DE20023785U1 - Composition useful for e.g. treating nervous system disorders, comprising botulinum neurotoxin, is free from natural complexing proteins and is not antigenic - Google Patents

Abstract

Pharmaceutical composition containing one or more botulinum neurotoxins (A) from Clostridium botulinum types A-G in which (A) is free of proteins that are naturally combined with (A) in complexes, is new. ACTIVITY : Antispasmodic; antimigraine. A patient, with torticollis spasmodicus had been treated with Botox (RTM for native neurotoxin-protein complexes) for 5 years and produced neutralizing antibodies, so no longer responded to treatment. He was treated with 145 units of uncomplexed (A), equivalent to the doses of Botox (RTM) previously given, and within 72 hr the muscles were relaxed, the head was held normally and muscular pain was alleviated. No adverse effects were observed. MECHANISM OF ACTION : Motor end plate nerve ending binder.

Description

The The present invention relates to pharmaceutical preparations which a botulinum neurotoxin of Clostridium botulinum, the neurotoxin being free of the complexing proteins that naturally occur in the complex available. The immediate consequence of this is that of the present one Invention underlying finding that the free neurotoxin in contrast to the complex in the patient to none or only one markedly reduced induction of neutralizing antibodies. The The present invention further relates to the use of botulinum neurotoxins of Clostridium botulinum for the manufacture of a medicament for Treatment of diseases of the nervous system. An other aspect The present invention relates to the use of botulinum neurotoxins of Clostridium botulinum for the cosmetic treatment.

Clostridium botulinum toxin complex type A (M _r 900,000) is used for the therapy of various dystonias for several years. Currently, two different preparations containing this complex are approved for the treatment of blepharospasm, hemifacial spasm and spasmodic torticollis: BOTOX ^® and DYSPORT ^® . The therapy of further diseases of the nervous system (eg spasticities, migraine, lumbalgia, cervical syndrome, hypersalivation) is currently being clinically tested. The preparations are also used in cosmetic indications such as hyperhidrosis and pronounced wrinkling. The other Clostridium botulinum toxin complexes (types B, C, D, E, F, G) are also suitable for these therapies. However, there is currently no approved product on the market that contains any of the B-G type toxins.

Botulinum toxin complexes are composed of a mixture of clostridial proteins. These are hemagglutinins with different molecular masses, a non-toxic non-hemagglutinating protein (M _r approx. 120,000) and a neurotoxin (M _r approx. 150,000). They form an acid-stable complex responsible for oral toxicity in food intoxications. In contrast to the pure neurotoxin, the complex resists the aggressive environment in the gastrointestinal tract and allows the enteral absorption of the neurotoxin, which reaches the target cells via the bloodstream or the lymphatic system, where it causes a blockade of the transmitter release. This leads to the paralysis of striated and smooth muscles and the drying up of various vegetative functions. Poisoned patients die from respiratory muscle insufficiency. Since the pure neurotoxin in the gastrointestinal tract is degraded and thus not enterally absorbed, it is not toxic after ingestion. Applied parenterally, the therapeutic effects of the neurotoxin and the complex do not differ because the complex breaks down into its components in the tissue, and only the neurotoxin is taken up in the target cells.

to therapeutic use is the complex to the present state the technique injected directly into dystone or spastic muscle, where the neurotoxin is released from the complex at physiological pH and the desired one pharmacological effect. Although the complex only in extremely low doses is applied (1-25 ng, depending on the indication and size of the affected Muscle), it comes after repeated injections at a considerable Number of patients to form specific, neutralizing antibodies which are also directed against the neurotoxin. Immediate consequence is that antibody-positive Patients no longer respond to the complex. But you could treated with other toxin types, none of which, however is approved for therapy. When the patient tries all toxin types and antibodies has formed against them is the further application of a botulinum toxin complex (no matter what type) no longer helpful. It is important to take into account each dose of the complex contributes to an increase in antibody titer up to the point at which another application of the complex no Makes more sense, because no effect is achieved. Until the antibody titer has dropped significantly, often pass years, so these patients over long periods not (with botulinum neurotoxin) be treated (can).

The formation of specific antibodies is favored by two factors. On the one hand, the neurotoxin, fixed in the complex, remains in the tissue for a longer period of time and can activate immune cells migrating into the tissue for antibody formation. The long residence time, however, does not lead to an increased uptake into the target cells, because poisoned target cells can no longer absorb toxin. The neurotoxin slowly dissociating from the complex is thus only immunologically effective. On the other hand, the proteins contained in the complex enhance an immune response. Hemagglutinins are lectins, proteins that are characterized by a high affinity for certain sugars. Due to their binding to sugar structures, lectins have an immunostimulating effect. It has thus been shown that the lectins concanavalin A, phytohemagglutinin and pokeweed mitogen activate the T and B lymphocytes. The hemagglutinins of the botulinum toxin complexes, which also bind to membrane-bound sugars, can therefore be described in a similar way Immunadjuvanzien act and contribute to antibody formation and thus to treatment failure.

therefore stood up for the inventors of the present invention, the task of an alternative Treatment route for to develop the above-mentioned diseases and disorders. Especially the inventors wanted to suggest a suitable active substance, with the patient who already formed neutralizing antibodies have, can be treated.

As a solution to the problem posed above, as an alternative to the two commercial preparations of botulinum toxin complex of types A, BOTOX ^® and DYSPORT ^® and as an alternative to those described in the prior art complexes of the other types (B, C, D, E , F, G), a new drug was developed which contains only pure neurotoxin (type A or B, C, D, E, F, G) and is free of hemagglutinins and other foreign proteins. Because of its lower molecular mass, it diffuses faster to the target cells it is taken into before activating immune cells attracted by hemagglutinins. In antigenicity studies, we found that the pure neurotoxin of all types - unlike commercial preparations of type A and the complexes of types B to G no or at most a very low formation of antibodies triggers. Therapeutic use of these newly developed drugs (pure neurotoxin types A, B, C, D, E, F, G), even after repeated applications to no antibody-related treatment failure. Furthermore, it was shown that the pure neurotoxins because of their immediate bioavailability continue to be suitable for the treatment of patients who after application of a botulinum toxin complex, eg. B. after treatment with BOTOX ^® or DYSPORT ^® , have developed an antibody titers against the corresponding type (so-called secondary non-responders), that are no longer accessible to further treatment with BOTOX ^® or DYSPORT ^® , since the administration of the commercial toxins no Relieving the symptoms brings more.

The provided according to the invention Medicines are suitable as a therapeutic especially in patients the one antibody titer against a botulinum toxin, especially against type A. Especially the pharmaceutical according to the invention is suitable (pure neurotoxin or mixture of several pure neurotoxins) in those patients who have an antibody titre of not greater than 50, preferably not greater than 30, more preferably not larger than 20, more preferably not more than 10, and most preferably not more than 5 mU / ml. In this case, 1 mU antibody is the one Amount of antibody, neutralizes the 10 U toxin.

on the other hand can the drug of the invention particularly advantageously be used in such persons, who have never, or not for many years, been treated with botulinum neurotoxin since their antibody titers is low or zero from the beginning. The advantage of The present invention then consists in that the titer of this Patients by treatment with the pure toxin according to the present Invention is not, or at most very negligible, increased. In other words, the therapeutic according to the invention can over long periods be administered without it losing its effect.

The Induction of antibodies in the therapy with a C. botulinum toxin is thus characterized prevents instead of the high molecular weight toxic complexes one pure neurotoxin is administered. That of the complex proteins Completely Separate neurotoxin is immediately bioavailable and can be used immediately bind the nerve endings of the motor end plates.

One Aspect of the present invention thus relates to a pharmaceutical Preparation containing at least one of the botulinum neurotoxins of Clostridium botulinum of types A, B, C, D, E, F or G (or a mixture of two or more of these neurotoxins) containing all the neurotoxins are free from the complexing proteins, naturally present in the complex.

According to one preferred embodiment it is a pharmaceutical preparation which is characterized is characterized in that the neurotoxin or the mixture of neurotoxins in the patient to no or, compared to the complexes, only leads to a reduced induction of neutralizing antibodies.

A further preferred embodiment provides a pharmaceutical preparation which can be used as neurotoxin or as a mixture of neurotoxins a natural neurotoxin or a Mixture of natural Contains neurotoxins.

Another preferred embodiment provides a pharmaceutical preparation which is considered new Rotoxin or as a mixture of neurotoxins a recombinant neurotoxin or a mixture of recombinant neurotoxins.

A furthermore preferred embodiment the pharmaceutical of the invention Preparation provides a preparation containing the neurotoxin as neurotoxin of Clostridium botulinum type A or B or as a mixture of neurotoxins a mixture of the neurotoxins of Clostridium botulinum type A and B has.

One Another aspect of the present invention relates to the Use of the botulinum neurotoxins of Clostridium botulinum der Types A, B, C, D, E, F or G or a mixture of two or more these neurotoxins for the manufacture of a medicament for treatment of diseases of the nervous system or of dystonia. At the diseases of the nervous system or the dystonia is according to one preferred embodiment to torticollis spasmodicus and blepharospasm, to spasticities like equinus, hämifasciale Spasms, Migraines, Lumbago, cervical syndrome or hypersalivation.

In turn Another aspect of the present invention relates to use the botulinum neurotoxins of Clostridium botulinum type A, B, C, D, E, F or G or a mixture of two or more of these Neurotoxins for the cosmetic treatment, being a cosmetic treatment for Treatment of hyperhidrosis and wrinkling, especially in the facial area, is particularly preferred.

All Particularly preferred according to the present invention is the Use of one of the neurotoxins alone or in admixture for preparation a drug for the treatment of the above-mentioned nerve diseases in such persons (preferably human, but also animal), the already neutralizing antibodies against a botulinum neurotoxin complex, especially against the Complex of Clostridium botulinum type A or B, or against several Complexes, in particular against the complexes of Clostridium botulinum Type A and B, have (so-called secondary non-responders).

The Neurotoxins, their mixtures or the pharmaceutical according to the invention Preparations can as watery Solution, especially as aqueous Solution for injection, but also be present as lyophilized products.

The Known pure neurotoxins of types A-G were prepared according to the protocols contained in the publications listed in the bibliography are included. Exemplary is the purification of two neurotoxins (Type A and B) in the following examples.

Example 1: Isolation of the pure neurotoxin

The pure neurotoxin from Clostridium botulinum type A is obtained by a method similar to the method of DasGupta & Sathyamoorthy. Clostridium botulinum type A is cultured in a 20 L fermentor in a medium consisting of 2% proteose peptone, 1% yeast extract, 1% glucose and 0.05% sodium thioglycolate. After 72 hours of growth, the toxin is precipitated by the addition of 3N H ₂ SO ₄ (final pH = 3.5). The precipitated and centrifuged biomass is extracted with 0.2 M sodium phosphate buffer pH 6.0.

After separation of the nucleic acids by precipitation with protamine sulfate, the toxin is precipitated by adding ammonium sulfate. The solubilized and dialyzed against 50 mM sodium phosphate pH 6.0 precipitate is bound on a DEAE Sephadex column at the same pH and replaced with 150 mM NaCl. Subsequently, a chromatography on a QAE-Sephadex column, which was equilibrated with a 50 mM Tris / HCl buffer pH 7.9. The toxin is eluted via a NaCl gradient. In the last step, the toxin is chromatographed over SP-Sephadex at pH 7.0. The bound toxin is thereby removed from the column by means of a NaCl gradient (0-300 mM). The purified toxin is analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and has a purity of 95 ± 5%. The biological activity is determined ₅₀ assay in mouse LD: LD ₅₀ unit correspond to 4.8 pg protein.

Example 2: Preparation a botulinum neurotoxin-containing finished medicinal product

The purified neurotoxin of Example 1 is made into a solution containing 200 mouse LD ₅₀ units, 10 mg sucrose and 2 mg human serum albumin per ml. 0.5 ml of the solution are filled into vials and freeze-dried. The lyophilisates are reconstituted with physiological saline and the biological activity is determined. The vials contain 100 ± 30 LD ₅₀ units.

Example 3: Isolation of pure neurotoxin B

Clostridium botulinum type B is in the same medium and under the same conditions cultivated as type A and worked up to Ammoniumsulfatfällung. Then done again a DEAE Sephadex chromatography at pH 6.0. With 150 mM NaCl from the column eluted fractions are pooled and against sodium phosphate pH 7.0 dialyzed followed by chromatography over QAE-Sephadex followed. The toxin-containing fractions are further purified by DEAE Sephadex chromatography at pH 8.5 (50 mM Tris / HCl pH 8.5).

Finally, the high purity botulinum toxin type B is obtained by chromatography on hydroxyapatite equilibrated with 10 mM Na phosphate pH 8.0. The adhered homogenous toxin is eluted with 80 mM Na phosphate, pH 8.0 followed by the biological activity in the LD ₅₀ assay determined (2-4 X 10 ⁷ LD ₅₀ units / mg protein).

Example 4: Detection of antibodies

20 rabbits were 25 U BOTOX ^® over a period of 12 weeks at an interval of 14 days (5 injections) intracutaneously. Serum was collected after 3 weeks and then every 14 days.

Antibodies against Clostridium botulinum neurotoxin A were obtained with an enzyme immunoassay detected by the homogeneous neurotoxin on a microtiter plate was immobilized. To the neurotoxin binding antibodies were quantified by a second, enzyme-labeled antibody.

The Result is shown in Table 1. Already 5 weeks after the first application antibodies could be detected in 5 rabbits. To For 11 weeks, sera contained 17 rabbits, ie 85% of those used Animals, antibodies against the neurotoxin In the biological activity test it was shown that 12 of the 17 sera neutralizing antibodies contained (Table 2).

Table 1 Determination of serum samples (diluted 1: 100) from rabbits treated with BOTOX ^® with an enzyme immunoassay. OD _490nm > 0.1 are specified. All OD values are corrected with the OD values of the preimmune sera (OD ca. 0.150).

• * Values were not corrected because no preimmune serum was present "-" means Optical Density (OD ₄₉₀ ) <0.1

Tab. 2 neutralization by sera from rabbits immunized with BOTOX ^® (week 11 after the first immunization) in the mouse hemidiaphragm assay (detection limit: 0.35 mU / ml antibody)

• n. d. = not determined

Example 5: Antigenicity test with market product and pure neurotoxin

After it was shown that the complex of neurotoxin and hemagglutinins and the non-toxic, non-hemagglutinating protein elicits the formation of neutralizing antibodies, the immunogenic effect of the pure neurotoxin (type A) was tested. For this 8 rabbits were treated with the toxin complex and 12 rabbits with the pure toxin. Following the procedure described above (see Example 1), 25 U of the respective preparation were administered intracutaneously. The amount of neurotoxin, measured by weight, was the same in both preparations (200 pg / dose) as detected in an ELISA. BOTOX ^® additionally contained complex proteins (about 800 pg / dose).

Four of the eight animals treated with BOTOX ^® showed an antibody titre in the ELISA, while in the 12 animals treated with pure neurotoxin no antibodies to the pure neurotoxin could be detected. The result was confirmed in the biological activity test. All four rabbit sera contained neutralizing antibody titers that prevented toxin activity (Table 3).

Tab. 3 neutralization by sera (diluted 1: 3) of rabbits treated with BOTOX ^® (week 11 after the first immunization) in the mouse hemidiaphragm assay (detection limit: 1 mU / ml antibody)

Example 6: (Comparative Example)

In this experiment, the antibody formation by BOTOX ^® was compared with that of DYSPORT. For this purpose, ten rabbits were either treated with BOTOX ^® (Group 1) with DYSPORT ^® (group 2) or with the pure neurotoxin (group 3) after-described scheme, respectively.

While in Group 1 and 2 more than 50% of the animals have a neutralizing antibody titer were formed, the sera from the animals of group 3 were free of antibodies.

Example 7: Clinical test

One patient (age 45 years) treated with BOTOX ^® for a period of 5 years for spasmodic torticollis had developed an antibody titer of 3 mU / ml serum. Neither BOTOX ^® nor DYSPORT ^® were therapeutically effective in this patient. A trial of pure botulinum neurotoxin at a dose of 145 U, which was equivalent to the last dose of BOTOX ^® injected, resulted in muscle relaxation, normalization of head posture, and disappearance of muscle pain within 72 hours. Undesirable effects did not occur.

Example 8: Clinical test

One patient (age 52 years) was treated with BOTOX ^® for 3 years for cerebral palsy. He had developed an antibody titer of 1 mU / ml serum, therefore the therapy had to be stopped. The injection of 200 U pure neurotoxin enabled a successful therapy.

literature

• The Gupta, B.R. & Sathyamoorthy, V. (1984), Purification and Amino Acid Composition of Type A Botulinum Neurotoxin; Toxicon 22 (3), p. 415-424
• De Jongh, K.S., Schwartzkoff, C.L. & Howden, M.E.H. (1989), Clostridium botulinum Type D Neurotoxin Purification and Detection; Toxicon 27 (2), p. 221-228
• Schmidt, J.J. & Siegel, L. S. (1986), Purification of Type E Botulinum Neurotoxin by qHigh-Performance Ion Exchange Chromatography; Analyte. Biochemistry 156, p. 213-219
• Nukina, M., Mochida, Y., Sakaguchi, S. & Sakaguchi, G. (1988), Purification of Clostridium botulinum Type G progenitor toxin; Zbl. Bact. Hyg. A 268, p. 220-227
• Terajima, J., Syuto, B., Ochanda, J.O. & Kubo, S. (1985), Purification and Characterization of Neurotoxin Produced by Clostridium botulinum Type C 6813; Infection and Immunity 48 (2), p. 312-317
• Wadsworth, J.D.F., Desai, M., Tranter, H.S. et al. (1990) Botulinum type F neurotoxin: Largescale Purification and Characterization of its binding to rat cerebrocortical synaptosomes; Biochem. J. 268, p. 123-128

Claims (8)

Use of the botulinum neurotoxins of Clostridium botulinum of types A, B, C, D, E, F, or G, or of a mixture of two or more of these neurotoxins, characterized in that the neurotoxin or mixture of neurotoxins is free from the complexing proteins that naturally form complexes with the botulinum neurotoxins, for the manufacture of a pharmaceutical for the treatment of dystonia or nervous system disorders in animal or human patients have already formed neutralizing antibodies against botulinum neurotoxin complexes. Use of botulinum neurotoxins from Clostridium botulinum type A, B, C, D, E, F, or G, or a mixture of two or more of these neurotoxins, characterized in that the neurotoxin or the mixture of neurotoxins is free from the Complexing proteins that naturally interact with the complex Botulinum neorotoxins, for the cosmetic treatment of animal or human patient. Use according to claim 2, characterized that the animal or human patient is already neutralizing antibody against botulinum neurotoxin complexes. Use according to claim 1 or 2, characterized that the animal or human patient is already neutralizing antibody against a complex of Clostridium botulinum type A or B, or have a complex of Clostridium botulinum type A and B. Use according to claim 2 to 4, characterized that the cosmetic treatment for the treatment of hyperhidrosis he follows. Use according to one of claims 2 to 4, characterized that the cosmetic treatment for the treatment of wrinkling he follows. Use according to claim 6, characterized that the cosmetic treatment for the treatment of wrinkling done in the facial area. Use according to claim 1, characterized that it is the diseases of the nervous system or the dystonia to torticollis spasmodicus, blepharospasm, spasticities like equinus, hämifasciale Spasms, Migraines, Lumbar, cervical syndrome or hypersalivation.