DE102020118520A1 - DEVICE AND METHOD FOR INJECTING AN AGENTS, PREFERRED NEUROTOXIN, MORE PREFERRED BOTULINUM TOXIN - Google Patents

Abstract

The invention relates to an injection device for injecting an active substance, preferably a neurotoxin, particularly preferably botulinum toxin, the injection device having a cavity, an outlet fluidically connected to the cavity and an ejection unit with which the active substance in the cavity can be ejected through the outlet from the injection device characterized in that the injection device has at least one coil which is set up to locally concentrate active substance located in the cavity and/or to align molecules of the active substance by means of a magnetic field generated by the coil. A corresponding method is also described.

Description

The invention relates to a device and a method for injecting an active substance, such as neurotoxin, in particular botulinum toxin, into defined regions of a face, with the depth and amount of the injected active substance being able to be determined. Advantageously, the amount of active ingredient can be precisely metered. A defined depot can be set at the nerve attachment of the muscle. When injecting, it is important to place a small amount of neurotoxin. This is very difficult with the usual method, i.e. a usual injection with a needle. A particular factor is also that with known injection methods through the cannula, cannula can cause pain to a patient; moreover, putting the syringe in the face is also perceived as very frightening. Studies have shown that injections in the facial area are extremely uncomfortable for patients. Nevertheless, many patients would prefer treatment with Botox to treatment with a cream if there was another option, e.g. injecting botulinum toxin without a needle.

An object of the invention is therefore to enable injection of neurotoxin without using a cannula. This goal is achieved by directly or indirectly accelerating the active ingredient and injecting it into the skin through a nozzle that centers the active ingredient. Several methods are conceivable for this.

There is also the possibility of expanding a commercially available syringe in such a way that it is coupled to a coil or motor so that at least the portioning of the active substance can be precisely controlled. The syringe can be a molecular syringe.

The advantage of using a coil is that the magnetic field that the coil creates has a positive effect on the drug molecules and can accelerate them.

Another option is to introduce the neurotoxin into microcapsules (nanocarriers) and accelerate them with a magnetic field. The capsules can be designed in such a way that they can be placed precisely and only then manipulated, e.g. from the outside, in such a way that the active ingredient is released. The transport of molecules such as biopharmaceuticals, e.g. neurotoxins, into human tissue without a cannula poses considerable challenges for pharmaceutical research. This problem is solved by the present invention with a molecular syringe capable of injecting bacterial toxins. For this purpose, magnetic nanocarriers can be loaded with neurotoxins.

Neuropharmaceuticals cannot be administered orally - as tablets or capsules - but must be injected at the site of action. The aim of the present invention is to transport proteins (e.g. botulinum toxin) and other biopharmaceutical agents to their target sites without a cannula. This goal is achieved by using nanocarriers. These are small particles, for example made of iron-containing lipid molecules or biodegradable magnetic polymers, which transport the biopharmaceutical active ingredients and only release them at their destination. The release can take place directly after the injection with magnetic waves, ultrasound or other methods. Advantageously, e.g. botulinum toxin is administered in very small amounts as an active ingredient, which is why nanocarriers can be used here for transport. Nanocarriers can range in size from 1-1000 nm. Carriers with a diameter of 200 nm are often used in nanomedicine. Because of their size, nanocarriers can deliver drugs to otherwise inaccessible locations throughout the body. Because nanocarriers are so small, they can often only deliver very small doses of drugs. The emulsion techniques used to prepare nanocarriers only work with low drug loading and drug encapsulation, which poses a difficulty for clinical use with other drugs. This method is ideal for the use of neurotoxins.

The term microencapsulation can mean a technique with which solid, liquid or gaseous substances are surrounded in the smallest portions with a shell and are thus immobilized. Microencapsulation is thus a form of inclusion immobilization. There are many areas of application for microencapsulation: in chemistry, in biotechnology, in pharmacy, in food technology, in cosmetics, in electronics and in environmental technology. Basically, microencapsulation is the embedding of at least one substance (active ingredient) with the help of at least one second substance (shell material). The former can often not be used directly for various reasons (solubility, reactivity, stability, etc.), or certain effects are to be achieved through the microencapsulation (e.g. release curves for controlled release, unique selling points, etc.).

In the case of matrix encapsulation, the active component(s) is/are homogeneously mixed with the shell component (“matrix”) and a particle is formed in which the active component(s) is/are evenly distributed. The release usually determines either the diffusion of the active component(s) into the environment or the rate of degradation of the matrix.

In core-shell encapsulation, the active ingredients ("core") are encased in a shell material. A true capsule is formed with one or more shells, e.g. B. a polymersome. The rupture of the hull usually produces a complete release of the core material in a short time (“burst”). However, it is also possible to produce an extremely slow release by choosing the right shell.

In particular, microencapsulation offers the following possibilities: transforming liquid substances into powder, easy encapsulation of volatile substances, protecting substances from the reaction, e.g. B. with air, light or liquids, release of active ingredients (e.g. drugs) in a certain period of time, fixation of poisons, separation of reactive substances, improvement of the tolerability of drugs, improvement of physical properties of powders, change of electrostatic properties of substances, Providing a shell of iron material, homogeneous mixture with iron components, small amounts of powdered neurutoxin.

The first industrial application was carbonless copy paper based on a patent from the NCR company in 1953. It was launched in the USA by the Appleton company in 1954. There are now a number of companies on the market that offer commercial microencapsulation using various processes such as dropping processes, Emulsion process, spray process, etc m. offer.

In the dripping process, regular droplets are formed when vibration is applied to a thin jet of liquid (with solution, sol, suspension or melt), which can possibly be charged with the help of high voltage. Due to the physical repulsion of the particles, almost “round droplets” are formed. These can be cooled in free fall (e.g. wax), dried (e.g. sugar) or exposed to chemical reactions. Binding agents are generally used for systems that do not exhibit any internal gelation (i.e. are solidified by cooling, for example). These solidify the droplets and can bring in additional properties, such as e.g. B. separate defined release profiles, additional protection against environmental influences or reactive components (e.g. oxygen from probiotics or similar). In the present invention, microencapsulated botulinum toxin molecules are to be enclosed in an iron-containing capsule and the physical properties of the Botox powder are thus to be changed in such a way that this capsule can perforate the human skin with the aid or using a device and can reach the site of action.

Botox (botulinum toxin) has been used for many years to smooth wrinkles in aesthetic medicine. Botulinum toxin is primarily suitable for correcting facial wrinkles, ie wrinkles caused by movement of the mimic muscles. In the upper third of the face, these are the so-called frown lines, transverse wrinkles on the forehead and wrinkles on the side of the eyes (crow's feet). These are well-known indication areas with extensive experience for many years. There are well-established injection schemes for this purpose, but they cannot be used unreservedly in all patients. Particular caution is z. B in patients with sagging eyebrows or asymmetry of the eyebrows. In the case of slippery limbs, a lot of experience is needed to determine whether and in what form treatment with Botox is an option. In addition to wrinkle treatment, Botox can be used to raise the eyebrows within certain limits (brow lift). A side effect appreciated by some patients is the reduction of tension headaches after a botox treatment of the forehead. In the lower part of the face, the use of botox in some indications is associated with more risks and side effects. Wrinkles in the upper lip can be smoothed out very effectively with small doses. Despite this, some patients report a certain weakness of the upper lip, which makes drinking from narrow glasses and whistling difficult for about 2-3 weeks. The patients must be informed about these circumstances beforehand; some patient groups, such as singers or musicians, may not be treated. Cleverly placed injection points make it possible to lift the corners of the mouth with Botox injections. Furthermore, by using botulinum toxin, a wrinkled chin can be smoothed out and a washed-out edge of the lower jaw can be emphasized again. The neck wrinkles (turkey neck) that often occur in old age due to protruding muscle bellies can be easily regressed. Transverse wrinkles on the neck or cleavage, on the other hand, are much more difficult to treat with the use of botulinum toxin. Other less well-established applications include Mesobotox, in which very small doses are injected under the skin so that the muscles under the skin relax and wrinkles are smoothed out. Botulinum toxin is a must bare method in aesthetic medicine with a wide range of applications. In the hands of the experienced practitioner, it is a safe and very effective tool for addressing many problem areas.

Botulinum toxin (BTX), also known as botulinum neurotoxin (BoNT), botulism toxin, botulinum toxin, botulin - also known under the trade name Botox for the first botulinum toxin ready-to-use preparation - is a collective term for several very similar neurotoxic proteins. The neurotoxins are formed and excreted by various strains of the bacterial species Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium argentinense; they are exotoxins. The toxic effect of these proteins is based on the inhibition of the transmission of stimuli from nerve cells, which, in addition to disorders of the autonomic nervous system, can result in muscle weakness up to and including cessation of lung function. Botulinum toxin is by far the deadliest poison known to living beings such as humans. The LD50 values for mice are 3 ng/kg for inhalation, up to 4 ng/kg for subcutaneous intake and for intravenous administration as little as 30 pg/kg (0.03 ng/kg) are lethal. Poisoning with botulinum toxins is called botulism and is a dreaded food poisoning, it can also occur as a result of intestinal infections and wound infections with C. botulinum. Since the 1980s, the toxic proteins produced by the bacterium have been used for medical purposes, primarily to treat neurological movement disorders (dystonia). Its use in cosmetic medicine for the temporary reduction of wrinkles (effective for 3-6 months) has been heavily criticized because of the massive increase in animal testing it has caused. There is also a risk of serious nerve damage without medical necessity for the procedure. Clostridium botulinum is ubiquitous in soil and aquatic sediments and forms resistant endospores, as does Clostridium tetani, which produces the structurally similar tetanus toxin. What evolutionary benefit such agents that are pathogenic for the host bring to the parasitizing microorganism is the subject of discussion. Botulism was first scientifically described in February 1815 by the Württemberg doctor and poet Justinus Kerner. In 1820 Kerner recognized the mechanism of action of the toxin as an inhibition of nerve conduction and in 1822 proposed the poison, which he called "fatty poison" and "fatty acid", in extremely low doses as a drug for various nervous disorders. In 1868, the physicians Rupprecht and Müller first described the poisoning as botulism. Clostridium botulinum (toxin type A), the bacterium responsible for the poisonings, was isolated in 1895-1897 by Emile van Ermengem, a Belgian bacteriologist, while studying ham that was responsible for three deaths. Van Ermengem initially called the microorganism Bacillus botulinus, today it is called Clostridium botulinum. As early as 1897, Walter Kemper produced the first antiserum against botulinum toxin A. After botulinum toxins could be obtained in large quantities from the 1920s, Carl Lammanna in Fort Detrick (Maryland) at USAMRIID succeeded in 1946 in producing the pure toxin type A. The previously suspected structure of two different protein chains was verified. In 1949 in London, Burgen, Dickens and Zatman demonstrated that botulinum toxin A inhibited acetylcholine secretion as the cause of muscle paralysis. Up to around 1970, a total of seven different neurotoxin serotypes (A, B, C1, D, E, F and G) were isolated. 1973-1978 the protein was first tested under Schantz according to an FDA-approved procedure on volunteers as a drug and was used in 1980 by Scott for the first time to treat strabismus (strabismus). In 1989, the US Food and Drug Administration granted the first drug approval for a botulinum toxin preparation. Under the name Oculinum, botulinum toxin A was approved for the indications nystagmus (trembling eyes) and blepharospasm (eyelid spasm). After the Allergan company took over the product from the manufacturer at the time in 1991, the trade name was changed to Botox in 1992. In 1993, Botox was approved in Germany, as was the competitor's product Dysport (Ipsen). Within a few years, spasms of the facial muscles and legs as well as spasmodic torticollis (cervical dystonia, “wry neck”) were added as further indications. In 1992, J. and A. Carruthers published a report on a transient attenuating effect of botulinum toxin A on skin folds (duration 3-6 months). Since then, the toxin has been used off-label in the cosmetics sector. Preparations for this use did not receive approval until 2002. Since 2001, botulinum toxin B has also been used to treat cervical dystonia. The names for the bacterium and the toxin are derived from the Latin word botulus 'sausage', as is the previously introduced term botulism for the clinical picture of sausage poisoning. The reason is that botulinum toxin poisoning used to be associated with sausage or canned sausage. This is also where the historical name Wurstgift (English sausage poison) comes from. The bacterium Clostridium botulinum, which mainly causes botulism, is found ubiquitously as a spore. The bacterium requires non-acidic, extremely low-oxygen conditions in order to germinate and produce the toxin. Such conditions are usually present in canned sausages and vegetables as well as vacuum-packed foods; In addition, the anaerobic core zone of meat and sausages is usually considered an excellent substrate for the growth of Clostridium. A surface model of botulinum toxin A has a neurotoxic light chain and a heavy chain. Botulinum toxins are high molecular weight pro complexes from the group of AB toxins, which consist of two parts: the light chain, the actual neurotoxin with a paralyzing (paralytic) effect (part A) and usually the heavy chain, a non-toxic protein (part B) that binds to nerve cells and conveyed a recording. The components are connected to one another via a disulfide bridge. The lighter neurotoxic part is a zinc-containing endopeptidase. The B part binds to the presynaptic membrane of nerve cells. After uptake into synaptic vesicles, the N-terminus of the heavy chain forms a membrane pore by lowering the pH. In addition, the disulfide bridge is cleaved, allowing the neurotoxic light chain to diffuse into the cytosol. The B part protects the neurotoxin from proteolytic degradation in the acidic environment of the stomach after oral administration, so that the toxin remains biologically available and can cause poisoning. However, the complex dissociates at neutral tissue pH and is no longer stable. The protein also becomes unstable if heated for a long time. In the therapeutic application of the botulinum toxin, the complex proteins therefore have no essential function and do not contribute to the effectiveness. However, it is being discussed whether differences in the clinical effectiveness of the approved drugs (e.g. the diffusion capacity or ability to spread) can be attributed to the different types of envelope proteins present. Serologically, botulinum toxins are divided into types A to G, of which A and B are used medicinally. Only types A, B, E, and F are toxic to humans. The botulinum toxins are very similar in terms of their structure and biochemical effect to the tetanus toxin, which is also produced by clostridia (Clostridium tetani). Botulinum toxin serotypes Type Some organisms in which the type is effective ^[20] Toxic to humans ^[19] UniProt / UNII Variants, Notes A Human (primarily USA), chick Yes UniProt P0DPI0 UniProt P0DPI1 (A1) UniProt Q45894 (A2) UNII: E211KPY694 The medically used BoNT/A exists in various native, ie complexed forms ^{[A 1] [A 2] [A 3] [A 4] [A 5]} as well as purified forms that are free of complex proteins ^{[A 6] [A 7 ] [A 8]} are. ^[21] B Human (mainly Europe), "child botulism", horses, cattle Yes UniProt P10844 (B) UniProt B1INP5 (B1) UNII: OY70779M1F Rimabotulinum toxin B ^{[A 9]} C waterfowl (C1); Cattle, horses, mink (C2) no UniProt P18640 UNII: FPM7829VMX Botulinum toxin C1 (botulinum neurotoxin C) ^{[A 10]} Botulinum toxin C2 ^{[A 11] [A 12]} D cattle, poultry no UniProt P19321 UNII: 331HTW151K E human Yes UniProt Q00496 UniProt P30995 UNII: T579M564JY f human Yes \ UniProt A7GBG3 UniProt P30996 UNII: U1R2P7107G G Poisoning cases not yet known no UniProt Q60393 UNII: 2C3DD06QGJ Produced by Clostridium argentinense

In addition to the seven generally recognized "classic" BoNT serotypes, several novel BoNT types have been postulated. In 2013, a group of researchers in the United States reported discovering another serotype in a child's stool sample, which they called "Type H''' (BoNT/H). Despite its decoding, the DNA structure was initially kept secret. Research by the Centers for Disease Control and Prevention (CDC) later showed that it is a hybrid type with structural similarities to serotypes A and F. BoNT/X is a new serotype that is also formed by C. botulinum. A variant that is similar to the botulinum neurotoxins at the molecular level is eBoNT/J (also called BoNT/En), which, however, is encoded by a gene from Enterococcus faecium.

Advances in DNA sequencing technology have also revealed differences within individual serotypes, leading to the introduction of subtypes. Such subtypes can differ by up to 36% within a serotype at the amino acid level. As of 2019, more than 40 subtypes have been described in the literature. It has been shown that the subtypes of a serotype differ in their biological activity, for example in the kinetics of uptake and substrate cleavage, the affinity for receptors or the overall activity. Botulinum toxin inhibits the transmission of impulses from nerve cells to other cells, especially at the synapses to muscle cells, which means that muscle contractions become weaker or stop altogether. Botulinum toxins are proteins that are first produced in the bacterium as long polypeptide chains and then cleaved by proteases and activated. They then consist of two protein subunits called light (L, approx. 50 kDa) and heavy (S, approx. 100 kDa) chains.

Different functional proteins involved in excitation transmission can be destroyed by different subtypes of botulinum toxin. The tissue specificity of the venom depends on structural features of the heavy chain. This is because botulinum toxin binds to the presynaptic part of a neuron that uses acetylcholine as a neurotransmitter - like a neuromuscular junction. Heavy chain portions also mediate venom entry into the presynaptic terminal by endocytosis. The light chain that has entered the interior of the nerve cell is the active part of the toxin. This is because it has structural features with catalytic activity and now acts as a zinc endopeptidase. This enables it to dissect certain functional proteins of the cell that are needed for synaptic vesicle fusion. Through this action, the venom impedes exocytosis, the process by which the messenger substance contained in vesicles is released into the synaptic cleft. The transmission of neuronal signals to muscle cells is thus blocked. The membrane protein synaptobrevin (VAMP2), which is an essential component of secretory vesicles, is already split by the presence of a few botulinum toxin molecules of types B, D, F and G due to the catalytic effect, without the botulinum toxin itself being consumed. Botulinum toxins A and E cleave the synaptic-associated protein SNAP-25; serotype C destroys syntaxins. When synaptic vesicles can no longer fuse with the membrane, their transmitter acetylcholine is no longer released into the synaptic cleft. The affected nerve cell can therefore no longer sufficiently stimulate the associated muscle fiber and this leads to paralysis of the muscle on which the poison is acting. When the interrupted neuromuscular transmission is reactivated, newly growing presynaptic endings temporarily take over the function of the disturbed axon terminals of the nerve cell process. Every year 20-40 cases of botulism are reported in Germany, 1-2 of which are fatal. Rarely, "visceral botulism" occurs, which occurs as "infant botulism" or "infantile botulism" in infants and as "adult infectious botulism" in adults with a rare predisposition to infection. The bacterium develops from spores ingested through food in the small intestine and produces the toxins there. As an antidote for all forms of botulism, either a trivalent (types A, B, E) or a polyvalent antiserum (types A-G) is used, in the USA also the heptavalent botulism antitoxin. All three are derived from horses.

In neurology, botulinum toxin has been used as an approved drug since the early 1980s, primarily in the treatment of specific movement disorders, so-called focal dystonia. These are diseases such as blepharospasm (eyelid spasm), oromandibular dystonia (spasm of the mouth, tongue and throat), spasmodic torticollis (wry neck) and other cervical dystonia, graphospasm (writer's cramp) and spasmodic dysphonia (spasm of the vocal cords). Furthermore, the focal symptoms of segmental or secondary dystonia and spasticity can be treated with botulinum toxin. These include hemifacial spasm, movement disorders after peripheral facial nerve affection and certain spastic syndromes in adults and children, for example spastic equinus, in patients with cerebral palsy, arm spasticity after a stroke or focal spasticity of the hand and wrist after a stroke. These indications are always injected intramuscularly or subcutaneously. A treatment success with improvement up to the temporary loss of symptoms is around 90% for eyelid spasms and between 60 and 80% for torticollis.

Other areas of application are, for example: strabismus (squinting), treatment of tension headaches and migraines, in the case of chronic migraines also approval at the expense of the GKV, reduction of hyperhidrosis (excessive sweat production), increased saliva production (e.g. in patients with M. Par kinson or amyotrophic lateral sclerosis), in the treatment of achalasia, narrowing of the esophagus, and diffuse esophagus spasm (spasm of the esophagus), in patients with sphincter-oddi dysfunction (SOD) type II, for the treatment of Hirschsprung's disease (aganglionosis of the large intestine) , for anal fissures, for bladder dysfunction (since 2018 benefit of the GKV).

Numerous other areas of application are being tested, e.g. B. orthopedic diseases (epicondylitis humeroradialis and humeroulnaris, plantar fasciitis, myofascial trigger points of the spine, etc.) or dermatological problems, each of which can be improved by muscle relaxation.

Certified medical training is u. a. offered by the botulinum toxin working group of the German Society for Neurology (DGN) and by the Pain Section (IGOST) of the German Society for Orthopedics and Orthopedic Surgery (DGOOC).

Botulinum toxins are exotoxins produced by bacteria. If they are injected into a muscle, they specifically block the release of the neurotransmitter acetylcholine by destroying protein complexes. As a result, the corresponding muscle can no longer be tensed as usual. Other nerve functions - such as feeling or touching - are not affected. After a therapeutic injection, the effect builds up slowly and - depending on the indication and dose - reaches its peak after about ten days. After two to six months, the nerve endings have stopped sprouting, allowing the muscles to be activated again. The injection can be carried out with simultaneous measurement of an electromyogram (EMG) in the muscle, the so-called stimulation technique, ultrasound-controlled or according to anatomical knowledge. There are a number of patients with neuromuscular diseases in which the body produces neutralizing antibodies against subtype A after previous long-term and high-dose use; the effectiveness of the medication decreases or is completely lost. Subtype B preparations (Neurobloc or Myobloc, approved in 2001 for torticollis) have also been available since 2001. However, these have a significantly shorter duration of action. The therapeutic index of the toxin is wide, with an experimentally determined LD50 value of approximately 2000 ng in monkeys when injected intraorbitally (into the eye socket); this corresponds to about 50 ampoules of Botox. In the event of an overdose, or if the toxin enters the bloodstream, a polyvalent equine botulism antitoxin is available. It is part of the emergency depot in larger hospitals. In most cases, however, the intravenous injection is given too late and the patient cannot fully recover immediately. Long-term artificial respiration is essential. Ptosis (in eyelid spasms), temporary muscle weakness, local pain resulting from the injection, dry mouth, disturbances in the accommodation of the eye and hematoma are mentioned relatively frequently as side effects of subcutaneous or intramuscular injection. When used cosmetically, the most common reports reported were swallowing disorders (dysphagia) and isolated cases, including sarcoidosis (a connective tissue disease) at the injection site and bruising from arterial damage (pseudoaneurysm). In a systematic review from 2015 on known complications from the cosmetic use of botulinum toxin, it was requested that patients be informed about the following possible, serious side effects before an operation: dry eye syndrome (keratoconjunctivitis sicca), squinting (strabismus), double vision (diplopia), pseudoaneurysm the superficial temporal artery (Arteria temporalis superficialis), neck weakness (neck weakness), voice disorder (dysphonia) and swallowing disorders. The local injection reduces the risk of side effects (in contrast to a systemic treatment that makes little sense), but still requires a high level of knowledge on the part of the user. A so-called titration is often used, i. In other words, too small a dose is initially injected, which is then supplemented by a larger amount every two to three weeks.

The first disadvantage is that the effect occurs after about 24 hours in the best case, usually after two to ten days, but then - depending on the dosage and indication - lasts for two to six months. If botulinum toxin paralyzes emotional facial expressions, then regions of the brain that process emotions - such as the left amygdala - also limit their activity. This prevents mimic imitation and thus the understanding of emotions (“embodied emotion”, “embodied feeling”). Systemic spread of botulinum toxin from the site of topical application to other areas of the body, particularly the nervous system, has been demonstrated. Although disturbances and damage have already been assigned to this spread, the underlying mechanisms of the spread and its consequences have so far (as of 2016) been almost unexplored. You are warned against using botulinum toxin to treat bruxism (teeth grinding), temporomandibular dysfunction (temporomandibular joint problems) and to slim the face visually, as it can lead to massive damage to the jawbone. Botulinum toxin is injected into the masseter muscle to reduce muscle tension. A period of time The three months between injections, which is usually observed, is not enough to regenerate the lost bone. Some of the results show that the jawbone no longer regenerates, even in the long term. Bone loss can lead to tooth loosening - even tooth loss - and increases the risk of fracture of the jawbone. The American Food and Drug Administration (FDA) has not (as of January 2017) approved the use of botulinum toxin in this area. In the current S3 guideline for bruxism as of 2019, on the other hand, the use of botulinum toxin is rated rather positively and laypersons are prohibited. In a case of years of violations of the ban on wrinkle injections for dentists, the Higher Administrative Court for the state of North Rhine-Westphalia affirmed the unreliability of a dentist, which is a prerequisite for the withdrawal of a license to practice medicine. The dentist was accused of his longstanding and stubborn behavior, which he could only have eliminated through a serious change in attitude.

The botulinum toxin is manufactured as a drug by various companies: Botulinum toxin drugs ^[5] designation active ingredient Manufacturer Approved areas of application Botox (D, A, CH, etc.) Toxin A allergy Symptomatic treatment of various types of spasticity (focal spasticity of the wrist and ankle in adults, focal spasticity in patients with cerebral palsy from the age of 2), spasms (blepharospasm, hemifacial spasm) and dystonia (cervical, focal); alleviating the symptoms of chronic migraines) Botox Cosmetics (USA) Vistabel (D, A, CH and others) Toxin A allergy Temporary improvement in the appearance of moderate to severe wrinkles on the upper half of the face (glabella lines, crow's feet, horizontal forehead lines) in adults with significant psychological stress Dysport (D) Toxin A ipsen Symptomatic treatment of various spasticities (upper extremities and ankles in adults, lower extremities in ambulatory patients with cerebral palsy from 2 years of age), spasms (blepharospasm, hemifacial spasm) and dystonia (cervical, focal) Azzalure (D) Toxin A ipsen Temporary improvement in the appearance of moderate to severe wrinkles on the upper half of the face (glabella lines, crow's feet) in adults under 65 years of age with significant psychological distress Xeomin (D, A, CH etc.) Toxin A Merz Wry neck (torticollis), eyelid spasm (blepharospasm) and spasticity of the upper extremities in adults Bocouture (D, A, CH, etc.) Toxin A Merz Temporary improvement in the appearance of moderate to severe wrinkles on the upper half of the face (glabella lines, crow's feet, horizontal forehead lines) in adults under 65 years of age with significant psychological stress Nuceiva (CA, EU) Jeuveau (USA) Myobloc (USA) Toxin A Toxin B Evolu's panache Treatment of wrinkles between the eyebrows (glabellar lines) in psychological stress Treatment of cervical dystonia in Adults to reduce the severity of abnormal head position and neck pain Neurobloc (EU) Toxin B Eisai Treatment of cervical dystonia (torticollis) in adults

As of April 2009, all botulinum toxin preparations sold in the US are required to carry a warning label, as determined by the FDA following reports of side effects. A Risk Evaluation and Mitigation Strategy (REMS) is still required for each approved product. The preparations concerned are Botox and Botox Cosmetic, Myobloc and Dysport. The manufacturer Eisai warns against off-label use of "Neurobioc" (toxin B) because it can lead to swallowing disorders and breathing difficulties if the neurotoxin spreads to neighboring structures. [59] Every batch of production units of a botulinum toxin drug must be routinely tested due to its high level of toxicity. Here, the classic LD ₅₀ test is permitted, in which at least 100 mice are injected with the poison into the abdomen. After 3 to 4 days, the LD ₅₀ value is determined from the number of animals killed. The animals suffer muscle paralysis, blurred vision and shortness of breath before suffocating. Around 600,000 mice are used worldwide every year.

The following alternative test methods are described in the European Pharmacopoeia: A phrenic nerve diaphragm preparation in which nerves are dissected out (ex vivo) from previously killed mice or rats and used as a test object; the endopeptidase test, an in vitro molecular biology test in which the venom cleaves a synthetic protein SNAP-25; a local muscle paralysis in which the poison mice in the skin fold between abdomen and hind limb and the extent of paralysis of the hind limb is assessed (determination of efficacy in the mouse with a paralytic endpoint).

Other methods are cell culture tests with a cell line from mouse cancer cells (neuroblastoma) and the immunochemical ELISA test with antibodies from laboratory animals[38]

The ban on animal testing for cosmetics does not apply, since botulinum toxin products are approved as medicines and are injected for use and not just applied. In animal studies with mice and rats, botulinum toxin was found to spread through the central nervous system. Botulinum toxin A is obtained from cultures of Clostridium botulinum. At pH 3.5, the protein is precipitated from the culture medium; the toxin is purified by a sequence of several centrifugation, precipitation and adsorption steps. Other botulinum toxins are obtained analogously from other Clostridium species and strains. The purified toxin can be stored at -70 °C for long periods and thawed without loss of activity. A solution for injection made up of the solid protein and sterile isotonic saline can be stored in the refrigerator for a maximum of four hours.

In medicine in particular, it is already known in principle to accommodate an active substance, for example a toxic substance, in an inert microsphere, ie in a preferably microscopically small nanocarrier, e.g. B. a microcapsule to enclose. For targeted, in particular local medication, for example in a very specific organ or organ area, the nanocarrier should release the active substance as far as possible when it is in this target area. The nanocarrier should therefore release the active substance contained in it according to plan after a certain time or after a certain path or at a certain place.

So far, the procedure for this purpose has been that the material of the nanocarrier wall was a material which after a certain time decomposes to such an extent that it then inevitably releases the active substance contained in it. The medication was therefore administered according to a schedule, with the time at which the nanocarrier would be at a specific location being calculated in advance in order to coordinate the decomposition of the material over time, for example with the selected wall thickness or the selected material itself, to this time.

However, this method is still relatively imprecise with regard to its local accuracy. In particular in the treatment of tumors or the like, it would be desirable if the active substance from the nanocarrier is definitely released only at the precise location of the discovered tumor, which cannot be calculated and predetermined with sufficient time.

Similar tasks and objectives could also arise in the non-medical field, for example in the transport and the precise delivery of substances in the case of highly sensitive, complex device material itself, to be coordinated with this time.

Targeted local release of the active substance can be or will be made possible.

After the active substance has been transported, the nanocarrier can be opened for the targeted release of the active substance when a planned, predetermined local release area is reached.

It can therefore advantageously be provided that the nanocarrier is actively opened when a local target area is reached, so that a targeted release of the active substance can be possible, with the unsafe method according to the prior art, the nanocarrier decomposing over a predeterminable period of time let, is avoided, because with the known method it is not guaranteed whether the predetermined local area is actually reached after the predetermined time. The predetermined period of time can also often only be calculated and set imprecisely. The transport routes cannot always be determined in advance.

A further development can be given in that the active substance in the nanocarrier can be admixed with an action substance which carries out or promotes the opening process through its activation and whose activation is planned and carried out locally in a targeted manner. An action substance can therefore be added to the nanocarrier, which is able to open the nanocarrier in a quasi-remote manner.

One way of opening the nanocarrier can be to increase the pressure inside the nanocarrier until it bursts or bursts, which would be possible by increasing the entropy of the active substance and/or by increasing the kinetic energy of the active substance .

However, another development can provide for the action substance to be stimulated to pierce through the nanocarrier shell, ie the nanocarrier can actually be opened micromechanically.

For all of the aforementioned methods, an action substance can be considered, which can consist of particles whose movement and/or structural formation can be influenced by a magnetic field, with the nanocarrier containing the action substance being able to be exposed to a locally limited magnetic field as planned. A locally arranged permanent magnet, which is implanted in a human body, for example, can be considered for generating such a magnetic field. However, it would also be possible to use electromagnetic magnetic fields, possibly also alternating fields, for example to form them locally with the aid of a computer tomograph. The kinetic energy of the active substance could primarily be increased by an alternating magnetic field, while the entropy could be increased by the static field and a mechanical effect of the magnetic particles could be triggered.

Magnetic particles can be used as the action substance, which form a more or less ring-shaped structure, i.e. can arrange themselves into a ring. On the other hand, when a magnetic field is applied, these particles can align themselves to form a linear chain parallel to the field lines of the magnetic field, so that the ring structure can be broken up. It is evident that the length of the chain structure can be greater than the diameter of the ring structure previously was, such that the wall material of the nanocarrier can be pierced through the formation of a spike-like linear chain. At least it can be mechanically stretched so much that it tears. At the same time, the linear chain structure can have a higher entropy, so that the internal pressure in the nanocarrier also increases, which can also promote the bursting of the nanocarrier. Particles of magnetite or other iron oxides come into consideration as preferably spherical magnetic particles. Ultimately, all highly paramagnetic substances are suitable, i.e. a large number of alloys that cannot be listed individually.

A nanocarrier can contain an action substance, which can consist of particles that can be influenced magnetically.

It can be provided that the magnetically unaffected particles are present in a ring structure, while the magnetically influenced particles are present in at least one linear chain aligned parallel to the magnetic field.

The nanocarrier can preferably have a diameter of about 5 × 10 -7 m, while the diameter of a particle of the action substance can be about 2 × 10 -8 m.

A plurality of particles, preferably 6 to 14 particles, can thus be accommodated in the nanocarrier, in addition to the active substance, so that a chain formed from these particles can certainly be able to open the nanocarrier at a predeterminable location bring. However, a chain of particles that is too long could form disadvantageous labyrinth structures. For their part, the small magnetic particles themselves can be produced and selected in such a way that they can also be completely harmless from a medical point of view.

In addition, the wall of the nanocarrier can be made of a material that is relatively easy to breach. For example, according to the invention, the nanocarrier can consist of cellulose. In principle, both biodegradable and nonbiodegradable substances can be considered as material for the wall of the nanocarrier. For example, albumin, poly(alkylcyanoacrylate) microspheres, ethylcellulose and polyglutaraldehyde microcapsules would be suitable. If possible, the nanocarrier itself can be dimensioned in such a way that it can easily be injected into a bloodstream, for example, and can be transported to a specific local location through the finest capillary vessels. It can be immaterial when the nanocarrier arrives at the target site, since it is only opened anyway when it comes within the sphere of influence of the very limited locally formed magnetic field.

Even substances that are actually toxic can therefore be used for targeted local therapy, for example to destroy carcinomas, tumors or metastases, without damaging the healthy environment of the organ.

If an external magnetic field is applied, the ring structure can break up and the magnetically influenced particles form a linear chain. As a result of the resulting lengthening of the particle chain, the nanocarrier can initially be elongated and deformed and finally opened or ruptured under the resulting pressure. In addition, the linear chain can have a higher entropy compared to the ring structure, so that a higher pressure can be generated within the nanocarrier due to the increase in entropy, which can also promote the bursting of the nanocarrier. The active substance, for example a liquid, can be released from the opened nanocarrier.

The linear chain can be aligned parallel to the field lines of the influencing magnetic field. Under certain circumstances, several parallel chains can also arise. However, since a relatively large number of particles that can be magnetically influenced can be accommodated in the nanocarrier in addition to the active substance, such partial chain strands can also reach such an extent that the nanocarrier can be made to tear.

DE 4 445 969 , EP 718 002 and U.S. 5,788,670 describe syringe barrels of two-chamber syringes for two components to be administered, a first part-cylinder, preferably the head-side part-cylinder, containing an active ingredient to be lyophilized, and a second part-cylinder, preferably the plunger-side part-cylinder, containing a liquid component. During administration, the powdered active ingredient mixes with the liquid component via a bypass.

The European patent application EP 520 618 describes a prefilled syringe consisting of two partial cylinders, the first partial cylinder, preferably the head-side partial cylinder containing the needle or the outlet, containing a lyophilized, powdered medicament, and the second partial cylinder, preferably the plunger-side partial cylinder, containing a second liquid component contains. During administration, the powdered drug mixes with the liquid component via a bypass. The needle-side partial cylinder contains the bypass and is sealed with a stopper at the proximal end. The partial cylinder on the piston side is closed with a plug each at the distal end and at the proximal end. Both part-cylinders are sealed separately by means of the plugs and connected to one another via flanges, with the proximal plug of the head-side part-cylinder and the distal plug of the piston-side part-cylinder being positively connected to each other after assembly and the total length of both plugs being shorter than the length of the bypass.

GB2010681 describes a two-chamber syringe for administering a liquid consisting of two part-cylinders, the first part-cylinder being designed as a needle holder and containing a channel to the outlet and the second, piston-side part-cylinder containing liquid. The first part-cylinder does not contain any substance to be administered. During administration, a stopper, with which the part-cylinder on the piston side is closed at the distal end, is moved in the direction of the syringe head, as a result of which the liquid can be administered via the channel of the first part-cylinder.

FR1099362 describes a two-chamber syringe consisting of two part-cylinders, the first, head-side part-cylinder containing a sterile powder or a sterile liquid, and the second, plunger-side part-cylinder containing a liquid. During administration, the sterile powder or sterile liquid in the first part-cylinder mixes with the liquid in the second part-cylinder via a bypass in the first part-cylinder on the needle side. The part cylinder on the piston side is closed with a stopper at the distal end and a piston at the proximal end. Both part-cylinders are connected to one another by gluing or welding via flanges or by means of a further (over)cylinder, the inside diameter of which corresponds to the outside diameter of the two part-cylinders.

The patent specification EP 0 397 977 A discloses a two-chamber carpule consisting of a cylinder and an attachment, the attachment being provided with radial through-holes so that the solvent released during lyphilization can escape through the through-holes.

Clostridium botulinum toxin complex type A (Mr 900,000) has been used for the therapy of various dystonia for several years. Two different preparations are currently approved for the treatment of blepharospasm, hemifacial spasm and spasmodic torticollis, namely BOTOX® and DYSPORT®. The therapy of other diseases of the nervous system (e.g. spasticity, migraine, lumbago, cervical syndrome, hypersalivation) is currently being clinically tested. The preparations are also used for cosmetic indications such. B. hyperhydrosis and pronounced wrinkling in the facial area.

Botulinum toxin complexes are composed of a mixture of clostridial proteins. These are hemagglutinins of different molecular masses, a non-toxic, non-hemagglutinating protein (Mr 120,000) and two neurotoxin molecules (Mr 150,000). They form a loose complex responsible for oral toxicity in food poisoning. In contrast to the pure neurotoxin, the complex withstands the aggressive environment in the gastrointestinal tract and enables enteral absorption of the neurotoxin, which reaches the target cells via the bloodstream or the lymphatic system and there triggers a blockage of the transmitter release. This leads to paralysis of the striated and smooth muscles and to the drying up of various vegetative functions. Poisoned patients die from insufficiency of the respiratory muscles. Since the pure neurotoxin is broken down in the gastrointestinal tract and is therefore not resorbed enterally, it is not toxic after ingestion. When applied parenterally, the effects of the complex do not differ from the effects of the neurotoxin, since the complex breaks down into its components in the tissue and only the neurotoxin is taken up into the target cells.

For therapeutic use, the complex is injected directly into dystonic or spastic muscle, where the neurotoxin is released from the complex and produces the desired pharmacological effect. Although the complex is applied in extremely small doses (1-25 ng, depending on the indication and the size of the affected muscle), after repeated injections, a significant number of patients develop specific, neutralizing antibodies also against the neurotoxin are directed. These patients no longer respond to either of the two preparations. However, they could be treated with other toxin subtypes, none of which are approved for therapy.

The formation of specific antibodies is favored by two factors. On the one hand, the neurotoxin, fixed in the complex, remains in the tissue for a long period of time and can activate immune cells migrating into the tissue to form antibodies. The long residence time does not lead to increased absorption in the target cells, because poisoned cells can no longer absorb the toxin. The toxin that slowly dissociates from the complex is therefore only immunologically effective. On the other hand, the proteins contained in the complex strengthen an immune response. Hemagglutins are lectins, proteins that are characterized by a high affinity for certain sugars. Due to their binding to sugar structures, lectins have an immune-stimulating effect. It could be shown that the lectins concanavalin A, phytohaemagglutinin and pokeweed mitogen activate T and B lymphocytes. The hemmagglutins of the botulinum toxin complex, which also bind to membrane-bound sugars, can therefore act in a similar way as immune adjuvants and contribute to the formation of antibodies and thus to therapy failure.

As an alternative to the two commercial preparations made from botulinum toxin complexes, BOTOX® and DYSPORT®, a new drug has been developed which contains only pure neurotoxin and is free from haemagglutinins and other exogenous proteins. Because of its lower molecular mass, it diffuses faster to the target cells into which it is taken up before immune cells attracted to hemagglutinins are activated. In antigenicity studies we found that this preparation - in contrast to commercial preparations - does not trigger the formation of antibodies. When this newly developed drug is used therapeutically, there should be no antibody-related therapy failure, even after repeated applications. Furthermore, it could be shown that the pure neurotoxin, because of its immediate bioavailability, is still suitable for the therapy of patients who have developed an antibody titer during treatment with BOTOX® or DYSPORT®.

A neurotoxin, e.g., a botulinum neurotoxin from Clostridium botulinum type A, can be free of other proteins of the botulinum toxin complex, in particular free of hemagglutins and non-toxic non-hemagglutinating proteins.

A neurotoxin, for example a botulinum neurotoxin from Clostridium botulinum type B, C, in particular C1, D, E, F and/or G, can be free from other proteins of the botulinum complex, in particular free from hemagglutins and non-toxic non-hemagglutinating be proteins.

This neurotoxin may be characterized by a Clostridium botulinum type B botulinum neurotoxin.

A neurotoxin can be characterized by a botulinum neurotoxin obtained in a manner known per se or by a recombinant botulinum neurotoxin.

Neurotoxin may be characterized by a botulinum neurotoxin of a grade (high purity botulinum neurotoxin) that results in no or reduced induction of neutralizing antibodies compared to its botulinum toxin complex.

Furthermore, a neurotoxin can be present as a lyophilized preparation with an optional content of a therapeutically customary carrier, diluent and/or adjuvant.

A neurotoxin can also be present as a liquid preparation, in particular as an infusion preparation, preferably on an aqueous basis, optionally obtainable by taking up a lyophilized preparation according to the invention in water or saline.

A neurotoxin may be intended for therapy of disorders of the nervous system.

Furthermore, a neurotoxin can be provided for the therapy of secondary non-responders.

Furthermore, a neurotoxin according to the invention can be provided for the therapy of dystonia, in particular spasmodic torticollis or blepharospasm, spasticity, in particular equinus foot, hemifascial spasms, migraine, lumbago, cervical syndrome or hypersalivation.

Furthermore, a neurotoxin according to the invention can be provided for cosmetic indications, in particular hyperhydrosis or pronounced wrinkling in the facial area.

Furthermore, a neurotoxin according to the invention can be provided for the therapy of patients with neutralizing antibodies against a botulinum complex.

Furthermore, a neurotoxin with a botulinum neurotoxin from Clostridium botulinum type A can be provided for the therapy of patients with neutralizing antibodies against a botulinum toxin complex from Clostridium botulinum type A.

Finally, a neurotoxin according to the invention with a botulinum neurotoxin from Clostridium botulinum type B can be provided for the therapy of patients with neutralizing antibodies against a botulinum toxin complex from Clostridium botulinum type B.

For example, 20 rabbits were injected intracutaneously with 25 U of BOTOX® over a period of 12 weeks at 14-day intervals (5 injections). Serum was obtained after 3 weeks and then at intervals of 14 days. Antibodies against Clostridium botulinum toxin A were detected using an enzyme immunoassay in which the homogeneous neurotoxin was immobilized on a microtiter plate. Antibodies binding to the neurotoxin were quantified using a second, enzyme-labeled antibody. Antibodies could be detected in sera from 17 animals after 11 weeks. The biological activity test showed that 12 of the 17 sera contained neutralizing antibodies.

After it was shown that the complex of neurotoxin and haemagglutinins and the non-toxic, non-haemagglutinating proteins triggers the formation of neutralizing antibodies, the immunogenic effect of the pure neurotoxin was tested in a further example. For this purpose, 8 rabbits were treated with the toxin complex and 12 rabbits with the pure toxin. According to the method described above, 25 U of the respective preparation were applied intracutaneously. The amount of neurotoxin measured by weight was the same in both preparations (200 pg/dose) as detected in an ELISA. BOTOX® also contained complex proteins (approx. 800 pg/dose). Four of the eight animals treated with BOTOX® showed an antibody titer in the ELISA, while no antibodies against the pure neurotoxin could be detected in the 12 animals treated with pure neurotoxin. The result was confirmed in the biological activity test. All four rabbit sera contained neutralizing antibodies that prevented toxin action.

In another experiment, the formation of antibodies by BOTOX® was compared with that by DYSPORT®. For this purpose, ten rabbits were treated either with BOTOX® (group 1), with DYSPORT® (group 2) or with the pure neurotoxin (group 3) according to the scheme described del. While more than 50% of the animals in groups 1, 2 formed a neutralizing antibody titer, the sera from the animals in group 3 were free of antibodies.

In another experiment, a patient (age 45 years) who had been treated with BOTOX® for a period of 5 years for spasmodic torticollis had developed an antibody titer of 3 mU/mL serum. Neither BOTOX® nor DYSPORT® were therapeutically effective in this patient. A therapy attempt with the pure botulinum neurotoxin in a dose of 145 units, which was equivalent to the last injected dose of BOTOX®, led to relaxation of the muscles, normalization of the head posture and disappearance of the muscle pain within 72 hours. Undesirable effects did not occur.

In yet another experiment, a patient (age 52 years) was treated with BOTOX for 3 years for cerebral palsy. He had developed an antibody titer of 10 mU/mL serum, so the therapy had to be discontinued. The injection of 200 units of pure neurotoxin enabled a successful therapy.

Botulinum toxin as a typical example of a neurotoxin secreted by bacteria is a proteotoxin produced by Clostridium botulinum. Botulinum toxin is classified into eight serum groups such as A, B, C1, C2, D, E, F and G depending on their antigenicity. Among them, the type A toxin was the first to be purified and crystallized (Lamnna, COE NcElroy and HW Eklund: Science, 103: 613-614, 1946).

Hitherto, the production process for crystalline type A botulinum toxin requires complicated procedures, which are performed in the order: acid precipitation, water washing, extraction with calcium chloride and acid precipitation, extraction with phosphate buffer, precipitation with ethanol, and extraction and settling in an ammonium sulfate solution. In addition, since the production is complicated, it requires skilled engineers skilled in observation and gives only low toxin recovery rates, and the required amount and the effect of this method fall short of expectations. In addition, although a method was used, e.g. B. has the combination of a protamine treatment, ion exchange chromatography and gel filtration, the crystallization effect is not beneficial.

One of the problems in crystallizing type A botulinum toxin is to obtain as much as possible of an easily crystallizable macromolecule, and the other problem is to avoid substances that prevent crystallization and mainly contain nucleic acid contained in a culture solution , without denaturing the toxin. The first problem can be solved by selecting a strain that secretes botulinum type A toxin with a molecular weight of 500,000 to 900,000, and the solution to the second problem depends on how to remove the nucleic acid under mild conditions.

Type A botulinum toxin can be crystallized without loss of toxicity using a nucleic acid scavenger which may contain a natural substance such as chitoxanent.

First, from strains secreting botulinum type A toxin, strains capable of secreting L toxin of molecular weight 500,000 and LL toxin of molecular weight 900,000 having a high hemoagglutination titer and high toxicity can be selected. The strains can be cultivated in a suitable culture medium, e.g. B. an NZ amine culture medium, an acid can be added to a culture filtrate to cause precipitation, and a phosphate buffer solution in which the molar concentration may be changed can be used instead of calcium chloride to eliminate the toxic components to be able to extract effectively from the precipitate. Further, the liquid extract may be repeatedly subjected to, e.g., pH control, overnight standing, centrifugation, dissolution in an acetate buffer solution, pH modification and centrifugation to obtain, e.g., a clear overlayer. A nucleic acid scavenger can be added to the overlayer under a very effective condition.

Traditionally, calcium and alcohol have been used to remove the nucleic acid. However, no remarkable result can be obtained even if the conditions such as concentration and pH of the nucleic acid eliminator are changed. Further, protamine is known as a nucleic acid remover, however, although it works excellently as a remover, it causes precipitation of not only nucleic acid but also necessary proteins. In addition, it creates a problem of uneven homogeneity between lots, which is a fatal disadvantage of natural products. Also must a procedure can be added to remove excess promatin from the resulting precipitate. On the other hand, the chitoxane (trade name of products produced by Dainippon Seiyaku Co.) is a natural high molecular weight substance, a natural substance produced by dividing chitin (poly-N-acyl-β-D- glucosamine) contained in the outer shells of e.g. B. contained by crab and shrimp is deacetylated, and C 7 and C 9 produced by reducing the molecular weight thereof can efficiently remove nucleic acid without loss of the toxin occurs. When salting out with ammonium sulfate is carried out after removing nucleic acid with chitoxan, the excess chitoxan does not precipitate, and after dissolving the salted out toxin in a phosphate buffer solution, ammonium sulfate was gradually added and left to crystallize the toxin easily .

In the production process according to Duff's method, the yield of crystalline type A botulinum toxin was only 1.4 - 3.1 taking the culture solution as 100%. Although it could be increased by about 5% by improving the extraction method, the yield of the toxic activity could be increased to 10% with a method in which the nucleic acid scavenger is chitoxan.

In view of the above, the nucleic acid can be efficiently removed after the generation of the crystalline type A botulinum toxin, the yield of the toxic activity can be improved, and as a result, a remarkable effect as a therapeutic drug for strabismus and blepharospasm can be achieved.

For example, a manufacturing method may include the following steps

Cultivation: Clostridium botulinum, Hall strain, NZ amine culture medium, culture left at 35°C for 4 days.

Acid precipitation 1: Sulphurous acid added: pH adjusted to 3.5 with 3N H2SO4, leave overnight at 4°C.

Overlayer removed, (siphon) centrifugation

Precipitation: Dissolved in distilled water, pH adjusted to 5.0 with 0.1N NaCl.

centrifugation extraction

Precipitation: 0.2 M phosphate buffer solution (pH 6.0) added.

centrifugation overlayer

Precipitation: 0.5 M NaCl containing 0.1 M Tris-HCl buffer solution (pH 7.5) added.

centrifugation acid precipitation 2

HCl added: pH adjusted to 3.7 with 2N HCl, leave overnight at 4°C.

Overlayer removed (siphon) centrifugation

Precipitation: Dissolved in 0.05 M acetate buffer solution (pH 5.0).

Centrifugation C-9 treatment

Overlayer: 5% C-9 solution added to 0.1% over 1 min, stirred over 10 min in an ice box.

centrifugation overlayer

Added Ammonium Sulfate: Solid ammonium sulfate added to make a 50% saturated solution. Leave overnight at 4°C.

centrifugation

Precipitation: Precipitation dissolved in 0.03 M phosphate buffer solution at pH 6.8.

Dialysis: Dialyzed with 0.03M phosphate buffer solution pH 6.8.

crystallization

Ammonium Sulfate Added Ammonium sulfate added until the solution exhibits a fishy silver color, being careful that the final concentration does not exceed a 0.9 M concentration.

Crystal Settlement: Leave at 4°C

Preparation of 5% C-9 solution

Distilled water is added to C-9 (Kurimover manufactured by Dainippon Seiyaku, Lot No.: K143) at a ratio of 10:1. Further, 1N HCl is supplied in a ratio of 2 and stirred with a stirrer for 2 hours. pH is adjusted to 4-6 with 1N sodium hydroxide. The solution is diluted with distilled water to a 5% solution.

Many urological medical conditions originate from spastic dysfunction of the sacral reflex arcs. Examples of such conditions include pelvic pain (e.g., interstitial cystitis, endometriosis, prostatodynia, urethral instability syndrome), pelvic myofascial elements (e.g., sphincter elevator, dysmenorrhea, anal fistula, hemorrhoids), urinary incontinence (e.g., unstable bladder, unstable sphincter), Prostate disease (eg, BPH, prostatitis, prostate cancer), recurrent infection (additional to spasticity) and urinary retention (additional to sphincter spasticity, bladder rejuvenation hypertrophy), and neurogenic bladder dysfunction (eg, Parkinson's disease, spinal cord injury, stroke, MS, spasm reflex).

The prostate is a part-glandular and part-fibromuscular gland of the male reproductive system. During the aging process, the prostate tends to enlarge (hypertrophy). This enlargement of the prostate can lead to urethral blockage and voiding dysfunction.

Enlarged prostate is common in older men. Lytton et al. (Lytton, B., Emery, J.M., and Harvard, B.M. (1973) 99:639-645) estimated that a 45-year-old male has a 10% risk of having prostate surgery by age 70. This proportion of the population is expected to increase sharply in the coming years. Therefore, the number of men with prostate enlargement will also increase. Based on estimates of population growth, the number of prostate resections performed annually will also increase.

The urethra passes through the prostate (prostatic urethra) on its way to the external urethral opening. The prostate has five distinct lobes which are visible in the human fetus at 12 weeks of age (Lowsley, O.S., Am. J. Anat (1912) 13:299-349). Although the branching of the lobes found in the fetus is not visible in the prepubertal prostate, the laterally medial anterior and posterior lobes are used to describe the enlarged prostate.

A somewhat recent finding is that the prostate also consists of several morphologically distinct zones (McNeal, J., Urol. Clin. North Am. (1990) 17(3): 477-486). The majority of the gland volume consists of the peripheral zone (approximately 70 to 75%). The remainder of the glandular volume is divided into the central zone (about 20 to 25%), the transitional zone (about 5 to 10%), and the glandular zone surrounding the urethra (about 1%).

McNeal (1990) reported that BPH develops in the transition zone and the glandular zone surrounding the urethra. BPH nodules develop either within or immediately adjacent to the anterior sphincteric zone. The transition zone is a small area near the urethra, closely associated with the proximal urethral sphincter. The stroma of the transition zone is dense and compact and is unusually prone to neurological growth control disorders. Their glands permeate the sphincter, while the muscle fibers of the sphincter do penetrate transitional stroma. The glandular zone surrounding the urethra has a similar urogenic cavity origin as the transitional zone.

BPH may be associated with increased amounts of stroma relative to the epithelium (Bartsch, G. Muller, H.R., Oberholzer, M., Rohr, H., P., J. Urol. (1979) 122: 487-491). A significant portion of the stroma is smooth muscle (McNeal, 1990) controlled by the sympathetic nervous system. The contractile properties of this smooth muscle may account for the dynamic component of block in BPH (Bruschini, H. et al. (1978) Invest. Urol. 15(4):288-90; Lepor, H. (1990) Urol. Clin. North Am. 17(3): 651-658).

In addition to the sympathetic control of the prostatic stroma, the prostate is highly innervated. The prostatic nerve fibers enter the prostate from the posterior lateral side, with a concentration of ganglia near the intersection of the prostate and seminal vesicles (Maggi, CA, 1993 edition, Nervous control of the Urogenital System, Harwood Academic Publishers; Higgins, JRA et al Gosling, JA (1989) Prostate Suppl. 2:5-16).

Acetylcholine (ACH), neuropeptide Y (NPY), vasoactive gut peptide (VIP), and norepinephrine fibers have been described in this gland. A strong neural plexus of ACH-positive neuron bodies is connected to secretory acini in all parts of the gland. Some of the ACH fibers also contain NPY neurons. VIP-containing neurons were found associated with ACH-containing nerve cell bodies. Occasionally, neurons were found between the ACH-contaminated nerve fibers, suggesting that both NPY and norepinephrine neurons supply smooth muscle (Higgins, J.R.A. and Gosling, J.A. (1989) Prostate Suppl. 2:5-16).

Autonomic nerves are evenly distributed between the central and peripheral zones of the prostate (Higgins, J.R.A. and Goslin, J.A. (1989) Prostate Suppl. 2:5-16). Peripheral neuron control is similar. In addition, there is no difference in the type of nerve fibers found associated with either the epithelial or stromal elements of the gland.

The anatomical studies of the nerve fiber types in the prostate linked to other studies of the innervation of the prostatic stroma (Brushing H., Schmidt, RA, Tanagho, EA (1978) Invest. Urol 15(4): 288-290; Watanabe, H., Shima, M., Kojima, M., Ohe, HL (1989) Pharmacol Res 21 (Suppl 2): 85-94) suggest that choline-mediated innervation influences epithelial behavior, while adrenaline-mediated innervation influences stromal atonus (excitability) affected. These observations gave reason to use e.g. B. Alpha blockers in the treatment of BPH. The effects of alpha-blockers (Downie, JW and Bialik, GJ (1988) J. Pharmacol. Exp. Ther. 246(1): 352-358) may also contribute to improvements in the symptoms of BPH as a result of dysfunctional behavior dulling striated sphincter by the alpha blockers.

Studies have also shown that there are several tachykinins (eg, substance P (SP), calcitonin gene-related peptides (CGRP), neurokinin A, bradykinin, and nerve growth factor (NGF)) that can affect smooth muscle tone ( Hakanson et al (1987) Neuroscience 21(3):943-950). Neurotransmitter receptors have been measured throughout the prostate (eg, NPY, VIP, SP, Leu-enkephalin (L-enk), met-enkephalin, 5-HT, somatostatin, acetylcholinesterase-positive fibers (ACTH), and dopamine beta -Hydroxylase (DBH) (Crowe, R., Chapple, CR, Burnstock, G., The Human Prostate Gland: A Histochemical and Immunohistochemical Study of Neuropeptides, Serotonins, Dopamine beta-Hydroxylase and Acetylcholinesterase in Autonomie Nerves and Ganglia). Fluctuations in receptor density at different sites in the prostate in benign prostatic hyperplasia.

Changes in electrophysiologically recorded cellular behavior and in the concentration of neuropeptides within the spinal cord have been shown to be secondary to mechanical clamping of rat tail muscles, catheter stimulation of the posterior urethra, and electrical stimulation of a peripheral nerve. Dyssynergia between the detrusor and the urethral sphincter is a prominent finding in prostatodynia patients. Prostate denervation has been shown to cause dramatic changes within the prostate epithelium. Thus, there is evidence that experimentally induced changes in neurological influences in the sacrum, spinal cord, bladder, or urethra can be produced by mechanical, electrical, chemical, or thermal (microwave, laser) methods to alter a stimulus state. However, no attempts have been known to date to use neurotoxins for therapeutic applications.

There is little association between the degree of prostate enlargement and the severity of symptoms. While 80% of men in their 70s have BPH on transrectal ultrasound, only 20% desire surgery (Coffey, DS and Walsh, PC (1990) Urol. Clin. North Am. 17(3): 461-475) , the treatment of choice for BPH (Fowler, FJ Jr., Wennberg, JE, Timothy, RP (1988) J. Amer. Med. Assoc. 259(20): 3022-3028). Symptoms of irritation can be far more severe than those expected based on the size of the prostate. Symptoms may worsen after surgical treatment of BPH by procedures such as transurethral resection of the prostate (TURP) (Christensen, Aagaard, MMJ, Madsen, PO (1990) Urol. Clin. North Am. 17(3): 621-629) , balloon dilatation (Dowd, JB and Smith, JJ III (1990) Urol. Clin. North Am. 17(3): 671-677) or prostatic hyperthermia (Baert, L., Ameye, F., Willemen, P et al (1990) J Urol 144:1383-1386). However, symptoms persist in 15% of all BPH patients (Beart, L., Ameye, F., Willemen, P., et al. (1990) J. Urol. 144:1383-1386); Wennberg JE, Mully AG, Hanley D, Timothy RP, Fowler FJ, Roos RP, Barry MJ et al (1988) J Amer. Med. Assoc. 259: 3027-3030). Up to 25% of BPH patients have add-on treatments in long-term follow-up studies, suggesting that the surgical approach does not address the fundamental mechanisms that produce BPH, ie, the defective neurological influence (controlling mechanism) on the integrity of the lower urinary tract.

The need for repeated surgeries, the morbidity and mortality associated with TURP, and the cost of surgery have led to the development of some non-surgical approaches, such as e.g., androgen ablation (McConnell, JD (1990) Urol. Clin. North Am. 17(3):661-670) and the use of the alpha-blockers discussed above, but few medical or surgical treatments have so far restored voiding behavior to normal (flow rate of about 25 cc/sec and void volume of about 400 cc).

The neurotoxin blocks synaptic function. Such blockage produces selective denervation and e.g. B. atrophy of the prostate and a reversal of irritation symptoms associated with prostate enlargement. The neurotoxin can cause dysfunction of the presynaptic neuronal terminal by specifically binding and blocking acetylcholine release at myoneural intersections. Such a neurotoxin can e.g. B. botulinum toxin type A (Botox, allergen).

Preferably, the neurotoxin is safe, highly selective, and easy to administer even when combined with other therapies. The neurotoxin can be administered by any means.

A therapeutically effective amount of neurotoxin can be that dose sufficient to block neuronal activity for at least a week, more preferably 1 month, more preferably about 6 to 8 months or longer. Dosing can be single or cumulative (series dosing) and can be readily determined by those skilled in the art. Neurotoxin can be administered in series (i.e., once a month, once every six months) so that the therapeutic effect can be optimized. Such a dosing schedule can easily be determined by a person skilled in the art based on e.g. B. patient size and the condition to be treated and depends on many factors including the selected neurotoxin, the condition to be treated, the degree of irritation and other variables. A suggested course of treatment for BPH may be 200 units every three days up to LD50 for botox or approximately 2500 units.

The above treatment methods should be particularly useful for the long-term control of urinary retention without the need for surgical intervention. Furthermore, some methods can provide control of urinary retention in a very selective manner, without the potential side effects and medical errors associated with current treatment modalities.

The basis for treatment of neurological-urological conditions can be the elimination or adaptation of the nerve base for dysfunctional regulation of the affected tissue. It may be preferred that neurotoxin causes a long-lasting blockage of synaptic function, preferably more than a week, more preferably more than a month, most preferably six to eight months or more. A preferred neurotoxin may be botulinum toxin, particularly botulinum toxin A, particularly botox (allergen).

The toxin can be formulated in any pharmaceutically acceptable formulation in any pharmaceutically acceptable dosage form. Such dosage forms and formulations include liquids, powders, creams, emulsions, pills, lozenges, suppositories, suspensions, solutions and the like chen. The toxin can also be used in any pharmaceutically acceptable dosage form supplied by any manufacturer.

The neurotoxin may be or include botulinum toxin type A. Therapeutically effective amounts of botulinum toxin can be any amount or dose below a toxic dose, e.g. B. less than about 3000 units/70 kg male, preferably between 100 units/70 kg male to 1200 units/70 kg male. Dosing can be as a single dose or as a divided dose, e.g. B. distributed over the course of 4 weeks.

For example, the neurotoxin can be injected urethroscopically into the prostate at 200 units in single or serial dosing. Preferably, the neurotoxin is injected every three days until a therapeutic effect is achieved or until about 2500 units are reached.

• Gewebevorbereitung für Lichtmikroskopie: Gewebe können in 6% Paraformaldehyd in 0,1 M Phosphatpuffer fixiert, bei pH 7,2 für 24 Stunden, in abgestuftem Alkohol und Xylen dehydriert und in Paraffin eingebettet werden. Querschnitte können geschnitten und mit passenden Färbemitteln, wie z. B. Hämatoxylin/Eosin, eingefärbt werden.
• Gewebevorbereitung für Elektronenmikroskopie: Gewebe können entnommen und in 2,5 Glutaraldehyd in 0,1 M Phosphatpuffer fixiert, bei pH 7,2 für 1 Stunde bei 4°C, dann mit 0,1% Osmium-Tetroxid für eine Stunde bebrütet und in EPON eingebettet werden. Extrem dünne Querschnitte (80 nm) können hergestellt und mit Bleicitrat/Uranylacetat eingefärbt und mit einem Elektronenmikroskop (Philips, Modell 201) untersucht werden.

The following techniques can be used:

• Tissue preparation for light microscopy: Tissues can be fixed in 6% paraformaldehyde in 0.1 M phosphate buffer, at pH 7.2 for 24 hours, dehydrated in graded alcohol and xylene, and embedded in paraffin. Cross-sections can be cut and colored with appropriate stains, e.g. e.g. hematoxylin/eosin.
• Tissue preparation for electron microscopy: Tissues can be removed and fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.2 for 1 hour at 4°C, then incubated with 0.1% osmium tetroxide for one hour and incubated in EPON be embedded. Extremely thin cross-sections (80 nm) can be made and stained with lead citrate/uranyl acetate and examined with an electron microscope (Philips, model 201).

Staining for Apoptosis: The tissue can be fixed and embedded as described above. Tissues can be deparaffinized and reacted with Proteinase K (Boehringer). They can be further treated with peroxidase and TDT enzyme and kept in a humidifier at 37°C for one hour. The cross sections can be washed and anti-digoxigenin peroxidase can be added for 30 minutes, followed by staining with nickel-DAB (di-aminobenzene).

Immuno-Tissue Chemistry Studies: The Presence of the Neuropeptides VIP, SP, NPY, L-Enk and Calcitonin Gene-Related Peptides (CGRP) and the Expression of Transforming Growth Factor-beta (TGF-beta), Transforming Growth Factor-alpha (TGF-alpha) , epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) can be determined in prostate tissues using appropriate monoclonal antibodies. The use of neurotoxins can lead to atrophy of the prostate, which should be reflected by lower levels of growth factors in the treated prostate tissue.

Cross sections can be incubated overnight at room temperature with primary antibodies, followed by immunostaining with avidin-biotin-peroxidase (Vectastain Elite ABC, Vector Labs, USA). Polyclonal rabbit antiserum against the neurotransmitters VIP, CGRP, SP, NPY and L-Enk (Peninsula Labs, USA) can be used in these formulations at dilutions from 1:8000 to 1:12000. Immunocytochemical controls can consist of pre-absorbing the primary antiserum with appropriate antigens, or substituting them with normal serum (Blasi, J., Chapman, ER, Yamaskai, S., Binz, T., Niemann, H., and Jahn, R (1993) The EMBO Journal 12:4821-4828 Black JD and Dolly JO (1986) J Cell Biol 103:535-544 Linial M (1995) Is J Med Sci 31 : 591-595). After mounting on slides, cross sections can be counterstained with eosin, dehydrated, and coverslipped.

Western Blot Analysis of Growth Factor Expression: Treated and untreated prostate cell homogenates can be assayed for growth factor expression by Western blot analysis. Cell homogenate protein can be separated by electrophoresis on SDS-PAGE (7, then electrophoretically transferred to nitrocellulose paper overnight (Towbin, H. et al. (1979) Proc. Nat. Acad. Sci. 76(9): 4350-4379). The Nitrocellulose paper can be soaked in 0.5% non-fat dry milk dissolved in phosphate-buffered saline for one hour at room temperature and further soaked in blocking solution (2% bovine serum albumin in 10mM Tris/0.15M NaCl /0.1 sodium acid, pH 7.4) The nitrocellulose membranes can be cleaned with antibodies (IgG fractions of anti-TGF-beta, anti-TGF-alpha, anti-EGF and antibFGF) using protein A (1× 106 cpm/ml) in a blocking buffer for 1 hour The membrane can be interleaved with PBS containing Nonidet P-40 be washed between incubations. XO-mat AR2 film (Kodak) can be exposed on the membrane at -70°C and the films developed to examine growth factor expression.

Determination of c-fos and c-myc expression: Expressions of c-fos and c-myc in treated and untreated prostate tissue can be determined by Northern blot analysis as follows. Tissue can be homogenized in lysis buffer for 15 seconds or until tissue is homogenized. Sodium acetate can be added and the solution mixed by stirring. An equal amount of water-saturated phenol can be added and mixed by inversion, followed by addition of chloroform/isoamyl alcohol. The solution can be vortexed vigorously for 30 seconds and placed on ice for 15 minutes. The solution can be centrifuged at 4°C for 10 to 20 minutes. After centrifugation, the aqueous phase can be carefully aspirated and placed in a new polypropylene tube. A volume of isopropanol can be added and the solution mixed by stirring. The solution can be placed in a -20°C freezer for at least 60 minutes to precipitate RNA. After precipitation, the tube can be centrifuged for 10 minutes and the supernatant poured off, allowing the RNA pellet to remain. One ml of ethanol can be added and the tube centrifuged for an additional 10 minutes. The aqueous phase can be separated and the precipitate washed with 100% ethanol by vortexing. The RNA precipitate can be redissolved in 0.4 ml lysis buffer. The RNA can be precipitated by adding 100% ethanol and incubating at -20°C in the freezer for at least 60 minutes. The solution can be centrifuged and the supernatant poured off. The RNA concentration can be determined by diluting 5 µL of the sample in 995 µL of DEPC water and measuring the uptake ratio at 260/280 nm.

For example, three patients with intractable voiding disorders were treated with botulinum toxin (Botox) injections as follows. Patient 1 was a 47-year-old male who was incontinent following a cervical spine injury (level C6-C7) sustained 14 months previously. Urodynamic studies at presentation showed a bladder capacity of 30 cm3 and a weak sphincter (peak urethral pressure of 40 cm of water). He has been unsuccessful with multiple pharmacological treatments and hypersensitive to penis clamps/condoms. He received 200-unit botulinum toxin injections into the narrowed bladder four times a week for a total dose of 800 units. After the injection, his bladder capacities between 300 and 400 cm ³ with oxybutinin and 150 to 200 cm ³ without oxybutinin. Peak bladder pressure was 200 cm of water before injection compared to a post-injection bladder pressure of 40 cm of water. The patient was continent with a penis clamp after treatment with botulinum toxin. In addition, walking ability and erections improved due to reduced bladder spasticity. Patient 2 was a 55-year-old T12 paraparetic woman secondary to a traumatic injury sustained 14 years earlier. The patient presented with urgent incontinence, and was scheduled to self-catheterize every 2 hours during the day and twice per night. The patient received injections into the lateral bladder wall in two weekly injections of 200 units each for a total of 400 units of botulinum toxin. Void diary data showed pre-injection capacities of 150 to 200 cm ³ . After the injection, the bladder capacity reported in the diary data increased to 300-400 cm ³ . In addition, the patient no longer had bothersome, constant pressure dysfunction, was able to sleep through the night, and was continent with self-catheterization every 4 hours. Patient 3 was a 65 year old male with disabling perineal pain following radiation treatment for prostate cancer. Medical therapy was unsuccessful in the patient. He was treated with a 200 unit injection of botulinum toxin into the external urethral sphincter. The patient experienced dramatic relief from testicular pain and significantly less severe penile shaft pain. Erections were not affected.

In another example, rats were injected with single and serial doses of botulinum toxin (Botox) in the abdominal lobes of the prostate. The prostates were "harvested" (taken out) at various time intervals to determine the smallest effective dose and the morphological and physiological changes that occur over time. The smallest effective dose is defined as the dose exhibiting a decrease in prostate volume. To assess response to electric field stimulation, preparations were mounted between two platinum electrodes placed in an organic bath. The tension of the preparations is adjusted. Stimulation through the wall of the nerves is performed using a Danted Neuromatic 2000 stimulator delivering single square wave pulses at supramaximal voltage of 0.8 millisecond duration at a frequency of 0.5 to 80 Hz. The polarity of the electrodes is changed after each pulse by means of a polarity changing unit. The length of a series is 5 seconds and the interval of the series is 120 seconds. Isometric tension is recorded using a Gould thermal field 8-channel recorder. Separate experiments are performed to determine the preload voltage that produces optimal responses. In addition, the We The effect of electric field stimulation in the presence of different concentrations of individual neuropeptides was determined. These neuropeptides are 10-20 µM adrenaline, 10 µM clonidine, 5-50 mM regitin, 10 nM-0.1 µM acetylcholine, 1-3 µM atropine, 1 nM-10 µM nifedipine, 1-10 nM VIP and 1-250 nM NPY. The effect of nitroprusside (a nitric oxide-releasing substance) and methylene blue (a guanylate cyclase inhibitor) on prostatic tone and contraction due to field stimulation will also be examined in these tissues.

In yet another example, in order to determine whether there is an interaction between the neurotoxin and testicular-derived hormones, studies examining the interaction of the neurotoxin with hormonal components were conducted. These studies compare botulinum toxin-treated prostate tissue collected from rats that have undergone orchiectomy (hormone-poor rats) and prostate tissue from botulinum toxin-treated rats that have not undergone orchiectomy. Fifty-two age-matched rats were treated as described below. Four healthy rats will undergo a sham operation consisting of anesthesia induction, exposure of the prostate and injection of 0.2 cm ³ saline into the left ventral lobe of the prostate. Three rats underwent bilateral orchiectomy without injection of the prostate (control of hormone elimination), five rats undergo orchiectomy and injection of 0.2 ml saline in the left abdominal lobe (control of hormone elimination and surgical load). Four groups of rats receive only botulinum injections of 0.5, 1.0, 1.5 and 2.5 units (hormonally intact experimental rats). Sixteen rats underwent bilateral orchiectomy. Eight of these rats are treated with a single injection of 2.5 units of botulinum toxin in the left abdominal lobe 5 weeks after surgery. All rats were sacrificed after six weeks and the excised prostate was prepared for examination as described above. A similar atrophic effect on the glandular cellular tissue is expected.

The problem with the injection of botulinum toxin is that even the smallest amounts are sufficient to paralyze nerves. In the case of a botox injection, for example, larger parts of human tissue can be unintentionally paralyzed.

It is therefore the object of the present invention to provide a device and a method with which botulinum toxin can be applied as precisely as possible.

This problem is solved by an injection device having the features of claim 1 and a method having the features of claim 9. The respective dependent claims relate to advantageous embodiments.

According to the invention, an injection device is provided which has at least one coil which is set up to locally concentrate active substance located in the cavity and/or to align molecules of the active substance by means of a magnetic field generated by the coil.

The active ingredient is thus already concentrated locally in the injection device, so that it is also present in a concentrated form in the injection jet ejected from the injection device. This means that the active ingredient can be injected precisely into human tissue, for example.

The coil may comprise an air coil, which is preferably wound around the cavity. Alternatively or additionally, the coil can have a permanent magnet.

The ejection unit can have a piston accommodated and movable in the cavity, a vacuum pump fluidly connected to the cavity, an air inlet fluidly connected to the cavity and/or an overpressure tank fluidly connected to the cavity.

The ejection unit can have a piston accommodated and movable in the cavity, wherein the piston can have a first part and a second part, wherein the first part and/or the second part can be movable such that when the piston moves into a first A fluidic connection between the cavity located in front of and behind the piston can be produced through the first part and the second part. When the piston moves in a second direction, preferably opposite to the first direction, the first part and/or the second part can be movable in such a way that the fluidic connection can be separated.

The outlet can be fluidically coupled to a preferably attachable nozzle or a preferably attachable injection needle and/or be or have such.

The injection device can have a magnetically shielding casing, so that the injection device can be shielded from external magnetic fields.

The active substance can have particles that can be influenced magnetically and/or be encapsulated with particles that can be influenced magnetically.

A membrane may fluidly seal the outlet, wherein the membrane may be configured to be pierced by a piercing cannula so that a fluidic connection may be established through the membrane.

1. a. Erzeugen eines Magnetfelds mit einer Spule der Injektionsvorrichtung, so dass durch das Magnetfeld in einem Hohlraum der Injektionsvorrichtung befindlicher Wirkstoff lokal konzentriert wird und/oder die Moleküle des Wirkstoffs ausgerichtet werden;
2. b. Ausstoßen des in dem Hohlraum befindlichen Wirkstoffs durch eine Ausstoßeinheit der Injektionsvorrichtung unter Aufrechterhaltung des Magnetfelds.

According to another aspect of the invention, a method for injecting an active substance, active substance, preferably a neurotoxin, particularly preferably botulinum toxin, with an injection device described has the following steps:

1. a. Generating a magnetic field with a coil of the injection device, so that active substance located in a cavity of the injection device is locally concentrated by the magnetic field and/or the molecules of the active substance are aligned;
2. b. Ejecting the active ingredient in the cavity by an ejection unit of the injection device while maintaining the magnetic field.

The magnetic field can be controlled and/or regulated by a control unit of the injection device connected to the coil, preferably depending on the type of active ingredient and/or a predetermined local concentration of the active ingredient in the cavity.

Provision can be made for the active substance and/or the neurotoxin to be accelerated in the cavity with a cartridge and/or a carpule without being compressed.

The active substance and/or the neurotoxin can be mixed with at least one excipient within the carpule or the cavity. Mixing can be done during the injection process.

The active substance and/or the neurotoxin can be in powder form. The drug and/or neurotoxin may be present in the cavity separate from the adjuvant. For example, the excipient and the active substance can be separated by a piston, a stopper and/or a partition. The adjuvant may be or include a diluent, e.g., NaCl. The cavity can be evacuated. The injection device can have the plunger, the stopper and/or the partition. The piston, the plug and/or the partition can be arranged in the cavity.

The dividing wall, the plug and/or the piston can consist of or comprise a material which has a shore hardness of 20-80.

The stopper and/or the plunger can be divided horizontally. At least one of the parts may be or comprise a disc or disc-shaped, or a disc may be at least partially connected to or abutting the plug and/or piston. The plug and/or piston can have a bore in the longitudinal direction, which can be closed by the disc. The disk may be shaped, designed or arranged to open or close the bore upon movement. It can be provided that the stopper and/or the piston can be moved inside the cavity from the outside, for example via a magnetic coupling, a slide or a mechanical connection.

A cable can be guided or connected through the plug and/or the plunger, so that the plug and/or plunger can be moved in the cavity with the cable under tension.

The plug and/or plunger may be made of or include a ferromagnetic material so that the plug and/or plunger can be moved within the cavity from the outside. The movement can be generated by a magnetic field. The magnetic field can be generated by an air coil will. The coil can consist of several segments. The segments of the coil can be controlled independently, for example by a control unit of the injection device.

The injection device may take the form and function of a conventional syringe. The injection device can have an injection needle for piercing human tissue, for example. However, alternatively or additionally, it can also be provided that the fluid jet ejected from the injection device penetrates into the tissue or substrate without having to pierce it with an injection needle. Provision can be made for the fluid jet, the active substance and/or the neurotoxin to be accelerated in such a way that it penetrates human tissue without causing any significant injuries.

Provision can be made for the dosing of the active substance and/or neurotoxin to be controlled by a control unit which forwards control signals to the coil. The coil may enclose the body of the injector. Provision can also be made for the coil to be wound and/or guided around the cavity.

The injection device may include a push rod coupled to the stopper and/or plunger. The push rod can be moved by a motor such as a stepper motor. The push rod can be or have a pulling element.

The magnetic field generated by the coil can align and position the drug molecules in the center of the cavity.

The active ingredient can be contained in a carrier fluid that can be expelled from the injection device.

The active substance can be contained in one or more microcapsules or nanocarriers. The microcapsules and/or nanocarriers can be contained in a carrier fluid.

The active substance and/or the neurotoxin can consist of or have particles that can be influenced magnetically. The magnetically affected particles can be in at least one linear chain aligning parallel to the magnetic field. The nanocarrier can have a diameter of about 5 x 10 ^-7 m. The diameter of a particle can be about 2 × 10 ^-8 m.

The active ingredient and/or the neurotoxin can be encapsulated, with the encapsulation in turn being encapsulated in an excipient and/or an NaCl solution.

Provision can be made for one or possibly all of the encapsulations to be able to dissolve in the magnetic field, so that the active substance and/or neurotoxin is mixed with the auxiliary substance and/or NaCl or a corresponding mixture is or is formed. The mixing can take place during the injection.

The nanocarrier can be activated within a transport encapsulation upon reaching a predetermined local release range.

The pressure inside the nanocarrier can be increased by ultrasound. The nanocarrier may be configured to rupture under the pressure imposed by the ultrasound. Alternatively or additionally, it can be provided that the encapsulation bursts or breaks open as a result of the kinetic energy applied during the injection.

The active substance and/or the neurotoxin can consist of particles or have particles whose movement and/or structure formation can be influenced by a magnetic field. The nanocarriers containing the active substance and/or the neurotoxin can be or will be exposed to a locally limited magnetic field, e.g. generated by an air coil.

The method may include the step of releasing an active agent and/or a neurotoxin in a controlled manner, wherein the active agent and/or the neurotoxin may be accommodated in a preferably microscopically small nanocarrier. The nanocarrier can be placed within an encapsulation. The nanocarrier can release the active substance contained in the nanocarrier in a controlled and controlled manner, in particular for medical therapy, preferably for the targeted placement of neurotoxin in aesthetic medicine.

The active ingredient and/or the neurotoxin can be used in particular for use in and for the treatment of dystonia, in particular spasmodic torticollis or blepharospasm, spasticity, in particular equinus foot, hemifascial spasms, migraine, lumbago, cervical syndrome or hypersalivation, strabismus, visual disturbances, hearing loss, ringing in the ears, dizziness, Difficulty breathing, abdominal pain, diarrhea, dry mouth, difficulty swallowing, hair loss, itching, perspiration, muscle pain, fever, general malaise and/or dry eyes.

The active substance and/or the neurotoxin can be designed for cosmetic indication, in particular hyperhydrosis or pronounced wrinkling in the facial area. However, the active ingredient does not necessarily have to be a neurotoxin.

A locally arranged permanent magnet can be used to generate the magnetic field. Alternatively or additionally, an air-core coil can be used. An electromagnetic alternating field can be used as the magnetic field or the magnetic field can be such.

• 1: eine Ausführungsform einer erfindungsgemäßen Injektionsvorrichtung;
• 2: eine schematische Darstellung des Wirkmechanismus von Botulinumtoxin;
• 3: eine weitere schematische Darstellung des Wirkmechanismus von Botulinumtoxin; und
• 4: eine weitere Ausführungsform einer erfindungsgemäßen Injektionsvorrichtung.

The invention is explained in more detail with reference to the following figures. The reference symbols are in each case based on the corresponding figure. It shows:

• 1 : an embodiment of an injection device according to the invention;
• 2 : a schematic representation of the mechanism of action of botulinum toxin;
• 3 : another schematic representation of the mechanism of action of botulinum toxin; and
• 4 : another embodiment of an injection device according to the invention.

1 shows a first embodiment of an injection device 3 according to the invention. The injection device 3 can be cylindrical and/or similar to a commercially available syringe. The injection device 3 has a cavity in which the active substance 11 to be injected can be accommodated. The cavity can be cylindrical. A piston 4 is arranged in various ways in the cavity. In particular, the piston 4 can be movably arranged in the cavity in the longitudinal direction of the injection device 3 . The piston 4 can consist of a soft material, preferably a silicone material, or can have such a material. The injection device 3 can have an attachment 1 for a cannula or a nozzle. The attachment 1 can be arranged at a distal end of the injection device 3 . The attachment 1, in particular an opening of the attachment 1, can be fluidly connected to the cavity. The opening can be configured to receive a cannula or a nozzle. The piston 4 can be set up to eject the active ingredient 11 received in the cavity through the attachment 1, for example its opening or the nozzle or cannula accommodated in the opening, when it moves in the cavity in the direction of the attachment 1.

The piston 4 can have a first and a second part or can be divided into two. The first part can face the distal end of the injection device 3 and/or the attachment 1 . The second part can face away from the distal end of the injection device 3 and/or the attachment 1 . The first part can be or have a membrane 5 . By actuating a pulling element 7, the plunger 4 can be pulled back in the cavity or pulled away from the distal end of the injection device 3 or the attachment 1. The front part and/or the membrane 5 or the first part of the piston 4 can be opened by the pulling movement, so that the front cavity between the piston 4 and the distal end or attachment 1 is fluidic with the rear cavity arranged behind the piston 4 is or will be connected. As a result, the excipient 10 accommodated in the rear cavity arranged behind the piston 4 can come into contact with or be mixed with the active ingredient 11 accommodated in the front cavity in front of the piston 4 . If the pulling element 7 is then pushed back in the direction of the distal end of the injection device 3 or the attachment 1 in order to inject the active substance 11 or the active substance 11 mixed with the excipient 10, the front part and/or the membrane 5 or the first part of the piston 4 should be closed again as a result of the movement of the pulling element 7 . When the pulling element 7 is pushed into the injection device 3, pressure is built up in the front cavity by the active substance 11 or the active substance 11 mixed with the excipient 10 from the injection device 3, in particular by the attachment 1 and/or the attachment in the attachment 1 captured cannula or nozzle, can be ejected.

The piston 4 may have one or more sealing elements 9 which fluidly separate the front cavity from the rear cavity. The sealing element 9 can be or have a sealing lamella. The dense lamella can have a low frictional resistance due to its small contact surface on the inner wall.

The active substance 11 can be or contain a botolinum toxin. The adjuvant 10 may be or include a diluent such as NaCl.

Provision can be made for the injection device 3 to have a coil body 2 with a coil winding 8 . The injection device 3 can have a coil holder 12 . The coil former 2 can be or have at least one air coil. If the bobbin 2 has a plurality of air-core coils, the air-core coils can be or will be connected in series. Provision can be made for aligning the molecules of the active ingredient 11 and/or the excipient 10 by means of the magnetic field generated by the coil former 2 in such a way that they are positioned in the center of the hollow body. In this way, the concentration of the active substance 11 and/or the auxiliary substance 10 can be significantly increased locally. If the active ingredient 11 centered or positioned in this way, possibly mixed with the excipient 10, is ejected from the injection device 3, the active ingredient 11 can be precisely and precisely at a desired injection site due to the active ingredient molecules lying close together in the jet exiting from the injection device 3 to be injected. In particular, the active ingredient 11 can be applied with pinpoint accuracy since scattering effects can be significantly minimized.

In one embodiment, the coil former 2 can have a ferromagnetic core, for example a soft-magnetic metal core, as an alternative or in addition to the air-core coil, in order to generate a stronger magnetic field. The ferromagnetic core can be or have the ferromagnetic element 6 . In this case it can be provided that the ferromagnetic element 6 is not connected to the piston. The ferromagnetic element 6 can be arranged in a stationary manner in the cavity. However, provision can also be made for the ferromagnetic element 6 to be movable in the cavity in order, for example, to locally intensify the generated magnetic field or to change it as a function of time.

It can also be provided that the piston 4 consists of or has a suitable material that interacts with the magnetic field generated by the coil body 2 in such a way that the piston 4 is or can be moved under the influence of the magnetic field. In this case, the coil body 2 can be provided as an alternative or in addition to the tension element 7 . For example, a ferromagnetic element can be connected to the piston 4 so that when the ferromagnetic element moves due to the magnetic field, the piston 4 is moved accordingly.

The injection device 3 can have at least one sensor 13 for measuring the thickness of the skin. The sensor 13 can be arranged at the distal end of the injection device 3 . Provision can also be made for the sensor 13 to be arranged on or in the attachment 1 . The sensor 13 can have a laser, for example. If the injection device 3 has more than one sensor 13, it can be provided to carry out a triangulation or trilateration (or generally multiangulation or multilateration) with the various sensors 13 in order to localize an exact position of the injection or a nerve attachment of a muscle.

The injection device 3 can have at least one ultrasound generator 14 and optionally at least one suitable receiver. The ultrasound generator 14 and/or the receiver can be arranged at the distal end and/or on or in the attachment 1. Active substances under the skin of the patient, for example active substances that have already been injected, can be localized by means of an ultrasound measurement.

The injection device 3 may have a closure 15 at a proximal end, which may be opposite the distal end. The closure 15 can fluidly seal the cavity of the injection device 3 against the environment. For the injection of active substance, the closure 15 can first be detached from the injection device 3 and the plunger 4 can be removed from the injection device 3 . Subsequent to this, active substance 11 can first be introduced into the cavity of injection device 3 thus exposed. Thereafter, the plunger 4 can be reinserted into the cavity and, if necessary, excipient 10 can be introduced into the rear cavity between the plunger 4 and the proximal end. Finally, the closure 15 can be placed on the proximal end of the injection device 3 in order to fluidly seal the cavity.

Further sensors or, for example, a drive 20 for example of the tension element 7 can be arranged on or in the closure 15 . The drive 20 can be coupled to the traction element 7 and this, for example, in the longitudinal direction of the injection device 3 method. It can also be provided that Pulling element 7 has a spindle 19 along which the piston 4 and/or the ferromagnetic element 6 can slide and thus be moved longitudinally while rotating in the cavity. In this case it can be provided that the motor 20 turns or rotates the pulling element 7 . At least one sensor 17, which can be arranged in or on the closure 15, can detect the movement, in particular the displacement or rotation, of the piston 4 and/or the ferromagnetic element 6. By rotating the piston 4 along the spindle 19, a mixture of the active substance 11 with the excipient 10 can in particular be promoted. The ferromagnetic element 6 can be a disk. The ferromagnetic element 6 can have holes and/or slots so that fluid can flow through the ferromagnetic element 6 .

A measuring strip 18, which can be arranged, for example, along an inner wall of the cavity, can alternatively or additionally detect the position of the ferromagnetic element 6.

The injection device 3 can have a control unit 16, which can be coupled, for example, to the sensors, e.g. The control unit 16 can be set up to receive and process measured data. The control unit 16 can be set up to control the motor 20 on the basis of the measured data, for example.

2 and 3 show schematically the mechanism of action of botulinum toxin. Botulinum toxin works by cleaving key proteins required for nerve activation. First, the toxin specifically binds to nerves that use the neurotransmitter acetylcholine. Once attached to the nerve endings, the neuron takes up the toxin into a vesicle by receptor-mediated endocytosis. As the vesicle penetrates further into the cell, it acidifies and activates part of the toxin, allowing it to enter the cell cytoplasm across the vesicle membrane. Once the toxin has arrived in the cytoplasm, the toxin cleaves SNARE proteins so that the acetylcholine vesicles cannot bind to the intracellular cell membrane. This prevents the cell from releasing neurotransmitter vesicles. This stops nerve signal transmission and thus leads to paralysis.

The toxin itself is released from the bacterium as a single chain and is then activated when cleaved by its own proteases. The active form consists of a two-chain protein composed of a 100 kDa heavy chain polypeptide linked by a disulfide bond to a 50 kDa light chain polypeptide. The light chain is a zinc metalloprotease of the M27 family and is the active part of the toxin. It is translocated to the host cell cytoplasm, where it cleaves the host protein SNAP-25, a member of the SNARE protein family. This prevents vesicle fusion and the release of the neurotransmitter acetylcholine from the axon endings.

Synaptobrevin plays an essential role in the release of the neurotransmitter acetylcholine and the associated conduction of excitation in the motor endplate of nerve cells. Even the presence of a single molecule of botulinum toxin can split the synaptobrevin through its catalytic effect until the affected nerve cell is destroyed. The connected muscle fiber can thus irreversibly no longer be controlled and can only be reactivated by the formation of new nerve cells.

In 2 acetylcholine 2, syntaxin 3, SNAP-25 4, vesicle 7, exocytosis 8 and SNARE complex 9 are shown at the synaptic cleft, as well as the targets for type C1 (reference 1), targets for type A, C, E (reference d 5) and attack points for type B, D, F, G (reference number 6).

In 3 nerve impulse 1, cleavage 2 of protein complexes by Botox molecules, inhibited release 3 of neurotransmitters and impulse transmission, synapse 4, neurotransmitter 5 and paralyzed muscle fiber 6 are shown.

4 shows a further embodiment of an injection device according to the invention. The injection device can have a distal end and a proximal end arranged opposite the distal end. The injection device has a cavity which can extend in the longitudinal direction of the injection device. The injection device and/or the cavity can be cylindrical.

A nozzle 1 can be arranged at the distal end, which nozzle can be fluidically connected to the cavity. The nozzle 1 can be an attachment nozzle that can be attached to the injection device. For example, the nozzle cross-section can easily be adjusted with this. It can be provided be to provide a membrane 3 between the intermediate nozzle 1 and the cavity, which initially separates the cavity fluidically from the nozzle 1 . A piercing cannula can be arranged on or near the nozzle 1 at the proximal end, which cannula can pierce the membrane 3 in order to establish a fluidic connection between the nozzle 1 and the cavity. For example, it can be provided that the membrane 3 is pierced with the puncture needle 2 when the nozzle 1 is placed on the injection device. This can be advantageous, for example, when the injection device is a disposable injection device or is designed for single use only. In this case, the cavity of the injection device can be filled with active substance and sealed by the membrane 3. The injection device can then become functional by putting on the nozzle 1.

The injection device can have a receptacle for a reservoir 10 in which the active substance and, if necessary, the auxiliary substance are accommodated. The receptacle can be located at or near the proximal end of the injection device. The active ingredient may be or include a botulinum toxin. The adjuvant may be or include a diluent, e.g., NaCl. The injection device can thus be set up to be used several times. The injection device may have a valve 13 separating the cavity from the receptacle for the reservoir 10 . It can be provided that when the reservoir 10 is inserted into the receptacle, the valve 13 is or will be opened, so that a fluidic connection between the reservoir 10 and the cavity is or will be established. It can be provided that the active substance and/or auxiliary substance taken up in the reservoir 10 is transferred from the reservoir 10 into the cavity by means of an overpressure 12 .

Alternatively or additionally, it can be provided that the injection device has a vacuum channel 5 and a vacuum pump 6, with which a negative pressure can be generated in the cavity. In this way, the active ingredient and/or possibly auxiliary substance contained in the reservoir 10 can be sucked out of the reservoir 10 into the cavity. If the injection device has a membrane 3, 15 arranged at the distal end, the membrane 15 can be transferred by the negative pressure in the cavity towards the cavity or away from the distal end into a bent-away position 14, compare the figure. The injection device can also have a valve 18 or an air inlet 18 which is connected to the cavity via an acceleration jerk 11 and through which air can be drawn into the cavity from the environment. In particular, when the cavity has been evacuated by the vacuum pump 6, the membrane can be transferred from its bent-away position 14 to a bent-away position 15 towards the distal end within a short time interval, with the puncture needle 2 being able to pierce the membrane during the transfer. In this case, the active ingredient and/or auxiliary substance, if any, located in the cavity can be ejected through the nozzle 1 from the injection device.

The injection device can have at least one coil 4 with a coil winding 16 . If the injection device has more than one coil, the respective coils can be separated from one another by a coil delimitation 17 . Through the coil 4, a magnetic field can be generated that as in relation to 1 described the active ingredient and possibly the excipient can concentrate locally. If the active ingredient centered or positioned in this way, possibly mixed with the excipient, is ejected from the injection device, the active ingredient can be injected exactly and precisely at a desired injection site due to the active ingredient molecules lying close together in the jet emerging from the injection device. In particular, the active ingredient can be applied with pinpoint accuracy, since scattering effects can be significantly minimized.

The active substance can be encapsulated. In particular, the active ingredient can be encapsulated with the excipient.

The injection device can have a control unit 7, sensors, a memory chip 8 and/or an energy supply 9. In this way, for example, the control unit 7 can control and/or regulate the vacuum pump 6 and/or the coil 4 . The control unit can be connected to the sensors, the memory chip and/or the power supply. The energy supply 9 can be or have a rechargeable battery, for example.

The features disclosed in the figures, the description and the claims can be essential individually or in combination for the implementation of the invention.

QUOTES INCLUDED IN DESCRIPTION

This list of documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent Literature Cited

• DE 4445969 [0049]
• US5788670 [0049]
• EP 520618 [0050]
• GB2010681 [0051]
• FR 1099362 [0052]
• EP0397977A [0053]

Claims (10)

Injection device for injecting active substance, preferably a neurotoxin, particularly preferably botulinum toxin, the injection device having a cavity, an outlet fluidically connected to the cavity and an ejection unit with which the active substance in the cavity can be ejected through the outlet from the injection device, characterized that the injection device has at least one coil which is set up to locally concentrate active substance located in the cavity by means of a magnetic field generated by the coil and/or to align molecules of the active substance. injection device claim 1 wherein the coil comprises an air coil, preferably wound around the cavity, or wherein the coil comprises a permanent magnet. Injection device according to one of the preceding claims, in which the ejection unit has a piston accommodated and movable in the cavity, a vacuum pump fluidly connected to the cavity, an air inlet fluidly connected to the cavity and/or a pressurized container fluidly connected to the cavity. Injection device according to one of the preceding claims, in which the ejection unit has a piston received and movable in the cavity, the piston having a first part and a second part, the first part and/or the second part being movable such that at a Movement of the piston in a first direction creates a fluidic connection between the cavity located in front of and behind the piston through the first part and the second part, with the first part and /or the second part is movable in such a way that the fluidic connection is separated. Injection device according to one of the preceding claims, in which the outlet is fluidically coupled to a preferably attachable nozzle or a preferably attachable injection needle and/or is or has such. An injector as claimed in any preceding claim, including a magnetically shielding enclosure such that the injector is shielded from external magnetic fields. Injection device according to one of the preceding claims, in which the active substance has particles that can be influenced magnetically and/or is encapsulated with particles that can be influenced magnetically. Injection device according to one of the preceding claims, in which a membrane fluidly seals the outlet, the membrane being adapted to be pierced by a piercing needle so that a fluidic connection is established through the membrane. Method for injecting an active substance, active substance, preferably a neurotoxin, particularly preferably botulinum toxin, with an injection device according to one of the preceding claims, the method having the following steps: a. Generating a magnetic field with a coil of the injection device, so that active substance located in a cavity of the injection device is locally concentrated by the magnetic field and/or the molecules of the active substance are aligned; b. Ejecting the active ingredient in the cavity by an ejection unit of the injection device while maintaining the magnetic field. Method for injecting an active ingredient claim 9 , in which the magnetic field is controlled and/or regulated by a control unit of the injection device connected to the coil, preferably depending on the type of active ingredient and/or a predetermined local concentration of the active ingredient in the cavity.

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