Muscle contraction is the physiological process in which muscles develop tension and are shortened or lengthened (or can also remain the same length) due to a prior excitation stimulus. These contractions produce the motive force of almost all superior animals to, for example, displace the contents of the cavity that they cover (smooth muscles) or move the organism through its environment or to move other objects (striated muscle).
Muscles are formed by contractile multinucleated muscle fibers (syncytes which result from the fusion of several myocytes), arranged forming portions which are innervated by the synaptic buttons in neuronal axons. The junction between neuron and muscle fiber is called a neuromuscular junction or NMJ. Neurons transmit stimuli in the form of action potentials and this causes the release of the neurotransmitter acetylcholine (ACh) to the synaptic cleft. In the postsynaptic part of NMJ there is an accumulation of acetylcholine receptors (AChRs) responsible for receiving the signal and causing the muscle to contract.
AChRs are classified into two large groups according to their binding with specific agonists: nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs). Nicotinic acetylcholine receptors are given this name due to the high affinity with which they bind to the alkaloid nicotine; the muscarinic acetylcholine receptors, however, bind with a high affinity to the alkaloid muscarine. The nicotinic acetylcholine receptors, in turn, can be classified according to their capacity or incapacity to bind α-bungarotoxin. nAChRs are ionotropic receptors, i.e., they are coupled to ionic channels whose pathway is opened by the ligand binding. mAChRs are metabotropic type receptors or receptors which are coupled to G proteins: in their case the circulation of ions depends on one or various metabolic pathways triggered by the ligand binding.
The neuromuscular junction has classically been used as a general neurotransmission model and therefore its functioning is described in detail. An action potential arrives at the terminal part of the motor neuron and causes the voltage-dependent calcium channels to open in the presynaptic membrane, with a resulting local intracellular increase in calcium. The Ca2+ ions bind to proteins which link the synaptic vesicles, full of neurotransmitter ACh, to the plasma membrane. In this case, the synaptobrevin protein binds strongly to syntaxin and SNAP-25, forming the SNARE complex (SyNaptosomal-Associated Protein Receptor), which enables the fusion between vesicular and plasma membranes and causes exocytosis of ACh to the synaptic cleft. This neurotransmitter spreads through the synaptic space and binds to the AChRs in the postsynaptic membrane. The binding of ACh (or another agonist) causes the channel to open and this enables the circulation of sodium ions towards to inside of the cell and the potassium to leave. The entrance of sodium causes a depolarization which is transmitted through the cell membrane until it arrives at the specialized areas that trigger the release of calcium from endoplasmic reticulum towards the cytosol which induces the contraction of the muscle.
At a molecular level, the nAChRs are pentameric proteins formed by 5 monomers arranged in radial symmetry and forming a pore 2.5 nm in diameter. These sub-units are generally found in a stoichiometry (α)2-β-γ-δ, and 17 types of subunits have been described: 10 types of α-called α1 to α10-, 4 types of subunit β-called β1 to β4-, γ, δ, and ε [Lindstrom J. (1997) “Nicotinic acetylcholine receptors in health and disease” Mol. Neurobiol. 15:193-222]. The molecular composition at a sub-unit level finely determines the functionality of the receptor: in the neuromuscular junction, for example, the nAChRs are heteropentameric (α1)2-β1-γ-δ in the embryonic phase, whilst in the adult phase they are heteropentameric (α1)2-β1-δ-ε. The change of sub-unit γ to ε has been related to the maturation of the neuromuscular junction, and with the maintenance of the structure in adult muscle [Yumoto N., Wakatsuki S. and Sehara-Fujisawa A. (2005) “The acetylcholine receptor gamma-to-epsilon switch occurs in individual endplates” Biochem. Biophys. Res. Commun. 331:1522-1527].
The neuromuscular junction is a specialized structure whose function is to ensure an efficient and rapid transmission of an action potential in order to cause depolarization in the postsynaptic muscle, which is the signal required for this to contract itself. To achieve a rapid transfer of information between neuron and muscle fiber a correct spatial disposition of the presynaptic zone with the postsynaptic membrane is required. The presynaptic part is characterized by the presence of ACh vesicles, whilst almost all the muscle fiber AChRs are concentrated and are densely packed in the postsynaptic part. The AChRs cluster in a complex process which is key in the formation of the NMJ and its maintenance [Hoch W. (1999) “Formation of the neuromuscular junction. Agrin and its unusual receptors” Eur. J. Biochem. 265:1-10].
AChR clustering requires the interaction of various proteins, of which three should be highlighted: agrin, MuSK (muscle-specific kinase) and rapsyn. The protein agrin is believed to be responsible for triggering the pathway which leads to the clustering of receptors [Mittaud P., Camilleri A. A., Willmann R., Erb-Vögtli S., Burden S. J. and Fuhrer C. (2004) “A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors” Mol. Cell Biol. 18:7841-7854], and which results in phosphorylation, clustering and stabilization of AChRs. The protein rapsyn is capable of inducing AChR clustering in culture and it has been demonstrated that in mutants deficient for rapsyn the formation of AChR clusters is inhibited [Apel E. D., Roberds S. L., Campbell K. P. and Merlie J. P. (1995) “Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex” Neuron 1:115-126]. The factor that links agrin and rapsyn is MuSK [Valenzuela D. M., Stitt T. N., DiStefano P. S., Rojas E., Mattsson K., Compton D. L., Nuñez L., Park J. S., Stark J. L., Gies D. R., Thomas S., Le Beau M. M., Fernald A. A., Copeland N. G., Jenkins N. A., Burden S. J., Glass D. J. and Yancopoulos G. D. (1995) “Receptor tyrosine kinase specific for the skeletal muscle lineage: Expression in embryonic muscle, at the neuromuscular junction, and after injury” Neuron 3:573-584]. MuSK is a receptor with kinase activity which phosphorylates the protein agrin in an interaction which requires an additional component called MASC (muscle associated specificity component). The activation of MuSK through the binding of this ligand is not sufficient on its own to cause AChR clustering, and an interaction between MuSK and rapsyn has been proposed which would be mediated by a factor called RATL (rapsyn associated transmembrane linking molecule), through which the AChR-rapsyn complex could interact with the agrin-MASC-MuSK complex and form the postsynaptic apparatus of mature NMJs [Zhou H., Glass D. J., Yancopoulos G. D. and Sanes J. R. (1999) “Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations” J. Cell Biol. 146:1133-1146]. It has been found that the gene which codifies MuSK is disturbed in severe muscular diseases such as congenital myasthenic syndrome, a disease caused by a decrease in neurotransmission in which patients suffer from muscle weakness [Chevessier F., Faraut B., Ravel-Chapuis A., Richard P., Gaudon K., Bauché S., Prioleau C., Herbst R., Goillot E., Ioos C., Azulay J. P., Attarian S., Leroy J. P., Fournier E., Legay C., Schaeffer L., Koenig J., Fardeau M., Eymard B., Pouget J. and Hantaî D. (2004) “MUSK, a new target for mutations causing congenital myasthenic syndrome” Hum. Mol. Genet. 13:3229-3240], and the relevant role of MuSK in this disease has been confirmed through the establishment of a mouse model in which the MuSK gene lacked its kinase domain and that also shows a phenotype with muscle weakness [Chevessier F., Girard E., Molgó J., Bartling S., Koenig J., Hantaî D. and Witzemann V. (2008) “A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions” Hum. Mol. Genet. 17:3577-3595].
Muscle contractions are controlled by the central nervous system; the brain controls voluntary contractions, whilst the spinal cord controls involuntary reflects. Both processes are mediated by the regulated release of the neurotransmitter acetylcholine. However, different disorders or neurological diseases are characterized by involuntary muscle spasms or dystonia, in which the muscles excessively contract in a sustained way [Fahn S. (1988) “Concept and classification of dystonia” Adv. Neurol. 50:1-8]. The appearance of the movements depends on the muscles involved and strength of the contraction. In its weakest form, dystonia can appear to be a simple exaggeration of a movement which would be considered normal, whilst in its most severe form dystonia can even cause abnormal postures of the limbs which are disabling. Depending on the parts of the body which are affected, dystonia is classified as (1) focal dystonia, localized in a specific part of the body, comprising, among others, dystonia of the periocular muscle or blepharospasm, cervical dystonia or spasmodic torticollis, laryngeal dystonia or spasmodic dysphonia, oromandibular dystonia, focal hand dystonia such as writer's or musician's cramp or dystonia of the feet, bruxism, hemifacial spasm, tics and strabismus; (2) generalized dystonia, which affects the greater part or the whole body; (3) multifocal dystonia, which involves two or more unrelated parts of the body; (4) segmental dystonia, which affects two or more adjacent parts of the body, comprising Meige's Syndrome, and (5) hemidystonia, which involves the arm and leg of the same side, comprising dystonia resulting from a stroke [Jinnah H. A. and Hess E. J. (2008) “Experimental therapeutics for dystonia” Neurotherapeutics 5:198-209; Brooke M. H. (1986) “A clinician's view of neuromuscular diseases” Baltimore, Williams & Wilkins; Layzer R. B. (1986) “Muscle pain, cramps and fatigue” in Myology, A G, Engel, B Q Banker, eds, New York, McGraw-Hill].
On the other hand, the repeated contraction of the facial muscles, which are those that transmit expression onto the face, lead to creases in specific areas. With age, from approximately thirty years of age, the appearance of wrinkles or expression lines start to become visible in these areas: furrows, more or less linear, arranged perpendicularly to the direction of the muscle fibers, caused by the disappearance of cells in the skin's dermal layer in these areas. Their depth depends on the individual's age, and the frequency and strength of the muscle contractions which cause the crease. The principle muscles involved in the appearance of expression lines are those surrounding the eyes and eyelashes, those on the forehead, the lip, mouth, cheek and neck muscles. These muscles are found in the subcutaneous connective frontal part of the face, from where they rise towards the skin and insert themselves in the deepest part of the dermal stratum. Their contraction can lead to raising, depressing, constricting or dilatory movements of the skin. The early appearance of wrinkles is the most characteristic sign of age and aging of the skin. Aging of the skin is a process which has two principal components: the chronological component, which is due to the passing of time, and the photo-induced or photoaging component, which is due to the level of exposure to ultraviolet radiation (UV). The sum of several environmental factors such as exposure to tobacco smoke, exposure to pollution, and climate conditions such as cold and/or wind also contributes to the skin's aging. The terms “aging” and “photoaging” of the skin refer to the visible changes in the appearance of the skin such as wrinkles, fine lines, roughness, expression lines, stretch marks, discontinuities, furrows, flaccidity, sagging of the skin such as sagging cheeks, loss of resilience, loss of firmness, elastosis, keratosis, and loss of smoothness.
Since the 1990s, the use of the toxins Clostridium botulinum (marketed as Botox® by Allergan) injected into the muscle to reduce muscle contraction and to treat associated diseases such as dystonia and/or pain. Neurotoxin injections have also been used to treat and/or care for the skin with the aim of reducing, delaying or preventing the signs of aging and/or photoaging and in particular to relax the facial expression and reduce the formation of wrinkles or minimize their appearance. Its action mechanism is based on blocking ACh release in the presynaptic terminal of the axon in the neuromuscular junction, thus avoiding nerve transmission and muscle contraction. The toxin binds to receptors in the presynaptic membrane, is internalized and becomes cytoplasm. Its activity is responsible for breaking the trimolecular synaptobrevin SNARE complex, SNAP-25 and syntaxin, which avoids the binding of synaptic vesicles to plasmalemma and releasing ACh to the synaptic cleft.
Although it has been demonstrated that the effects of the botulinum neurotoxin are very lasting, since they take several weeks to reestablish a completely functional innervation of the muscle after the irreversible destruction of the SNARE complex, its administration is not risk free. The main risk is that of its toxicity: even doses lower than 50 μg are lethal for an adult human being. The botulinum neurotoxin causes muscle paralysis, and can even cause death due to cardiac arrest. Administration of the botulinum toxin should, therefore, be carried out by a specialist due to the intrinsic danger of the substance as well as the administration method: direct injection into the muscle. The blocking of the nerve transmission at AChR level through botulinum toxin is not specific enough to just inhibit muscle contraction, but it has other activities which may be undesired by the patient and can cause secondary effects such as nausea, pain or erythema.
The pharmaceutical field has also used other compounds for the inhibition of muscle contraction, including modulators of the gamma-aminobutyric receptor (GABA) such as baclofen or benzodiazepines, agonists of the α2-adrenergic receptors such as tizanidine or clonidine, modulators of AChRs such as the alkaloids derived from curare, specific antibodies against AChRs such as those described in U.S. Pat. No. 6,780,413 B2 or agonists of the ryanodine receptor such as dantrolene. None of these treatments is free from secondary effects, since many of them act at a central nervous system level and may cause dizzy spells, sedation and even addiction in continued treatments.
The cosmetic industry has also carried out different efforts to develop new compounds for the treatment of expression lines and, therefore, to treat and/or care for the skin with the aim of reducing, delaying or preventing the signs of aging and/or photoaging through topical application which avoids the potential secondary effects which occur after injecting botulinum toxin and are, therefore, safer. Different products aimed at the inhibition of the neuromuscular junction at a synaptic level to avoid the appearance or to soften expression lines are described in the prior art. The patents EP 1180524 B1 and WO9734620 describe the use of peptides derived from the protein SNAP-25 which act pre-synaptically competing with SNAP-25 in the formation of the SNARE complex, causing a reduction in the release of ACh and inhibiting the neuronal transmission in the NMJ. Patent EP 1809652 A2 describes antagonist peptides of AChRs which act post-synaptically with a mechanism of action similar to waglerin-1 to block the nerve transmission and prevent the appearance of wrinkles. The active cosmetic pentapeptide-3 also acts post-synaptically by inhibiting AChRs, with a mechanism of action similar to tubocurarine to block the nerve transmission and prevent the appearance of wrinkles.
However, none of the compounds developed by the cosmetic or pharmaceutical field is capable of inhibiting muscle contraction with effectiveness similar to that of botulinum toxin, but in a way that is risk free. Thus there is still a need to identify new agents capable of inhibiting muscle contraction for their co-administration with existing agents in order to achieve better results in the treatment of dystonias as well as in the reduction and/or softening of wrinkles, and in particular, expression lines and, therefore, treat and care for the skin with the aim of reducing, delaying or preventing the signs of aging and/or photoaging.
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USPTO Backgrounds