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Title:  Promoters of neural regeneration

United States Patent:  6,512,004

Issued:  January 28, 2003

Inventors:  Song; Hong-jun (La Jolla, CA); Poo; Mu-ming (La Jolla, CA); Ming; Guo-li (La Jolla, CA); Tessier-Lavigne; Marc (San Francisco, CA); He; Zhigang (San Francisco, CA)

Assignee:  The Regents of the University of California (Oakland, CA)

Appl. No.:  900268

Filed:  July 6, 2001


The invention provides methods and compositions for promoting neural cell growth and/or regeneration. The general methods involve contacting with an activator of a cyclic nucleotide dependent protein kinase a neural cell subject to growth repulsion mediated by a neural cell growth repulsion factor. The activator may comprise a direct or an indirect activator of the protein kinase; the repulsion factor typically comprises one or more natural, endogenous proteins mediating localized repulsion or inhibition of neural cell growth; and the target cells are generally vertebrate neurons, typically injured mammalian neurons. The subject compositions include mixtures comprising a neural cell, an activator of a cyclic nucleotide dependent protein kinase and a neural cell growth repulsion factor.


The general methods involve contacting with an activator of a cyclic nucleotide dependent protein kinase a neural cell subject to growth repulsion mediated by a neural cell growth repulsion factor. Preferred activators enhance the activity of at least one of PKA or PKG. A wide variety of direct and indirect activators of cyclic nucleotide dependent protein kinases are known in the art, or readily identified in assays such as immuno, kinase and cell based assays. Indirect activators are agents which increase the activity of the protein kinase without directly interacting with the kinase, and include any agent which increases the functional activity of the corresponding cyclic nucleotide (e.g. by increasing its synthesis, increasing its availability, decreasing its degradation, etc.). Exemplary activators include cyclic nucleotide analog agonists, activators of cyclic nucleotide cyclases, NO inducers, inhibitors of cyclic nucleotide phosphodiesterases, drugs such as KT5720, etc. Additional activators are readily made by screening candidate agents for activation of the targeted protein kinase, inhibition of a targeted phosphodiesterase (e.g. cAMP or cGMP phosphodiesterase), activation of a targeted cyclase (e.g. guanylate or adenylate cyclase), etc. in conventional in vitro or cell based assays.

The repulsion factor typically comprises one or more natural, endogenous agents mediating localized repulsion or inhibition of the targeted neural cell growth, which repulsion or inhibition is reversible by increasing the activity of a cyclic nucleotide dependent protein kinase in the cell. Such factors are generally present at the site of neuronal cells in situ, particularly at the cite of CNS axons, and provide an endogenous inhibition to nerve cell growth and/or regeneration. A wide variety of such factors are known or are readily identified in cell based assays, such as described herein. Exemplary agents capable of acting as repulsion factors include neural cell guidance proteins such as some semaphorins, netrins, CNS myelin fractions or components thereof such as MAG, etc.

The target cells are generally vertebrate neurons, typically injured mammalian neurons in situ. A wide variety of methods may be used to effect the contacting of the cell with the activator. For example, for CNS administration, a variety of techniques are available for promoting transfer of therapeutic agents across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. The compositions may also be amenable to direct injection or infusion, intraocular administration, or within/on implants e.g. fibers such as collagen fibers, in osmotic pumps, grafts comprising appropriately transformed cells, etc.

In a preferred embodiment, the activator is delivered locally and its distribution is restricted. For example, a particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic agents, see also Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Another particular embodiment is adapted from treatment of spinal cord injuries, e.g. Schulz M K, et al., Exp Neurol. 1998 Feb; 149(2): 390-397; Guest J D, et al., J Neurosci Res. 1997 Dec 1; 50(5): 888-905; Schwab M E, et al., Spinal Cord. 1997 Jul; 35(7): 469-473; Tatagiba M, et al., Neurosurgery. 1997 Mar; 40(3): 541-546. For example, the subject compositions improve corticospinal tract (CST) regeneration following thoracic spinal cord injury by promoting CST regeneration into human Schwann cell grafts in the methods of Guest et al. (supra). For these data, the human grafts are placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. Activators (see Table 1) incorporated into a fibrin glue are placed in the same region. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), are performed. Thirty-five days after grafting, the CST response is evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provide information about axonal die-back. In control animals (graft only), the CST do not enter the SC graft and undergo axonal die-back. As shown in Table 1, the activators dramatically reduce axonal die-back and cause sprouting.

                             TABLE I
    In Vivo Neuronal Regeneration with Exemplary Activator Formulations
                                              Reduced   Promote
    Activator                    Formulation Die-Back  Sprouting
     1. Forskolin                    5 uM     + + + +   + + + +
     2. 7.beta.-Deaceyl-7.beta.-[.gamma.-     5 uM     + + + +   + + + +
     3. 6.beta.-[.beta.'-(Piperidino)-     5 uM     + + + +   + + + +
     4. 3-Isobutyl-1-methylxanthine  25-100 uM   + + + +   + + + +
     5. Rolipram                     2 uM     + + + +   + + + +
     6. 8-bromo-cAMP               100 uM     + + + +   + + + +
     7. 8-chloro-cAMP              100 uM     + + + +   + + + +
     8. 8-(4-chlorophenylthio)-cAMP   100 uM     + + + +   + + + +
     9. Dibutyryl-cAMP             100 uM     + + + +   + + + +
    10. Dioctanoyl-cAMP            100 uM     + + + +   + + + +
    11. Sp-cAMPS                    20 uM     + + + +   + + + +
    12. Sp-8-bromo-cAMPS            20 uM     + + + +   + + + +
    13. 8-br-cGMP                  100 uM     + + + +   + + + +
    14. 8-(4-chlorophenylthio)-cGMP   100 uM     + + + +   + + + +
    15. Dibutyryl-cGMP             100 uM     + + + +   + + + +
    16. Glyco-SNAP-1               300 uM     + + + +   + + + +
    17. Glyco-SNAP-2               300 uM     + + + +   + + + +
    18. S-Nitroso-N-acetylpenicill-   300 uM     + + + +   + + + +
    19. NOC-18                     100 uM     + + + +   + + + +
    20. NOR-3                      100 uM     + + + +   + + + +
    21. Protoporphyrin-9            10 uM     + + + +  + + + +

In another demonstration of in vivo therapeutic activity, the subject activators are incorporated in the implantable devices described in U.S. Pat. No. 5,656,605 and tested for the promotion of in vivo regeneration of peripheral nerves. Prior to surgery, 18 mm surgical-grade silicon rubber tubes (I.D. 1.5 mm) are prepared with or without guiding filaments (four 10-0 monofilament nylon) and filled with test compositions comprising the activators of Table 1. Experimental groups consist of: 1. Guiding tubes plus Biomatrix 1.TM. (Biomedical Technologies, Inc., Stoughton, Mass.) ; 2. Guiding tubes plus Biomatrix plus filaments; 3-23. Guiding tubes plus Biomatrix 1.TM. plus activators 1-21 of Table 1 (supra).

The sciatic nerves of rats are sharply transected at mid-thigh and guide tubes containing the test substances with and without guiding filaments sutured over distances of approximately 2 mm to the end of the nerves. In each experiment, the other end of the guide tube is left open. This model simulates a severe nerve injury in which no contact with the distal end of the nerve is present.

After four weeks, the distance of regeneration of axons within the guide tube is tested in the surviving animals using a functional pinch test. In this test, the guide tube is pinched with fine forceps to mechanically stimulate sensory axons. Testing is initiated at the distal end of the guide tube and advanced proximally until muscular contractions are noted in the lightly anesthetized animal. The distance from the proximal nerve transection point is the parameter measured. For histological analysis, the guide tube containing the regenerated nerve is preserved with a fixative. Cross sections are prepared at a point approximately 7 mm from the transection site. The diameter of the regenerated nerve and the number of myelinated axons observable at this point are used as parameters for comparison.

Measurements of the distance of nerve regeneration document the therapeutic effect of groups 3-23. Similarly, plots of the diameter of the regenerated nerve measured at a distance of 7 mm into the guide tube as a function of the presence or absence of one or more activators of the device demonstrate a similar therapeutic effect of all 21 activators tested. No detectable nerve growth is measured at the point sampled in the guide tube with the matrix-forming material alone. The presence of guiding filaments plus the matrix-forming material (no activator) induces only very minimal regeneration at the 7 mm measurement point, whereas dramatic results, as assessed by the diameter of the regenerating nerve, are produced by the device which consisted of the guide tube, guiding filaments and activator compositions. Finally, treatments using guide tubes comprising either a matrix-forming material alone, or a matrix-forming material in the presence of guiding filaments, result in no measured growth of myelinated axons. In contrast, treatments using a device comprising guide tubes, guiding filaments, and matrix containing activator compositions consistently result in axon regeneration, with the measured number of axons being increased markedly by the presence of guiding filaments.

The amount of activator administered depends on the activator, formulation, route of administration, etc. and is generally empirically determined. For example, with cyclic nucleotide activators delivered locally in a solid matrix or semi-solid phase, the administered dose is typically in the range of about 2 mg up to about 2,000 mg, although variations will necessarily occur depending on the target, the host, and the route of administration, etc.

In one embodiment, the invention provides the subject activators combined with a pharmaceutically acceptable excipient suitable for contacting target neuronal cells in situ, such as CNS administration, including as sterile saline or other medium, gelatin, an oil, etc. to form pharmaceutically acceptable compositions. The compositions and/or compounds may be administered alone or in combination with any convenient carrier, solid or semi-solid matrix, diluent, etc. and such administration may be provided in single or multiple dosages. Useful carriers and matrices include solid, semi-solid or liquid media including water and non-toxic organic solvents. In another embodiment, the invention provides the subject compounds in the form of a pro-drug, which can be metabolically converted to the subject compound by the recipient host. A wide variety of pro-drug formulations are known in the art. The compositions may be provided in any convenient form including tablets, capsules, fibers, guides, osmotic pumps, etc. (see, e.g. U.S. Pat. Nos. 5,656,605; 5,660,849 and 5,735,863 for delivery systems particularly suited for CNS administration). As such the compositions, in pharmaceutically acceptable dosage units or in bulk, may be incorporated into a wide variety of containers and materials. For example, dosage units may be included in a variety of containers including microcapsules, pumps, fibers, etc.

The compositions may be advantageously combined and/or used in combination with other therapeutic or prophylactic agents, different from the subject compounds. In many instances, administration in conjunction with the subject compositions enhances the efficacy of such agents. For example, the compounds may be advantageously used in conjunction with other neurogenic agents, neurotrophic factors, growth factors, anti-inflammatories, antibiotics etc.; and mixtures thereof, see e.g. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9h Ed., 1996, McGraw-Hill, esp. Chabner et al., Antineoplastic Agents at pp.1233.

The subject compositions include ex vivo mixtures comprising a neural cell, an activator of a cyclic nucleotide dependent protein kinase and a neural cell growth repulsion factor. Such mixtures may be used in in vitro screens for identifying suitable activators, optimizing formulations, delivery concentrations, etc., etc.

Claim 1 of 85 Claims

What is claimed is:

1. A method for promoting growth of a mammalian central nervous system neural cell subject to growth inhibition by an endogenous neural cell growth repulsion factor, the method comprising the steps of contacting the cell with an effective amount of an activator of a cyclic nucleotide dependent protein kinase, whereby the growth of the cell is promoted, and detecting a resultant promotion of the growth of the cell, wherein the activator comprises an active component selected from a cyclic nucleotide analog and an activator of a cyclic nucleotide cyclase.

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