WO2013134177A1

WO2013134177A1 - Methods and compositions for improving spinal surgery outcomes

Info

Publication number: WO2013134177A1
Application number: PCT/US2013/028975
Authority: WO
Inventors: John M. HOLLIMAN; Randolph C. Steer; Randall W. PORTER
Original assignee: Capstone Therapeutics Corp.
Priority date: 2012-03-05
Filing date: 2013-03-05
Publication date: 2013-09-12

Abstract

The present invention provides methods and compositions for improving outcomes in spinal surgery by administration of an AZXIOO peptide.

Description

METHODS AND COMPOSITIONS FOR IMPROVING SPINAL SURGERY

OUTCOMES

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Application No.

61/606,763, filing date March 5, 2012, which is incorporated herein by reference in its entirety.

BACKGROUND

Back pain is a significant health problem. However, clinical interventions, including surgery, often do not provide satisfactory outcomes or sustained relief from back pain. In fact, surgical interventions can sometimes result in worse outcomes than non-surgical intervention and the chance of improved outcomes from surgery decrease with subsequent surgical interventions. Movement can cause tearing of adhesions developed after surgery resulting in repeated bleeds at the surgical site, diminishing the duration of good surgical outcomes. Therefore, once a poor outcome is achieved, currently there is little chance of improving the outcome.

Failed back surgery syndrome (FBSS) is a term embracing a constellation of conditions that describes persistent or recurring low back pain, with or without sciatica following one or more spine surgeries. FBSS can be characterized by any of a number of post-surgical symptoms including, for example, the presence of persistent, disabling pain in the hip, thigh, leg, and/or lower back; nerve root compression and tethering resulting in pain, numbness, and muscle weakness; chronic pain of the back and/ or extremities; and neuropathic back pain. The incidence of patients that will develop FBSS following lumbar spinal surgery is commonly quoted in the range of 10% to 40%, depending on the study population and evaluation criteria. Moreover, as the success rate of spine surgeries drops with repeated surgery, the importance of obtaining a good outcome after the first surgery is critical (Chan and Peng, Pain Medicine 2011 ; 12: 577-606). Factors that can result in unsuccessful lumbar spine surgeries can also effect outcomes in surgery on the thoracic, cervical, and sacral spine. SUMMARY OF THE INVENTION

It has now been found that administration of a peptide disclosed herein to rabbits undergoing laminectomy reduces adhesion formation and/ or reduces the fibrous tissue at or between the dura and scar tissue (see Example 1). Reduction of the formation of spinal adhesions and/or reduction of the formation of fibrous tissue at or between the dura and scar tissue can result in reducing pressure on, tethering of and compression of spinal nerve root(s) that often follows spinal surgery. Based on this discovery, methods of reducing post-operative numbness and pain, including neuropathic pain, which may result from pressure on spinal nerves or spinal adhesions, are disclosed herein.

The invention provides methods for reducing the likelihood of developing or reducing the severity of post-operative pain, numbness, or loss of mobility after spinal surgery comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

B 1 -X 1 - A(X2) APLP-Z 1 -Z2-Z3 -B2 (SEQ ID NO : 1 ) wherein XI is a fragment comprising at least amino acids 11-14 of a peptide of the amino acid sequence MEIPVPVNPSWLRR (SEQ ID NO: 2);

X2 is selected from the group consisting of S, T, Y, D, E, hydroxylysine, hydroxyproline, phosphoserine, and phosphotyrosine;

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K;

B 1 and B2 are independently absent or a transduction domain, wherein at least one of Bl and B2 is a transduction domain; and

the peptide is optionally acylated at the N-terminus and optionally amidated at the C-terminus.

The invention further provides methods for reducing the likelihood of developing or reducing the severity of post-operative neuropathic pain after spinal surgery comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

B 1 -X 1 -A(X2) APLP-Z 1 -Z2-Z3 -B2 (SEQ ID NO: 1) wherein XI is a fragment comprising at least amino acids 11-14 of a peptide of the amino acid sequence MEIPVPVNPSWLRR (SEQ ID NO: 2);

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K;

the peptide is optionally acylated at the N- terminus and optionally amidated at the C-terminus.

The invention also provides methods for reducing the likelihood of developing or reducing the severity of failed back surgery syndrome (FBSS) comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

B1-X1-A(X2)APLP-Z1-Z2-Z3-B2 (SEQ ID NO: 1) wherein XI is a fragment comprising at least amino acids 11-14 of a peptide of the amino acid sequence MEIPVPVNPSWLRR (SEQ ID NO: 2);

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K; and

B 1 and B2 are independently absent or a transduction domain, wherein at least one of B 1 and B2 is a transduction domain, and

In certain embodiments, post-operative pain, numbness, or loss of mobility after spinal surgery, neuropathic pain, and/or FBSS result from one or more of compression of, tethering of, or pressure on a spinal nerve. Compression of, tethering of, or pressure on a spinal nerve can result, for example, from formation of spinal adhesions and/or formation of fibrous tissue at or between the dura and scar tissue. All of the methods comprise administering an effective amount of a polypeptide described herein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Failed back syndrome (FBS), also called "failed back surgery syndrome" (FBSS), refers to chronic back and/or leg pain that occurs after back (spinal) surgery. It is characterized as a chronic pain syndrome. Common symptoms associated with FBSS include diffuse, dull and aching pain involving the back and/or legs. Abnormal sensibility may include sharp, pricking, and stabbing pain in the extremities. The term "post-laminectomy syndrome" is used by some doctors to indicate the same condition as failed back syndrome. Causes of FBSS

include persistent post-operative pressure on a spinal nerve, tethering of a spinal nerve root resulting from the back surgery, and/or compression of a spinal nerve root in the back resulting from the back surgery. Pressure on, tethering of and compression of a spinal root after spinal surgery can, in some instances, result from peridural or epidural scarring or fibrosis or spinal adhesion formation. As such, the invention is also a method of reducing or reducing the likelihood of developing peridural or epidural scarring or fibrosis resulting from spinal surgery. All of these methods comprise administering an effective amount of a polypeptide described herein.

Failed back surgery syndrome (FBSS) is characterized by one or more of cervical, thoracic, or lumbar spine pain; pain or conditions associated with or present as a result of spine dysfunction; neck, shoulder, shoulder blade, and arm pain; hip, thigh, leg, lower back, neck and arm weakness; neck, back, and arm limited range of mobility; headache, dizziness, nausea, neck stiffness, pins and needles, and numbness, reduced quality of life, persistent hip, thigh, leg, or lower back pain, disabling hip, thigh, leg, or lower back pain; nerve root compression pain, nerve root tethering pain, pressure from pain on a spinal nerve; hip, thigh, leg, or lower back numbness; back chronic pain; extremity chronic pain; and neuropathic back pain, epidural or peridural scarring and fibrosis, adhesion formation, and inflammation. FBSS can be understood as a condition wherein the subject does not achieve an improvement in back function after surgery relative to before surgery.

Signs or symptoms of back pain include pain as assessed by any of a number of clinically known or acceptable pain assessment scales such as Visual Analog Scales (VAS), Numeric Pain Intensity Scale, Simple Descriptive Pain Intensity Scale, Graphic Rating Scale, Verbal Rating Scale, Pain Faces Scale, Numeric Pain Intensity & Pain Distress Scales, Brief Pain Inventory, an Memorial Pain Assessment Card (from Memorial Sloan-Kettering Cancer Center), or as an aspect of a Quality of Life Inventory® assessment, and can further include the use of pain diaries to track pain levels over time with improvement including shorter duration of pain after surgery, see also e.g. , pain-topics.org/clinical_concepts/assess.php; strength and range of motion using standard methods including those provided by e.g. , Davidson and De Nardis, 2011 (Spine. 36:E307-E312, incorporated herein by reference).

As used herein, the "dura mater" is understood as the tough fibrous membrane that envelops the brain and spinal cord external to the arachnoid and pia mater.

"Epidural" is understood as situated upon or administered or placed outside the dura mater, whereas "peridural" is understood as occurring or applied about the dura mater. Epidural and peridural are used interechangably herein.

As used herein, "spinal surgery" is understood as surgery on the neck or any portion of the vertebral column, i.e., surgery on one or more of the cervical, thoracic, lumbar, or sacral spine. Spinal surgery includes contact with peridural tissue. A spinal surgery can be a first surgery to correct spinal dysfunction, or a revision surgery after a first surgery did not result in the desired outcome.

As used herein, a "spinal adhesion" is understood as fibrous bands that form between the dura and scar tissue or the spine, often as a result of injury during spinal surgery. Adhesions can prevent the dura from moving freely from the spine and nerves, potentially resulting in poor outcomes.

An appropriate "control" or "control group" can readily be selected by one of skill in the art, e.g., the subject before and after surgery, or a group matched as appropriate for age, gender, weight, severity of condition, surgical intervention, related or unrelated diseases or symptoms, and other considerations in the art. A "control level" of pain, range of motion, numbness, scarring or adhesion formation, etc. is understood as a defined, clinically acceptable amount of any sign or symptom associated with back pain or surgical outcomes in subjects that have undergone back surgery that is used to compare to the outcome of a subject who has undergone treatment using the compositions and methods provided herein. A control level can also be understood as the level of back and/ or limb pain, back and/ or limb mobility, back and/ or limb numbness, nerve compression, quality of life, scarring or adhesion formation in the subject prior to back surgery and after back surgery. Determination of statistical significance from a control is within the ability of those skilled in the art, e.g. , the number of standard deviations from the mean that constitute a positive or negative result.

The methods provided herein for reducing of the likelihood of developing and/or reducing the severity of post-operative pain, numbness, lack of mobility, epidural or peridural fibrosis, neuropathic pain; or FBSS after spinal surgery are understood as reducing the likelihood of developing or the severity of one or more signs or symptoms of a disease or condition, diminishing the extent of disease, stability (i.e. , not worsening) state of disease, amelioration or palliation of the disease state, or delaying or diminishing the onset or progression of a disease or condition. The methods do not require that all signs or symptoms of a disease or condition need to be eliminated or prevented, either temporarily or indefinitely. The methods can include administration of multiple and/ or continuous doses of a therapeutic agent. Administration of the peptides can be initiated prior to the onset of the disease or condition, e.g., prior to back surgery or during or after back surgery, but prior to the onset of symptoms of FBSS. Administration of the peptide can be initiated after the onset of symptoms of FBSS, e.g., prior to revision back surgery, during revision back surgery, and/or after revision back surgery. In certain embodiments, administration of the peptide can be initiated after the onset of FBSS, but prior to a revision surgery to correct FBSS.

An "effective amount" is that amount of one or more polypeptides sufficient to treat a disease in a subject. An effective amount of the polypeptides that can be employed ranges generally between about 0.01 μg/kg body weight and about 10 mg/kg body weight, preferably ranging between about 0.05 μg/kg and about 5 mg/kg body weight. The invention includes the administration of the polypeptides of the invention at various doses and various routes of administration. For example, a loading dose of the peptide may be administered intravenously prior to surgery, topically during surgery, at a first dose for a period of time immediately after surgery. Dosage levels are based on a variety of factors, including the type of surgery, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. An effective amount can be administered in one or more doses. An effective amount can be administered at one or more times before, during, or after spinal surgery.

The terms "administer", "administering" or "administration" include any method of delivery of a pharmaceutical composition or agent into a subject's system or to a particular region in or on a subject. In certain embodiments of the invention, the pharmaceutical composition is administered by injection, either systemically, or at or adjacent to the surgical site. For example, the agent is administered intravenously, intramuscularly, subcutaneously, intradermally, transcutaneously, or intrathecally. In other embodiments, the agent can be delivered intranasally, orally, mucosally, or topically, e.g., at the surgical site. In a preferred embodiment, an agent is

administered intravenously, optionally by bolus injection, sustained administration, infusion pump. Administration can occur at multiple times optionally by multiple routes, e.g. , prior to, during, or after spinal surgery. Administration can occur at one or more times including prior to, during, or after surgery by one or more routes. As used herein, the term "benefit" refers to something that is advantageous or good, or an advantage. Similarly, the term "benefiting", as used herein, refers to something that improves or advantages, e.g. , decreased pain including decreased neuropathic pain, decreased numbness, increased range of mobility, increased quality of life, decreased adhesion formation, decreased nerve or dura tethering, decreased nerve compression or damage, and/or improved histological outcome.

As used herein, the term "subject" refers to human and non-human animals, including veterinary subjects. The term "non-human animal" includes all vertebrates, e.g. , mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dog, cat, horse, cow, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human and may be referred to as a patient.

The term "or" is understood to be conjunctive, and used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with the preferred embodiments, it will be understood that it is not intended to limit the invention to those preferred embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

I. Dosages and Modes of Administration

Techniques and dosages for administration vary and are well known to those skilled in the art or are readily determined.

Therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.

Administration may be parenteral, intravenous, subcutaneous, intramuscular, intrathecal, oral or local by direct administration at the surgical site.

The composition can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; or a liquid for intrathecal, intravenous, subcutaneous, or parenteral administration; intermittent or sustained delivery using an infusion pump; or a polymer or other sustained release vehicle for local

administration, particularly during surgery.

Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy (20th ed., Ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, PA). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g. , biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g. , sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g. , magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.

The dosage and the timing of administering the compound depends on various clinical factors including the overall health of the subject and the severity of the symptoms of disease, the likelihood of having a poor outcome from spinal surgery, and the extent of spinal surgery. II. Methods of the Invention

As demonstrated in the Examples, the methods and compositions provided herein result in a decrease in the formation of epidural or peridural scaring and adhesions after back surgery which would result in an improved clinical outcome, e.g. , reduction of back pain, preferably sustained reduction of back pain including neuropathic pain, and related conditions for subjects undergoing spinal surgery. The methods include administration of a peptide provided herein which includes and HSP20 phosphopeptide covalently linked to a peptide transduction domain to promote delivery of the HSP20 phosphopeptide into the cell. The peptide is demonstrated to reduce scarring and adhesion formation which would be expected to reduce the likelihood of a poor outcome, and improve the likelihood of a positive outcome and the duration of a positive outcome after spinal surgery.

Peptides for use in the methods and compositions of the invention include a polypeptide comprising or consisting of a sequence a polypeptide represented by general formula I:

Zl is selected from the group consisting of G and D; Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K;

B 1 and B2 are independently absent or a transduction domain, wherein at least one of Bl and B2 is a transduction domain; and wherein the peptide is optionally acylated at the N-terminus and optionally amidated at the C-terminus.

In certain embodiments, XI, the fragment comprising at least amino acids 11- 14 of a peptide of the amino acid sequence MEIPVPVNPSWLRR (SEQ ID NO: 2) is WLRR (SEQ ID NO: 3). In certain embodiments, XI includes, SWLRR (SEQ ID NO: 4), PSWLRR (SEQ ID NO: 5), NPSWLRR (SEQ ID NO: 6), VNPSWLRR (SEQ ID NO: 7), PVNPSWLRR (SEQ ID NO: 8), VPVNPSWLRR (SEQ ID NO: 9), PVPVNPSWLRR (SEQ ID NO: 10), IP VP VNPSWLRR (SEQ ID NO: 11), EIPVPVNPSWLRR (SEQ ID NO: 12), or MEIPVPVNPSWLRR (SEQ ID NO: 13). According to various embodiments of the polypeptides of general formula I, X2 is S, T, Y, D E, phosphoserine (pS), or phosphotyrosine (pY). In a preferred embodiment, X2 is pS, S, T, or Y; more preferred that X2 is pS, S, or T, and most preferred that X2 is pS. In these embodiments where X2 is S, T, or Y, it is most preferred that X2 is phosphorylated. When X2 is D or E, these residues have a negative charge that mimics the phosphorylated state. The polypeptides of general formula I are optimally effective in the methods of the invention when X2 is phosphorylated, is a phosphoserine or a phosphotyrosine analog

Acylation means CH₃(Cj¾)nC(0)-, wherein n is an integer from 0-10; and amidation means -C(0)-NR_aRb, wherein R_a and R are independently -H or - (CH2)_nCH₃ wherein n is an integer from 0-10, preferably -C(0)-N]¾.

Thus, according to these various embodiments, a representative sample of polypeptides according to the X1-A(X2)APLP-Z1-Z2-Z3 (SEQ ID NO: 14) portion of general formula I for use in the methods of the invention include, but are not limited to, polypeptides comprising or consisting of the following sequences:

WLRRApSAPLPGLK (SEQ ID NO: 15); WLRRApSAPLPGLS (SEQ ID NO: 16); WLRRApSAPLPGLT (SEQ ID NO: 17); WLRRApSAPLPGKS (SEQ ID NO: 18); WLRRApSAPLPGKT (SEQ ID NO: 19); WLRRApSAPLPDLS (SEQ ID NO: 20); WLRRApSAPLPDLT (SEQ ID NO: 21); WLRRApSAPLPDKS (SEQ ID NO: 22); WLRRApSAPLPDKT (SEQ ID NO: 23); WLRRApYAPLPGLS (SEQ ID NO: 24); WLRRApYAPLPGLT (SEQ ID NO: 25); WLRRApYAPLPGKS (SEQ ID NO: 26); WLRRApYAPLPGKT (SEQ ID NO: 27); WLRRApYAPLPDLS (SEQ ID NO: 28); WLRRApYAPLPDLT (SEQ ID NO: 29); WLRRApYAPLPDKS (SEQ ID NO: 30); and WLRRApYAPLPDKT (SEQ ID NO: 31), wherein pS is phosphoserine and pY is phosphotyrosine. Other specific polypeptides falling within the scope of the XI- A(X2)APLP-Z1-Z2-Z3 (SEQ ID NO: 14) portion of general formula I will be readily apparent to one of skill in the art based on the teachings herein.

The polypeptides according to general formula I further comprise one or more transduction domains. As used herein, the term "transduction domain" (PTD), also known as a "cell penetrating peptide" (CPP) are a class of small cationic peptides of approximately 10-30 amino acids in length that have been shown to engage the anionic cell surface through electrostatic interaction and induce their own cellular internalization through various forms of endocytosis. Some of the most well characterized PTDs include the HIV TAT peptide (Vives et al,m 1997, /. Biol. Chem., 272: 16010-7), penetratin (Derossi et al., 1994, /. Biol. Chem., 10444-50), transportan (Pooga, 2001, FASEB J. 15: 1451-53), polyarginine (Futaki et al., 2001. /. Biol.

Chem., 276:5836-40), and MPG (Morris et al., 1997. Nuc. Acids Res. 25:2730-36). As used herein, a protein transduction domain is understood as an amino acid sequence that can transport the HSP20 polypeptide into a cell. These domains can be linked to other polypeptides to direct movement of the linked polypeptide across cell membranes. In some cases the transducing molecules do not need to be covalently linked to the active polypeptide. In a preferred embodiment, the transduction domain is linked to the rest of the polypeptide via peptide bonding. Examples of such transduction domains include, but are not limited to YARAAARQARA (SEQ ID NO: 32); YGRKKRRQRRR (SEQ ID NO: 33); THRLPRRRRRR (SEQ ID NO: 34); GGRRARRRRR (SEQ ID NO: 35); SYGRKKRRQRRR (SEQ ID NO: 36); (R)₈-io (SEQ ID NO: 37); GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 38); and WLRIKKA (SEQ ID NO: 39).

In a preferred embodiment, one or both of B 1 and B2 comprise or consist of the amino acid sequence of YGRKKRRQRRR (SEQ ID NO: 33) and/or

YARAAARQARA (SEQ ID NO: 32). In a preferred embodiment, the polypeptide according to the general formulas disclosed herein comprises or consists of a polypeptide YGRKKRRQRRRWLRRApSAPLPGLK (SEQ ID NO: 40) or

YARAAARQARAWLRRApSAPLPGLK (SEQ ID NO: 41), wherein "pS" represents a phosphorylated serine residue. In certain embodiments, the polypeptide is acylated at the N-terminus. In certain embodiments, the polypeptide is amidated at the C-terminus. In certain embodiments, the polypeptide is acylated at the N-terminus and amidated at the C-terminus. In preferred embodiments, the acylation is -COOH. In preferred embodiments, the amidation is -NH₂. For example, in certain embodiments, the polypeptide is NH₂-YARAAARQARAWLRRApSAPLPGLK- COOH (SEQ ID NO: 41).

The polypeptides described herein may be chemically synthesized or recombinantly expressed using methods well known in the art such as those provide in US Patent No.7,875,588 which is incorporated herein by reference. The method of synthesizing the polypeptides of the invention is not a limitation of their use.

The polypeptides may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

For administration, the polypeptides are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, dextran sulfate, heparin-containing gels, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

The polypeptides or pharmaceutical compositions thereof may be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. Preferred embodiments for administration vary with respect to the condition being treated or the stage of treatment. In a preferred embodiment, the polypeptides or pharmaceutical compositions are disposed on or in a wound dressing or other topical administration. Such wound dressings can be any used in the art, including but not limited to films (e.g. , polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (nonwoven composites of fibers from calcium alginate), cellophane, and biological polymers such as those described in US patent application publication number 20030190364, published October 9, 2003.

The polypeptides may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The polypeptides of the invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the polypeptides, and have no known drug related serious adverse effects.

Example 1 - AZX100 Improved Laminectomy Outcomes in a Rabbit Model

The objective of this study was to assess the effectiveness of AZX100 (NH₂- YARAAARQARAWLRRASAPLPGLK-COOH, (SEQ ID NO: 41)), wherein the S is phosphorlylated, in improving surgical outcomes after laminectomy in a rabbit model. 1.0 Abstract

Methods: According to the protocol, fourteen female New Zealand white rabbits, divided into five groups, had a laminectomy performed in the lamina T4-5. Animals in Groups 2 through 5 had either the drug vehicle or AZX100 delivered by an Alzet® osmotic pump between days 8 through 28. Group 1 animals received no intervention. Four weeks post-surgery, all animals were sacrificed and a gross evaluation of the surgical sites was performed. Protocol specified tissues were collected and forwarded to a clinical research organization where the tissues were processed, embedded in paraffin, and sectioned. Two slides were produced for each tissue; one slide was stained with hematoxylin and eosin (H&E) while the other was stained with Masson's trichrome. All H&E and Masson's trichrome slides were evaluated by a single individual.

Conclusion: The objective of this study was to assess the effectiveness of AZX100 in improving surgical outcomes after laminectomy in the rabbit model. A notable improvement surgical outcome was noted for laminectomy site specimens from Group 5 (AZX100 at 500 μg/day) as compared to their appropriate controls. According to the grading scheme employed for histological observation, the mean score value was 0.33 for Group 5 (AZX100 at 500 μg/day) as compared to mean scores of 2.50 for Group 1 (sham, no treatment) and 3.00 for Group 2 (drug vehicle). Regarding fibroblast density and inflammatory cell density, indicators of local inflammation, there were no meaningful difference in the mean score values for AZXIOO-treated groups as compared to the sham and drug vehicle control groups.

2.0 Introduction

2.1 Protocol This report presents the results of histopathological evaluation of the spine of fourteen animals at a clinical research organization using routine methods. All surgical procedures, tissue harvests, and microscopic evaluation of the slides were preformed by individuals of skill in the art.

2.2 Objective The objective of this study was to assess the effectiveness of AZX100 in improving surgical outcomes after laminectomy in the rabbit model.

3.0 Methods

3.1 Compliance Statement

The histology and pathology portions of the study were conducted in a manner consistent with US FDA GLP: 21 CFR 58, 1987, although quality assurance inspections were neither required nor performed.

3.2 Study Design

A total of fourteen female New Zealand white rabbits were enrolled in the study according to the study test protocol. Study design is further described in Table 1 below.

2 3 Vehicle 0

3 3 AZX100 5.0 μg/day

(0.11 μg total)

4 3 AZX100 50 μg/day

(1.1 μ total)

5 3 AZX100 500 μg/day

(11.5 μ total)

3.3 Necropsy

According to the test protocol, four weeks post-surgery, all animals were euthanized and a gross evaluation of the surgical sites was performed. The spinal column was resected in bloc from Tl to LI, including the overlying soft tissue and the spinal columns, then fixed in 10% buffered formalin.

3.4 Histological Processing

Collected tissues were forwarded to a clinical research organization where the tissues were processed, embedded in paraffin, and sectioned. Two slides were produced for each tissue; one slide was stained with hematoxylin and eosin (H&E) while the other was stained with Masson's trichrome. All H&E and Masson's trichrome slides were evaluated in a blinded fashion.

3.5 Microscopic Evaluation Scoring

Each specimen was scored for histological observations, density of fibroblasts, inflammatory cell density and bone regeneration by the scales noted below. A grade was assigned to each specimen to assess the surgical and treatment outcomes as follows:

Table 2. Histological Observations

laminectomy site

4 Large scar affecting more than two-thirds of the laminectomy site, or adherence extends to the nerve roots

A grade was assigned to each specimen to assess the fibroblast and inflammatory density:

Table 3. Fibroblast and Inflammatory Density

4.0 Results

4.1 Animal Mortality

There were no unscheduled deaths among the animals submitted to HSRL for histopathological evaluation. 4.2 Macroscopic Observations

There were no macroscopic observations reported by the Testing Facility.

4.3 Microscopic Observations Histological observations: According to the grading scheme employed, the histological observations indicated improved surgical outcomes for Group 5 (AZXIOO at 500 μg/day) with a mean score value of 0.3 as compared to Group 1 (sham, no treatment) and Group 2 (drug vehicle) with mean histological score values of 2.5 and 3.0, respectively. There was no improvement in the histological score for Group 3 (5 μg/day) and Group 4 (50 μg/day) as compared to their appropriate controls; the mean histological score was 2.3 for Group 3 and the mean histological score was 2.7 for Group 4.

Fibroblast/inflammatory density: There was no meaningful difference in the mean score values across groups for fibroblast density and inflammatory cell density. The degree of both fibroblast density and inflammatory cell density was invariably a Grade 1 (fewer than 100 cells in each field at 400 times magnification). For fibroblast density, the mean score values were 1.0 for the sham control (Group 1), 1.3 for drug vehicle (Group 2), 1.0 for AZXIOO at 5 μg/day (Group 3), 1.3 for AZXIOO at 50 μg/day (Group 4), and 1.0 for AZXIOO at 500 μg/day (Group 5). For inflammatory cell density, the mean score values were 1.0 for the sham control (Group 1), 1.0 for drug vehicle (Group 2), 1.3 for AZXIOO at 5 μg/day (Group 3), 1.0 for AZXIOO at 50 μg/day (Group 4), and 1.0 for AZXIOO at 500 μg/day (Group 5).

Bone regeneration: In the specimens examined, bone regeneration was either noted as Grade 1 (bone regeneration at the edges of the laminectomy defect) or as Grade 2 (bone regeneration closing the laminectomy defect). The mean score values for bone regeneration were 2.0 for the sham control (Group 1), 1.0 for drug vehicle (Group 2), 2.0 for AZXIOO at 5 μg/day (Group 3), 1.7 for AZXIOO at 50 μg/day (Group 4), and 1.0 for AZXIOO at 500 μg/day (Group 5).

Table 5: Summary of Results

Bone Regeneration 2.0 1.0 2.0 1.7 1.0

5.0 Summary and Conclusions

The objective of this study was to assess the effectiveness of AZXIOO in improving surgical outcomes after laminectomy in the rabbit model. A notable improvement in histological outcome was noted for laminectomy site specimens from Group 5 (AZXIOO at 500 μg/day) as compared to their appropriate controls.

According to the grading scheme employed for the histological observations, the mean score value was 0.3 for Group 5 (AZXIOO at 500 μg/day) as compared to mean scores of 2.5 for Group 1 (sham, no treatment) and 3.0 for Group 2 (drug vehicle). Regarding fibroblast density and inflammatory cell density, there were no meaningful difference in the mean score values for AZXIOO-treated groups as compared to the sham and drug vehicle control groups.

Example 2 -- AZXIOO Improves Functional Laminectomy Outcomes in a Rabbit Model

Laminectomies are performed on rabbits as in the previous example and are subsequently treated with AZXIOO at appropriate doses or with vehicle control. At appropriate intervals, rabbits are tested for their response to various pain stimuli and the responses graded. Rabbits are also observed for general mobility and range of motion. Rabbits treated with AZXIOO have less pain and greater mobility than rabbits not treated with AZXIOO.

Example 3 - Prevention of Poor Laminectomy Outcomes by Prophylactic Administration of AZXIOO Prior to performance of laminectomy, rabbits are treated with AZXIOO at various doses for various time intervals (e.g., 1 day, 3 days, 1 week, 2 weeks). A laminectomy is performed as above. Optionally during the surgery, the surgical site is irrigated with AZXIOO in rabbits that have or have not been pretreated with AZX 100. Optionally, rabbits are treated with AZXIOO post-operatively.

After an appropriate interval to allow for healing, rabbits are observed for pain levels and mobility. At the end of the experiment, the animals are euthanized and tissues harvested for analyses as in Example 1. Samples are graded for histological outcomes, fibroblast and inflammatory cell density, and bone regeneration.

Pretreatment with AZXIOO and/ or irrigation at the surgical site are demonstrated to result in an improved surgical outcome as compared to control rabbits not treated with AZXIOO.

Example 4 -- Clinical Trial to Determine Outcomes of Subjects Undergoing Spinal Surgery

Subjects are recruited to participate in a clinical trial to demonstrate the beneficial effect of AZXIOO on spinal surgery outcomes. Subjects are identified based on appropriate criteria and randomized into at least one treatment group and a control group. Treatment groups can include, for example, different dosing regimens (e.g., amount and frequency of dosing) and administration of AZXIOO before, during, and/ or after surgery. The groups are matched for factors such as age, gender, disease severity and duration, surgical location, etc. Studies can be open label, but are preferably blinded as the assessment criteria are at least partially subjective (e.g., pain, mobility). A pre-surgical assessment is made to determine pain, mobility, and values for other criteria that will be assessed after surgery. At predetermined post-surgical intervals, subjects are assessed for pain, mobility, and other factors. Treatment regimen can be selected based on the outcome of the study.

Incorporation by Reference

All publications, patents, and patent applications mentioned in this

specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more that routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method for reducing the likelihood of developing or for reducing the severity of post-operative pain, numbness, or loss of mobility after spinal surgery comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K;

2. A method for reducing the likelihood of developing or for reducing the severity of post-operative neuropathic pain after spinal surgery comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K;

3. The method of claim 1 or 2, wherein XI is WLRR (SEQ ID NO: 3).

4. The method of claim 1 to 3, wherein Z1-Z2-Z3 is GLK.

5. The method of any of claims 1 to 4, wherein XI -A(X2)APLP-Z1 -Z2- Z3 (SEQ ID NO: 14) is the polypeptide WLRRASAPLPGLK (SEQ ID NO: 15), wherein the S residue is phosphorylated.

6. The method of any of claims 1 to 5, wherein the transduction domain is a polypeptide selected from the group consisting of YARAAARQARA (SEQ ID NO: 32) and YGRKKRRQRRR (SEQ ID NO: 33).

7. The method of any of claims 1 to 6, wherein the amino acid sequence of the polypeptide is YARAAARQARA WLRRASAPLPGLK (SEQ ID NO: 41), wherein the S residue is phosphorlylated.

8. The method of any of claims 1 to 7, wherein the polypeptide is NH₂- YARAAARQARAWLRRASAPLPGLK-COOH (SEQ ID NO: 41), wherein the S residue is phosphorlylated.

9. The method of any of claims 1 to 7, wherein the polypeptide is acylated at the N-terminus.

10. The method of any of claims 1 to 7, wherein the polypeptide is amidated at the C-terminus.

11. The method of any of claims 1 to 10, wherein the spinal surgery comprises removal of at least a portion of a vertebral bone.

12. The method of any of claims 1 to 11, wherein the surgery is selected from the group consisting of laminectomy, spinal fusion, lumbar disectomy, microdisectomy, facetectomy, decompressive surgery; and any combination thereof.

13. The method of any of claims 1 to 12, wherein pain, numbness, weakness, or loss of mobility is in at least one body part selected from the group consisting of back, thigh, neck, hip, shoulder blade, shoulder, and arm.

14. The method of any of claims 1 to 13, wherein reducing the likelihood of developing or reducing the severity of post-operative pain, numbness, loss of mobility, or neuropathic pain after spinal surgery is characterized by a decrease in the likelihood or presence of one or more of cervical, thoracic, lumbar, or sacral spine pain; pain or conditions associated with or present as a result of spine dysfunction; neck, shoulder, shoulder blade, and arm pain; hip, thigh, leg, lower back, neck and arm weakness; neck, back, and arm limited range of mobility; headache, dizziness, nausea, neck stiffness, pins and needles, and numbness, reduced quality of life, persistent hip, thigh, leg, or lower back pain, disabling hip, thigh, leg, or lower back pain; nerve root compression pain, nerve root tethering pain, hip, thigh, leg, or lower back numbness; back chronic pain; and neuropathic back pain, spinal scarring, adhesion formation, and inflammation.

15. The method of any of claims 1-14, wherein the post-operative pain, numbness, loss of mobility, or neuropathic pain after spinal surgery results from one or more of compression of, tethering of, or pressure on a spinal nerve; or spinal adhesion formation.

16. A method for reducing the likelihood of developing or for reducing the severity of failed back surgery syndrome (FBSS) comprising administering to a subject an effective amount of a polypeptide represented by general formula I:

Zl is selected from the group consisting of G and D;

Z2 is selected from the group consisting of L and K; and

Z3 is selected from the group consisting of S, T, and K; and B 1 and B2 are independently absent or a transduction domain, wherein at least one of B 1 and B2 is a transduction domain, and

17. The method of claim 16, wherein XI is WLRR (SEQ ID NO: 3).

18. The method of claim 16 or 17, wherein Z1-Z2-Z3 is GLK.

19. The method of any of claims 16 to 18, wherein the polypeptide fragment X1-A(X2)APLP-Z1-Z2-Z3 (SEQ ID NO: 14) is the sequence

WLRRASAPLPGLK (SEQ ID NO: 15), wherein the S residue is phosphorylated.

20. The method of claim any of claims 16 to 19, wherein the transduction domain is a polypeptide selected from the group consisting of YARAAARQARA

(SEQ ID NO: 32) and YGRKKRRQRRR (SEQ ID NO: 33).

21. The method of any of claims 16 to 19, wherein the amino acid sequence of the polypeptide is YARAAARQARAWLRRASAPLPGLK (SEQ ID NO: 41), wherein the S residue is phosphorlylated.

22. The method of any of claims 16 to 19, wherein the polypeptide is NH₂- YARAAARQARAWLRRASAPLPGLK-COOH (SEQ ID NO: 41), wherein the S residue is phosphorlylated.

23. The method of any of claims 16 to 20, wherein the polypeptide is acylated at the N-terminus.

24. The method of any of claims 16 to 21, wherein the polypeptide is amidated at the C-terminus.

25. The method of any of claims 16 to 24, wherein the spinal

comprises removal of at least a portion of a vertebral bone.

26. The method of any of claims 16 to 25, wherein the surgery is selected from the group consisting of laminectomy, spinal fusion, lumbar disectomy, microdisectomy, facetectomy, decompressive surgery; and any combination thereof.

27. The method of any of claims 16 to 26, wherein FBSS is characterized by one or more of cervical, thoracic, or lumbar spine pain; pain or conditions associated with or present as a result of spine dysfunction; neck, shoulder, shoulder blade, and arm pain; hip, thigh, leg, lower back, neck and arm weakness; neck, back, and arm limited range of mobility; headache, dizziness, nausea, neck stiffness, pins and needles, and numbness, reduced quality of life, persistent hip, thigh, leg, or lower back pain, disabling hip, thigh, leg, or lower back pain; nerve root compression pain, nerve root tethering pain, hip, thigh, leg, or lower back numbness; back chronic pain; extremity chronic pain; and neuropathic back pain, spinal scarring, adhesion formation, and inflammation.

28. The method of any of claims 16 to 27, wherein the FBSS results from one or more of compression of, tethering of, or pressure on a spinal nerve, or spinal adhesion formation.

29. The method of claim 1 to 28, wherein the polypeptide is administered locally.

30 The method of claim 1 to 28, wherein the polypeptide is administered systemically.