JP2012125180A - Lower limb ischemia model animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lower limb ischemia model of monkey, a method for producing the same, and a method for screening a pharmaceutical agent or the like for treating peripheral artery diseases by using the same.SOLUTION: There are provided the lower limb ischemia model of monkey prepared by ligating and/or surgically removing the proximal part to the distal part of a thigh artery and blood vessel branches existing therebetween, the method for producing the same and the method for screening a material having therapeutic action for peripheral artery diseases or therapeutic angiogenesis action, including administration of a candidate material to the lower limb ischemia model.

Description

  The present invention relates to a monkey lower limb ischemia model, a method for producing the model, and a method for screening a drug or the like for treating peripheral arterial disease using the model.

Peripheral arterial disease (PAD) is a collective term for several diseases associated with peripheral arterial blood circulation disorders. They are classified into several grades according to their severity and clinical findings. For example, the Fontaine classification is widely used as a combination of stage and symptoms. In other words, the Fontine classification is classified into degrees I to IV according to the following symptoms.
I) Asymptomatic to cold sensation and color change of the lower limbs,
IIa) Mild lameness,
IIb) moderate to severe lameness,
III) Ischemic resting pain,
IV) Ulcers and gangrene.

  In general, as the severity increases, various complications, poor prognosis, dropout of the lower limbs, and risk of life are caused. In the pharmacotherapy of intermittent claudication classified into Fontane classification IIa and IIb, cilostazol having vasodilatory action and antiplatelet action, naphthidrofuryl having improved muscular metabolism and erythrocyte and platelet aggregation inhibitory action are used in clinical practice. Yes. Moreover, L-carnitine, propionyl-L-carnitine, etc. which have a skeletal muscle metabolism improvement effect are known as a pharmaceutical with clinical usefulness. However, these drug therapies are not expected to be as effective as successful cases of supervised exercise therapy or revascularization. Furthermore, in the treatment of severe limb ischemia classified into Fontane classification III and IV, there is almost no effect by the said drug. For patients with severe limb ischemia, the primary goals of treatment are reducing ischemic pain, healing of ischemic ulcers, prevention of amputation, improving quality of life (QOL), and extending survival. There is no satisfactory drug therapy for all of them. There are reports that treatments using prostanoids that have antiplatelet activity and leukocyte activation inhibitory activity are effective against some events, but only in a limited number of patients (non-patented) Document 1, Non-Patent Document 2). In addition, long-term combined treatment with the antiplatelet drugs aspirin and ticlopidine has been carried out for the purpose of reducing the risk of systemic vascular events, but no reports have been obtained on improving severe limb ischemia.

  Therefore, as a new treatment strategy for severe limb ischemia, research on therapeutic angiogenesis therapy aimed at promoting angiogenesis around the ischemic site and restoring the blood flow of the ischemic tissue has been actively conducted. . In the early 1990s, basic research data was reported that growth factors such as basic fibroblast growth factor (bFGF) and vascular endowmental growth factor (VEGF) promote blood flow recovery in collateral blood vessels, and their clinical application is expected. It was. If it is actually applied to clinical practice, it is expected to improve the patient's QOL and improve the life prognosis. Therefore, it has attracted worldwide attention and many clinical trials have been conducted. Specifically, a method of locally administering vascular growth factor bFGF, VEGF, hepatocyte growth factor (HGF) or the like to an ischemic tissue using a protein or a vector, or secreting various vascular growth factors A method of administering expected endothelial progenitor cells, autologous bone marrow cells, or peripheral blood mononuclear cells (factors attracting cells) to the lower limb ischemic tissue was performed. However, they are contrary to expectation and do not have the effect obtained in large-scale clinical practice or are minor, and have not spread as a definitive treatment method (Non-patent Document 3). The reasons are as follows: 1) The half-life of vascular growth factor is short, so that even if vascular growth factor is administered directly, the effect is insufficient. 2) Even if the dose of vascular growth factor is increased, the effect is enhanced. 3) Administration of vascular growth factor by gene therapy has the risk of side effects such as edema, worsening of vascular disease, development and exacerbation of cancer, and 4) reduction of angiogenic potential of patients And the like.

  Another factor is that non-clinical efficacy studies of these treatments were conducted using a mouse acute lower limb ischemia model. Species differences are too large between mice and humans, and there are large anatomical gaps. Therefore, it has been pointed out that extrapolation to humans is difficult. That is, in the case of mice, ischemic tissue that requires the formation of microvasculature and functional collateral circulation is extremely small, so that ischemia is improved even with a small amount of angiogenic factor. On the other hand, in the case of humans, the blood flow is not sufficiently recovered no matter how many microvascular vessels are born, and it is essential to extend the collateral circulation of blood vessels that are somewhat thick. There has also been a basic study in which a rabbit chronic lower limb ischemia model reproduced in a rabbit, which is a non-rodent middle animal, was surgically produced and VEGF and bFGF proteins were administered intravascularly (Non-patent Document 4). However, in this case as well, even if good results are obtained with model animals, clinical application in humans has not been achieved. In other words, for the development of new therapeutic angiogenesis therapy, the extension of collateral circulation, which is important for clinical therapeutic angiogenesis, can be evaluated using the same measuring equipment as humans such as contrast-enhanced computed tomography (contrast-enhanced CT) Therefore, it is important to conduct pharmacological tests and pharmacokinetic tests using a more appropriate model of lower limb ischemia. Dogs have often been used in safety studies in animals other than rodents and rabbits. However, when conducting research and development of a novel angiogenic factor, dogs are known to have more collateral circulation than other animals, and are inappropriate as an ischemic model. In a model using monkeys, production of a model for evaluating restenosis of a blood vessel after placement of a stent (Non-Patent Document 5) and a model for evaluating a thrombolytic action (Non-Patent Document 6) has been reported. However, there is no model optimized to evaluate the collateral circulation growth-promoting action, which is important for microvascularization and clinical therapeutic angiogenesis, using the same measurement equipment as humans, such as contrast-enhanced CT .

Diabetes Metab Res Rev. 2000; 16 (suppll): S37-S41 Eur J Vasc Endovasc Surg. 2004; 28 (1): 9-23 Circulation. 2003; 107; 1359-1365 Circulation. 1995; 92: 365-371 Gene Therapy (2004) 11: 1273-1282 Arterioscler Thromb Vascular Biol. Vol.16, No.1, January 1996: 34-43

  An object of the present invention is to provide a monkey lower limb ischemia model, a preparation method thereof, and a screening method for drugs and the like for treatment of peripheral arterial disease using the same.

As a result of intensive studies, the present inventors evaluate therapeutic angiogenesis such as microvascular neovascularization or collateral circulation expansion by ligating and / or excising the monkey femoral artery. Thus, the present invention has been completed.
That is, the present invention is as follows.
[1] A model of monkey leg ischemia in which a blood vessel branch existing between and between the proximal part and the distal part of the femoral artery is ligated and / or excised.
[2] The lower limb ischemia model according to [1], wherein the lower limb ischemia is severe lower limb ischemia,
[3] A lower limb ischemia model according to any one of [1] or [2], wherein the monkey is a cynomolgus or rhesus monkey,
[4] A method for producing a monkey lower limb ischemia model comprising ligating and / or excising a blood vessel branch existing between and from the proximal part to the distal part of the femoral artery,
[5] A method for producing a monkey leg ischemia model according to [4], wherein the leg ischemia is severe leg ischemia,
[6] A method for producing a lower limb ischemia model according to any of [4] or [5], wherein the monkey is a cynomolgus monkey or a rhesus monkey, and [7] lower limb ischemia according to [1] to [3] A method for screening a substance having a therapeutic action for peripheral arterial disease or a therapeutic angiogenesis action, comprising administering a candidate substance to a model.

Since the model animal of the present invention is an animal obtained by ligation and / or excision of the femoral artery of a monkey which is the same primate as a human, the anatomical features and pharmacokinetics of the lower limb can be a model closest to humans. . By providing this disease model animal, at the stage of non-clinical trials, using a device such as contrast-enhanced CT similar to that of humans, the neovascularization and collateral circulation extension and maturation by candidate drugs can be accurately evaluated. It becomes possible. In addition, in model animals prepared using rodents and rabbits, ischemic tissue is extremely small, so ischemia can be improved even with microvascularization and clinically insufficient collateral circulation. On the other hand, since the model animal of the present invention has a large thigh volume, a therapeutic effect cannot be found unless sufficient treatment is given (for example, repeated administration for 6 days). Therefore, clinical trials (clinical trials) can be conducted on the basis of easy pharmacological effects in lower limb ischemia models using rodents and rabbits to reduce the risk of ineffectiveness in humans. It becomes possible.
According to the model animal production method of the present invention, it is possible to reliably provide the model animal described above.
According to the evaluation method of the present invention, at the stage of non-clinical trial, using a device such as contrast-enhanced CT similar to that of humans, it is possible to accurately evaluate the formation of microvasculature and the extension and maturation of collateral circulation by candidate drugs Is possible. According to the screening method of the present invention, a candidate substance that improves peripheral arterial disease, particularly severe lower limb ischemia, can be accurately selected.

FIG. 3 is a diagram of arteries and muscles in the monkey leg. It is the result of contrast CT examination. It is a result of a motor ability score. It is a result of an arteriovenous oxygen saturation difference test.

  The present invention is a model animal for evaluation of peripheral arterial disease using monkeys which are physiologically related animal species. The monkey used in the present invention is a monkey raised for a laboratory animal. Specific examples include cynomolgus monkeys (Macaca fascicularis) and rhesus monkeys (Macaca mulatta), preferably cynomolgus monkeys, which are bred for laboratory animals of the genus Cercopithecidae (Macaca). These monkeys can be commercially available (Japan Clare, Japan Institute for Medical Animal Research, Gaoyao Kangda Laboratory Animal Science & Technology, Omori Primate Experimental Animal Breeding Company, Covance, etc.).

  The monkey used in the present invention is preferably an individual of 24 to 38 months old, and more preferably an individual of 32 to 36 months old. If it is less than 24 months of age, the formation of microvessels and the extension and maturation of collateral blood vessels occur easily, which is not preferable for pharmacological studies in view of extrapolation to humans. Moreover, if it is over 38 months of age, it is not preferable because it becomes severe after surgical treatment and the possibility of necrosis or loss of the lower limb tissue increases. The sex of the monkey used in the present invention may be male or female, but male is preferred. The body weight is preferably 2.5 kg to 3.8 kg, and more preferably 3.0 kg to 3.5 kg. In addition, the monkeys used in the present invention are preferably healthy, and are preferably kept and managed properly. Moreover, about the feed to provide, the feed marketed as a monkey feed generally is preferable.

In the monkey lower limb ischemia model in the present invention, the femoral artery is ligated and / or excised by a surgical procedure.
The manufacturing method includes at least the following steps. A diagram of arteries and muscles in the lower extremities of monkeys is shown in FIG.
Step 1) Anesthesia of the monkey Step 2) Incision of either the right thigh or left thigh of the monkey and separation of the femoral artery from the surrounding tissue Step 3) Distal portion from the proximal portion of the femoral artery , And ligating and / or excising the blood vessel branch existing therebetween, and blocking the blood flow of the femoral artery

  In Step 1, when anesthetizing the monkey, either local anesthesia or general anesthesia at the surgical site may be used, and local anesthesia and general anesthesia may be used in combination. In this case, the anesthetic is one or two selected from the group consisting of cocaine, procaine, chloroprocaine, tetracaine, lidocaine, mepivacaine, dibucaine, bupivacaine, ropivacaine, levobupivacaine, xylocaine and their salts as local anesthetics. A combination of more than one species, the group consisting of ketamine, fentanyl, remifentanil, diazepam, flunitrazepam, midazolam, flumazenil, thiopental, thiamylal, pentobarbital, propofol, nitrous oxide, isoflurane, sevoflurane and their salts 1 type or 2 or more types of combinations chosen from these are mentioned.

  Prior to administration of the above anesthetics, as pre-anesthetic drugs, penicillins, cephems, carbapenems, monobactams, penems, β-lactams such as β-lactamase inhibitors, aminoglycosides, macrolides Antibiotics such as ketolide, ketolide, lincomycin, streptogramin, tetracycline, glycopeptide, fosfomycin, chloramphenicol, polypeptide, quinolone, new quinolone, oxazolidinone, sulfa, ST combination Administration of substances or synthetic antibacterial agents; anticholinergics such as atropine or its salts; tranquilizers; sedatives; These anesthetics or pre-anesthetic drugs can be appropriately administered by transdermal, oral, intravenous injection, intramuscular injection or the like.

  In Step 2, when the femoral region is incised and the femoral artery is separated from the surrounding tissue, the skin and muscle tissue at the site may be incised with a scalpel or an electric knife.

In step 3, when the distal part is ligated from the proximal part of the femoral artery, a silicone band can be placed around the origin of the femoral artery and the vicinity of the saphenous artery before ligation.
In the present invention, the proximal part of the femoral artery means from the origin of the femoral artery (somatic ligament) to 1.0 cm upstream, and the distal part of the femoral artery means the distal end of the femoral artery. That is, it means from the origin of the saphenous artery to the downstream side of 3.0 cm.
At the time of ligation, it is preferable to ligate the branch vessel first, and finally ligate the distal part of the femoral artery and the proximal part of the femoral artery. Branched vessels include at least the deep femoral artery, the popliteal artery, and the uppermost knee artery. In addition, depending on the individual, there may be other branch blood vessels that are not anatomically described, and these are included. For ligation, 4-0 blade silk, 6-0 blade silk, 4-0 nylon, 6-0 nylon, No. 2 silk thread, No. 1 silk thread and the like can be used.

  In the ligation of the distal part of the femoral artery, it is preferable to ligate any part up to the distal side (downstream) 3.0 cm from the origin of the saphenous artery, and ligate any part up to 1.5 cm. More preferably.

  The ligation of the proximal part of the femoral artery may be a ligation of a blood vessel upstream from the origin of the femoral artery, that is, an arbitrary part located 1.0 cm upstream from the ligament ligament by pulling the femoral artery downstream. Preferably, it is more preferable to ligate any part located up to 0.8 cm.

  After ligating all necessary blood vessels, the ligated blood vessels can be cut and removed (removed). When cutting a blood vessel, in order to reduce the amount of bleeding, it is preferable to ligate two places 0.1 cm to 0.5 cm upstream and downstream of the cutting site and cut the center. In addition, when bleeding does not stop, it is preferable to stop bleeding appropriately.

  After the blood flow of the femoral artery is blocked by the above process, the treatment site is washed with physiological saline for injection, and it is confirmed that there is no bleeding, and then the skin is sutured to finish the treatment. At that time, it is preferable not to sew the incised muscle tissue because it affects the general state score.

  In addition, after the above-mentioned treatment, for prevention of infection with bacteria and viruses, penicillins, cephems, carbapenems, monobactams, penems, β-lactams such as β-lactamase inhibitors, aminoglycosides, Macrolide, ketolide, lincomycin, streptogramin, tetracycline, glycopeptide, fosfomycin, chloramphenicol, polypeptide, quinolone, new quinolone, oxazolidinone, sulfa, ST mixture, etc. Antibiotics or synthetic antimicrobials can be administered. These antibiotics or synthetic antibacterial agents can be appropriately administered by transdermal, oral, intravenous injection, intramuscular injection or the like.

  The monkey lower limb ischemia model of the present invention thus prepared can evaluate the degree of ischemia by the following method.

Evaluation Method 1 Contrast CT Examination By taking and analyzing two-dimensional and / or three-dimensional images of microvessels and collateral blood vessels by contrast CT, the degree of extension of collateral blood vessels can be evaluated. In some cases, the extent of collateral circulation can be evaluated by photographing and analyzing the area and volume of the semi-membranous muscle by contrast CT. The contrast agent used for contrast CT is not particularly limited, but examples include iohexol injection solution, iopamidol injection solution, ioversol kit, iomeprol injection solution, iopromide injection solution, ioxirane injection solution, and oxaglucic acid injection solution. It is done. Commercially available products may be used, such as Omnipark 240 (Daiichi Sankyo), Omnipark 300 (Daiichi Sankyo), Omnipark 350 (Daiichi Sankyo), Iopamilon 300 (Bayer Pharmaceutical), Iopamilon 370 (Bayer) Pharmaceutical), Optiray 320 (Covidien Japan), Iomeron 300 (Blackco Eisai), Iomeron 350 (Blackco Eisai), ProScope 300 (Mitsubishi Tanabe Pharma), ProScope 370 (Mitsubishi Tanabe Pharma), Imagineil 300 (Gelve Japan) ), Imaginil 350 (Gerbe Japan), Hexabrix 320 (Gerbe Japan) and the like. The method of administering the contrast agent is not particularly limited, but 5 mL is administered from the brachial vein of the model animal in 5 seconds using a 23-gauge winged needle and a 5 mL syringe with luer lock (Terumo). Since it has been confirmed that the venous plexus shifts to the arterial plexus 3 seconds after administration, imaging is started at that timing. However, since the transition time to the arterial plexus varies depending on the weight of the animal and the like, it is preferable to carry out preliminary studies in advance. The contrast CT apparatus used in the present invention is preferably used for human examination. For example, Aquilion ^™ CX Edition (Toshiba Medical Systems), Aquilion ^™ 32-row system (Toshiba Medical Systems), Aquilion ^™ 16-row system (Toshiba) Medical Systems), Aquilion ^™ LB system (Toshiba Medical Systems), SOMATOM Spirit (Siemens Japan), SOMATOM Emotion 6-Slice configuration (Siemens Japan), SOMATOM Emotion 16-Slice configuration M Siemens Japan), SOMATOM Defi ition AS (Siemens Japan), SOMATOM Definition AS + (Siemens Japan), Brilliance iCT (Phillips Electronics Japan), Brilliance iCT SP (Phillips Electronics Japan), Brilliance CT16 (Philips Electronics Japan ce) Etc.

Evaluation method 2 Oxygen saturation Using a device such as a pulse oximeter, red light and infrared light are applied to the big toe of a model animal, and the ratio of hemoglobin is measured from the intensity of the light passing through to evaluate the degree of ischemia. be able to. The pulse oximeter used in the present invention is not particularly limited, and examples thereof include OxiHeart OX-700 (Nippon Seimitsu Sokki Co., Ltd.), Parxy (SND Co., Ltd.) and the like.

Evaluation Method 3 Blood Flow Measurement Since blood flow is reduced by lower limb ischemia, blood flow in the lower limb is directly measured using a laser-Doppler blood flow meter or electromagnetic flow meter, or indirectly by microsphere method. The degree of ischemia can be evaluated by measuring the blood flow in the lower limbs.
These blood flow meters are not particularly limited, and commercially available products can be used. When measuring blood flow by the microsphere method, the microspheres may be administered to the left ventricle of the model animal, and the microspheres trapped in tissues or microvessels downstream from the treatment site may be quantified. The microspheres to be administered may be those having a diameter of 2 μm to 25 μm, and preferably 10 μm to 15 μm. The microspheres used can be radiolabeled microspheres or fluorescently labeled microspheres.

Evaluation Method 4 Arterial-venous oxygen difference test The treadmill exercise test in patients with lower limb ischemia is applied to an animal model. After applying electrical stimulation to the skeletal muscle and forcibly applying exercise load, the oxygen concentration in the arteries and veins is measured, and the degree of ischemia can be evaluated by the difference. Specifically, the following method is desirable.

  For example, it is performed under anesthesia at 5 and 9 weeks after surgically ligating and / or excising part or all of the right femoral artery. An 18 gauge surf flow indwelling needle or the like is placed in the right femoral vein and the left femoral artery. For exercise load, use an electrical stimulator (Nihon Kohden, etc.), pierce a right gauge muscle, rectus femoris muscle, right medial vastus muscle or right hemigastric muscle and a right gastrocnemius muscle with a 21 gauge needle etc. And The exercise load voltage is determined visually so that a sufficient exercise load is applied. Arterial blood and venous blood are collected from the indwelling needle before the start of exercise load and 15 minutes and 30 minutes after the start of exercise load, and each blood oxygen saturation is measured to calculate an arteriovenous oxygen range. If severe lower limb ischemia occurs, the difference between the arteriovenous oxygen range before exercise and the arteriovenous oxygen range after exercise is large. If the functional collateral circulation is sufficiently developed, the difference is small .

Evaluation method 5 Evaluation of exercise ability Since the exercise ability of an animal is reduced by lower limb ischemia, the degree of ischemia can be evaluated by scoring the following evaluation items visually.
Assess the lower limb paralysis and posture / behavior using the 6-point score table as follows. As the degree of lower limb ischemia becomes severe, the motor performance score increases, and the total score of lower limb paralysis and posture / behavior is higher than 5 in Fontane classification III to IV. If the total score is 3 to 5, it is determined as Fontane classification I degree to II degree.
Lower limb paralysis
5 = Do not use the ischemic foot at all
4 = May use the ischemic foot, but does not have power
3 = use the ischemic foot, but can't grab the cage,
2 = I often use the ischemic foot, but I can't grab the cage,
1 = Use the ischemic foot relatively freely,
0 = use the ischemic leg normally.
Posture and action 5: = sitting posture,
4 = walk around from sitting to sitting,
3 = sometimes climbing into the cage,
2 = walk around a lot,
1 = Climb well,
0 = act normally

Evaluation method 6 Pathological evaluation The body weight is measured before dissection. The animal is pre-anaesthetized with ketamine 15 mg / kg by intramuscular injection or the like, the animal is placed in the dorsal position, exsanguinated from the brachial artery, etc., and euthanized. After exsanguination, the left and right hemiplegic muscles are removed, weighed, and corrected for body weight. The other side is the normal control and the weight ratio is the data. Ischemia causes atrophy and necrosis of the left and right hemiplegic muscles, so if weight is reduced and functional collateral circulation is sufficiently developed, it is expected to be comparable to normal controls on the opposite side. The
In addition, the left and right hemiplegic muscles that have been removed can be fixed with 10% neutral buffered formalin or 4% paraformaldehyde after weight measurement. Alternatively, it can be frozen with a frozen tissue embedding agent (OCT compound) or physiological saline to prepare a frozen section.

Paraffin sections of fixed semi-membranous muscle were prepared to obtain pathological findings, and immunohistochemically using anti-CD31 antibody, anti-von Willebrand factor (vWF) antibody, anti-alpha smooth muscle cell antibody, etc. By staining the microvasculature and the collateral circulation, the degree of microvascular neovascularization and collateral circulation extension per unit area can be quantitatively evaluated. For example, quantitative evaluation can be performed by randomly selecting 1 field of view of 1 mm ² and counting the number of positive blood vessels.

Evaluation Method 7 Temperature Evaluation of the Plantar Part Using Thermography The degree of ischemia can be evaluated by evaluating the temperature difference between the right and left plantar parts using thermography.

Evaluation method 8 Outer surface findings If an ischemic ulcer occurs, take a photograph. Record site, depth and shape of necrosis. The size of the ulcer is evaluated by major axis × minor axis. The ulcer depth is scored according to the following criteria.
1 = superficial defect;
2 = reach subcutaneous tissue;
3 = tendon or bone exposure;
4 = tendon or bone necrosis.

In addition, a therapeutic drug for peripheral arterial disease can be screened using the model animal of the present invention. That is, the present invention can also provide a method for screening a drug, gene, protein or cell for the treatment of peripheral arterial disease, and the method is achieved by including the following steps.
Step 1: A step of administering a candidate substance to a model animal of the present invention.
Step 2, the degree of ischemia in the candidate substance administration group is evaluated by one or more selected from the group consisting of the evaluation methods 1 to 8, and the degree of ischemia before and after administration of the candidate substance, or the control group And comparing the degree of ischemia.

  The candidate substance may be any known substance or novel substance, for example, a candidate drug, gene, protein for the treatment of peripheral arterial disease having a microvascular neovascularization effect or a collateral circulation extension effect. (Such as HGF, VEGF, aFGF or bFGF), physiologically active peptides (such as adrenomedullin, AG-30 or apelin) or cells (stem cells, endogenous producer cell (EPC), Induced pluripotent stem cells, iPS cells) including artificially synthesized peptides Autologous peripheral blood cells, autologous bone marrow cells, etc.).

The peripheral arteries in the present invention mean arteries other than the heart and coronary arteries. The internal thoracic artery, carotid artery, pulmonary artery, aorta, brachiocephalic artery, common carotid artery, arteriole, abdominal aorta, superior phrenic artery Inferior phrenic artery, lumbar artery, midline sacral artery, celiac artery, superior mesenteric artery, inferior mesenteric artery, gastric artery, gastric coronary artery, gastric omental artery, gastroduodenal artery, jejunal artery, ileal artery, Colon artery, middle colon artery, ileocolon artery, sigmoid colon artery, appendix artery, superior pancreaticoduodenal artery, inferior pancreaticoduodenal artery, marginal artery, straight artery, short gastric artery, splenic artery, adrenal artery, renal artery, carotid artery Femoral artery, inferior knee artery, inferior abdominal artery, common iliac artery, superficial iliac artery, iliac arteries, superior bladder artery, inferior bladder artery, uterine artery, coronary artery, superior rectal artery, midrectal artery, inferior rectal artery, umbilical cord Artery, superficial iliac artery, deep iliac rotator artery, vulva artery, internal pudendal artery, lateral sacral artery Upper gluteal artery, lower gluteal artery, closed artery, external iliac artery, internal iliac artery, intrinsic hepatic artery, common hepatic artery, gallbladder artery, interlobar artery, interlobular artery, imported arteriole, export arteriole, straight arteriole , Testicular artery, vas deferens, superficial penile dorsal artery, deep penile dorsal artery, penile deep artery, spiral artery, anterior scrotal artery, posterior scrotal artery, ovarian artery, superior adrenal artery, middle adrenal artery, inferior adrenal artery, Subclavian artery, axillary artery, superficial carotid artery, transverse carotid artery, dorsal scapular artery, thyroid carotid artery, subscapular artery, thoracic arterial artery, brachial artery, brachial artery, brachial artery, upper thoracic artery, Lateral thoracic artery, common iliac artery, anterior interosseous artery, radial artery, ulnar artery, dorsal digital artery, thumb main artery, common palmar artery, superior collateral artery, inferior collateral artery , Superior thyroid artery, dorsal metacarpal artery, deep femoral artery, popliteal artery, femoral artery, dorsal artery, medial plantar artery, lateral plantar artery, radial artery, calf Pulse, anterior tibial artery, posterior tibial artery, descending knee artery, medial superior knee artery, lateral superior knee artery, medial inferior knee artery, lateral inferior knee artery, middle knee artery, penetrating artery, arcuate artery, medial rotational artery, and Including the lateral circumflex artery.
In addition, peripheral arterial diseases in the present invention include, for example, abnormal vascularization of peripheral arteries, vascular injury, acute arterial occlusion, obstructive arteriosclerosis, carotid artery disease (carotid artery stenosis), vertebral artery disease (cranium) External vertebral artery stenosis), abdominal visceral artery disease (acute mesenteric artery occlusion, chronic mesenteric artery occlusion, celiac artery compression syndrome, mesenteric insufficiency), renal artery disease (renal artery stenosis) ), Takayasu arteritis, Behcet's disease, Buerger's disease, collagen disease (nodular polyarteritis, allergic granulomatous vasculitis, Wegener's granulomatosis, SLE, rheumatoid arthritis), diabetic foot disease, functional disease (limbs) Crimson pain disease, Raynaud phenomenon, causalgia), thoracic outlet syndrome, subclavian artery steal syndrome, popliteal epicardial cyst, popliteal artery capture syndrome, blue toe syndrome, and residual sciatic artery disease, Acute limb ischemia caused by them each time, intermittent claudication, and critical limb ischemia (critical limb ischemia).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Example 1 Production of a model animal A commercially available male cynomolgus monkey (Gaoyao Kangda Laboratory Animal Science & Technology) was used as a model animal.

  Confirm that there are no abnormalities in bacterial tests (Salmonella, Shigella, Mycobacterium tuberculosis), virus antibody tests (B-VIRUS), endoparasite tests (roundworms, helminths) and ectoparasite tests (microscopic tick and flea tests) Then, a male cynomolgus monkey 36 months old (body weight 3.0-3.5 kg) that had been quarantined for 2 weeks or more was intramuscularly injected with a penicillin preparation for injection (400,000 units) about 30 minutes before the operation. . Thereafter, about 5 minutes before the operation, ketamine hydrochloride (15 mg / kg) was intramuscularly injected and anesthetized. The right thigh was shaved and then disinfected using isodine. The right thigh skin was incised with a scalpel so as not to bleed, and the right femoral artery was detached. When bleeding was observed when the skin and subcutaneous fat were incised, hemostasis was performed by pressing the blood or coagulating with an electric knife. Silicon bands (vesseloops, Maximim Medical) were respectively placed around the origin of the femoral artery and the saphenous artery.

  Next, 0.5 cm to 0.7 cm of the deep femoral artery was peeled off from the origin of the deep femoral artery, and both ends thereof were ligated with 4-0 blade silk. Further, the branch vessels including the popliteal artery and the uppermost knee artery were peeled from 0.2 cm to 0.5 cm from their origins, and both ends thereof were ligated with 4-0 blade silk.

  Finally, the distal femoral artery and proximal femoral artery were ligated. As for the ligation of the distal part of the femoral artery, the origin of the saphenous artery and the distal side (downstream) 0.7 cm to 1.0 cm from the origin of the saphenous artery were ligated. The ligation of the proximal part of the femoral artery is the origin of the femoral artery and the part located upstream from the femoral artery origin, i.e., pulling the femoral artery downstream and 0.5 cm upstream from the ligament ligament The part located at 0.7 cm was ligated.

  For each blood vessel, the center of the portion sandwiched between the two ligations was cut, and the cut blood vessel was removed so that there was no bleeding. The treatment site was washed with a physiological saline solution, and after confirming that there was no bleeding, the skin was sutured and the treatment was terminated. Following treatment, 400,000 units of penicillin for injection were injected intramuscularly for 3 days.

Example 2 Evaluation of ischemia of model animal The following evaluation was performed on a model animal having the right hind limb obtained in Example 1 as an ischemic limb.
Example 2-1 Scoring of motor ability Scoring of motor ability was performed 21 days after the operation. As a result, the motility score was 0 in normal animals, whereas the motility score deteriorated to 6.2 ± 0.5 in model animals. Therefore, model animals corresponding to Fontine classification III to IV were obtained.
Example 2-2 Evaluation by contrast-enhanced CT A contrast-enhanced CT examination was performed 35 days after the operation, and the degree of ischemia was observed.
Measurements were taken with 16-row helical CT (Siemens), CT images of the abdomen and lower limbs before and after contrast imaging. For evaluation of these CT examinations, 3D images of blood vessels were created using image analysis software (Osirix for Mac) based on the obtained CT images, and the number of blood vessels and the value of CT values were calculated from the two-dimensional images. When imaging with contrast-enhanced CT was performed, it was observed that there was no blood flow in the femoral artery.
Example 2-3 Arteriovenous Oxygen Difference Test Further, an arteriovenous oxygen difference after exercise was measured.
An 18-gauge Surfflow indwelling needle was inserted as far as possible into the right femoral vein and left femoral artery under ketamine anesthesia at 5 weeks after surgery for all animals. For the exercise load, an electrical stimulation device (Nihon Kohden) was used, and a 21 gauge needle was inserted into the right thin muscle and the right gastrocnemius muscle to form an electrode. Arterial blood and venous blood are collected from the indwelling needle before the start of exercise load, 15 minutes after the start of exercise load, and 30 minutes after the start of exercise load, and the blood oxygen saturation is measured using i-Stat analyzer 300F (Fulso Pharmaceutical). It was measured.
The arterial-venous oxygen difference was calculated as follows.
Arterial oxygen saturation after loading (%)-venous oxygen saturation after loading (%).
As a result, while it was 25% before exercise load, it increased to 50% after 15 minutes of exercise load and 55% after 30 minutes of exercise load.
From the above results, it was confirmed that the obtained model animal exhibited sufficient lower limb ischemia.

Example 3 Drug Screening Pitavastatin PLGA nanoparticles were used as candidate drugs. One week after the model was prepared, the candidate drug was administered to the semi-membranous muscle of the model animal obtained in Example 1 so as to give 4 mg / kg of pitavastatin per body weight. Randomly divided into a solvent control group (saline), a single administration group of the drug, a continuous administration group for 3 days, and a continuous administration group for 6 days, and the degree of ischemia of the lower limbs in each group was compared. The candidate drug was administered by intramuscular injection at the ischemic site. In view of clinical application, a surgical tape marked in a lattice shape was stretched so that the administration sites on the thigh did not overlap, and the administration sites were marked for each administration.

  Here, pitavastatin PLGA nanoparticles used as candidate drugs were produced according to the method described in Examples i) to iii) of WO2008 / 026702. Pitavastatin PLGA nanoparticles are known to have microvascular neovascularization and collateral circulation extension in mice and rabbits (Arterioscler Thromb Vasc Biol. 2009 Jun; 29 (6): 796-801, J Vasc Surg. 2010 Aug; 52 (2): 412-20.).

  Pitavastatin PLGA nanoparticles are disclosed in, for example, Journal of Powder Engineering 42 (11), 765-772 (2005), JP 2003-275281 A, JP 2004-262810 A, JP 2006-321863 A, and the like. It can also be produced by referring to the method described.

Example 3-1 Contrast CT Examination After Drug Administration All animals were subjected to 5 weeks and 9 weeks after surgery (4 and 8 weeks after drug administration). Measurements were taken with 16-row helical CT (Siemens), CT images of the abdomen and lower limbs before and after contrast imaging. In the evaluation of these CT examinations, 3D images of blood vessels were created using image analysis software (Osirix for Mac) based on the obtained CT images, and the degree of therapeutic angiogenesis was compared using two-dimensional images. The measurement item was the number of collateral circulations from the heel portion on each side to the knee. The results are shown in FIG. There was no statistically significant difference in the single administration group at 8 weeks after administration compared to the solvent control group, whereas in the 3 day continuous administration group and the 6 day continuous administration group, the number of collateral circulations An increase was observed (Figure 2). Therefore, extension of the collateral circulation was confirmed by contrast-enhanced CT examination similar to that of humans.

Example 3-2 Motor Ability Score It was carried out 14 days after drug administration for all animals. The results are shown in FIG. Compared to the solvent control group, there was no statistically significant difference between the single administration group and the 3 day continuous administration group. However, improvement of the motor performance score was observed in the 6-day continuous administration group (FIG. 3).

Example 3-3 Arteriovenous Oxygen Difference For all animals, an 18-gauge Surfflow indwelling needle was inserted as far as possible into the right femoral vein and left femoral artery under ketamine anesthesia at 5 weeks after surgery. For the exercise load, an electrical stimulation device (Nihon Kohden) was used, and a 21 gauge needle was inserted into the right thin muscle and the right gastrocnemius muscle to form an electrode. Arterial blood and venous blood are collected from the indwelling needle before the start of exercise load, 15 minutes after the start of exercise load, and 30 minutes after the start of exercise load, and the blood oxygen saturation is measured using i-Stat analyzer 300F (Fulso Pharmaceutical). It was measured. The results are shown in FIG. In the solvent control group, the single administration group, and the 3 day continuous administration group, there was a difference in the arteriovenous oxygen difference before and after exercise. On the other hand, in the 6-day continuous administration group, the difference in the arteriovenous oxygen difference after 15 minutes and 30 minutes of exercise load was reduced. This indicates that sufficient blood is delivered to the ischemic tissue due to the formation of functional microvessels and the development of mature collateral circulation.
From the above results, it was shown that this screening method can be used for drug evaluation for monkey leg ischemia model.

  The animal model of the present invention can be used for drug evaluation of drugs, genes, peptides or cells having angiogenesis and collateral circulation growth promoting action.

Claims (7)

  A monkey lower limb ischemia model in which vascular branches existing between and from the proximal femoral artery to the distal femoral artery are ligated and / or excised.   The monkey leg ischemia model according to claim 1, wherein the leg ischemia is severe leg ischemia.   The monkey lower limb ischemia model according to claim 1, wherein the monkey is a cynomolgus monkey or a rhesus monkey.   A method for producing a monkey lower limb ischemia model, comprising ligating and / or excising a blood vessel branch existing between and between a proximal part and a distal part of a femoral artery.   The method for producing a monkey leg ischemia model according to claim 4, wherein the leg ischemia is severe leg ischemia.   The method for producing a monkey lower limb ischemia model according to any one of claims 4 or 5, wherein the monkey is a cynomolgus monkey or a rhesus monkey.   A method for screening a substance having a therapeutic effect on peripheral arterial disease or a therapeutic angiogenesis action, comprising administering a candidate substance to the lower limb ischemia model according to claim 1.