Background of the invention Low back pain (LBP) : 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 eco- nomically 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, disequilib- rium, 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 abnor- malities in the majority of patients, the characteristics of dizziness due to whip- lash can be elucidated by means of ElectroNystagmoGraphic (ENG) evalua- tion. 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 com- monly viewed as suspect.

Whiplash injuries can be quite complex and may include a variety of re- lated 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.

Acuteconditions : 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). Vari- ous medical conditions such as intraspinal tumors, rheumatic diseases, infec- tions as well as injuries to muscles or ligaments in the spine, have all be sug- gested to induce low back pain (1). The physician may recognize these condi- tions 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 stabiliza- tion 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 su- perficial 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 sub- stance 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 sug- gested 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 syn- dromes 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 angiogene- sis (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 fi- brosus 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 neoinner- vation of the disk, and the development of discogenic, chronic pain.

There are also pharmacological substances that act as general angio- genesis 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 neovasculariza- tion and neoinnervation of injured discs.

A third possibility would be to use pharmacological substances that spe- cifically 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 im- portant. 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 in- jury has healed. Since healing of the disc may be difficult to determine clini- cally, 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 sub- stance for the production of a pharmaceutical preparation for prevention of ne- ovascularization 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 neovas- cularization 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 in- tervertebral 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 pharmacologi- cal 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 tendini- tis. 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- cadeTM) D2E7, and CDP-870 Polyclonal antibodies Soluble cytokine recep- tors such as: etanercept, lenercept, pegylated TNF re- ceptor type I, and TBP-1 TNF receptor antago- nists 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 com- positions comprising them described in WO 99/15535, and methotrexate Declopramide DPC 333 DWP 205297 DY 9973 Edodekin alfa Flt 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 de- scribed 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 Modi- fied 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 (chinolones) 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 recep- tors 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 En- zyme-inhibitors) Tetracyclines such as: Doxycy- cline, Trovaflox- acin, Lymecy- cline, Oxitetracy- cline, Tetracy- cline, and Mino- cycline Synthetic tetracy- cline derivates (CMT = Chemically Modi- fied 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 syn- thesis inhibitors includ- ing non-steroidal anti- inflammatory drugs, NSAID) such as: Acetylsalicylic acid, Monoclonal antibod- ies, Polyclonal antibod- ies, 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, Genistein, Human tumor inhibitors, IL-12, Irsogladine, Kringle 5 of plasmino- gen, 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 in- hibitor. 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. Accord- ing to an other especially preferred embodiment the substance used is a binu- clear DNA threading transition metal complex with anti-cancer effect. Accord- ing to an other especially preferred embodiment the substance used is a lacto- ferrin derivable peptide. According to an other especially preferred embodi- ment 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 sub- stance used is or TTP.

For the purpose of this disclosure the expression anti-angiogenetic sub- stance relates to all substances, compounds, preparations, drugs, and medica- ments 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-a.

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 ob- tained. The substance or pharmaceutical composition according to the inven- tion 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 accept- able 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 inven- tion may be administered in any efficacious way. The substances or pharma- ceutical 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 admin- istered orally (per os), e. g. in the form of oral preparations, such as pills, syr- ups, 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 3rd 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 3rd month epidurally 1-100 mg Examples of suitable doses for different substances according to the in- vention 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, uglkg body weightlday) 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 mglkg body weightlday) HP-228 i. v. 5-100 10-50 20-40 (all doses given in µg/kg body weight) Ariflo (E) SB 207499 Per os 10-100 30-60 30-45 (all doses given in mglday) KB-R7785 s. c. 100-500 100-300 150-250 (all doses given in mglkg body weightlday) Prinomastat (AG3340) Per os 1-250 5-100 10-50 (all doses given in mg for administration twicedaily) Batimastat Per os 1-250 5-100 10-50 (all doses given in mg for administration twicedaily)

Marimastat Per os 1-250 5-100 10-50 (all doses given in mg for administration twicedaily) CDC-501 Per os 50-1200 200-800 400-600 (all doses given in mglday) CDC-801 Per os 50-1200 200-800 400-600 (all doses given in mglday) 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 adminis- tered 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 inven- tion.

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 anti- bodies 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 indo- methacin 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 un- treated 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 in- jection 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 retrop- eritoneal fat was placed in 3 pigs serving as control. Of the 12 pigs which re- ceived 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 visual- ized 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 visual- ized 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 Quantity of Nerve vessels 1) vessels 2) fibers 3) Fat (control tissue) 0 0 (+) 0 0 0 NP + saline + ++ + ++ ++ (+) ++ ++ (+) NP + methotrexate + + + (+) (+) (+) + (+) + NP + doxycycline ++ + 0 0 0 0 0 0 (+) NP + infliximab (+) 0 (+) 0 0 0 0 0 0 Quality of vessels : 0 = no vessels (+) = small with no lumen + = larger vessels with lumen, also longitudinally cut ++ = vessels like in previous control experiments 2) Quantity of vessels : 0 = no vessels (+) = only few vessels + = some vessels ++ = many vessels +++ = abundant vessels 3) 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

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