RU2633487C2 - Method for treatment of lower limbs ischemic diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method includes intramuscular administration of a suspension of autologous peripheral blood stem cells (PBSC) into several points of the muscle mass in the ischemic zone. Three to four days after PBSC administration, vascular endothelial growth factor (VEGF) is introduced into the same muscular mass, depth, place of injection in the muscle mass and the fact of PBSC and VEGF spread is determined and fixed by ultrasound control.

EFFECT: use of the invention makes it possible to reduce the number of complications with introduction of peripheral blood stem cells.

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Description

The inventive method relates to medicine, namely to therapeutic treatment of ischemic disease of the lower extremities. It can be used in clinics and centers of cardiovascular surgery.

Coronary disease of the lower extremities is associated with a sharp decrease in blood flow as a result of occlusion of arteries and arterioles. The frequency of critical lower limb ischemia, indicating complete decompensation of regional blood circulation, is 500-1000 patients per 1 million population per year [Savelyev B.C., Koshkin V.M. Critical lower limb ischemia. - M .: Medicine, 1997. - 40 p.]. Progressive ischemia of the peripheral arteries of the extremities is manifested by pain during walking (intermittent claudication), at rest, trophic tissue changes, which leads to disability of the patient and high mortality among patients in this category.

The most effective treatment for this category of patients today is vascular reconstruction - an operation aimed at restoring arterial blood flow in the affected limb [Vascular surgery. National leadership. Moscow, GEOTAR-Media Publishing Group, 2014, p. 139-140].

However, in a number of patients, performing reconstructive interventions on the arteries is either technically impossible (for example, with distal forms of arterial damage), or is associated with too high operational risk due to the presence of a pronounced concomitant pathology. Therefore, adequate surgical revascularization of the arterial bed of the lower extremities is possible only in 37-58% of patients [SV. Lebedev, A.V. Karasev, V.V. Kungurtsev et al. // Vestnik RAMS. - 2013. - No. 3. - S. 1-44]. In other cases, you have to resort to complex conservative therapy, but standard conservative therapy is not effective enough. In the short term from the start of treatment, only half of patients have a positive result, and a third of patients are candidates for amputation, since the main problem is not solved - the lack of adequate blood supply in the distal parts of the affected limb.

A more effective method of treating chronic lower limb ischemia is to stimulate blood circulation in the distal limb using cell and gene technologies due to the development of collaterals [E. Tateishi-Yuyama, N. Matsubara, T. Murohara et al. // Lancet. - 2002. - Vol. 360. - P. 427-435; Shoikhet Y.N., Khoreev N.G. Cellular technologies in the treatment of peripheral arterial diseases // KTTI. - 2011. - No. 3. - S. 15-23; A. Kawamura, H. Horie, Isuda et al. // J. Artif. Organs. - 2006. - Vol. 9. - P. 226-233]. The introduction of autologous stem cells leads to stimulation of collateral blood flow and an overall improvement in blood circulation in the ischemic limb.

A known method of treating terminal limb ischemia [RU 2369399, IPC A61K 35/28, 2009], according to which (in an experiment on rats), the mononuclear fraction of syngenic bone marrow was introduced in the form of a suspension in a 0.9% aqueous solution of sodium chloride in two equal portions: half was introduced intraarterially into the affected artery, half - intramuscularly into the ischemic zone with 10 injections. It is shown that the combined method of administration, in contrast to purely intramuscular and purely intra-arterial, promotes faster and more pronounced restoration of collateral circulation and microcirculation.

However, the intra-arterial route of administration has several drawbacks: puncture of the femoral artery with a modified wall is technically difficult, there is a risk of its post-traumatic occlusion, as well as dispersion of the cellular material along the collaterals and failure to reach the ischemic zone.

A method for treating lower limb ischemia is also known [RU 2546007, IPC A61K 35/28, 2015], according to which a suspension of mononuclear autologous bone marrow cells was injected via a microcatheter directly into the ischemic zone immediately after angioplasty of peripheral arterial stenosis. It is indicated that, if angioplasty is not possible for occlusion of the leg artery on the affected lower limb, the microcatheter was inserted into the distal end of the passable artery or its terminal branches, microperforation of the artery wall and the suspension of autologous cells were performed. According to the authors, the method ensures the delivery of monocytes, as probable inducers of arteriogenesis, directly to the ischemic focus, minimizes the risk of transplanted cell death in the ischemic zone and duplicates the production of endotheliocyte growth factor due to the inclusion of pericytes, which provides the necessary proliferative potential of endotheliocytes.

The implementation of this method is associated with the risk of thrombosis of the microvasculature by cellular aggregates of the affected area, and also requires the implementation of expensive equipment.

In the experiment, it was shown that the safety of stem cells obtained from adipose tissue after their introduction into the calf muscles of mice and rats was 50-70% after 1 day and 5-10% after 1 week [Autologichnye stvolovye kletki. Eksperimental΄nye issledovaniya i perspektivy klinicheskogo primeneniya. Rukovodstvo dlya vrachei. Pod red. V.A. Tkachuka. M .: "Litterra". 2009. 448 s.].

The closest in technical essence and the achieved effect to the claimed method is a method of intramuscular injection of a suspension of autologous peripheral blood stem cells at several points in the ischemic zone, followed by the introduction of vascular endothelial growth factor (VEGF) in the same zone [P.P. Huang, S.Z. Li, M.Z. Ha et al. // Thromb. Haemost. - 2004. - Vol. 791. - P. 606-609]. The specified method allows to achieve quite positive results in the treatment of coronary disease, namely the stimulation of collateral blood flow and reduce the risk of large amputations. However, with a puncture, a needle may enter the blood vessel, which causes the formation of a hematoma, as well as the ingress of a therapeutic agent into epiphascial tissues, which reduces the angiotropic effect of stem cell transplantation and causes an additional pain syndrome.

The result, which is achieved by the claimed method, is to achieve a combined effect and to prevent complications that are possible during the implementation of stem cell transplantation.

This result is achieved by the fact that in a method of treating ischemic disease of the lower extremities, including intramuscular injection of a suspension of autologous peripheral blood stem cells (SCPC) at several points in the muscle array in the ischemic zone, vascular endothelial growth factor (VEGF) is introduced 3-4 days after the administration of SCPC ) into the same muscle mass, depth, place of introduction in the muscle mass and the fact of the spread of SCPC and VEGF in it is determined and recorded by ultrasound control.

The method is as follows.

To obtain autologous patient peripheral blood stem cells (SCPC), they are mobilized using a granulocyte colony-stimulating factor, such as filgrastim, which is administered to the patient subcutaneously for 3 consecutive days at a dose of 10 μg per kilogram of patient body weight.

The patient’s hematological response to stimulation is assessed by hemogram indices and the content of CD34 + cells according to flow cytometry. The study is carried out on a flowing laser cytofluorimeter "Cytomics FC 500" ("BeclmianCoulter", CIIIA).

For explantation of mobilized hematopoietic stem cells from the patient’s peripheral blood, a leukocytapheresis procedure is performed. It is carried out on the 4th day from the start of filgrastim administration on a Haemonetics® MCS® + LN 900-220Е apparatus with standard parameters for collection via venous access (cubital vein). Testing of hematopoietic CD4 + stem cells in leukocytapheresis cell concentrate is performed using flow laser cytofluorimetry.

The implantation of the obtained peripheral blood stem cells to a patient with chronic lower limb ischemia is as follows.

Premedication is preliminarily performed - 1.0 ml of ketonal is intramuscularly injected. The procedure is carried out in the operating room in compliance with the requirements of asepsis and antiseptics. The patient’s position on the abdomen, with a roller placed under the ankle joint. The projection of the small saphenous vein and 30 puncture points of the gastrocnemius muscle are marked for the introduction of a CD4 + stem cell suspension. The introduction of suspension of SCPC is carried out intramuscularly in the calf muscles of the problem limb. The procedure is performed by injection of 1 ml of a cell suspension, under the control of an ultrasound scan (ultrasound, Aloka 1700, MS program, linear sensor 7.5 MHz). The distance between the punctures and their depth is determined under the supervision of ultrasound. G29 injection needles are used without laser echo amplification. The suspension of SCPC is introduced strictly subfascially, to the depth of administration (from 10 mm to 15 mm), which is controlled by ultrasound for uniform distribution of the SCPC in muscle tissue. The average dose to the patient is 2,337 × 10 ⁷ / l of CD34 + cells.

Taking into account the data of experimental studies, which showed that the safety of implanted stem cells in tissues by day 5 after administration is only 5-10% and, consequently, the level of endothelial vascular growth factor (VEGF) in the ischemic zone decreases sharply, to prolong the angiotrophic effect after 3 -4 days after cell surgery shows the introduction of the drug VEGF "Neovasculgen" at a dose of 1.2 mg.

In a clean dressing room after treatment with an antiseptic, the drug is injected from 5-6 injections into the calf muscle using the method of cell implantation.

The method is further illustrated by examples, but not limited to.

Example 1

Patient I., 75 years old, has been ill since 2010 with peripheral arterial arteriosclerosis obliterans with progression of chronic lower limb ischemia (HINK). In 2012, stenting of the iliac arteries was performed, followed by endarterioectomy and femoral-popliteal bypass surgery of the left lower limb. In 2014, the deterioration of the left lower limb, night pain, painless walking distance (DBH) was 100 m. A duplex study revealed occlusion of the superficial femoral and popliteal arteries, and a decrease in collateral blood flow in the distal vessels. Conservative therapy with the use of angiotropic drugs was ineffective. In connection with the progression of HINC, the patient is offered treatment with the integrated use of gene-cell therapy.

After signing the informed consent for this method of treatment, the patient was mobilized with stem cells using granulocyte colony-stimulating factor (GCSF) filgrastim at the rate of 10 μg / kg subcutaneously for 3 consecutive days. On the 4th day, a method of leukopheresis obtained a concentrate of autologous stem cells from peripheral blood in a volume of 40 ml and, under operating conditions, transplantation under ultrasound control into the calf muscle area from 30 points in a volume of 35-40 ml was performed. In the field of the surgical field, ultrasound monitoring is used with the addition of a 7.5 MHz linear sensor of the Aloka 1700 apparatus, the MS program visualizing the underlying soft tissues, including muscle arrays with the determination of their structure, fascial membranes, epiphascial tissues and blood vessels. Puncture needles are traced as echo-negative artifacts controlled by operator movements; the administration of drugs (SCPC and the preparation of endothelial growth factor) is visualized as a cloud-shaped echo-negative formation, topically localized by anatomical formations. The drug is administered strictly intramuscularly.

After 4 days at 5 points, the nevasculgen angiogenesis inducer at a dose of 1.2 mg was intramuscularly injected into the same anatomical loci under ultrasound control. 2 months after therapy, there was an increase in DBH from 100 m to 300 m, an increase in the ankle-brachial index (LII) from 0.4 to 0.5 with a recovery period 14 minutes after passing the distance of 320 m. The patient was stopped night pains, saved working capacity. Adverse complications during gene-cell therapy were not noted.

Example 2

Patient A., 58 years old, is sick with peripheral arterial arteriosclerosis obliterans with progression of chronic lower limb ischemia (HINC) for 15 years. In 2001, a lumbar sympathectomy + femoral-popliteal bypass grafting was performed. In 2007 - decunting on the left. Deterioration of the condition of the left lower limb in 2013, when aortic-femoral bifurcation bypass surgery was performed. In 2014, the progression of ischemia, pain at rest, DBH less than 100 m. In connection with the progression of limb ischemia, the patient was offered gene-cell therapy, to which the patient agreed.

After a standard procedure for mobilizing stem cells with filgrastim at a dose of 10 μg / kg for 3 days subcutaneously, a stem cell concentrate of peripheral blood in a volume of 42 ml was obtained by leukopheresis. Aseptic cell transplant was administered intramuscularly at 30 points in the calf muscle array under operating conditions under ultrasound control. On the 4th day, in order to potentiate distal angiogenesis, a VEGF inducer was introduced - the Neovasculgen preparation at a dose of 1.2 mg intramuscularly at 5 points in the same region of the calf muscles of the problem leg.

When examined 9 months after gene-cell therapy, the patient continues to improve the condition of the diseased limb: relief of night pain, seizures; DBH increased from 50-100 m to 250 m. The LPI index increased from 0.35 to 0.7 with a recovery period after exercise after 15 minutes, which may indicate an increase in perfusion of the distal parts of the lower limb. The patient continues to work.

As follows from the above, the use of ultrasound control allowed us to avoid the formation of hematomas that cause additional pain syndrome, and, most importantly, to avoid the therapeutic agent getting into epiphascial tissues, which reduces the angiotropic effect of transplantation due to irrational and ineffective administration of stem cells and endothelial growth factor.

Claims (1)

A method of treating ischemic disease of the lower extremities, including intramuscular injection of a suspension of autologous peripheral blood stem cells (SCPC) at several points in the muscle mass in the ischemic zone, characterized in that vascular endothelial growth factor (VEGF) is introduced 3-4 days after the administration of SCPC the muscle mass, depth, place of introduction in the muscle mass and the fact of the spread of SCPC and VEGF in it are determined and recorded by ultrasound monitoring.