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Definitions

• this releasing hormone Today, the importance of this releasing hormone is widely recognized for its regulatory role in human development and growth. Furthermore, this releasing hormone may be the basis of various crippling illnesses. Commonly, this particular releasing hormone is referred to as the gonadotrophin-releasing hormone (GnRH) .
• GnRH gonadotrophin-releasing hormone
• a normal production of GnRH beneficially regulates the body's level of LH and FSH (also known as gonadotrophins) .
• LH together with FSH stimulates the release of estrogens from the maturing follicles in the ovary and induces the process of ovulation in the female.
• LH stimulates the interstitial cells and is, for that reason, also called interstitial cell stimulating hormone (ICSH) .
• ICSH interstitial cell stimulating hormone
• FSH induces maturation of the follicles in the ovary and together with LH, plays an important role in the cyclic phenomena in the female. FSH promotes the development of germinal cells in the testes of the male.
• GnRH gonadotrophin secretion
• a high level of circulating gonadotrophin is known to cause, for example, precocious puberty, endometriosis, breast cancer, prostate cancer, pancreatic cancer and endometrial cancer. These illnesses may be treated by reducing the level of gonadotrophin secretion.
• GnRH agonists and antagonists are existing drugs that act to decrease gonadotrophin secretion.
• GnRH agonists act by initially increasing the quantity of gonadotrophin secreted by the pituitary. However, with treatment of the agonist over a period of time, gonadotrophin secretion will decrease. (Presently, the mechanism behind how the agonist reduces gonadotrophin secretion is not fully understood.)
• GnRH antagonists act by binding competitively to the
• GnRH receptors on the pituitary thereby preventing GnRH from exerting its stimulatory effect on pituitary cells.
• GnRH antagonists and agonists have proven effective in the treatment of certain conditions which require a reduction of gonadotrophin release. For example, they have proven effective in the treatment of endometriosis, uterine fibroids, polycystic ovarian disease, precocious puberty and several gonadal steroid-dependent neoplasia, most notably cancers of the prostate, breast and ovary. GnRH agonists and antagonists have also been investigated as a potential contraceptive in both men and women.
• GnRH agonist and antagonist have been useful, their continual administration may be problematic.
• treatment using GnRH agonists is normally limited to a six-month duration because of the negative effects that GnRH agonist therapy can have on bone mineral density (BMD) .
• Women of reproductive age who undergo GnRH agonist therapy often show as much as 2.3% loss in BMD, comparable to the loss typically experienced by women in the first several years of menopause. This loss in women of reproductive age is particularly noteworthy, because bone density in women of this age group is still often increasing.
• Use of GnRH antagonists in the clinical setting is a relatively new event .
• Nett et al . in U.S. Patent 5,631,229 further discloses a potential method of reducing GnRH secretion by administering to a patient a cytotoxin conjugate, for example a diphtheria toxin-GnRH.
• a cytotoxin conjugate for example a diphtheria toxin-GnRH.
• the disclosure of Nett et al . is incorporated in its entirety herein by reference
• Such conjugate may reduce GnRH secretion, its long-term administration may amount to a continual destruction of cells in the brain, which may be detrimental .
• Clostridiu The bacterial genus Clostridiu includes more than one hundred and twenty seven species, grouped according to morphology and function.
• Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes the neuroparalytic illness in humans and animals referred to as botulism.
• the spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism.
• the effects of botulism typically appear 18 to 36 hours after eating food infected with a Clostridium botulinum culture or spores.
• the botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor nerves . Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
• Botulinum toxin type A is the most lethal natural biological agent known to man. About 50 picograms of a commercially available botulinum toxin type A (purified neurotoxin complex) 1 is a LD 50 in mice (i.e. 1 unit) . One unit of BOTOX ® contains about 50 picograms (about 56 attomoles) of botulinum toxin type A complex. Interestingly, on a molar basis, jbotulinum toxin type A is about 1.8 billion times more lethal than diphtheria, about 600 million times more lethal than sodium cyanide, about 30 million times more lethal than cobra toxin and about 12 million times more lethal than cholera. Singh, Cri tical Aspects of Bacterial Protein Toxins, pages 63-84
• botulinum toxin is defined as the LD S0 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each.
• botulinum neurotoxin serotypes A, B, C ⁇ , D Seven generally immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C ⁇ , D,
• botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B.
• botulinum toxin type B has been determined to be non- toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD 50 for botulinum toxin type A.
• Botulinum toxin Type B Experimental and Clinical Experience, being chapter 6, pages 71-85 of "Therapy With Botulinum Toxin", edited by Jankovic, J. et al . (1994), Marcel Dekker, Inc.
• Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine .
• serotype the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages.
• the toxin binds to the presynaptic membrane of a motor neuron through a specific interaction between the heavy (or H) chain of the botulinum toxin and a neuronal cell surface receptor.
• the receptor is believed to be different for each type of botulinum toxin and for tetanus toxin.
• the carboxyl end segment of the H chain, H c appears to be important for targeting of the toxin to the surface of the motor neuron.
• the toxin crosses the plasma membrane of the motor neuron.
• the toxin is first engulfed
• the last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain, H chain, and the light (or
• the L chain is a zinc (Zn ++ ) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane.
• Tetanus neurotoxin, Jotulinum toxin types B, D, F, and G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP) ) , a synaptosomal membrane protein.
• VAMP vesicle-associated membrane protein
• Botulinum toxin serotype A and E cleave SNAP-25.
• Botulinum toxin serotype C cleaves syntaxin and SNAP-25.
• Each of the botulinum toxins specifically cleaves a different bond, except botulinum toxin type B and tetanus toxin which cleave the same bond.
• Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles.
• a botulinum toxin type A complex has been approved by the U.S.
• Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A. Most if not all of the botulinum toxins can, upon intramuscular injection, produce significant muscle paralysis within .one day of the injection, as measured, for example, by the mouse Digit Abduction Score (DAS) .
• DAS Digit Abduction Score
• pancreatic islet B cells contains at least SNAP-25 and synaptobrevin
• the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins.
• the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms.
• Botulinum toxin types B and C 1 are apparently produced as only a 700 kD or 500 kD complex.
• Botulinum toxin type D is produced as both 300 kD and 500 kD complexes.
• botulinum toxin types E and F are produced as only approximately 300 kD complexes.
• the complexes i.e. molecular weight greater than about 150 kD
• These two non-toxin proteins may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested.
• botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
• vi tro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue.
• botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine (Habermann E., et al . , Tetanus Toxin and Botulinum A and C Neurotoxins Inhibi t Noradrenaline
• Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form.
• botulinum toxin serotypes C l t D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture.
• Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form.
• the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture.
• a certain percentage of any preparation of, for example, the jbotulinum toxin type B toxin is likely to be inactive, possibly accounting for the known significantly lower potency of botulinum toxin type B as compared to botulinum toxin type A.
• the presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy.
• botulinum toxin type B has, upon intramuscular injection, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level .
• High quality crystalline Jbotulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of >3 X IO 7 U/mg, an
• the known Shantz process can be used to obtain crystalline botulinum toxin type A, as set forth in Shantz, E.J., et al , Properties and Use of Botulinum Toxin and Other Microbial Neurotoxins in
• the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium.
• the known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1-2 X 10 s LD 50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2 X 10 s LD S0 U/mg or greater, and; purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1-2 X 10 7 LD 50 U/mg or greater.
• Botulinum toxins and/or botulinum toxin complexes can be obtained from List Biological Laboratories, Inc., Campbell, California; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wisconsin) as well as from Sigma Chemicals of St Louis, Missouri.
• Both pure Jbotulinum toxin and botulinum toxin complexes can be used to prepare a pharmaceutical composition.
• Both pure botulinum toxin and botulinum toxin complexes, such a toxin type A complex are susceptible to denaturation due to surface denaturation, heat, and alkaline conditions.
• Inactivated toxin forms toxoid proteins which may be immunogenic.
• the resulting antibodies can render a patient refractory to toxin injection.
• botulinum toxin type A is detoxified by heat, various chemicals surface stretching and surface drying. Additionally, it is known that dilution of the toxin complex obtained by the known culturing, fermentation and purification to the much, much lower toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution. Since the toxin may be used months or years after the toxin containing pharmaceutical composition is formulated, the toxin can stabilized with a stabilizing agent such as albumin and gelatin.
• a stabilizing agent such as albumin and gelatin.
• a commercially available botulinum toxin containing pharmaceutical composition is sold under the trademark BOTOX ® (available from Allergan, Inc., of Irvine,
• BOTOX ® consists of a purified botulinum toxin type A complex, albumin and sodium chloride packaged in sterile, vacuum-dried form.
• the botulinum toxin type A is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine and yeast extract .
• the botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemaglutinin protein.
• the crystalline complex is re-dissolved in a solution containing saline and albumin and sterile filtered (0.2 microns) prior to vacuum drying.
• BOTOX ® can be reconstituted with sterile, non-preserved saline prior to intramuscular injection.
• Each vial of BOTOX ® contains about 100 units (U) of Clostridium botulinum toxin type A purified neurotoxin complex, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride in a sterile, vacuum-dried form without a preservative .
• BOTOX To reconstitute vacuum-dried BOTOX" , sterile normal saline without a preservative; (0.9% Sodium Chloride Injection) is used by drawing up the proper amount of diluent in the appropriate size syringe. Since BOTOX" may be denatured by bubbling or similar violent agitation, the diluent is gently injected into the vial.
• BOTOX is preferably administered within four hours after the vial is removed from the freezer and reconstituted. During these four hours, reconstituted BOTOX" can be stored in a refrigerator at about 2° C. to about 8°C. Reconstituted, refrigerated
• botulinum toxin type A has been used in clinical settings as follows:
• BOTOX ® per intramuscular injection (multiple muscles) to treat cervical dystonia
• BOTOX ® per intramuscular injection to treat glabellar lines (brow furrows)
• glabellar lines brown furrows
• BOTOX ® to treat constipation by intrasphincter injection of the puborectalis muscle
• biceps brachii 50 U to 200 U.
• Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX ® by intramuscular injection at each treatment session.
• pericranial injected injection of 25 U of BOTOX ® has showed significant benefit as a prophylactic treatment of migraine compared to vehicle as measured by decreased measures of migraine frequency, maximal severity, associated vomiting and acute medication use over the three month period following the 25 U injection.
• intramuscular botulinum toxin has been used in the treatment of tremor in patients with
• Parkinson's disease although it has been reported that results have not been impressive. Marjama-Jyons, J. , et al . , Tremor- Predominant Parkinson ' s Disease, Drugs &
• botulinum toxin type A can have an efficacy for up to 12 months (European J. Neurology 6 (Supp 4): S111-S1150 :1999) , and in some circumstances for as long as 27 months.
• botulinum toxin type A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes .
• Botulinum toxin preparations were injected into the head of the right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscle weakness was assessed using the mouse digit abduction scoring assay (DAS) .
• ED 50 values were calculated from dose response curves. Additional mice were given intramuscular injections to determine LD 50 doses. The therapeutic index was calculated as LD 50 /ED 50 .
• BOTOX ® (5.0 to 10.0 units/kg) or botulinum toxin type B
• BOTOX ® (1.5 or 6.5 ng/kg for botulinum toxin type B or 0.15 ng/kg for BOTOX ® ) . Peak muscle weakness and duration were dose related for all serotypes.
• DAS ED S0 values (units/kg) were as follows: BOTOX ® : 6.7, Dysport ® : 24.7, botulinum toxin type B: 27.0 to 244.0, botulinum toxin type F: 4.3.
• BOTOX ® had a longer duration of action than botulinum toxin type B or botulinum toxin type F.
• Therapeutic index values were as follows: BOTOX ® : 10.5, Dysport ® : 6.3, botulinum toxin type B: 3.2.
• botulinum toxin type A was greater than botulinum toxin type B, and botulinum toxin type B duration of effect was greater than botulinum toxin type F.
• botulinum toxin type A was greater than botulinum toxin type B, and botulinum toxin type B duration of effect was greater than botulinum toxin type F.
• BOTOX the two commercial preparations of botulinum toxin type A
• Dysport the two commercial preparations of botulinum toxin type A
• the increased water consumption behavior observed following hind limb injection of Jbotulinum toxin type B indicates that clinically significant amounts of this serotype entered the murine systemic circulation.
• the results also indicate that in order to achieve efficacy comparable to botulinum toxin type A, it is necessary to increase doses of the other serotypes examined. Increased dosage can comprise safety.
• botulinum toxins may also have inhibitory effects in the central nervous system. Work by Weigand et al, Nauny-Schmiedeberg' s Arch . Pharmacol . 1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg ' s
• botulinum toxin is able to ascend to the spinal area by retrograde transport.
• a botulinum toxin injected at a peripheral location for example intramuscularly, may be retrograde transported to the spinal cord.
• the neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic) , by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system.
• acetylcholine has an excitatory effect .
• acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of heart rate by the vagal nerve .
• the efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system.
• the preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord.
• the preganglionic sympathetic nerve fibers, extending from the cell body synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons .
• Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors.
• the muscarinic receptors are found in all effector cells stimulated by the postganglionic, neurons of the parasympathetic nervous system as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system.
• the nicotinic receptors are found in the adrenal medulla, as well as within the autonomic ganglia, that is on the cell surface of the postganglionic neuron at the synapse between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic systems. Nicotinic receptors are also found in many nonautonomic nerve endings, for example in the membranes of skeletal muscle fibers at the neuromuscular junction.
• Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane.
• non-neuronal secretory cells such as, adrenal medulla
• pancreatic islet cells release catecholamines and parathyroid hormone , respectively, from large dense-core vesicles.
• the PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development .
• Botulinum toxin inhibits the release of both types of compounds from both types of cells in vi tro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell.
• Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
• a neuromuscular junction is formed in skeletal muscle by the proximity of axons to muscle cells.
• a signal transmitted through the nervous system results in an action potential at the terminal axon, with activation of ion channels and resulting release of the neurotransmitter acetylcholine from intraneuronal synaptic vesicles, for example at the motor endplate of the neuromuscular junction.
• the acetylcholine crosses the extracellular space to bind with acetylcholine receptor proteins on the surface of the muscle end plate. Once sufficient binding has occurred, an action potential of the muscle cell causes specific membrane ion channel changes, resulting in muscle cell contraction.
• the acetylcholine is then released from the muscle cells and metabolized by cholinesterases in the extracellular space. The metabolites are recycled back into the terminal axon for reprocessing into further acetylcholine .
• “About” means approximately or nearly and in the context of a numerical value or range set forth herein means +10% of the numerical value or range recited or claimed.
• “Local administration” means direct administration of a pharmaceutic at or to the vicinity of a site on or within an animal body, at which site a biological effect of the pharmaceutic is desired. Local administration excludes systemic routes of administration, such as intravenous or oral administration.
• “Intracranial” means within the cranium or at or near the dorsal end of the spinal cord and includes the medulla, brain stem, pons, cerebellum and cerebrum. The neurohumoral pathway and the pituitary are both considered to be intracranial.
• Clostridial toxins include botulimum toxin, butyricum toxin and tetani toxins .
• Light chain component comprises a light chain and/or a fragment thereof of a Clostridial toxin.
• the light chain has a molecular weight of about 50 kDa, and may be referred to as L chain or L.
• a light chain or a fragment thereof may have proteolytic activity.
• Heavy chain component comprises a heavy chain and/or a modified heavy chain of a Clostridial toxin.
• the full-length heavy chain has a molecular weight of about 100 kDa and can be referred to as H chain or as H.
• a heavy chain comprises an H c and an H N .
• a modified heavy chain may be a fragment of a heavy chain, for example, H N .
• H c means a fragment derived from the H chain of a
• H N means a fragment derived from the H chain of a Clostridial toxin which is approximately equivalent, for example functionally equivalent, to the amino end segment of the H chain, or the portion corresponding to that fragment in the intact H chain involved in the translocation of at least the L chain across an intracellular endosomal membrane into a cytoplasm of a cell.
• An H N may result from an H c being removed from an H chain.
• An H N may also result from an H chain being modified such that its H c no longer binds to cholinergic cell surfaces .
• LH N means a fragment derived from a Clostridial toxin that contains the L chain, or a functional fragment thereof coupled to the H N fragment .
• LH N can be obtained from the intact Clostridial toxin by chemical modification or removal of the H c domain by methods known to those skilled in the art .
• Targeting component means a chemical moiety which is able to preferentially bind to a cell surface receptor, for example, a GnRH receptor, under physiological conditions.
• GnRH gonadotrophin-releasing hormone
• GnRH-A means an analog of GnRH.
• variable region means the part of an antibody that varies extensively from one antibody to another as a result of alternative subunit sequences.
• the variable region can specifically bind to an antigen, for example, a GnRH receptor.
• Spacer means a molecule or set of molecules which physically separate and add distance between the components. One function of a spacer is to prevent steric hindrance between the components.
• an agent of the present invention may be: L-lin er-spacer- linker-H N -linker-GnRH.
• Linker means a molecule which couples two or more other molecules or components together.
• Variant means a molecule or peptide which is substantially the same as that of the referenced molecule or peptide in its identity and function.
• a variant of a referenced light chain has slight and non- consequential sequence variations from the referenced light chain. In one embodiment, variants are considered to be equivalent to the disclosed sequences and as such are within the scope of the invention.
• an agent comprising (1) a light chain component which comprises a light chain or a fragment thereof of a botulimum toxin, a butyricum toxin, a tetani toxin or variants thereof, (2) a translocation component which comprises a heavy chain or a modified heavy chain of a botulimum toxin, a butyricum toxin, a tetani toxin or variants thereof; and (3) a targeting component which selectively binds to a GnRH receptor.
• the agent may be useful for decreasing gonadotrophin secretion in a mammal, for example, a human being.
• the agent of the invention is used to treat the symptom of a pituitary hormone related disease, particularly gonadotrophin related illnesses, for example, breast cancer, prostate cancer, pancreatic cancer, endometriosis, • endometrial cancer or precocious puberty.
• the light chain component is a light chain or a fragment of a jbotulinum toxin type A, B, C 1( D, E, F, G or variants thereof.
• the light chain component decreases the release of hormones from a cell.
• the effeet (s) of the light chain component is/are reversible.
• the translocation component comprises a heavy chain or a modified heavy chain of a botulinum toxin type
• the translocation component facilitates the transfer of the light chain component into the cytoplasm of a cell.
• the targeting component is an amino acid component that can selectively bind to a GnRH receptor under physiological conditions.
• the amino acid component is the variable region of an antibody.
• the amino acid component is a peptide.
• the peptide may be a GnRH or an analog thereof (hereinafter "GnRH-A") represented by the amino acid sequence : pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Z wherein X is an amino acid selected from the group consisting of glycine, lysine, D-lysine, ornithine, D- ornithine glutamic acid, D-glutamic acid, aspartic acid, D-aspartic acid, cysteine, D-cysteine, tyrosine and D- tyrosine; and Z is a substituent selected from the group consisting of Gly-NH 2 , ethylamide, and Aza-Gly-NH 2 .
• the agent may comprise only a portion of the GnRH or GnRH-A.
• an agent of the present invention may comprise a polypeptide having 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids or 5 consecutive amino acids of GnRH or GnRH- A.
• the agent is linked to a facilitator component.
• the facilitator component is able to facilitate the transfer of the agent across a blood brain barrier.
• FIG 1 is a schematic representation of the tetani toxin (hereinafter "TeTx") and the DNA construct (pMAL-L) used to express the fusion proteins comprising a light chain and a maltose binding protein, referred to herein as the MBP-L chain fusion proteins.
• TeTx tetani toxin
• pMAL-L DNA construct
• the single-letter code in the first part of the figure represents the amino acid sequence of the first several residues of the purified recombinant L chain determined by N-terminal microsequencing.
• the second part of the figure shows the H chain is disulfide bonded to the L chain.
• the location of the zinc-binding domain is also diagrammed.
• an agent of this invention comprises a light chain component, a translocation component, and a targeting component.
• the light chain component may include a light chain of a botulinum toxin, a butyricum toxin, a tetani toxin or biologically active variants of these toxins.
• the light chain component may also include a fragment of the mentioned light chains, providing that the fragments are biologically active in a physiological environment . That is, these fragments can substantially interfere with the release of hormones from a cell.
• the light chain component includes a light chain of a botulinum toxin type A, B, C ⁇ ; D, E, F, G or biologically active variants of these serotypes.
• the light chain component may even be fragments of the botulinum toxin type A, B,
• the light chain component of this invention is not cytotoxic, that is their effect (s) is/are reversible.
• the light chain component can exert its effect from inside a cell, for example, from inside a pituitary gonadotroph.
• an agent with a light chain exerting its effect from inside a cell further comprises a translocation component.
• the translocation component is able to facilitate the transfer of at least a part of the agent into the cytoplasm of the target cell .
• the translocation component comprises a heavy chain.
• the translocation component comprises a modified heavy chain.
• the modified heavy chain may comprise an H N component.
• a modified heavy chain may include an amino terminal of a botulinum toxin, a butyricum toxin, a tetani toxin or variants thereof.
• the modified heavy chain includes an amino terminal of a botulinum toxin type A, B, C 17 D, E, F, G or variants thereof. More preferably, the modified heavy chain comprises an amino terminal of a botulinum toxin type A.
• the modified heavy chain comprises an amino terminal fragment of a heavy chain of botulinum toxin type A, which is capable of facilitating the translocation of at least part of the agent, for example the therapeutic component, from inside a vesicle into the cytoplasm of a cell.
• an agent according to this invention comprises a light chain component comprising a light chain of a botulinum toxin type A and the translocation component comprising an H N of a botulinum toxin type A, wherein the H N can assist in the translocation of at least the therapeutic component into a cytoplasm of a cell.
• an agent according to this invention comprises a therapeutic component comprising a light chain of one type of botulinum toxin and a translocation component comprising an H N , or a fragment of an H N , of another botulinum toxin, constituting a chimeric protein.
• an agent in accordance with the invention comprises LH N whereof the L chain is derived from botulinum toxin type B and the H N is derived from jbotulinum toxin type A.
• an H N fragment of the botulinum toxin type A is produced according to the method described by Shone et al . (1987, Bur. J " .
• the L chain of botulinum toxin type B is produced according to the method of Sathyamoorthy and DasGupta (1985, J " . Biol . Chem . 260, 10461-10466) .
• the free cysteine on the amine end segment of the H chain fragment of botulinum toxin type A is then derivatized by the addition of a ten- fold molar excess of dipyridyl disulphide followed by incubation at 4 degree C. overnight.
• the excess dipyridyl disulphide and the thiopyridone by product are then removed by desalting the protein over a PDIO column (Pharmacia) into PBS.
• the derivatized H N is then concentrated to a protein concentration in excess of 1 mg/ml before being mixed with an equimolar portion of L chain from Jbotulinum toxin type B (>1 mg/ml in PBS) . After overnight incubation at room temperature the mixture is separated by size exclusion chromatography over Superose 6 (Pharmacia) , and the fractions analyzed by SDS-PAGE. The chimeric LH N is then available for dramatization to produce a targeted conjugate .
• the light chain component and the translocation component are originally derived from a botulinum toxin, preferably botulinum toxin type A.
• an LH N may be produced by recombinant techniques or chemically modifying the heavy chain of a di-chain jbotulinum toxin to eliminate the H c portion: it is well known in the art that the H c of the neurotoxin molecule, for example botulinum toxin type A, can be removed from the other segment of the H chain, the H N , such that the H N fragment remains disulphide linked to the L chain of the neurotoxin molecule to provide a LH N fragment. This fragment may be covalently coupled to a targeting component forming an agent of the present invention.
• a targeting component of this invention is able to bind to a specific target cell receptor, for example, a GnRH receptor, preferably the pituitary GnRH receptor.
• the targeting component comprises an amino acid component.
• the amino acid component comprises an antibody which will specifically bind a GnRH receptor, preferably a pituitary GnRH receptor.
• the targeting component comprises an Fab portion of an antibody which binds to a GnRH receptor.
• the targeting component comprises a variable region of an antibody. The variable region may be produced recombinantly in accordance with techniques which are well known in the art.
• the amino acid component comprises a peptide.
• the peptide may include, for example, a GnRH.
• GnRH is a decapeptide and has the following chemical structure:
• the peptide may also include analogs of GnRH (GnRH-A) .
• GnRH-A GnRH-A
• the general structure of a GnRH-A is:
• X is any amino acid, but preferably the amino acids glycine, lysine, D-lysine, ornithine, D- ornithine glutamic acid, D-glutamic acid, aspartic acid, D-aspartic acid, cysteine, D-cysteine, tyrosine or D- tyrosine; and Z is a substituent selected from the group consisting of Gly-NH 2 , ethylamide, and Aza-Gly-NH 2 .
• GnRH-A is :
• a targeting component may be linked to a botulinum toxin, preferably a botulinum toxin without the H c (such as LH N ) , to form an agent of the present invention.
• the targeting component is a GnRH-A.
• a GnRH-A may be linked to a Jbotulinum toxin or an LH M to form an agent of the present invention.
• the GnRH-A molecule is linked to an LH N in a manner as to not substantially interfere with the therapeutic function of L and the translocation function of H N .
• an LH N is linked to position 6 of GnRH-A to form an agent of the present invention.
• a linkage to position 6 of the GnRH-A allows for the GnRH-A to effectively bind to a GnRH receptor, preferably a pituitary GnRH receptor.
• spacers may be used to physically further separate components of the present invention.
• an agent of the present invention may comprise an LH N connected to a GnRH through a spacer.
• a spacer functions to create a distance between the components to minimize or eliminate steric hindrances to the components. Even more preferably, the minimization or elimination of steric hindrances allows the respective components to function more effectively.
• a spacer comprises a proline, serine, threonine and/or cysteine-rich amino acid sequence similar or identical to a human immunoglobulin hinge region.
• the spacer comprises the amino acid sequence of an immunoglobulin gl hinge region.
• Such a sequence has the sequence: Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys- Pro (SEQ ID 41) .
• Spacers may also comprise hydrocarbon moieties.
• hydrocarbon moieties are represented by the chemical formulas:
• Linker Y may be used to link together two or more molecules, components and/or spacers.
• a Linker Y may be used to link a GnRH-A to a LH N .
• a Linker Y may be employed to link an LH N to a spacer; in turn, that spacer may then be linked to GnRH by another Linker Y, forming an agent comprising the structure:
• Linker Y may be selected from the group consisting of 2-iminothiolane, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , 4-succinimidyloxycarbonyl-alpha- (2- pyridyldithio) toluene (SMPT) , m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) , N-succinimidyl (4- iodoacetyl) aminobenzoate (SIAB) , succinimidyl 4- (p- maleimidophenyl) butyrate (SMPB) , l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) , bis- diazobenzidine and glutaraldehyde .
• SPDP N-succinimidyl-3- (2-pyridyldithio) prop
• Linker Y may be attached to an amino group, a carboxylic group, a sulf ydryl group or a hydroxyl group of an amino acid group of a component .
• a Linker Y may be linked to a carboxyl acid group of amino acid of GnRH-A, preferably the amino acid at the 6 position.
• an agent of the present invention may further be conjugated to a facilitator component.
• a facilitator component is at least effective to assist the transportation of the agent across the blood brain barrier.
• a facilitator component includes a cationic lipid molecule.
• a cationic lipid molecule may be attached to an agent of the present invention, for example LH N -GnRH molecule, through a covalent bond using methods known to those familiar with the art, including the use of Linker Y. Examples of these cationic lipids are disclosed in U.S. patent 5,459,127, which is incorporated in its entirety herein by reference.
• a facilitator component includes molecules which can undergo receptor mediated transcytosis at the blood brain barrier. Examples of these molecules include, without limitation, insulin, IGF-II and transferrin, Shoichet and Winn (2000, Advanced Drug Delivery Reviews 42, 81-102), Laszlo et al . (2000,
• the facilitator component is a carrier peptide which can facilitate the transport of an agent of the invention across the blood brain barrier.
• carrier peptides include, for example, a penetratin peptide represented by the formula:
• Agents of the present invention have potential utility in human medicine.
• prostate cancer remains an important cause of cancer deaths and represents the second leading cancer in males .
• a present method of treating prostate cancer is to castrate the patient to reduce his level of circulating testosterone/DHT, which are thought to propagate the cancer.
• Agents of the present invention may be administered, instead, to treat the prostate cancer.
• an effective dose of LH W -GnRH-insulin may be systemically administered to the patient to reduce serum testosterone/DHT levels to adequately treat the illness.
• Agents of the present invention may also be administered to treat endometriosis.
• Endometriosis is a condition wherein the uterus produces painful growth of endometrial tissue in the female peritoneum and pelvis . This condition may also be treated by reducing the level of circulating gonadotrophin in the patient. As such, an effective dose of the present agent may be administered to reduce the level of gonadotrophin to treat endometriosis .
• an agent of this invention may also be administered to treat medical conditions which will benefit from a decrease of gonadotrophin levels in the body. Additional, non-limiting, examples are further provided herein below.
• an effective dose of an agent to be administered may be about 1 U to about 500 U of the botulinum toxin.
• the amount of the agents administered can vary widely according to the particular disorder being treated, its severity and other ' various patient variables including size, weight, age, and responsiveness to therapy. Such determinations are routine to one of ordinary skill in the art (see for example, Harrison's Principles of Internal Medicine (1998) , edited by Anthony Fauci et al . , 14th edition, published by McGraw Hill) .
• the amount of agent administered is able to lower the secretion of gonadotrophin from the pituitary by about 5% to about 80%, preferably about 30% to about 50%.
• the amount of agent administered is able to lower the circulating gonadotrophin level by about 5% to about 80%, preferably about 30% to about 50%.
• the routes of administration of the present invention include, but are not limited to, direct injection into the central nervous system. Such injection includes direct intraspinal injection and intracranial injection. It is preferred that the treating physician intracranially apply the agent, for example, LH N -GnRH-A, directly to the pituitary, preferably anterior pituitary.
• Intracranial injection methods are widely known in the art. For example, U.S. Patent Application No. 09/692,811, filed November 20, 2000 discloses various intracranial methods of administering a drug, which may be readily adopted to intracranially administer an agent of the present invention. (See also PCT/US99/17880 (WO 00/07652, which discloses a device useful for chronic intracranial delivery of a drug) .
• routes of administration include, without limitation, transdermal, peritoneal, subcutaneous, intramuscular, intravenous and intrarectal . If it is determined that an agent of the present invention by itself may be unable to pass through the blood brain barrier to reach its target site, preferably the anterior pituitary, it is preferred that a these agents be conjugated to a facilitator component prior to administration .
• recombinant techniques are used to produce at least one of the components of the agents. See, for example International Patent Application Publication WO 95/32738, the disclosure of which is incorporated in its entirety herein by reference.
• the technique includes steps of obtaining genetic materials from DNA cloned from natural sources, or synthetic oligonucleotide sequences, which have codes for one of the components, for example the therapeutic, translocation and/or targeting component
• the genetic constructs are incorporated into host cells for amplification by first fusing the genetic constructs with a cloning vector, such as a phage, plasmid, phagemid or other gene expression vector.
• a cloning vector such as a phage, plasmid, phagemid or other gene expression vector.
• the recombinant cloning vectors are transformed into a mammalian, yeast or bacterial host.
• the preferred host is E. coli.
• resultant proteins can be isolated using conventional techniques .
• the protein expressed may comprise all three components of the agent.
• the protein expressed may include a light chain of botulinum toxin type A (the therapeutic component) , an H N of a botulinum toxin type A (the translocation component) , and a GnRH-A, which binds a GnRH receptor, preferably an anterior pituitary GnRH receptor, under physiological conditions (a targeting component) .
• the protein expressed may include less than all three components of the agent. In such case, the components may be chemically joined, preferably through linker Y.
• toxin from anaerobic Clostridium cultures is a cumbersome and time consuming process including a multi-step purification protocol involving several protein precipitation steps and either prolonged and repeated crystallization of the toxin or several stages of column chromatography.
• the high toxicity of the product dictates that the procedure must be performed under strict containment (BL3) .
• the folded single chain neurotoxins are activated by endogenous Clostridial proteases through a process termed nicking. This involves the removal of approximately 10 amino acid residues from the single-chain to create the dichain form in which the two chains remain covalently linked through the intra-chain disulfide bond.
• the nicked toxin is more active than the unnicked form.
• the amount and precise location of nicking varies with the serotypes of the bacteria producing the toxin or with the modification made in the outer loop.
• the differences in single-chain toxin activation and, hence, the yield of nicked toxin are due to variations in the type and amounts of proteolytic activity produced by a given strain. For example, greater than 99% of Clostridium botulinum type A single-chain toxin is activated by the Hall A Clostridium botulinum strain, whereas type B and E strains produce toxins with lower amounts of activation (0% to 75% depending upon the fermentation time) . Thus, the high toxicity of the mature toxin plays a major part in the commercial manufacture of toxins as therapeutic agents.
• Clostridial toxins such as botulinum toxin and tetanus toxin could be expressed, recombinantly, in high yield in rapidly-growing bacteria (such as E. coli cells) as relatively non-toxic single-chains (or single chains having reduced toxic activity) which are safe, easy to isolate and simple to convert to the fully-active form.
• Jbotulinum toxins these isolated chains are, by themselves, nontoxic; see Li et al . (1994, Biochemistry
• H and L subunits can be combined by oxidative disulphide linkage to form the neuroparalytic di-chains.
• an agent comprising a therapeutic component and a translocation component is recombinantly produced as an unnicked single chain. See Dolly et al . US 09/648,692, the disclosure of which is incorporated in its entirety by reference herein.
• the agent includes an amino acid sequence that is susceptible to specific cleavage in vitro following expression as a single chain.
• proteins may include Clostridial toxins and derivatives thereof, such as those proteins disclosed in U.S. Patent 5,989,545 and International Patent Application W095/32738, both incorporated in their entirety by reference herein.
• the agents, or toxins of the invention are expressed as their low activity (or inactive) single-chain pro-forms, then, by a carefully controlled proteolytic reaction in vitro, they are activated, preferably to the same potency level as the native neurotoxin from which they were derived.
• the engineered proteolytic cleavage sites can be designed to occur in a specially designed loop between the H and L portions of the single amino acid chain that promotes accessibility of the protease to the holotoxin substrate.
• the amino acid sequences of the cleavage site are preferably designed to have a high degree of specificity to proteolytic enzymes which do not normally occur in humans
• proteases (as either human proteases or occurring in part of the foreseeable human fauna and flora) .
• a non-exclusive list of examples of such proteases includes bovine enterokinase, which cleaves the amino acid sequence DDDDK; tobacco etch virus (TEV) protease, which cleaves the sequence EXXYXQS/G; GENENASE ® from Bacillus a yliquifaciens, which cleaves the sequence HY or YH; and PRESCISSION ® protease from human rhinovirus 3C, which cleaves the amino acid sequence LEVLFQGP .
• TEV tobacco etch virus
• GENENASE ® from Bacillus a yliquifaciens
• PRESCISSION ® protease from human rhinovirus 3C, which cleaves the amino acid sequence LEVLFQGP .
• the letter X indicates any amino acid. All amino acid sequences shown in the present specification are in the
• the interchain loop region of the C. botulinum subtype E toxin which is normally resistant to proteolytic nicking in the bacterium and mammals, is modified to include the inserted proteolytic cleavage site, and this loop region used as the interchain loop region in the single-chain toxin or modified toxin molecules of the present invention. It is believed that using the loop from C. botulinum subtype E will stabilize the unnicked toxin molecule in vivo, making it resistant to undesired cleavage until activated through the use of the selected protease.
• a DNA sequence encoding the H c is included in the recombinant DNA sequence which encodes the Clostridial toxin contained in the heterologous gene expression system. Therefore, the corresponding H c portion of an H chain will not be produced. This too will lower the safety risk associated with Clostridial toxin production.
• GnRH may be produced by similar heterologous recombinant DNA expression systems as is familiar to one skilled in the art.
• a GnRH or GnRH-A may be produced by standard t-Boc/Fmoc technologies in solution or solid phase as is known to those skilled in the art. Similar synthesis techniques are also covered by the scope of this invention, for example, methodologies employed in Milton et al.
• Endometriosis A 34-year-old woman seeks medical attention after undergoing a spontaneous abortion. The patient reports that she has been suffering from dyspareunia (painful intercourse) , dysmenorrhea (painful menstruation) , and dyschezia (painful bowel evacuation) as early as two years prior to her pregnancy. Physical examination reveals the presence of endometrial tissue outside the lining of the uterine cavity and multiple tender nodules on her uterosacral ligaments. A preliminary diagnosis of endometriosis is confirmed during a laparoscopy-an examination of the peritoneum. The endometriosis is diagnosed as stage III endometriosis indicating a severe case of the disease with stage I being a mild case and stage IV an extensive case.
• agents of the present invention for example LH N - GnRH.
• the agent is administered intracranially to the anterior pituitary.
• a method for intracranial administration is set forth in co-pending patent application serial number 09/692811, "Method for Treating Endocrine Disorders" filed October 20, 2000, incorporated herein by reference in its entirety.
• PSA prostate specific antigen
• the PSA test was administered during a routine physical examination.
• the patient suffers no symptoms of prostate cancer.
• a fine needle aspiration biopsy is performed on the patient confirming an early stage prostatic cancer.
• the patient is treated by intracranial injection of LH N -GnRH administered directly to the anterior pituitary.
• the dose of LH N -GnRH is sufficient to reduce the patient ' s level of circulating gonadotrophin by 80% to 30%, preferably 50%.
• the patient is monitored closely for advance of the cancer. Over the next 24 months there is no spread of the cancer. Also, there is no detectable further enlargement of the prostate. The treatment is repeated at 27 months. At 36 months from the initial, diagnosis, the patient no longer tests positive for PSA.
• a 5 -year-old female is diagnosed with precocious puberty. Her physical symptoms are development of breasts and growth of pubic hair. The patient has also begun to menstruate in the past month. Vaginal smears have detected abnormally high estrogen levels for the patient's age. Urinary tests also show abnormally high levels of gonadotrophins . X-rays of the child's hands, knees, wrists and hips show the beginning signs of epiphyseal closure. The patient's height is in the normal range for a 5 year old child. However, if the condition is left untreated epiphyseal closure will completely stunt the child's growth. The patient is injected with LH N -GnRH. The dose of
• LH N -GnRH is sufficient to reduce the patient's level of circulating gonadotrophin by 80% to 30%, preferably 50%.
• the injection is intracranial and the drug is administered to the anterior pituitary.
• breast size reduction occurs in the patient.
• the patient After passage of three months, the patient has not menstruated nor does she show any signs of having reached puberty.
• the patient grows to approximately 80% the height of a normal child her age.
• a partial hysterectomy is performed on the patient to remove the cancer.
• the patient is injected intracranially with LH N -GnRH.
• the dose of LH N - GnRH is sufficient to reduce the patient's level of circulating gonadotrophin by 80% to 30%, preferably 50%. After 24 months the cancer does not reoccur. The injection is repeated after 27 months.
• a 42 year old woman seeks a physical medical exam after discovering a lump in her left breast during a routine self breast examination.
• the patient has a family history of breast cancer and has never conceived a child. These two factors put the patient in a high-risk group for breast cancer.
• the medical diagnosis consists of a mammography and a needle biopsy.
• the cancer appears restricted within a nodule contained within a duct. This, combined with the patient's resistance to a mastectomy procedure, makes her an excellent candidate for a lumpectomy combined with post-operative drug treatment.
• the lump is surgically removed and the patient is treated with LH N -GnRH by intracranial injection.
• the dose of LH N -GnRH is sufficient to reduce the patient's pituitary secretion gonadotrophin by about 80% to about 30%, preferably 50%.
• the patient is checked monthly for the spread of cancer for the first 6 months after surgery and every two months thereafter. Two years after surgery there is no sign of the cancer.
• Example 6 Subcloninq the BoNT/A-L Chain Gene
• the DNA sequence encoding the BoNT/A-L chain may be amplified by a PCR protocol that employs synthetic oligonucleotides having the sequences, 5'- AAAGGCCTTTTGTTAATAAACAA-3 ' (SEQ ID 43) and 5'- GGAATTCTTACTTATTGTATCCTTTA-3 ' (SEQ ID 44).
• Use of these primers allows the introduction of Stu I and EcoR I restriction sites into the 5 ' and 3 ' ends of the BoNT/A-L chain gene fragment, respectively. These restriction sites may be subsequently used to facilitate unidirectional subcloning of the amplification products.
• Chromosomal DNA from C. botulinum may serve as a template in the amplification reaction.
• the PCR amplification is performed in a 0.1 mL volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KC1 , 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate
• dNTP dNTP
• 50 pmol of each primer 200 ng of genomic DNA and 2.5 units of Taqpolymerase (Promega).
• the reaction mixture is subjected to 35 cycles of denaturation (1 minute at 94° C) , annealing (2 minutes at 37°C) and polymerization (2 minutes at 72 °C) . Finally, the reaction is extended for an additional 5 minutes at 72 °C.
• the PCR amplification product may be digested with Stu I and EcoR I, purified by agarose gel electrophoresis, and ligated into Sma I and EcoR I digested pBluescript II SK* to yield the plasmid, pSAL.
• Bacterial transformants harboring this plasmid may be isolated by standard procedures. The identity of the cloned L chain polynucleotide is confirmed by double stranded plasmid sequencing using SEQUENASE (United States Biochemicals) according to the manufacturer's instructions . Synthetic oligonucleotide sequencing primers are prepared as necessary to achieve overlapping sequencing runs. The cloned sequence is found to be identical to the sequence disclosed by Binz, et al., in J " . Biol . Chem. 265, 9153 (1990), and Thompson et al . , in Eur. J. Biochem . 189, 73 (1990). Site-directed mutants designed to compromise the enzymatic activity of the BoNT/A-L chain may also be created.
• This example describes the methods to verify expression of the wild-type L chains, which may serve as a therapeutic component, in bacteria harboring the pCA-L plasmids.
• Well isolated bacterial colonies harboring either pCAL are used to inoculate L-broth containing 0.1 mg/ml ampicillin and 2% (w/v) glucose, and grown overnight with shaking at 30°C. The overnight cultures are diluted 1:10 into fresh L-broth containing 0.1 mg/ml of ampicillin and incubated for 2 hours. Fusion protein expression is induced by addition of IPTG to a final concentration of 0.1 mM. After an additional 4 hour incubation at 30°C, bacteria are collected by centrifugation at 6,000 x g for 10 minutes.
• a small-scale SDS-PAGE analysis confirmed the presence of a 90 kDa protein band in samples derived from IPTG-induced bacteria. This Mr is consistent with the predicted size of a fusion protein having MBP ( ⁇ 40 kDa) and BoNT/A-L chain ( ⁇ 50 kDa) components. Furthermore, when compared with samples isolated from control cultures, the IPTG-induced clones contained substantially larger amounts of the fusion protein.
• the MBP-L chain fusion proteins encoded by the pCAL and pCAL-TyrU7 expression plasmids are purified from bacteria by amylose affinity chromatography. Recombinant wild-type or mutant L chains are then separated from the sugar binding domains of the fusion proteins by sitespecific cleavage with Factor X 2 . This cleavage procedure yielded free MBP, free L chains and a small amount of uncleaved fusion protein. While the resulting L chains present in such mixtures have been shown to possess the desired activities, we have also employed an additional purification step. Accordingly, the mixture of cleavage products is applied to a second amylose affinity column that bound both the MBP and uncleaved fusion protein. Free L chains are not retained on the affinity column, and are isolated for use in experiments described below.
• the lysates are cleared by centrifugation at 15,000 x g for 15 minutes at 4°C.
• Supematants are applied to an amylose affinity column [2x10 cm, 30 ml resin] (New England BioLabs; Hitchin, UK) . Unbound proteins are washed from the resin with column buffer until the eluate is free of protein as judged by a stable absorbance reading at 280 nm.
• the bound MBP-L chain fusion protein is subsequently eluted with column buffer containing 10 mM maltose.
• Fractions containing the fusion protein are pooled and dialyzed against 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM, CaCl2 and 1 mM DTT for 72 hours at 4°C.
• Fusion proteins may be cleaved with Factor X 2 (Promega; Southampton, UK) at an enzyme: substrate ratio of 1:100 while dialyzing against a buffer of 20 mM Tris- HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM, CaCl 2 and 1 mM DTT. Dialysis is carried out for 24 hours at 4°C. The mixture of MBP and either wild-type or mutant L chain that resulted from the cleavage step is loaded onto a 10 ml amylose column equilibrated with column buffer. Aliquots of the flow through fractions are prepared for SDS-PAGE analysis to identify samples containing the L chains. Remaining portions of the flow through fractions are stored at -20°C. Total E. coli extract or the purified proteins are solublized in SDS sample buffer and subjected to PAGE according to standard procedures. Results of this procedure indicated the recombinant toxin fragment accounted for roughly 90% of the protein content of the sample.
• a quantitative assay may be employed to compare the abilities of the wild-type and their recombinant analogs to cleave a SNAP-25 substrate.
• the substrate utilized for this assay is obtained by preparing a glutathione-S-transferase (GST) -SNAP-25 fusion protein, containing a cleavage site for thrombin, expressed using the pGEX-2T vector and purified by affinity chromatography on glutathione agarose.
• GST glutathione-S-transferase
• SNAP-25 is then cleaved from the fusion protein using thrombin in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 2.5 mM CaCl 2 (Smith et al . Gene 67, 31 (1988) at an enzyme : substrate ratio of 1:100. Uncleaved fusion protein and the cleaved glutathione-binding domain bound to the gel.
• the recombinant SNAP-25 protein is eluted with the latter buffer and dialyzed against 100 mM HEPES (pH 7.5) for 24 hours at 4°C. The total protein concentration is determined by routine methods.
• Rabbit polyclonal antibodies specific for the C- terminal region of SNAP-25 are raised against a synthetic peptide having the amino acid sequence, CANQRATKMLGSG
• This peptide corresponded to residues 195 to 206 of the synaptic plasma membrane protein and an N- terminal cysteine residue not found in native SNAP-25.
• the synthetic peptide is conjugated to bovine serum albumin (BSA) (Sigma; Poole, UK) using maleimidobenzoyl-
• N-hydroxysuccinimide ester (MBS) as a cross-linking agent
• Affinity purification of the anti-peptide antibodies is carried out using a column having the antigenic peptide conjugated via its N- terminal cysteine residue to an aminoalkyl agarose resin
• Reaction mixtures include 5 ⁇ l recombinant SNAP-25 substrate (8.5 ⁇ M final concentration) and either 20 ⁇ l reduced BoNT/A or recombinant wild-type L chain. All samples are incubated at 37 °C for 1 hour before quenching the reactions with 25 ⁇ l aqueous 2% trifluoroacetic acid (TFA) and 5 mM EDTA, Foran et al . (1994, Biochemistry 33, 15365) . Aliquots of each sample are prepared for SDS-PAGE and Western blotting with the polyclonal SNAP-25 antibody by adding SDS-PAGE sample buffer and boiling. Anti-SNAP-25 antibody reactivity is monitored using an ECL detection system and quantified by densitometric scanning.
• TFA trifluoroacetic acid
• H chain is combined with an equimolar amount of either native L chain or recombinant wild-type L chain. Reconstitution is carried out by dialyzing the samples against a buffer consisting of 25 mM Tris (pH 8.0), 50 ⁇ M zinc acetate and 150 mM NaCl over 4 days at 4°C. Following dialysis, the association of the recombinant L chain and native H chain to form disulfide linked 150 kDa dichains is monitored by SDS-PAGE and quantified by densitometric scanning. The proportion of dichain molecules formed with the recombinant L chains is lower than that obtained when native L chain is employed.
• Example 11 Expression of TeTx Fusion Proteins and Purification of Wild-Type L Chain Proteins
• This Example describes the techniques to produce and purify recombinant L chain fusion proteins encoded by the plasmid constructs described in the previous Example B.
• E. coli clones harboring plasmids pMAL-L are grown to densities of roughly 2 x IO 8 cells/ml (A 5oonm ⁇ 0.5) at 37°C in L-broth that is made lO ⁇ g/ml ampicillin and 2 mg/ml glucose. Induction is initiated by the addition of IPTG to a final concentration of 0.3 mM. Cells are harvested 2 hours later by centrifugation at 6000 x g for 30 minutes.
• MBP-L fusion proteins are eluted with column buffer containing 10 mM maltose according to the procedure described by Maina et al . , in Gene 74, 365 (1988) .
• the isolated fusion proteins are concentrated to 0.5-1 mg/ml using an Amicon CENTRICON. Protein samples are then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, using anti-MBP polyclonal and anti-L chain monoclonal antibodies.
• both MBP-L chain preparations are cleaved at 23 °C for 24 hours with factor X, at an enzyme : protein ratio of 0.5-1:100 (w/w) .
• This cleavage gave complete conversion of the fusion proteins to the respective wild-type L chain with the liberation of MBP, as confirmed by SDS-PAGE.
• L chain is further purified by reabsorption onto a new affinity column. The desired product from this purification step is found in the column wash fraction. Fractions of the column wash are monitored for A 280nm and checked again by SDS-PAGE and Western blotting.
• Measurement of the zinc-dependent protease activity of native L chain is employed as an assay for the activity of the recombinant L chain proteins.
• Two different protein substrates are used in this assay.
• bovine small synaptic vesicles (SSVs) are used in the first case.
• the assay for proteolytic cleavage of the substrate is based on Coomassie staining and Western blotting of protein gels.
• TeTx dichains that incorporates either native L chain or recombinant wild-type L chain.
• Native H chain purified from TeTx as detailed by Weller et al . in Eur. J. Biochem . 182, 649 (1989), is combined with an equimolar amount of either native L chain or recombinant wild-type L.
• the mixtures are dialyzed against 2 M urea, 20 mM DTT, 1 M NaCl, and 50 mM Tris-HCl (pH 8.4) with stirring for 18 hours and then further dialyzed without agitation against 50 mM Tris-HCl and 600 mM glycine (pH 8.4) for 72 hours.
• dichain species The reassociation of dichain species is confirmed by virtue of the presence of stained high Mr protein bands that comigrated with native TeTx. With respect to recombinant wild-type and mutant L chains, the relative amounts of the dichain species are 55.1 and 56.8%, respectively, as determined by densitometric ' scanning of the silver-stained gel. Native H chain and L chain gave similar levels of reconstitution. The latter involved interchain disulfide formation as the toxin is converted back to free H chain and L chain upon reduction by DTT.

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