Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Facilitation of repair of neural injury with CM101/GBS toxin

United States Patent:  6,476,001

Issued:  November 5, 2002

Inventors:  Hellerqvist; Carl G. (Brentwood, TN); Wamil; Artur W. (Nashville, TN); Wamil; Barbara D. (Nashville, TN)

Assignee:  Vanderbilt University (Nashville, TN)

Appl. No.:  364620

Filed:  July 29, 1999

Abstract

Neural injury may be advantageously treated with CM101, a polysaccharide toxin isolated from Group B .beta.-hemolytic Streptococcus bacteria. CM1O1 treatment aids in the re-establishment of neuronal connectivity, at least partially inhibits scar formation, and increases the probability of survival during the critical period following injury to the central nervous system. Preexisting neural injuries having scar tissue are ameliorated by surgical excision of the scar tissue in conjunction with administration of CM101.

DESCRIPTION OF SPECIFIC EMBODIMENTS

CM101, a GBS toxin, is a polysaccharide molecule isolated from group B .beta.-hemolytic Streptococcus (GBS). Specifically, pathogenic group B .beta.-hemolytic Streptococcus produces a polysaccharide exotoxin. This exotoxin is the putative agent for early onset disease in neonatal humans. It is believed that receptors for CM101 are present primarily on the lungs of newborns, making them susceptible to early onset disease, but that the receptors are lost approximately four to seven days after birth.

Isolated and purified CM101 has been shown to have toxic effects on sheep experimental models that mimic GBS infant pneumonia (Hellerqvist, C. G. et al., Studies on group B .beta.-hemolytic streptococcus I. Isolation and partial characterization of an extra-cellular toxin., Pediatr. Res., 12:892-898 (1981)). In the sheep model for neonatal early onset disease, GBS toxin causes pulmonary hypertension, increased pulmonary vascular permeability, granulocytopenia, and pulmonary sequestration of granulocytes.

CM101 has a molecular weight of approximately 300,000 Daltons and comprises N-acetyl-galactosamine, N-acetyl-glucosamine, glucose, galactose, and mannose residues. Carboxylic acid residues are also believed to be an integral part of the molecule. Repeating active epitopes most likely play an important role in the pathophysiological response to CM101 by crosslinking receptors on target endothelium (Hellerqvist, C. G. et al., Early Results of a Phase I Trial of CM101 in Cancer Patients., Proceedings of the American Association of Cancer Research Annual Meeting (1995)).

A method of purification of a GBS toxin is provided in U.S. Pat. No. 5,010,062. Preferably, however, the CM101 is purified according to the method taught in International Application No. PCT/US97/17535, incorporated herein by reference.

A source for GBS starting material for use in the method of the present invention may be obtained by culturing strains of Group B .beta.-hemolytic Streptococcus bacteria that have recently infected or are capable of infecting newborn infants. Isolates of such strains may be obtained from the blood or cerebrospinal fluid of infected infants.

Without limitation to a particular theory, it is believed that GBS toxin, and specifically CM101, plays an important role in the treatment of neural injury because it facilitates the regeneration of neuronal connectivity by inhibiting glial cell proliferation. Trauma to the CNS is known to cause hypoxia (Liu et al., 1997), and this causes the release of vascular endothelial growth factor (VEGF). VEGF stimulates endothelial cells to dedifferentiate and begin to form new vasculature. The newly formed vasculature facilitates glial cell proliferation which gives rise to scars that physically interfere with the reformation of neuronal contacts. CM101 treatment impairs the proliferation of glial cells, thereby lessening gliosis (the formation of scars in the healing CNS). CM101-mediated impairment of glial cell proliferation is indirect and occurs by binding of CM101 to dedifferentiated angiogenic endothelial cells, thus targeting them for complement-mediated destruction, and by down-regulation of VEGF transcription (Wamil, B. D. et al., CM101 inhibits VEGF induced tumor neovascularization as determined by MRI and RT-PCR, AACR Proceedings, 38:237 (1997)). Since glial scars sterically interfere with neuronal reconnection of damaged axons, treatment with CM101 fosters the reestablishment of neuronal connectivity and permits recovery of vital organ function as well as motor and sensory function.

The present invention is useful for treating central nervous system injuries and peripheral nervous system injuries. It facilitates post-injury re-establishment of neuronal connectivity by inhibiting scar formation and by enhancing neuronal regeneration. In addition to treatment of trauma, surgery, and ischemia patients in the minutes or hours preceding or following the infliction of the injury, CM101 finds use in patients with pre-existing neural injury, an injury that has been in existence for more than two weeks. These patients receive CM101 to inhibit the formation of new scars after microsurgical excision of existing scar tissue. These patients may receive infusions of nerve growth factors such as IGF-1, bFGF or TGF.beta.S (Houle, J. et al., Axonal regeneration by chronically injured supraspinal neurons can be enhanced by exposure to insulin-like growth factor, basic fibroblast growth factor or transforming growth factor beta, Restorative Neurol. Neurosci., 10(4):205-215 (1996)), or BDNF, NT-3 (Grill, R. et al., Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury, J. Neurosci., 17:5560-5572 (1997)), CNTF (Ye, J-H. and Houle, J., Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons, Exp. Neurol., 143(1):70-81 (1997); Grill et al., 1997) or transplants of neural progenitor cells (Lundberg, C. et al., Conditionally immortalized neural progenitor cell lines integrate and differentiate after grafting to the adult rat striatum: A combined autoradiographic and electron microscopic study, Brain Res., 737(1-2):295-300 (1996)) to increase neuronal regeneration at the excision site. For patients with head trauma, CM101 treatment decreases the likelihood of posttraumatic epilepsy associated with cerebromeningeal scarring.

Since CM101 promotes neuronal repair and facilitates the re-establishment of neuronal connectivity, it provides a method for treating persons with diseases characterized by neuronal degeneration or impairment of neuronal connectivity. These diseases include Alzheimer's disease, Pick's disease, Parkinson's disease, striatonigral degeneration, Shy-Drager syndrome, Hallervorden-Spatz syndrome, progressive supranuclear palsy, olivopontocerebellar atrophy, Friedreich's ataxia, ataxia-telangiectasia, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, Werdnig-Hoffman disease, Kugelberg-Welander syndrome, multiple sclerosis, and perivenous encephalomyelitides. The benefit of CM101-mediated enhanced neuronal repair to patients suffering from diseases such as these is appreciable.

One of the methods used to determine CM101's effect on neuronal repair is an in vitro assay of GABA-mediated alteration of neuron membrane potential. This assay is based on the observation that, although healthy neurons hyperpolarize when exposed to GABA, neurons that have an unrepaired injury depolarize when GABA is applied. Since the amplitude of the depolarization reflects the severity of the neuronal damage, response to GABA over time provides a measure of the kinetics of neuronal sprouting and repair. This assay demonstrates that CM101 enhances neuronal regeneration. The GABA assay has utility for the testing of other compounds for their abilities to positively or negatively affect repair of neuronal injury.

GBS toxin as used herein is defined as any fraction or component isolated from natural or lysed GBS bacteria, or derived from media supernatants of lysed and/or autoclaved GBS bacteria, and which has a biological activity as evidenced by induction of respiratory distress in the sheep assay (Hellerqvist, C. G. et al., Studies on group B .beta.-hemolytic streptococcus I. Isolation and partial characterization of an extra-cellular toxin., Pediatr. Res., 12:892-898 (1981)) or activation of, complement and binding to neovasculature as demonstrated by a peroxidase-antiperoxidase (PAP) assay of a tumor tissue specimen (Hellerqvist, C. G. et al., Anti-tumor effects of GBS toxin: a polysaccharide exotoxin from group B .beta.-hemolytic streptococcus, J. Canc Res. Clin. Oncol., 120:63-70 (1993); and Hellerqvist, C. G. et al., Early Results of a Phase I Trial of CM101 in Cancer Patients., Proceedings of the American Association of Cancer Research Annual Meeting (1995)). GBS toxin also means any synthetic polysaccharide with the same structure or function as any GBS-derived molecule with the aforementioned activity.

Substantially pure GBS toxin means a preparation in which GBS toxin is greater than 40% pure (e.g., present in a concentration of at least about 40% by weight), preferably at least approximately 60% pure, more preferably at least approximately 90% pure, and most preferably at least approximately 95% pure. Dosages of lower purity GBS toxin should be altered accordingly. The purity of GBS toxin is discussed in greater detail in International Application No. PCT/US97/17535.

The CM101 or other GBS toxin is preferably combined with a pharmaceutically acceptable carrier and administered to a patient systemically. The carrier is preferably one that is readily mixed with CM101 to form a composition that is administrable by intravenous (IV) means. Thus, the carrier is preferably saline, which may have other pharmaceutically acceptable excipients included to ensure its suitability for intravenous administration. The resulting composition will be sterile and will have acceptable osmotic properties. In general, a suitable IV formulation is prepared in accordance with standard techniques known to one of skill in the art. For example, Chapter 85 entitled "Intravenous Admixtures" by Salvatore J. Turco in the Eighteenth Edition of Remington's Pharmaceutical Sciences, Mach Publishing Co. (1990), incorporated herein by reference, provides standard techniques for preparing a pharmaceutically acceptable IV composition useful in accordance with this invention. Other dosage forms to administer CM101 may also be used. As an alternative to systemic administration, CM101 may be administered locally to a site. Administration of CM101 to the patient may occur before, during, and/or after infliction of the neural injury. Preferably, CM101 is administered within an appropriate temporal window following the injury. Administration of CM101 soon after the injury occurs is most preferred. For example, administration within 1 day, or preferably within six hours is best. Administration of CM101 soon after CNS injury is necessary to increase the probability (relative to patients with similar injuries who do not receive CM101 ) that the patient will survive the first few days following the injury. While the length of this "critical post-injury period" varies according to the type and magnitude of the injury, survival at 72, 98 and 120 hours following injury are significant milestones.

The amount of CM101 that is administered to a patient is an amount that is sufficient to aid in the reestablishment of neuronal connectivity and to minimize scarring. A preferred dosage range is 1 to 100 .mu.g/kg body weight. A more preferred dosage range, however, is 1 .mu.g/kg to 50 .mu.g/kg body weight, and most preferred is a dosage in the range of 1 .mu.g/kg to 25 .mu.g/kg. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the age, body weight, general health, sex, diet, and severity of the neural injury. Each dosage is preferably administered in an infusion of up to 120 minutes, with 5 to 60 minutes being the preferred duration range, and 5 to 30 minutes being the most preferred dosage range. Once weekly treatment is preferred, and is likely to be all that is necessary for evidence of results.

Neural injury exists when a portion of a neuron or nerve, a bundle of fibers or processes, is punctured, torn, severed, crushed, bruised, or otherwise incapacitated in its abilities to transmit or receive electrochemical signals. Re-establishment establishment of neuronal connectivity involves the reformation of normal synaptic structure or the resumption of normal synaptic function. This can be assessed by neurological examination, neurologic diagnostic procedures such as electroencephalography, magnetic resonance imaging and CT scan, or electrophysiological recordings, or synaptic visualization.

Another aspect of the present invention is an article of manufacture, such as a kit, and a method for making the article of manufacture. The article includes a pharmaceutical composition comprising a GBS toxin, and particularly CM101, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be placed in a suitable container, as is well known in the art. Also included are instructions for treatment of patients according to the methods of the present invention.

Claim 1 of 32 Claims

What is claimed is:

1. A method of treating a patient with a neutral injury, which method comprises:

administering to the patient a polysaccharide toxin from group B .beta.-hemolytic Streprococces (GBS) bacteria in a quantity sufficient to at least partially inhibit scar formation by impairing glial cell proliferation.
 


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]