MXPA97008124A - Botulinum toxin derivatives capable of modifying perfery affording functions - Google Patents

Abstract

The invention relates to a specific agent for peripheral sensory afferents. The agent can inhibit the transmission of signals between a primary sensory afferent and a projection neuron by controlling the release of at least one neurotransmitter or neuromodulator from the primary sensory afferent. The agent can be used in or as a pharmacist for the treatment of pain, particularly chronic pain.

Description

BOTULINUM TOXIN DERIVATIVES CAPABLE OF MODIFYING PERIPHERAL AFFORDING FUNCTIONS. Technical Field This invention relates to a novel agent that is capable of modifying peripheral afferent function. The agent can inhibit neurotransmitter release from discrete populations of neurons and therefore reduces, or preferably avoids, the transmission of pain signals afferent peripheral and central pain fibers. The agent can be used in or as a pharmacist for the treatment of pain, particularly chronic pain. Background The sense of touch has traditionally been considered as one of the five classical senses, but in reality it is highly complex, transducing a number of different sensations. These sensations are detected in the periphery by a variety of specialized nerve endings and associated structures. Some of these are specific for mechanical stimuli of various kinds such as touch, pressure, vibration and deformation of hair or whiskers. Another class of nerves is able to detect temperatures, being activated different fibers by heat and cold. An additional population of nerve endings is not normally excited by moderate stimuli, but only by strong stimuli. Sensory nerves in this category often respond to more than one stimulus and are known as high-threshold poiimodal fibers. They can be used to capture potentially harmful situations or objects. Polymodal fibers also transduce chemical signals so that the "burning" sensation evoked by acid. Therefore, the sense of touch can convey a very detailed description of objectives and serve both to inform and to notify events. The transduction of sensory signals from the periphery to the sensation itself is achieved by a multineuronal path and the information processing centers of the brain. The first nerve cells in the pathway involved in the transmission of sensory stimuli are called primary sensory afferents. The cell bodies for the primary sensory afferents of the head and some internal organs reside in several of the ganglia associated with the cranial nerves, particularly the trigeminal nuclei and the nuclei of the solitary tract. The cell bodies for the primary sensory afferents for the rest of the body lie in the dorsal root ganglia of the spinal column. The primary sensory afferents and their processes have been classified histologically, cell bodies fall into two classes: Type A are long (60-120 μm in diameter) while type B are smaller (14-30 μm) and more numerous. Similarly, the processes fall into two categories: C fibers lack the myelin layer of the A fibers. Fibers can be further subdivided into Aß fiber, which are large diameter with well developed myelin and Ad and C fibers arise from B-type cell bodies. These classifications can be further extended and subdivided by studying the selective expression of a scale of molecular markers. Functional analyzes indicate that under normal circumstances, Aß fibers transmit the senses of touch and moderate temperature discrimination, whereas C fibers are mainly equivalent to the polymodal high-threshold fibers mentioned above. The role of Ad fibers is less clear since they seemed to have a variety of responsive modes, with both high and low thresholds. After the activation of the primary sensory afferents the next step in the transduction of sensory signals is the activation of the projection neurons, which carry the signal to upper parts of the central nervous system such as the thalamic nuclei. The cell bodies of these neurons (different from those related to the cranial nerves) are located in the dorsal keratin of the spinal cord. This is also where the synapses between the primary afferents and the projection neurons are located. The dorsal keratin is organized in a series of sheets that are stacked, lamina I being more dorsal followed by lamina II, etc. The different classes of primary afferents maine synapses in different sheets. For primary skin afferents, C fibers synapse in sheets I and II, Ad fibers in sheets I, II, and V, and Aβ fibers in sheets III, IV, and V. The deeper sheets (V-VII). , X) is thought to be involved in sensory pathways that come from deeper tissues such as muscles and viscera. The predominant neurotransmitter in the synapse between primary afferents and projection neurons is glutamate, although importantly C fibers contain several neuropeptides such as substance P and peptide related to the calcitonin gene (PRGC). Also fiber A expresses neuropeptides such as neuropeptide Y under some circumstances. The transmission efficiency of these synapses can be altered via descending pathways and local interneurons in the spinal cord. These odulator neurons release a number of mediators that are either inhibitory (eg, opioid peptides, glycine) or excitatory (eg, nitric oxide, cholecystokinin) to provide a mechanism to increase or decrease the warning of sensations. A category of sensation that requires such physiological modulation is pain. Pain is a sensation that can warn of damage or illness and as such is essential in daily life. However, sometimes there is a need to be able to ignore it and physiologically this is a function of, for example, opioid peptides. Unfortunately, in spite of this physiological mechanisms, pain can continue to be experienced during the illness or after long damages after its usefulness has passed. In these cases, the pain becomes a symptom of illness that could be alleviated better.

Clinically, pain can be divided into three categories: (1) Usually, acute pain of damage or surgery that is expected to disappear when the damage heals occurs. (2) Chronic pain arising from malignant disease; Most people with metastatic cancer have moderate to severe pain and this is resolved by successfully treating the disease or by killing the patient. (3) Chronic pain not caused by malignant disease; this is a heterogeneous complaint, caused by a variety of diseases, including arthritis and peripheral neuropathies, which usually do not threaten life but can last for decades increasing pain levels. The physiology of pain resulting from tissue damage is better understood than that caused by defects of the central nervous system. Under normal circumstances the sensations that can lead to pain are first translated by fibers Ad and C can carry high threshold signals. Therefore the synapses in sheets I and II are involved in the transmission of pain signals, using glutamate and the peptides released by the C fibers to produce the activation of the neurons. However, there is evidence that in some chronic pain states other A fibers (including Aß fibers) can carry pain signals and therefore act as primary nociceptive afferents, for example, in aloidine hyperalgesia associated with neuropathic pain. These changes have been associated with the expression of peptides such as neuropeptide Y in A fibers. During various conditions of chronic pain the synopsis of several sensory afferents with projection neurons can be modified in various ways there can be changes in the morphology leading to an increase in the number of synapses, the levels and relationships of the different peptides may change and the sensitivity of the projection neuron may change Given the enormity of the clinical problem presented by pain, considerable efforts have been made to find methods for its relief Pharmacists most commonly used for pain relief fall into two categories (1) non-spheroidal anti-inflammatory drugs (FNEAI) including aspirin and ibuprofen, (2) Opioids, including morphine The FNEAI have their main analgesic action in the periphery by inhibiting the production of prostaglandins by feared damaged It has been shown that the prostaglandi are peripheral mediators of pain and inflammation and a reduction in their concentration provides relief to patients This is especially the case in medium arthritic disease, where inflammation is a major cause of pain It has been suggested that prostaglandins are involved in the mediation of pain in the spinal cord of the brain, this may be explained by the fact that FNEAI have analgesic effects in some pain states that do not involve inflammation or peripheral tissue damage. However, like prostaglandins, there are only several FNEAI mediators of pain alone and only They are effective in reducing some types of moderate pain to acceptable levels. They are considered because they have an upper limit of activity above which increasing doses does not increase pain relief. They also have side effects that limit their usefulness in chronic complaints. The use of FNEAI is associated with irritation of the gastrointestinal tract and prolonged use can lead to the development of extensive bowel ulceration. This is particularly true in older patients who make up the largest group of patients with, for example, arthritis. Opioids act at the level of the spinal cord to inhibit the efficiency of neurotransmission between the primary nociceptive fibers (mainly C fibers) and the projection neurons. They achieve this by causing prolonged hyperpolarization of both elements of these synapses. The use of opioids is effective in relieving most types of acute pain and chronic malignant pain. However, there are a number of chronic malignant pain conditions that are partially or completely refractory to opioid analgesia, particularly those involving nerve compression, e.g., by tumor formation. Unfortunately, opioids also have unwanted side effects including: (1) depression of the respiratory system at the level of respiratory centers in the brainstem; (2) the induction of constipation by a variety of effects on the smooth muscles of the gastrointestinal tract; (3) psychoactive effects including sedation and the induction of euphoria. These side effects occur at doses similar to those that produce analgesics and therefore limit the doses that can be given to patients.

The supply of opioids at the spinal level may reduce the profile of side effects, but requires frequently repeated spinal injections or adaptation of a catheter, both of which may carry increased risks to the patient. The adaptation of a catheter requires that the patient be confined essentially to the bed, also restricting their quality of life. The use of opioids for the treatment of some other types of chronic pain is usually ineffective or undesirable. Examples include the pain associated with rheumatoid arthritis and neurons that develop after nerve damage. The undesirable nature of opioid treatment in these patients is related not only to the aforementioned side effects and the probable duration of the disease but also to the fourth major side effect of opioids: dependence. Opioids such as morphine and heroin are well-known drugs of abuse that lead to physical dependence, this latter side effect involves the development of tolerance > the dose of a drug required to produce the same analgesic effect increases with time. This can lead to a condition in which the dose required to alleviate the pain threatens life due to the first three side effects. Other treatments are also used, particularly the treatment of chronic severe pain including surgical lesions of the pain routes at various levels in peripheral nerves through dorsal root section and cordotomy to destroy the pituitary. However, these are the most severe operations that are associated with significant risk to the patient. Therefore, it can be observed that there remains a significant need for the development of new classes of pharmacists for the treatment or pain of many types. The desired properties of such new therapies can be briefly expressed as follows (1) the ability to provide significant pain relief including severe pain, (2) the lack of systemic side effects that significantly impair the patient's quality of life; (3) durable actions that do not require frequent injections or long-term catheterization of patients; (4) provision of agents that does not lead to tolerance and associated dependency. Declaration of Invention The present invention relates to an agent that can reduce and preferably prevent the transmission of pain signals from the periphery to the central nervous system, thus relieving the sensation of pain. Specifically, the invention can provide an agent that can reduce and preferably prevent the transmission of pain signals from nociceptive afferents to the projection neurons. More specifically, the invention can provide an agent that can inhibit the exocytosis of at least one neurotransmitter or neuromodulatory substance of at least one category of nociceptive afferents. In a first aspect of the invention, an agent that can be administered systematically is provided, and can specifically target defined populations of nonceptive afferents to inhibit the release of at least one neurotransmitter or neuromodulator from the synaptic terminals of nerves. In a second aspect of the invention, there is provided an agent that can be administered locally in the periphery and that is capable of inhibiting the release of at least one neurotransmitter or neuromodulator from the synaptic terminals of nociceptive afferents transmitting the pain signal of the periphery. In a third aspect of the invention, an agent is provided which can be administered in the spinal cord and which can inhibit the release of at least one neurotransmitter or neuromodulador from the synaptic terminals of nociceptive afferents ending in that region of the spinal cord. spinal. In a fourth aspect of the invention, a person is provided who can specifically target defined populations of afferent neurons, so that the effect of the agent is limited to that cell type. In a fifth aspect of the invention, a pain treatment method is provided which comprises administering an effective dose of the people according to the invention. In a sixth aspect of the invention, the agent can be recombinantly expressed as a fusion protein that includes the required components of the agent. Definitions Without wishing to be limited by the established definitions, it is intended in this description that the following terms have the following meanings: Light chain means the smallest of the two polypeptide chains that form Clostridial neurotoxins; has a molecular mass of approximately 50 kDa and is commonly referred to as an L chain or simply L. Heavy chain means the longest of the two polypeptide chains that form clostridial neurotoxins, has a molecular mass of approximately 100 kDa and is commonly referred to as a chain H or simply H. Fragment of Hc means a fragment derived from the H chain of a Clostridial neurotoxin approximately equivalent to the amino terminal half of the H chain, or the domain corresponding to the intact fragment in the H chain. It contains a domain involved in the translocation of the L chain through endosomal membranes. LHN means a fragment derived from a Clostridial neurotoxin containing the L chain, or a functional fragment thereof coupled to the HN fragment. It is commonly derived from intact neurotoxin by proteolysis. The "White Portion" (PB) means any chemical structure of an agent that functionally interacts with a binding site that causes a physical association between the agent and the surface of a primary sensorial afferent.

The binding site (SU) means a structure on the surface of a cell with which exogenous molecules are able to interact in such a way that it reaches a physical association with the cell. The primary sensory afferent is a nerve cell that can carry sensory information from the periphery to the central nervous system. The primary nociceptive afferent is a nerve cell that can carry sensory information from the periphery to the central nervous system, where that information can result in a sensation of pain. Brief Description of the Drawings Figure 1 shows a Coomassie strain of an SDS-PAGE analysis of the size exclusion chromatography fractions of the products of the coupling reaction between the derived Nerve Growth Factor (FCN) and LHN derived from BoNT. /TO. Figure 2 shows a Coomassie strain of an SDS-PAGE analysis of the conjugate of FCN and LHN under reducing and nonreducing conditions. Figure 3 shows a Western plot of extracts of PC12 cells with the conjugate of FCN and LHN tested with an antibody that recognizes the product of proteolysis of SNAP-25 by the L chain of BoNT / A. Figure 4 shows a Western plot of extracts of dorsal root ganglion neurons from rats treated with the FCN and LHN conjugate, tested with an antibody that recognizes the product of SNAP-25 proteolysis by the L chain of BoNT / A Detailed Description of the Invention It can be seen that an agent to reduce or prevent the transmission of pain signals from peripheral nociceptive afferent neurons to the projection neurons has many potential applications in reducing the sensation of pain, particularly chronic severe pain. According to the invention, an agent is provided that can inhibit the release of at least one neurotransmitter or neuromodulator or both from the synaptic terminals of nociceptive afferents The agent has a number of discrete functions 1) It binds to a surface structure (the Union site [SU]) which is characteristic of, and has a degree of specificity for, nociceptive afferent neurons 2) It enters the neuron The entry of molecules into a cell can occur through a process of endocytosis Only certain SU cell surfaces suffer endocytosis and preferably the SU to which the agent binds is one of these In one aspect of this invention, the SU is present in the peripheral sensory fibers of the afferent nociceptive neuron and, after internalization, suffers from retrograde transport to the cell body and central process of the neuron, in such a way that the agent is also supplied to these regions of the neuron. another aspect of this invention the SU to which the agent binds is present in the central processes or cell body of the afferent nociceptive neuron. 3) The agent enters the cytosol. 4) The agent modifies components of the exocytotic machinery present in the synaptic terminals of the central processes of those neurons, so that the release of at least one neurotransmitter or neuromodulator from the synaptic terminal is reduced or preferably avoided. Surprisingly, an agent of the present invention can be produced by modifying a clostridial neurotoxin or fragment thereof. Clostridial neurotoxins are proteins with molecular masses in the order of 150 kD. They are produced by several species of the genus Clostridum, most importantly C. tetani and several strains of C. botulinum. Currently there are eight different classes of known neurotoxins, tetanus toxin and botulinum neurotoxin in their serotypes A, B, C1, D, E, F and G and they all share similar structures and modes of action Clostridial neurotoxins are synthesized by the bacterium as a single polypeptide that is modified post-translationally to form two polypeptide chains linked by a disulfide bond. The two chains are called the heavy chain (H) which has a molecular mass of approximately 100 kDa, and the light chain L), which has a molecular mass of approximately 50 kDa. Clostpdial neurotoxins bind to an acceptor site in the cell membrane of the motor neuron at the neuromuscular junction and are internalized by an endocytotic mechanism. The internalized clostridial neurotoxins have an endopeptidase activity that depends on highly specific zinc that hydrolyzes a specific peptide binding in at least one of three proteins, synaptobrevin, syntaxin or SNAP-25, which are crucial components of the neurosecretory machinery and activity. Clostridial toxins result in prolonged muscle paralysis The zinc-dependent endopeptidase activity of Clostridial neurotoxins is found to reside in the L chain. Clostridial neurotoxins are highly selective for motoneurons due to the specific nature of the acceptor site in those neurons It is known that the specific neuromuscular binding activity of clostridial neurotoxins resides in the carboxy terminal portion of the heavy chain component of the two-chain neurotoxin molecule, a region known as Hc. Surprisingly, by covalently binding a Clostridial neurotoxin or a hybrid of two Clostridial neurotoxins in which the Hc region of the H chain has been removed or modified to a new molecule or portion, the White Portion (PB), which binds to a SU on the surface of sensory neurons, a novel agent is produced capable of inhibiting the release of at least one neurotransmitter or neuromodulator of nociceptive afferents. A further surprising aspect of the present invention is that if the L chain of a clostridial neurotoxin, or a fragment of the L chain containing the endopeptidase activity, is covalently linked to a PB that can also effect the internalization of the L chain or fragment thereof, in the cytoplasm of a sensory neuron, this also produces a novel agent capable of inhibiting the release of at least one neurotransmitter or neuromodulator. The covalent ligatures used to couple the component parts of the agent may include appropriate spacer regions. PB provides specificity for SU in the afferent nociceptive neuron. The PB component of the agent may comprise one of many cells that bind to the molecules, including, but not limited to, antibodies, monoclonal antibodies, antibody fragments (Fab, F (ab) '2, Fv, ScFv etc. ), lecithins and ligands to receptors for hormones, cytokines, growth factors or neuropeptides. A list of the PB is given in Table 1, this list is illustrative and is not intended to limit the scope of the PB that could meet the requirements of this invention. In one embodiment of the invention, the PB joins an SU that suffers from retrograde transport. It is known in the art that the Hc portion of the neurotoxin molecule can be removed from the other portion of the heavy chain, known as HN, so that the HN fragment remains attached in the disulfide to the light chain (L-chain). ) of the neurotoxin molecule to provide a fragment known as LHN. Therefore, in one embodiment of the present invention, the LHN fragment of a clostridial neurotoxin is covalently linked, using ligatures that can include one or more spacer regions for a PB. In another embodiment of the invention, the Hc domain of a clostridial neurotoxin is mutated or modified, e.g., by chemical modification, to reduce or preferably impair its ability to bind the neurotoxin to receptors in the neuromuscular junction. This modified clostridial neurotoxin is covalently linked, using ligatures that can include one or more spacer regions to a PB. In another embodiment of the invention, the heavy chain of a clostridial neurotoxin in which the Hc domain is mutated or modified, e.g., by chemical modification in order to reduce or preferably impair its ability to bind the neurotoxin to receptors. in the neuromuscular junction it is combined with the L chain of a different clostridial neurotoxin. The modified Clostridial neurotoxin binds covalently, using ligatures that may include one or more spacer regions, to a PB. In another embodiment of the invention, the HN portion of a clostridial neurotoxin is combined with the L chain of a different clostridiai neurotoxin. The hybrid LHN is then linked covalently, using ligatures that can include one or more spacer regions to a PB. In another embodiment of the invention, the light chain of a Clostridial neurotoxin or a fragment of the light chain containing the endopeptidase activity, is bound, using ligatures that can include one or more separation regions, to a PB that can also effect the internalization of the light chain, or fragment thereof containing endopeptidase activity, in the cytoplasm of the cell. In another embodiment of the invention, the agent is recombinantly expressed as a fusion protein that includes an appropriate fragment of a White Portion in addition to any desired separation domain. The recombinantly expressed agent can be derived completely from the gene encoding a neurotoxin serotype or it can be a chimera derived from the genes encoding two different serotypes. In another embodiment of the invention, the required LHN which may be a hybrid of an L and HN of different types of clostridial toxin was recombinantly expressed as a fusion protein with the PB and may also include one or more separation regions. In another embodiment of the invention, the light chain of a clostridial neurotoxin, or a fragment of the light chain containing the endopeptidase activity, was recombinantly expressed as a fusion protein with a PB that may also affect the internalization of the light chain , or fragment thereof containing the activity of endopeptidase, in the cytoplasm of the cell. The expressed fusion protein may also include one or more separation regions.

The basis of this description is the creation of novel agents with very specific and defined activities against a limited and defined class of neurons (primary sensory afferents) and as such the agents can be considered to represent a form of neurotoxins The therapeutic use of botulinum neurotoxins native is well known in the prior art The mode of action of botulinum neurotoxins, as described in the prior art, however is by a mechanism, inhibition of acetylcholine secretion and against a category of white neurons, afferent motoneurons, clearly Other than the agents described in this disclosure The prior art teaches neither the activity nor the chemical structure of the described agents. Therefore, although, as discussed in this application, the prior art teaches a lot about clotspial neurotoxins the clostpdial neurotoxins unmodified native are not subject of this description The agent of this invention requires modification of the clostpdial neurotoxins so that the white property taught in the prior art is removed. The modified neurotoxin is coupled to a new white function (the PB), to give a novel agent with new, different biological properties. of those of the native clostpdial neurotoxins are taught in the prior art. It is a novel agent with novel properties that is the subject of this disclosure. Exploitation in Industry The agent described in this invention can be used in vivo, either directly or as a pharmaceutically acceptable salt for the treatment of pain. For example, an agent according to the invention can be used systematically for the treatment of severe chronic pain.

A specific example of this is the use in the treatment of clinical pain associated with rheumatoid arthritis that affects multiple joints. In another example, an agent according to the invention can be applied locally for the treatment of pain. A specific example of this treatment by local injection in a joint affected by inflammatory pain. In a further example, an agent according to the invention can be administered by spinal injection (epidural or intrathecal) at the level of the spinal segment involved in the innervation of an affected organ for the treatment of pain. This is, for example, applicable to the treatment of deep tissue pain, such as chronic malignant pain. The present invention will now be illustrated by reference to the following non-limiting examples: Example 1, Synthesis of an FCN conjugate and the lyophilized murine fragment 2.5 S FCN was dissolved by the addition of water and dialysed in MES pH buffer (0.1 M MES; 0.1 M sodium chloride pH 5.0). To this solution (at a concentration of about 0 3 mg / ml) was added PDPH (100 mg / ml in DMF) at a final concentration of 1 mg / ml. After mixing, solid EDAC was added to produce a final concentration approximately 0.2 mg / ml The reaction was allowed to proceed for at least 30 minutes at room temperature Excess of pDPH was then removed by desalting on a PD-10 column (Pharmacia) previously equilibrated with MES pH buffer The LHN fragment of BoNT / A was produced essentially by the method of Shone CC, Hambleton, P, and Melling, J 1987, Eur. J. Biochem., 167, 175-180 An amount of LHN equivalent to half the weight of FCN used dissolved in pH regulator solution of ttentenolamine (0 02 M tpetenolamine / HCl, 0 1 M sodium chloride, pH 78) at a concentration of approximately 1 mg / ml, was reacted with the Traut reagent (100 mM stock solution in ttentenolamine / HCI 1M, pH 80) at a concentration end of 2 mM After one hour the LHN was desalted in PBSE (phosphate buffered saline with 1 mM EDTA) using a PD-10 column (Pharmacia) The protein peak of the eluate of the column was concentrated using a Microcon 50 (Amicon) at a concentration of approximately 2 mg / ml The derivatized FCN was subjected to a final concentration step resulting in a reduction in volume to less than 10% of the starting volume and then mixed with the LHN depvatized at room temperature The reaction products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). The conjugate resulting from the above reaction was partially purified by size exclusion chromatography on Bio-Gel P-100 (Bio-Rad). The elution profile was followed by measuring the optical density at 280 nm and SDS-PAGE analysis of the fractions. This allowed the separation of conjugate from free FCN and by-products of the reaction. Figure 1, shows the SDS-PAGE analysis of the fractions of a Bio-Gel P-100 column. Free and conjugated LHN (Mr 100 kDa and more) are separated from most free FCN (M, 13 kDa). As 2.5S FCN is a homodimer formed by non-covalent interactions, it is dissociated by treatment with SDS. Therefore, molecules that have formed covalent crosslinks to LHN through the subunit will only dissociate during the SDS PAGE analysis and originate the free FCN band seen in fractions 4-6. This result demonstrates that the homodimeric structure of FCN remains intact after derivatization. The free LHN seen in these fractions represents a minor component that has not been coupled to FCN. Fractions 4-6 were combined before further analysis. Figure 2 shows an analysis of the conjugate by SDS-PAGE under reducing conditions and without reduction. Line 1 is free LHN under non-reducing conditions, line 2 is the same amount of LHN reduced with 50 mM dithiothreitol. Lines 3 and 4 show the conjugate after size exclusion chromatography either without (line 3) or with (line 4) by dithiothreitol. Similarly, lines 5 and 6 show FCN without or with reduction respectively. The results clearly show that the material in line 5 with an evident molecular mass greater than 100 kDa produces, by reduction, the constituent bands of LHN and FCN only. In addition, the intensity of the bands after reduction is such that they must be derived from material other than the small amounts of free LHN and FCN observed in the non-reduced sample. The only available source for the excess is the material with an evident molecular mass >100 kDa. The conjugate in the fractions obtained after size exclusion chromatography thus represents FCN and LHN covalently bound by reducible disulfide ligands. Fractions containing conjugate were stored at 4 ° C until required. Example 2. Activities of an FCN conjugate and LHN in PC-12 cells PC12 cells are a neuroectodermal derivatization cell line that are commonly used as a model system for the study of nerve function. As a model system for testing the function of a conjugate of FCN and LHN have two necessary aspects: first they are well known for providing cell surface receptors for FCN which have been shown to be involved in a differentiation process that contains the exocytotic machinery for neurotransmitter release that importantly includes in this example, SNAP-25 PC12 cells were seeded into plates in a 24-well plate that was coated with MATRIGEL base membrane matrix (Collaborative Biomedical Products) at a density of approximately 5 x 10 5 cells per well After a few days in culture (RPMI 1640 with 2 mM glutamine, 10% horse serum and 5% fetal sheep serum, 37 ° C, 5% C02) the medium was replaced with fresh medium containing conjugate added (prepared as described in Example 1) or LHN or without addition After it was kept in culture overnight the medium was removed and the The cells were washed once with fresh medium. The cells were lysed by the addition of 045 ml of sodium hydroxide (02 M) for 30 minutes. After this time the solutions were neutralized by the addition of 045 ml of hydrochloric acid (02M) followed by 0 1 ml of HEPES / NaOH (1M pH 74) To extract membrane proteins from these mixtures Tpton-X-114 (10%, v / v was added and incubated at 4 ° C for 60 minutes, the material Insoluble was removed by centrifugation and the supernatants were heated at 37 ° C for 30 minutes. The two resulting phases were separated by centrifugation and the upper phase was discarded. The proteins in the lower phase were precipitated with chloroform / methane! for analysis by Western blotting Samples were separated by SDS-PAGE and transferred to nitrocellulose The proteolysis of SNAP-25, a crucial component of the neurosecretor process and the substrate of the zinc-dependent endopeptidase activity of BoNT / A, was it then detected by testing with an antibody that recognizes the newly revealed carboxy terminus of the separated SNAP-25 (the antibody was described in Patent Application PCT / GB95 / 01279) Figure 3 shows an example of said Western plot No immunoreactivity was observed significant in the samples of the control cells (lines 1 and 2) while a band corresponding to a molecular mass of 29 kDa was observed weakly in the samples incubated with 10 mg / ml of the conjugate of FCN and LHN (lines 3 and 4) Therefore incubation of PC12 cells with the conjugate leads to the marked proteohsis of SNAP-25 including that the conjugate has introduced the z-dependent proteolytic activity. inc of the L chain of BoNT / A in the cytoplasm of the cells Little or no activity was seen with the constituent components of the conjugate Incubation of cells with the conjugate in the presence of an excess of free FCN resulted in a reduced production in the proteolytic product of SNAP-25 than in the incubation with the conjugate alone. This indicates that the action of the conjugate occurs by means of of FCN targeting the portion that interacts with cell surface receptors for NFG Example 3 The activity of a conjugate of FCN and LHN in primary cultures of dorsal root ganglia neurons The dorsal root ganglia contain nociceptive afferent cell bodies Primary It is well established that neurons in the primary in vitro cultures of this tissue retain many of the characteristics of nociceptive afferents. These characteristics include the ability to release neuropeptides such as substance P in response to chemical stimuli known to cause pain in viro (e.g., capsaicin). In addition, it is known that neurons have receptors for FCN. The primary cultures of dorsal root ganglia neurons were established following the dissociation of the dissected ganglia of embryos from rats (embryological age 12-13 days). The cells were plated in 12 well plates at an initial density of 3 x 10 cells / well in a medium containing FCN (100 mM) was added to kill non-neuronal cells. The cytosine arabinoside was removed after 2-4 days. After several more days in culture, the medium was replaced with fresh medium containing conjugate or LHN in the absence of FCN. After incubation overnight at 37 ° C the medium was removed, the cells were used and the hydrophobic proteins were extracted using Triton-X 114 as described in Example 2. The samples were analyzed by Western blot analysis as described in Example 2 with the antibody recognizing the SNAP-25 BoNT / A proteolysis product. No immuno-reactivity was observed in the control cell samples (line 4) while a band corresponding to a molecular mass of 29 kDa was weakly observed in samples incubated with 10 mg / ml LHN (line 3) and strongly in the samples incubated with 10 mg / ml of the conjugate of FCN and LHN (lines 1 and 2). This result indicates that the conjugate can supply the proteolytically active L chain of BonNT / A in the cytoplasm of the neuronal cells which, in vivo, form the primary nociceptive afferents. Example 4. The production of a chimeric LHN from which the Bont / B chain L and the HM fragment of BoNT / A are derived. The HN fragment of BoNT / A was produced according to the method described by Shone CC, Hambleton, P., and Melling, J. (1987, Eur. J. Biochem, 167, 175-180) and the L chain of BoNT / B according to the method of Sathyamoorthy, V., and DasGupta, BR (1985, J. Biol. Chem, 260, 10462-10466). The cysteine released in the HN fragment of BoNT / A is then derived by the addition of a ten-fold molar excess of dipyridyl disulfide followed by incubation at 4 ° C overnight. The excess dipyridyl disulfide and the thiopyridone per product are then removed by desalting the protein on a pD10 column (Pharmacia) in PBs. The derivatized HN was then concentrated to a protein concentration in excess of 1 mg / ml before being mixed with an equimolar portion of the L chain of BoNT / B (> 1 mg / ml in PBS). After incubation at room temperature the mixture was separated by size exclusion chromatography on SuperScope 6 (Pharmacia) fractions were analyzed by SDS-PAGE. The chimeric LHN is available for derivatization to produce a white conjugate as described in Example 1. The examples described above are only illustrative of the invention. To synthesize the coupling agent of the PB of the modified clostridial neurotoxin or fragment thereof, it is achieved via chemical coupling using reagents and techniques known to those skilled in the art. Therefore, although the examples given use exclusively PDPH / EDAC and Traur's reagent chemistry, any other coupling chemistry capable of covalently binding the PB component of the agent to the clostyridial neurotoxin derived the component and those skilled in the art know that it is covered by the scope of this application. Similarly, it is also evident to those skilled in the art that both the DNA encoding all the agent or fragments of the agent could be easily constructed and when expressed in an appropriate organism, they can be used to recombinantly produce the agent or fragment of the agent. Said genetic constructions of the agent of the invention obtained by the techniques known to those skilled in the art were also covered within the scope of this invention.

Table 1 - White Portions (PB) Possible Growth Factors: 1. Nervous growth factor (NGF); 2. Leukemia inhibitory factor (FIL); 3. Basic fibroblast growth factor (FCFb); 4. Brain-derived neurotrophic factor (FNDC) . Neurotrophin-3 (NT-3); 6. Hydrated head activating peptide (PACH); 7. Transformation growth factor 1 (FCT-1); 8. Transformation growth factor 2 (FCT-2); 9. Transformation growth factor (FCT-); 10. Epidermal growth factor (EGF); 11. Ciliary neurotrophic factor (FNTC). Cytokines: 1. Tumor necrosis factor (TNF-); 2. Interleukin-1 (IL-1); 3. interleukin-1 (IL-1); 4. Interleukin-8 (IL-8). Peptides: 1. -Endorfina; 2. Methionine-enkephalin; 3. D-Ala2-D-Leu3-enkephalin; 4. Bra diquinine. Antibodies: 1. Antibodies against carbohydrate epitopes of lactoseries found on the surface of dorsal root ganglion neurons (e.g., monoclonal antibodies 1 B2 and LA4); 2. Antibodies against any of the receptors for the ligands given above. 3. Antibodies against the Thy1 antigen expressed on the surface (e.g., MRC OX7 monoclonal antibody).

Claims (51)

1. CLAIMS 1. A non-cytotoxic agent exhibiting specificity for peripheral sensory afferents comprising a White (PB) portion coupled to a Clostridial neurotoxin modified with PB comprises a binding to a cell surface binding site in a primary sensory afferent and is capable of functionally interacting with a binding site that causes a physical association between the agent and the surface of a primary sensory afferent; and the heavy chain (H chain) of the clostridial neurotoxin was removed or modified by chemical derivation, mutation or proteolysis to reduce or remove its native binding affinity for motor neurons; and the light chain (L chain) of a clostridial neurotoxin or a fragment thereof retains a protease activity specific to the components of the neurosecretory machinery; the PB and the modified H chain (if present) forming a molecule that introduces the L chain or fragment thereof into the cytosol of a primary sensory afferent, and therefore inhibits the transmission of signals between a primary sensory afferent and a projection neuron controlling the release of at least one neurotransmitter or neuromodulador of the primary sensory afferent. An agent according to claim 1, comprising a White Portion (PB) coupled to a clostridial neurotoxin in which the Hc part of the h chain is removed or modified. 3. An agent according to claim 1 or 2 wherein the modified h-chain is the HN fragment of a clostridial neurotoxin. 4. An agent according to any of the preceding claims in which the clostridial neurotoxin component is obtained from botulinum neurotoxin. 5. An agent according to any of claims 1-4, wherein the clostridial neurotoxin component is obtained from botulinum neurotoxin type A. 6. An agent according to any of claims 1-4, wherein the clostridial neurotoxin component is obtained from botulinum neurotoxin type B. 7. An agent according to any of claims 1-4, wherein the clostridial neurotoxin component is obtained from botulinum neurotoxin type C1. An agent according to claim 5, which is formed by coupling a PB to the LHN fragment of botulinum neurotoxin type A. 9. An agent according to claim 6, which is formed by the coupling of a PB to the LHN fragment of botulinum neurotoxin type B. 10. An agent according to claim 7, which is formed by the coupling of a PB to the LHN fragment of botulinide neurotoxin type C1. 11. An agent according to any of claims 1-7 wherein the H chain is obtained from a clostyridial neurotoxin different from that from which the L chain is obtained. 12. A people according to claim 11, wherein the chain h is obtained from botulinum neurotoxin type A and the L chain of botulinum neurotoxin type B. 13. An agent according to claim 12, which is composed of a PB linked to the HN fragment of botulinum neurotoxin type A and the L chain of botulinum neurotoxin type B. 14. An agent according to any preceding claim wherein the L chain or L chain fragment is attached to the H chain by a direct covalent linkage. 15. An agent according to any of claims 1-13 wherein the L chain or L chain fragment is attached to the H chain by a covalent linkage that includes one or more separation regions. 16. An agent according to any preceding claim wherein the PB is capable of delivering the L chain or L chain fragment in the cytosol of a primary unassisted sensory afferent. 17. An agent according to any preceding claim wherein the ability to deliver the L chain or L chain fragment in the cytosol of a primary sensory afferent is contained completely within the PB. 18. An agent according to any preceding claim wherein PB binds to a binding site that is characteristic of a particular defined population of primary sensory afferents. 19. An agent according to any preceding claim wherein the PB binds to a binding site that is characteristic of a particular defined population of primary non-selective afferents. 20. An agent according to any of the preceding claims in which the PB is bound to a binding site that is subjected to retrograde transport within a primary sensory afferent. 21. An agent according to any of the preceding claims in which the PB is bound to a binding site that is subjected to retrograde transport within a primary nociceptive afferent. 22. An agent according to any of the preceding claims wherein the PB comprises a ligand to a cell surface receptor in a primary sensory afferent. 23. An agent according to any of the preceding claims wherein the PB comprises a ligand to a growth factor receptor in a primary sensory afferent. 24. An agent according to any of the preceding claims wherein the PB comprises a ligand to a neuropeptide receptor in a primary sensory afferent. 25. An agent according to any of the preceding claims in which the PB comprises a ligand to a cytokine receptor in a primary sensory afferent. 26. An agent according to any of the preceding claims wherein the PB comprises a ligand to a hormone receptor in a primary sensory afferent. 27. An agent according to any of the preceding claims wherein the PB comprises a monoclonal antibody or is derived from a monoclonal antibody to a surface antigen in a primary sensory afferent. 28. An agent according to claim 23, wherein the PB comprises a ligand to a de facto nerve growth receptor. 29. An agent according to claim 28, wherein the PB comprises nerve growth factor. 30. An agent according to claim 29, which comprises nerve growth factor bound to the LHN fragment of botulinum neurotoxin type A. 31. An agent according to any preceding claim wherein the PB is bound to the component derived from Clostridial neurotoxin by a direct covalent ligature. 32. An agent according to any preceding claim wherein the PB is linked to the clostridial neurotoxin-derived component by a covalent binding that includes one or more separation regions. 33. An agent according to any preceding claim that prevents the release of a neurotransmitter and neuromodulator from a primary sensory afferent. 34. An agent according to any preceding claim that inhibits the release of a neurotransmitter or neuromodulator from a primary nociceptive afferent. 35. A method for obtaining an agent according to any preceding claim comprising the covalent attachment of a PB to the modified clostridial neurotoxin or a fragment of a clostridial neurotoxin. 36. A method for obtaining an agent according to any of claims 1-34 comprising the covalent attachment of a PB to a modified clostridial neurotoxin or a fragment of a clostidial neurotoxin with the inclusion of one or more separation regions. 37. A method for obtaining an agent according to any of claims 1-34 comprising constructing a genetic construct that encodes a modified clostridial neurotoxin or a fragment of a clostridial neurotoxin, incorporating said construct into a host organism, expressing the construction for produce the modified Clostridial neurotoxin or fragment of a Clostridial neurotoxin and the covalent attachment of the modified Clostridial neurotoxin or fragment of a Clostridial neurotoxin to a PB. 38. A method for obtaining an agent according to any of claims 1-34 comprising constructing a genetic construct that encodes a modified Clostridial neurotoxin or a fragment of a Clostridial neurotoxin, incorporating said construction into a host organism, expressing the construction to produce the modified clostridial neurotoxin or fragment of a clostridial neurotoxin and the covalent attachment of the modified Clostridial neurotoxin or fragment of a Clostridial neurotoxin to a PB with the inclusion of one or more regions separators. 39. A method for obtaining an agent according to any of claims 1-34, comprising constructing a genetic construct encoding the agent, incorporating said construct into a host organism and expressing the construct to produce the agent. 40. A method for controlling the release of a neurotransmitter or neuromodulator from a primary sensory afferent by applying the agent of any of claims 1-14. 41. A method for controlling the release of a neurotransmitter or neuromodulator from a nociceptive afferent by applying the agent to any of claims 1-34. A method for controlling the transmission of sensory information from a primary sensory afferent to a projection neuron by applying the agent to any of claims 1-34 43 A method for controlling the transmission of sensory information from a primary nociceptive afferent to a neuron projection by applying the agent to one of claims 1-34 A method for controlling the chlorine sensation by applying the agent to any of claims 1-34 The use of the agent according to any of claims 1-34 or a salt pharmaceutically acceptable thereof as a medicament for pain relief 46 Use of the agent according to any of claims 1-34, or a pharmaceutically acceptable salt thereof as a medicament for the prevention of pain 47 Use of the agent in accordance with any of claims 1-34, in the manufacture of a medicament for pain relief 48 Use of the agent e according to any of claims 1-34 in the manufacture of a medicament for the prevention of pain. A method for alleviating pain which comprises administering an effective dose of the agent according to any of claims 1-34. 50. A method for preventing pain which comprises administering an effective dose of the agent according to any of claims 1-34. 51. An agent that exhibits specificity for peripheral sensory afferent that can inhibit the release of at least one neurotransmitter or neuromodulator or both from the synaptic terminals of nociceptive afferents, the agent having the following discrete functions: 1) It binds to a surface structure (the binding site [SU]) that is characteristic of, and has a degree of specificity for afferent nociceptive afferent neurons; 2) Enter the neuron; 3) Enter the cytosol; and 4) Modifies components of the exocytotic machinery present in the synaptic terminals of the central processes of the neurons, so that the release of at least one neurotransmitter or neuromodulator from the synaptic terminal is reduced or prevented.