RU2563983C2 - Analysis for quantitative determination of clostridial neurotoxin - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry, namely to a method of determining an unknown concentration of a clostridial neurotoxin in the first sample with respect to a known concentration of the clostridial neurotoxin in the second sample and to a method for determining the relative activity of the clostridial neurotoxin in the first sample relative to the activity of the clostridial neurotoxin in the second sample, where the method includes the following stages: (a) contact of a cell culture with the said second sample, containing the clostridial neurotoxin in the known concentration; (c) measurement of the second effect, caused in the said cell culture by the said clostridial neurotoxin in the known concentration; (d) repetition of stages (a)-(c) with different concentrations of the said clostridial neurotoxin; (e) registration of the measured second effect of stage (d) depending on the concentration of the clostridial neurotoxin with the registration, in such a way, of the second set of data; (f) contact of the cell culture with the said first sample, containing the said clostridial neurotoxin in the unknown concentration; (h) measurement of the first effect, caused in the said cell culture by the said clostridial neurotoxin in the unknown concentration; (k) determination of the concentration of the clostridial neurotoxin, at which the said first and the said second effects are identical; and (l) comparison of the concentration of the clostridial toxin, determined in (k), with the said unknown concentration of the clostridial neurotoxin; where before the said measurement at stage (c) and stage (h) and after contact at stage (a) and stage (f) the said cell structure is subjected for 0.5 to 100 h to contact with a water medium, which does not contain the clostrdial neurotoxin, and where stage (c) is performed in case the said second sample is missing, and stage (h) is performed if the said first sample is missing. Application of the method for the determination of the unknown concentration of the clostridial neurotoxin in the first sample relative to the known concentration of the clostridial neurotoxin in the second sample is described.

EFFECT: method makes it possible to increase the accuracy of determining an unknown concentration of the clostridial neurotoxin and relative activity of the clostridial neurotoxin in a sample.

8 cl, 1 dwg, 1 ex

Description

FIELD OF THE INVENTION

The present invention relates to an ex vivo method for determining an unknown concentration of clostridial neurotoxin in a sample in relation to a known concentration of clostridial toxin in a standard sample. The method may include electrical stimulation of muscle tissue that has been in contact with said samples and comparing the corresponding effects induced in said muscle tissue, thereby determining said unknown concentration. The method can also be used to assess the relative activity of clostridial neurotoxin in a sample relative to a standard sample.

State of the art

In recent years, botulinum neurotoxins have become the standard treatment for focal dystonia and spasmodic symptoms. Medicines are available from companies such as Ipsen Ltd. (Dysport ^® ) or Allergan Inc. (Botox ^® ). A highly purified neurotoxin that does not contain other clostridial proteins, for example, is produced by Merz Pharmaceuticals (Xeomin ^® ). Another drug was registered by Solstice Neurosciences, Inc (Myobloc ^® ). Another drug has been registered by Mentor Corporation (PurTox ^® ). The listed drugs either differ in the type of botulinum toxin used, or in biological effectiveness or activity.

Treatment of patients usually involves the injection of neurotoxin into the affected muscle tissue with the introduction of funds near the neuromuscular end plate, that is, next to the cell receptor, which mediates its absorption by the nerve cell that controls the specified affected muscle. Various degrees of neurotoxin spread were observed. It is believed that such distribution correlates with the amounts administered, and in particular with the administered neurotoxin preparation. As a result of the spread, systemic side effects can be observed due to inhibition of acetylcholine secretion in closely located muscle tissue. Cases of unintended paralysis of normal muscles can be largely avoided by reducing the administered doses to a therapeutically relevant level. Exceeding dosing can also be a problem with the patient’s immune system, since administration of a neurotoxin can cause the formation of neutralizing antibodies. If this happens, the toxin will be inactivated with the loss of ability to reduce involuntary muscle contractions.

Mismatch of dose equivalents or differences in the determined activity of drugs, such as commercial products or batches produced during the manufacturing process, pose an increased risk for patients associated with possible side effects and the development of immunity. Thus, reliable (that is, without significant discrepancy) and the most accurate determination of the concentration of clostridial neurotoxin contained in these commercial products or batches of products is critical in bringing the concentration of toxin to a confirmed effective dose for the benefit of the patient. It can also serve as an incentive for manufacturers to propose dosage forms that ensure optimal use of biological activity for various therapeutic purposes.

EP 1597584 B1 proposes an ex vivo method for determining the amount of a substance blocking presynaptic neuromuscular transmission in a sample, such as a sample containing botulinum neurotoxin. The method includes electrical stimulation of muscle tissue, preferably the costal muscle of the mouse, in the presence of a sample containing a substance that blocks presynaptic neuromuscular transmission, and comparing the effect caused by the sample with the effect caused by a standard substance, thus determining the amount of substance that blocks presynaptic neuromuscular transmission, in the sample.

GB 2416849 A and GB 2398636 A provide an ex vivo method for determining the amount of a substance that blocks presynaptic neuromuscular transmission in a sample, such as a sample containing botulinum neurotoxin. The method includes electrical stimulation of smooth muscle tissue, preferably of the costal muscle of a mouse or rat, in the presence of a sample containing a substance that blocks presynaptic neuromuscular transmission, and comparing the effect caused by the sample with that caused by a standard substance, thus determining the amount of substance blocking presynaptic neuromuscular transmission, in the sample.

US 2003/0032891 A1 proposes an in vivo method for measuring the activity of a substance such as a clostridial toxin, wherein said substance is administered to a mammal, the mammal is stimulated and the reflex response of the ear of said mammal to said stimulation is monitored.

EP 2015065 A1 proposes a method for quantifying the effectiveness of a neurotoxin, such as a Clostridium neurotoxin, where the toxin is introduced into the hind leg of a non-human mammal, an electrical stimulus is applied to the specified non-human mammal, the contraction of said hind leg is measured and compared to the contraction of another hind legs.

In Pearce et al., Toxicon, Vol. 35, No. 9, pp. 1373-1412, 1997, describes the suitability of the rat / mouse phrenic diaphragmatic nerve hemidiaphragm for binding of botulinum neurotoxin.

Wohlfahrt et al., Naunyn-Schmiedeberg's Arch Pharmacol 355, 335-340 (1997) compare the efficacy of two commercial botulinum toxin formulations using dose-response curves using a mouse diaphragm.

In an article by Chang et al., Naunyn-Schmiedeberg's Arch. Pharmacol 282, 129-142 (1974), the presynaptic effects of botulinum toxin type A and β-bungarotoxin on isolated neuromuscular preparations, such as mouse and rat diaphragms, are compared.

In an article by Sheridan et al., J. Appl. Toxicol. 19, S29-S33 (1999) describes the determination of the effectiveness of botulinum antagonists based on classical bioassays of toxin concentration.

In an article by James et al., Am. J. Physiol. Gastrointest. Liver Physiol. 285, G291-G297 (2003) describe the inhibitory effects of botulinum toxin on pyloric and antrum smooth muscles.

In an article by Goschel et al., Exp. Neurol., Vol. 147, 1, 1997, concentration-effect curves are described to determine the relative activity of botulinum toxin in a sample relative to the activity of a sample containing a known amount of toxin. Various botulinum toxin preparations were tested on mouse hemidiaphragms.

The above methods of quantitative analysis from the prior art, however, have insufficient accuracy required for certification by regulatory authorities. Thus, the methods disclosed in the above sources cannot be applied for normative purposes, and instead, it is still necessary to perform an outdated analysis on mice with their killing.

OBJECTS OF THE INVENTION

One objective of the invention is to improve the methods of the prior art and to develop a reliable and accurate method for determining the activity or, accordingly, the concentration of clostridial neurotoxin in a sample giving the indicated activity, which could also be used for control purposes. Such an improved method could also serve to satisfy the high need for a safe and effective administration.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for measuring the effect caused by clostridial neurotoxin in muscle tissue, comprising:

(a) contacting muscle tissue with a sample comprising said clostridial neurotoxin;

(c) measuring said effect induced in said muscle tissue by said clostridial neurotoxin;

where step (c) is performed in the absence of said sample.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method after step (a) comprises step (b):

(b) electrical stimulation of said muscle tissue obtained in step (a).

In another embodiment, step (b) is performed in the absence of said sample.

In another aspect, the invention relates to a method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample, comprising:

(a) contact of muscle tissue with said second sample;

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contact of muscle tissue with said first sample;

(h) measuring the first effect caused in said muscle tissue;

(k) determining a concentration at which said first and said second effects are identical;

(l) equating the indicated concentration in (k) with the indicated unknown concentration.

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method after step (a) includes step (b), and after step (f), step (g):

(b) electrical stimulation of said muscle tissue obtained in step (a);

(g) electrical stimulation of said muscle tissue obtained in step (f).

In another aspect, the invention relates to a method for determining the relative activity of clostridial neurotoxin in a first sample with respect to the activity of clostridial neurotoxin in a second sample, comprising:

(a) contact of muscle tissue with said second sample;

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contact of muscle tissue with said first sample;

(h) measuring the first effect caused in said muscle tissue;

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method after step (a) includes step (b), and after step (f), step (g):

(b) electrical stimulation of said muscle tissue obtained in step (a);

(g) electrical stimulation of said muscle tissue obtained in step (f).

In one embodiment, the method further includes steps (m) and (n):

(m) selecting said various concentrations from the concentration range that best fits the first and second data set;

(n) determining the indicated best fit using a statistical criterion including the following sub-steps (α) - (δ):

(α) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In one embodiment, the statistical criterion is an F-test or a χ ² criterion, or a t-test.

In one embodiment, the probability of a false deviation for each sub-step (a) - (δ) is ≤5 (expressed in%).

In one embodiment, the method further includes the step (ε):

(ε) calculating, based on the displacement of linearized and parallelized empirical curves relative to each other, the relative activity of the first sample with respect to the second sample.

In one embodiment of the invention, according to the methods of the second and third aspect, steps (b) or (g) are performed in the absence of a second or first sample, or steps (b) and (g) are performed in the absence of a second and first sample.

In one embodiment, in accordance with any of the methods of the three aspects of the invention, the duration of the muscle tissue exposure period to the specified clostridial neurotoxin, i.e., the period of muscle tissue contact with the sample or, accordingly, the first or second sample comprising clostridial neurotoxin, according to step (a ) until the specified sample or, respectively, the first or second sample, or, respectively, measuring the specified effect in accordance with step (c) or step (h), or step (c) and step (h) is from 1 to 60 minutes

In one embodiment, in accordance with any of the methods of the three aspects of the invention, prior to said measurement in step (c) or step (h), or step (c) and step (h), said muscle tissue is contacted with said clostridial toxin in flow from 5 to 30 minutes

In another embodiment, in accordance with any of the methods of the three aspects of the invention, the exposure period of said muscle tissue to said neurotoxin is about 15 minutes.

In one embodiment, in accordance with any of the methods of the three aspects of the invention, said muscle tissue is already subjected to electrical stimulation prior to step (a) and / or step (f).

In another embodiment, said muscle tissue has already undergone electrical stimulation during step (a) and / or step (f).

In another embodiment, said muscle tissue has already undergone electrical stimulation prior to step (a) and during step (a) and / or before step (f) and during step (f).

In one embodiment, said registration of said measured second effect is done by plotting a concentration of said second effect and said registration of said second data set is done by registering a calibration curve.

In one embodiment, said second effect is determined at least at a concentration expressed in murine LD ₅₀ units / ml of at least 10.

In another embodiment, said concentration is from 10 to 1000, or from 10 to 70, or from 15 to 60, or from 20 to 45.

In another embodiment, said concentration is from 20 to 400, or from 100 to 800.

In one embodiment, said mouse LD ₅₀ units are units Xeomin ^®.

In one embodiment, said effect, or, respectively, said first and second effect, is selected from the group consisting of time before paralysis of said muscle tissue, change in contractions of said muscle tissue, changes in contraction distance of said muscle tissue, changes in contraction strength of said muscle tissue , changes in the potential of the end plate or miniature potential of the end plate of the specified muscle tissue.

In one embodiment, said effect, or, respectively, said first and second effect, is the time until paralysis occurs.

In one embodiment, said muscle tissue is selected from intercostal muscle, hind limb muscle and long extensor muscle of the hind limb finger, plantar muscles of the hind paw, diaphragmatic nerve-hemidiaphragm, long lifting muscle of the ear, neuromuscular connection of a frog, biceps of the chicken neck, costal muscles, brain tissue or electrical organ of stingray.

In one embodiment, said diaphragmatic nerve hemidiaphragm is rat or murine.

In one embodiment, said clostridial neurotoxin is a botulinum toxin.

In another embodiment, a serotype of said botulinum neurotoxin is selected from the group consisting of A, B, C, D, E, F, and G; or botulinum neurotoxin is a chemically or genetically modified derivative of botulinum neurotoxin, a serotype of which is selected from the group consisting of A, B, C, D, E, F and G.

In one embodiment, the neurotoxin does not contain complexing proteins.

In another embodiment, said neurotoxin is serotype A or B.

In one embodiment, said electrical stimulation is carried out in a buffer comprising an antifoam agent.

In one embodiment, said antifoam agent is selected from silicon-containing compounds.

In one embodiment, said buffer is flushed with oxygen.

In another aspect, the invention relates to a computer program product comprising a computer program comprising software for implementing the method of the invention.

In another aspect, the invention relates to a kit comprising:

(A) a device for stimulating muscle tissue that has been exposed to clostridial neurotoxin to select an effect caused by said neurotoxin in said muscle tissue;

- a device for measuring and recording the specified effect; and

(B) a computer program product comprising a computer program including software for implementing the method according to the invention.

In another aspect, the invention relates to the use of muscle tissue in any of the methods of the invention.

In another aspect, the invention relates to the use of the method of the invention according to any of the three aspects of the invention for controlling the activity of a sample comprising clostridial neurotoxin.

In one embodiment, the sample is a stored sample.

In one embodiment, the sample is a lyophilized sample or a reconstituted sample.

In one aspect, the invention relates to the use of the method according to the first aspect of the invention for determining an unknown concentration of clostridial neurotoxin in a first sample relative to a known concentration of clostridial neurotoxin in a second sample; or to determine the relative activity of clostridial neurotoxin in the first sample relative to the activity of clostridial neurotoxin in the second sample, for example, during quality control during the production of clostridial neurotoxin.

DETAILED DESCRIPTION OF THE INVENTION

It was found that the variability observed in relation to methods of quantitative analysis of the prior art, can be greatly reduced to a small extent using the methods disclosed in this application.

According to a first aspect, the invention relates to a method for measuring an effect caused in a muscle tissue by a clostridial neurotoxin, comprising:

(a) contacting muscle tissue with a sample comprising said clostridial neurotoxin;

(c) measuring the effect caused in said muscle tissue by said neurotoxin;

where step (c) is performed in the absence of said sample.

The term “contact of muscle tissue with said sample (which may be a first or second sample according to methods in accordance with other aspects of the invention)” means that at least a portion of said neurotoxin of said sample is taken by said muscle tissue during said contact, i.e. at least a portion of the neurotoxin contained in said sample binds to corresponding receptors contained in said muscle tissue.

The term “absence of sample” means that the measurement of the effect in step (c) is performed in an environment, usually in a suitable buffer that contains 10% by weight or less, for example, does not contain a sample or, in other words, a neurotoxin sample.

In one embodiment, said muscle tissue is not in constant contact with a sample (which may be a first or second sample according to methods in accordance with other aspects of the invention), including clostridial neurotoxin, but only temporary.

This means that after a predetermined period of exposure of said muscle tissue to a neurotoxin, that is, contact in step (a) to obtain a response of said muscle tissue to an effect, a corresponding measurement of the effect (or, accordingly, the first or second effect according to methods in accordance with other aspects of the invention ), where, for example, said muscle tissue is subjected to electrical stimulation, performed in the absence of said sample (which may be said first or said second sample according to the methods in in accordance with other aspects of the invention) using the methods described below.

In one embodiment, prior to said measurement, said muscle tissue, for example, is removed from an organ chamber containing said sample and transferred to an organ chamber containing components without neurotoxin, as described below. Then conduct electrical stimulation and measure the magnitude of the effect (which may be the first or second effect when the sample is the first or second sample). This means that electrical stimulation and a reaction to said stimulation are performed with muscle tissue containing the resulting neurotoxin.

In another embodiment, components containing a neurotoxin, i.e., a sample (which may be a first or second sample), are replaced with components containing no neurotoxin. After replacement, an effect magnitude is measured (which may be the first or second effect when the sample is the first or second sample).

The term "clostridial neurotoxin (or clostridial toxin)" encompasses complexes of clostridial toxin, as well as high purity neurotoxin, that is, a neurotoxin preparation that does not contain any other clostridial proteins.

In one embodiment, said clostridial neurotoxin is a botulinum neurotoxin.

In another embodiment, a serotype of said botulinum neurotoxin is selected from the group consisting of A, B, C, D, E, F, and G.

The term “botulinum toxin complex” encompasses botulinum toxin bound to at least another non-toxic protein. Obviously, the term botulinum toxin complex used in this application includes 450 kDa and 900 kDa botulinum toxin complex, which, for example, can be obtained from cultures of C. botulinum . Such preparations based on the type A botulinum toxin complex are commercially available, for example, Ipsen Ltd. (Dysport ^® ) or Allergan Inc. (Botox ^® ). Another type B botulinum complex drug is manufactured by Solstice Neurosciences, Inc. (Myobloc ^® ). A highly purified type A neurotoxin that does not contain other clostridial proteins is available from Merz Pharmaceuticals (Xeomin ^® ). This is the drug of choice, designed to improve some forms of focal dystonia.

In another embodiment, said botulinum neurotoxin is a chemically or genetically modified derivative of a serotype selected from the group consisting of A, B, C, D, E, F and G.

A chemically modified derivative of said neurotoxin may be a derivative that is modified by pyruvation, phosphorylation, sulfation, lipidation and / or glycosylation.

A genetically modified derivative of said neurotoxin is one that has been modified by deletion, addition, or substitution of one or more amino acids contained in the proteins of said serotype.

Such a modified toxin is preferably biologically active.

A biologically active toxin is a toxin that is capable of being absorbed by the cell and produces proteolytic cleavage of one or more polypeptides that are part of the SNARE complex.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method further includes:

(b) electrical stimulation of said muscle tissue obtained in step (a).

In one embodiment, step (b) is performed in the absence of said sample.

It was unexpectedly discovered that electrical stimulation and measurement of the indicated effect in the absence of the indicated sample, after the contact of the specified muscle tissue with neurotoxin, biases the corresponding dose-response curves of the effect so that the sensitivity of the method according to the invention increases significantly. Sensitivity is especially increased at low concentrations expressed in murine LD ₅₀ units / ml of the specified clostridial neurotoxin in the specified sample.

For example, if the time before the onset of paralysis is set as an effect or, accordingly, the reaction, the indicated time before the onset of paralysis is increased compared to the method in which the indicated effect is measured in the presence of a sample. This leads to an advantageous increase in the sensitivity of the method, which is especially used in the field of lower concentrations of neurotoxin. If the activity is determined at a lower concentration, neurotoxins can usually show the greatest difference, while at fairly high concentrations of activity converge to each other.

Such an increase in sensitivity allows a more accurate and more reliable analysis of the corresponding dose-response curves of the effect. This in turn can significantly reduce the number of laboratory animals, for example, mice, which otherwise would have to be killed in order to perform any of the methods according to the invention. Thus, this embodiment of the invention is progressive not only within the technical aspects, but also within the ethical aspects.

The term "sensitivity" is used in the present description in the standard meaning in which it is usually used in physiology, that is, sensitivity determines the ability of muscle tissue to respond to external stimuli. In this case, external stimuli are produced by the contact of muscle tissue with clostridial neurotoxin. It is within the scope of the invention to select a certain concentration range, for example, a concentration range at a relatively low concentration of clostridial neurotoxin, where the indicated sensitivity increases, that is, the reaction can be determined that under other conditions it is either impossible or, accordingly, can be determined only within the limits of an unacceptable deviation .

According to a second aspect, the invention relates to a method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample, comprising:

(a) contact of muscle tissue with said second sample;

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contact of muscle tissue with said first sample;

(h) measuring the first effect caused in said muscle tissue;

(k) determining a concentration at which said first and said second effects are identical;

(l) equating the indicated concentration in (k) with the indicated unknown concentration.

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method after step (a) includes step (b), or step (b) after step (a) and step (g) after step (f):

(b) electrical stimulation of said muscle tissue obtained in step (a);

(g) electrical stimulation of said muscle tissue obtained in step (f).

In another embodiment, steps (b) or (g) are performed in the absence of a second or first sample, or steps (b) and (g) are performed in the absence of a second and first sample.

Thus, in one embodiment, the determination of the second and / or first effect is performed in the absence of said second and / or first sample.

In another embodiment, electrical stimulation of said muscle tissue is performed in the absence of said second and / or first sample. This means that after step (a) and before step (b), and / or after step (f) and before step (g), said muscle tissue is separated from the second and / or first sample, as described above.

The term "determination of the concentration at which said first and said second effects are identical" (steps (k) and (l)) means that said first and second effect are qualitatively and quantitatively identical, that is, the induced effect is, for example, the time before paralysis , and that these effects have the same measured value.

In one embodiment, in order to obtain results that can be reliably compared, the exposure time of muscle tissue to the neurotoxin contained in the second or, respectively, first sample should be comparable.

In one embodiment, said exposure periods are identical.

In one embodiment, said registration of said measured second effect in step (e) is performed by measuring said second effect at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thus recording a calibration curve.

If the effect caused by said second sample in said muscle tissue is determined on the basis of various concentrations expressed in murine LD ₅₀ units / ml, a calibration curve can be obtained as described above.

For example, you can determine the specified effect caused in stages from ten LD ₅₀ units / ml or from five LD ₅₀ units / ml within the selected concentration range.

Accordingly, using a second data set recorded in step (e), a calibration curve is constructed by which an unknown concentration of said clostridial neurotoxin in said first sample is determined according to steps (k) and the next step (l).

In one embodiment, a calibration curve is built and the indicated determination and equalization steps according to steps (k) to (l) are performed using graphical analysis.

The specified unknown concentration of the first sample can be determined by determining from the calibration curve the concentration at which the specified first and specified second effects have the same value, for example, the same time until paralysis occurs, and equating the specified concentration to the specified unknown concentration according to step (l )

A prerequisite for this determination is that an unknown concentration of clostridial toxin in the first sample causes an effect in muscle tissue that can be quantified using the specified calibration curve. A person skilled in the art is aware that it may be necessary to dilute or concentrate the first sample with an unknown concentration one or more times, if necessary, to obtain a range of concentrations in which comparison with the second sample can be made, that is, to obtain identical first and second effects . Then, taking into account the dilution factor or concentration, the concentration of neurotoxin initially present in the undiluted or non-concentrated first sample can be calculated.

In another embodiment, said determination and equating are not carried out by measuring at one point only one concentration in step (h) and subsequent steps (k) and (l), but by measuring at a variety of different concentrations. This is especially important given regulatory requirements.

According to another aspect of the invention, it is desirable to optimize the concentration range in which reliable comparison of said second and first samples is possible. This applies not only to the possibility of comparison regarding the biological effectiveness of the currently known and commercial preparations of clostridial neurotoxins, but also to preparations that could be developed in the future or are already under development.

In one embodiment, in order to optimize the concentration range expressed in murine LD ₅₀ units / ml, in which a reliable comparison of said second and first samples is possible, it is desirable, firstly, to determine the standard deviation of the calibration curve recorded in step (e) and / or in step (h). Using a suitable stepwise regression analysis, you can create a regression model to predict the activity of an unknown toxin sample based on the dose-response curve.

Using this method, it is possible to identify the concentration range for the first and second samples representing two different sets of data in which the correlation between the corresponding dose-response curves reaches a maximum, that is, the best fit is established.

In one embodiment, the criterion can be further adjusted by presenting the range of values of the respective data sets of the first and second sample by empirical curves according to a given regression model, respectively, as well as linearizing and parallelizing said empirical curves within a specified confidence interval.

Thus, according to a third aspect, the invention relates to a method for determining the relative activity of clostridial neurotoxin in a first sample with respect to the activity of clostridial neurotoxin in a second sample, comprising:

(a) contact of muscle tissue with said second sample;

(b) electrical stimulation of said muscle tissue obtained in step (a);

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contact of muscle tissue with said first sample;

(g) electrical stimulation of said muscle tissue obtained in step (f);

(h) measuring the first effect caused in said muscle tissue obtained in step (g);

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

In one embodiment, the method further includes steps (m) and (n):

(m) selecting the indicated various concentrations from the concentration range that best fits the first and second data set;

(n) determining the indicated best fit using a statistical criterion including the following sub-steps (α) - (δ):

(α) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In one embodiment, said muscle tissue is subjected to electrical stimulation.

In one embodiment, the method after step (a) includes step (b), and after step (f), step (g):

(b) electrical stimulation of said muscle tissue obtained in step (a);

(g) electrical stimulation of said muscle tissue obtained in step (f).

In one embodiment, steps (b) or (g) are performed in the absence of a second or first sample, or steps (b) and (g) are performed in the absence of a second and first sample.

Thus, in one embodiment, the determination of the second and / or first effect is performed in the absence of said second and / or first sample.

In another embodiment, electrical stimulation of said muscle tissue is performed in the absence of said second and / or first sample. This means that after step (a) and before step (b), and / or after step (f) and before step (g), said muscle tissue is separated from the second and / or first sample, as described above.

Statistical criteria suitable for performing the above sequence are well known, for example, likelihood ratio criteria. An example of such a likelihood ratio criterion is the well-known F-criterion. A criterion such as a χ ² criterion (a chi-square test or a χ ² distribution criterion) or a t-test can also be used. These criteria are also known in the art.

In one embodiment, the specified statistical criterion is an F-test.

Using this criterion, one can decide, within the established confidence interval, whether two random samples taken from two different populations differ significantly, taking into account their deviation. Thus, this criterion serves to verify the differences within the two statistical samples, in this case the second and first samples.

In one embodiment, the confidence interval should be wide to obtain reliable results, that is, the probability of a false bias should be relatively low.

In one embodiment, the probability of a false deviation is ≤5 (expressed in%; (or 0.05)) or, accordingly, the confidence interval is ≥95 (expressed in%; (or 0.95)).

In one embodiment, the probability of a false deviation for each sub-step (a) - (δ) is ≤5 (expressed in%).

In one embodiment, the linearization in step (γ) is performed by presenting the corresponding data sets as a direct best fit.

In one embodiment, parallelization in step (δ) is performed by determining the overall slope of the straight lines of best fit.

After step (δ), based on the displacement of the linearized and parallelized empirical curves relative to each other, the relative activity of the first sample relative to the second sample is determined.

Thus, in one embodiment, the method after step (δ) further includes step (ε):

(ε) calculating, based on the displacement of linearized and parallelized empirical curves relative to each other, the relative activity of the first sample with respect to the second sample.

In one embodiment, the term “relative activity” means that the activity of the first sample with respect to the second sample is determined at the same concentration or, correspondingly, identical concentrations, based on linearized and parallelized empirical curves.

In one embodiment, the activity of the second sample is equated to 100% and the relative activity of the first sample is expressed as%. For example, for the first sample, activity is obtained, for example, 110% or 90% with respect to the second sample. By appropriate dilution of the first sample, having 110% activity, to 100% activity, an effective concentration of clostridial neurotoxin in the first sample is obtained, which until now was not known, applying the rule of proportion. The unit of measurement is now relative activity, and the value is expressed as the unit of activity defined in relation to the activity of the reference standard (second sample).

In another embodiment, relative activity is expressed as the ratio of activity of the first and second sample.

In one embodiment, the above model is used to predict the logarithmic value of the applied dose of neurotoxin.

In another embodiment, the amount of the stimulated effect and the dose of neurotoxin in the sample are recorded on a logarithmic scale.

In one embodiment, the second effect, or, accordingly, the first effect, is measured at least at three different concentrations of clostridial neurotoxin in the second sample or, accordingly, the first sample.

In one embodiment, said registration of said data sets or, respectively, said registration of a calibration curve or, accordingly, calibration curves, is performed in the form of a semi-log graph.

In another embodiment, a double (on both axes) logarithmic plot is used.

A method for determining relative activity is described in the European Pharmacopoeia.

In one embodiment, starting from a concentration of, for example, 10 murine LDs of ₅₀ units / ml, a method for determining said relative activity is applied over the full range of the data set. Values in excess of 10 murine LD ₅₀ units / ml are then used as starting points, for example, 11, 12, 13, 14, 15, 16, 17 murine LD ₅₀ units / ml. The specified iteration is continued for as long as the applied model does not provide the desired and necessary accuracy.

In one embodiment, after determining the best fit and thus the concentration range using a statistical criterion, any first sample with an unknown concentration (relative to the effective concentration) of clostridial neurotoxin can be compared with a known concentration of said clostridial neurotoxin in a second sample within the specified concentration range identified according to the method of the invention.

In one embodiment, said registration of said measured second effect is performed by plotting the concentration of said second effect, said registration of said second data set being performed by registering a calibration curve.

The use of relative activity estimates, as well as the inclusion of a reference standard (second sample) in the analysis, gives more accurate and more reproducible estimates, which reduces the use of animals.

In one embodiment, according to any of the methods in accordance with the three aspects of the invention, before said measurement in step (c) or step (h), or step (c) and step (h), said muscle tissue is contacted with said clostridial toxin within 5 to 30 minutes

In one embodiment, according to any of the methods in accordance with the three aspects of the invention, said muscle tissue has already been electrically stimulated prior to step (a) and / or step (f).

In another embodiment, said muscle tissue has already been electrically stimulated during step (a) and / or step (f).

In another embodiment, said muscle tissue has already been subjected to electrical stimulation prior to step (a) and during step (a) and / or before step (f) and step (f).

Statistical analyzes are usually performed using a suitable computer program and a suitable computer.

In one embodiment, statistical analysis is performed using a suitable computer program including suitable software for performing statistical analysis.

Thus, in one embodiment, the invention relates to a computer program product comprising a computer program including software for implementing the method according to the invention.

In one embodiment, the second sample is selected from a commercial and registered botulinum toxin preparation. Since such products are registered and authorized as a medicine or, respectively, a medicine, they include a clearly defined amount or, accordingly, the concentration of botulinum toxin.

In another embodiment, any botulinum toxin preparation that has been manufactured under standard conditions may be used.

In one embodiment, the commercial preparations mentioned above can be used as a second sample. Thus, the second sample may be Xeomin ^® , Botox ^® , Dysport ^® , Myobloc ^® or PurTox ^® . These preparations differ in the type of botulinum toxin used, or in biological effectiveness / activity, for example, in the concentration of botulinum neurotoxin or in the type of botulinum toxin contained in them.

A mouse unit expressed in mouse LD ₅₀ is the standard unit for determining the concentration of clostridial neurotoxin contained in a sample. The LD ₅₀ value determines the lethal dose at which 50% of the mouse population dies if the specified amount is applied to the mice of the specified mouse population. A method for determining this value is known to a person skilled in the art. This method is described in the European Pharmacopoeia.

As you know, the units LD ₅₀ on the label of products based on botulinum neurotoxin may be characteristic of the product or, accordingly, for the manufacturer, and may not be equivalent due to the lack of a standard.

In one embodiment, the LD unit _50, referred to in this application are the units defined herein and Xeomin ^® label. For example, the second sample is Xeomin ^® . Therefore, units that are related to a certain activity are Xeomin ^® units. Thus, the analysis system of the present invention can be used to compare the activity of any sample, including clostridial neurotoxin, compared with Xeomin ^® . In addition, the method allows you to directly compare the first samples, including clostridial neurotoxin (in unknown concentration), in units of Xeomin ^® .

Xeomin ^® and Botox ^® show approximately comparable efficacy or activity. To obtain the same potency or activity as Xeomin ^® and Botox ^® , approximately 2.5 times the amount of Dysport ^® or, respectively, 10 times the amount of Myobloc ^{® should be used} .

In one embodiment, said commercial preparations are diluted or concentrated to predetermined concentrations of the botulinum neurotoxin contained therein, and said second effect is measured depending on various concentrations of said clostridial neurotoxin in said second sample. The indicated measured effect is plotted against the concentration of botulinum toxin, thus recording a calibration curve. Using the specified second data set or, respectively, the specified calibration curve, an unknown concentration of botulinum neurotoxin in the first example can be determined.

It was found that when the concentration of clostridial neurotoxin in the sample (which may be the first or second sample), expressed in murine LD ₅₀ units / ml and equal to at least 10, the methods according to the invention can be useful. It should be noted that all concentrations cited in the framework of this application are indicated in murine LD ₅₀ units / ml.

In one embodiment, said concentration is at least 15.

In another embodiment, said concentration is at least 20.

In another embodiment, said concentration is from 10 to 1000.

In one embodiment, the concentration is from 10 to 70.

In another embodiment, the concentration is from 15 to 60.

In yet another embodiment, the concentration is from 20 to 45.

In one embodiment, the second sample is Xeomin ^®.

In one embodiment, it was found that in that case, if the second sample is applied Xeomin ^®, most reliable results can be obtained if the second effect is determined at least at a concentration of from 10 to 70. In another embodiment, the concentration is from 15 to 60. In yet another embodiment, the concentration is from 25 to 45.

In one embodiment, it was found that in that case, if the second sample is used Botox ^®, reliable results can be obtained if the second effect is determined at least at a concentration of from 10 to 70. In another embodiment, the concentration ranges from 15 to 60. In yet another embodiment, the concentration is from 25 to 45.

If the second sample is used to determine the calibration curve according to step (e), while the second sample has a lower concentration or contains a less effective or active botulinum neurotoxin than Xeomin ^® or Botox ^® , higher concentrations of neurotoxin, i.e. higher LD ₅₀ values units / ml are required to achieve a second effect strength comparable to that produced by Xeomin ^® or Botox ^® .

In the embodiment wherein the second sample has a low concentration or activity of the botulinum neurotoxin than Xeomin ^® or Botox ^®, the second effect is determined at at least one concentration of from 20 to 400, or from 100 to 800.

In one embodiment, wherein the second sample is Dysport ^®, the second effect is determined at at least one concentration of from 20 to 400 or 25 to 300 or from 30 to 250.

In another embodiment, in which the second sample is Myobloc ^® , the second effect is determined at least at a concentration of from 100 to 800 or from 150 to 700, or from 200 to 600.

In other embodiments, the implementation of the concentration may vary from 30 to 600, or from 30 to 400, or from 30 to 200, or from 30 to 100, or from 30 to 80, or from 40 to 500, or from 40 to 400, or from 40 to 300, or from 40 to 200, or from 40 to 100, or from 40 to 90, or from 50 to 300, or from 50 to 200, or from 50 to 100, or from 60 to 100, depending on the concentration of the effectiveness or activity of the neurotoxin in the second sample compared to Xeomin ^® or Botox ^® .

In one embodiment, the LD unit ₅₀ are units Xeomin ^®.

According to a first embodiment of the invention, the effect used to determine said unknown concentration is the time until muscle paralysis occurs. Time can be measured, for example, in seconds or minutes. According to the sub-options, the time to paralysis can be determined based on the contraction distance of the muscle (paralysis is achieved as soon as the contraction distance becomes equal to 0), or by the frequency of contractions of the muscle (paralysis is achieved as soon as the contraction frequency becomes 0). The contraction distance, for example, can be measured in centimeters or millimeters.

“Time to paralysis” can be defined as the period that has elapsed before reaching half of the maximum reduction. It strictly depends on the concentration of the toxin.

According to other variants of the invention, the induced effect is a change in the frequency of contractions of muscle tissue or a change in contraction of muscle tissue, or a change in the force of contraction of muscle tissue, or a change in the potential of the end plate or miniature potential of the end plate of muscle tissue. These methods are known in the art and are disclosed, for example, in EP 1597584 B1.

In one embodiment, the effect, or, respectively, the first and second induced effect, is the time before paralysis of muscle tissue.

In fact, any muscle tissue that exhibits neuromuscular characteristics, namely, responds to electrical stimulation, can be selected for the method of the invention. By muscle tissue is meant a preparation comprising one or more muscle fibers to which a nerve cell or nerve cells are attached, or a nerve that can be electrically stimulated. Smooth and striated muscle tissue can be used.

According to the description of the present invention, muscle tissue includes the intercostal muscle, the hind limb muscle and the long extensor muscle of the hind limb finger, for example, mice and rats, the plantar muscles of the hind paw, for example, a mouse or rat, the phrenic nerve-hemidiaphragm, for example, a rat or mouse, a long raising muscle of the ear, for example, mice and rats, the neuromuscular junction of the frog, biceps muscle of the neck of chickens. Can also be used rib muscles or brain tissue, for example, mice and rats, or the electric organ of stingrays.

In addition, in one embodiment, experiments showed that the use of the mouse diaphragmatic nerve hemidiaphragm is a suitable tool for measuring clostridial toxicity. Thus, this can be used as an analysis to determine clostridial toxicity.

In one embodiment, due to the reliability of said assay on the mouse hemidiaphragm, compliance with regulatory requirements and the need for the safe and effective administration of botulinum toxin, such as serotype A or serotype B toxin, can be guaranteed.

In one embodiment, the hemidiaphragm is the hemidiaphragm of a rodent, such as a rat or mouse.

In one embodiment, the hemidiaphragm is a mouse hemidiaphragm.

The term "mouse or rat hemidiaphragm" means the diaphragmatic nerve hemidiaphragm of a rat or mouse.

In yet another embodiment, said clostridial toxin in said first sample and said clostridial toxin in said second sample are the same clostridial toxins.

In yet another embodiment, said clostridial toxin or neurotoxin in a first sample and said clostridial toxin or neurotoxin in said second sample are different from each other.

For the experimental implementation of the method, as a rule, muscle tissue with attached motor neurons is removed in an animal, such as a mouse or rat, and placed in an organ or tissue chamber containing a buffer, such as a physiological buffer, in which conditions such as the ionic composition are controlled glucose, temperature, pH and oxygen saturation to optimize tissue viability and function. Measurements of muscle contraction force after electrical excitation can be made when the muscle is attached to a force sensor, which makes it possible to directly measure the effect of the toxin in relation to neuromuscular function.

In one embodiment, the temperature in the buffer is from 35 to 39 ° C or from 36 to 38 ° C. In another embodiment, the temperature is from 36.5 to 37.5 ° C.

In yet another embodiment, the temperature is equal to or approximately equal to 37 ° C.

In one embodiment, said pH in said buffer is from 7 to 8, or from 7.2 to 7.8. In one embodiment, said pH is equal to or approximately equal to 7.5.

In one embodiment, oxygenation is carried out with a gas mixture comprising oxygen. In one embodiment, oxygenation is carried out with a mixture of carbon dioxide and oxygen. In one embodiment, a gas mixture is used consisting of 95 parts of oxygen (by volume) and 5 parts of carbon dioxide (by volume). Commercial mixtures are known as carbogen.

In order to conduct electrical stimulation in order to measure the effect or, accordingly, the second and first effect, essentially methods of the prior art can be applied.

In one embodiment, the method is carried out such that electrical stimulation in step (b) or (g) is performed at a voltage of at least equal to an overmaximum voltage. Supermaximal stress is understood as the minimum stress necessary to obtain the maximum contractile reaction of muscle tissue. As a rule, such an experiment is repeated several times, and the results are averaged to obtain a reliable result.

Electrical stimulation can be performed so that when the voltage is at least equal to the supermaximal voltage of the specified tissue, the stimulation is carried out by pulses at certain time intervals. Pulse stimulation is understood as stimulation lasting for some time, divided by certain periods during which no stimulation is carried out. This approach is described, for example, in Goschel et al., Exp. Neurol., Vol. 147, 1, 1997, Wohlfahrt et al., Naunyn-Schmiedeberg's Arch Pharmacol (1997) 355: 335-340.

In the alternative, electrical stimulation may be sequential pulse stimulation. Such a method is disclosed in EP 1597584 B1.

In one embodiment of pulsed stimulation, the duration of stimulation may vary from 10 μs to 1 ms. The duration of periods in which stimulation is not carried out can vary from 0.1 to 10 s. The supermaximum voltage can vary, for example, between 1 mV and 15 V. Muscle tissue, for example, is subjected to continuous electrical stimulation by pulses with a frequency of, for example, 1 Hz, using two electrodes.

Microelectrodes can be placed on or near neuromuscular junctions, and intracellular recording of spontaneous and induced membrane potentials can be performed. These membrane potentials are produced upon activation of ligand-regulated ion channels with acetylcholine, which in turn serve as a target for the toxin. Endplate potential analysis can be used to obtain information on the effect of the toxin on the quantitative release of acetylcholine.

In particular, suitable muscle tissue, for example, the left phrenic nerve hemidiaphragm ( nervus phrenicus ), can be excised, for example, from a male or female mouse and placed in an organ chamber. In one embodiment, said organ chamber is a chamber containing Krebs-Ringer solution or Earl's balanced salt solution (EBSS), or physiological saline. These solutions are known to those skilled in the art. Muscle tissue is then stimulated through the phrenic nerve in the presence of the first or, respectively, second sample according to known methods. The induced effects are recorded and evaluated, also using known methods, for example, methods described in the prior art.

Muscle tissue can be immersed in a buffer, such as physiological buffer. The buffer may include an energy source. The energy source may be an ATP-based energy source, for example, one or more of the following: ATP, sugar, such as glucose, and / or creatine, fatty acid, amino acid, glycogen, surfactant, and pyruvic acid.

The buffer can be saturated with oxygen, especially in the case of a longer analysis. Preferably, oxygen and glucose (or another ATP source) can be added to the organ chamber to increase the lifespan of said muscle tissue. The addition of a surfactant can be advantageous in particular to reduce the formation of bubbles, which may have an undesirable effect on the method of the invention.

In one embodiment, the surfactant is an anti-foaming agent.

The term “antifoaming agent” includes all substances that affect the surface tension of gas bubbles that are contained in a liquid.

One type of antifoaming agent reduces the surface tension of gas bubbles contained in a liquid, thus destroying gas bubbles.

However, it is also possible that antifoaming agents can increase the surface tension of gas bubbles, causing the bubbles to merge into larger bubbles that exit the liquid more easily than small bubbles.

The effect of surface tension can be measured by methods that are known to the person skilled in the art, such as measuring the contact angle and the contact angle.

Thus, an anti-foaming agent is a substance that prevents the formation of foam or destroys the already formed foam.

Standard anti-foaming agents are insoluble oils, dimethyl polysiloxanes and other silicones, alcohols, stearates and glycols.

In one embodiment, the antifoaming agent is selected from at least one silicon-containing compound.

In another embodiment, the at least one silicon-containing compound is siloxane.

The term "siloxane" includes oligosiloxanes and polysiloxanes. In one embodiment, said siloxanes are substituted with alkyl groups and / or aryl groups. Such siloxanes are known in the art. Silicon-containing compounds can be used in the form of a separate compound or in the form of a mixture of more than one silicon-containing compound.

Examples of suitable silicon compounds or, accordingly, suitable siloxanes, without limitation, are α- (trimethylsilyl) -ω-methylpoly [hydroxy (dimethylsilylene)] and polydimethyl-siloxane. Such compounds are available on the market and are used in medicines or as them, for example, under the names simethicone and dimethicone.

One skilled in the art knows that other compounds having similar activity, such as dimethicone and simethicone, can also be used in the method of the present invention.

In another aspect, the invention relates to a kit comprising an organ chamber in which stimulation of muscle tissue exposed to a Clostridial neurotoxin is performed, and where the effect of said stimulation is measured (for example, as described above), as well as a computer program product by which the statistical analysis, thus optimizing the range of concentrations in which the effect caused by a neurotoxin must be measured in order to obtain reliable results.

Thus, in one embodiment, the invention relates to a kit comprising:

(A) a device for stimulating muscle tissue that has been exposed to clostridial neurotoxin to select an effect caused by said neurotoxin in said muscle tissue;

- a device for measuring and recording the specified effect; and

(B) is a computer program product comprising a computer program including software for implementing the method according to the invention.

According to a fourth aspect, the invention also provides an improved method for determining a concentration range in which the activity of a first sample comprising clostridial neurotoxin can be determined with respect to a second sample comprising clostridial neurotoxin within a specified confidence interval or probability of false rejection.

In one embodiment, such a method for determining a concentration range in which activity of a first sample comprising clostridial neurotoxin can be determined with respect to a second sample comprising clostridial neurotoxin comprises the following steps:

(a) contact of muscle tissue with said second sample;

(b) electrical stimulation of said muscle tissue obtained in step (a);

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) recording the indicated measured second effect of step (d) as a function of concentration, thereby recording a second data set;

(f) contact of muscle tissue with said first sample;

(g) electrical stimulation of said muscle tissue obtained in step (f);

(h) measuring the first effect caused in said muscle tissue by said neurotoxin;

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where the indicated concentration is selected from the concentration range that best fits the first and second data set, and where the indicated best fit is determined using a statistical criterion that includes the following sub-steps (a) - (δ):

(a) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In said embodiment, said second and said first effect are qualitatively identical. To clarify the method, the methods described above with reference to the method according to the third aspect of the invention can be applied.

In a further aspect of the invention, the methods of the invention can preferably be used for quality control, that is, the activity of a sample comprising a clostridial neurotoxin, as compared to a reference standard, as required in the manufacturing process.

Thus, in this aspect, the invention relates to the use of the method of the invention for quality control, that is, the activity of a sample comprising clostridial neurotoxin.

In one embodiment, the activity of the sample in storage is determined. In one embodiment, the sample was stored for at least one hour or at least one day.

In one embodiment, the sample is a lyophilized sample or a reconstituted sample.

According to another aspect, the invention relates to the use of the method according to the first aspect of the invention for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample; or to determine the relative activity of clostridial neurotoxin in the first sample with respect to the activity of clostridial neurotoxin in the second sample.

According to another aspect, the invention relates to the use of muscle tissue, in particular a mouse or rat hemidiaphragm, for determining clostridial activity in any of the methods of the invention, or for determining clostridial activity using a kit according to the invention.

The following embodiments also relate to the present invention, it being understood that the embodiments described above are mutually applicable to the following methods.

Thus, the invention also relates to an ex vivo method for determining an unknown concentration of clostridial neurotoxin in a first sample in relation to a known concentration of clostridial neurotoxin in a second sample, comprising:

(a) contact of muscle tissue with said second sample;

(b) electrical stimulation of said muscle tissue obtained in step (a);

(c) measuring a second effect induced in said muscle tissue by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contact of muscle tissue with said first sample;

(g) electrical stimulation of said muscle tissue obtained in step (f);

(h) measuring the first effect caused in said muscle tissue obtained in step (g);

where the specified second effect is determined at least at one concentration expressed in mouse LD ₅₀ units / ml equal to at least 10.

In one embodiment, the concentration at which said first and said second effects are identical is identified and equated to an unknown concentration of said clostridial neurotoxin in said first sample.

Thus, in one embodiment, the method further includes steps (k) and (l):

(k) determining a concentration at which said first and said second effects are identical;

(l) equating the indicated concentration in (k) with the indicated unknown concentration.

In one embodiment, said muscle tissue has already been electrically stimulated prior to step (a) and / or step (f).

In another embodiment, said muscle tissue has already been electrically stimulated during step (a) and / or step (f).

In another embodiment, said muscle tissue has already been electrically stimulated before step (a) and during step (a) and / or before step (f) and during step (f).

Said electrical stimulation of said muscle tissue may be performed in the absence or presence of a second and / or first sample if said muscle tissue has been exposed to said clostridial neurotoxin present in said second and / or first sample.

In one embodiment, the invention relates to an ex vivo method for determining an unknown concentration of clostridial neurotoxin in a first sample in relation to a known concentration of clostridial neurotoxin in a second sample, comprising:

(i) electrically stimulating muscle tissue in the presence of said second sample and selecting a second effect caused by said second sample in said muscle tissue,

(ii) measuring said second effect in (i) at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thus recording a second data set,

(iii) electrical stimulation of said muscle tissue in the presence of said first sample,

(iv) selecting a first effect caused by said first sample in said muscle tissue,

(v) determining a concentration at which said first and said second effects are identical, and

(vi) equating said concentration in (v) with said unknown concentration,

where the specified second effect is determined at least at one concentration expressed in mouse LD ₅₀ units / ml equal to at least 10.

In one embodiment, said registration of said measured second effect in step (e) or step (ii) is performed by measuring said second effect at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect with registration, thus a calibration curve.

Thus, by means of a second data set recorded in step (e) or (ii), a calibration curve is constructed by which an unknown concentration of said clostridial neurotoxin in said first sample is determined according to steps (k) and the subsequent step (l) or, respectively , step (v) and subsequent step (vi).

In one embodiment, a calibration curve is constructed, and the indicated determination and equalization steps according to steps (k) to (l) or, respectively, step (v) and the subsequent step (vi) are carried out using graphical analysis.

In one embodiment, said concentration is at least 15 or at least 20.

In another embodiment, said concentration is from 10 to 1000.

In one embodiment, the concentration of the second sample is from 10 to 70.

In another embodiment, the concentration of the second sample is from 15 to 60.

In yet another embodiment, the concentration is from 20 to 45.

In one embodiment, the above commercial preparations may be used as the second sample. Thus, the second sample can be Xeomin ^® , Botox ^® , Dysport ^® , Myobloc ^® or PurTox ^® .

In one embodiment, the units used are units Xeomin ^®.

In one embodiment, said commercial preparations are diluted or concentrated to predetermined concentrations of the botulinum neurotoxin contained therein, and said second effect is measured depending on various concentrations of said clostridial neurotoxin in said second sample. A graph is plotted of the indicated measured effect on the concentration of botulinum toxin, thus recording a calibration curve. Using the specified second data set or, respectively, the specified calibration curve, determine the unknown concentration of botulinum neurotoxin in the first example.

In one embodiment, it was found that in that case, if the second sample is used Xeomin ^®, the most reliable results are obtained if the second effect define at least one at a concentration of from 10 to 70. In another embodiment, the concentration is from 15 to 60. In yet another embodiment, the concentration is from 25 to 45.

In one embodiment, it was found that if Botox ^{® is} used as the second sample, reliable results are obtained if the second effect is determined at least at a concentration of from 10 to 70. In another embodiment, the concentration is from 15 to 60 In yet another embodiment, the concentration is from 25 to 45.

If the second sample is used to determine the calibration curve according to step (ii) and the second sample has a lower concentration or contains a less effective or active botulinum neurotoxin than Xeomin ^® or Botox ^® , higher concentrations of neurotoxin, i.e. higher LD values ₅₀ units / ml are required to produce a second effect of such strength that is comparable to the strength of the effect caused by Xeomin ^® or Botox ^® .

In the embodiment wherein the second sample has a low concentration or activity of the botulinum neurotoxin than Xeomin ^® or Botox ^®, the second effect is determined at least at one concentration of from 20 to 400 or from 100 to 800.

In one embodiment, wherein the second sample is Dysport ^®, the second effect is determined at at least one concentration of from 20 to 400 or 25 to 300 or from 30 to 250.

In another embodiment, wherein the second sample is Myobloc ^®, the second effect is determined at at least one concentration from 100 to 800 or 150 to 700 or from 200 to 600.

In other embodiments, the concentration may range from 30 to 600, or from 30 to 400, or from 30 to 200, or from 30 to 100, or from 30 to 80, or from 40 to 500, or from 40 to 400, or from 40 to 300, or from 40 to 200, or from 40 to 100, or from 40 to 90, or from 50 to 300, or from 50 to 200, or from 50 to 100, or from 60 to 100, depending on concentration of the effectiveness or activity of neurotoxin in the second sample compared to Xeomin ^® or Botox ^® .

If the effect caused by said second sample in said muscle tissue is determined based on various concentrations expressed in murine LD ₅₀ units / ml, a calibration curve can be obtained as described above.

For example, you can determine the specified effect caused in stages, ten LD ₅₀ units / ml or five LD ₅₀ units / ml within the specified concentration ranges.

The specified unknown concentration of the first sample can be determined by determining the concentration on the basis of a calibration curve at which the specified first and specified second effect have the same value, for example, the same time until paralysis, and equating the specified concentration to the specified unknown concentration according to step ( l).

A prerequisite for this determination is that an unknown concentration of clostridial toxin in the first sample causes an effect in muscle tissue that can be quantified using the specified calibration curve. A person skilled in the art is aware that it may be necessary to dilute or concentrate a first sample having an unknown concentration, one or more times if necessary, in order to obtain a concentration range in which comparison with the second sample becomes possible, i.e., to obtain an identical first and second effects. Then, knowing the dilution or concentration factor, a calculation of the concentration of neurotoxin present initially in an undiluted or non-concentrated sample can be performed.

In one embodiment, the method is carried out such that electrical stimulation in step (b) or (g), or, respectively (i) and (iii), is carried out at a voltage of at least equal to a supramaximal voltage using methods of the prior art as described above.

In one embodiment, the muscle tissue is the diaphragm of the mouse.

Thus, a method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample comprises:

(i) electrically stimulating the mouse hemidiaphragm in the presence of said second sample and selecting a second effect caused by said second sample in said mouse hemidiaphragm,

(ii) measuring said second effect in (i) at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thereby recording a calibration curve,

(iii) electrical stimulation of said muscle tissue in the presence of said first sample,

(iv) measuring a first effect caused by said first sample in said muscle tissue,

(v) determining a concentration at which said first and said second effects are identical, and

(vi) equating said concentration in (v) with said unknown concentration,

where the specified second effect is determined at least at one concentration expressed in mouse LD ₅₀ units / ml equal to at least 10.

In one embodiment, said muscle tissue is a rat or mouse phrenic nerve hemidiaphragm, the effect is time to paralysis, and said clostridial botulinum toxin is serotype A botulinum neurotoxin.

In a specific embodiment, the method encompasses a method for determining an unknown concentration of serotype A botulinum neurotoxin in a first sample with respect to a known concentration of botulinum toxin in a second sample, wherein said method comprises:

(i) electrically stimulating the mouse hemidiaphragm in the presence of said second sample and selecting a second time before paralysis caused by said second sample in said mouse hemidiaphragm,

(ii) measuring said second effect in (i) at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thereby recording a calibration curve,

(iii) electrical stimulation of said muscle tissue in the presence of said first sample,

(iv) measuring a first effect caused by said first sample in said muscle tissue,

(v) determining a concentration at which said first and said second effects are identical, and

(vi) equating said concentration in (v) with said unknown concentration,

wherein said second time before the onset of paralysis is determined at at least one concentration, expressed in mouse LD ₅₀ units / ml, of from 10 to 70 or 15 to 60 or 20 to 45, and wherein the second sample is Xeomin ^® or Botox ^® .

In one embodiment, said concentration is in the range of 16.6 murine LD ₅₀ units / ml to 56.3 murine LD ₅₀ units / ml.

In another embodiment, said concentration ranges from 20 murine LD ₅₀ units / ml to 55 murine LD ₅₀ units / ml.

In yet another embodiment, said concentration is in the range of 25 murine LD ₅₀ units / ml to 50 murine LD ₅₀ units / ml.

In another specific embodiment, the method encompasses a method for determining an unknown concentration of serotype A botulinum toxin in a first sample with respect to a known concentration of botulinum toxin in a second sample, wherein said method comprises:

(i) electrically stimulating the mouse hemidiaphragm in the presence of said second sample and selecting a second time before paralysis caused by said second sample in said mouse hemidiaphragm,

(ii) measuring said second effect in (i) at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thereby recording a calibration curve,

(iii) electrical stimulation of said muscle tissue in the presence of said first sample,

(iv) measuring a first effect caused by said first sample in said muscle tissue,

(v) determining a concentration at which said first and said second effects are identical, and

(vi) equating said concentration in (v) with said unknown concentration,

wherein said second time before the onset of paralysis is determined at at least one concentration, expressed in mouse LD ₅₀ units / ml, of 20 to 400 or 25 to 300 or 30 to 250, and wherein the second sample is Dysport ^®.

In another specific embodiment, the method encompasses a method for determining an unknown concentration of botulinum neurotoxin serotype B in a first sample with respect to a known concentration of botulinum B toxin or botulinum A toxin in a second sample, wherein said method comprises:

(i) electrically stimulating the mouse hemidiaphragm in the presence of said second sample and selecting a second time before paralysis caused by said second sample in said mouse hemidiaphragm,

(ii) measuring said second effect in (i) at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thereby recording a calibration curve,

(iii) electrical stimulation of said muscle tissue in the presence of said first sample,

(iv) measuring a first effect caused by said first sample in said muscle tissue,

(v) determining a concentration at which said first and said second effects are identical, and

(vi) equating said concentration in (v) with said unknown concentration,

wherein said second time to paralysis is determined at least at a concentration expressed in murine LD ₅₀ units / ml from 100 to 800 or from 150 to 700, or from 200 to 600, and where the second sample is Myobloc ^® .

However, an analysis to determine the concentration of neurotoxin or the activity of neurotoxin can be based not only on tissue, as described above, but also on cell cultures.

According to another aspect, the invention relates to an assay for determining the activity of a clostridial neurotoxin based on cell cultures, for determining an unknown concentration of a clostridial neurotoxin in a sample relative to a known concentration of clostridial toxin in a standard sample. The method uses a quantitative analysis of proteins, such as SNAP25, which are formed upon cleavage of the SNARE complex upon contact of cell cultures sensitive to clostridial botulinum neurotoxin with the specified neurotoxin. The method can also be used to assess the relative activity of clostridial neurotoxin in a sample when compared with a reference standard.

Pellet, S., et al., Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type A determination, Journal of Pharmacological and Toxicological Methods (2010), doi: 10.1016 / j. vascn.2010.01.003, a cell analysis has been proposed to determine the activity of purified botulinum neurotoxin serotype A as an alternative to bioassay in mice.

Keller, JE, et al., Persistence of botulinum neurotoxin action in cultured spinal cord cells, FEBS Letters 456 (1999) 137-142, discloses the mechanism underlying differences in the persistence of botulinum neurotoxin (BoNT / A) and botulinum neurotoxin activity neurotoxin E (BoNT / E).

Another objective of the invention is to improve these methods of the prior art and to develop a reliable and accurate method for determining the activity or, accordingly, the concentration of clostridial neurotoxin in a sample that causes the specified activity, which could be used for control purposes. Such an improved method could also serve to satisfy the high need for a safe and effective administration.

This other objective is achieved by a method in which a cell culture is exposed to or in contact with a sample comprising clostridial neurotoxin, wherein before measuring the effect that is induced in the cell culture cells by said clostridial neurotoxin, said sample is replaced with an aqueous medium such as a buffer or such as a neutral buffer that does not contain clostridial neurotoxin or said clostridial neurotoxin, and said cell culture is exposed to said aqueous medium for a certain period, for example, a period of more than 1 hour or more than 2 hours, or more than 3 hours, or more than 4 hours, or more than 5 hours. Before measurement, the cell culture may be in contact with the specified aqueous medium for a period of up to 100 hours or even more.

It was unexpectedly found that measuring this effect in the absence of the specified sample and subsequent contact with an aqueous medium that does not contain Clostridial botulinum neurotoxin, after the contact of the specified cell culture with a sample comprising neurotoxin, biases the corresponding dose-response curves so that the sensitivity of the method according to the invention increases significantly. Sensitivity is especially increased at low concentrations expressed in murine LD ₅₀ units / ml of the specified clostridial neurotoxin in the specified sample.

Thus, in a first aspect, the invention relates to a method for measuring the effect induced in a cell culture by clostridial neurotoxin, including:

(a) contacting the cell culture with a sample comprising said clostridial neurotoxin;

(c) measuring said effect caused in said cell culture by said clostridial neurotoxin;

where step (c) is performed in the absence of said sample; and

prior to the measurement in step (c) and after contact in step (a), the cell culture is contacted for 0.5 to 100 hours with an aqueous medium that does not contain clostridial toxin.

In a second aspect, the invention relates to a method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample, comprising:

(a) contacting the cell culture with said second sample;

(c) measuring a second effect induced in said cell culture by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contacting the cell culture with said first sample;

(h) measuring a first effect induced in said cell culture;

(k) determining a concentration at which said first and said second effects are identical;

(l) equating the indicated concentration in (k) with the indicated unknown concentration,

where step (c) and / or step (h) is performed in the absence of said second and / or first sample; and

before the specified measurement in step (c) or step (h), or step (c) and step (h), and after contacting in step (a) or step (f), or step (a) and step (f), the specified cell culture is contacted for 0.5 to 100 hours with an aqueous medium that does not contain clostridial toxin.

In a third aspect, the invention relates to a method for determining the relative activity of clostridial neurotoxin in a first sample with respect to the activity of clostridial neurotoxin in a second sample, comprising:

(a) contacting the cell culture with said second sample;

(c) measuring a second effect induced in said cell culture by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contacting the cell culture with said first sample;

(h) measuring a first effect induced in said cell culture;

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where step (c) and / or step (h) is performed in the absence of said second and / or first sample; and

before the specified measurement in step (c) or step (h), or step (c) and step (h), and after contacting in step (a) or step (f), or step (a) and step (f), the specified cell culture is contacted for 0.5 to 100 hours with an aqueous medium that does not contain clostridial toxin.

In one embodiment, the method further includes steps (m) and (n):

(m) selecting said various concentrations from the concentration range that best fits the first and second data set;

(n) determining the indicated best fit using a statistical criterion including the following sub-steps (α) - (δ):

(α) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In one embodiment, the statistical criterion is an F-test or a χ ² criterion, or a t-test.

In one embodiment, the probability of a false deviation for each sub-step (a) - (δ) is ≤5 (expressed in%).

In one embodiment, the method further includes the step (ε):

(ε) calculating, based on the displacement of the linearized and parallelized curves with respect to each other, the relative activity of the first sample with respect to the second sample.

In one embodiment, said effect (including the first and / or second effect) is the cleavage of a protein from the SNARE complex.

In one embodiment, the protein is SNAP25.

In one embodiment, prior to said measurement in step (c) or step (h), or step (c) and step (h), said cell culture is contacted with said clostridial toxin for 5 to 45 hours or 15 to 40 h, or from 25 to 35 hours

In one embodiment, prior to said measurement in step (c) or step (h), or step (c) and step (h), and after contact in step (a) or step (f), or step (a) and step (f) said cell culture is contacted for from 0.5 to 100 hours, or from 1 to 95 hours, or from 6 to 90 hours, or from 7 to 80 hours, or from 8 to 70 hours, or from 9 to 60 hours, or from 10 to 50 hours, or from 11 to 50 hours, or from 12 to 40 hours, or from 15 to 40 hours, with an aqueous medium that does not contain clostridial toxin.

In one embodiment, prior to said measurement in step (c) or step (h), or step (c) and step (h), and after contact in step (a) or step (f), or step (a) and step (f) the cell culture is lysed.

In another embodiment, the cell culture is lysed to contact in step (a) or step (f), or step (a) and step (f).

In one embodiment, said measurement is carried out using Western blot analysis or ELISA.

In one embodiment, said cell culture is selected from cell cultures of nerve cell lines or primary nerve cells.

In one embodiment, said registration of said measured second effect is done by plotting the concentration of said second effect and said registration of said second data set is done by recording a calibration curve.

In one embodiment, said second effect is determined at least at a concentration expressed in murine LD ₅₀ units / ml of at least 10.

In another embodiment, said concentration is from 10 to 1000, or from 10 to 70, or from 15 to 60, or from 20 to 45.

In another embodiment, said concentration is from 20 to 400, or from 100 to 800.

In one embodiment, said units are mouse LD ₅₀ units Xeomin ^®.

In one embodiment, said clostridial neurotoxin is a botulinum toxin.

In another embodiment, a serotype of said botulinum neurotoxin is selected from the group consisting of A, B, C, D, E, F, and G; or it is a chemically or genetically modified derivative of a botulinum neurotoxin serotype selected from the group consisting of A, B, C, D, E, F and G.

In another embodiment, said neurotoxin is serotype A or C, or E.

In one embodiment, the neurotoxin does not contain complexing proteins.

In another aspect, the invention relates to a computer program product comprising a computer program comprising software for implementing the method of the invention.

In another aspect, the invention relates to the use of cell culture in any of the methods of the invention.

In another aspect, the invention relates to the use of the method of the invention according to any of the first, second and third aspects of the invention for controlling the activity of a sample comprising clostridial neurotoxin.

In one embodiment, the sample is a stored sample.

In one embodiment, the sample is a lyophilized sample or a reconstituted sample.

In another aspect, the invention relates to the use of the method according to the first aspect of the invention for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample; or to determine the relative activity of clostridial neurotoxin in the first sample relative to the activity of clostridial neurotoxin in the second sample, for example, during quality control during the production process of clostridial neurotoxin.

Compared with methods known from the prior art that use cell cultures, the methods of the invention provide a significant increase in the accuracy of quantifying the biological activity of clostridial botulinum neurotoxin. The methods of the invention satisfy regulatory requirements.

It was found that the instability observed in relation to the methods of quantitative analysis of the prior art, which use cell cultures, can be significantly reduced to a small extent, by applying the methods disclosed in this application.

In one embodiment, the invention relates to a method for measuring the effect caused in a cell culture by clostridial neurotoxin, including:

(a) contacting the cell culture with a sample comprising said clostridial neurotoxin;

(c) measuring the effect caused in said cell culture by said neurotoxin;

where step (c) is performed in the absence of said sample.

The term “contact of a cell culture with said sample (which may be a first or second sample according to methods in accordance with other aspects of the invention)” means that at least a portion of said neurotoxin of said sample was obtained by said cell culture during said contact, i.e. at least a portion of the neurotoxin contained in said sample was bound by corresponding receptors contained in said cell culture cells.

The term “absence of sample” means that the measurement of the effect in step (c) is carried out in an environment, usually in a suitable buffer that contains 10% by weight or less, for example, does not contain at all, the sample or, in other words, the neurotoxin of the sample.

In one embodiment, said cell culture is not subjected to continuous exposure (contact with) to a sample (which may be a first or second sample according to methods in accordance with other aspects of the invention), including clostridial neurotoxin, but only temporary.

This means that after a specified period of exposure of the indicated cell culture to neurotoxin, i.e., contact in step (a), in order to obtain the response of the specified cell culture to the effect, an appropriate measurement of the effect (of the first or, accordingly, second effect, according to methods in accordance with aspects of the invention), in the absence of said sample (which may be said first or said second sample according to methods in accordance with other aspects of the invention), using sobov as described below.

In one embodiment, prior to said measurement, said cell culture, for example, is removed from a chamber containing said sample and transferred to a chamber containing components without neurotoxin, as described below. Then measure the magnitude of the effect (which may be the first or second effect when the sample is the first or second sample), that is, the effect is quantified. This means that the reaction to this stimulation is carried out in a cell culture containing the resulting neurotoxin.

In another embodiment, components containing a neurotoxin, i.e., a sample (which may be a first or second sample), are replaced with components containing no neurotoxin. In one embodiment, the sample is removed from the cell culture, for example, by filtration, and replaced with components that do not contain neurotoxin, as described below. After replacement, measure the magnitude of the effect (which may be the first or second effect when the sample is the first or second sample).

The term "clostridial neurotoxin (or clostridial toxin)" encompasses complexes of clostridial toxin as well as high purity neurotoxin, that is, a neurotoxin preparation that does not contain any other clostridial proteins.

In one embodiment, said clostridial neurotoxin is a botulinum neurotoxin.

In another embodiment, said botulinum neurotoxin is a serotype selected from the group consisting of A, B, C, D, E, F, and G.

The term “botulinum toxin complex” encompasses botulinum toxin bound to at least another non-toxic protein. Obviously, the term botulinum toxin complex used in this application includes 450 kDa and 900 kDa botulinum toxin complex, which can be obtained, for example, from C. botulinum cultures. Such preparations based on a complex of botulinum toxin type A are available, for example, Ipsen Ltd. (Dysport ^® ) or Allergan Inc. (Botox ^® ). Another type B botulinum complex-based drug is Solstice Neurosciences, Inc. (Myobloc ^® ). High purity type A neurotoxin, free of other clostridial proteins, is available from Merz Pharmaceuticals (Xeomin ^® ). This is the drug of choice for improving with several forms of focal dystonia.

In another embodiment, said botulinum neurotoxin is a chemically or genetically modified derivative of a serotype selected from the group consisting of A, B, C, D, E, F and G.

A chemically modified derivative of said neurotoxin may be a derivative that is modified by pyruvation, phosphorylation, sulfation, lipidation and / or glycosylation.

A genetically modified derivative of the specified neurotoxin is a derivative that has been modified by deletion, addition or substitution of one or more amino acids contained in the proteins of the specified serotype.

Such a modified toxin is preferably biologically active.

A biologically active toxin is a toxin capable of being absorbed by the cell and causing proteolytic cleavage of one or more polypeptides, such as SNAP25, which is part of the SNARE complex. If the concentration of a proteolytically cleavable polypeptide, such as SNAP25, is measured and quantified, then the concentration or activity of the toxin used can be calculated.

In one embodiment, according to any of the methods in accordance with the three aspects of the invention, prior to said measurement in step (c) or step (h), or step (c) and step (h), said cell culture is exposed (contact with) the specified clostridial toxin for from 5.0 to 45 hours or from 15 to 40 hours, or from 25 to 35 hours

In another embodiment, prior to said measurement in step (c) or step (h), or step (c) and step (h), and after contact in step (a) or step (f), or step (a) and step (f) said cell culture is contacted for from 0.5 to 100 hours, or from 1 to 95 hours, or from 6 to 90 hours, or from 7 to 80 hours, or from 8 to 70 hours, or from 9 to 60 hours, or 10 to 50 hours, or 11 to 50 hours, or 12 to 40 hours, or 15 to 40 hours, with an aqueous medium that does not contain clostridial toxin.

The term "aqueous medium" defines a liquid or fluid including water.

In one embodiment, said aqueous medium is a buffer.

In one embodiment, said buffer is a neutral buffer. The term “neutral” encompasses a pH range of from 6 to 8, or from 6.5 to 7.5, or about 7.

In one embodiment, said buffer is a phosphate buffer.

In one embodiment, the temperature of said aqueous medium is from 20 to 40 ° C or from 25 to 40 ° C, or from 30 to 40 ° C. In one embodiment, the temperature is about 37 ° C.

In one embodiment, prior to said measurement in step (c) or step (h), or step (c) and step (h), and after contact in step (a) or step (f), or step (a) and step (f), the cell culture is lysed.

The term "lysis" refers to the destruction of cells, for example, viral, enzymatic or osmotic mechanisms that violate its integrity. A fluid containing components of lysed cells is called a "lysate." For example, lysis can be used in Western and Southern blots to analyze the composition of certain proteins, lipids and nucleic acids, individually or in complexes. Standard lysis buffers may be used for lysis.

In another embodiment, the cell culture is lysed to contact in step (a) or step (f), or step (a) and step (f).

Surprisingly, it was found that measuring said effect in the absence of said sample and subsequent contact with an aqueous medium that does not contain Clostridial botulinum neurotoxin after contact or exposure to said cell culture of neurotoxin biases the corresponding dose-response curves so that the sensitivity of the method according to the invention increases significantly. Sensitivity is especially increased at low concentrations expressed in murine LD ₅₀ units / ml of the specified clostridial neurotoxin in the specified sample.

For example, if the cleavage of a protein, such as SNAP25 from the SNARE complex, is determined as an effect or a reaction, the method leads to an advantageous increase in the sensitivity of the method, which is especially used in the field of lower concentrations of neurotoxin. If the activity is determined at a lower concentration, neurotoxins, as a rule, can show the greatest difference, whereas at fairly high concentrations of activity converge to each other.

This increase in sensitivity provides a more accurate and more reliable analysis of the corresponding dose-response curves of the effect. This in turn can significantly reduce the number of laboratory animals used, such as mice, which otherwise would need to be killed to implement any of the methods according to the invention. Thus, this embodiment of the invention is progressive not only within the technical aspects, but also within the ethical aspects.

The term "sensitivity" is used in the present description in the standard meaning in which it is usually used in physiology, that is, sensitivity determines the ability of muscle tissue to respond to external stimuli. In this case, external stimuli are produced by the contact of muscle tissue with clostridial neurotoxin. It is within the scope of the invention to select a certain concentration range, for example, a concentration range at a relatively low concentration of clostridial neurotoxin, where the indicated sensitivity increases, that is, the reaction can be determined that under other conditions it is either impossible or, accordingly, can be determined only within the limits of an unacceptable deviation .

In another embodiment, the invention relates to a method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample, comprising:

(a) contacting the cell culture with said second sample;

(c) measuring a second effect induced in said cell culture by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contacting the cell culture with said first sample;

(h) measuring a first effect induced in said cell culture;

(k) determining a concentration at which said first and said second effects are identical;

(l) equating the indicated concentration in (k) with the indicated unknown concentration;

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

Thus, in one embodiment, the determination of the second and / or first effect is performed in the absence of said second and / or first sample. This means that after step (a) and / or after step (f), said cell culture is separated from the second and / or first sample, or, respectively, the second and / or first sample is separated from the cell culture, as described above.

The term "determining the concentration at which the first and second effects are identical" (steps (k) and (l)) means that the first and second effects are qualitatively and quantitatively identical, that is, the effect caused is, for example, the cleavage of a protein or polypeptide such as SNAP25 from the SNARE complex, and that these effects have the same measured value.

In one embodiment, in order to obtain results that can be reliably compared, the periods of exposure of the cell culture to the neurotoxin contained in the second or, respectively, first sample should be comparable.

In one embodiment, said exposure periods are identical.

In one embodiment, said registration of said measured second effect in step (e) is carried out by measuring said second effect at various concentrations of said clostridial neurotoxin in said second sample and plotting the concentration of said measured second effect, thereby recording a calibration curve .

If the effect caused by the specified second sample in the specified cell culture is determined based on various concentrations expressed in murine LD ₅₀ units / ml, a calibration curve can be obtained as described above.

For example, you can determine the specified effect caused in stages, ten LD ₅₀ units / ml or five LD ₅₀ units / ml within the selected concentration range.

Thus, by means of the second data set recorded in step (e), a calibration curve is constructed by which an unknown concentration of said clostridial neurotoxin in said first sample is determined according to steps (k) and the subsequent step (l).

In one embodiment, a calibration curve is constructed and the indicated determination and equalization steps according to steps (k) to (l) are carried out using graphical analysis.

The specified unknown concentration of the first sample can be determined by determining the concentration from the calibration curve at which the specified first and specified second effect have the same value, for example, the same concentration of the resulting SNAP25, and equating the specified concentration to the specified unknown concentration according to step (l).

A prerequisite for this determination is that an unknown concentration of clostridial toxin in the first sample produces an effect on the cell culture, which can be quantified using the specified calibration curve. One skilled in the art is aware that it may be necessary to dilute or concentrate a first sample having an unknown concentration, one or more times if necessary, in order to obtain a concentration range in which it is possible to carry out a comparison with the second sample, that is, achieve identical to the first and second effects. Then, knowing the dilution or concentration factor, the concentration of neurotoxin originally present in the undiluted or non-concentrated first sample can be calculated.

In another embodiment, said identification and equalization is performed not by measuring at one point with only one concentration in step (h) and subsequent steps (k) and (l), but by measuring at different concentrations. This is especially important given regulatory requirements.

According to another embodiment of the invention, it is desirable to optimize the concentration range in which a reliable comparison of said second and first sample is possible. This applies not only to the possibility of comparing the biological effectiveness of currently known and commercial preparations of clostridial neurotoxins, but also to preparations that could be developed in the future or are already in the process of development.

In one embodiment, in order to optimize the concentration range expressed in murine LD ₅₀ units / ml, in which a reliable comparison of said second and first samples is possible, it is desirable, firstly, to determine the standard deviation of the calibration curve recorded in step (e) and / or in step (h). Using a suitable stepwise regression analysis, you can create a regression model to predict the activity of an unknown toxin sample based on the dose-response curve.

Using this method, it is possible to identify the concentration range for the first and second samples, representing two different sets of data in which the correlation between the corresponding dose-response curves reaches a maximum, that is, the best similarity is established.

In one embodiment, the criterion can be further adjusted by presenting the range of values of the respective data sets of the first and second sample by empirical curves according to a given regression model, respectively, as well as linearizing and parallelizing said empirical curves within a specified confidence interval.

Thus, according to a third aspect, the invention relates to a method for determining the relative activity of clostridial neurotoxin in a first sample with respect to the activity of clostridial neurotoxin in a second sample, comprising:

(a) contacting the cell culture with said second sample;

(c) measuring a second effect induced in said cell culture by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contacting the cell culture with said first sample;

(h) measuring the first effect caused in the specified cell culture obtained in step (g);

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where step (c) and / or step (h) is performed in the absence of said second and / or first sample.

In one embodiment, the method further includes steps (m) and (n):

(m) selecting said various concentrations from the concentration range that best fits the first and second data set;

(n) determining the indicated best fit using a statistical test including the following sub-steps (a) - (δ):

(a) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In one embodiment, the determination of the second and / or first effect is performed in the absence of said second and / or first sample.

In another embodiment, an effect measurement is performed in the absence of said second and / or first sample. This means that after step (a) and / or after step (f), said cell culture is separated from the second and / or first sample, as described above, or the second and / or first sample is separated from the cell culture.

Statistical criteria suitable for performing the above sequence are well known, for example, likelihood ratio criteria. An example of such a likelihood ratio criterion is the well-known F-criterion. A criterion such as a χ ² criterion (a chi-square test or a χ ² distribution criterion) or a t-test can also be used. These criteria are also known in the art.

In one embodiment, the specified statistical criterion is an F-test.

Using this criterion, one can decide, within the established confidence interval, whether two random samples taken from two different populations differ significantly, taking into account their deviation. Thus, this criterion serves to verify the differences within the two statistical samples, in this case the second and first samples.

In one embodiment, the confidence interval should be wide to obtain reliable results, that is, the probability of a false bias should be relatively low.

In one embodiment, the probability of a false deviation is ≤5 (expressed in%; (or 0.05)) or, accordingly, the confidence interval is ≥95 (expressed in%; (or 0.95)).

In one embodiment, the probability of a false deviation for each sub-step (a) - (δ) is ≤5 (expressed in%).

In one embodiment, the linearization in step (γ) is performed by presenting the corresponding data sets as a direct best fit.

In one embodiment, parallelization in step (δ) is performed by determining the overall slope of the straight lines of best fit.

After step (δ), based on the displacement of the linearized and parallelized empirical curves relative to each other, the relative activity of the first sample relative to the second sample is determined.

Thus, in one embodiment, the method after step (δ) further includes step (ε):

(ε) calculating, based on the displacement of the linearized and parallelized empirical curves relative to each other, the relative activity of the first sample relative to the second sample.

In one embodiment, the term “relative activity” means that the activity of the first sample with respect to the second sample is determined at the same concentration or, correspondingly, identical concentrations, based on linearized and parallelized empirical curves.

In one embodiment, the activity of the second sample is equated to 100% and the relative activity of the first sample is expressed as%. For example, for the first sample, activity is obtained, for example, 110% or 90% with respect to the second sample. By appropriate dilution of the first sample, having 110% activity, to 100% activity, an effective concentration of clostridial neurotoxin in the first sample is obtained, which until now was not known, applying the rule of proportion. The unit of measurement is now relative activity, and the value is expressed as the unit of activity defined in relation to the activity of the reference standard (second sample).

In another embodiment, relative activity is expressed as the ratio of activity of the first and second sample.

In one embodiment, the above model is used to predict the logarithmic value of the applied dose of neurotoxin.

In another embodiment, the amount of the stimulated effect and the dose of neurotoxin in the sample are recorded on a logarithmic scale.

In one embodiment, the second effect, or, accordingly, the first effect, is measured at least at three different concentrations of clostridial neurotoxin in the second sample or, accordingly, the first sample.

In one embodiment, said registration of said data sets or, respectively, said registration of a calibration curve or, accordingly, calibration curves, is performed in the form of a semi-log graph.

In another embodiment, a double (on both axes) logarithmic plot is used.

A method for determining relative activity is described in the European Pharmacopoeia.

In one embodiment, starting from a concentration of, for example, 10 murine LDs of ₅₀ units / ml, a method for determining said relative activity is applied over the full range of the data set. Values in excess of 10 murine LD ₅₀ units / ml are then used as starting points, for example, 11, 12, 13, 14, 15, 16, 17 murine LD ₅₀ units / ml. The specified iteration is continued for as long as the applied model does not provide the desired and necessary accuracy.

In one embodiment, after determining the best fit and thus the concentration range using a statistical criterion, any first sample with an unknown concentration (relative to the effective concentration) of clostridial neurotoxin can be compared with a known concentration of said clostridial neurotoxin in a second sample within the specified concentration range identified according to the method of the invention.

In one embodiment, said registration of said measured second effect is performed by plotting the concentration of said second effect, said registration of said second data set being performed by registering a calibration curve.

The use of relative activity estimates, as well as the inclusion of a reference standard (second sample) in the analysis, gives more accurate and more reproducible estimates, which reduces the number of animals used.

Statistical tests are usually performed using a suitable computer program and a suitable computer.

In one embodiment, a statistical test is performed using a suitable computer program including suitable software for performing the statistical test.

Thus, in one embodiment, the invention relates to a computer program product comprising a computer program including software for implementing the method according to the invention.

In one embodiment, the second sample is selected from a commercial and registered botulinum toxin preparation. Since such products are registered and authorized as a medicine or, respectively, a medicine, they include a clearly defined amount or, accordingly, the concentration of botulinum toxin.

In another embodiment, any botulinum toxin preparation that was produced under standard conditions may be used.

In one embodiment, the commercial preparations mentioned above can be used as a second sample. Thus, the second sample may be Xeomin ^® , Botox ^® , Dysport ^® , Myobloc ^® or PurTox ^® . These preparations differ in the type of botulinum toxin used, or in biological effectiveness / activity, for example, in the concentration of botulinum neurotoxin or in the type of botulinum toxin contained in them.

A mouse unit expressed in mouse LD ₅₀ is the standard unit for determining the concentration of clostridial neurotoxin contained in a sample. The LD ₅₀ value determines the lethal dose at which 50% of the mouse population dies if the specified amount is applied to the mice of the specified mouse population. A method for determining the indicated value is known to a person skilled in the art. This method is described in the European Pharmacopoeia.

As you know, the units LD ₅₀ on the label of products based on botulinum neurotoxin may be characteristic of the product or, accordingly, for the manufacturer, and may not be equivalent due to the lack of a standard.

In one embodiment, the LD unit _50, referred to in this application are the units defined herein and Xeomin ^® label. For example, the second sample is Xeomin ^® . Therefore, units that are related to a certain activity are Xeomin ^® units. Thus, the analysis system of the present invention can be used to compare the activity of any sample, including clostridial neurotoxin, compared with Xeomin ^® . In addition, the method allows you to directly compare the first samples, including clostridial neurotoxin (in unknown concentration), in units of Xeomin ^® .

Xeomin ^® and Botox ^® show approximately comparable efficacy or activity. To obtain the same potency or activity as Xeomin ^® and Botox ^® , approximately 2.5 times the amount of Dysport ^® or, respectively, 10 times the amount of Myobloc ^{® should be used} .

In one embodiment, said commercial preparations are diluted or concentrated to predetermined concentrations of the botulinum neurotoxin contained therein, and said second effect is measured depending on various concentrations of said clostridial neurotoxin in said second sample. The indicated measured effect is plotted against the concentration of botulinum toxin, thus recording a calibration curve. Using the specified second data set or, respectively, the specified calibration curve, an unknown concentration of botulinum neurotoxin in the first example can be determined.

It was found that when the concentration of clostridial neurotoxin in the sample (which may be the first or second sample), expressed in murine LD ₅₀ units / ml and equal to at least 10, the methods according to the invention can be useful. It should be noted that all concentrations cited in the framework of this application are indicated in murine LD ₅₀ units / ml.

In one embodiment, the sample includes water in addition to the neurotoxin. In one embodiment, the sample comprises a solution or suspension of neurotoxin in water.

In one embodiment, said concentration of neurotoxin in said sample is at least 15.

In another embodiment, said concentration is at least 20.

In another embodiment, said concentration is from 10 to 1000.

In one embodiment, the concentration is from 10 to 70.

In another embodiment, the concentration is from 15 to 60.

In yet another embodiment, the concentration is from 20 to 45.

In one embodiment, the second sample is Xeomin ^®.

In one embodiment, it was found that in that case, if the second sample is applied Xeomin ^®, most reliable results can be obtained if the second effect is determined at least at a concentration of from 10 to 70. In another embodiment, the concentration is from 15 to 60. In yet another embodiment, the concentration is from 25 to 45.

In one embodiment, it was found that in that case, if the second sample is used Botox ^®, reliable results can be obtained if the second effect is determined at least at a concentration of from 10 to 70. In another embodiment, the concentration ranges from 15 to 60. In yet another embodiment, the concentration is from 25 to 45.

If the second sample is used to determine the calibration curve according to step (e), while the second sample has a lower concentration or contains a less effective or active botulinum neurotoxin than Xeomin ^® or Botox ^® , higher concentrations of neurotoxin, i.e. higher LD ₅₀ values units / ml are required to achieve a second effect of such strength that is comparable to the effect caused by Xeomin ^® or Botox ^® .

In the embodiment wherein the second sample has a low concentration or activity of the botulinum neurotoxin than Xeomin ^® or Botox ^®, the second effect is determined at least at one concentration of from 20 to 400 or from 100 to 800.

In one embodiment, wherein the second sample is Dysport ^®, the second effect is determined at at least one concentration of from 20 to 400 or 25 to 300 or from 30 to 250.

In another embodiment, in which the second sample is Myobloc ^® , the second effect is determined at least at a concentration of from 100 to 800 or from 150 to 700, or from 200 to 600.

In other embodiments, the concentration may range from 30 to 600, or from 30 to 400, or from 30 to 200, or from 30 to 100, or from 30 to 80, or from 40 to 500, or from 40 to 400, or from 40 to 300, or from 40 to 200, or from 40 to 100, or from 40 to 90, or from 50 to 300, or from 50 to 200, or from 50 to 100, or from 60 to 100, depending on concentration of the effectiveness or activity of neurotoxin in the second sample compared to Xeomin ^® or Botox ^® .

In one embodiment, the LD unit ₅₀ are units Xeomin ^®.

In one embodiment, due to the reliability of said assay in cell culture, regulatory requirements of regulatory authorities can be met and the need for the safe and effective administration of botulinum toxin, for example, serotype A or serotype C, or serotype E, can be met.

In yet another embodiment, said clostridial toxin in said first sample and said clostridial toxin in said second sample are the same clostridial toxins.

In yet another embodiment, said clostridial toxin or neurotoxin in a first sample and said clostridial toxin or neurotoxin in said second sample are different from each other.

For the experimental implementation of the method, as a rule, they use a cell culture that responds to the effects of botulinum toxin, that is, botulinum toxin has an effect on the cell culture, for example, causes the cleavage of a protein or polypeptide in the SNARE complex.

The term "cell culture" encompasses cells that are grown under controlled conditions outside the body.

In one embodiment, the term "cell culture" refers to the cultivation of cells derived from multicellular eukaryotes, especially animal cells. However, this term also covers cell cultures of plants, fungi and microorganisms, including viruses, bacteria and protozoa.

Cell culture methods are known in the art. In one embodiment, cells can be isolated from tissues to obtain an ex vivo culture. In one embodiment, tissue fragments can be placed in culture media and grown cells become available for culture. In another embodiment, the cells can be cleared of soft tissues by enzymatic cleavage by enzymes such as collagenase, trypsin, or pronase, which destroy the extracellular matrix. If immortalized cell lines are used, such cell lines are often capable of unlimited proliferation as a result of accidental mutation or directed modification. Cells can be grown in suspension or adhesion cultures. Depending on the type of cells, cells can naturally live in suspension, remaining unattached to the surface. Adhesion cells require a surface, for example, tissue culture plastic or a microcarrier, which can be coated with extracellular matrix components to enhance adhesive properties, as well as provide other signals necessary for growth and differentiation.

In one embodiment, for the experimental implementation of the method according to the invention, the cells can be grown and maintained at a suitable temperature and composition of the gas mixture, for example, at 37 ° C and 5% CO ₂ in a cell incubator. Culturing conditions can vary significantly for each type of cell, and changing conditions for a specific type of cell can lead to the manifestation of various phenotypes. In addition to temperature and gas mixture, the most often variable factor in crop systems is the nutrient medium. The composition of the culture media may vary in pH, glucose concentration, growth factors and the presence of other nutrients, and the like. A person skilled in the art is familiar with these various cell culture methods.

After harvesting, the grown cells can be used in any of the methods of the invention.

In one embodiment, the cells are selected from nerve cell lines or primary nerve cell cultures.

The term "cell line" encompasses cells of the same type, which proliferate indefinitely.

The term "primary cells" encompasses an unimortalized cell line that has been obtained directly from tissue.

In one embodiment, cell culture cells include spinal cord cells.

In one embodiment, cells, for example, spinal cord cells, cell culture obtained from a rodent. In one embodiment, the cell culture cells are mouse spinal cord cells or rat spinal cord cells.

In one embodiment, cell cultures used in the prior art section (see Pellet, S. et al .; Keller, J.E. et al.) Can be used in the framework of the present invention.

According to one aspect, the invention also provides an improved method for determining a concentration range in which the activity of a first sample comprising clostridial neurotoxin with respect to a second sample comprising clostridial neurotoxin can be determined within a specified confidence interval or probability of false bias.

In one embodiment, such a method for determining a concentration range in which activity of a first sample comprising clostridial neurotoxin can be determined with respect to a second sample comprising clostridial neurotoxin comprises the following steps:

(a) contacting the cell culture with said second sample;

(c) measuring a second effect induced in said cell culture by said neurotoxin;

(d) repeating steps (a) to (c) at various concentrations of said clostridial neurotoxin;

(e) registering said measured second effect of step (d) as a function of concentration, thereby registering a second data set;

(f) contacting the cell culture with said first sample;

(h) measuring a first effect induced in said cell culture by said neurotoxin;

(i) repeating steps (f) to (h) at various concentrations of said clostridial neurotoxin;

(j) recording the indicated measured first effect of step (i) as a function of concentration, thereby recording the first data set;

where the indicated concentration is selected from the concentration range that best fits the first and second data set, and where the indicated best fit is determined using a statistical test that includes the following sub-steps (a) - (δ):

(a) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;

(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;

(γ) linearization of empirical curves, respectively;

(δ) parallelization of linearized empirical curves.

In said embodiment, said second and said first effects are qualitatively identical. To clarify the method, the methods described above with reference to the method according to the third aspect of the invention can be applied.

In another aspect of the invention, the methods of the invention can preferably be used to control the quality, that is, the activity of a sample comprising a clostridial neurotoxin, compared to a reference standard, as required in the manufacturing process.

Thus, in this aspect, the invention relates to the use of the method of the invention for quality control, that is, the activity of a sample comprising clostridial neurotoxin.

In one embodiment, the activity of the sample in storage is determined. In one embodiment, the sample was stored for at least one hour or at least one day.

In one embodiment, the sample is a lyophilized sample or a reconstituted sample.

According to another aspect, the invention relates to the use of the method in accordance with the first aspect of the invention for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample; or to determine the relative activity of clostridial neurotoxin in the first sample with respect to the activity of clostridial neurotoxin in the second sample.

According to another aspect, the invention relates to the use of a cell culture, especially a cell culture, including spinal cord cells, such as rat or mouse cells, for determining clostridial activity in any of the methods of the invention.

The following embodiments also relate to the invention, it being understood that the embodiments described above are mutually applicable to the methods listed below.

In FIG. Figure 1 shows a graph of the time to paralysis (the time required to achieve half the initial force to reduce the hemidiaphragm), expressed in minutes, on the concentration of botulinum neurotoxin NT, expressed in murine LD ₅₀ units, introduced into the organ chamber (semi-logarithmic scale). Curve ■ represents the sample where the induced effect was measured in the presence of neurotoxin, and curve ♦ represents the sample where the tissue was exposed to the sample containing neurotoxin for 15 minutes. The muscle tissue was then removed from the bath and the sample was replaced with components that did not contain neurotoxin. After electrical stimulation, the induced effect was measured. Curves are empirical lines determined according to the method of the invention.

Example 1

For standard measurement, the mouse hemidiaphragm was prepared and transferred to an organ chamber filled with Earl's balanced salt solution. The diaphragmatic nerve ( nervus phrenicus ) of the hemidiaphragm was placed on a platinum electrode, with which electrical stimulation of the nerve was performed, which subsequently caused a reduction in the hemidiaphragm. The hemidiaphragm was fixed with clamps in the organ chamber. During fixation, the stimulation was turned off, but immediately turned on after it. The intensity of the electric current for stimulation was selected so that it was possible to measure the contraction force of the hemidiaphragm. After it was possible to measure the constant force of contraction, the medium was replaced with a medium containing botulinum neurotoxin. The time required to achieve half the contraction force (paralysis time) was determined for each concentration (at least for the period per concentration) and plotted against the concentration of botulinum neurotoxin introduced into the organ chamber.

Claims (8)

1. A method for determining an unknown concentration of clostridial neurotoxin in a first sample with respect to a known concentration of clostridial neurotoxin in a second sample, comprising:
(a) contacting the cell culture with said second sample comprising said clostridial neurotoxin in a known concentration;
(c) measuring a second effect induced in said cell culture by said clostridial neurotoxin at a known concentration;
(d) repeating steps (a) to (c) at various concentrations of clostridial neurotoxin;
(e) recording the measured second effect of step (d) depending on the concentration of clostridial neurotoxin, thereby recording a second data set;
(f) contacting the cell culture with said first sample containing said clostridial neurotoxin at an unknown concentration;
(h) measuring the first effect induced in said cell culture by said clostridial neurotoxin at an unknown concentration;
(k) determining a concentration of clostridial neurotoxin at which said first and said second effects are identical; and
(l) comparing the concentration of clostridial neurotoxin defined in (k) with the indicated unknown concentration of clostridial neurotoxin;
where before the specified measurement in step (c) and step (h), and after contact in step (a) and step (f), said cell culture is contacted for 0.5 to 100 hours with an aqueous medium that does not contain clostridial neurotoxin, and where step (c) is performed in the absence of said second sample, and step (h) is performed in the absence of said first sample. 2. A method for determining the relative activity of clostridial neurotoxin in the first sample with respect to the activity of clostridial neurotoxin in the second sample, comprising:
(a) contacting the cell culture with said second sample containing said clostridial neurotoxin in a known concentration;
(c) measuring a second effect induced in said cell culture by said clostridial neurotoxin at a known concentration;
(d) repeating steps (a) to (c) at various concentrations of clostridial neurotoxin;
(e) recording the measured second effect of step (d) as a function of the concentration of clostridial neurotoxin, thereby recording a second data set;
(f) contacting the cell culture with said first sample containing said clostridial neurotoxin at an unknown concentration;
(h) measuring the first effect induced in said cell culture by said clostridial neurotoxin at an unknown concentration;
(i) repeating steps (f) to (h) at various concentrations of clostridial neurotoxin;
(j) recording the measured first effect of step (i) depending on the concentration of clostridial neurotoxin, thereby registering the first data set;
where before the specified measurement in step (c) and step (h), and after contact in step (a) and step (f), said cell culture is contacted for 0.5 to 100 hours with an aqueous medium that does not contain clostridial neurotoxin, and where step (c) is performed in the absence of said second sample, and step (h) is performed in the absence of said first sample. 3. The method of claim 2, further comprising steps (m) and (n):
(m) selecting said various concentrations of clostridial neurotoxin from the concentration range that best fits the first and second data sets;
(n) determining the indicated best fit using a statistical criterion including the following sub-steps (α) - (δ):
(α) presenting the range of values of the second data set obtained in step (e) in the form of an empirical curve;
(β) presenting the range of values of the first data set obtained in step (j) as an empirical curve;
(γ) linearization of empirical curves, respectively;
(δ) parallelization of linearized empirical curves. 4. The method according to p. 3, further comprising the step (ε):
(ε) calculating, based on the displacement of linearized and parallelized empirical curves relative to each other, the relative activity of the first sample with respect to the second sample. 5. The method according to any one of paragraphs. 1-4, where the indicated effect is the cleavage of a protein from the SNARE complex. 6. The method according to any one of paragraphs. 1-4, where before the specified measurement in step (c) and step (h), said cell culture is contacted with said clostridial neurotoxin for 5 to 45 hours, or 15 to 40 hours, or 25 to 35 hours; or
where prior to the specified measurement in step (c) and step (h), said cell culture is contacted with said clostridial neurotoxin for 5 to 45 hours, or 15 to 40 hours, or 25 to 35 hours; and before said measurement in step (c) and step (h), and after contact in step (a) and step (f), said cell culture is contacted for 0.5 to 100 hours, or 1 to 95 hours or from 6 to 90 hours, or from 7 to 80 hours, or from 8 to 70 hours, or from 9 to 60 hours, or from 10 to 50 hours, or from 11 to 50 hours, or from 12 to 40 hours, or from 15 to 40 hours with an aqueous medium that does not contain clostridial neurotoxin. 7. The method according to any one of paragraphs. 1-4, where the specified registration of the specified measured second effect is carried out by plotting the dependence of the specified second effect on the concentration of the specified clostridial neurotoxin, and the specified registration of the specified second data set is carried out by recording the calibration curve. 8. The application of the method according to claim 1 for determining the unknown concentration of clostridial neurotoxin in the first sample in relation to the known concentration of clostridial neurotoxin in the second sample.

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