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Title:  Compositions and methods for treating gonadotrophin related illnesses

United States Patent:  6,831,059

Issued:  December 14, 2004

Inventors:  Donovan; Stephen (Capistrano Beach, CA)

Assignee:  Allergan, Inc. (Irvine, CA)

Appl. No.:  810601

Filed:  March 15, 2001

Abstract

The present invention relates to an agent comprising a neurotoxin, methods for making the agents and methods for treating endocrine disorders, for example gonadotrophin related illnesses. Preferably, the agent comprises at least a portion of a botulinum toxin.

Description of the Invention

BACKGROUND OF THE INVENTION

In 1971, after years of intense research, Andrew Schally finally was able to identify the structure of the releasing hormone responsible for stimulating the secretion of luteinizing hormones (LH) and follicle-stimulating hormones (FSH) from the pituitary gland. This releasing hormone is produced by the hypothalamus and reaches the pituitary gland by a neurohumoral pathway.

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).

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. In the male, LH stimulates the interstitial cells and is, for that reason, also called interstitial cell stimulating hormone (ICSH). 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.

However, an abnormally high production of GnRH by the hypothalamus may cause an increased gonadotrophin secretion, which may deleteriously harm the body. 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. They have also shown possible utility in the treatment of pituitary gonadotroph adenomas, sleep disorders such as sleep apnea, irritable bowel syndrome, premenstrual syndrome, benign prostatic hyperplasia, hirsutism, as an adjunct to growth hormone therapy in growth hormone deficient children, and in murine models of lupus.

Although GnRH agonist and antagonist have been useful, their continual administration may be problematic. For example, 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. Pat. No. 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. (The disclosure of Nett et al. is incorporated in its entirety herein by reference). Although 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.

Botulinum Toxin

The bacterial genus Clostridium includes more than one hundred and twenty seven species, grouped according to morphology and function. The anaerobic, gram-positive bacterium 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 LD50 in mice (i.e. 1 unit). One unit of BOTOX.RTM. contains about 50 picograms (about 56 attomoles) of botulinum toxin type A complex. Interestingly, on a molar basis, botulinum 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, Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1976) (where the stated LD50 of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng of BOTOX.RTM. equals 1 unit). One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each.

Seven generally immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that 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. Additionally, 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 LD50 for botulinum toxin type A. Moyer E et al., 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.

Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, 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, HC, appears to be important for targeting of the toxin to the surface of the motor neuron.

In the second step, the toxin crosses the plasma membrane of the motor neuron. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This step may be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin (or at a minimum the light chain) then translocates through the endosomal membrane into the cytoplasm.

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 L) chain of the toxin. The entire toxic activity of botulinum toxin and of the tetanus toxin is contained in the L chain of the holotoxin. 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, botulinum toxin types B, D, F, and G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytoplasmic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. 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. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm. 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). Aoki K. R., Preclinical Update on BOTOX (Botulinum Toxin Type A)--Purified Neurotoxin Complex Relative to Other Botulinum Toxin Preparations, Eur J. Neur 1999, 6 (suppl 4):S3-S10. Maximal clinical effect may not result for several days. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months. Apparently, a substrate for a botulinum toxin can be found in a variety of different cell types. See e.g. Biochem ,J 1;339 (pt 1):159-65:1999, and Mov Disord, 10(3):376:1995 (pancreatic islet B cells contains at least SNAP-25 and synaptobrevin).

The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, 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 C1 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. Finally, 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) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) 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.

In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that 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 Inhibit Noradrenaline Release From Cultured Mouse Brain, J Neurochem 51(2); 522-527:1988) CGRP, substance P and glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks Glutamate Exocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J. Biochem 165;675-681:1897. Thus, when adequate concentrations are used, stimulus-evoked release of most neurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterization of Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9); 1373-1412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons in Culture, Brain Research 360; 318-324:1985; Habermann E., Inhibition by Tetanus and Botulinum A Toxin of the release of [3 H]Noradrenaline and [3 H]GABA From Rat Brain Homogenate, Experientia 44; 224-226:1988, Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Release and Uptake of Various Transmitters, as Studied with Particulate Preparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's Arch Pharmacol 316;244-251:1981, and; Jankovic J. et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 5.

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. In contrast, botulinum toxin serotypes C1, 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. However, even 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. Therefore, a certain percentage of any preparation of, for example, the botulinum 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. Additionally, it is known that 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 botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of .gtoreq.3x107 U/mg, an A260 /A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. 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 Medicine, Microbiol Rev. 56; 80-99:1992. Generally, 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 1-2x108 LD50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2x108 LD50 U/mg or greater, and; purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1-2x107 LD50 U/mg or greater.

Botulinum toxins and/or botulinum toxin complexes can be obtained from List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals of St Louis, Mo.

Both pure botulinum 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.

As with enzymes generally, the biological activities of the botulinum toxins (which are intracellular peptidases) is dependant, at least in part, upon their three dimensional conformation. Thus, 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 commercially available botulinum toxin containing pharmaceutical composition is sold under the trademark BOTOX.RTM. (available from Allergan, Inc., of Irvine, Calif.). BOTOX.RTM. 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. The vacuum-dried product is stored in a freezer at or below -5oC. BOTOX.RTM. can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each vial of BOTOX.RTM. 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.

To reconstitute vacuum-dried BOTOX.RTM., 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.RTM. may be denatured by bubbling or similar violent agitation, the diluent is gently injected into the vial. For sterility reasons BOTOX.RTM. is preferably administered within four hours after the vial is removed from the freezer and reconstituted. During these four hours, reconstituted BOTOX.RTM. can be stored in a refrigerator at about 2oC. to about 8oC. Reconstituted, refrigerated BOTOX.RTM. has been reported to retain its potency for at least about two weeks. Neurology, 48:249-53:1997.

It has been reported that botulinum toxin type A has been used in clinical settings as follows:

(1) about 75-125 units of BOTOX.RTM. per intramuscular injection (multiple muscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX.RTM. per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);

(3) about 30-80 units of BOTOX.RTM. to treat constipation by intrasphincter injection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX.RTM. to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 units of BOTOX.RTM., the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).

(6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX.RTM. into five different upper limb flexor muscles, as follows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radialis: 15 U to 60 U

(e) 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.RTM. by intramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically into glabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX.RTM. 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.

Additionally, 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 & Aging 16(4);273-278:2000.

It is known that 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. The Laryngoscope 109:1344-1346:1999. However, the usual duration of an intramuscular injection of Botox.RTM. is typically about 3 to 4 months.

The success of botulinum toxin type A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes. A study of two commercially available botulinum type A preparations (BOTOX.RTM. and Dysport.RTM.) and preparations of botulinum toxins type B and F (both obtained from Wako Chemicals, Japan) has been carried out to determine local muscle weakening efficacy, safety and antigenic potential. 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). ED50 values were calculated from dose response curves. Additional mice were given intramuscular injections to determine LD50 doses. The therapeutic index was calculated as LD50 /ED50. Separate groups of mice received hind limb injections of BOTOX.RTM. (5.0 to 10.0 units/kg) or botulinum toxin type B (50.0 to 400.0 units/kg), and were tested for muscle weakness and increased water consumption, the later being a putative model for dry mouth. Antigenic potential was assessed by monthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg for botulinum toxin type B or 0.15 ng/kg for BOTOX.RTM.). Peak muscle weakness and duration were dose related for all serotypes. DAS ED50 values (units/kg) were as follows: BOTOX.RTM.: 6.7, Dysport.RTM.: 24.7,botulinum toxin type B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX.RTM. had a longer duration of action than botulinum toxin type B or botulinum toxin type F. Therapeutic index values were as follows: BOTOX.RTM.: 10.5, Dysport.RTM.: 6.3, botulinum toxin type B: 3.2. Water consumption was greater in mice injected with botulinum toxin type B than with BOTOX.RTM., although botulinum toxin type B was less effective at weakening muscles. After four months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of 4 (where treated with 6.5 ng/kg) rabbits developed antibodies against botulinum toxin type B. In a separate study, 0 of 9 BOTOX.RTM. treated rabbits demonstrated antibodies against botulinum toxin type A. DAS results indicate relative peak potencies of botulinum toxin type A being equal to botulinum toxin type F, and botulinum toxin type F being greater than botulinum toxin type B. With regard to duration of effect, 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. As shown by the therapeutic index values, the two commercial preparations of botulinum toxin type A (BOTOX.RTM. and Dysport.RTM.) are different. The increased water consumption behavior observed following hind limb injection of botulinum 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. Furthermore, in rabbits, type B was more antigenic than was BOTOX.RTM., possibly because of the higher protein load injected to achieve an effective dose of botulinum toxin type B. Eur J Neurol 1999 Nov;6(Suppl 4):S3-S10.

In addition to having pharmacologic actions at the peripheral location, 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 Arch. Pharmacol. 1974; 281, 47-56 showed that botulinum toxin is able to ascend to the spinal area by retrograde transport. As such, a botulinum toxin injected at a peripheral location, for example intramuscularly, may be retrograde transported to the spinal cord.

Acetylcholine

Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. 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. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic as most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, 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. A wide variety of non-neuronal secretory cells, such as, adrenal medulla (as well as the PC12 cell line) and 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 vitro, 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.

As indicated above, the drugs presently being used to treat illnesses related to gonadotrophins are often accompanied by detrimental side effects. There continues to be a need for an improved agent and method for treating gonadotrophin related illnesses.

SUMMARY OF THE INVENTION

In accordance with the present invention, an agent is featured 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.

Further in accordance with the present invention, the agent may be useful for decreasing gonadotrophin secretion in a mammal, for example, a human being. In one embodiment 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.

Still further in accordance with the present invention, the light chain component is a light chain or a fragment of a botulinum toxin type A, B, C1, D, E, F, G or variants thereof. The light chain component decreases the release of hormones from a cell. Preferably, the effect(s) of the light chain component is/are reversible.

Still further in accordance with the present invention, the translocation component comprises a heavy chain or a modified heavy chain of a botulinum toxin type A, B, C1, D, E, F, G or variants thereof. The translocation component facilitates the transfer of the light chain component into the cytoplasm of a cell.

Still further in accordance with the present invention, the targeting component is an amino acid component that can selectively bind to a GnRH receptor under physiological conditions. In one embodiment, the amino acid component is the variable region of an antibody. In a preferred embodiment, the amino acid component is a peptide. In one embodiment, the peptide may be a GnRH or an analog thereof (hereinafter "GnRH-A") represented by the amino acid sequence:

pyroGlu-His-Trp-Ser-Try-X-Leu-Arg-Pro-Z (SEQ ID NO: 46)

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-NH2, ethylamide, and Aza-Gly-NH2.

Still further, in accordance with the present invention, the agent may comprise only a portion of the GnRH or GnRH-A. For example, 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.

Still further in accordance with the present invention, 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.

Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to agents and methods for treating sex hormone related diseases, for example gonadotrophin related illnesses. Without wishing to limit the invention to any theory or mechanism of operation, it is believed that the agents of the present invention are effectively used to lower the level of gonadotrophin secretion in a mammal and thereby treat or alleviate illnesses associated with an elevated level of gonadotrophin.

In a broad embodiment, 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. In a preferred embodiment, the light chain component includes a light chain of a botulinum toxin type A, B, C1, D, E, F, G or biologically active variants of these serotypes. In another preferred embodiment, the light chain component may even be fragments of the botulinum toxin type A, B, C1, D, E, E, F, G or the biologically active variants of these serotypes, provided that the fragments themselves are biologically active, for example the fragment is able to interfere with the release of hormones from a cell. In one preferable embodiment, the light chain component of this invention is not cytotoxic, that is their effect(s) is/are reversible.

In one embodiment, the light chain component can exert its effect from inside a cell, for example, from inside a pituitary gonadotroph. In one embodiment, 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.

In a broad embodiment, the translocation component comprises a heavy chain. In one embodiment, the translocation component comprises a modified heavy chain. The modified heavy chain may comprise an HN component. For example, a modified heavy chain may include an amino terminal of a botulinum toxin, a butyricum toxin, a tetani toxin or variants thereof. Preferably, the modified heavy chain includes an amino terminal of a botulinum toxin type A, B, C1, D, E, F, G or variants thereof. More preferably, the modified heavy chain comprises an amino terminal of a botulinum toxin type A. Even more preferably, 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.

In a preferred embodiment, 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 HN of a botulinum toxin type A, wherein the HN can assist in the translocation of at least the therapeutic component into a cytoplasm of a cell.

In another embodiment, 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 HN, or a fragment of an HN, of another botulinum toxin, constituting a chimeric protein. For example, in one preferred embodiment, an agent in accordance with the invention comprises LHN whereof the L chain is derived from botulinum toxin type B and the HN is derived from botulinum toxin type A. In this example, an HN fragment of the botulinum toxin type A is produced according to the method described by Shone et al. (1987, Eur. J. Biochem. 167, 175-180). 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 HN is then concentrated to a protein concentration in excess of 1 mg/ml before being mixed with an equimolar portion of L chain from botulinum 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 LHN is then available for dramatization to produce a targeted conjugate.

In one embodiment, the light chain component and the translocation component are originally derived from a botulinum toxin, preferably botulinum toxin type A. For example, an LHN may be produced by recombinant techniques or chemically modifying the heavy chain of a di-chain botulinum toxin to eliminate the HC portion: it is well known in the art that the HC of the neurotoxin molecule, for example botulinum toxin type A, can be removed from the other segment of the H chain, the HN, such that the HN fragment remains disulphide linked to the L chain of the neurotoxin molecule to provide a LHN fragment. This fragment may be covalently coupled to a targeting component forming an agent of the present invention.

In a broad embodiment, 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.

In a broad embodiment, the targeting component comprises an amino acid component. In one embodiment, the amino acid component comprises an antibody which will specifically bind a GnRH receptor, preferably a pituitary GnRH receptor. In a preferred embodiment, the targeting component comprises an Fab portion of an antibody which binds to a GnRH receptor. In an even more preferred embodiment, 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.

In one embodiment, 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:

pyro Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ ID 1)

In one embodiment, the peptide may also include analogs of GnRH (GnRH-A). In a preferred embodiment, the general structure of a GnRH-A is:

pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Z (SEQ ID NO: 46)

wherein 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-NH2, ethylamide, and Aza-Gly-NH2.

Within the possibilities of this general structure, a particularly preferred GnRH-A is:

PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-ethylamide (SEQ ID NO: 9)

Table 1 identifies the various, non-limiting, examples of the GnRH-A.

    TABLE 1
    SEQ ID 1    PyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly
    SEQ ID 2    PyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
    SEQ ID 3    PyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-ethylamide
    SEQ ID 4    PyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 5    PyroGlu-His-Trp-Ser-Tyr-Lys-Leu-Arg-Pro-Gly-NH2
    SEQ ID 6    PyroGlu-His-Trp-Ser-Tyr-Lys-Leu-Arg-Pro-ethylamide
    SEQ ID 7    PyroGlu-His-Trp-Ser-Tyr-Lys-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 8    PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2
    SEQ ID 9    PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-ethylamide
    SEQ ID 10   PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 11   PyroGlu-His-Trp-Ser-Tyr-Ornithine-Leu-Arg-Pro-Gly-NH2
    SEQ ID 12   PyroGlu-His-Trp-Ser-Tyr-Ornithine-Leu-Arg-Pro-
                ethylamide
    SEQ ID 13   PyroGlu-His-Trp-Ser-Tyr-Ornithine-Leu-Arg-Pro-Aza-
                Gly-NH2
    SEQ ID 14   PyroGlu-His-Trp-Ser-Tyr-D-Ornithine-Leu-Arg-Pro-Gly-
                NH2
    SEQ ID 15   PyroGlu-His-Trp-Ser-Tyr-D-Ornithine-Leu-Arg-Pro-
                ethylamide
    SEQ ID 16   PyroGlu-His-Trp-Ser-Tyr-D-Ornithine-Leu-Arg-Pro-Aza-
                Gly-NH2
    SEQ ID 17   PyroGlu-His-Trp-Ser-Tyr-Glu-Leu-Arg-Pro-Gly-NH2
    SEQ ID 18   PyroGlu-His-Trp-Ser-Tyr-Glu-Leu-Arg-Pro-ethylamide
    SEQ ID 19   PyroGlu-His-Trp-Ser-Tyr-Glu-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 20   PyroGlu-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-Gly-NH2
    SEQ ID 21   PyroGlu-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-ethylamide
    SEQ ID 22   PyroGlu-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 23   PyroGlu-His-Trp-Ser-Tyr-Asp-Leu-Arg-Pro-Gly-NH2
    SEQ ID 24   PyroGlu-His-Trp-Ser-Tyr-Asp-Leu-Arg-Pro-ethylamide
    SEQ ID 25   PyroGlu-His-Trp-Ser-Tyr-Asp-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 26   PyroGlu-His-Trp-Ser-Tyr-D-Asp-Leu-Arg-Pro-Gly-NH2
    SEQ ID 27   PyroGlu-His-Trp-Ser-Tyr-D-Asp-Leu-Arg-Pro-ethylamide
    SEQ ID 28   PyroGlu-His-Trp-Ser-Tyr-D-Asp-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 29   PyroGlu-His-Trp-Ser-Tyr-Cys-Leu-Arg-Pro-Gly-NH2
    SEQ ID 30   PyroGlu-His-Trp-Ser-Tyr-Cys-Leu-Arg-Pro-ethylamide
    SEQ ID 31   PyroGlu-His-Trp-Ser-Tyr-Cys-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 32   PyroGlu-His-Trp-Ser-Tyr-D-Cys-Leu-Arg-Pro-Gly-NH2
    SEQ ID 33   PyroGlu-His-Trp-Ser-Tyr-D-Cys-Leu-Arg-Pro-ethylamide
    SEQ ID 34   PyroGlu-His-Trp-Ser-Tyr-D-Cys-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 35   PyroGlu-His-Trp-Ser-Tyr-Tyr-Leu-Arg-Pro-Gly-NH2
    SEQ ID 36   PyroGlu-His-Trp-Ser-Tyr-Tyr-Leu-Arg-Pro-ethylamide
    SEQ ID 37   PyroGlu-His-Trp-Ser-Tyr-Tyr-Leu-Arg-Pro-Aza-Gly-NH2
    SEQ ID 38   PyroGlu-His-Trp-Ser-Tyr-D-Tyr-Leu-Arg-Pro-Gly-NH2
    SEQ ID 39   PyroGlu-His-Trp-Ser-Tyr-D-Tyr-Leu-Arg-Pro-ethylamide
    SEQ ID 40   PyroGlu-His-Trp-Ser-Tyr-D-Tyr-Leu-Arg-Pro-Aza-Gly-NH2

In one embodiment, a targeting component may be linked to a botulinum toxin, preferably a botulinum toxin without the HC (such as LHN), to form an agent of the present invention.

In a preferred embodiment, the targeting component is a GnRH-A. For example, a GnRH-A may be linked to a botulinum toxin or an LHN to form an agent of the present invention. Preferably the GnRH-A molecule is linked to an LHN in a manner as to not substantially interfere with the therapeutic function of L and the translocation function of HN. In one embodiment, an LHN is linked to position 6 of GnRH-A to form an agent of the present invention. Without wishing to limit the invention to any particular theory or mechanism of operation, it is believed that 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.

In a broad embodiment, spacers may be used to physically further separate components of the present invention. For example, an agent of the present invention may comprise an LHN connected to a GnRH through a spacer. Preferably, 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.

In one embodiment, a spacer comprises a proline, serine, threonine and/or cysteine-rich amino acid sequence similar or identical to a human immunoglobulin hinge region. In a preferred embodiment, the spacer comprises the amino acid sequence of an immunoglobulin g1 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. For example, such hydrocarbon moieties are represented by the chemical formulas:

HOOC--(CH2)n --COOH, where n=1-12 or,

HO--(CH2)n --COOH, where n>10

In a broad embodiment, linkers (hereinafter "Linker Y" or "Y") may be used to link together two or more molecules, components and/or spacers. For example, a Linker Y may be used to link a GnRH-A to a LHN. In another embodiment, a Linker Y may be employed to link an LHN to a spacer; in turn, that spacer may then be linked to GnRH by another Linker Y, forming an agent comprising the structure:

LHN -Y-spacer-Y-GnRH.

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), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidine and glutaraldehyde.

In one embodiment, Linker Y may be attached to an amino group, a carboxylic group, a sulfhydryl group or a hydroxyl group of an amino acid group of a component. For example, 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.

Although the described chemistry may be used to couple the components of the described invention, any other coupling chemistry known to those skilled in the art capable of chemically attaching a targeting component to another component of an agent of the invention is covered by the scope of this invention.

In a broad embodiment, 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.

In one embodiment, a facilitator component includes a cationic lipid molecule. A cationic lipid molecule may be attached to an agent of the present invention, for example LHN -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. Pat. No. 5,459,127, which is incorporated in its entirety herein by reference.

In one embodiment, 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, Targeting Drugs to the Brain by Redox Chemical Delivery Systems 377-416).

In a preferred embodiment, the facilitator component is a carrier peptide which can facilitate the transport of an agent of the invention across the blood brain barrier. Such carrier peptides include, for example, a penetratin peptide represented by the formula:

(SEQ ID 42)

Arg-Gly-Gly-Arg-Lys-Ser-Trp-Ser-Arg-

Arg-Arg-Phe-Ser-Thr-Ser-Thr-Gly-Arg

Agents of the present invention have potential utility in human medicine. For example, 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. However, such surgical intervention may be too drastic. Agents of the present invention may be administered, instead, to treat the prostate cancer. For example, an effective dose of LHN -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.

Those skilled in this art will also appreciate that 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.

The dose of the agent to be administered depends on many factors. For example, the better each one of the components is able to perform its respective function, the lower the dose of the agent is required to obtain a desired therapeutic effect. One of ordinary skill will be able to readily determine the specific dose for each specific agent. For agents employing a natural, mutated or recombinant botulinum toxin A comprising the therapeutic, translocation and targeting component, an effective dose of an agent to be administered may be about 1 U to about 500 U of the botulinum toxin.

Furthermore, 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). In one embodiment, 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%. In another embodiment, 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, LHN -GnRH-A, directly to the pituitary, preferably anterior pituitary. Intracranial injection methods are widely known in the art. For example, U.S. patent application Ser. No. 09/692,811, filed Nov. 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).

Other 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.

According to a broad aspect of this invention 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 (s). 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. The recombinant cloning vectors are transformed into a mammalian, yeast or bacterial host. The preferred host is E. coli. Following expression of recombinant genes in host cells, resultant proteins can be isolated using conventional techniques. The protein expressed may comprise all three components of the agent. For example, the protein expressed may include a light chain of botulinum toxin type A (the therapeutic component), an HN 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). In one embodiment, 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.

There are many advantages to producing these agents recombinantly. For example, production of 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. Significantly, the high toxicity of the product dictates that the procedure must be performed under strict containment (BL3). During the fermentation process, 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.

The degree of activation of engineered Clostridial toxins is, therefore, an important consideration for manufacture of these materials. It would be a major advantage if 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.

With safety being a prime concern, previous work has concentrated on the expression in E. coli and purification of individual H and L chains of tetanus and botulinum toxins; these isolated chains are, by themselves, nontoxic; see Li et al. (1994, Biochemistry 33, 7014-7020) and Zhou et al. (1995, Biochemistry 34, 15175-15181), hereby incorporated by reference herein. Following the separate production of these peptide chains and under strictly controlled conditions the H and L subunits can be combined by oxidative disulphide linkage to form the neuroparalytic di-chains.

In one embodiment, an agent comprising a therapeutic component and a translocation component is recombinantly produced as an unnicked single chain. See Dolly et al. U.S. Ser. No. 09/648,692, the disclosure of which is incorporated in its entirety by reference herein. In a preferred embodiment, the agent includes an amino acid sequence that is susceptible to specific cleavage in vitro following expression as a single chain. Such proteins may include Clostridial toxins and derivatives thereof, such as those proteins disclosed in U.S. Pat. No. 5,989,545 and International Patent Application W095/32738, both incorporated in their entirety by reference herein.

To minimize the safety risk associated with handling neurotoxin, the agents, or toxins of the invention the agent precursors 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. To improve the efficiency and rate of proteolytic cleavage 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.

To reduce the risk of unintentional activation of the toxin by human or commonly encountered proteases, 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 (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 (SEQ ID NO: 50); tobacco etch virus (TEV) protease, which cleaves the sequence EXXYXQS/G (SEQ ID NO: 51); GENENASE.RTM. from Bacillus amyliquifaciens, which cleaves the sequence HY or YH; and PRESCISSION.RTM. protease from human rhinovirus 3C, which cleaves the amino acid sequence LEVLFQGP (SEQ ID NO: 52). As used above, the letter X indicates any amino acid. All amino acid sequences shown in the present specification are in the direction from amino terminus to carboxyl terminus, and all nucleotide sequences from 5'to 3', (from left to right) unless otherwise indicated.

In one embodiment, 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.

In one embodiment, a DNA sequence encoding the HC is included in the recombinant DNA sequence which encodes the Clostridial toxin contained in the heterologous gene expression system. Therefore, the corresponding HC portion of an H chain will not be produced. This too will lower the safety risk associated with Clostridial toxin production.

In one embodiment, GnRH may be produced by similar heterologous recombinant DNA expression systems as is familiar to one skilled in the art. In another embodiment, 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. (1992, Biochemistry 31, 8799-8809) and Swain et al. (1993, Peptide Research 6, 147-154).

Claim 1 of 6 Claims

I claim:

1. A method for treating a gonadotrophin related illness in a mammal, said method comprises the steps of:

(I) administering to the mammal a therapeutically effective amount of an agent, the agent comprises:

(a) an LHN which comprises (i) a light chain component, an L-chain of a botulinum toxin, a butyricum toxin, or a tatani toxin and (ii) a translocation component, an HN of a botulinum toxin, a butyricum toxin, or a tatani toxin; and

(b) a targeting component which comprises a gonadotrophin-releasing hormone (GnRH) or GnRH analog, wherein the LHN is covalently coupled to the GnRH or GnRH analog, and wherein the targeting component selectively binds to a GnRH receptor; and

(II) alleviating the gonadotrophin related illness by lowering the level of a gonadotrophin secretion, wherein the gonadotrophin related illness is selected from the group consisting of breast cancer, prostate cancer, pancreatic cancer, and endometrial cancer.


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