WO2002100387A1

WO2002100387A1 - Prevention of neovascularization of intervertebral discs and/or of tissues with local inflammation

Info

Publication number: WO2002100387A1
Application number: PCT/SE2002/001115
Authority: WO
Inventors: Kjell Olmarker
Original assignee: A+ Science Invest Ab
Priority date: 2001-06-11
Filing date: 2002-06-10
Publication date: 2002-12-19
Also published as: SE0102067D0

Abstract

The use of an anti-angiogenetic substance for the production of a pharmaceutical preparation for prevention of neovascularization and/or neoinnervation of intervertebral discs and/or of tissues with local inflammation, in particular for prevention of chronic pain, such as chronic low back pain and chronic whiplash associated disorder, is disclosed. Said neovascularization and/or neoinnervation of intervertebral discs may be caused by spinal trauma. Also a method for prevention of neovascularization and/or neoinnervation of intervertebral discs and/or of tissues with local inflammation wherein a therapeutically effective amount of an anti-angiogenetic substance is administered to an individual is disclosed.

Description

PREVENTION OF NEOVASCULARIZATION OF INTERVERTEBRAL DISCS AND/OR OF TISSUES WITH LOCAL INFLAMMATION

Field of the invention The present invention relates to prevention of neovascularization and neoinnervation of intervertebral discs and/or of tissues with local inflammation, in particular in order to prevent chronic pain, such as chronic low back pain and chronic whiplash associated disorder.

Background of the invention Low back pain flLBP):

Low back pain affects approximately 80% of the population during their lifetime in most countries. Except for being extremely common, it is also one of the most costly disorders for the society. In Sweden alone, low back pain was estimated to cost USD 320.000.000 in 1997 (1). The major part of the cost relates to indirect costs such as sick-compensation and reduced productivity, and only a minor part is related to direct costs such as medical care and phar- macological substances.

In a minority of the cases (5%), there may be a known cause for the pain such as intraspinal tumors, rheumatic diseases, infections and more (2). In these cases the treatment may be specifically aimed at the cause. However, in the majority of the cases of low back pain, the cause remains unknown. At pre- sent there is no direct way to treat low back pain with an unknown cause and existing treatment modalities only aim at symptomatic relief.

Regarding the societal expenses for low back pain it is well known that 80% of the costs is related to the 10% of the patients that will develop chronic low back pain. The major socioeconomic effects thus relates rather to chronic low back pain patients than to patients with episodes of acute low back pain. It must therefore be considered that it may be even more rewarding, both economically and for the patients, to be able to prevent episodes of acute low back pain developing into chronic conditions, than to treat the acute pain per se. Whiplash and Whiplash associated disorders (WAD):

About 10% to 20% of the occupants of a stricken vehicle in rear-end car collisions suffer from whiplash injury. The injury may also occur as a result of other types of accidents, such as train accidents, and sudden retardations. This injury is defined as a non-contact acceleration-deceleration injury to the head- neck system. It is most often caused by a rear-end car collision and there is no direct impact on the neck.

Presenting symptoms usually include neckpain, headaches, disequilibrium, blurred vision, paraesthesiae, changes in cognition, fatigue, insomnia and hypersensitivity to light and sound. Dizziness described in a variety of terms such as imbalance, light-headedness and vertigo also occur frequently and these symptoms may be associated with long-term disability.

Although neurologic and orthopedic examinations do not reveal abnormalities in the majority of patients, the characteristics of dizziness due to whip- lash can be elucidated by means of ElectroNystagmoGraphic (ENG) evaluation. This examination is a method that is suitable for proving pathology in the oculo-vestibular system of whiplash-patients.

Until recently, the reason for the extent of injury was poorly understood. In addition, due to the legal and insurance issues, the veracity of complaints of neck pain and other symptoms by people who suffer from whiplash is commonly viewed as suspect.

Whiplash injuries can be quite complex and may include a variety of related problems, such as joint dysfunction, and faulty movement patterns, chronic pain and cognitive and higher center dysfunction.

Pathophysiologic aspects of low back pain and whiplash associated disorders. Acute conditions:

Little is known regarding the exact causes of acute low back pain. This may be due to the fact that LBP is not a specific disease but rather a symptom of various conditions causing pain in the lumbar region of the spine (2). Various medical conditions such as intraspinal tumors, rheumatic diseases, infections as well as injuries to muscles or ligaments in the spine, have all be suggested to induce low back pain (1). The physician may recognize these conditions and a proper diagnosis may be achieved. However, in the majority of the cases (95%) the underlying cause remains unknown. It was recently suggested that a significant part of these cases of acute low back pain may be due to disk- related substances leaking through the annulus fibrosus, thus irritating the nerve endings located in the superficial layers of the annulus. This leakage may either be seen as annular tears or like silent disk herniations (3, 4). Silent in this regard only implies that the disc herniation did not induce sciatica. Chronic conditions:

The knowledge regarding the exact cause of the chronic form of low back pain is just as limited as that of acute low back pain (2, 5, 6). However, degeneration of the intervertebral disc has been considered to be one of the most important factors (7, 8). Degeneration of the intervertebral disc may be seen as a "black disc" and may in some cases be treated by surgical stabilization of the involved spinal segment (9). However, the results of such surgery have been debated. It is not known why a tissue like the intervertebral disc may produce pain although there are no nerves in the disc, except for the most superficial layers of the annulus fibrosus (10-12). It is, however, known that nerve endings may be found in the deeper layers, reaching all the way into the nucleus pulposus in injured discs (13, 14). Nerves do not enter new tissues readily, particularly not into cartilage-tissue such as the nucleus pulposus (15). However, it is known that nerves may join newly formed vessels that grow into a scar (16-21). It is therefore assumed that nerves are found in the deeper parts of the disc due to the ingrowth of newly formed blood vessels. These nerves are not just involved in regulating vascular tonus but nerves containing substance P and CGRP have been found in degenerated discs (13, 14, 17, 22-24). Substance P is a neurotransmitter that mainly transmits pain and it is therefore assumed that these nerves induce pain. The pain derived from discs in this fashion is called "discogenic pain" (25).

Tissues with local inflammation:

Similar to the situation of the intervertebral discs, various tissues and structures with an ongoing local inflammatory process are known to be in- vaded by newly formed vessels and nerves. Such neoinnervation has been suggested to be the main factor for chronic pain and disability in these structures. For instance, it is known that local inflammation in the supraspinatus muscle tendon in the shoulder will lead to neoinnervation of the tendon and that these nerves will produce chronic pain. Attempts have been made to remove these newly formed nerves and blood vessels by surgery. Summary of the invention A novel approach to prevent the development of chronic pain syndromes such as chronic low back pain and whiplash associated disorders is to use pharmacological intervention to inhibit the ingrowth of newly formed ves- sels, and subsequently nerves, into the intervertebral discs after injury. This can be achieved by various approaches; by blocking the activity of substances known to promote angioneogenesis and neovascularization and by using an- giogenesis-inhibitors.

It has recently been found that there are various cytokines in the nucleus pulposus (26-30). Some of these substances are known to promote angiogenesis (vessel formation) such as for instance TNF, IL-2, IL-6, IL-8, VEGF, TGF- beta, Basic-FGF and prostaglandins (31, 32). It is known that the annulus fibrosus per se may induce neovascularization (33), but the relationship between the nucleus pulposus-related substances and vascular ingrowth into the disk, in particular into the nucleus pulposus, has never been considered. There are pharmacological substances that may be used for inhibiting these substances. By using such substances it would thus be possible to significantly reduce the risk of newly formed vessels growing into the disk, with subsequent neoinnervation of the disk, and the development of discogenic, chronic pain. There are also pharmacological substances that act as general angiogenesis inhibitors. They may both inhibit these angiogenetic substances or may also inhibit the formation of new blood vessels in other ways. The use of such angiogenesis-inhibitors would be equally useful for preventing neovascularization and neoinnervation of injured discs. A third possibility would be to use pharmacological substances that specifically inhibit nerve growth, thereby preventing the neoinnervation of the disc at the site of disc injury.

The timing of the preventive treatment according to the invention is important. The neovascularization is initiated by the disc injury. The injury may present clinically as low back or neck pain. The preventive treatment according to the present invention should thus be started as soon as pain is experienced and continued either until the pain has resolved or preferable until the disc injury has healed. Since healing of the disc may be difficult to determine clinically, one may suspect that treatment for 2-6 months is advisable. In patients were it may be assumed that a disc injury may have been induced, as in the case of trauma, the treatment should be started as soon as possible after the time of injury.

Detailed description of the present invention The present invention relates to the use of an anti-angiogenetic substance for the production of a pharmaceutical preparation for prevention of neovascularization and/or neoinnervation of intervertebral discs and/or of and/or of tissues with local inflammation. The invention also relates to the resulting pharmaceutical preparation. Furthermore, the invention relates to a method for prevention of neovascularization and/or neoinnervation of intervertebral discs and/or of tissues with local inflammation wherein a therapeutically effective amount of an anti- angiogenetic substance is administered to an individual.

The neovascularization and/or neoinnervation of intervertebral discs that can be prevented according to the present invention may be caused by spinal trauma. By preventing this neovascularization and/or neoinnervation of intervertebral discs, it is possible to prevent chronic low back pain and/or chronic whiplash associated disorder.

As stated above, similar to the situation of the intervertebral discs, vari- ous tissues and structures with an ongoing local inflammatory process are known to be invaded by newly formed vessels and nerves. Such neoinnervation has been suggested to be the main factor for chronic pain and disability in these structures. For instance, it is known that local inflammation in the supraspina- tus muscle tendon in the shoulder will lead to neoinnervation of the tendon and that these nerves will produce chronic pain. In analogy with the pharmacological prevention of neovascularization/neoinnervation of intervertebral discs as discussed, it would thus be possible also to prevent the neovasculariza- tion/neoinnervation of tissues with local inflammation with pharmacological inhibition. By preventing neovascularization and/or neoinnervation of tissues with local inflammation it is possible to prevent chronic pain, such as tendinitis. The treatment should be initiated as soon as possible after the injury and continued for 1-6 months.

The anti-angiogenetic substance used according to the invention can either have an indirect anti-angiogenetic effect by inhibiting an angiogenetic substance or a direct anti-angiogenetic effect. The anti-angiogenetic substance used according to the invention may also have an anti-angiogenetic effect by inhibiting a neurotrophic factor.

The anti-angiogenetic substance used according to the invention may be one of the following substances, or a combination of two or more of the fol- lowing substances, as well as pharmaceutically acceptable salts thereof.

ANTI-ANGIOGENETIC SUBSTANCES WITH INDIRECT ANTI- ANGIOGENETIC EFFECT BY INHIBITION OF ANGIOGENETIC

SUBSTANCES

Angiogenin inhibitors

Beta-fibroblast growth factor inhibitors

GM-CSF inhibitors

Interleukin 2 (IL-2) inhibitors

Interleukin 6 (IL-6) inhibitors

Interleukin 8 (IL-8) inhibitors

Prostaglandin inhibitors

TGF-beta inhibitors

TNF inhibitors

Vascular Endothelial Growth

Factor inhibitors such as

P1C11,

C225,

SU6668, and anti-KDR monoclonal antibodies Vascular P factor inhibitors

ANTI-ANGIOGENETIC SUBSTANCES WITH DIRECT ANTI- ANGIOGENETIC EFFECT TNF inhibitors

Specific TNF inhibitors

Monoclonal antibodies such as: infliximab, CDP-571 (Humi- cade™), and CDP-870

Polyclonal antibodies Soluble cytokine receptors such as: etanercept, lenercept, pegylated TNF receptor type I, and TBP-1

TNF receptor antagonists

Antisense oligonucleo- tides such as: ISIS-104838

Non-specific TNF inhibitors 5,6-dimethyl- xanthenone-4-acetic acid (acemannan) AGT-1 ANA 245 AWD 12281 BN 58705 Caspase inhibitors CBP-1011 CC 1069 CC 1080 CDC 801 CDDO CH-3697 CLX 1100 CM 101 CT3

CT 2576 CPH 82 CV 1013 Cyclosporin Compounds used in anti-cancer treatment such as: the binuclear DNA threading transition metal complexes and pharmaceutical compositions comprising them described in WO 99/15535, and methotrexate

Declopramide

DPC 333

DWP 205297

DY 9973

Edodekin alfa

Fit ligand (available from Immunex)

Gallium nitrate

HP 228

Hydroxamic acid deri- vates

IL-12

IL-18

Ilodekacin

Ilomastat

ITF-2357

JTE 607

Lactoferrin

Lactoferrin derived or derivable peptides such as those described in WO 00/01730

Lazaroids; nonglucocor- ticoid 21-aminosteroids such as: U-74389G (16- desmethyl tirilazad), U-74500

LPS agonist Esai

Melancortin agonists such as HP-228

Mercaptoethyl- guanidine

Metoclopramide

MMP inhibitors

(i.e. matrix metallopro- teinase inhibitors or

TACE inhibitors, i.e. such as:

TNF Alpha Converting Tetracyclines such as:

Enzyme-inhibitors) Doxycy- cline,

Lymecy- cline,

Oxitetracy- cline,

Tetracy- cline, and

Mino- cycline

Synthetic tetracy- cline derivates (CMT = Chemically Modified Tetracyclines) KB-R7785 TIMP1 and TIMP2 adTIMP2 and adTIMP2

M-PGA

Napthopyrans

NCS-700

Nimesulide NR58-3.14.3 p38 kinase inhibitors such as VX-702

VX-745 (Pralnaca- san),

VX-850,

SB-202190,

SB-203580, and pyridinyl imidazoles

PCM-4

PD-168787 Pentoxifyllin derviates Pharma projects no. 6181, 6019 and 4657 Phosphodiesterase I, II, III, IV, and V-inhibitors such as: CC-1088, Ro 20-1724, rolipram, amrinone, pimobendan, vesnarinone, and SB 207499

Piclamastat

PMS-601

Prostglandins such as:

Iloprost (prostacy- clin)

Quinolones (chmolones) such as:

Norfloxacin,

Levofloxacin,

Enoxacin,

Sparfloxacin,

Temafloxacin,

Moxifloxacin,

Gatifloxacin,

Gemifloxacin, Grepafloxacin, Trovafloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin, and Temafloxacin

RDP-58 RIP-3

Sch-23863

SH-636

Solimastat

SR-31747

Tasonermin

Thalidomid derivates (or

SelCID = Selective Cy- tokin inhibitors, e.g. tha- lidomide derivate) such as: CC-1088 CDC-501, and CDC-801

TNF alpha proteinase inhibitor available from

Immunex

TNF-484A

Tristetraproline (TTP)

(available from Astra-

Zeneca)

VRCTC 310

Yissum project no.

11649

Zanamivir

Eicosanoid synthesis inhibitors

Specific eicosanoid synthesis inhibitors Monoclonal antibodies Polyclonal antibodies Soluble cytokine receptors

Receptor antagonists Antisense oligonucleo- tides Non-specific eicosanoid synthesis inhibitors

Betalactames such as: penicillium, fenoximethylpenicil- lium, and cephalosporin

Cyclosporin Macrolids such as: sirolimus, spiramycin, tilmicosin, tylosin, kitasamicin, josamicin, erythromycin, and oleandomycin

MMP inhibitors (or TACE-inhibitors =TNF Alpha Converting Enzyme-inhibitors) Tetracyclines such as:

Doxycy- cline,

Trovaflox- acin,

Lymecy- cline,

Oxitetracy- cline,

Tetracy- cline, and Mino- cycline

Synthetic tetracy- cline derivates (CMT

= Chemically Modified Tetracyclines)

KB-R7785 Quinolones (chinolones) such as:

Norfloxacin,

Levofloxacin,

Enoxacin,

Sparfloxacin,

Temafloxacin,

Moxifloxacin,

Gatifloxacin,

Gemifloxacin,

Grepafloxacin,

Trovafloxacin,

Ofloxacin,

Ciprofloxacin,

Pefloxacin,

Lomefloxacin, and

Temafloxacin Thalidomid derivates (SelCID = Selective Cy- tokin inhibitors, e.g. tha- lidomide derivate) such as:

CC-1088,

CDC-501, and

CDC-801

Inhibitors of enzymes related to eicosanoid synthesis Inhibitors of phospholi- pase A2 (PLA A2) such as: monoclonal antibod- ies polyclonal antibodies Lidocaine Mepacrine Pyrrophenone Inhibitors of cyclooxy- genase (COX) 1 and 2 (i.e. prostaglandin synthesis inhibitors including non-steroidal anti- inflammatory drugs, NSAID) such as:

Acetylsalicylic acid, Monoclonal antibodies,

Polyclonal antibodies,

Celecoxib, Corticosteroides, Diclofenac, Ibuprofen, Indomethacin, Meloxicam, Naproxen, and Refecoxib Various inhibitors of angioneogenesis 2-methoxyestradiol, AG3340 (prinomastat), Angiostatin, Anti Integrin alpha- vbeta3, Batimastat, Captopril,

Carboxyamido-triazole, CM101, Combretastatin, Contortrostatin, Curcumin, Diphenylureas, Endostatin, Flavone Acetic Acid, Geni stein,

Human tumor inhibitors, IL-12, Irsogladine, Kringle 5 of plasminogen,

Latent antithrombin, LM-609, Marimastat, Mitoxantrone, Neovastat Aetherna, Nigella sativa, P53 gene therapy, Pentosan polysulfate, Peptide delivery system, PF-4, PI-88,

Prelatent antithrombin, PSK,

Recombinant Platelet Factor 4, Retinoids, Scatter factor, Spironolactone, Squalamine, Suramin and suramin analogues, Tamoxifen, Taxol, Tecogalan, Tie2 pathway, Thrombospondin 1 and

2,

TIP-1,

TNP-470 (AGM-1470),

Vinblastine,

Vitamin E, and

Vitaxin®

SUBSTANCES THAT INHIBIT NEUROTROPHIC FACTORS Substances that inhibit nerve growth factor (NGF)

Monoclonal antibodies,

Polyclonal antibodies

Soluble receptors,

Receptor antagonists, and

Antisense oligonucleo- tides Substances that inhibit brain derived nerve growth factor (BDNF)

Monoclonal antibodies,

Polyclonal antibodies

Soluble receptors,

Receptor antagonists, and

Antisense oligonucleo- tides Substances that inhibit insulin-like growth factor (IGF-1)

Monoclonal antibodies,

Polyclonal antibodies

Soluble receptors,

Receptor antagonists, and

Antisense oligonucleo- tides

According to a preferred embodiment, the substance used is a TNF inhibitor. According to an especially preferred embodiment the substance used is a monoclonal antibody directed against TNF, such as infliximab, CDP-571, D2E7 or CDP-870. According to an other especially preferred embodiment the substance used is a soluble cytokine TNF receptor, such as etanercept. According to an other especially preferred embodiment the substance used is a binu- clear DNA threading transition metal complex with anti-cancer effect. According to an other especially preferred embodiment the substance used is a lactoferrin derivable peptide. According to an other especially preferred embodiment the substance used is an MMP inhibitors, such as doxycycline. According to an other especially preferred embodiment the substance used is a p38 kinase inhibitor. According to yet another especially preferred embodiment the substance used is or TTP.

For the purpose of this disclosure the expression anti-angiogenetic substance relates to all substances, compounds, preparations, drugs, and medicaments that inhibit vascular tissue growth and neovascularization. Furthermore, for the purpose of this disclosure, an inhibitor may be an inhibitor, a blocking agent, a blocking substance, an antagonist, an antibody, a soluble receptor, and/or any other substance that prevents transcription and/or expression.

Also for the purpose of this disclosure, TNF relates to what formerly was called TNF-α.

The term "individual", as it is used herein, relates to any human or non- human mammal in need of preventive treatment according to the invention.

The term "chronic" relates to a condition or pain which has lasted for at least one month. The substance or pharmaceutical composition according to the invention is administered once or repeatedly until the desired therapeutical effect is obtained. The substance or pharmaceutical composition according to the invention is administered in a therapeutically effective amount, i.e. an amount that will lead to the desired therapeutical effect. The pharmaceutical composition according to the invention may also comprise other substances, such as an inert vehicle, or pharmaceutical acceptable adjuvants, carriers, preservatives etc., which are well known to persons skilled in the art.

According to one preferred embodiment of the invention, the pharmaceu- tical composition is formulated as a sustained- release preparation. The sub- stance according to the invention may then, for example, be encapsulated in a slowly-dissolving biocompatible polymer.

The substances or pharmaceutical compositions according to the invention may be administered in any efficacious way. The substances or pharmaceutical compositions according to the invention may for example be injected via intra-articular, intravenous (i.v.), intramuscular (i.m.), intraperitoneal (i.p.), intrathecal (i.t.), epidural, intracerebroventricular (i.c.v.) or subcutaneous (s.c.) routes by bolus injections or by continuous infusion. They may also be administered orally (per os), e.g. in the form of oral preparations, such as pills, syrups, or lozenges. Furthermore, they may be administered by inhalation. They may also be administered intranasally. Moreover, they may be administered transepidermally, e.g. in the form of topical preparations such as lotions, gels, sprays, ointments or patches. They may also be administered as suppositories. Finally, they may also be administered by genetical engineering.

Examples of suitable doses for different administration routes are given below.

Per os 10-300 mg i.m. 25-100 mg i.v. 2.5-25 mg i.t. 0.1-25 mg daily - every 3^r month inhalation 0.2-40 mg transepidermally 10-100 mg intranasally 0.1-10 mg s.c. 5-10 mg i.c.v. 0.1-25 mg daily - every 3 r^rd month epidurally 1-100 mg

Examples of suitable doses for different substances according to the invention are given below. Preferred More Most dosage preferred preferred dosage dosage

Iloprost i.v. 0.1-2000 1-1500 100-1000

(all doses given in μg/kg body weight/day) intranasally 50-250 100-150 100-150

(all doses given in μg/day) CC-1088 Per os 50-1200 200-800 400-600

(all doses given in mg/day) Linomide

(Roquinimex®)

Per os 0.1-25 5-20 10-15

(all doses given in mg/kg body weight/day)

HP-228 i.v. 5-100 10-50 20-40

(all doses given in μg/kg body weight)

Ariflo® SB 207499 Per os 10-100 30-60 30-45

(all doses given in mg/day)

KB-R7785 s.c. 100-500 100-300 150-250

(all doses given in mg/kg body weight/day)

Prinomastat (AG3340) Per os 1-250 5-100 10-50

(all doses given in mgfor administration twice daily)

Batimastat Per os 1-250 5-100 10-50

(all doses given in mgfor administration twice daily) Marimastat

Per os 1-250 5-100 10-50

(all doses given in mgfor administration twice daily)

CDC-501

Per os 50-1200 200-800 400-600

(all doses given in mg/day)

CDC-801

Per os 50-1200 200-800 400-600 (all doses given in mg/day)

It is possible to use either one ore two or more substances according to the invention. When two or more substances are used they may be administered either simultaneously or separately. The substances according to the invention may also be administered in combination with other drugs or compounds, provided that these other drugs or compounds do not eliminate the effects desired according to the present invention.

It is understood that the response by individual patients to the sub- stances according to the invention or combination therapies, may vary, and the most efficacious combination of drugs for each patient will be determined by the physician in charge.

The invention will now be further explained in the following examples. These examples are only intended to illustrate the invention and should in no way be considered to limit the scope of the invention.

Example 1 The intervertebral disc (L4-5) is incised in 60 rats following a left-sided facet- tectomy. One group of rats receives infliximab 5 mg/kg intraperitoneally (n=10); one group receives a peptide derived from lactoferrin consisting of the amino acid residues in positions 20-31 of human lactoferrin wherein the amino acid residue in position 24 has been replaced with lysine and the amino acid residue in position 26 has been replaced with alanine, i.e. the peptide with the sequence C-F-Q-W-K-R-A-M-R-K-V-R (corresponding to the peptide of Ex- periment 2 in Example 24 of WO 00/01730) (n=10), one group receives 0.2 ml of 2.5 mg/ml of methotrexate intraperitoneally once a week (n=10), one group receives indomethacin 2 mg/kg/day (n=10), one group receives 10 mg/kg of a monoclonal antibody towards VEGF after surgery and two weeks after surgery (n=10). The remaining 10 rats do not receive any treatment. After three weeks the rats are killed and the incised discs are harvested. Tissue sections of the discs are immunostained with von Willebrand factor and Ulex europaeus antibodies to reveal the presence of blood vessels, and with substance-P, C- flanking peptide of neuropeptide Y and synaptophysin antibodies to detect nerve endings. Using light microscopy the number of blood vessels and nerves is calculated. The number of blood vessels, as well as nerve fibers, is then found to be significantly lower in all five treatment groups when compared to the non-treated group.

Example 2 The tendon of the semimembranosus muscle on the left side in 60 pigs is injected by carrageenan approximately 30 mm from its insertion to the bone. This results in a local inflammation of the tendon. Ten of the pigs receive the lactoferrin derived peptide described in Example 1, ten pigs receive 5 mg of methotrexate at surgery and one week after surgery, ten pigs receive indomethacin 2 mg/kg/day, ten pigs receive 10 mg/kg of a monoclonal antibody towards VEGF after surgery and two weeks after surgery. The remaining ten pigs do not receive any treatment. After 14 days the tendons are harvested and processed for light microscopy. The tendons are immunostained with von Willebrand factor and Ulex europaeus antibodies to reveal the presence of blood vessels, and with substance-P, C-flanking peptide of neuropeptide Y and synaptophysin antibodies to detect nerve endings. It is then found that the number of blood vessels as well as nerve endings is significantly less in all the tendons from the treated groups as compared to the tendons from the ten untreated pigs.

Example 3

Fifteen pigs (each with a body weight of approximately 25 kg) received an intramuscular injection of 20 mg/kg body weight of Ketalar (ketamine 50 mg/ml; Parke-Davis, Morris Plains, New Jersey), an intravenous injection of 20 mg/kg body weight of Hypnodil (methomidate chloride 50 mg/ml; AB Leo, Helsingborg, Sweden), and 0.1 mg/kg body weight of Stresnil (azaperon 2 mg/ml; Janssen Pharmaceutica, Beerse, Belgium). Anesthesia was maintained by additional intravenous injections of 2 mg/kg body weight of Hypnodil and 0.05 mg/kg body weight of Stresnil. The pigs also received an intravenous injection of 0.1 mg/kg of Stesolid Novum (diazepam; Dumex, Helsingborg, Sweden) after surgery. Nucleus pulposus or retroperitoneal fat was harvested from the fifth lumbar disk by a retroperitoneal approach. Approximately 40 mg the nucleus pulposus was placed subcutaneously in 12 pigs and the same amount of retroperitoneal fat was placed in 3 pigs serving as control. Of the 12 pigs which received nucleus pulposus, 3 received treatment with methotrexate in a low dose of 2 ml of 25 mg/ml i.v., 3 received treatment with doxycycline in a does of 100 mg i.v., and 3 received treatment with infliximab in a does 100 mg. The remaining 3 pigs only received an infusion of saline.

After one week the pigs were killed and the transplanted tissues were excised and snap frozen in methylbuturate chilled with liquid nitrogen. Frozen sections were stained for blood vessels using rabbit anti-human von Willebrand factor (Code A0082, Lot 105, Dako, Glostrup, Denmark) and biotinylated goat anti-rabbit Ig (Code E 0432, Lot 029, Dako, Glostrup, Denmark) and visualized using DAB (diaminobenzidin tetrahydrochloride, Sigma- Aldrich, St Louis, MO, USA). The sections were also stained for nerve tissue using mono- clonal mouse anti-human neurofilament protein Clone 2F11 (Code M0762, Lot 089, Dako, Glostrup, Denmark) and biotinylated F(ab')2 fragment of rabbit anti-mouse Ig (Code E 0413, Lot 065, Dako, Glostrup, Denmark) and visualized using DAB.

The data are shown in Table 1 and clearly demonstrate that there was no neovascularization and minimal neoinnervation of the transplanted fat tissue. However, in nucleus pulposus in pigs treated with saline both new vessels and new small nerves were clearly present already one week after transplantation. Treatment with methotrexate reduced the quantity and quality of new vessels but not the number of newly formed nerves. However, treatment with doxycy- cline (metalloproteinase inhibitor) or infliximab (monoclonal antibody to TNF) dramatically reduced the number of both newly formed vessels and nerves. Table 1

' Quality of vessels:

0 = no vessels (+) = small with no lumen

+ = larger vessels with lumen, also longitudinally cut ++ = vessels like in previous control experiments ' Quantity of vessels:

0 = no vessels (+) = only few vessels

+ = some vessels ++ = many vessels +++ = abundant vessels ' Nerve fibers:

0 = no nerve fibers (+) = 1-2 nerve fibers in transplanted tissue

+ = 3-5 nerve fibers in transplanted tissue ++ = > 5 nerve fibers in transplanted tissue References:

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Claims

1. Use of an anti-angiogenetic substance for the production of a pharmaceutical preparation for prevention of neovascularization and/or neoinnervation of intervertebral discs.

2. Use of an anti-angiogenetic substance for the production of a phar- maceutical preparation for prevention of neovascularization and/or neoinnervation of tissue with local inflammation.

3. Use according to claim 1, wherein said neovascularization and/or neoinnervation of intervertebral discs is caused by spinal trauma.

4. Use according to claim 1 or 3 for prevention of chronic low back pain.

5. Use according to claim 1 or 2 for prevention of chronic whiplash associated disorder.

6. Use according to claim 2 for prevention of chronic pain.

7. Use according to claim 2 or 6 for treatment of tendinitis.

8. Use according to any one of the claims 1-7, wherein said anti- angiogenetic substance has an indirect anti-angiogenetic effect by inhibition of an angiogenetic substance.

9. Use according to claim 8, wherein said anti-angiogenetic substance is a substance that inhibits a substance selected from the group consisting of vas- cular endothelial growth factor, vascular P factor, IL-2, 11-6, IL-8, beta- fibroblast growth factor, angiogenin, GM-CSF, TNF, TGF-beta and pros- taglandins.

10. Use according to claim 9 for treatment of a low back pain and/or whiplash associated disorder.

11. Use according to any one of the claims 1-7, wherein said anti- angiogenetic substance has a direct anti-angiogenetic effect.

12. Use according to claim 11, wherein said anti-angiogenetic substance is a TNF inhibitor.

13. Use according to claim 12, wherein said TNF inhibitor is a specific TNF inhibitor.

14. Use according to claim 13, wherein said specific TNF inhibitor is selected from the group consisting of antibodies, soluble cytokine receptors, TNF receptor antagonists and antisense oligonucleotides.

15. Use according to claim 14, wherein said specific TNF inhibitor is selected from the group consisting of the monoclonal antibodies infliximab, CDP-571, D2E7 and CDP-870.

16. Use according to claim 14, wherein said specific TNF inhibitor is se- lected from the group consisting of the soluble cytokine receptors etanercept, lenercept, pegylated TNF receptor type I and TBP-1.

17. Use according to claim 12, wherein said TNF inhibitor is a nonspecific TNF inhibitor.

18. Use according to claim 17, wherein said non-specific TNF inhibitor is lactoferrin or a peptide derivable thereof.

19. Use according to claim 17, wherein said non-specific TNF inhibitor is a binuclear DNA threading transition metal complex with anti-cancer effect.

20. Use according to claim 17, wherein said non-specific TNF inhibitor is TTP.

21. Use according to claim 17, wherein said non-specific TNF inhibitor is a p38 kinase inhibitor.

22. Use according to claim 11, wherein said anti-angiogenetic substance is an eicosanoid synthesis inhibitor.

23. Use according to claim 22, wherein said eicosanoid synthesis inhibi- tor is a specific eicosanoid synthesis inhibitor.

24. Use according to claim 22, wherein said eicosanoid synthesis inhibitor is a non-specific eicosanoid synthesis inhibitor.

25. Use according to claim 24, wherein said non-specific eicosanoid synthesis inhibitor is a non-steroidal anti-inflammatory drug.

26. Use according to claim 22, wherein said eicosanoid synthesis inhibitor is an inhibitor of an eicosanoid synthesis enzyme.

27. Use according to any one of the claims 19-26 for treatment of a low back pain and/or whiplash associated disorder.

28. Use according to any one of the claims 1-7, wherein said anti- angiogenetic substance is a substance that inhibits a neurotrophic factor.

29. Use according to claim 28, wherein said substance that inhibits a neurotrophic factor is a substance that inhibits nerve growth factor.

30. Use according to claim 28, wherein said substance that inhibits a neurotrophic factor is a substance that inhibits brain derived nerve growth fac- tor.

31. Use according to claim 28, wherein said substance that inhibits a neurotrophic factor is a substance that inhibits insulin-like growth factor.

32. Use according to any one of the claims 28-31 for treatment of a low back pain and/or whiplash associated disorder.

33. A method for prevention of neovascularization and/or neoinnervation of intervertebral discs wherein a therapeutically effective amount of an anti-angiogenetic substance is administered to an individual.

34. A method for prevention of neovascularization and/or neoinnervation of tissue with local inflammation wherein a therapeutically effective amount of an anti-angiogenetic substance is administered to an individual.

35. The method according to claim 33, wherein said neovascularization and/or neoinnervation of intervertebral discs is caused by spinal trauma.

36. The method according to claim 33 used for prevention of chronic low back pain.

37. The method according to claim 33 used for prevention of chronic whiplash associated disorder.

38. The method according to claim 34 used for prevention of chronic pain.

39. The method according to claim 34 used for treatment of tendinitis.

40. The method according to any one of the claims 33-39, wherein said anti-angiogenetic substance has an indirect anti-angiogenetic effect by inhibiting an angiogenetic substance.

41. The method according to claim 40, wherein said anti-angiogenetic substance is a substance that inhibits a substance selected from the group con- sisting of vascular endothelial growth factor, vascular P factor, IL-2, 11-6, IL-8, beta- fibroblast growth factor, angiogenin, GM-CSF, TNF, TGF-beta and pros- taglandins.

42. The method according to claim 41, for treatment of low back pain or whiplash associated disorder.

43. The method according to any one of the claims 33-39, wherein said anti-angiogenetic substance has a direct anti-angiogenetic effect.

44. The method according to claim 42, wherein said anti-angiogenetic substance is a TNF inhibitor.

45. The method according to claim 44, wherein said TNF inhibitor is a specific TNF inhibitor.

46. The method according to claim 45, wherein said specific TNF inhibitor is selected from the group consisting of monoclonal antibodies, soluble cytokine receptors, TNF receptor antagonists and antisense oligonucleotides.

47. The method according to claim 46, wherein said specific TNF in- hibitor is selected from the group consisting of the monoclonal antibodies infliximab, CDP-571, D2E7 and CDP-870.

48. The method according to claim 46, wherein said specific TNF inhibitor is selected from the group consisting of the soluble cytokine receptors etanercept, lenercept, pegylated TNF receptor type I and TBP-1.

49. The method according to claim 44, wherein said TNF inhibitor is a non-specific TNF inhibitor.

50. The method according to claim 49, wherein said non-specific TNF inhibitor is lactoferrin or a peptide derivable thereof.

51. The method according to claim 49, wherein said non-specific TNF inhibitor is a binuclear DNA threading transition metal complex with anti- cancer effect.

52. The method according to claim 49, wherein said non-specific TNF inhibitor is TTP.

53. The method according to claim 49, wherein said non-specific TNF inhibitor is a p38 kinase inhibitor.

54. The method according to claim 43, wherein said anti-angiogenetic substance is an eicosanoid synthesis inhibitor.

55. The method according to claim 54, wherein said eicosanoid synthesis inhibitor is a specific eicosanoid synthesis inhibitor

56. The method according to claim 54, wherein said eicosanoid synthesis inhibitor is a non-specific eicosanoid synthesis inhibitor.

57. The method according to claim 56, wherein said non-specific eicosanoid synthesis inhibitor is a non-steroidal anti-inflammatory drug.

58. The method according to claim 54, wherein said eicosanoid synthe- sis inhibitor is an inhibitor of an eicosanoid synthesis enzyme.

59. The method according to any one of the claims 54-58 for treatment of a low back pain or whiplash associated disorder.

60. The method according to any one of the claims 33-39, wherein said anti-angiogenetic substance is a substance that inhibits a neurotrophic factor.

61. The method according to claim 60, wherein said substance that inhibits a neurotrophic factor is a substance that inhibits nerve growth factor.

62. The method according to 60, wherein said substance that inhibits a neurotrophic factor is a substance that inhibits brain derived nerve growth factor.

63. The method according to claim 60, wherein said substance that in- hibits a neurotrophic factor is a substance that inhibits insulin-like growth factor.

64. The method according to any one of the claims 60-63 for treatment of a low back pain or whiplash associated disorder.