RU2787368C2 - Deproteinized preparation of veal blood for use in prevention or treatment of peripheral arterial disease (pad) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to the use of a deproteinized preparation of veal blood for prevention or treatment of a peripheral arterial disease stage IIB by Fontaine and a treatment method. The use of a deproteinized preparation of veal blood for prevention or treatment of a peripheral arterial disease stage IIB by Fontaine is proposed, where the specified deproteinized preparation of veal blood is intravenously injected at least once by means of infusion, at a dose of 1200 mg, once a day for a period of 14 sequential days, with subsequent period of oral administration of a dose in the form of two tablets by 200 mg, taken three times a day, for a period of 70 sequential days. The method for the prevention or treatment of a peripheral arterial disease stage IIB by Fontaine in a patient who needs it includes certain stages.

EFFECT: above-described group allows for efficient prevention and treatment of a peripheral arterial disease stage IIB by Fontaine in a patient.

18 cl, 1 dwg, 3 tbl, 1 ex

Description

Technical field

The present invention relates to a deproteinized bovine blood preparation for use in the prevention or treatment of peripheral arterial disease. More specifically, the present invention relates to a deproteinized bovine blood preparation for use in the prevention or treatment of peripheral arterial disease, wherein the deproteinized bovine blood preparation is administered at least once parenterally, preferably intravenously, followed by a period of enteral administration, preferably oral administration. In a preferred embodiment of the present invention, the deproteinized calf blood preparation is Actovegin®.

State of the art

Peripheral arterial disease (PAD, PAD), otherwise known as "occlusive peripheral arterial disease", is a manifestation of systemic atherosclerosis and is defined by progressive stenosis or occlusion in the arteries of the lower extremities [1]. BPA affects approximately 202 million adults worldwide [2]. The prevalence of PAD increases with age and the number of vascular risk factors (eg, diabetes, smoking, hypertension, hypercholesterolemia, age, gender, family history) [1, 3].

More importantly, it is a marker of atherosclerotic disease and is associated with increased mortality from cardiovascular and cerebrovascular causes [1, 3].

Decreased blood flow to the legs caused by BPA can be moderate to severe and is associated with a wide range of symptoms. Patients may not experience noticeable limb symptoms, but may also experience intermittent claudication (IC, IC) or may present with symptoms of severe limb ischemia. PH, the most common symptom of PD, is defined as fatigue, cramps, or overt muscle pain in the buttocks, thighs, or calves that is regularly triggered by exercise and reproducibly relieved by rest.

Patients with IC are often limited in their daily activities due to gait impairment and therefore experience a reduced quality of life. With continued exposure to risk factors for atherosclerosis, PA may progress to critical limb ischemia, which portends a severe decline in quality of life and is associated with a high likelihood of amputation and a significant increase in short-term mortality. Thus, PPA is a common manifestation of atherosclerosis associated with a range of symptoms, various impacts on quality of life, and an increased risk of cardiovascular ischemic events [1].

Actovegin® is a deproteinized hemoderivative from calf blood, which was purified from pyrogens and antigens during the production process using staged ultrafiltration.

Actovegin® contains physiological blood components, including vitamins, amino acids, lipids, oligosaccharides and oligopeptides, nucleosides, carbohydrate and fatty acid metabolism intermediates, and cell membrane components such as glycosphingolipids. It has pleiotropic metabolic, neuroprotective and regenerative properties [4]. Experimental studies have shown that Actovegin® improves the use and absorption of oxygen, as well as energy metabolism and glucose uptake [5-7]. It has been shown that Actovegin® has neuroprotective effects and increases the survival of neurons under conditions of ischemia [8]. It also reduces apoptosis induced by beta-amyloid protein, along with a decrease in the formation of reactive oxygen species [9], modulates the activity of nuclear factor kappa B [10], and inhibits the nuclear enzyme poly(ADP-ribose) polymerase, which may also partially explain its neuroprotective effects. properties [11]. Actovegin® has a positive effect on microcirculation and microvascular endothelium, increasing the rate of capillary blood flow and reducing the pericapillary zone and arteriovenous shunting of blood flow [12].

Data on the clinical efficacy of Actovegin® indicate the possibility of treating cerebrovascular disorders, including dementia [13, 14] and post-stroke cognitive impairment [15]. Several randomized controlled trials and many uncontrolled studies have been conducted for these indications. Available clinical data also support the use of Actovegin® for the treatment of diabetic polyneuropathy [16]. The effectiveness of Actovegin® for these indications is further supported by non-clinical pharmacodynamic studies. Efficacy data also support the possibility of treating peripheral perfusion disorders, suggesting positive research findings regarding maximum pain-free walking distance in BPA and healing time in venous ulcers [17-21].

Actovegin® was first approved in the Federal Republic of Germany in 1976 and remains on the market in more than 20 countries (including several countries in the Commonwealth of Independent States and several countries in Europe and Asia). It is available in a variety of oral, intravenous (IV) and intramuscular formulations. Actovegin® has been approved for the following indications (which vary by country): symptomatic treatment of cognitive impairment, including post-stroke cognitive impairment and dementia; symptomatic treatment of peripheral perfusion disorders and their consequences; and symptomatic treatment of diabetic polyneuropathy.

Actovegin® has been used in clinical practice for over 40 years. Due to the fact that it consists of biological components present under normal physiological conditions and a strictly controlled manufacturing process, the drug has an excellent safety profile, which reflects clinical experience equivalent to drug exposure corresponding to more than 1,200,000 patient years (Periodical Safety Report [PSUR] 2014 submitted to the Austrian Agency for Health and Food Safety). The adverse drug reaction (ADR) profile was stable across all PSURs, showing no specific target organ toxicity.

Actovegin® has been used since the 1970s to treat BPA of varying severity. Several studies have evaluated the efficacy of Actovegin® (IV) in patients with predominantly Fontaine stage II and III BPA.

A randomized, open, 8-week study was conducted to investigate the effect of Actovegin® on lower limb ischemic syndrome in patients with type 1 and type 2 diabetes compared with sulodexide [17]. Patients were randomized to receive either IV infusions of Actovegin® (2000 mg once daily, n=12) or sulodexide (1200 lipoprotein lipase units [LSU] per day, n=14) for 2 weeks, followed by oral administration of both funds for another 6 weeks (1200 mg/day and 1000 LSU per day, respectively). Both agents increased the maximum duration of pain-free walking after 8 weeks of treatment (p<0.05). But treatment with Actovegin® significantly increased the maximum duration of pain-free walking after 2 weeks of treatment compared with baseline (p <0.05), in contrast to treatment with sulodexide. In addition, the relative increase in pain-free walking time was greater in the Actovegin® group compared to the sulodexide group (95.1% vs. 38.1%, respectively, p<0.05).

A randomized, double-blind, placebo-controlled, parallel-group study was conducted, including 60 patients with stage II CAD [18]. All patients had participated in a 12-week regular exercise program for at least 4 weeks prior to randomization. Patients then received daily IV infusions of Actovegin® 20% (250 ml/day) or placebo (saline 250 ml/day) for 4 weeks. The primary endpoint, pain-free walking distance on a treadmill, increased by 27% (from 73 to 93 meters) in the Actovegin® group and decreased by 12% (by 10 meters) in the placebo group. The difference between the two groups was statistically significant (p<0.001). A similar result was obtained for maximum walking distance (+25% compared to -10.4% for Actovegin® and placebo, respectively, p<0.01).

Horsch et al. conducted a randomized, double-blind, placebo-controlled trial including 138 patients with stage II CAD [19]. Actovegin® 20% (250 ml) and a matching placebo were administered as daily intravenous infusions over a 3-week period. Efficacy was assessed by patient response analysis, where response was defined as a 35% increase in pain-free walking distance from baseline measured on a treadmill. In the Actovegin® group, there were 57% responders compared to 33% in the placebo group (p=0.01).

In another double-blind study, Müller-Bühl et al. evaluated the efficacy and compatibility of infusion of Actovegin® 20% (250 ml) compared with placebo (0.9% saline 250 ml) in patients with stage II CAD [20]. In this study, Actovegin® and placebo were injected intra-arterially into the femoral artery of the walking distance-limiting leg; patients had to have a stable walking distance prior to randomization to receive either Actovegin® (n=40) or placebo (n=40).

Infusions were administered daily, except on weekends, for 4 weeks, and training was stopped at the start of the treatment phase. As a result of therapy with Actovegin®, an increase in pain-free walking distance from 112 to 162 meters (49%) and a maximum distance from 171 to 266 meters (59%) was observed. In the placebo group, the pain-free walking distance increased from 114 to 135 meters (23%), and the maximum distance from 176 to 201 meters (17%). Differences between the two groups were significant for both parameters (p<0.05). After discontinuation of treatment with Actovegin® and placebo, an 8-week follow-up showed that the walking distance decreased markedly in the placebo group compared with patients previously treated with Actovegin®.

A randomized, single-blind study was also conducted to compare the effectiveness of Actovegin® and bencyclane in stage III BPAD [21]. Fifty-eight patients were randomized to receive either an infusion of Actovegin® 20% (20 intra-arterial infusions) or 250 mg of bencyclane in 250 ml of 0.9% saline for 4 weeks. Endpoint analysis showed that after 4 weeks of patients treated with Actovegin®, 23.1% experienced pain at night, compared with 61.5% of patients treated with benciclane (p<0.05). None of the patients treated with Actovegin® reported persistent pain after 4 weeks, while 16% of patients treated with bencyclane complained of it (p<0.05). The use of painkillers for 4 weeks was also lower in patients treated with Actovegin® compared with patients treated with benciclane (28.7% vs. 42.3%, respectively, p<0.05). Finally, both agents increased pain-free walking distance (29.2 ± 14.6 m and 25.8 ± 18.7 m for the Actovegin® and benciclane groups, respectively), but there was no statistically significant difference between the two groups.

Thus, IV treatment with Actovegin® has been shown to increase initial walking distance and relieve pain symptoms in patients with PD in small, double-blind, controlled trials. However, there is an urgent need for a new and effective regimen for the treatment of BP.

Brief description of the invention

The present invention provides in a first aspect a deproteinized bovine blood preparation, such as the drug Actovegin®, for use in the prevention or treatment of peripheral arterial disease, wherein the deproteinized bovine blood preparation is administered at least once parenterally, preferably intravenously, followed by an enteral period. administration, preferably oral.

In another aspect, the present invention relates to the use of a deproteinized calf blood preparation, such as the drug Actovegin®, for the prevention or treatment of peripheral arterial disease, wherein said deproteinized calf blood preparation is administered at least once parenterally, preferably intravenously, followed by a period enteral administration, preferably oral.

In another aspect, the present invention provides a method for the prevention or treatment of peripheral arterial disease in a patient in need thereof, said method comprising the steps of: a) administering at least once parenterally, preferably intravenously, to said patient a therapeutically effective amount of a deproteinized preparation from calf blood, such as the drug Actovegin®, followed by b) administering at least once enterally, preferably orally, to said patient a therapeutically effective amount of said deproteinized calf blood preparation.

The present invention may be further briefly described in the following paragraphs:

1. A deproteinized calf blood preparation, such as the drug Actovegin®, for use in the prevention or treatment of peripheral arterial disease, wherein said deproteinized calf blood preparation is administered at least once parenterally, preferably intravenously, followed by an enteral period, preferably - oral.

2. The deproteinized calf blood preparation for use according to claim 1, wherein said deproteinized calf blood preparation is administered parenterally once a day.

3. A deproteinized calf blood preparation for use according to claim 1 or 2, wherein said deproteinized calf blood preparation is administered parenterally for at least about 10 consecutive days.

4. A deproteinized calf blood preparation for use according to claim 1 or 2, wherein said deproteinized calf blood preparation is administered parenterally for about 10 to about 18 consecutive days.

5. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-4 wherein said deproteinized calf blood preparation is administered parenterally for about 14 days.

6. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-5, where parenteral administration is intravenous administration.

7. The deproteinized bovine blood preparation for use according to claim 6, wherein said deproteinized calf blood preparation is administered intravenously by infusion.

8. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-7 wherein the total daily dose of a parenterally administered deproteinized bovine blood preparation is in the range of about 1000 mg to about 1400 mg.

9. The deproteinized bovine blood preparation for use according to claim 8, wherein the total daily parenteral dose of the deproteinized bovine blood preparation is about 1200 mg.

10. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-9, wherein said deproteinized calf blood preparation is administered intravenously once daily at a dose ranging from about 1000 mg to about 1400 mg.

11. A deproteinized calf blood preparation for use according to claim 10, wherein said deproteinized calf blood preparation is administered intravenously once a day at a dose of about 1200 mg.

12. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-11, wherein said deproteinized calf blood preparation is administered enterally at least once a day.

13. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-12 wherein the deproteinized calf blood preparation is administered enterally at least twice a day.

14. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1 to 13, wherein the deproteinized calf blood preparation is administered enterally three times a day.

15. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-14 wherein the deproteinized bovine blood preparation is administered enterally for at least about 63 consecutive days.

16. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-15 wherein the deproteinized bovine blood preparation is administered enterally for a period of about 63 to about 77 consecutive days.

17. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-16 wherein the deproteinized calf blood preparation is administered enterally for about 70 consecutive days.

18. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-17, where enteral administration is oral administration.

19. The deproteinized calf blood preparation for use according to claim 18, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet.

20. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-19 wherein the total daily dose of enterally administered deproteinized bovine blood preparation is in the range of about 400 mg to about 2000 mg.

21. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-20, where the total daily dose of enterally administered deproteinized calf blood preparation is about 1200 mg.

22. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-19 wherein the deproteinized bovine blood preparation is administered orally three times a day at a dose in the range of about 300 to about 500 mg each time.

23. A deproteinized calf blood preparation for use according to claim 22, wherein the deproteinized calf blood preparation is administered orally three times a day at a dose of about 400 mg each time.

24. A deproteinized calf blood preparation for use according to claim 23, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet containing about 200 mg of said deproteinized calf blood preparation.

25. Deproteinized preparation from calf blood for use according to any one of paragraphs. from 1 to 24, where the specified deproteinized preparation from calf blood is the drug Actovegin®.

26. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1-25, wherein said peripheral arterial disease is Fontaine stage II peripheral arterial disease.

27. A deproteinized bovine blood preparation for use according to claim 26, wherein said peripheral arterial disease is Fontaine stage IIB peripheral arterial disease, e.g., confirmed by ultrasound color duplex scanning.

28. Deproteinized preparation from calf blood for use according to any one of paragraphs. 1 to 27, where the patient is a human, such as a human suffering from or affected by diabetes mellitus.

29. A deproteinized bovine blood preparation for use according to claim 28, wherein the human patient has a resting Doppler ankle-brachial index ≤ 0.9.

30. A deproteinized bovine blood preparation for use according to 28 or 29 where the human patient has intermittent claudication with an ICD <200 meters.

31. Deproteinized preparation from calf blood for use according to any one of paragraphs. 28-30, wherein the human patient is at least about 40 years of age.

32. Deproteinized preparation from calf blood for use according to any one of paragraphs. 28-31, where the human patient is male.

33. Deproteinized preparation from calf blood for use according to any one of paragraphs. 28-32, where the human patient is a woman.

34. The use of a deproteinized bovine blood preparation in the prevention or treatment of peripheral arterial disease, wherein the deproteinized bovine blood preparation is administered at least once parenterally, preferably intravenously, followed by a period of enteral administration, preferably oral administration.

35. Use according to claim 34, wherein the deproteinized calf blood preparation is administered parenterally once a day.

36. Use according to claims 34 or 35 wherein the deproteinized calf blood preparation is administered parenterally for at least about 10 consecutive days.

37. The use of claims 34 or 35, wherein the deproteinized bovine blood preparation is administered parenterally over a period of about 10 to about 18 consecutive days.

38. Application according to any one of paragraphs. 34-37 wherein the deproteinized bovine blood preparation is administered parenterally for about 14 consecutive days.

37. Application according to any one of paragraphs. 34-36, where parenteral administration is intravenous administration.

38. Use according to claim 37, wherein the deproteinized calf blood preparation is administered intravenously by infusion.

39. Application according to any one of paragraphs. 34-38 wherein the total daily dose of a parenterally administered deproteinized calf blood preparation is in the range of about 1000 mg to about 1400 mg.

40. Use according to claim 39, wherein the total daily dose of the parenterally administered deproteinized calf blood preparation is approximately 1200 mg.

41. Use according to any one of claims 34-40, wherein the deproteinized bovine blood preparation is administered intravenously once a day at a dose of from about 1000 mg to about 1400 mg.

42. Use according to claim 41, wherein the deproteinised bovine blood preparation is administered intravenously once a day at a dose of about 1200 mg.

43. Application according to any one of paragraphs. 34-42 wherein the deproteinized calf blood preparation is administered enterally at least once a day.

44. Application according to any one of paragraphs. 34-43 wherein the deproteinized calf blood preparation is administered enterally at least twice a day.

45. Application according to any one of paragraphs. 34-44 wherein the deproteinized calf blood preparation is administered enterally three times a day.

46. Application according to any one of paragraphs. 34-45 wherein the deproteinized bovine blood preparation is administered enterally for at least about 63 consecutive days.

47. Application according to any one of paragraphs. 34-46 wherein the deproteinized calf blood preparation is administered enterally for 63 to 77 consecutive days.

48. Application according to any one of paragraphs. 34-47 wherein the deproteinized calf blood preparation is administered enterally for about 70 consecutive days.

49. Application according to any one of paragraphs. 34-48, where enteral administration is oral administration.

50. Use according to claim 49, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet.

51. Application according to any one of paragraphs. 34-50, wherein the total daily dose of enterally administered deproteinized calf blood preparation is in the range of about 400 mg to about 2000 mg.

52. Application according to any one of paragraphs. 34-51, where the total daily dose of enterally administered deproteinized calf blood preparation is approximately 1200 mg.

53. Use according to any one of claims 34-50, wherein the deproteinized bovine blood preparation is administered orally three times a day at a dose of about 300 to about 500 mg each time.

54. The use of claim 53, wherein the deproteinized bovine blood preparation is administered orally three times a day at a dose of about 400 mg each time.

55. Use according to claim 54, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet containing about 200 mg of said deproteinized calf blood preparation.

56. Application according to any one of paragraphs. 34-55, where the specified deproteinized preparation from calf blood is the drug Actovegin®.

57. Application according to any one of paragraphs. 34-56, wherein said peripheral arterial disease is Fontaine stage II peripheral arterial disease.

58. The use of claim 57, wherein said peripheral arterial disease is Fontaine stage IIB peripheral arterial disease, eg, confirmed by ultrasound color duplex scanning.

59. Application according to any one of paragraphs. 34-58, wherein the patient is a human, such as a human afflicted with or suffering from diabetes mellitus.

60. Use according to claim 59, wherein the human patient has a resting Doppler ankle-brachial index ≤ 0.9.

61. Use according to paragraphs 59 or 60, where the human patient has intermittent claudication with an ICD <200 meters.

62. Application according to any one of paragraphs. 59-61, wherein the human patient is at least about 40 years of age.

63. Application according to any one of paragraphs. 59-62, where the human patient is male.

64. Application according to any one of paragraphs. 59-62, where the human patient is a woman.

65. In another aspect, the present invention provides a method for preventing or treating peripheral arterial disease in a patient in need thereof, said method comprising the steps of: a) administering at least once parenterally, preferably intravenously, to said patient a therapeutically effective an amount of a deproteinized bovine blood preparation, such as the drug Actovegin®, followed by b) administering at least once enterally, preferably orally, to said patient a therapeutically effective amount of said deproteinized calf blood preparation.

66. The method of claim 65, wherein the deproteinized calf blood preparation is administered parenterally once a day.

67. The method of claim 65 or 66, wherein the deproteinized calf blood preparation is administered parenterally for at least about 10 consecutive days.

68. The method of claim 65 or 66 wherein the deproteinized calf blood preparation is administered parenterally for about 10 to about 18 consecutive days.

69. The method according to any one of paragraphs. 65-68 wherein the deproteinized calf blood preparation is administered parenterally for about 14 consecutive days.

70. The method according to any one of paragraphs. 65-69, where parenteral administration is intravenous administration.

71. The method of claim 70, wherein the deproteinized calf blood preparation is administered intravenously by infusion.

72. The method according to any one of paragraphs. 65-71 wherein the total daily dose of a parenterally administered deproteinized bovine blood preparation is in the range of about 1000 mg to about 1400 mg.

73. The method of claim 72, wherein the total daily dose of the parenterally administered deproteinized calf blood preparation is about 1200 mg.

74. The method according to any one of paragraphs. 65-71 wherein the deproteinized bovine blood preparation is administered intravenously once daily at a dose of about 1000 mg to about 1400 mg.

75. The method of claim 74, wherein the deproteinized calf blood preparation is administered intravenously once a day at a dose of about 1200 mg.

76. The method according to any one of paragraphs. 65-75 wherein the deproteinized calf blood preparation is administered enterally at least once a day.

77. The method according to any one of paragraphs. 65-76 wherein the deproteinized calf blood preparation is administered enterally at least twice a day.

78. The method according to any one of paragraphs. 65-77 wherein the deproteinized calf blood preparation is administered enterally three times a day.

79. The method according to any one of paragraphs. 65-78 wherein the deproteinized bovine blood preparation is administered enterally for a period of at least about 63 consecutive days.

80. The method according to any one of paragraphs. 65-79 wherein the deproteinized bovine blood preparation is administered enterally for about 63 to about 77 consecutive days.

81. The method according to any one of paragraphs. 65-80 wherein the deproteinized calf blood preparation is administered enterally for a period of approximately 70 consecutive days.

82. The method according to any one of paragraphs. 65-81, where enteral administration is oral administration.

83. The method of claim 82, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet.

84. The method according to any one of paragraphs. 65 to 83, wherein the total daily dose of enterally administered calf blood deproteinized preparation is in the range of about 400 mg to about 2000 mg.

85. The method according to any one of paragraphs. 65-84, where the total daily dose of enterally administered deproteinized bovine blood preparation is approximately 1200 mg.

86. The method of any one of claims 65-83, wherein the deproteinized bovine blood preparation is administered three times a day orally each time at a dose of about 300 to about 500 mg.

87. The method of claim 86 wherein the deproteinized bovine blood preparation is administered three times a day orally each time at a dose of about 400 mg.

88. The method of claim 87, wherein the deproteinized calf blood preparation is administered orally in the form of a tablet containing about 200 mg of said deproteinized calf blood preparation.

89. The method according to any one of paragraphs. 65-88, where the specified deproteinized preparation from calf blood is the drug Actovegin®.

90. The method according to any one of paragraphs. 65-89, wherein said peripheral arterial disease is Fontaine stage II peripheral arterial disease.

91. The method of claim 90, wherein said peripheral arterial disease is Fontaine stage IIB peripheral arterial disease, eg, confirmed by ultrasound color duplex scanning.

92. The method according to any one of paragraphs. 65-191, wherein the patient is a human, such as a human afflicted with or suffering from diabetes mellitus.

93. The method of claim 92, wherein the human patient has a resting Doppler ankle-brachial index ≤ 0.9.

94. The method of claim 92 or 93 wherein the human patient has intermittent claudication with an ICD <200 meters.

95. The method according to any one of paragraphs. 92-94, wherein the human patient is at least 40 years of age.

96. The method according to any one of paragraphs. 92-95, where the human patient is male.

97. The method according to any one of paragraphs. c 92-95 where the human patient is a woman.

Brief description of the drawings

Figure 1: Outline of study design

Detailed description of the invention

Except as specifically defined herein, all technical and scientific terms used have the same meaning as is generally understood by a qualified medical and pharmaceutical professional.

All methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the present invention; suitable methods and materials are described herein. All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. In the event of a conflict, the present description, including definitions, will govern. In addition, the materials, methods, and examples are illustrative only and are not restrictive unless otherwise indicated.

In cases where a numeric limit or range is specified here, the extreme points are also considered included. In addition, all values and subranges within any numerical limit or range are considered to be specifically included as if they were explicitly written out.

Below, the present invention will be described in more detail.

In accordance with one aspect, the present invention provides a deproteinized bovine blood preparation for use in the prevention or treatment of peripheral arterial disease (PAD).

Peripheral arterial disease (PAD), also known as "peripheral arterial occlusive disease", is a well-known manifestation of systemic atherosclerosis defined by progressive stenosis or occlusion. Peripheral artery disease most often affects the arteries of the lower extremities, but other arteries can also be affected. The classic symptom is pain in the legs when walking that resolves with rest, known as intermittent claudication. Other symptoms may also occur in the affected leg, including skin ulcers, bluish skin, cold skin, or poor nail and hair growth. Complications may include infection or tissue death, which may require amputation; ischemic heart disease or stroke. Up to 50% of PAD cases are asymptomatic. Well-known risk factors include smoking, diabetes mellitus, dyslipidaemia, and hypertension. The risk of PAD also increases in people over 50 years of age, men, those who are obese, who have had a heart attack or after a stroke, or who have a family history of vascular disease.

More specifically, the present invention provides a deproteinized calf blood preparation, such as the drug Actovegin®, for use in the prevention or treatment of peripheral arterial disease, wherein said deproteinized calf blood preparation is administered at least once parenterally, preferably intravenously, followed by period of enteral administration, preferably oral administration.

The present invention also provides the use of a deproteinized bovine blood preparation, such as the drug Actovegin®, in the prevention or treatment of peripheral arterial disease, wherein the deproteinized calf blood preparation is administered at least once parenterally, preferably intravenously, followed by a period of enteral administration. , preferably oral administration.

The present invention also provides a method for the prevention or treatment of peripheral arterial disease in a patient in need thereof, said method comprising the steps of: a) administering at least once parenterally, preferably intravenously, to said patient a therapeutically effective amount of a deproteinized bovine blood preparation, such as the drug Actovegin® followed by b) administering at least once enterally, preferably orally, to said patient a therapeutically effective amount of said deproteinized calf blood preparation.

As used herein, the term "parenteral" or "parenteral" refers to a route of drug administration that does not involve the gastrointestinal (GI) tract and includes any appropriate form of administration such as intravenous (injection into a vein), subcutaneous (injection under the skin ), intramuscular (injection into a muscle), inhalation (infusion through the lungs), or transdermal (absorption through intact skin).

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally once a day.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally for at least about 10 consecutive days, for example, over a period of from about 10 to about 18, from about 11 to about 17, from about 12 to about 16 or 13 to 15 consecutive days.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally over a period of about 10 to about 18 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally over a period of about 11 to about 17 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally over a period of about 12 to about 16 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered parenterally over a period of about 13 to about 15 consecutive days.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered parenterally for about 14 consecutive days.

In accordance with some variants of implementation of the present invention, parenteral administration is intravenous administration, for example, by infusion.

Typically, an infusion volume of from about 100 ml to about 500 ml is administered, containing a deproteinized bovine blood preparation, for example, about 200 ml, 250 ml, 280 ml, 300 ml, 350 ml, 400 ml, or 450 ml. According to some embodiments, an infusion volume of 200 to 400 ml is administered containing the deproteinized bovine blood preparation. In some embodiments, an infusion volume of about 250 to about 300 ml is administered containing the deproteinized bovine blood preparation. In some embodiments, an infusion volume of about 280 ml is administered containing the deproteinized bovine blood preparation.

The recommended total daily dose for a parenterally administered deproteinized bovine blood product, such as the drug Actovegin®, is in the range of about 800 to about 2000 mg. Thus, in accordance with some embodiments of the present invention, the total daily dose of a parenterally administered deproteinized calf blood preparation is in the range of about 800 to about 2000 mg.

In accordance with some embodiments of the present invention, the total daily dose of a parenterally administered deproteinized bovine blood preparation is in the range of about 900 mg to about 1800 mg. In accordance with some embodiments of the present invention, the total daily dose of a parenterally administered deproteinized calf blood preparation is in the range of about 1000 mg to about 1400 mg. In accordance with some embodiments of the present invention, the total daily dose of a parenterally administered deproteinized bovine blood preparation is in the range of about 1100 to about 1300 mg.

In accordance with some embodiments of the present invention, the total daily dose of parenterally administered deproteinized bovine blood preparation is about 1200 mg.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered intravenously once daily, eg, by infusion, at a dose of from about 1000 mg to about 1400 mg.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered once daily intravenously, eg, by infusion, at a dose of about 1200 mg.

As described above, the parenteral administration period is followed by the enteral administration period.

As used herein, the term "enteral" or "enteral" refers to the route of administration of a drug through the gastrointestinal tract and includes any appropriate form of administration such as oral, sublingual (dissolving the drug under the tongue), or rectal.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally at least once a day. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally at least twice a day.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered enterally three times a day.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally for at least about 63 consecutive days, e.g., for a period of about 65 to about 75, about 66 to about 74, about 67 to about 73 , 68 to 72, or 69 to 71 consecutive days.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered enterally for a period of 63 to 77 consecutive days. In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered enterally for a period of 65 to 75 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally for a period of from about 66 to about 74 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally for a period of 67 to 73 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally for a period of from about 68 to about 72 consecutive days. In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered enterally for a period of 69 to 71 consecutive days.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered enterally for about 70 consecutive days.

In accordance with some embodiments of the present invention, enteral administration is oral administration.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered orally in the form of a tablet.

The recommended total daily dose for an enterally administered deproteinized bovine blood product, such as the drug Actovegin®, is in the range of about 400 mg to about 2000 mg. Thus, in accordance with some embodiments of the present invention, the total daily dose of a parenterally administered deproteinized bovine blood preparation is in the range of about 400 mg to about 2000 mg.

In accordance with some embodiments of the present invention, the total daily dose of enterally administered deproteinized calf blood preparation is in the range of about 600 mg to about 1800 mg. In accordance with some embodiments of the present invention, the total daily dose of enterally administered deproteinized calf blood preparation is in the range of about 800 to about 1600 mg. In accordance with some embodiments of the present invention, the total daily dose of enterally administered deproteinized calf blood preparation is in the range of about 1000 mg to about 1400 mg. In accordance with some embodiments of the present invention, the total daily dose of enterally administered deproteinized bovine blood preparation is in the range of about 1100 mg to about 1300 mg.

In accordance with some embodiments of the present invention, the total daily dose of enterally administered deproteinized bovine blood preparation is about 1200 mg.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered orally three times a day at a dose in the range of about 300 to about 500 mg each time.

In accordance with some embodiments of the present invention, the deproteinized bovine blood preparation is administered orally three times a day at a dose of about 400 mg each time.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is administered orally in the form of a tablet containing about 200 mg of said deproteinized calf blood preparation.

Deproteinized calf blood preparations are well known to those skilled in the art. Examples of the preparation of such formulations are described in detail in Examples 1-3 of International Patent Application WO 2010/034315, the contents of which are incorporated herein by specific reference. A preferred bovine blood preparation is available on the market as Actovegin®.

In accordance with some embodiments of the present invention, the deproteinized calf blood preparation is Actovegin® drug.

In accordance with some embodiments of the present invention, the peripheral arterial disease is Fontaine stage II peripheral arterial disease.

In accordance with some embodiments of the present invention, the peripheral arterial disease is Fontaine stage IIB peripheral arterial disease.

Fontaine stage II peripheral arterial disease, and more specifically Fontaine stage IIB peripheral arterial disease, can be confirmed by ultrasound color duplex scanning.

In accordance with some embodiments of the present invention, the patient to be treated is a human.

In accordance with some embodiments of the present invention, the human patient is male.

In accordance with some embodiments of the present invention, the human patient is a female.

In accordance with certain embodiments of the present invention, the human patient has a Doppler ankle-brachial index ≤ 0.9.

In accordance with some embodiments of the present invention, a human patient has intermittent claudication with an ICD <200 meters.

In accordance with some embodiments of the present invention, the human patient is an adult.

In accordance with some embodiments of the present invention, the human patient is at least 40 years of age. In accordance with some embodiments, the human patient is at least 45 years of age. In accordance with some embodiments, the human patient is at least 50 years of age. In accordance with some embodiments, the human patient is at least 55 years of age. In accordance with some embodiments, the human patient is at least 60 years of age. In accordance with some embodiments, the human patient is at least 65 years of age. In accordance with some embodiments, the human patient is at least 70 years of age.

In accordance with some embodiments of the present invention, the age of the human patient is from 40 to 75 years. In accordance with some embodiments, the human patient is between 45 and 75 years of age. In accordance with some embodiments, the human patient is between about 50 and 75 years of age. In accordance with some embodiments, the human patient is between about 55 and 75 years of age. In accordance with some embodiments, the human patient is between 60 and 75 years of age. In accordance with some embodiments, the age of the human patient is about 65-75 years.

In some embodiments, the human patient has diabetes mellitus, such as type 1 diabetes mellitus or type 2 diabetes mellitus. In some embodiments, the human patient has type 1 diabetes mellitus. In accordance with some embodiments, the human patient has type 2 diabetes mellitus.

In certain embodiments, the human patient is at least 40 years old, such as at least 50 years old, and is diabetic.

In certain embodiments, the human patient is at least 40 years old, such as at least 50 years old, and is diabetic and has at least one cardiovascular risk factor, such as smoking.

In some embodiments, the human patient is at least 40 years old, such as at least 50 years old, and is diabetic and has at least one other atherosclerosis risk factor, such as another atherosclerosis risk factor selected from the group including smoking, dyslipidemia, hypertension, or hyperhomocysteinemia.

List of abbreviations

ACD - absolute distance walked with intermittent claudication

NLR - unwanted drug reaction

AE - an undesirable phenomenon

ALT - alanine aminotransferase

AST - aspartate aminotransferase

CPMP - Committee on Patent Medicines

ECG - electrocardiogram

eCRF - electronic individual registration card

EMA - European Medicines Agency

FSH - follicle stimulating hormone

GCP - Rules for Conducting Quality Clinical Research

HbA1c - glycosylated hemoglobin

hCG - human chorionic gonadotropin

IC - intermittent claudication

ICD - initial distance walked with intermittent claudication

ICH - International Conference on Harmonization

ILP - investigational drug

IP - researched product

IRT - interactive communication technology

in / in - intravenously

LFT - liver function test

LSU - lipoprotein lipase unit

MedDRA - Regulatory Medical Dictionary

Med ID - drug identification number

PAD - peripheral artery disease

PSUR - Periodic Drug Safety Report

PTE is an adverse event occurring before the start of treatment

SAE is a serious adverse event

SAP - Statistical Analysis Plan

SF-36 - 36 item short health assessment questionnaire

SUSAR - Suspected Unexpected Serious Adverse Reaction

TID - 3 times a day

ULN - upper limit of normal

Given the general description of this invention, additional understanding can be obtained by referring to some specific examples, which are given here for illustration only and are not restrictive, unless otherwise indicated.

Examples

Example 1 - Randomized international multicenter, parallel group, double-blind, placebo-controlled study to evaluate the efficacy and safety of 12-week treatment with Actovegin®, administered first intravenously and subsequently orally in patients with Fontaine stage IIB occlusive peripheral arterial disease

1. OBJECTIVES AND END POINTS OF THE STUDY

1.1 Goals

1.1.1 Main purpose

The main goal is to study the effectiveness of Actovegin® for the symptomatic treatment of BPA stage IIB according to Fontaine.

1.1.2 Secondary goals

To study the effect of Actovegin® on sustained improvement in walking distance with lameness in patients with Fontaine stage IIB BPA.

Study of the effect of Actovegin® on the quality of life of patients.

1.1.3 Safety goal

Safety assessment of Actovegin® compared with placebo.

1.2 Endpoints

1.2.1 Primary endpoint

The primary endpoint is the percentage change in initial lameness walking distance (ICD) from baseline to 12 weeks of treatment.

1.2.2 Secondary endpoints

Percent change in ICD from baseline to 2 and 24 weeks after randomization.

Change in absolute distance in intermittent claudication (ACD) from baseline to 2, 12, and 24 weeks after randomization.

Proportion of patients with pain at rest at 12 and 24 weeks after randomization.

Percentage of patients undergoing revascularization procedures at 24 weeks after randomization.

Change in 36-item Short Health Questionnaire (SF-36) scores at 12 and 24 weeks after randomization.

1.2.3 Additional endpoints: security

The safety and tolerability of Actovegin® will be assessed by assessing adverse events (AEs), clinical laboratory safety tests, vital signs, weight, electrocardiogram (ECG), and physical examination.

2. DESIGN AND DESCRIPTION OF THE STUDY

2.1 Study design

This is a randomized, multicenter, parallel-group, double-blind, placebo-controlled phase 3b study to evaluate the efficacy and safety of 12 weeks of treatment with Actovegin® administered initially intravenously and subsequently orally in patients with Fontaine stage IIB CAD.

A total of 366 Fontaine stage IIB CAD patients will be enrolled in the study at approximately 20-25 centers in 5 countries (Russia, Ukraine, Belarus, Kazakhstan and Georgia).

The study will consist of a 1-2 week screening period, randomization, a 12-week treatment period, and a 12-week follow-up period. The total duration of the study will be from 25 to 26 weeks.

Patients will undergo a 1-2 week screening period during which the patient's stability will be checked, the diagnosis of PAD will be confirmed, and patients with high walking instability will be identified and excluded. For this purpose, 2 treadmill tests will be performed ≥1 week and ≤2 weeks apart. Patients showing a change in ACD of more than 25% during the screening period will be excluded from the study.

Patients with a history of intermittent claudication of at least 6 months prior to enrollment will be eligible for enrollment. The diagnosis of BPA (ICD < 200 meters) will be confirmed with an ultrasound color duplex scan and a treadmill test.

Eligible patients will be randomized in a 1:1 ratio to receive either Actovegin® or placebo. The treatment period will include 2 weeks of IV infusion of Actovegin® (a deproteinized hemo-derivative) at a dose of 1200 mg/day, followed by 10 weeks of oral tablets at a dose of 1200 mg/day (two 200 mg tablets three times daily [TID ]). Throughout the treatment period, a matching placebo (in ampoules and tablets) will be used to maintain blinding. The total duration of treatment will be 12 weeks.

The 12-week treatment period will be followed by a 12-week observation period without treatment with the investigational drug (ILP) designed to study persistent efficacy after treatment, as well as safety after discontinuation of treatment with Actovegin®.

The outline of the study design is shown in Figure 1. The score chart is shown in Table 1.

Table 1: Schedule of study procedures

Footnotes are transcribed on the last page of the table.

V = visit.

(a) Early termination visit for patients who withdraw from the study before the completion of the oral treatment period.

(b) Early termination visit for patients who withdraw from the study during the follow-up period.

(c) On Day 0, Visit 1, AEs should be checked post-infusion. Starting on Day 1, AEs should be checked pre-infusion [AEs since last visit] and post-infusion.

(d) Height and weight will be measured at visit V1. At visits V5 and V8, only weight will be measured.

(e) After the final infusion.

(f) only in women of childbearing potential.

(g) Evaluation of contraceptive compliance is not required at this visit.

(h) The time span from the first test must be ≥ 1 week and ≤ 2 weeks.

2.2 Rationale for study design, dose and endpoints

This randomized, parallel-group, double-blind, placebo-controlled study is in accordance with the recommendations of CPMP/EWP/714/98 "Note on guidelines for clinical trials of medicinal products for the treatment of peripheral arterial occlusive disease".

The dosing regimen for this study is consistent with the product label, except that oral treatment will be longer than labeled. The recommended dose for Actovegin® is 800 to 2000 mg (20-50 ml) intravenously for 4 weeks. The recommended dose for tablets is 1 to 2 tablets (each tablet containing 200 mg of Actovegin®) TID with an average treatment duration of 4 to 6 weeks. The oral regimen in this study is 2 tablets (200 mg) TID (1200 mg/day) for 10 weeks.

The efficacy of Actovegin® 1000 mg IV was demonstrated in a comparative randomized trial in patients with Fontaine stage IIB BPA. Maximum walking distance increased after 4 weeks of treatment by 100.7% in the Actovegin® group compared to 67% and 8.1% in the naftidrofuryl and vitamin B groups, respectively [22]. In another open controlled study, Actovegin® was administered at 1000 mg IV daily for 10 days and showed an increase in pain-free walking distance comparable to the pentoxifylline group. Analysis of microcirculation in the skin using laser Doppler flowmetry showed that Actovegin® has a number of significant effects that are more pronounced compared to the pentoxifylline group [‎23].

Such a long period of oral treatment has demonstrated its safety and efficacy in previous trials of Actovegin®.

Intermittent claudication was chosen as the main endpoint of the change in ICD from baseline, as it is a key symptom of stage II of ABP and is recommended in the guidelines of the European Medicines Agency (EMA) "Note to the guidelines for clinical trials of drugs for the treatment of peripheral occlusive disease arteries".

3. SELECTION AND TERMINATION OF TREATMENT/TERMINATION OF PATIENT PARTICIPATION

All inclusion criteria, including test results, must be validated prior to randomization or the first dose.

3.1 Inclusion criteria

Patient eligibility for participation is determined according to the following criteria prior to study entry:

1. The patient is male or female and aged 40 to 75 inclusive.

2. History of stable intermittent claudication lasting more than 6 months prior to screening.

3. The diagnosis of BPA (code I70.2 according to the International Classification of Diseases 10th revision) stage IIB according to Fontaine was confirmed using ultrasound color duplex scanning.

4. Newly diagnosed with ABP or history of stable ABP therapy (including smoking status, exercise habits, and medications) for at least 3 months prior to randomization.

5. Doppler ankle-brachial index=0.9.

6. Intermittent claudication with ICD <200 meters.

7. In the opinion of the investigator, the patient is able to understand and comply with the requirements of the protocol.

8. The patient or, where applicable, the patient's legal representative, signs and dates the written informed consent form and any necessary authorization for the processing of personal data prior to initiating any study procedures.

9. A male patient who is not spayed and is sexually active with a fertile partner agrees to use a barrier method of contraception (eg, a condom with or without spermicide) from the time of signing informed consent throughout the study or within 30 days after the last doses in case of premature discontinuation of treatment. The male partner of the patient should also be advised to use a highly effective/effective method of contraception throughout the study or for 30 days after the last dose in case of premature termination.

10. A female patient of childbearing potential who is sexually active with an unsterilized male partner agrees to use an effective or highly effective method of contraception from the time of signing informed consent throughout the study or within 30 days of the last dose in case of early termination. Definitions of effective/highly effective contraceptive methods are provided in Section 5.1.11 and the reporting responsibilities are defined in Section 5.1.12.

3.2 Exclusion criteria

Any patient meeting any of the following criteria after the screening period is ineligible for study participation:

1. BPA Fontaine stage III or IV (pain at rest, ulceration that does not heal, or gangrene).

2. Signs of non-atherosclerotic BPA.

3. ACD variability >25% as measured by treadmill test during the screening period.

4. Lower limb artery reconstruction (surgical or endovascular) or sympathectomy within 3 months prior to screening.

5. Candidate for revascularization or angioplasty.

6. Patient had a myocardial infarction or cardiac surgery within 3 months prior to screening.

7. Congestive heart failure (class III / IV according to the classification of the New York Heart Association).

8. Uncontrolled diabetes mellitus (glycosylated hemoglobin [HbA1c > 9%]) or diabetic polyneuropathy.

9. Any other illness that significantly limits exercise capacity or other medical condition, including any mental disorder that limits participation (in the opinion of the investigator).

10. Taking any prohibited drug (section 7.3) within 14 days prior to randomization (Day 0).

11. Taking any experimental compound within 30 days prior to screening.

12. Taking Actovegin® as a therapeutic agent within 30 days prior to screening.

13. The patient is an immediate family member of the employee, an employee of the research center, a relative of the employee of the center who participates in this research (for example, is a spouse, parent, child, sibling), or may agree to participation under duress.

14. Clinically significant abnormal hematologic parameters of hemoglobin, hematocrit, or erythrocytes at screening, in the opinion of the investigator.

15. History of hypersensitivity or allergy to Actovegin® or similar drugs or excipients.

16. History of substance abuse (defined as any use of illegal drugs) or alcohol abuse in the 6 months prior to screening.

17. For female patients, pregnancy or lactation, or intention to become pregnant prior to participation in this study and during the study, or intention to become an egg donor during this period of time.

18. Patient participation in another clinical trial within 30 days prior to screening.

3.3 Excluded drugs and therapeutic procedures

Concomitant and prior medicines should be carefully reviewed and documented in an electronic individual registration card (eCRF).

The following are drugs that are prohibited during the study:

Alprostadil and other prostaglandins

Angiogenic growth factors

Chronic use of non-steroidal anti-inflammatory drugs

Cilostazol

Ginkgo biloba extract

L-carnitine

L-lysine aescinate

Naftidrofuryl

A nicotinic acid

Pentoxifylline

Sulodexide

Vincamine

Hyperbaric oxygen therapy and physical therapy of any type that could potentially affect peripheral circulation are not allowed during the course of the study.

The use of a concomitant medication for the treatment of any other acute or chronic disease may be initiated or continued as directed by a physician. Patients should be instructed not to take any medication, including over-the-counter medication, without prior consultation with the investigator. For more information on concomitant therapy with DMARDs, see section 4.1.1.2.

3.4 Diet, Fluid Intake, Activity Control, Treatment Facilities and General Recommendations and Limitations

All patients will receive advice on lifestyle changes to minimize vascular risk factors (eg, smoking cessation and regular exercise). Smoking and exercise habits must be documented and stable for at least 3 months prior to randomization.

In addition, the treatment and glycemic control status of patients with diabetes, as well as the occasional use of non-steroidal anti-inflammatory agents, may also affect DMARDs. These drugs should be reviewed and documented in the eCRF.

During the period of IV treatment, hospitalization or day care is required to ensure that treatment procedures are followed.

3.5 Criteria for discontinuation of treatment or patient participation

The main reason for discontinuation of study drug treatment or the patient's participation in the study must be recorded in the eCRF using the following categories listed below. Cases of non-screened patients are discussed in Section 5.1.14.

1. Pre-Treatment Adverse Event (PTE) or AE. The patient has experienced a PTE or AE requiring early disenrollment because continued participation imposes an unacceptable risk to the patient's health or the patient is unwilling to continue participation due to the PTE or AE.

PTE is defined as any adverse medical event in a clinical trial patient who signed an informed consent form to participate in the study, but prior to the administration of any investigational drug; it does not have to be causally related to participation in the study.

An AE is defined as any adverse medical event in a clinical trial patient who has been administered a drug; it need not be causally related to that treatment.

Thus, an AE is any adverse and unintended indicator (eg, a clinically significant abnormal laboratory value), symptom, or disease temporally associated with the use of a drug, whether or not it is thought to be drug-related.

Abnormal liver function tests (LFT)

The study drug should be discontinued immediately with appropriate clinical monitoring (including repeat laboratory tests until the patient's laboratory profile returns to normal/baseline, see Section 5.1.10) if the following conditions occur at any time during study drug treatment:

- alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 8 × upper limit of normal (ULN) or

- ALT or AST > 5 × ULN and persists for more than 2 weeks, or

- ALT or AST > 3 x ULN in combination with elevated total bilirubin > 2 x ULN, or

- ALT or AST > 3 x ULN with at least 1 of the following: fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and/or eosinophilia (> 5%).

2. Significant deviation from the protocol. Finding after randomization that a patient did not meet the inclusion criteria according to the protocol or did not comply with the requirements of the protocol, and continued participation carries an unacceptable risk to the patient's health.

3. Impossibility of follow-up due to loss of communication with the patient. The patient did not return to the clinic and attempts to contact him were unsuccessful. Attempts to contact the patient should be documented in the patient's original documents.

4. Voluntary termination of participation. The patient (or the patient's legal representative) wishes to withdraw from the study. The reason for disenrollment, if provided, must be recorded in the eCRF.

Note: Every attempt should be made to determine the root cause of exit and, if possible, to capture the underlying cause (i.e. exit due to AE should not be recorded in the “voluntary exit” category. Similarly, lack of effectiveness should not be recorded in the “voluntary exit” category. ").

5. Termination of the study. The sponsor, ethics committee, or regulatory agency terminates the study.

6. Pregnancy. Detection of pregnancy in a patient.

Note. If a patient is found to be pregnant, the patient should be removed from the study immediately. The procedure is described in section 5.1.12.

7. Disease progression. Clinical signs of critical limb ischemia (pain at rest, ulceration and gangrene) were revealed, confirmed by instrumental methods, requiring a revascularization procedure.

8. Other.

Note. Specific reasons must be specified in the "Specify" field in the eCRF.

3.6 Procedures for terminating treatment or patient participation

An Investigator may terminate a patient's participation in the study at any time during the course of the study if the patient meets the study termination criteria described in Section 3.5. In addition, the patient may terminate their participation without explanation at any time during the course of the study. In the event of termination of a patient's participation, the primary termination criterion must be recorded by the investigator. In addition, efforts must be made to complete all procedures scheduled for the Early Termination Visit. Patients who stop treatment or participation are not eligible for replacement.

4. Management of material resources of a clinical trial

This section contains information about all medicinal products and materials supplied directly by the sponsor and/or by other means as required by the study protocol, including important sections describing the management of study materials.

4.1 Medicinal products and study materials

4.1.1 Dosage form, manufacturer, packaging and labeling

In this protocol, the term "investigational drug" refers to the active agent Actovegin® (400 mg/10 ml and 200 mg) and the corresponding placebo, as described below. Investigational drugs will be blind packaged.

4.1.1.1 Investigational product

Actovegin®, ampoules (400 mg/10 ml) and tablets (200 mg)

The investigational drug (Actovegin®) will be supplied as ampoules (400 mg/10 ml) and tablets (200 mg). Actovegin® (deproteinized hemoderivat) is available on the market both in ampoules and tablets. Actovegin® ampoules and tablets available on the market will be packaged and blind labeled by a contract manufacturing organization (Fisher Clinical Services, Switzerland), and thus both of these products are considered an investigational drug.

Takeda GmbH, Austria, is the original manufacturer of Actovegin® 400 mg/10 ml ampoules. One ampoule contains 400 mg of Actovegin® in 10 ml of water for injection. The solution is clear, yellowish and contains almost no particles. The color may change to a more intense yellow during the expiration date. Taking into account the color of the Actovegin® solution and the different design of the ampoules, a pharmacist will be included in the research team of the center, from whom information about the drug will not be hidden, who will be responsible for the preparation of the investigational product (PI) during the IV treatment period.

Ampoules will be packaged in InPatient ("hospital") kits for 7 days ((21 ampoules for 1 week, plus 3 ampoules for 1 day). Dosing includes 3 ampoules per day.

Each ampoule will be supplied with a multilingual instruction leaflet or a monolingual instruction leaflet that includes relevant information about the study. Each set of Actovegin® 400 mg/10 ml ampoules will be supplied with a multilingual instruction leaflet or a monolingual instruction leaflet that includes relevant information about the study along with a warning about precautions for use.

The main manufacturer of Actovegin® 200 mg tablets is Takeda GmbH, Germany. Tablets are round, biconvex, bright greenish-yellow color, film-coated, shiny without engraving.

The tablets will be packaged in child-resistant bottles containing 50 tablets. Dosage includes 6 tablets per day.

Each bottle of Actovegin® 200 mg tablets will be supplied with a multilingual instruction leaflet or a single language instruction leaflet that includes relevant study information along with a warning. Each set of bottles will also be provided with a multilingual instruction leaflet or a single language instruction leaflet that includes relevant information about the study along with a warning about precautions for use.

The instructions will be in the appropriate language, depending on the country in which the investigational drug was issued.

Placebo ampoules and tablets

Placebo ampoules mimicking Actovegin® 400 mg ampoules are commercially available ampoules containing 10 ml of 0.9% NaCl solution for infusion.

The original manufacturer of placebo ampoules is Fresenius Kabi, Spain. The ampoules will be supplied, packaged and labeled by Fisher Clinical Services, Switzerland.

Placebo ampoules will be packaged in 7-day In-Patient kits (21 ampoules for 1 week, plus 3 ampoules for 1 day). Dosage includes 3 ampoules per day.

Each ampoule will be supplied with a multilingual instruction leaflet or a monolingual instruction leaflet that includes relevant information about the study. Each set of placebo ampoules will be supplied with a multilingual instruction leaflet or a monolingual instruction leaflet that includes relevant information about the study along with a warning about precautions for use.

Placebo tablets simulating Actovegin® 200 mg tablets will be manufactured by Globopharm Pharmazeutische Produktions- und Handelsgesellschaft mbH, Austria and then packaged and labeled by Fisher Clinical Services, Switzerland.

The tablets will be packaged in child-resistant bottles containing 50 tablets. Dosage includes 6 tablets per day.

Each bottle of placebo tablets will be supplied with a multilingual instruction leaflet or a monolingual instruction leaflet that includes relevant study information along with a warning. Each set of bottles will also be provided with a multilingual instruction leaflet or a single language instruction leaflet that includes relevant information about the study along with a warning about precautions for use.

The instructions will be in the appropriate language, depending on the country in which the investigational drug was issued.

4.1.1.2 Concomitant medicines

Concomitant Therapy

Patients can continue with their current treatment for ABP. These treatments must be optimized and stable for at least 3 months at the time of randomization. Smoking status, exercise habits and medications will be assessed and documented. These treatments must be maintained at a stable level throughout the study.

Therapy for the treatment of BPA includes:

- Antiplatelet drugs and anticoagulants.

- Means that reduce lipid levels.

- Antihypertensive drugs.

- Physical exercise.

- To give up smoking.

In addition to the medications for DMARDs listed, DMARDs can also be affected by the treatment and glycemic control status of diabetic patients and the occasional use of non-steroidal anti-inflammatory drugs. These drugs should be reviewed and documented in the eCRF.

4.1.1.3 Supporting materials

The sponsor will also provide the centers with 250 ml 0.9% NaCl infusion bags, protective bags for the infusion bags, and accessory labels to attach to the protective bags at study centers.

The original manufacturer of 0.9% NaCl solution for infusion bags, 250 ml is Fresenius Kabi, Germany.

4.1.1.4 Sponsored product

The Sponsor will supply Actovegin® in ampoules (400 mg/10 ml) and tablets (200 mg), placebo ampoules and placebo tablets. In addition, the sponsor will supply infusion bags containing 250 ml of 0.9% NaCl solution for infusion, oversleeves and accessory labels for the infusion bags.

4.1.2 Storage

Investigational drug, i.e. Actovegin® ampoules (400 mg/10 ml), Actovegin® tablets (200 mg), placebo ampoules, placebo tablets and auxiliary materials i.e. NaCl 0.9% solution for infusion bags, protective bags and accessory labels must be stored in an appropriate, secure, restricted area until used or returned to the sponsor or designated person for destruction. The study drug and excipients should be stored under the conditions specified on the label (15°C to 25°C), protected from light, and kept in their original packaging until dispensed. For more information, see the Pharmaceutical Guide. The daily temperature log of the drug storage area must be completed every working day.

4.1.3 Dose and regimen

4.1.3.1 Period of IV treatment (weeks 0-2)

Each patient who meets the eligibility criteria will be randomized to receive either Actovegin® or placebo using an interactive online communication system. Following randomization (Visit 1, Day 0), patients will receive 3 ampoules of either Actovegin® 400 mg/10 ml or placebo/10 ml in 250 ml 0.9% NaCl (total infusion volume 280 ml) as a single infusion. The infusion rate will be approximately 2 ml per minute (40 to 50 drops per minute). Patients will receive 1 infusion per day for 14 days. The investigator or designated person must ensure that the drug supplied by the sponsor is prepared and administered in accordance with the Pharmaceutical Manual.

PI infusions should be administered at the study center under medical supervision. During the first 15 minutes of the first infusion, the investigator (or designated physician) should monitor for possible allergic reactions and take prompt action to prevent or treat them.

4.1.3.2 Period of oral therapy (weeks 2-12)

At the end of the IV treatment period (Visit 2), patients will be given a box of 4 bottles containing 200 tablets of Actovegin® 200 mg or placebo. Patients will be instructed to take 2 tablets of Actovegin® 200 mg or placebo TID, for a total of 6 tablets per day. Patients will receive study drug for 4 weeks of treatment and will be reminded to bring their study drug bottle (including unused tablets) to their next visit.

At the third visit (Visit 3), the patient will be given another box of 4 bottles containing a total of 200 tablets of Actovegin® 200 mg or placebo for an additional 4 weeks of treatment. Patients will be instructed to take 2 tablets of Actovegin® 200 mg or placebo TID, for a total of 6 tablets per day. The patient will also be reminded to bring the study drug bottle (including unused tablets) to the next visit.

At the fourth visit (Visit 4), the patient will be given the last box containing 2 bottles, a total of 100 tablets of Actovegin® 200 mg or placebo, for 2 weeks of treatment. Patients will be instructed to take 2 tablets of Actovegin® 200 mg or placebo TID, for a total of 6 tablets per day. The patient will also be reminded to bring the study drug bottle (including unused tablets) to the next visit (Visit 5).

Patients will then enter the follow-up phase where they will not receive any study drug.

Table 2 describes the dose and number of ampoules/tablets to be given to each group.

4.1.4 Overdose

An overdose is defined as the known intentional or accidental administration of an investigational drug, to a study patient or study patient, at a dose in excess of the dose administered to that particular patient in accordance with the study protocol.

All cases of overdose (with or without associated AEs) will be documented on the eCRF Overdose page in order to consistently capture this important safety information in the database.

Cases of overdose without obvious signs or symptoms are not considered AEs. AEs associated with overdose will be documented in the AE section of the eCRF.

Serious adverse events (SAEs) associated with overdose are subject to mandatory reporting.

There is no experience of an overdose of Actovegin®. According to a non-clinical study, Actovegin® does not show a toxic effect at doses up to 30-40 times higher than the dose recommended for human use.

There are no specific instructions for the treatment of overdose. Based on the pharmacology of the drug, undesirable effects are not expected.

4.2 Prescribing and dosing procedures

The Investigator, or Investigator's designee, logs into the Interactive Communications System (IRT) at screening to obtain a patient identification number for the study ("Patient Number").

The investigator or investigator's designee will use the IRT system to randomize a patient in the study. During this communication, the investigator or designee will provide the necessary information to identify the patient, including the patient number assigned at screening.

During dispensing visits, the investigator or designee will contact the IRT system again to record visits. At visits 1, 2, 3, and 4, all patients in both treatment groups will be assigned a drug identification number (Med ID) by the IRT system, which will be provided to the center's pharmacist/nurse from whom the information is not masked, by e-mail.

To maintain blinding, the IRT system provides that the investigator or designee is not aware of the assigned Med IDs.

If a sponsor-supplied drug is lost or damaged, the pharmacist/nurse of the non-masked center may request a replacement through the IRT system.

4.3 Generating and storing the randomization code

The Takeda Randomization Officer or designee will generate a randomization schedule for the study; the IRT system will be used centrally to randomize patients and prescribe study medication.

All randomization information will be stored in a secure area accessible only to authorized personnel.

4.4 Maintaining study drug masking

Investigational drug masking will be maintained using the IRT system. The Principal Investigator at each center will be instructed to receive medication orders through the IRT system.

Information regarding the type of therapy will be hidden (masked) from researchers, clinical center staff involved in patient care or clinical examinations, statistical group, data management group and patients, until the database is locked. Information will not be masked from pharmacy staff (nurses, pharmacists). All infusion bags and infusion sets will be sealed and the sealed material will not allow unauthorized opening to maintain the masking.

Study staff designated by the center will store drug information in blind mode.

During regular scheduled follow-up visits, the monitor, from whom the information is not masked, from the sponsor or designated person, will perform an inventory of boxes/bottles of undistributed and dispensed investigational drug. All unallocated and allocated packages will be reconciled and returned to the sponsor or designee prior to termination of the study.

4.5 Unmask procedure

Investigational drug masking should not be violated by the investigator unless information about the investigational product is required for patient care. If possible, the sponsor/designee should be notified prior to unmasking the product. In the event of an emergency requiring immediate release, the investigator (or designee) at the center will contact the sponsor or designee to assess the need for release.

To unmask information about the drug prescribed for a particular patient, it can be obtained by contacting the IRT system. If the product is unmasked, the sponsor/nominee should be notified immediately. The date, time and reason for unmasking must be recorded in the original documents and in the appropriate section of the eCRF.

If any staff member of the center has received unmasked information about the drug prescribed for a particular patient, the study drug should be immediately discontinued and the patient should be withdrawn from the study. The reason for the withdrawal should be recorded as "Major protocol deviation".

4.6 Reporting and destruction of sponsored products

Drugs must be accounted for and reconciled at the center before being returned to the sponsor or designated person.

The investigator or designated person must ensure that the sponsored drug (Actovegin®/placebo ampoules, Actovegin®/placebo tablets) and auxiliary material (0.9% NaCl solution for infusion, protective bags and accessory labels) are used in accordance with protocol and were issued only to patients included in the study. To document the proper use of a sponsored drug, the investigator or designee must maintain a record of all deliveries of the sponsored drug to the center, center inventory, dispensing and use for each patient, and return to the sponsor or designee.

Upon receipt of the sponsored medicinal product, the investigator or designated person should check the contents of the shipment with the packing list. The inspector must ensure that the quantity matches and that the medicine is in good condition. If the quantity and condition are acceptable, the investigator or designee must acknowledge receipt of the shipment by signing the bottom half of the packing list and emailing/faxing it in accordance with the instructions provided on the form/by registering with the IRT system. If there is any discrepancy between the packing list and the actual product received, Takeda or the designated person should be contacted to resolve the issue. The packing list must be included in the investigator's main document file.

The investigator or designee must maintain 100% records of all drugs received from the sponsor and dispensed during the entire duration of the sponsor's participation in the study. Proper drug reporting includes, but is not limited to:

Continuous monitoring of expiration dates (via the IRT system).

Regular reconciliation of actual stocks with documents.

Checking the completion of the journal indicating the Identifier / other data of the drug used to prepare each dose.

Verify that all used containers are documented correctly in the log.

Checking the correctness and readability of the filled fields.

A representative from a non-masked center or a non-masked monitor will review the randomization schedule and patient dosing log to ensure that all patients receive the appropriate dose of study drug.

If any errors or inconsistencies are found, the sponsor should be notified immediately.

The IRT system will contain all the necessary information in a separate record for each patient who is given a sponsored drug.

The investigator or designee must record the current stocks of all sponsor-supplied drugs in a sponsor-approved drug log book. At a minimum, the following information will be recorded: protocol number and title, investigator name, site ID and number, description of sponsored drugs, expiration date, and quantity dispensed, including the initials, seal or signature of the dispenser, and the date and quantity returned. to the center by the patient, including the initials, seal, or signature of the recipient of the sponsored drug. The journal should contain all the necessary information in the form of a separate entry for each patient (by patient ID) who is given a drug supplied by the sponsor.

The investigator or designated center staff member who administers the investigational drug (Actovegin®/placebo infusion) must complete an individual patient reporting log to document whether the infusion was or was not performed and the drugs were returned to the pharmacy, including date and amount, returned to the pharmacy, including the initials, seal or signature of the person performing the infusion.

All non-returns of an investigational drug to a center by a patient should be investigated at the center and properly documented in the drug log.

Prior to center closure or at regular intervals, the sponsor's representative or designee will report and verify the sponsor's supplied drug prior to returning the drug to the sponsor or designee for destruction. The investigator or designee will retain a copy of the documentation relating to reporting, return and/or disposal of the sponsored drug and the originals will be sent to the sponsor or the sponsor's designee.

During the course of the study, the investigator will be notified of the expiration date of the sponsored medicinal product. Upon receipt of an expiration notice from the sponsor or designee, the center must comply with all instructions in the notice, including isolating the expired drug provided by the sponsor for return to the sponsor or designee for destruction.

5. RESEARCH PLAN

5.1 Study procedures

The following sections describe the survey procedures and the data that will need to be collected. For each procedure, patients should be evaluated, if possible, by the same investigator or center staff. A schedule of study procedures is shown in Table 1 above.

5.1.1 Informed consent procedure

Informed consent must be obtained prior to enrolling a patient in the study and before performing any protocol procedures.

Each patient will be assigned a unique patient identification number (patient number) at the time informed consent is obtained; this patient number will be used throughout the study.

5.1.2 Procedure for collecting demographic data, clinical history and drug history

The demographic information collected will include date of birth, gender, race reported by the patient, height, weight, and smoking status of the patient at screening.

The medical history collected will include a determination of whether the patient has any significant conditions or illnesses relevant to the study disease that resolved at or before informed consent was signed, including a psychiatric history (any psychiatric disorder is an exclusion criterion). Current pathologies are considered comorbidities (section 5.1.9).

Drug history information collected includes any drugs relevant to selection criteria (section 4.1.1.2) discontinued 14 days prior to randomization (day 0).

5.1.3 Physical examination procedure

The baseline physical examination (defined as evaluation prior to the first dose of study drug) will include the following body systems: (1) eyes; (2) ears, nose, throat; (3) cardiovascular system; (4) respiratory system; (5) gastrointestinal system; (6) dermatological system; (7) limbs; (8) musculoskeletal system; (9) nervous system; (10) lymph nodes; and (11) other. All subsequent physical examinations should evaluate for clinically significant changes from those assessed prior to the first dose.

5.1.4 Weight and height

Weight and height of patients should be measured in room clothes and without shoes. Values must be specified with 1 decimal place.

5.1.5 Smoking status

Smoking status will be classified as follows:

1. Never smoked.

2. Active smoker (information is collected on the number of years of smoking and the current number of cigarettes per day).

3. Former smoker (information is collected on the number of years of smoking, the approximate number of cigarettes previously smoked per day, and the date of quitting).

For the purposes of the study, e-cigarette use should not be considered in determining smoking history. One gram of tobacco = 1 cigarette.

Smoking status will be checked at each visit.

5.1.6 Procedure for determining vital signs

Vital signs include blood pressure (systolic and diastolic) and pulse (beats/minute) after at least 5 minutes of rest in a sitting position. When a vital sign collection is scheduled at the same time as a blood draw, the blood draw will be prioritized and the vital sign collection should be performed 0.5 hours before or after the scheduled blood draw.

5.1.7 Performance measurements

5.1.7.1 Treadmill testing

A treadmill is a device used to walk or run in place. In this study, this method will be used to determine distances covered with lameness. Distance covered with lameness will be measured using a constant workload protocol (3.0 km/h constant speed and 10% incline on the treadmill).

From the moment of signing the informed consent, the treadmill test will be performed twice. The first test must be performed during the screening period. A second treadmill test should be repeated at intervals of =1 to =2 weeks and should be performed on Day 0 prior to randomization.

Patients with high variability in ACD values [>25% for maximum walking distance with lameness] will be excluded. If a patient is randomized to study therapy, Day 0 test results will be used as baseline.

5.1.7.2 SF-36

The SF-36 is a 36-item patient-reported health-related quality of life measurement tool. SF-36 has previously been extensively evaluated and tested with different populations and allows distinguishing between groups with different quality of life status in clinical trials [24, 25]. The SF-36 consists of 8 sections (vitality, physical functioning, pain intensity, general health, role physical functioning, role emotional functioning, role social functioning, and mental health), which are weighted sums of questions in each health domain. Each scale is directly converted to a scale from 0 to 100, based on the assumption that each question carries equal weight. The lower the score, the greater the disability, and the higher the score, the less disability, i.e. a score of 0 is equivalent to maximum disability, and a score of 100 is equivalent to no disability.

A validated version of the SF-36 in the respective language will be available for each participating country; each patient must be provided with a version of the SF-36 in their own language. Patients must complete the SF-36 at Visits 1, 5, and 8 (or end-of-treatment visit) prior to any study-related procedures. The researcher must check that all questions are answered.

5.1.8 Documentation of related procedures and medications

A concomitant drug is any drug given in addition to an investigational drug. They can be prescribed by a doctor or purchased by a patient without a prescription. The co-medication is not provided by the sponsor. Patients will be asked at each visit if they have taken any medications other than the study drug (used from the time of signing the informed consent until the end of the study), and all medications, including vitamin supplements, over-the-counter medications, and oral herbal medications, must be recorded in the eCRF. In addition, all related procedures will be taken into account and recorded in the eCRF.

5.1.9 Documentation of medical comorbidities

Comorbidities are those significant current conditions or illnesses that are present at the time of signing the informed consent. This includes clinically significant laboratory, ECG, or physical examination abnormalities noted at the screening examination, as determined by the investigator.

Color duplex ultrasonography will be used at screening to confirm the diagnosis of PAD and rule out other pathology that may lead to peripheral arterial occlusion. The location of narrowing and occlusion of the artery will also be identified, and the aortoiliac or femoropopliteal lesion will be documented on an individual record card. This examination will be carried out on certified equipment by a qualified ultrasound specialist in accordance with generally accepted methodology. This survey will be conducted in accordance with local routine practice. The results (at least a written opinion) should be kept together with other source documents.

5.1.10 Procedures for clinical laboratory specimens

All specimens will be taken on an empty stomach and in accordance with acceptable local laboratory procedures. The maximum volume of blood collected at each visit will be approximately 10 ml, and the approximate total volume of blood collected during the study will be 30 ml.

Timing of sample collection for clinical laboratory testing is presented in Table 1 and Table 3.

(a) HbA1c at screening for study eligibility.

(b) If the values are above the normal range, the determination of direct and free bilirubin is necessary.

(c) At screening.

Laboratory tests for hematology, serum biochemistry and urinalysis will be performed by the local laboratory. Laboratory test results will be returned to the investigator, who is responsible for reviewing and archiving those results.

If a patient has ALT or AST > 3 x ULN, additional laboratory tests (at least serum alkaline phosphatase, ALT, AST, total bilirubin and gamma-GTP) should be performed for a maximum of 7 days and preferably within 48-72 hours after an abnormality is detected (see section 3.5 for appropriate guidance on reporting abnormal liver function test results).

The investigator should keep a copy of the laboratory's accreditation and reference ranges for the laboratory used at all times.

5.1.11 Procedure for contraception and prevention of pregnancy

5.1.11.1 Male patients and their partners

From the time of signing the informed consent and throughout the study or within 30 days after the last dose of study drug (in case of early termination of the study), unsterilized male patients who are sexually active with a partner of childbearing potential must use barrier contraception (eg, a condom with with or without spermicidal cream or gel). In addition, they should not be sperm donors during this period. Women of childbearing potential who are partners with male patients are also recommended to use supplemental contraception, according to the list of highly effective/effective contraception (section 5.1.11.3).

5.1.11.2 Female patients and their male partners

From the time of signing the informed consent and throughout the study or within 30 days after the last dose of study drug (in case of early termination of the study), women of childbearing potential who are sexually active with an unsterilized male partner should use a highly effective/effective method of contraception ( section 5.1.11.3).

In addition, they should not be egg donors during this period.

5.1.11.3 Definitions and procedures for contraception and prevention of pregnancy

The following definitions apply to contraceptive and pregnancy prevention procedures.

Postmenopausal status is defined as the absence of menstruation for 12 months without an alternative medical reason. High levels of follicle-stimulating hormone (FSH) in the postmenopausal range (FSH > 40 IU/L) may be used to confirm postmenopausal status in young women (eg, those under 45 years of age) or women not using hormonal contraception or hormone replacement therapy.

However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient.

Sterilized males must be at least 1 year post-bilateral vasectomy, with documented ejaculate-free semen, or undergo bilateral orchidectomy.

The following procedures are used for contraception and pregnancy prevention.

1. Highly effective contraceptive methods are defined as methods that, alone or in combination, produce a low failure rate (i.e. less than 1% of conceptions per year with consistent and correct use). In this study, which includes medications and devices containing hormones, the accepted highly effective methods of contraception are:

Non-hormonal methods:

o Intrauterine device.

o Bilateral tubal occlusion.

o Vasectomized partner (provided that this partner is the study participant's only sexual partner and that the vasectomy partner has obtained a medical report that the operation was successful).

Hormonal methods:

Combination (estrogen and progestogen) hormonal contraception associated with ovulation inhibition started at least 3 months before the first dose of study drug OR in combination with a barrier method (male condom, female condom, or diaphragm), at a shorter duration of hormonal contraception, until this period will not reach 3 months:

- Oral

- Intravaginal (eg ring)

- Transdermal

Progestogen-only hormonal contraception associated with ovulation suppression started at least 3 months prior to the first dose of study drug OR in combination with a barrier method (male condom, female condom, or diaphragm), for a shorter duration of hormonal contraception, until then will not reach 3 months:

- Oral

- Injectable

- Implantable

2. Effective contraceptive methods are defined as methods that provide a low failure rate, which can be higher than 1% of conceptions. In this study, which includes medications and devices containing hormones, the accepted effective methods of contraception are:

Double barrier method (contraceptive sponge, diaphragm or cervical cap with spermicidal gels or creams PLUS a male condom).

Progestogen-only hormonal contraception where ovulation inhibition is not the primary mode of action PLUS a condom with or without spermicide.

3. Unacceptable methods of contraception are:

Periodic abstinence (for example, calendar, ovulation, symptomatic, postovulation methods).

Only spermicides.

Interrupted intercourse.

Lack of any method.

Sharing female and male condoms.

Cap/diaphragm/sponge without spermicide and without condom.

Sexual abstinence is NOT an acceptable method of contraception.

4. Patients will be provided with information on highly effective and effective contraceptive methods during the patient informed consent process and will be asked to sign a consent form stating that they understand the requirements for preventing pregnancy, egg donation and sperm donation during the course of the study.

5. The study will conduct regular pregnancy tests for hCG urine, only in women of childbearing potential, and all patients (male and female) will receive reminder guidelines for preventing pregnancy and sperm donation as part of the study procedures. Such guidance should include a reminder of the following:

a. Research requirements for contraception.

b. Assess patient compliance with these requirements using the following questions:

I. Have you used contraception consistently and properly since your last visit?

II. Have you forgotten to use contraception since your last visit?

III. Do you have a delay in menstruation? (if the answer is yes, even women with irregular or slow menstrual cycles should have a pregnancy test done)

IV. Is there a possibility that you may be pregnant?

6. In addition to a negative serum hCG pregnancy test at female screening, patients of childbearing potential must also confirm menstruation within the month prior to first dosing (no missed period) and show a negative hCG pregnancy test at the randomization visit (baseline visit). ) prior to receiving any dose of study drug.

5.1.12 Pregnancy

If pregnancy is detected in any patient during the course of the study, her participation in the study and the use of any drug supplied by the sponsor should be stopped immediately. In addition, any pregnancies in the partner of a male patient during the course of the study or within 30 days of the last dose (in the event of an early termination of the study) must also be reported after obtaining permission from the patient's partner.

If pregnancy occurs while taking study drug, such as after Visit 1 or within 30 days of the last dose of study drug, the pregnancy should be reported immediately using the pregnancy notification form.

If pregnancy occurs during or after the use of a blinded drug, the investigator should inform the patient of his right to receive information about the treatment received. If the patient chooses to receive information about an unmasked treatment, the investigator must disclose the treatment.

If the female patient and/or partner of the male patient agrees to informing their PCP, the investigator should notify the PCP that the female patient or partner of the male patient was participating in the clinical trial at the time of the pregnancy and provide him with details of study drug received by the patient (blinded or open, as appropriate).

All pregnancies, including pregnancies of female partners of a male patient, in patients treated with the active drug will be followed up to their outcome using the pregnancy form. Pregnancies will remain blind to the research team. The outcome of the pregnancy, including any early termination, must be communicated to the sponsor. There will also be an assessment after the baby is born.

5.1.13 ECG procedure

A standard 12-lead ECG will be recorded. The investigator (or qualified observer at the study site) will interpret the ECG according to one of the following categories: within normal limits, abnormal but not clinically significant, or abnormal and clinically significant. A copy of the ECG should be kept with the patient's original study documentation.

5.1.14 Documentation of non-screeners

Investigators should consider all patients who have signed informed consent. If a patient is excluded from the screening visit, the investigator must complete the eCRF.

The data that should be collected in the eCRF for non-screeners includes at a minimum: patient number and initials, date of informed consent, demographic data, inclusion/exclusion criteria, PTE/SAE, date of non-screening.

The root cause of screening failure is recorded in the eCRF using the following categories:

• PTE/AE.

• Not meeting inclusion criteria or meeting exclusion criteria <specify reason>.

• Significant protocol deviation.

• Impossibility of follow-up due to loss of communication with the patient.

• Voluntary withdrawal < state reason >.

• Termination of the study.

• Other <specify reason>.

Patient IDs assigned to non-screeners should not be reused.

5.1.15 Documenting randomization

Only patients who meet all of the inclusion criteria and none of the exclusion criteria may participate in randomization to treatment.

If a patient is determined to be ineligible for randomization, the investigator should record the root cause of rejection in the appropriate section of the eCRF.

5.2 Monitoring patient compliance

During the IV treatment period, daily IV infusions will be recorded in the infusion log. The following data will be recorded:

• Infusion set data.

• Date of infusion.

• Start time.

• Termination time.

• The initials of the person who administered the ILP.

• Infusion completed successfully; Yes or no.

• Comments.

During the oral treatment period, the investigator should check patient compliance by counting the number of returned tablets at the time points specified in the clinical study review. Patients will be required to bring their assigned bottles of investigational drug to each visit.

Treatment adherence will be calculated by the investigator at Visits 3, 4, and 5 using the formula:

Compliance with the regime in %=

(number of tablets taken since last visit/number of tablets to be taken since last visit)*100

This data will subsequently be entered into the eCRF by the center's staff.

In the event of a deviation from the treatment regimen, the patient should be re-instructed regarding dosing requirements at the time of contact with the investigator. Authorized study personnel conducting the retraining should document this process in the original records.

If treatment compliance is outside the 70%-130% range for the treatment period since the last visit, it should be reported as a protocol deviation.

If a patient consistently fails to comply with the study drug regimen (eg, compliance for two consecutive visits outside the range of 70% to 130%), it may be appropriate (based on the investigator's judgment) to withdraw the patient from the study.

5.3 Schedule of observations and procedures

A schedule of all study-related procedures for all assessments is shown in Table 1. Assessments must be completed at the scheduled visit/time points.

5.3.1 Screening period

Screening is considered started from the moment of signing the ICF. Patients should be screened within 1-2 weeks prior to randomization. Patients will be screened according to predefined inclusion and exclusion criteria as described in Section 3. See Section 5.1.14 for procedures for documenting non-screeners.

The following procedures are provided during screening (Visit 0):

• Informed consent.

• Demographic data.

• History and comorbidities.

• Drug history.

• Concomitant medications.

• PTE.

• Physical examination.

• Vital Signs.

• Smoking status and exercise habits.

• ECG in 12 leads.

• Clinical laboratory tests.

• Urine pregnancy test (only for women of childbearing potential) and oral confirmation of menstruation within a month before the first dosing (without delaying menstruation).

• Ultrasonic color duplex scanning.

• Treadmill testing (first testing during the screening period).

• Criteria for inclusion/exclusion.

5.3.2 Randomization (baseline), Day 0, Visit 1

Randomization will take place on Day 0.

The following procedures are foreseen at randomization (Visit 1):

• Criteria for inclusion/exclusion.

• SF-36 (before all procedures).

• PTE.

• Smoking status and exercise habits.

• Treadmill testing (the second test during the screening period to verify that the results of both tests do not differ by more than 25%) (sections 3.2 and 5.1.7.1, table 1).

• Urine pregnancy test (only for women of childbearing potential).

• Assessment of the patient's compliance with contraceptive requirements.

• Physical examination.

• Vital Signs.

• Weight and height.

• Concomitant medications.

• Drug history.

If a patient meets all of the inclusion criteria and none of the exclusion criteria for randomization, the patient should be randomized using the IRT system as described in section 4.2.

5.3.3 Period of treatment with infusions (i.v.)

After randomization, patients enter a period of IV treatment (Day 0, Visit 1 - Day 14, Visit 2), during which they receive daily infusions of Actovegin® or placebo (up to 14 infusions).

PI infusions should be performed at the study center under medical supervision. During the first 15 minutes of the first infusion, such monitoring should be carried out by the Investigator (or designated physician) in order to immediately detect possible allergic reactions and take urgent measures to prevent or treat them.

The following data will be collected and recorded during the IV treatment period:

• AE (day 0 after first infusion). Starting on Day 1, AEs should be checked before (AE since last visit) and after the infusion.

• Concomitant medications.

• Smoking status and exercise habits.

• Urine pregnancy test (only for women of childbearing potential).

• Assessment of patient compliance with contraceptive requirements.

5.3.4 End of IV treatment, Day 14, Visit 2

The end of IV treatment is defined as the day of the last infusion (Day 14) and before the start of oral treatment. Assessments required at this visit should be done after the last infusion.

An IV end-of-treatment visit and subsequent transition to oral treatment can be performed either while the patient is still hospitalized or after discharge, depending on the patient's need.

The following procedures are included in the End of IV Treatment Visit (Visit 2):

• AEs (before infusion [AE since last visit] and after infusion).

• Concomitant medications.

• Smoking status and exercise habits.

• Testing on a treadmill.

• Urine pregnancy test (only for women of childbearing potential).

• Assessment of patient compliance with contraceptive requirements.

• Issuance of an investigational drug for oral treatment.

5.3.5 Period of oral treatment

The oral treatment period is defined as the period from the day after the last infusion to 12 weeks after randomization (days 15-84). During the oral treatment period, the patient should visit the center on Day 42 (± 2 days), Visit 3 and Day 70 (± 2 days), Visit 4.

The following treatments are foreseen during the oral treatment period (Visits 3 and 4):

AE (since last visit).

Concomitant medications.

Smoking status and exercise habits.

Urine pregnancy test (only for women of childbearing potential).

Assessment of patient compliance with contraceptive requirements.

Issuance of an investigational drug for oral treatment.

Returning unused study drug for oral treatment.

Verify patient compliance with study medication regimen.

5.3.6 End of Oral Treatment, Day 84, Visit 5/Early Stop Visit

The oral treatment end visit will take place on the last day of oral administration 12 weeks later (Day 84 ± 2 days). This will also be an Early Discontinuation Visit for patients who withdraw from the study before the completion of the oral treatment period.

The following treatments are provided for at the End of Oral Treatment Visit (Visit 5):

• AE (since last visit).

• Physical examination.

• Vital Signs.

• The weight.

• Concomitant medications.

• Smoking status and exercise habits.

• SF-36 (before all procedures).

• Clinical laboratory tests.

• Urine pregnancy test (only for women of childbearing potential).

• Assessment of patient compliance with contraceptive requirements.

• Testing on a treadmill.

• Returning an unused study drug for oral treatment.

• Verification of patient compliance with the study drug regimen.

5.3.7 Follow-up period

The 12-week follow-up period will begin on the first day after the oral treatment end visit (Visit 5). During this period, patients will visit the center every 4 weeks, on Day 112 (± 5 days), Visit 6 and Day 140 (± 5 days), Visit 7.

During each follow-up visit, the following data will be collected (Visits 6 and 7):

AE (since last visit).

Concomitant medications.

Smoking status and exercise habits.

Urine pregnancy test (only for women of childbearing potential).

Assessment of patient compliance with contraceptive requirements.

5.3.8 Final Follow-up Visit/Early Termination Visit

The final follow-up visit will take place on the last day of the follow-up period (Day 168 ± 2 days). This will also be an early termination visit for patients who leave the study during the follow-up period.

The final follow-up visit (Visit 8) includes the following:

AE (since last visit).

Physical examination.

Vital signs.

The weight.

Concomitant medications.

Smoking status and exercise habits.

Clinical laboratory tests.

Urine pregnancy test (only for women of childbearing potential).

Assessment of patient compliance with contraceptive requirements.

Treadmill testing.

SF-36 (before all procedures).

For all patients who received investigational drug, the investigator must complete the end of study page in the eCRF.

5.3.9 Post-study therapy

The study drug will not be available after the patient's participation in the study ends. The patient should be returned for treatment to their doctor, with standard treatments continued as needed.

6 STATISTICAL METHODS

6.1 Statistical and analytical plans

A Statistical Analysis Plan (SAP) will be prepared and finalized prior to the release of treatment for patients. This document will provide additional information regarding the definition of analysis variables and analysis methodology to address all research objectives.

The analysis of masked data will be carried out before the treatment prescribed for patients is unmasked. This review will assess the accuracy and completeness of the study database, the ability to evaluate patients, and the adequacy of the planned statistical methods.

6.1.1 Analysis populations

The complete analysis population (FAS) will include all patients who were randomized, received at least 1 dose of study drug, and have at least 1 significant value after baseline for the primary endpoint. In the FAS performance summaries and analyzes, patients will be analyzed according to the type of treatment to which they were randomized.

The safety evaluation population will include all patients who were randomized and received at least 1 dose of study drug in a double-blind manner. In the safety briefs, patients will be analyzed according to the treatment they actually received. In the event that a patient receives more than one type of treatment, the actual treatment will be determined as the most commonly used. If the two most common treatments are used with equal frequency, then randomized treatment will be used as the actual treatment.

6.1.2 Analysis of demographics and other background characteristics

Baseline characteristics will be listed and summarized by demographics (sex, age, and race), physical examination data, and medical history, including psychiatric history, smoking history, diabetic status, and concomitant medications.

Baseline values for efficacy and safety parameters will be provided in standard tables summarizing visit data; however, baseline efficacy data will also be presented separately for all randomized patients.

For continuous variables, the comparability of treatment groups will be assessed using analysis of variance, with treatment and study center as factors. For discrete variables, comparability will be assessed using the overall Cochran-Mantel-Haensel relationship stratified by centers. P-values will be given as descriptive comparability statistics.

6.1.3 Performance analysis

Primary and secondary endpoints will be analyzed in all randomized patients who received at least one dose of study drug.

Percent change in ICD from baseline and change in SF-36 score from baseline will be analyzed using a mixed model for repeated measures covariance analysis, with treatment, center, week, treatment interaction by week, baseline value interaction by week as fixed effects, the original value as a covariate, and a fully unstructured covariance matrix. Comparisons between Actovegin® and placebo will be made at all evaluation points. Based on the assumption of random omissions, this analysis will be carried out using data from observed cases only. The effect at each time point for each treatment is free to vary, and an unstructured covariance matrix is assumed.

The proportion of patients with pain at rest and the proportion of patients undergoing revascularization will be analyzed at all time points by treatment-adjusted logistic regression.

6.1.4 Security analysis

6.1.4.1 AEs

AE reports will be provided throughout the study.

A definition of AEs occurring during treatment will be provided by SAP. AEs will be coded using MedDRA and summarized by system and organ class and preferred term over the main treatment period and throughout the study.

AEs repeatedly identified in a patient during the same period will be counted only once for that patient and the period with the highest severity.

6.1.4.2 Clinical assessments

Absolute values and changes from screening/baseline in clinical laboratory safety tests, vital signs and weight will be summarized for each treatment group using descriptive methods. Values outside the normal ranges and potentially clinically significant values will be specifically flagged and tabulated. Physical examination results will also be summarized for each treatment group.

6.2 Interim analysis and early termination criteria

Interim analysis is not planned.

6.3 Determining the sample size

Assuming an 85% standard deviation for ICD percent change from baseline and a 20% dropout rate, a total of 366 patients (183 per treatment group) is sufficient to achieve a minimum of 80% power to detect a 28% difference in ICD percent change from baseline between Actovegin® and placebo using a two-sample t-test with a two-sided significance level of 0.05.

The rationale for the sample size is based on published data for the ICD for naftidrofuril, which show that the difference in percentage change in ICD from baseline between naftidrofuril and placebo is approximately 28%, while the corresponding standard deviation is approximately 85% [‎26].

List of some documents cited in the description

1. Bordeaux L, Reich L, Hirsch A. The epidemiology and natural history of peripheral arterial disease. In: Coffman JD, Eberhardt RT, editors. Contemporary Cardiology: Peripheral Arterial Disease: Diagnosis and Treatment. Totowa, NJ: Humana Press Inc.; 2010, p. 21-34.

2. Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott MM, et al. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis. Lancet 2013;382(9901):1329-40.

3. Dhaliwal G, Mukherjee D. Peripheral arterial disease: epidemiology, natural history, diagnosis and treatment. Int J Angiol 2007;16(2):36-44.

4. Buchmayer F, Pleiner J, Elmlinger MW, Lauer G, Nell G, Sitte HH. Actovegin®: a biological drug for more than 5 decades. Wien Med Wochenschr 2011;161(3-4):80-8.

5. Kuninaka T, Senga Y, Senga H, Weiner M. Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract. J Cell Physiol 1991;146(1):148-55.

6. Bachmann W, Forster H, Mehnert H. Experimental studies in animals on the effect of a protein-free blood extract on the metabolism of glucose [in German]. ArzneimittelForschung/Drug Research 1968;18:1023-7.

7. Jacob S, Dietze GJ, Machicao F, Kuntz G, Augustin HJ. Improvement of glucose metabolism in patients with type II diabetes after treatment with a hemodialysate. Arzneimittelforschung 1996;46(3):269-72.

8. Meilin S, Machicao F, Elmlinger M. Treatment with Actovegin® improves spatial learning and memory in rats following transient forebrain ischaemia. J Cell Mol Med 2014;18(8):1623-30.

9. Elmlinger MW, Kriebel M, Ziegler D. Neuroprotective and anti-oxidative effects of the hemodialysate Actovegin® on primary rat neurons in vitro. Neuromolecular Med 2011;13(4):266-74.

10. Machicao F, Muresanu DF, Hundsberger H, Pfluger M, Guekht A. Pleiotropic neuroprotective and metabolic effects of Actovegin®'s mode of action. J Neurol Sci 2012;322(1-2):222-7.

11. Dieckmann A, Kriebel M, Andriambeloson E, Ziegler D, Elmlinger M. Treatment with Actovegin® improves sensory nerve function and pathology in streptozotocin-diabetic rats via mechanisms involving inhibition of PARP activation. Exp Clin Endocrinol Diabetes 2012;120(3):132-8.

12. Fedorovich A.A. Non-invasive evaluation of vasomotor and metabolic functions of microvascular endothelium in human skin. Microvasc Res 2012;84(1):86-93.

13. Herrmann WM, Bohn-Olszewsky WJ, Kuntz G. Actovegin infusion treatment in patients with primarily degenerative dementia of the Alzheimer type and multi-infarct dementia 46-55. Zeitschrift für Geriatrie 1992;5:46-55.

14. Kanowski S, Kinzler E, Lehmann E, Schweizer A, Kuntz G. Confirmed clinical efficacy of Actovegin® in elderly patients with organic brain syndrome. Pharmacopsychiatry 1995;28(4):125-33.

15 Guekht A, Skoog I, Edmundson S, Zakharov V, Korczyn AD. ARTEMIDA Trial (A Randomized Trial of Efficacy, 12 Months International Double-Blind Actovegin®): A Randomized Controlled Trial to Assess the Efficacy of Actovegin® in Poststroke Cognitive Impairment. Stroke 2017;48(5):1262-70.

16. Ziegler D, Movsesyan L, Mankovsky B, Gurieva I, Abylaiuly Z, Strokov I. Treatment of symptomatic polyneuropathy with Actovegin® in type 2 diabetic patients. Diabetes Care 2009;32(8):1479-84.

17 Gurieva IV, Begma IV, Kuzina IV, et al. [Actovegin pathogenetic treatment of neuroischemic pain syndrome in the lower extremities in diabetes mellitus. (In Russ.)]. RMJ 2009;17:8-11.

18. Angelkort B, Blume J, de la Haye R, Kuntz G. Metabolically active calves blood extract in welltrained patients with AVD in stage II. A placebo-controlled doubleblind study. VASA - J Vasc Dis 1988(Suppl 23):141-3.

19. Horsch S, Claeys L, Diehm C, Rieger H, Cachovan M, Baitsch C. [Assessment of the clinical effectiveness of actihaemyl in Fontaine stage IIb intermittent claudication]. Vasa Suppl 1992;37:64-5.

20. Muller-Buhl U, Ruhlmann KU, Meister K, Diehm C, Kuntz G. Increase of walking distance by intra-arterial infusions of hemodialysate. Therapiewoche 1991;41:188-96.

21. Angelkort B, Ruhlmann KU, de la Haye R, Kuntz G. Influence of deproteinized hemodialysate on rest pain and walking distance in the presence of peripheral chronic arterial occlusive disease. Angiology 1992;43(1):47-58.

22. Blume J. Peripheral arterial occlusive disease - Intravenous and intraarterial infusion therapy with Actovegin® (In German). Therapiewoche 1986;36:5355-58.

23. Uchkin IG, Zudin AM, Bagdasarian AG, Fedorovich AA. Effect of drug therapy for chronic obliterating diseases of lower-limb arteries on the state of the microcirculatory bed. Angiol Sosud Khir 2014;20(2):27-36.

24. Garratt AM, Ruta DA, Abdalla MI, Buckingham JK, Russell IT. The SF36 health questionnaire survey: an outcome measure suitable for routine use within the NHS? BMJ 1993;306(6890):1440-4.

25. Ware J, Kosinski M. Improvements in the content and scoring of the SF-36 Health Survey Version 2. Accessed 16 July 2014. Available at: http://www.sf-36.org/.

26. Adhoute G, Bacourt F, Barral M, Cardon JM, Chevalier JM, Cuny A, et al. Naftidrofuryl in chronic arterial disease. Results of a six month controlled multicenter study using Naftidrofuryl tablets 200 mg. Angiology 1986;37(3 Pt 1):160-7.

Claims (18)

1. Use of a deproteinized bovine blood preparation for the prevention or treatment of Fontaine stage IIB peripheral arterial disease, wherein said deproteinized bovine blood preparation is administered at least once intravenously by infusion at a dose of 1200 mg once daily for a period of 14 consecutive days , followed by an oral dosing period in the form of two 200 mg tablets taken three times a day for a period of 70 consecutive days. 2. Use according to claim 1, where the deproteinized preparation from calf blood is the drug "Actovegin®". 3. Use according to claim 1 or 2, wherein said peripheral arterial disease is Fontaine stage IIB peripheral arterial disease, eg, confirmed by ultrasound color duplex scanning. 4. Application according to any one of paragraphs. 1-3, where the patient is a human, such as a human affected or suffering from diabetes. 5. Use according to claim 4, wherein the human patient has a Doppler ankle-brachial index at rest ≤ 0.9. 6. Use according to claim 4 or 5, wherein the human patient has intermittent claudication with an ICD < 200 meters. 7. Application according to any one of paragraphs. 4-6, wherein the human patient is at least about 40 years of age. 8. Application according to any one of paragraphs. 4-7, where the human patient is male. 9. Application according to any one of paragraphs. 4-7, where the human patient is a woman. 10. A method for preventing or treating Fontaine stage IIB peripheral arterial disease in a patient in need thereof, comprising the steps of: a) administering at least once intravenously by infusion to said patient a therapeutically effective amount of a 1200 mg deproteinised bovine blood preparation once a day for a period of 14 consecutive days, followed by b) administering at least once orally to said patient a therapeutically effective amount of said deproteinized bovine blood preparation at a dose in the form of two 200 mg tablets taken three times daily for period of 70 consecutive days. 11. The method according to p. 10, where the specified deproteinized preparation from calf blood is the drug Actovegin ^® . 12. The method of claim 10 or 11, wherein said Fontaine stage IIB peripheral arterial disease is, for example, confirmed by ultrasound color duplex scanning. 13. The method according to any one of paragraphs. 10-12, where the patient is a human, such as a human afflicted with or suffering from diabetes mellitus. 14. The method of claim 13, wherein the human patient has a resting Doppler ankle-brachial index ≤ 0.9. 15. The method of claim 13 or 14, wherein the human patient has intermittent claudication with an ICD < 200 meters. 16. The method according to any one of paragraphs. 13-15, wherein the human patient is at least 40 years of age. 17. The method according to any one of paragraphs. 13-16, where the human patient is male. 18. The method according to any one of paragraphs. 13-16, where the human patient is a woman.

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