Title:  Cytotoxin (non-neurotoxin) for the treatment of human headache disorders and inflammatory diseases

United States Patent:  6,429,189

Issued:  August 6, 2002

Inventors:  Borodic; Gary E. (Canton, MA)

Assignee:  Botulinum Toxin Research Associates, Inc. (Qunicy, MA)

Appl. No.:  458784

Filed:  December 10, 1999

Pharmaceutical applications of a chemodenervating agent reduce pain by altering release of pain and inflammation-mediating autocoids, with a duration of action between 12-24 weeks. The limiting factor in dosing for this application is weakness and paralysis created by higher doses of the chemodenervating pharmaceutical. This weakness and paralysis is mediated by action of the neurotoxin component of the chemodenervating pharmaceutical. The invention described herein represents a novel mechanism and pharmaceutical formulation which eliminates the neurotoxin component of the chemodenervating pharmaceutical, while retaining the cytotoxin component which provides an essential bioeffect for the relief of pain and inflammation. The invention allows for improvement in administering the pharmaceutical agent for the reduction of pain and/or inflammation without causing muscular weakness and paralysis.

DETAILED DESCRIPTION OF THE INVENTION

Botulinum toxin exists as multiple immunotypes (A-G), which have been investigated as to specific medical properties. The immunotypes share little cross reactivity and have been shown to differ in chemical composition and biological behavior when injected at sublethal doses to produce a regional dose-dependent effect. Differences in biological activity between the various immunotypes include (1) differing durations of action at the injection site and (2) differing regional denervation potencies, measured as the relative quantity of LD 50 units required to produce a given regional or clinical effect.

Botulinum toxin application to human essential headache disorders was originally identified in myofascial tension type headaches (see Acquardro and Borodic, Treatment of myofascial pain with botulinum A toxin. Anesthesiology. 1994 Mar;80(3):705-6), and later, as a coincident finding, for migraine headache. The coincident finding and application for migraine was noted as botulinum toxin was being used as a neurotoxin to remove forehead wrinkles. Regional facial muscular weakening effaces facial and forehead dynamic lines associated with aging. Such dynamic lines are produced by facial muscle tone as forces are transmitted from facial muscles to dermal attachments of these muscles in facial skin. The neurotoxin component was thought active in the treatment of headaches and pain during this period. Prior art U.S. Pat. No. 5,714,468 teaches that the neurotoxin (that component of botulinum toxin which causes neuromuscular paralysis) is the active agent and mechanism by which botulinum toxin is effective for the treatment of migraine and other forms of pain.

Of the immunotypes of botulinum toxin, a mutant and unusual derivative subtype of botulinum toxin is botulinum toxin C2, which exhibits no neurotoxin properties. Botulinum toxin C2 possibly represents a mutated gene derivative which demonstrates no neurotoxin capability, yet is toxic by other cytopathic mechanisms (Ohishi, I. Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components. Infect Immun 1983 May;40(2):691-5). The proteinaceous materials derived from botulinum C2 strains are, however, biologically active and have been demonstrated to cause lethal effects by mechanisms other than neuromuscular paralysis. The materials are described as cytotoxic in nature. Described herein is the demonstration of botulinum type C2 as a specific inhibitor of inflammation in a sensitized animal model, and reduction to practice utilizing botulinum C2 as a (1) therapy for inflammation and (2) therapy for migraine and tension headache treatment.

Botulinum toxin has been used for the past 16 years to treat various forms of facial movement disorders, including Blepharospasm, hemifacial spasm, bruxism, and synkinetic facial movements after chronic facial palsy. This substance has also proven substantial utility for the treatment of spasmodic torticollis, spasticity associated with cerebral palsy, stroke, occupational hand cramping, and speech disorders (spasmodic dysphonia). In each of these applications, the mechanism of action had been postulated to involve weakening of a fixed volume of muscle around the area injected for a period of 10-18 weeks, with complete reversal of the weakening effect after that time. During the period after the injection, the weakening is correlated to (1) blockage of release of acetylcholine from the presynaptic nerve terminal at the neuromuscular junction, (2) atrophy similar to motor nerve denervation atrophy in the area over which the toxin diffuses, (3) decreased contractility within the muscles over which the toxin diffuses, (4) motor nerve terminal sprouting from the motor axon terminal, (5) spread of acetylcholinesterase and acetylcholine receptors from the post synaptic membrane, and (6) reversibility of the above findings within the denervation field after 10-18 weeks.

Collectively the above describes a cycle that has been well characterized in the observations of Duchenne (Scott AB Botulinum toxin injection of eye muscles to correct strabismus. Trans Am Ophthalmol Soc 1981;79:734-70). Additionally, it has been well established that botulinum toxin has local effects on autonomic nerve ganglion and nerve function. (MacKenzie I, Burnstock G, Dolly JO The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission.Neuroscience 1982 Apr;7(4):997-1006).

The medical utility of botulinum toxin has been based primarily on the neuromuscular effects of botulinum neurotoxin, as the neurotoxin generates the cycle described in steps (1)-(6) above. The definition of a neurotoxin is an agent capable of producing death by action on a portion of the central or peripheral nervous system in such a manner as to destroy or critically impair organism function. In the case of botulinum neurotoxin, the action is at the level of the neuromuscular junction, leading to disseminated weakness with paralysis of critical muscles such as the muscles driving respiratory ventilation. The lethal effect, which occurs at a critical point of muscular weakness, is asphyxiation and suffocation. The pharmacological principle governing the utility of botulinum toxin in the treatment of human diseases is that a regional effect occurs at a diluted concentration-dose remote from the lethal concentration-dose. Stated another way, this principle is the property of neurotoxin that allows a regional effect at a neurotoxin dilution and concentration substantially lower than that concentration that would cause a lethal systemic effect for the various types of botulinum neurotoxin used. That lower concentration allows for regional muscular weakening, which has been thought to be the sole mechanism by which the neurotoxin exerts its beneficial action in diseases involving spastic or involuntary movement.

Despite this scientific understanding of botulinum toxin as a neurotoxin, there remains insufficient understanding of the biological tissue effects to explain observed utility for other medical conditions such as the treatment of human pain such as occurs in essential headache disorders, myofascial pain, and certain pain components associated with dystonias. Also, there exists no explanation of the mechanism by which botulinum toxin is effective in reducing inflammation within the denervation field. The action of botulinum toxin as a neurotoxin, a substance acting at the neuromuscular junction causing muscular weakness, fails to provide a sufficient basis for the mechanism by which utility is achieved for conditions which are not associated with abnormality in movements.

Described herein is the bioeffect thought critical to the property of botulinum toxin that is directly or indirectly related to its ability to relieve human pain. Also described are methods by which this property can be chemically dissociated from the neurotoxin component (muscle-weakening component) of the botulinum toxin pharmaceutical agent, thereby generating a new perfected botulinum-derived pharmaceutical agent capable of eliminating the undesirable muscle weakness associated with injection of prior-art botulinum toxin preparations into a diseased area.

Efforts to explain the critical property of botulinum toxin capable of causing an improvement in pain associated with essential headache disorders and migraine headaches initially came from observations of the patient seen in FIG. 1. This 53 year old woman experienced flushed face and disseminated itching following physical exertion. The face demonstrated hives, associated with the flushing. Her past medical history was significant for Bell's palsy for which she received a botulinum type A injection for the treatment of forehead asymmetry. It was noted that after the botulinum toxin was injected into the forehead, there would be white blotches appearing on the forehead in which there was no flushing, and no hive formation (blocked urticaria within the denervation field). (See FIG. 1). This patient exhibited this effect after physical exertion consistently for a period of three months after the botulinum type A injections and the effect slowly faded thereafter. This duration of effect is typical for botulinum type A injections.

The syndrome of cholinergic urticaria is typically associated with urticarial eruption after exertion. Sometimes the condition is also associated with symptoms of asthma. The pathophysiology has been linked to increased release of circulating histamine, as well as mast cell degranulation. As the above-noted bioeffect appeared novel and not well explained by existing understanding of botulinum toxin efficacy, efforts were made to confirm the effect on human mast cells in an in vivo laboratory experiment. A Hart Bartley guinea pig (a guinea pig prone to type 1 hypersensitivity reactions) was sensitized to pollen spores (short ragweed pollen-Ambrosia artemisfolia), with aerosolized spores sprayed into the conjunctiva of the animals for a period of two weeks. Prior to this exposure, the animals had no reaction to the pollen, with the conjunctival membranes appearing white and quiet after exposure. After two weeks however, animals were again exposed to the short ragweed pollen, which caused acute edema, erythema, itching, flame hemorrhages within the conjunctiva, and distortion of the eyelids. This animal model has been pathologically characterized as being associated with measurable mast cell degranulation histologically, when pollen spores were exposed to sensitized conjunctiva.

The typical reaction is seen in FIG. 2. FIG. 3 shows the protection by botulinum toxin from the inflammatory response after exposure to the short ragweed pollen. The duration of the protective effect is demonstrated in FIG. 4 for a series of 6 animals followed for 6 months. Given the demonstrated efficacy in cholinergic urticaria and demonstrated anti-inflammatory effect in the allergic animal model measuring immediate hypersensitivity reactions, reactions thought to represent mast cell degranulation phenomenon, it appears that botulinum toxin either directly or indirectly is influencing the system which involves mast cells, histamine, possibly serotonin, and other related autocoids in such a fashion to cause a blocked physiological response important to the pathogenesis of certain forms of inflammation and pain. Due to release of autocoids, such as various forms of prostaglandins and leukotrienes as well as other formed and generated local mediators, and as an obvious clinical observation, it is expected that the inflammatory response will be associated with pain degeneration by mechanisms relating to alterations of mast cell secretion or degranulation.

In a known physiological assay, the relationship between mast cell degranulation and pain is clearly demonstrated. After a type 1 hypersensitivity response is demonstrated on the forearm of a person with known allergy to an introduced allergen, there appears to be a typical wheal and flare response associated with the sensory perception of itching. This is known as the immediate response. After a period of 6-8 hours, a late response is occasionally noted, characterized not by itching but rather tenderness and pain. The immediate response is thought to be associated and effected by preformed mediators such as histamine, whereas the late response is thought to be associated with the leukotrienes and prostaglandins. The prostaglandins and leukotrienes are important in the late phase reaction and are associated with pain generation. Compounds known to block prostaglandin derivatives such as indomethacin and corticosteroids will also block the late phase reactions associated with mast cell degranulation. In cellular systems, dependent on the adhesion of mast cells, there has been observed an increase or decrease in secretion induced by C. botulinum C2 toxin. In suspended mast cells, pretreatment with botulinum C2 toxin causes inhibition of secretion. In contrast, in adherent mast cells, the destruction of the cytoskeleton by botulinum C2 toxin causes increase in secretion. Thus, the signaling is largely effected by adhesion of mast cells in cellular in vitro studies, and mast cells have the capability of being influenced by the non-neurotoxin botulinum C2.

There exists a relationship between mast cell activity and migraine and other forms of essential headaches. The pathophysiolgy of essential headaches and migraine has been thought to relate to mast cell function and mast cells degranulation. (Theoharides, TC. The mast cell: a neuroimmunoendocrine master player. Int J Tissue React 1996;1 8(1):1-21; Moskowitz, Ma. Neurogenic inflammation in the pathophysiology and treatment of migraine. Neurology 1993 Jun;43(6 Suppl 3):S 16-20; Delepine, L., Aubineau, P. Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion. Exp Neurol 1997 Oct;147(2):389-400.)

Authors cited above have found that a relationship exists between mast cells and the possible mechanism by which pain is generated in headache disorders, postulating that mast cells play a functional role in the generation of pain nerve adaptation at C-fibers. Although postulated, it appears that no absolute proof relating mast cells to pain generation has been totally established.

Clinical observations have also linked allergy and mast cell function to the syndrome of migraine headache. The following factors indicate the relationship between mast cells and migraine based on the relationship between type I hypersensitivity reactions and migraine. (1) Hayfever allergy season brings out migraines. (2) Stress can be associated with both migraine and urticarial reactions. (3) Patients born to mothers with common migraine are more likely to have offspring with allergic asthma, a mast cell related disease. (4) A known migraine patient receiving a forehead bee sting experienced violent migraine headache within two minutes of the sting. (5) Forms of food allergy are thought to precipitate migraine headaches. (6) Components of headaches (light sensitivity) can also be associated with migraine. (7) Patients with migraine often have elevated blood histamine levels. (8) Mast cells are responsive to cytotoxins.

In each case, mast-cell-generated inflammation is conceived as a form of inflammation and/or tissue change that provokes genetically predisposed individuals to develop a violent painful sensory experience. Described herein is a cytotoxic botulinum-derived compound capable of blocking inflammation without causing a neurotoxic (neuromuscular) effect.

There exist both advantages and limitations of botulinum neurotoxins in the treatment of human essential headache disorders and human inflammation. Botulinum toxin has many advantages over existing therapy for the treatment of essential headache disorders. These include (1) lack of systemic side effects, particularly compared to the krypton class of drugs, (2) long duration of action (3-5 months), (3) maintenance free therapy (no pills, no autoinjections), (4) high degree of efficacy.

The major limiting factor is that the prior-art medication produces weakness. In a series of 104 patients treated with type A botulinum neurotoxin, the major side effect was ptosis from diffusion of the botulinum toxin into the orbital space (Borodic). Diffusion of botulinum toxin and attendant weakening effect is not seen only with the treatment of human pain syndromes, but also has been noted with treatment of movement diseases (blepharospasm) causing drooping lids (ptosis), and treatment of cervical dystonia (torticollis) causing difficulty swallowing food (dysphagia).

Hence for the treatment of movement disease the neurotoxin and weakening bioeffects of botulinum toxin are both helpful and a cause of complication. In diseases in which there is no involuntary muscular movements or tone, such as tension or migraine headaches, or forms of human inflammation, the neurotoxin effect would be more detrimental to human clinical applications, causing weakness solely as a complication. Here lies the fundamental utility of the present invention. Botulinum toxin exists as immunotypes A-G. Each immunotype is a neurotoxin and causes neuromuscular blockade and weakness when locally injected. However one strain of botulinum toxin, perhaps a mutant or derivative strain, has produced a non-neurotoxin protein which demonstrates a selective interaction with the mast cell, does not interact with the neuromuscular junction (neurotoxin), and does not produce weakness (as shown in FIG. 5). This botulinum protein is biologically active and theorized to act at important tissue sites relative to human pain, migraine headaches, tension headaches, and headaches involving human inflammation-involving mast cells, or mast-cell-contained mediators of inflammation.

This protein is characterized by those skilled in botulinum toxin technology as a cyototoxin, which causes intestinal inflammation as a cause of toxicity, without inducing muscular weakness. Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin act on ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the intracellular microfilament network (intracellular actin cytoskeleton). Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These cytoskeletal modifications may contribute to the cytopathic action of this toxins.

The botulinum toxin used to practice the present invention may be prepared as follows. A preparation is made consisting of a Clostridium botulinum strain which produces solely C2 cytotoxin. Culture is accomplished with appropriate agents to procure the maximum number of LD 50 units per ml of culture solution. LD 50 units are determined using the mouse bioassay, and the preparation may be freeze-dried for the purpose of preservation and stability. A known quantity of bioactivity as determined by LD 50 is injected into the area in which pain, headache, or inflammation have been diagnosed by the clinician. A quantity for injection is chosen by the clinician based on LD 50 units.

Botulinum C2 is characterized as a non-neurotoxin, capable of causing increased vascular permeability, fluid accummulation in ligated intestine, and rounding of tissue cultured cells. Bioassay for the activity can be accomplished by a "time to mouse death method" after intravenous infusion, or by intraperitoneal injection. Anti-neurotoxin sera to botulinum toxin may be used to confirm complete neutralization of the neurotoxin prior to bioassay. Preparation may be accomplished by biochemical isolation from spent cultures, or by recombinant production using either E coli or a Clostridium expression system, given that genes encoding the C2 protein have been elucidated.

Botulinum C2 cytotoxin has been shown to consist of two protein components not covalently bound, segment I and segment II, both required for the biological activity of the toxin. The following represents examples of preparations that can be used to isolate the C2 property from neurotoxin properties of botulinum toxin. Because the biological activity of component II has not been recovered after freeze-drying, a liquid formulation of the material would be preferred.

Claim 1 of 29 Claims

What is claimed is:

1. A pharmaceutical composition for treatment of inflammation or pain comprising purified protein, wherein said purified protein is derived from Clostridium botulinum, and wherein said purified protein exhibits no neurotoxin properties.
 

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