,6-triiod-N, N-bis (2,3-dihydroxypropyl)), polypropylene glycols (such as ARCOL ^® (polypropylene glycol 4000), silica-based compositions (such as RediGrad ^® ).

It is difficult to completely avoid some extraction of the group 1 group 1 allergen from tick bodies during the centrifugation described above. According to the invention, liquids that do not extract group 1 and group 2 allergens from tick bodies are liquids that do not extract group 1 allergen and group 2 allergen from tick bodies at the temperature used in steps (ii) and (iv) described above, in an amount that has a significant effect on the final yield and the ratio of the allergen of group 1 and group 2.

The extraction of group 1 and group 2 allergens from tick bodies during centrifugation can be reduced by centrifugation at low temperature, for example, at freezing temperature.

In a preferred embodiment, the liquid having a density higher than tick bodies and lower than particles of the culture medium is an anhydrous liquid, for example, anhydrous glycerin.

Glycerol can be removed from mite bodies by washing with ethanol, preferably anhydrous ethanol.

The corresponding steps (ii) and (iii) are carried out once or centrifugation in glycerol is repeated until the obtained fraction of tick bodies contains at least 70% v / v. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. mite bodies, and the mite body fraction contains less than 20% v / v. fragments of ticks, fecal particles and particles of the medium and less than 10% of the particles of the medium.

Instead of centrifuging in a density gradient with glycerol as described in steps (ii) and (iii), the tick body fraction can be cleaned by separating with saturated NaCl as the first liquid until the tick body fraction contains at least 70% vol. ./about. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. mite bodies, and contains less than 20% vol./about. fragments of ticks, fecal particles and particles of the medium and less than 10% of the particles of the medium without the need for stage (iv).

Instead of centrifuging in a density gradient, the tick body fraction can be cleaned by sieving until the tick body fraction contains at least 70% v / v. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. mite bodies, and the mite body fraction contains less than 20% v / v. fragments of ticks, fecal particles and particles of the medium and less than 10% of the particles of the medium.

Mite bodies may be 400 μm long in one dimension, but may nevertheless be able to pass through a sieve with a hole size of 300 μm and 400 μm when they are dried before sieving.

Ticks have limbs and hair, and they can be distinguished from the tick medium on a shaker with a sieve with horizontal circular motion. The method has the advantage of the ability of ticks to form tangled tangles formed by their hair and limbs during horizontal movement on a sieve. In a sieving tower that moves horizontally in circles, the mite bodies are captured at the top of the sieves with openings of 200, 300 and / or 400 μm, and as a result they are separated from other particles of the tick culture with sizes of 150 and 300 μm.

Thus, in a method alternative to centrifugation, a mite-rich fraction with a particle size in the range of 90 to 350 μm isolated in the sieving method described above can be purified in the following way:

(1) sifting Der f or Der p mite cultures in a shaker with a vibrating sieve with a tower with openings of 300, 150 and 80 μm in size, followed by isolation of a body-rich mite fraction ranging in size from 150 to 300 μm and a fecal-rich mite fraction below 80 microns then

(2) sifting the body-rich mite fraction (150 μm and 300 μm) in a shaker with a horizontal and circular motion sieve and a tower with sieves with openings of 200, 300 and / or 400 μm, and isolating the body-rich mite fraction (s) captured by sieves with openings of 200, 300 and / or 400 microns, then

(3) sieving the mite-rich fraction in a shaker with a sieve having a 3D torsion movement and with a sieve with openings of 300, 200, 150 and / or 100 μm to obtain a fraction of mite bodies ranging in size from 150 to 300 μm and a fecal fraction particles below 100 microns in size; and

(4) optionally combining the fecal particle fraction below 80 μm in size with a vibrating sieve and the fecal particle fraction below 100 μm in size from a sieve with 3D torsion movement, and sieving the combined fecal particle fraction on a sieve with 3D 3D torsion movement and a tower with sieves with size holes 100, 80, 50 microns.

Tick body fractions (b) obtained as described above can be purified by steps 2 and 3 in the method described above.

Extraction

The fractions of tick bodies and fecal tick particles are extracted in a more or less the same way, but in different processes, since the optimal conditions for the extraction of tick bodies are not the same as for fecal tick particles.

To extract allergens from the bodies of ticks and fecal particles, the bodies must be ground to release allergens. Grinding can be achieved by applying external force to the suspension of tick bodies.

According to the invention, it was found that a mixer with a high shear force is particularly effective for grinding and releasing allergens from mite bodies and fecal particles.

Thus, according to one embodiment of the invention, the extraction of allergens is carried out by suspending the bodies of mites or fecal particles of mites in a buffer using a mixer with a high shear force (usually for 15-30 minutes) and then extraction without stirring (usually for 1 -6 h).

Allergen release varies for bodies and fecal mite particles. Allergens in fecal particles are extracted after 1 hour of extraction, while at least 3 hours are required for the extraction of allergens from tick bodies.

Buffers for the extraction of tick allergens are well known in the art.

The allergen source material (tick body and / or tick fecal particles), for example, can be extracted with an extraction buffer containing 0.15 M NaCl, 0.012 M NaHCO ₃ and a mixture of NaOH / HCl / water to adjust the pH. Extraction, respectively, is carried out using a ratio of the buffer for extraction to the source material of allergens equal to 1:10 (wt./wt.) PH 7-8, temperature within 4-12ºС and extraction time 1-6 hours.

These extracts can be further purified before being used in a pharmaceutical product.

The remaining insoluble material can, for example, be removed from the extract of fecal particles of ticks by centrifugation and clarification. Then, the extract of fecal mite particles can be subjected to ultrafiltration, carried out in three stages: first concentration by removal of water and other small molecules by tangential flow filtration, then by diafiltration to remove small molecules and salts, and then brought to the desired solids content.

Then, the allergen extract can be clarified by clarification filtration.

Further purification of the extracts of tick bodies accordingly includes the following processes: separation using diafiltration, leaving an insoluble substance on the membrane and continuously adding water to the extract on the other side of the membrane. Then the extract is subjected to ultrafiltration and clarification, as described above.

The extracts can be stabilized by the formation of frozen extract granules (cryogranules), as described in international application WO 05/058474.

The concentration of the allergen of group 1 and group 2 in extracts of tick bodies and fecal particles of ticks can be determined by a number of methods commonly used to determine the content of the allergen, including, for example, ELISA, RID / SRID (simple radial immunodiffusion) or MS (mass spectrometry, for example, described in international application WO 2007/031080), also see above.

The principle of the RID method is that proteins (antigens) placed on a round hole diffuse outward, forming a concentration gradient in an agarose gel containing the corresponding antibody. The antibody used in this method is a monospecific antibody (i.e., produced against a specific antigen) and a polyclonal antibody, which allows the formation of precipitates of antigen-antibody complexes. The antibody is in excess, and antigen diffusion will continue until the equilibrium point is reached, and an antigen-antibody precipitate ring is formed. Then the precipitation rings are stained. The area of the precipitate ring is determined, and then the antigen concentration in unknown samples can be interpolated from a calibration curve constructed with the reference standard. The content of the main allergen is determined relative to the reference standard (see, for example, Allergy Methods and Protocols, Jones and Lympany (2008), 138-141, p. 152-153 and p. 159-161).

You can use various options sandwich ELISA (solid phase immunosorbent analysis) to determine the content of the main allergen in Der p and Der f. One for the determination of Der f 1, one for the determination of Der p 1 and one for the determination of Der group 2, since the two main allergens of group 2 are immunochemically identical.

Monoclonal antibodies specific for the main allergen (Der f 1, Der p 1 and Der group 2) are adsorbed in the wells of a 96-well microtiter plate. mAbs recognize epitopes on the primary allergen and bind the allergen present in the sample. The antibody-antigen complex is washed out and peroxidase-labeled rabbit IgG antibody against the main allergen is added. When an enzyme substrate is added, a color reaction will develop whose intensity is directly proportional to the content of the bound main allergen. A standard curve is also analyzed in the analytical cycle, and the activity of the main allergen is interpolated on the calibration curve. After using the internal standard, the Center for Biologics Evaluation and Research under FDA is used for competitive ELISA as described in Potency Limits for Standardized Dust Mite and Grass Allergen Vaccines: A revised protocol. 2000 ( http://www.fda.gov/BiologicsBloodVaccines/GuidanceComlianceRegulatoryInformation/Glidances/Allergenics/ucm01931.htm ) for determining the activity of allergen products.

In yet another embodiment, the concentration of the allergen of group 1 and group 2 is determined by MS (mass spectrometry), as described in international application WO 2007/031080 using the following standard peptides for calibration:

TNACSINGNAPAEIDLR (Dep p 1) SEQ ID NO: 1

NSWDTTWGDSGYGYFQAGNNLMMIEQYPYVVIM (Der f 1) SEQ ID NO: 2

VLVPGCHGSEPCIIHR (Dep p 2) SEQ ID NO: 3

GKPFTLEALFDANQNTK (Der f 2) SEQ ID NO: 4 and

GIEYIQQNGVVEER (Der f 1) SEQ ID NO: 5

When the allergen concentration of group 1 and group 2 is determined for a number of tick extracts, the extracts of tick bodies and extracts of fecal particles are mixed to achieve the desired mass ratio of the allergen of group 1 and group 2.

Determining which extract and how much of these extracts should be mixed is an iterative process in which the weighted amounts of extracts from different batches to be used are determined and entered into the following formula for Der f until the desired ratio is reached:

the ratio of the main allergen = common for group 1 / common for group 2 = mf1 × Cf1grp1 + mb1 × Cb1grp1 + mf2 × Cf2grp1 + ... /

mf1 × Cf1grp2 + mb1 × Cb1grp2 + mf2 × Cf2grp2 + ...

where mf1 represents the mass of the first used fecal extract extract Der f, mf2 represents the mass of the second used fecal particle extract Der f, etc ... Cf1grp1 is the concentration of group 1 allergen in mf1, Cf2grp1 is the concentration of group 1 allergen in the second used extracts of fecal particles, mb1 is the first used tick body extract, and Cb1grp1 is the concentration of group 1 allergen in mb1. Cf1grp2 is the concentration of group 2 allergen in mf1, Cb1grp2 is the concentration of allergen in mb1, and Cf2grp2 is the concentration of group 2 allergen in mf2, etc.

A similar formula for Der p is used to determine which and how much extract should be mixed to obtain an extract with the desired allergen ratio of group 1 to group 2.

The individual extracts are thawed and mixed according to the formula above.

The mixture of allergen extracts can be stabilized and stored as frozen drops, as described in international application WO 05/0058474, or used directly in the composition.

Typically, extracts are subjected to other standardization processes, including a series of tests, for example, an activity determination test, before they are released for formulation.

A mixture of tick allergen extracts can be used as the active ingredient in solid dosage form for sublingual administration, as described in international application WO 04/77994.

Alternatively, a mixture of allergen extracts may be formulated as a liquid for sublingual administration in the form of drops. Typically, such a composition contains about 50% glycerol, NaCl and NaOH.

The mixture can also be formulated as a liquid composition for subcutaneous injection. The extract can be administered as an aqueous liquid composition without adjuvants or with an adjuvant such as aluminum hydroxide.

Allergoids can be prepared by crosslinking extracts using a crosslinking agent, which is typically an aldehyde such as glutaraldehyde. Methods for producing allergoids are known in the art.

Additional embodiments of the invention:

Embodiment 1. A pharmaceutical product containing an allergen extract or an allergoid thereof, which contains at least one tick body extract selected from the following groups (a) to (b):

(a) the extract of the bodies of ticks Der p,

(b) Der f tick body extract,

and at least one tick culture extract selected from the following groups (c) to (g):

(c) an extract of fecal particles Der p,

(d) extract of fecal particles Der f,

(f) an extract of a whole Der mite tick culture,

(f) an extract of an entire Der p tick culture,

(g) a combination of extracts (c) - (f).

Embodiment 2. A pharmaceutical product containing an allergen extract or an allergoid thereof, which contains at least one tick body extract selected from the following groups (a) to (b):

(a) the extract of the bodies of ticks Der p,

(b) Der f tick body extract,

and at least one extract of fecal particles selected from the following groups (c) to (g):

(c) an extract of fecal particles Der p,

(d) extract of fecal mite particles Der f.

An implementation option 3. The pharmaceutical product according to one of embodiments 1-2,

where Der p mite body extract is prepared from a single fraction or combined mite culture fraction (s), wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p, and / or

mite body extract Der f is prepared from a single fraction or combined fraction (s) of tick cultures, wherein said fractions contain more than 70% v / v. tel Der f, preferably more than 75% v / v tel Der f, preferably more than 80% v / v tel Der f, preferably more than 85% v / v tel Der f, preferably more than 90% v / v tel Der f, more preferably more than 95% vol./about. tel Der f and preferably more than 98% vol./about. tel Der f.

Embodiment 4. A pharmaceutical product containing an allergen extract that includes allergens selected from Der f 1, Der f 2, Der p 1 and Der p 1, and wherein the allergen is in the lowest concentration (w / v) in allergen extract, is in a concentration that is higher than 50% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 60% the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferred up to 70% concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 at the highest concentration (w / v), more preferably above 80% concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 85% of the concentration of allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 being in the highest concentration (w / v), more preferably above 90% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 being in the highest concentration (wt. / vol.), more preferably above 95% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (wt./vol.), preferably above 98% of the concentration of the allergen, selected from Der f 1, Der f 2, Der p 1 and Der p 2, which is in the highest concentration (w / v) or most preferably above 99% of the concentration of allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v).

An implementation option 5. The pharmaceutical product according to one of embodiments 1-4, containing the extract of the bodies of ticks Der p and the extract of fecal particles Der p.

Embodiment 6. The pharmaceutical product according to one of Embodiments 1-4, comprising Der f tick body extract and Der f fecal particle extract.

An implementation option 7. The pharmaceutical product according to one of embodiments 1-4, containing the extract of bodies of ticks Der p and extract of bodies of Der f.

Embodiment 8. The pharmaceutical product of Embodiment 1-7, comprising Der p tick body extract, Der f fecal particle extract, Der f tick body extract and Der f 2 fecal particle extract.

Embodiment 9. The pharmaceutical product according to one of Embodiments 4-8, wherein the Der p tick body extract is prepared from one fraction or a combined tick culture fraction (s), wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p, and / or

Der f mite body extract is prepared from the fraction or pooled fraction (s) of Der f mite cultures, wherein said fractions contain more than 70% v / v. mites of Der f mites, preferably greater than 75% v / v mites of Der f mites, preferably greater than 80% v / v mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mite bodies Der f, more preferably greater than 95% v / v. mite bodies Der f and preferably more than 98% v / v. mite bodies Der f.

Embodiment 10. The pharmaceutical product according to one of embodiments 1-9, wherein the Der p tick body extract is prepared from a single Der p tick body fraction or the combined Der p tick body fraction, and where the extraction of said tick body fraction (s) causes that the extract contains equal weight amounts of a group 1 and group 2 allergen, or more of a group 2 allergen than a group 1 allergen by weight and / or Der f mite body extract is prepared from one or more of the combined Der f mite body fractions, and where the extraction of the specified fraction ( ) Mites bodies leads to the fact that the extract comprises equal amounts by weight of the allergen group 1 and group 2 allergen or more Group 2 than in Group 1 allergen.

An implementation option 11. The pharmaceutical product according to one of embodiments 1-10, where the extract of the bodies of ticks Der p is prepared from one fraction or the combined fraction (s) of tick cultures, where the specified fraction (s) contains more than 70% vol./about. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p, and / or

mite body extract Der f is prepared from a single fraction or combined fraction (s) of tick cultures, wherein said fractions contain more than about 70% v / v. mites of Der f mites, preferably greater than 75% v / v mites of Der f mites, preferably greater than 80% v / v mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mites of Der f mites, preferably greater than 95% v / v mite bodies Der f and preferably more than 98% v / v. mite bodies Der f.

An implementation option 12. The pharmaceutical product according to one of embodiments 1-10, where

extract of fecal mite particles Der p is prepared from a single fraction or combined fraction (s) of tick cultures, where said fraction (s) contains more than 70% v / v. fecal mite particles Der p, preferably greater than 75% v / v. fecal mite particles Der p, preferably greater than 80% v / v. fecal mite particles Der p, preferably greater than 85% v / v. fecal mite particles Der p, preferably greater than 90% v / v. mite bodies of Der p, more preferably greater than 95% v / v. fecal mite particles Der p, most preferably more than 98% v / v. fecal mite particles Der p, and / or

extract of fecal mite particles Der f is prepared from a single fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than about 70% v / v. fecal mite particles Der f, preferably greater than 75% v / v. fecal mite particles Der f, preferably greater than 80% v / v. fecal mite particles Der f, preferably greater than 85% v / v. fecal mite particles Der f, preferably greater than 90% v / v. fecal mite particles Der f, preferably greater than 95% v / v. fecal mite particles Der f, more preferably greater than 98% v / v. fecal particle mite Der f.

Embodiment 13. The pharmaceutical product of Embodiment 12 wherein the fraction or combined fractions of tick bodies and / or where the fraction or combined fractions of fecal tick particles each contains less than 10% v / v. culture medium for ticks.

Embodiment 14. The pharmaceutical product of Embodiment 13 wherein the tick body fraction or the combined tick body fraction contains up to 20% v / v. tick fragments (e.g. limbs), fecal particles, and tick media.

Embodiment 15. The pharmaceutical product according to one of embodiments 12-14, wherein the tick fecal particle fraction or the combined fecal particle fraction contains up to 20% v / v. tick fragments (e.g. limbs) and tick media.

Embodiment 16. The pharmaceutical product of one of Embodiments 12-15, wherein the weight amount of group 1 allergen is lower than the weight amount of group 2 allergen in tick extracts and the weight amount of group 1 allergen is higher than the weight amount of group 2 allergen in fecal extracts mite particles.

Embodiment 17. The pharmaceutical product of Embodiment 1-16 comprising an allergen extract that includes allergens selected from Der f 1, Der f 2, Der p 1 and Der p 1, and wherein the allergen is in the lowest concentration (w / v) in the allergen extract is in a concentration which is higher than 50% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v) .), more preferably above 60% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 70% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably higher than 80% concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 at the highest concentration (w / v), more preferably above 85% concentration of an allergen selected from Der f 1 , Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 90% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, finding which is in the highest concentration (w / v), more preferably above 95% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v) ), preferably above 98% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2, which is in the highest concentration (w / v) or most preferably above 99% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v).

An implementation option 18. The pharmaceutical product according to one of embodiments 1-17, where the pharmaceutical product is a compressed or unpressed sublingual tablet (s) containing the extract (s) or allergoids prepared from the extract (s), a liquid sublingual product containing the extract ( s) or allergoids prepared from extract (s), or a liquid product for injection containing extract (s) or allergoids prepared from extract (s).

Embodiment 19. The pharmaceutical product of Embodiment 18 wherein the pharmaceutical product is a rapidly disintegrating sublingual tablet that contains an extract.

Embodiment 20. The pharmaceutical product of Embodiment 1-19, further comprising an extract of a whole tick culture or body fraction (s) containing less than 70% v / v. bodies of ticks.

Embodiment 21. The pharmaceutical product of Embodiment 1-20 for the treatment and / or prevention of allergies or allergic asthma caused by house dust mites and / or allergy diagnosis.

Option exercise 22. The use of the pharmaceutical product according to one of embodiments 1-21 to obtain a medical product for the treatment and / or prevention of allergies or allergic asthma caused by house dust mites, and / or for the diagnosis of allergies.

Embodiment 23. Tick body fraction containing more than 70% v / v mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. bodies of ticks.

Embodiment 24. The tick body fraction of Embodiment 23, wherein the tick bodies are Der p mite bodies or Der f mite bodies.

An implementation option 25. The fraction of bodies of ticks according to one of embodiments 23-24, containing less than 10% of the particles of the culture medium.

An implementation option 26. The fraction of bodies of ticks according to one of embodiments 23-25, containing less than 20% vol./about. tick fragments, fecal particles, and tick media.

Embodiment 27. The tick body fraction extract according to one of embodiments 23-26 for use in a pharmaceutical product for treating and / or preventing allergies and allergic asthma caused by house dust mites.

Embodiment 28. A fecal mite particle fraction containing more than 70% v / v. fecal particles, preferably more than 75% v / v fecal particles, preferably more than 80% v / v. fecal particles, preferably more than 85% v / v fecal particles, preferably more than 90% v / v. fecal particles, more preferably more than 95% v / v. fecal particles, most preferably more than 98% v / v. fecal particles.

Embodiment 29. The fecal mite particle fraction of Embodiment 28, wherein the fecal particles are Der p particles or fe f Der particles.

Option exercise 30. The fraction of fecal particles of mites according to one of embodiments 28-29, containing up to 20% vol./about. tick fragments (e.g. limbs) and tick media.

Embodiment 31. An extract of a fecal mite particle fraction according to one of embodiments 28-30 for use in a pharmaceutical product for treating and / or preventing allergies or allergic asthma caused by house dust mites.

An implementation option 32. The use of the extract according to one of embodiments 27 or 31 to obtain a pharmaceutical product for use in a pharmaceutical product for the treatment and / or prevention of allergies or allergic asthma caused by house dust mites.

Embodiment 33. A method for producing a tick allergen extract containing an allergen Der p 1 and Der p 2, wherein the method comprises the following steps:

(a) isolating fraction (s) of Der p mite bodies from Der p mite cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mite bodies of Der p, preferably more than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p;

(b) optionally combining several of these fractions of the bodies of ticks Der p; and

(c) extracting allergens from said fraction (s) of Der p mite bodies obtained in step (a) or step (b), as described above, to obtain said tick mite allergen extract.

An implementation option 34. A method of manufacturing a tick allergen extract containing allergen Der f 1 and Der f 2, where the method includes the following stages:

(a) isolating fraction (s) of Der f mite bodies from Der f mite cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies Der f, more preferably greater than 75% v / v. mite bodies Der f, more preferably greater than 80% v / v. mite bodies Der f, preferably more than 85% v / v mites of Der f mites, preferably greater than 90% v / v mites of Der f mites, preferably greater than 95% v / v mite bodies Der f and most preferably more than 98% v / v. mite bodies Der f;

(b) optionally combining several of said fractions of Der f mite bodies; and

(c) extracting allergens from said fraction (s) of Der f mite bodies obtained in step (a) or step (b), as described above, to obtain said tick mite allergen extract.

Embodiment 35. The method of one of Embodiments 33-34, wherein the method further comprises the following step: mixing one or more tick allergen extracts of Embodiment 33 with one or more tick allergen extracts of Embodiment 34 to produce a mixture of tick allergen extracts, containing allergen Der p 1, Der p 2, Der f 1 and Der f 2.

Embodiment 36. The method of one of embodiments 33-35, wherein the method further comprises the steps of:

a. isolating fraction (s) of fecal Der p tick particles from Der p tick cultures;

b. optionally combining several of these fractions of fecal mite particles Der p;

from. extraction of allergens from the fraction (s) of fecal mite particles Der p obtained in stage (a) or stage (b), as described above, and

d. mixing one or more tick allergen extracts according to one of embodiments 33-35 with one or more fecal particle extracts obtained in step (c), as described above, to obtain a mixture of tick allergen extracts.

Embodiment 37. The method according to one of embodiments 33-36, wherein the method further comprises the following steps:

a. isolating fraction (s) of fecal mite particles Der f from mite cultures Der f;

b. optionally combining several of these fractions of fecal mite particles Der f;

from. extraction of allergens from fraction (s) of fecal mite particles Der f obtained in step (a) or step (b) as described above, and

d. mixing one or more tick allergen extracts according to one of embodiments 33-35 with one or more fecal particle extracts obtained in step (c), as described above, to obtain a mixture of tick allergen extracts.

Embodiment 38. The method according to one of embodiments 33-37, wherein the method further comprises the following step: mixing one or more tick allergen extracts according to embodiment 33 and embodiment 34 with one or more tick allergen extracts from step (c) of embodiment exercise 36 and from step (c) of embodiment 37 to obtain a mixture of tick allergen extracts containing the allergen Der p 1, Der p 2, Der f 1 and Der f 2.

An implementation option 39. The method according to one of embodiments 33-38, where the obtained tick allergen extract is converted to its allergoid.

An implementation option 40. The method according to one of embodiments 33-39, where the method includes an additional step

a. determining the concentration (w / v) of the group 1 and group 2 allergen in the tick allergen extracts obtained in step (c) according to one of embodiments 33-34 and 36-37.

Embodiment 41. The method of Embodiment 40, wherein the method comprises an additional step

(e) mixing one or more extracts of tick bodies from step (c) according to one of embodiments 33-34 with one or more extracts of fecal particles from step (c) according to one of embodiments 36-37 to obtain a mixture of tick allergen extracts with a predetermined weight amount of a group 1 allergen to a group 2 allergen.

Embodiment 42. The method according to one of embodiments 33-41, wherein the mixture of tick allergen extracts comprises Der p tick body extract (s), Der p fecal particle extract (s), Der f tick body extract (s) and extract (s) ) fecal particles Der f.

Embodiment 43. The method of one of Embodiments 33-42, wherein more than one tick body fraction is combined in step (b), followed by extraction in step (c) of embodiment 33 and / or 34.

An implementation option 44. The method according to one of embodiments 33-43, where more than one fraction of fecal particles is combined in stage (b), followed by extraction in stage (c) according to embodiment 36 and / or 37.

Embodiment 45. The method of one of Embodiments 33-44, wherein one or more tick body extracts obtained in step (c) of Embodiment 33 and / or 34 are mixed in step (e) with one or more fecal extracts mite particles obtained in step (c) of Embodiment 36 and / or 37.

Embodiment 46. The method of one of Embodiments 33-45, wherein one or more tick body extracts obtained in step (c) of Embodiment 33 and / or 34 are mixed in step (e) with one or more fecal extracts mite particles obtained in step (c) of Embodiment 36 and / or 37.

Embodiment 47. The method of one of Embodiments 33-46, wherein one tick body extract obtained in step (c) of Embodiment 33 and / or 34 is mixed in step (d) with one or more extracts of fecal tick particles obtained in step (c) of Embodiment 36 and / or 37.

An implementation option 48. The method according to one of embodiments 33-47, where the extract (s) of the bodies of ticks enriched by the weight of the extract of fecal particles, the extract of bodies from the fraction of bodies with less than 70% vol./about. or an extract of the whole culture, which is sufficient to achieve the desired weight ratio of group 1 allergen to group 2 allergen in a mixture of tick allergen extracts.

An implementation option 49. The method according to one of embodiments 33-48, where

extract of fecal mite particles Der p in step (c) according to embodiment 36 is prepared from one fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 70% v / v. fecal mite particles Der p, preferably greater than 75% v / v. fecal mite particles Der p, preferably greater than 80% v / v. fecal mite particles Der p, preferably greater than 85% v / v. fecal mite particles Der p, preferably greater than 90% v / v. fecal mite particles Der p, more preferably greater than 95% v / v. fecal mite particles Der p, most preferably more than 98% v / v. fecal mite particles Der p, and / or

the extract of fecal tick particles Der f in step (c) of Embodiment 37 is prepared from one fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 70% v / v. fecal Der f particles, preferably greater than 75% v / v. fecal Der f particles, preferably greater than 80% v / v. fecal Der f particles, preferably greater than 85% v / v. fecal Der f particles, preferably greater than 90% v / v. fecal Der f particles, preferably greater than 95% v / v. fecal particles Der f, more preferably more than 98% v / v. fecal particles Der f.

Embodiment 50. The method according to one of embodiments 33-49, wherein the tick body fraction in step (a) or the combined tick body fraction in step (b) of Embodiment 33 and / or 34, and / or where the fecal particle fraction is stage (a) or combined fractions of fecal mite particles in stage (b) according to a variant implementation 36 and / or 37 each contain less than 10% vol./about. culture medium for ticks.

Embodiment 51. The method of Embodiment 50, wherein the tick body fraction in step (a) or the combined tick body fractions in step (b) of Embodiment 33 and / or 34 comprise up to 20% v / v. tick fragments (e.g. limbs), fecal particles, and tick media.

Embodiment 52. The method of Embodiment 50, wherein the tick fecal particle fraction in step (a) or the combined fecal particle fraction in step (b) of Embodiment 36 and / or 37 comprise up to 20% v / v. tick fragments (e.g. limbs) and tick media.

Embodiment 53. The method according to one of embodiments 33-52, wherein the tick body fraction (s) of embodiment 33 and / or 34, and / or the fecal tick fraction (s) of step mites in step (a) of embodiment 36 and / or 37 is isolated from the tick culture (p) by sieving the tick culture (p) of Der p and / or Der f mites to obtain the tick body fraction (b) and the fecal tick fraction (d).

Embodiment 54. The method of Embodiments 33-53, wherein the tick body fraction (s) and the tick fecal particles fraction (s) are obtained by sieving the Der p and / or Der f tick culture (p) to obtain four fractions:

a. large particles of the medium> 350 microns;

b. fraction of tick bodies 350-90 microns;

from. fecal agglomerates and tick fragments 90-50 microns;

d. fecal particles <50 μm;

e. fraction (a) and (c) are discarded, fraction (d) and (b) are optionally further purified or directly used for extraction.

Embodiment 55. The method of Embodiment 54, wherein fraction (b) is:

(ii) subjected to density gradient centrifugation in a first liquid medium having a higher density than mite bodies and lower density than particles of the culture medium; then

(iii) collecting mite bodies from said first liquid medium and repeating steps (ii) and (iii) until the desired mite bodies are clean, optionally then

(iv) washing the tick bodies to remove said first liquid with a second liquid.

Embodiment 56. A method for isolating and purifying tick bodies, comprising the steps of:

(i) isolating the tick body fraction (s) from the tick culture (p) of Der p and / or Der f mites; and

(ii) centrifuging one or more Der p1 mite body fractions or one or more Der f mite body fractions in a density gradient in a first liquid medium having (iia) higher density than mite bodies and lower density than culture medium particles; and

(iii) collecting tick bodies from said first liquid medium

and repeating steps (ii) and (iii) until the desired mite bodies are clean, optionally then

(iv) washing the tick bodies to remove said first liquid with a second liquid.

An implementation option 57. The method according to one of embodiments 55-56, where the first and second liquids are anhydrous, preferably pharmaceutically acceptable liquids.

An implementation option 58. The method according to one of embodiments 55-57, where the first liquid is anhydrous glycerin.

An implementation option 59. The method according to one of embodiments 55-58, where the second liquid is anhydrous ethanol.

An implementation option 60. The method according to one of embodiments 55-59, where stage (ii) and (iii) is carried out once.

An implementation option 61. The method according to one of embodiments 55-60, where stage (ii) and (iii) is repeated until the obtained fraction of tick bodies contains at least 70% v / v. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. bodies of ticks.

Embodiment 62. The method of Embodiments 55-61, wherein purification of the tick body fraction is continued until the tick body fraction contains less than 10% of the culture medium particles.

Embodiment 63. The method of Embodiment 62, wherein purification of the tick body fraction is continued until less than 20% v / v is present. tick fragments, fecal particles, and tick media.

An implementation option 64. The method according to one of embodiments 33-63, where the fractions of the bodies of mites Der f, the fractions of the bodies of mites Der p, the fraction (s) of fecal particles Der p and / or the fractions of fecal particles Der f at stage (a) one at a time of embodiments 33-34 and 36-37 are isolated by sieving, including:

(1) sieving Der f or Der p mite cultures in a shaker with a vibrating sieve with a tower with openings of 300, 150 and 80 μm in size, followed by isolation of a body-rich mite fraction ranging in size from 150 to 300 μm and a fecal-rich mite fraction below 80 microns then

(2) sieving the body-rich tick fraction (150 μm and 300 μm) in a shaker with a horizontal and circular motion sieve and a tower with sieves with openings of 200, 300 and / or 400 μm, and isolating the body-rich tick fraction (s) captured by sieves with openings of 200, 300 and / or 400 microns, then

(3) sieving the mite-rich fraction in a shaker with a sieve having a 3D torsion movement and a sieve with a mesh size of 300, 200, 150 and / or 100 μm to obtain a fraction of mite bodies ranging in size from 150 to 300 μm and a fecal fraction particles below 100 microns in size; and

(4) optionally combining the fecal particle fraction below 80 μm in size with a vibrating sieve and the fecal particle fraction below 100 μm in size from a sieve with 3D torsion movement, and sieving the combined fecal particle fraction on a sieve with 3D 3D torsion movement and a tower with sieves with size holes 100, 80, 50 microns, and the allocation of fecal particles rich in size below 50 microns.

An implementation option 65. The method according to one of embodiments 33-64, where the obtained extracts or mixture of extracts are formulated into a pharmaceutical product, such as compressed or unpressed sublingual tablet (s) containing the extract (s) or allergoids prepared from the extract (s) , a liquid sublingual product containing the extract (s) or allergoids prepared from the extract (s), or a liquid injection product containing the extract (s) or allergoids prepared from the extract (s).

Embodiment 66. The method of Embodiment 65, wherein the pharmaceutical product is a rapidly disintegrating sublingual tablet that contains an extract.

An implementation option 67. The pharmaceutical product obtained by the method according to one of embodiments 33-66.

Embodiment 68. The pharmaceutical product of Embodiment 67 for the treatment and / or prevention of allergies or allergic asthma caused by house dust mites.

Embodiment 69. An allergen extract from a series of allergen extracts for use in a pharmaceutical allergen product for treating and / or preventing allergies and allergic asthma caused by house dust mites, wherein each allergen extract contains at least one tick body extract, and, at least one extract of fecal mite particles, or an extract of mite bodies from a body fraction containing less than 70% v / v. mite bodies or an entire tick culture extract, wherein said allergen extract has a predetermined ratio of group 1 to group 2 allergen, and where the variability coefficient of said ratio is not more than 25, 20, 15, 10, or 7.5% for a series of extracts.

Embodiment 70. An allergen extract from a series of allergen extracts for use in a pharmaceutical allergen product for diagnosing allergies caused by house dust mites, where each allergen extract contains at least one tick body extract and at least one fecal particle extract ticks, or tick body extract from a fraction of bodies containing less than 70% v / v mite bodies or an entire tick culture extract, wherein said allergen extract has a predetermined ratio of group 1 to group 2 allergen, and where the variability coefficient of said ratio is not more than 25, 20, 15, 10, or 7.5% for a series of extracts.

Embodiment 71. The allergen extract according to one of embodiments 69-70, wherein the weight amount of group 1 allergen is lower than the weight amount of group 2 allergen in tick extracts and the weight amount of group 1 allergen is higher than the weight amount of group 2 allergen in extracts fecal particles of ticks.

Embodiment 72. An allergen extract according to one of embodiments 69-71, wherein the allergen extract is an allergen according to one of embodiments 1-32.

An implementation option 73. The allergen extract according to one of embodiments 69-71, where the allergen extract is obtained according to one of embodiments 33-66.

Additional embodiments of the invention:

Embodiment 1. A pharmaceutical product containing an extract of an allergen (I) or an allergoid thereof for treating and / or preventing allergies and allergic asthma caused by house dust mites, comprising at least one tick body extract and optionally at least one extract of fecal mite particles selected from the following groups (a) - (d):

a) the extract of the bodies of ticks Der p,

b) extract of fecal particles Der p,

c) Der f tick body extract, and

d) extract of fecal particles Der f.

Embodiment 2. A pharmaceutical product containing an allergen (I) extract for treating and / or preventing allergies and allergic asthma caused by house dust mites, containing allergens selected from Der f 1, Der f 2, Der p 1 and Der p 1, and where the allergen in the lowest concentration (w / v) in the allergen extract is in a concentration that is higher than 50% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 located at the highest concentration (w / v), more preferably above 60% of the concentration of the allergen selected and h Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 70% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 80% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 85% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably higher 90% concentration a Lergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 95% concentration of an allergen selected from Der f 1, Der f 2 , Der p 1 and Der p 1, which is in the highest concentration (w / v), preferably above 98% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2, located in the a high concentration (w / v) or most preferably above 99% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v).

An implementation option 3. The pharmaceutical product according to one of embodiments 1-2, containing at least one extract of tick bodies and at least one extract of fecal particles of ticks.

Embodiment 4. The pharmaceutical product of Embodiment 3, comprising Der p tick body extract, Der p fecal particle extract, Der f tick body extract and Der f 2 fecal particle extract.

Embodiment 5. The pharmaceutical product of Embodiment 1-2, comprising Der p tick body extract and Der p fecal particle extract, and containing Der f tick body extracts and Der f fecal particle extracts.

Embodiment 6. The pharmaceutical product of Embodiment 1-2, comprising Der f tick body extract and Der f fecal particle extract, and Der p mite body extracts and Der p fecal particle extracts.

Embodiment 7. The pharmaceutical product of Embodiment 1-2, comprising Der p mite body extract and Der f mite body extract, and not containing fecal mite particle extract.

Embodiment 8. The pharmaceutical product of Embodiment 1-2, comprising Der p mite body extract and not Der f mite body extract and fecal mite particle extract.

Embodiment 9. The pharmaceutical product of Embodiment 1-2, comprising Der f mite body extract and Der p mite body extract and fecal mite particle extract.

An implementation option 10. The pharmaceutical product according to one of embodiments 1-9, where

extract of tick pit bodies Der p is prepared from a single fraction of tick pit bodies Der p or combined fractions of tick pit bodies Der p, and where extraction of said tick pit fraction (s) causes the extract to contain equal weight amounts of allergen of group 1 and group 2, or more group 2 allergen than group 1 allergen by weight, and / or

Der f tick body extract is prepared from one or more combined fractions of Der f tick bodies, and where the extraction of said tick body fraction (s) causes the extract to contain equal weight amounts of group 1 and group 2 allergen, or more group allergen 2 than group 1 allergen by weight.

An implementation option 11. The pharmaceutical product according to one of embodiments 1-10, where

the tick pit body extract Der p is prepared from a single fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p,

Der f mite body extract is prepared from the fraction or pooled fraction (s) of Der f mite cultures, wherein said fractions contain more than 70% v / v. mites of Der f mites, preferably greater than 75% v / v mites of Der f mites, preferably greater than 80% v / v mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mite bodies Der f, more preferably greater than 95% v / v. mite bodies Der f and preferably more than 98% v / v. mite bodies Der f,

The Der p faecal particle extract is prepared from a single fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 70% v / v. fecal Der p particles, preferably greater than 75% v / v. fecal Der p particles, preferably greater than 80% v / v. fecal Der p particles, preferably greater than 85% v / v. fecal Der p particles, preferably greater than 90% v / v. fecal Der p particles, more preferably greater than 95% v / v. fecal Der p particles, most preferably greater than 98% v / v. fecal particles Der p, and / or

Der f faecal particle extract is prepared from the Der f mite culture fraction or pooled fraction (s), wherein said fraction (s) contains more than 70% v / v. fecal Der f particles, preferably greater than 75% v / v. fecal Der f particles, preferably greater than 80% v / v. fecal Der f particles, preferably greater than 85% v / v. fecal Der f particles, preferably greater than 90% v / v. fecal Der f particles, preferably greater than 95% v / v. fecal particles Der f, more preferably more than 98% v / v. fecal particle mite Der f.

Embodiment 12. The pharmaceutical product of Embodiment 11 wherein the fraction or combined fractions of tick bodies and / or where the fraction or combined fractions of fecal tick particles each contains less than 10% v / v. culture medium for ticks.

Embodiment 13. The pharmaceutical product of Embodiment 12 wherein the body fraction or combined tick body fractions comprise up to 20% v / v. tick fragments (e.g. limbs), fecal particles, and tick media.

Embodiment 14. The pharmaceutical product of Embodiment 12 wherein the fecal particle fraction or the combined tick fecal particle fraction contains up to 20% v / v. tick fragments (e.g. limbs) and tick media.

Embodiment 15. The pharmaceutical product of one of Embodiments 1-14, wherein the weight amount of group 1 allergen is lower than the weight amount of group 2 allergen in tick extracts and the weight amount of group 1 allergen is higher than the weight amount of group 2 allergen in extracts fecal particles of ticks.

Embodiment 16. A pharmaceutical product according to one of Embodiments 1, 3-15, containing an allergen (I) extract for treating and / or preventing allergies and allergic asthma caused by house dust mites, allergens selected from Der f 1, Der f 2 , Der p 1 and Der p 1, and where the allergen in the lowest concentration (w / v) in the allergen extract (I) is in a concentration that is higher than 50% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 60% con the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 70% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), more preferably above 80% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, located in the highest concentration (w / v), more preferably above 85% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v) more pre significantly higher than 90% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1 in the highest concentration (w / v), more preferably above 95% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (w / v), preferably above 98% of the concentration of an allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2, which is in the highest concentration (w / v) or most preferably above 99% of the concentration of the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 1, which is in the highest concentration (Wt. / Vol.).

Embodiment 17. The pharmaceutical product according to one of embodiments 1-16, wherein the pharmaceutical product is a compressed or unpressed sublingual tablet (s) containing extract (I) or allergoids prepared from extract (I) in solid form, a liquid sublingual product, or a liquid product for injection containing extract (I) or allergoids prepared from extract (I), preferably the pharmaceutical product is a rapidly disintegrating sublingual tablet that contains extra CT (I) in solid form.

Embodiment 18. A method for producing a tick allergen extract (I) or allergoid thereof for a pharmaceutical product according to one of embodiments 1-10 and 15-16, wherein said allergen extract (I) has a predetermined and controlled weight amount of allergens selected from allergens Der f 1, Der f 2, Der p 1 and Der p 2, and comprising the following steps:

(a) isolating a Der p mite body fraction (s) and / or Der p mite fecal particle fractions from Der p mite cultures and / or isolating a Der f mite body fraction (s) and / or Der f mite fecal particle fractions from mite cultures Der f;

(b) optionally combining several fractions of Der p tick mites, optionally combining several fractions of fecal tick Der p mites, optionally combining several fractions of Der f tick mites, and optionally combining several fractions of Der f tick fecal particles

(c) extracting allergens from the fraction of tick bodies Der p of the tick obtained in step (a) or step (b) as described above, and / or extracting the allergens from the fraction (s) of fecal tick particles obtained in step (a) ) or step (b) as described above; and then

(d) determining the concentration (w / v) of the group 1 and group 2 allergen in the tick allergen extracts obtained in step (c) as described above; and optional

(e) mixing one or more tick body extracts with one or more fecal particle extracts obtained in step (c) as described above, to obtain a mixture of allergen extracts (I) with a predetermined weight amount of a group 1 allergen to a group 2 allergen, and optional

(f) converting the extract (I) to its allergoid.

Embodiment 19. The method of Embodiment 18 wherein one or more extracts of tick bodies and one or more extracts of fecal particles are mixed to form a mixture of allergen (I) extracts.

Option exercise 20. The method according to one of embodiments 18-19, where the mixture of tick allergen extracts (I) contains the extract of tick mites Der p, extract (s) of fecal particles Der p, extract (s) of mites Der f and extract (s) of fecal particles Der f 2.

Embodiment 21. The method of one of Embodiments 18-20, wherein more than one tick body fraction is combined in step (b), followed by extraction in step (c) of embodiment 33 and / or 34.

Embodiment 22. The method according to one of embodiments 18-21, wherein more than one fraction of fecal particles is combined in step (b) followed by extraction in step (c).

Embodiment 23. The method according to one of embodiments 18-22, wherein one or more tick body extracts obtained in step (c) are mixed in step (e) with one or more tick fecal particle extracts obtained in step (c) .

Embodiment 24. The method of one of Embodiments 18-23, wherein one or more tick body extracts obtained in step (c) are mixed in step (e) with one fecal tick extract obtained in step (c).

Option exercise 25. The method according to one of embodiments 18-24, where one extract of tick bodies obtained in stage (C), is mixed in stage (e) with one or more extracts of fecal particles of ticks obtained in stage (C).

Embodiment 26. The method according to one of embodiments 18-25, wherein the tick body extract (s) are enriched with a weight quantity of fecal particles extract, which is sufficient to achieve the desired weight ratio of group 1 allergen to group 2 allergen in a mixture of tick allergen extracts (I) .

An implementation option 27. The method according to one of embodiments 14-26, where

extract of mites of Der p mites in step (c) is prepared from one fraction or combined fraction (s) of mite cultures, where said fraction (s) contains more than 70% v / v. mite bodies of Der p, more preferably greater than 75% v / v. mite bodies of Der p, more preferably greater than 80% v / v. mites of Der p mites, preferably greater than 85% v / v mites of Der p mites, preferably greater than 90% v / v mites of Der p mites, preferably greater than 95% v / v mite bodies of Der p and most preferably more than 98% v / v. mite bodies Der p;

extract of mites of Der f mites in step (c) is prepared from one fraction or a combined fraction (s) of mite cultures, where said fractions contain more than 70% v / v. mites of Der f mites, preferably greater than 75% v / v mites of Der f mites, preferably greater than 80% v / v mites of Der f mites, preferably greater than 85% v / v mites of Der f mites, preferably greater than 90% v / v mite bodies Der f, more preferably greater than 95% v / v. mite bodies Der f and preferably more than 98% v / v. mite bodies Der f,

extract of fecal mite particles Der p in stage (C) is prepared from a single fraction or a combined fraction (s) of tick cultures, where said fraction (s) contains more than 70% v / v. fecal mite particles Der p, preferably greater than 75% v / v. fecal mite particles Der p, preferably greater than 80% v / v. fecal mite particles Der p, preferably greater than 85% v / v. fecal mite particles Der p, preferably greater than 90% v / v. fecal mite particles Der p, more preferably greater than 95% v / v. fecal mite particles Der p, most preferably more than 98% v / v. fecal mite particles Der p;

the extract of fecal tick mites Der f in step (c) is prepared from one fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 70% v / v. fecal mite particles Der f, preferably greater than 75% v / v. fecal mite particles Der f, preferably greater than 80% v / v. fecal mite particles Der f, preferably greater than 85% v / v. fecal mite particles Der f, preferably greater than 90% v / v. fecal mite particles Der f, preferably greater than 95% v / v. fecal mite particles Der f, more preferably greater than 98% v / v. fecal particle mite Der f.

Embodiment 28. The method of Embodiment 27, wherein the tick body fraction in step (a) or the combined tick body fraction in step (b), and / or where the fecal particle fraction in step (a) or the combined tick fecal particle fraction in step (b) each contains less than 10% v / v. culture medium for ticks.

Embodiment 29. The method of Embodiment 28, wherein the tick body fraction in step (a) or the combined tick body fraction in step (b) contain up to 20% v / v. tick fragments (e.g. limbs), fecal particles, and tick media.

Embodiment 30. The method of Embodiment 29, wherein the tick fecal particle fraction in step (a) or the combined fecal particle fraction in step (b) contain up to 20% v / v. tick fragments (e.g. limbs) and tick media.

Option exercise 31. The method according to one of embodiments 18-30, where the fraction (s) of tick bodies and / or the fraction (s) of fecal mite particles in step (a) of embodiment 18 are isolated from the tick culture (p) by sieving the culture ( p) Der p and / or Der f ticks to obtain the tick body fraction (b) and the fecal tick fraction (d).

Embodiment 32. The method of Embodiment 31, wherein the tick body fraction (s) and the tick fecal particles fraction (s) are obtained by sieving the Der p and / or Der f tick culture (p) to obtain four fractions:

(a) large particles of the medium> 350 μm;

(b) a tick body fraction of 350 to 90 microns;

(c) fecal agglomerates and tick fragments 90-50 microns;

(d) fecal particles <50 μm;

fraction (a) and (c) are discarded, fraction (d) is directly used for extraction, and fraction (b) is further purified.

Embodiment 33. A method for isolating and purifying tick bodies, comprising the steps of:

(i) isolating the tick body fraction (s) from the tick culture (p) of Der p and / or Der f mites; and

(ii) centrifuging one or more fractions of mites of Der p mites or one or more fractions of mites of Der f mites in a density gradient in a first liquid medium having (iia) higher density than mite bodies and lower density than particles of the culture medium, and (iib) does not extract group 1 and group 2 allergen from tick bodies; and

(iii) collecting mite bodies from the surface of said first liquid medium

and repeating steps (ii) and (iii) until the desired mite bodies are clean, optionally then

(iv) washing the tick bodies to remove said first liquid with a second liquid that does not extract the group 1 and group 2 allergen from the tick bodies.

An implementation option 34. The method according to one of embodiments 31-32, where the fraction of tick bodies (b) is:

(ii) density gradient centrifuged in a first liquid medium having (iia) higher density than mite bodies and lower density than culture medium particles, and (iib) which does not extract group 1 and group 2 allergen from mite bodies ; then

(iii) collecting mite bodies from the surface of said first liquid medium and repeating steps (ii) and (iii) until the desired mite bodies are clean, optionally then

(iv) washing the tick bodies to remove said first liquid with a second liquid that does not extract the group 1 and group 2 allergen from the tick bodies.

An implementation option 35. The method according to one of embodiments 33 or 34, where the first and second liquids are anhydrous, preferably pharmaceutically acceptable liquids.

Embodiment 36. The method of Embodiment 35, wherein the first liquid is anhydrous glycerin.

Embodiment 37. The method of Embodiment 36 wherein the second liquid is anhydrous ethanol.

Embodiment 38. The method of Embodiments 33-37, wherein step (ii) and (iii) are performed once.

Embodiment 39. The method of one of Embodiments 33-37, wherein step (ii) and (iii) is repeated until the obtained tick body fraction contains at least 70% v / v. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. bodies of ticks.

Embodiment 40. The method of Embodiments 31-33 and 38-39, wherein purification of the tick body fraction is continued until the tick body fraction contains less than 10% of the culture medium particles.

Embodiment 41. The method of Embodiment 40, wherein cleaning the tick bodies is continued until less than 20% v / v is present. tick fragments, fecal particles, and tick media.

An implementation option 42. The fraction of bodies of ticks containing more than 70% vol./about. mite bodies, more preferably greater than 75% v / v. mite bodies, more preferably more than 80% v / v. mite bodies, preferably greater than 85% v / v mite bodies, preferably greater than 90% v / v mite bodies, preferably greater than 95% v / v mite bodies and most preferably more than 98% v / v. bodies of ticks.

Embodiment 43. The tick body fraction of Embodiment 42 containing less than 10% particles of culture medium.

Embodiment 44. The tick body fraction of Embodiment 43 comprising less than 20% v / v. tick fragments, fecal particles, and tick media.

Embodiment 45. The method according to one of embodiments 18-30, wherein the Der f tick body fractions, Der p tick body fractions, Der p fecal particle fraction (s) and / or Der f fecal particle fractions in step (a) are isolated by sieving including:

(1) sieving Der f or Der p mite cultures in a shaker with a vibrating sieve with a tower with openings of 300, 150 and 80 μm in size, followed by isolation of a body-rich mite fraction ranging in size from 150 to 300 μm and a fecal-rich mite fraction below 80 microns then

(2) sieving the body-rich tick fraction (150 μm and 300 μm) in a shaker with a horizontal and circular motion sieve and a tower with sieves with openings of 200, 300 and / or 400 μm, and isolating the body-rich tick fraction (s) captured by sieves with openings of 200, 300 and / or 400 microns, then

(3) sieving the mite-rich fraction in a shaker with a sieve having a 3D torsion movement and a sieve with a mesh size of 300, 200, 150 and / or 100 μm to obtain a fraction of mite bodies ranging in size from 150 to 300 μm and a fecal fraction particles below 100 microns in size; and

(4) optionally combining the fecal particle fraction below 80 μm in size with a vibrating sieve and the fecal particle fraction below 100 μm in size from a sieve with 3D torsion movement, and sieving the combined fecal particle fraction on a sieve with 3D 3D torsion movement and a tower with sieves with size holes 100, 80, 50 microns, and the allocation of fecal particles rich in size below 50 microns.

Embodiment 46. The method according to one of embodiments 18-45, 45, wherein the obtained mixture of extracts (I) is formulated as a compressed or unpressed sublingual tablet (current) containing extract (I) or allergoids prepared from extract (I) in a solid form, a liquid sublingual product or a liquid product for injection containing extract (I) or allergoids prepared from extract (I), preferably the pharmaceutical product is a rapidly disintegrating sublingual tablet that contains extract (I) in solid form.

Experimental Section

Example 1

Tick cultivation

Mites Der p and Der f are individually cultured in a suitable medium with medium particles that are of sufficient size and integrity to withstand the effects of subsequent processing and can be easily removed during initial screening. The tick medium accordingly contains a source of proteins, carbohydrates, minerals, oils, vitamins and preservatives.

Ticks are killed by freezing at a temperature of -20 ° C ± 5 ° C, when optimal growth is achieved.

1.1. Cleaning mite bodies and fecal mite particles

The tick culture (Der p or Der f) is dried to a moisture content of less than 15% and sieved to obtain four fractions:

(1) large particles of the medium> 350 microns;

(2) the fraction of tick bodies 350-90 microns;

(3) fecal agglomerates and tick fragments 90-50 microns;

(4) fecal particles <50 μm.

For sieving a dried tick culture, an automatic sieving plant is used. The sifter uses rotation at set angles and speeds to produce fractions. A sieve that gives fractions 1, 2 and 3 described above can be cleaned during sieving. Fine particles (fraction 4) Collected on the bottom pan of the screening device.

Large particles of the medium in fraction 1 and fecal agglomerates, and tick fragments in fraction 3 are discarded.

The quality of the fecal particles is tested (to ensure that the fraction contains predominantly fecal particles and meets the requirements of the specification), and then the question is solved of how suitable the fraction is for the extraction of Der p and Der f allergens.

Determine% vol./about. whole mite bodies, mite body fragments, fecal particles, mite media and impurities by specimen microscopy, see pages 13-14.

Several fecal fractions can be combined before extraction.

1.2. Purification of the tick body fraction 2 by centrifugation with glycerin

Centrifugation of fraction 2 is carried out in a centrifuge (Beckman J6M1).

Not more than 900 g of fraction 2 is slowly added to anhydrous glycerin, the US Pharmacopoeia (purity 99.0-101.0%) in an amount that exceeds the mass of fraction 2 by five times.

A tick culture mixed with anhydrous glycerin is poured into a centrifuge glass so that all glasses contain the same volume. The glasses are placed in a rotor and centrifuged at 3000 rpm for 7 min at -5 ° C. A precipitate of mite bodies is removed from the surface of anhydrous glycerol and weighed. Mite bodies are mixed with anhydrous glycerin, the amount of which is 3 times greater than the mass of the mite body fraction, and centrifugation is repeated.

The layer of tick bodies is carefully removed from the surface of anhydrous glycerol and weighed. The mass of tick bodies is multiplied by 2.5 to determine the minimum weight quantity of anhydrous ethanol required for the formation of a suspension. Mite bodies and ethanol suspension are mixed for 5 minutes in a mixing tank equipped with a low shear knife. The suspension is poured onto two sieves with a hole size of 106 microns with collection in a collecting pan.

The bodies of ticks are scraped off from the sieves and the mass of ticks is recorded. Repeat the final purification step with ethanol once more and dry the mite bodies using vacuum or allow to dry on a sieve 106 in a fume hood.

Determine% vol./about. whole mite bodies, mite body fragments, fecal particles, mite media and impurities by specimen microscopy, see pages 13-14. The resulting fraction of tick bodies, as a rule, contains 98-99% vol./about. whole bodies of ticks.

You can mix fractions of tick bodies from different tick cultures.

1.3. Purification of the tick body fraction 2 by centrifugation with saturated NaCl solution

The body fraction of ticks 2 is mixed with saturated NaCl solution (in an amount that is five times the mass of fraction 2) to obtain a suspension. The suspension is centrifuged at 3000 rpm for 7 min (15 ° C in a Sorvall centrifuge). The layer of tick bodies is carefully removed from the surface and resuspended in an amount of NaCl, which is five times the mass, and centrifugation is repeated. Determine% vol./about. whole mite bodies, mite body fragments, fecal particles, mite media and impurities by specimen microscopy, see pages 13-14.

The resulting fraction of tick bodies, as a rule, contains 98-99% vol./about. whole bodies of ticks.

Table 1b centrifuged # 1 and 2 with saturated aqueous solution of salt 1, centrifuged # 3 and 4 with saturated aqueous solution of salt 2 Volume percent composition Sample Number Body Fecal particles Wednesday 1 98,200 1,180 0.620 2 98,100 1,230 0.660 3 99,890 0,110 0,000 4 99,880 0.140 0,000

Example 2

2.0. Cleaning mite bodies and fecal mite particles

Frozen culture is placed on trays in the room for cleaning. The culture is evenly distributed on trays and the culture is thawed and dried at room temperature for 4-14 days.

Then the culture is turned into a liquid for drying and separation into two parties, see figure 1. Apply air flow, connecting the vacuum pump on one side and the air inlet on the other.

The glass containers of FIG. 1 are designated left to right as fraction “A” with a whole tick culture, fraction “B” represents the fraction rich in body particles, and fraction “C” represents fecal particles.

Two fractions (B) and (C) are isolated and screened.

2.1. Cleaning tick bodies

2.1.1. Coarse sieve

Fractions B are introduced into an industrial shaker with a vibrating sieve with a continuous feed of Filtra FT 500. The sieve has a sieving tower with three types of holes: 300, 150 and 80 μm and operates at 1500 rpm.

A material with a particle size greater than 300 microns mainly represents particles of a nutrient material and is discarded.

Material with a particle size of less than 80 microns is further purified on a small sieve to isolate the fecal fraction.

Material with a particle size of less than 300 microns and more than 150 microns is isolated as a fraction rich in mite bodies and sieved on an average sieve.

2.1.2. Medium screen

Ticks have a shape that is irregular, and their sizes can reach 400 microns in one dimension, but usually they are smaller in dry form. Ticks have limbs and hair, and in the case when horizontal circular motion is used during sifting, the tick bodies are oriented horizontally, which leads to the formation of tangled tangles. Only a smaller part of the non-oriented particles or smaller particles not participating in the formation of the fragment of the coil pass into the hole, and the sieve does not clog so quickly.

The sieve used is a shaker with a Retsch AS 400 sieve with a sieving tower with holes of 400, 300 and 200 microns. The horizontal circular screening mode is used, and the screening process mainly contributes to the formation of tangles of ticks having a size of more than 200, 300 and / or 400 microns.

Material from a large sieve is divided into portions weighing 150-200 g and placed on a second sieve (medium sieve).

The sieve operates at speeds ranging from 210 to 240 rpm, and each batch is sieved for 1-2 minutes.

Fractions of particles larger than 200 and 300 microns are subjected to repeated processing 2-6 times.

The number of mite bodies captured on sieves with openings of 200, 300 and / or 400 μm is determined by microscopic examination of the fractions and the body-rich fraction is separated and subjected to additional sieving, as described below.

2.1.3. Small sieve

The tick-rich fraction extracted from the medium sieve is then screened on a small sieve (Retsch AS 200) using a 3D torsion motion. The average amount of the mite-rich fraction of the mites loaded onto the sieve is 60 g, and a tower with openings of 300, 200, 150 and 100 μm in size is used.

The speed is set at 240-60 rpm, the amplitude is set for 30 seconds and each load is sieved for 5-15 minutes. Each download is re-processed 1-6 times.

In-process controls: during the sieving process, the purity of the fractions is tested using a microscope.

A fraction of particles with a size of less than 100 μm is separated as a fraction of fecal particles, which is purified as described below.

Fractions of particles larger than 100 μm and 150 μm are separated as fractions of tick bodies.

2.2. Fecal fraction purification

The fractions rich in fecal particles from the previous stages are combined (fraction C from the pretreatment stage, a fraction of particles with a size of <80 μm from a shaker with a large sieve, and a fraction of particles with a size of <100 μm from a shaker with a small sieve).

A small sieve shaker (Retsch AS 200) with a tower with openings of 100, 80, and 50 μm was used, and the operating conditions are the same as described above for this sieve.

A fraction of particles with a size of less than 50 μm is separated as a fecal fraction.

In-process controls: during the sieving process, the purity of the fractions is tested using a microscope.

Example 3

3.0 Tick body extraction

Tick bodies are suspended using a high speed mixer (20 min). The extraction is carried out in a buffer containing 0.012 M NaHCO ₃ , 0.154 M NaCl at pH 7.5 for 3 hours at a temperature of 8 ° C. The ratio of tick bodies to buffer is 1 + 10 (w / w).

3.0.1. Branch

The remaining insoluble source material is separated from the extract solution by centrifugation.

After centrifugation, the resulting centrifugate is drained from the solid fraction and clarified by filtration in a filtrate solution. After filtering the allergen extract, the filter is washed with extraction buffer and combined with the extract to increase the yield.

3.0.2 Ultrafiltration

Ultrafiltration is carried out in three stages: the concentration stage, the diafiltration stage and the stage of bringing to the desired dry matter content. First, the filtrate solution is concentrated by removal of water and other small molecules by tangential flow filtration. Second, small molecules and salts are removed from the solution of the allergen extract by diafiltration against purified water. Third, the concentration of dry matter (CB) of the allergen extract is adjusted by further concentration or by adding purified water. During ultrafiltration, temperature and transmembrane pressure (TMP) are monitored and maintained within the acceptance criteria.

In-process controls:

temperature: 3-15ºС

transmembrane pressure: ≤3 bar

CB concentration: 40-60 mg / ml

3.0.3. Brightening filtering

The allergen extract solution is clarified by passing through a filter with a pore size of 0.2 μm.

3.0.4. Stabilization

After filtration, the extract is sprayed in the form of freely falling drops into a container filled with liquid nitrogen, in which the drops immediately freeze. Then, liquid nitrogen is evaporated. During the stabilization process, the feed stream (product stream) is controlled so that it is within the acceptance criteria.

In-process control: product flow: 8-12 ml / min / spray hole.

3.1. Fecal particles

Fecal mite particles are suspended using a high shear mixer (20 min). The extraction is carried out in a buffer containing 0.012 M NaHCO ₃ , 0.154 M NaCl at pH 7.5 for 1 h at a temperature of 8 ° C. The ratio of tick bodies to buffer is 1 + 10 (w / w).

3.1.1. Branch

After extraction, the remaining insoluble source material is separated from the allergen solution by a diafiltration step. Diafiltration is carried out using a vibrating system for membrane filtration (TFF method). The allergen extract passes through the membrane like a permeate, while the insoluble source material will remain on the membrane. During diafiltration, a predetermined volume of purified water is constantly added to the recycle solution of the extract at the same rate as the allergen (permeate) extract is collected on the other side of the membrane.

In-process control:

Permeate flow: 18-22 l / m ² / h.

Temperature: 4-12 ° C.

3.1.2. Ultrafiltration

Ultrafiltration is carried out in three stages: the concentration stage, the diafiltration stage and the stage of bringing to the desired dry matter content. First, the filtrate solution is concentrated by removal of water and other small molecules by tangential flow filtration. Second, small molecules and salts are removed from the solution of the allergen extract by diafiltration against purified water. Third, the concentration of dry matter (CB) of the allergen solution is adjusted by additional concentration or the addition of purified water. During ultrafiltration, temperature and transmembrane pressure (TMP) are monitored as in-process controls and maintained within the acceptance criteria.

In-process control:

temperature: 3-15 ° C

transmembrane pressure: 3 bar

CB concentration: 40-60 mg / ml.

3.1.3. Brightening filtering

The allergen extract solution is clarified by passing through a filter with a pore size of 0.2 μm.

3.1.4. Stabilization

After filtration, the extract is sprayed in the form of freely falling drops into a container filled with liquid nitrogen, in which the drops immediately freeze. Then, liquid nitrogen is evaporated. During the stabilization process, the feed stream (product stream) is controlled so that it is within the acceptance criteria.

In-process control:

product flow: 8-12 ml / min / spray hole.

Example 4

The allergen content of group 1 and group 2 is determined by MS (mass spectrometry), as described in international application WO 2007/031080 using the following standard peptides for calibration:

TNACSINGNAPAEIDLR (Dep p 1) SEQ ID NO: 1

NSWDTTWGDSGYGYFQAGNNLMMIEQYPYVVIM (Der f 1) SEQ ID NO: 2

VLVPGCHGSEPCIIHR (Dep p 2) SEQ ID NO: 3

GKPFTLEALFDANQNTK (Der f 2) SEQ ID NO: 4 and

GIEYIQQNGVVEER (Der f 1) SEQ ID NO: 5.

Example 5

5.0. Mixing Extracts

To the frozen granules of the extract of tick bodies, frozen granules of the extract of fecal particles are added to achieve the desired allergen ratio of group 1 and group 2.

Determining which batches of frozen pellets and how many frozen pellets should be used is an iterative process.

A mixture is prepared with the required ratio according to the calculation of the weight amount of allergen, which will be ensured by mixing the number of granules of at least one batch of extracts of tick bodies and at least one batch of fecal mite particles. The weight amount of the group 1 allergen is calculated as the weight amount of any batch by multiplying by the concentration of the group 1 allergen in the batch. The weight amount of group 2 allergen provided by mixing the same batches is calculated in a similar way.

Add or remove more batches or parts of batches according to the calculation described on page 27 above, until the desired ratio is achieved.

Example:

table 2 Extract (IM) Name (m _B1 , m _F2 , m _B2 , etc.) Production batch number The main allergen of group 1 [μg group 1 / ml IM] The main allergen of group 2 [mcg / ml IM] Body m _B1 IMPTE2001-B 991 1223 Fecal particles m _F2 IMPTE2001-F 2393 792

The number of extracts from each batch is calculated as equal to 140 g mf1 and 966 g mb1.

The ratio of the main allergen = total for group 1 / total for group 2 = mf1 × Cf1grp1 + mb1 × Cb1grp1 / mf1 × Cf1grp2 + mb1 × Cb1grp2 = 2393 × 140 + 991 × 966/792 × 140 + 1223 × 966 = 1.

The necessary frozen granules are then thawed and mixed, and optionally again frozen in the form of granules. Thawed mixtures can be used in the formulation process.

Example 6

Allergens of group 1 and group 2 are determined as described in example 5.

The consignment FAR1 FAR2 FAR3 FAR4 FAR5 FAR6 FAR6 FAR7 CV (In%) ^* Ratio (Der 1 / Der 2) 0.96 0.97 1.00 0.95 0.89 0.88 0.95 0.95 6 The consignment FAR1 FAR2 FAR3 FAR4 FAR5 FAR6 FAR6 FAR7 CV (In%) ^* Ratio (Der 1 / Der 2) 1,04 1.17 0.94 1.01 1.00 1,03 1.10 1.01 6.5 * CV = relative standard deviation (SD / mean) with 95% CI

Example 7

7.1. Preparation of a rapidly disintegrating pharmaceutical composition containing an extract

Composition of a tablet with an allergen extract of house dust mites before sublimation of water during lyophilization:

Table 3 Ingredient name Weight ratio of group 1 allergen to group 2 allergen Function Active ingredient Der p tick body extract
Extract of fecal mite particles Der p
Mite Body Extract Der f
Extract of fecal mite particles Der f 1: 1: 1: 1 Active ingredient Other ingredients Gelatin (fish) 15 mg Forming agent of lyophilized tablet structure Mannitol 12.7 mg Forming agent of lyophilized tablet structure Sodium hydroxide to pH 7.5 qs PH adjustment Purified water up to 250 mg Solvent Total wet weight for dosing 250 mg

Cooking pills

Gelatin, mannitol and purified water are mixed, and the resulting premix is heated to the appropriate temperature. After cooling, the pH of the solution was adjusted using sodium hydroxide solution (≈6% w / w). To the premix add the substance of the drug in the form of frozen drops. Check and, if necessary, adjust the pH of the mixture. Calculate the additional amounts of purified water needed to complete the formulation using a production recipe and transfer to a mixture solution. The solution is dispensed into preformed blister trays. After dosing, the filled blister packs are passed through a tunnel for freezing with liquid nitrogen. All frozen products are immediately placed in a freezer for storage in a frozen state before lyophilization. The compositions are lyophilized and stored in a dry chamber for storage in a controlled humidity environment until the foil is sealed. Then the lyophilized composition is sealed with foil.

Claims (23)

1. A pharmaceutical composition for treating allergies caused by house dust mites, comprising an allergen extract of Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) as an active ingredient that contains an allergen extract: mite allergens of group 1 Der f 1 and Der p 1 and allergens of ticks of group 2 Der f 2 and Der p 2 and where the allergens Der p 1, Der f 1, Der p 2 and Der f 2 are present in predetermined controlled amounts, so that the amount by weight of the group 1 allergens is Der f 1 and Der p 1, to the group 2 allergens are Der f 2 and Der p 2, is in a ratio of 1: 0.7 to 1: 1.5; and where the pharmaceutical composition is a rapidly dissolving sublingual tablet. 2. The pharmaceutical composition according to claim 1, wherein the allergen selected from Der f 1, Der f 2, Der p 1 and Der p 2, which is present in the lowest concentration, w / v, in the allergen extract, is present in the concentration above 70% of the concentration of the allergen Der f 1, Der f 2, Der p 1 or Der p 2 present in the highest concentration wt./about. 3. The pharmaceutical composition according to any one of claims 1, 2, where the specified pharmaceutical composition has a mass ratio of allergen of ticks of group 1 to group 2 in the range from 1: 0.9 to 1: 1.3. 4. The pharmaceutical composition according to any one of claims 1 to 3, including a) extract from the bodies of mites Der p, where the extract from the bodies of mites Der p is obtained from one fraction or a combined fraction (s) of tick cultures, said fraction (s) containing more than 90% v / v. mite bodies Der p, b) Der f mite bodies extract, where Der f mite bodies extract is obtained from a single fraction or a combined fraction (s) of tick cultures, said fraction containing more than 90% v / v. mite bodies Der f, c) an extract from fecal particles of Der p, where an extract from fecal particles of Der p is obtained from one fraction or a combined fraction (s) of tick cultures, said fraction (s) containing more than 90% v / v. fecal particles Der p, and d) an extract from fecal particles of Der f, where an extract from fecal particles of Der f is obtained from a single fraction or a combined fraction (s) of tick cultures, said fraction (s) containing more than 90% v / v. fecal particles Der f. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the Der p mite body extract is obtained from one Der p mite body fraction or the combined Der p mite body fractions and where the extraction of said tick body fraction (s) of mites results in an extract comprising, by weight, equal amounts of a group 1 allergen and a group 2 allergen or more of a group 2 allergen than a group 1 allergen, and a Der f tick body extract is obtained from one Der mite body fraction f or combined fractions of tick mites Der f and extracting said fraction (s) of tick mites, an extract is obtained containing equal mass amounts of group 1 allergen and group 2 allergen or more group 2 allergen than group 1 allergen by mass. 6. The pharmaceutical composition according to any one of claims 1 to 5, where extract from the bodies of ticks Der p obtained from a single fraction or combined fraction (s) of tick cultures, and the specified fraction (s) contains more than 95% vol./about bodies of ticks Der p; extract from the bodies of mites Der f obtained from a single fraction or combined fraction (s) of tick cultures, and the specified fraction (s) contains more than 95% vol./about. mite bodies Der f; an extract from the fecal particles Der p is obtained from a single fraction or a combined fraction (s) of tick cultures, wherein said fraction (s) contains more than 95% v / v. fecal particles Der p; and extract from fecal particles Der f is obtained from a single fraction or combined fraction (s) of tick cultures, said fraction (s) contains more than 95% vol./about. fecal particles Der f. 7. The pharmaceutical composition according to any one of claims 1 to 6, where the daily dose of the specified pharmaceutical composition contains from 3 to 8 μg Der p 1, from 3 to 8 μg Der p 2, from 3 to 8 μg Der f 1 and from 3 to 8 mcg Der f 2. 8. The pharmaceutical composition according to any one of claims 1 to 7, where the pharmaceutical composition is an unpressed, rapidly dissolving sublingual tablet. 9. The use of the pharmaceutical composition according to any one of claims 1 to 8 for obtaining a medical product for the treatment of allergies caused by house dust mites. 10. A medical product for treating allergies caused by home dust mites, said medical product containing a pharmaceutical composition according to any one of claims 1 to 8 as an active ingredient. 11. The use of the pharmaceutical composition according to any one of claims 1 to 8 for the treatment of allergies caused by house dust mites. 12. A method of treating allergies caused by home dust mites, said method comprising administering to a patient an effective amount of a pharmaceutical composition according to any one of claims 1 to 8.

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