IMPROVEMENT OF ANKLE-BRACHIAL INDEX BY DLBS1O33 TREATMENT IN DIABETIC PATIENTS WITH PERIPIIERAL ARTERIAL DISEASE.

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IMPROVEMENT OF ANKLE-BRACHIAL INDEX BY DLBS1033

TREATMENT IN DIABETIC PATIENTS WITH PERIPHERAL

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APPROVAL OF FINAL PROTOCOL

Clinical Data Management:

_______________________________________________ __________________

Liana W. Susanto, MBiomed Date

Clinical Project Management:

_______________________________________________ __________________

dr. Prihatini Hendri Date

Director:

_______________________________________________ _________________

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LIST OF KEY STAFF AND RELEVANT DEPARTMENTS

Principal Investigator:

Investigator Team Members:

Study Coordinator

Trial Site:

Clinical Laboratory:

Prof. Dr. dr. Ketut Suastika, SpPD-KEMD

dr. Wira Gotera, SpPD

dr. A. A. Gede Budhitresna, SpPD dr. I Made Pande Dwipayana, SpPD dr. Made Ratna Saraswati, SpPD

dr. Pande Made Oka Arimas, S.Ked

Department of Internal Medicine,

Faculty of Medicine, University of Udayana/Sanglah Hospital Jl. Diponegoro, Denpasar, Bali

Phone: +62 361 227 911 Fax: +62 361 224 206

PT Prodia DiaCRO Laboratories Prodia Tower 3rd Floor

Jl. Kramat Raya No. 150 Jakarta 10430, Indonesia Phone: +6221 3190 3065

Fax: +6221 314 4182 / 3190 5036

Prodia Laboratory Denpasar Jl. Diponegoro No. 192 Denpasar 80114, Indonesia Phone: +62 361 261 001 Fax: +62 361 236 349

Monitors: Dr. Prihatini Hendri Nurul Hidayah, Pharm.

Jalan Boulevard Bintaro Block B7/B1 No. 5 Bintaro Jaya Sector 7

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Data Management:

Sponsor:

Liana W. Susanto, MBiomed Fenny, Pharm.

Tangerang 15224 - Indonesia Phone: +62 21 7454 111 Fax: +62 21 7452 623

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investigational drug’s efficacy and safety will be performed at baseline and at interval of 6 weeks over the 12 week-course of therapy, while drug dispensing and accountability will be carried out at interval of 3 weeks. Study medication will be standard antiplatelet, aspirin at a dose of 80 mg once daily and either the investigational product, DLBS1033 at a dose of 490 mg three times daily or its placebo for 12 weeks. Concomitant medication allowable during study treatment will be any antidiabetes treatment currently used by each individual subjects and lifestyle modification (or intervention). The schematic design of the study is shown in Figure 1.

This study will enrol 114 diabetic patients with PAD, allocated equally in two groups (57 subjects in each group), i.e. The Control Group (will receive aspirin and placebo DLBS1033) and The DLBS Group (will receive aspirin and DLBS1033). In this study, the change (augmentation) of resting ankle-brachial index (ABI) will be measured as the primary efficacy endpoint.

Study Population

Study population will be diabetic patients having PAD with resting ABI of 0.41–0.90.

Key inclusion criteria include:

1. Signed informed consent before any trial related activities. 2. Male or female subjects of 40 – 65 years of age.

3. Diagnosis of diabetes mellitus defined as HbA1c level of  6.5% (for newly diagnosed diabetes) or based on medical history.

4. Presence of peripheral arterial disease with resting ankle-brachial index (ABI) of 0.41–0.90 inclusive (i.e. mild to severe PAD, without critical limb ischemia).

(Note: the reference leg should be determined at this point. Henceforth, ABI improvement will be based on measurement on the same leg as reference).

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6. Patients or patients’ legally acceptable representatives are able and willing to record adverse events in a diary.

7. Patients or patients’ legally acceptable representatives have the ability to comply with the trial protocol.

Key exclusion criteria include:

1. Females of childbearing potential: pregnancy, breast-feeding, and the intention of becoming pregnant.

 Patients must accept pregnancy tests during the trial if menstrual cycle is missed.

 Fertile patients must use a reliable and effective contraceptive.

2. Recent stroke attack, myocardial infarction/unstable angina/acute coronary syndrome, coronary artery bypass surgery (CABG) or percutaneous transluminal coronary angioplasty (PTCA)/stent within 3 (three) months prior to screening.

3. Impaired liver function: serum ALT > 2.5 times upper limit of normal. 4. Impaired renal function: serum creatinine ≥ 1.5 times upper limit of normal.

5. Concomitant use of other antithrombosis drugs, such as oral anticoagulants, dypiridamole, thienopyridines (such as clopidogrel), cilostazol or glycoprotein IIb/IIIa antagonist or any antiplatelets other than the study medication.

6. Subjects with concurrent herbal (alternative) medicines or food supplements suspected to have effect on hemostasis parameters.

7. Subjects with any other disease state, including chronic or acute systemic infections, uncontrolled illnesses or other chronic diseases, which judged by the investigator, could interfere with trial participation or trial evaluation.

8. Subjects with high risk of bleeding:

 Subjects with history of acquired or congenital bleeding disorder, coagulopathy, or platelet disorder;

 Subjects with evidence of active pathological bleeding or history of bleeding such as gastrointestinal or genitourinary bleeding unless the cause has been definitely corrected;

 History or presence of intracranial bleeding or hemorrhagic retinopathy, unstable angina, severe valvular heart disease, severe anemia, suspected or known dissecting aneurysm, acute myocarditis or pericarditis, severe aortic stenosis, severe hypertrophic obstructive cardiomyopathy,

 Severe or uncontrolled arterial hypertension (systolic blood pressure > 160 mmHg, diastolic > 100 mmHg).

9. Subjects with prior experience with DLBS1033 or other oral lumbrokinase products. 10. Subjects with known or suspected allergy to any of study medications used in the

study, including other lumbrokinase products.

11. Subjects with known or suspected allergy or resistant to aspirin.

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Withdrawal criteria:

The patient may be withdrawn from the trial at the discretion of the investigator or the sponsor if judged non-compliant with trial procedures or due to safety concerns. A patient must be withdrawn if the following applies:

1. Pregnancy or intention of becoming pregnant.

2. Allergy to any of study medications used in the study.

3. Treatment with any antiplatelets, antithrombosis, anticoagulants, and/or fibrinolytic agents, other than the study medication, including other oral lumbrokinase products, during the course of the study.

4. Any events occurring during the course of the study that might jeopardize the health of the patient.

5. Participation in any other clinical trial during the course of the trial.

Assessments: Efficacy endpoint:

Efficacy endpoint will be measured at baseline, Week 6th of treatment, and at the end of study (Week 12th of treatment).

Primary endpoint:

 Change (augmentation) of resting ankle-brachial index (ABI) from baseline to the end of study (week 12th).

Secondary endpoints:

 Change of resting ABI from baseline to week 6th of treatment.

 Change of hs-CRP from baseline to week 6th and 12th of treatment.

 Change of thromboxane B2 (TXB2) from baseline to week 6th and 12th of treatment.

 Change of fibrinogen level from baseline to week 6th and 12th of treatment.

 Change of d-dimer level from baseline to week 6th and 12th of treatment.

Safety endpoint:

Unless specified time of measurement is mentioned, all safety parameters will be measured at baseline, week 6th, and week 12th of treatment:

 Vital signs: blood pressure, heart rate, respiratory rate.

 Electrocardiography (ECG).

 Routine hematology: Hb, Ht, RBC, WBC, differential of WBC, platelet count.

 Liver function:

o_{Serum ALT (SGPT)}

o_{Serum AST (SGOT)}

o_{Alkaline phosphatase}

Liver function will be examined at baseline and at the end of study (week 12th).

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Page : 11 of 69 o_{Serum creatinine}

o_{Blood Urea Nitrogen (BUN)}

Renal function will be examined at baseline and at the end of study (week 12th).

 Hemostasis parameters:

o_{Prothrombin Time (PT)}

o_{Activated Partial Thromboplastin Time (aPTT).}

 Adverse event will be observed and carefully evaluated along the course of the study.

Other Examinations at Screening:

1. Demography data, including: age, sex, weight and height (for BMI calculation), smoking and alcohol status.

2. Interview on medical history, including first-time diagnosis of diabetes (and the type

of DM), family and subject’s history of cardiovascular diseases (coronary arterial disease, peripheral arterial disease, venous thromboembolism, and other atherothrombotic states necessitating medical treatment) or CV events (myocardial infarction, stroke, heart failure).

3. Clinical examination (in addition to laboratory examination) on the presence and degree of the existing microangiopathy complications, such as: retinopathy, neuropathy, and nephropathy.

4. Physical examinations.

5. Vital signs, including blood pressure, heart rate, and respiratory rate. 6. Pregnancy test (for female subjects).

7. Concomitant illnesses and concomitant medications.

8. Diabetes parameters: FPG, HbA1c, and 2-hour plasma glucose.

9. Lipid profile: HDL cholesterol, LDL cholesterol, total cholesterol, and triglycerides. 10. Assessment of Framingham point score.

Trial Products: Investigational drug:

 DLBS1033 bioactive protein fraction enteric-coated tablet @ 490 mg

Investigational product will be given one tablet three times daily for twelve weeks.

Standard drug:

 Aspirin enteric-coated tablet @ 80 mg (Thrombo Aspilets®, Medifarma) Aspirin will be given one tablet once daily for twelve weeks.

Concomitant medication:

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 Beta-blockers, ACE inhibitors, calcium antagonists, other blood pressure lowering agents for indication of hypertension, heart failure or any other cardiovascular diseases.

 Statins or other cholesterol-lowering agents.

 Insulin and/or any oral antidiabetes agents.

Anticoagulant, other fibrinolytic, or antithrombosis agents, any medications other than study medication and permitted concomitant medications, including herbals or food supplements which may affect hemostasis parameters are not allowed during study participation.

Study Allocation and Study Conduct:

There will be two groups of treatment, each consists of 57 subjects receiving either study treatment as follows:

Three times daily Once daily Three times daily

I: treatment group  

II: control group  

Eligible subjects will be enrolled in the study, randomly allocated into either study group, and instructed to come for study visit at interval of six weeks for efficacy and safety evaluation. Subject should take study medication everyday over twelve weeks of study period.

Diabetic subjects who have been being under therapy with aspirin can directly start with study treatment. For those who are currently not under therapy with aspirin, though, i.e. newly-diagnosed diabetic subjects (with PAD) who have never been on aspirin therapy or those who have been being treated with antiplatelet(s) other than aspirin, there will be a run-in period during which they will receive (or be switched to) aspirin treatment, without any other antiplatelets, for two weeks. After then, they will receive study medication. Such a run-in period with aspirin is set to make a matching or comparable baseline hemostasis condition of all subjects and the period of two weeks is based on the average lifespan of a platelet, which is normally 7–10 days.

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Inclusion and exclusion criteria √ √**)

Withdrawal criteria √ √ √ √

Medical history (anamnesis) √ √**)

Demographics data √ √**)

Physical exam √ √ √ √ √ √

Electrocardiography (ECG) √ √ √

General clinical condition and vital signs √ √ √ √ √ √

Drug dispensing and accountability √ (aspirin)

√ √ √ √ √

*)

Run-in period will be applicable for newly diagnosed diabetes subjects with PAD who have never been on aspirin therapy or those who have been being treated with antiplatelet(s) other than aspirin. See section 7.1 for further explanation.

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1. INTRODUCTION

Peripheral arterial disease (PAD) broadly encompasses the vascular diseases caused primarily by atherosclerosis and thromboembolic pathophysiology processes that alter the normal structure and function of the aorta, its visceral arterial branches, and the arteries of the lower extremity. PAD is the preferred clinical term that should be used to denote stenotic, occlusive, and aneurysmal disease of the aorta and its branch arteries, exclusive of the coronary arteries.1 Patients with PAD may have symptoms, but can also be asymptomatic. The most common symptom of PAD is intermittent claudication, defined as pain, cramping, or aching in the claves, thighs, or buttocks that appears reproducibly with walking exercise and is relieved by rest.2 Patients diagnosed as having PAD, including those who are asymptomatic, have an increased risk of mortality, myocardial infarct, and stroke.3

The major risk factors for PAD are older age (over 40 years), cigarette smoking, and diabetes mellitus. Hyperlipidemia, hypertension, and hyperhomocysteinemia are also important risk factors.4,5,6 Other potential risk factors for PAD include elevated levels of C-reactive protein (CRP), fibrinogen, apolipoprotein B, lipoprotein (a), and plasma viscosity. An inverse relationship has been suggested between PAD and alcohol consumption.2 It is important to note that diabetes is most strongly associated with femoral–popliteal and tibial (below the knee) PAD, whereas other risk factors (e.g. smoking and hypertension) are associated with more proximal disease in the aorto–ilio– femoral vessels.2

In people with diabetes, the risk of PAD is increased by age, duration of diabetes, and presence of peripheral neuropathy. Levels of CRP are also abnormally elevated in patients with impaired glucose tolerance and diabetes; then elevated levels of CRP are strongly associated with the development of PAD.7 Finally, CRP may increase the local production of compounds impairing fibrinolysis, such as plasminogen activator inhibitor (PAI)-1. Studies also showed that PAI-1 levels are elevated in insulin-resistant subjects and associated with increased cardiovascular risk of atherothrombosis.8,9,10 PAI-1 also has positive correlation with body mass index, which is usually high in diabetes patient.10,11 Whereas, evidence shows that in diabetic patients fibrinogen may be a cardiovascular risk factor because it is correlated to increased thrombin formation.12 The existence of both increased fibrinogen and thrombin activation in diabetes has been hypothesized to play a role in the pathogenesis of atherosclerosis in this disease. Therefore, people with diabetes have increased risk of PAD.

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An accurate estimation of the prevalence of PAD in diabetes should rely upon a validated and reproducible test. Such a test is the ankle–brachial index (ABI), which involves measuring the systolic blood pressure in the ankles (dorsalis pedis and posterior tibial arteries) and arms (brachial artery) using a hand-held Doppler and then calculating a ratio. This test has been reported to have a sensitivity above 90% and a specificity of 95% for the diagnosis of peripheral arterial disease.13 Using the ABI, one recent survey found a prevalence of PAD in people with diabetes > 40 years of age to be 20%.14 Moreover, another survey of patients with diabetes > 50 years of age showed a prevalence of PAD of 29%.15 The ABI greater than 1.30 suggesting a non-compressible, calcified vessel. In this condition, the true pressure at that location cannot be obtained and additional tests are required to diagnose PAD. The value of between 0.91–1.30 is considered as normal condition.2,13,16 Patients with leg pain on exertion who have such ABI values should be considered for an exercise test. An ABI value that is over 0.90 at rest, but decreasing by 20% after exercise, is diagnostic of PAD. If the initial ABI value is 0.90 or less at rest, the patient probably has PAD and no additional tests are necessary.17,18

Because of the presence of above-mentioned risk factors, the systemic nature of atherosclerosis and the high risk of ischemic events, diabetic patients with PAD should be considered candidates for secondary-prevention strategies that include aggressive risk-factor modification and antiplatelet drug therapy.2,3,19,20,21 At the present time, it is recommended that patients with PAD be treated with antiplatelet and lipid-lowering agents, along with management of their hypertension, hyperglycemia, and lifestyle modification in order to reduce the risk of cardiovascular complications.

To date, there are three different classes of platelet-inhibiting drugs which are mostly used for the prevention and treatment of atherothrombotic disorders, i.e. cyclooxygenase-1 (COX-1) inhibitors (aspirin), ADP P2Y12 receptor antagonists

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Even though aspirin therapy for diabetic patients at increased CVD risk is recommended,20 its benefit to prevent cardiovascular events and death in diabetic patients with PAD is considered equivocal.25,26 There is also a reduced clinical efficacy (aspirin resistance) of aspirin in diabetic compared with a non-diabetic population.19 In CAPRIE study, it was shown that in patients with PAD, clopidogrel was associated with a risk reduction of 24% compared with aspirin. Clopidogrel was also superior to aspirin therapy in PAD subjects who had diabetes. In terms of safety, clopidogrel was as well tolerated as aspirin.27 Based on these results, the Food and Drug Administration (FDA) approved clopidogrel for the reduction of ischemic events in all patients with PAD.2 However, resistance to the agent was also observed in patients with diabetes receiving long-term treatment with clopidogrel, especially in those under insulin therapy.22 Glycoprotein IIb/IIIa inhibitors (abciximab, tirofiban, lamifiban, eptifibatide) are generally used via the parenteral route within the multiple antithrombosis treatment panel for acute coronary syndromes. These agents seem highly effective in diabetes and are recommended in diabetic patients with acute coronary syndromes undergoing percutaneous interventions.22 Unfortunately, these agents failed to show efficacy in oral preparations, making them unsuitable for long-term prevention in a lifelong disease, such as diabetes.

Through relentless effort of searching for a new agent that may benefit diabetic patients with PAD, DLBS1033, a bioactive protein fraction derived from the local earthworm, Lumbricus rubellus, through a patented technology of extraction, was discovered. The earthworm extract possesses eight major proteins with molecular weight below 100 kDa. This specific pattern of proteins possess unique characteristic of DLBS1033, named as LLP (Lumbricus Low molecular weight Proteins). DLBS1033 has been shown to have antithrombosis activities due to its fibrinogen degradation assay, antiplatelet aggregation, and ex vivo antithrombosis assay; and thrombolytic activities demonstrated by fibrin plate assay and clot lysis assay.28 In vitro studies demonstrated reduced expression of several genes involves in inflammatory and atherogenic reaction by DLBS1033, such as NF-B, TNF-, VCAM-1, and P-selectin.29 In the same study, it was shown that DLBS1033 suppressed the expression of MMP-9 gene, a marker of plaque instability, suggesting that this bioactive protein fraction has the ability to control plaque stabilization. Besides, DLBS1033 also down-regulated JAK/STAT1 system which is responsible for vascular smooth muscle cell proliferation.

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Safety profile of DLBS1033 has also been demonstrated in healthy adult subjects.35,36 Based on the evaluation of safety parameters, which included hematology parameters, blood chemistry, urine and stool occult blood evaluation, and ECG interpretation, DLBS1033 was proven safe in healthy subjects. To date, DLBS1033 has been approved by National Agency of Drug and Food Control (NADFC), Republic of Indonesia, to be marketed as Indonesian standardized herbal medicine. Since then, no clinically significant adverse drug reactions have been reported.

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2. STUDY RATIONALE

The prevalence of PAD in diabetes appears higher than previously estimated.14,15 People with both PAD and diabetes mellitus are at increased risk of subsequent cardiovascular events.

At the present time, it is recommended that patients with PAD be treated with antiplatelet and lipid-lowering agents, along with management of their hypertension, hyperglycemia, and lifestyle modification in order to reduce the risk of cardiovascular complications. More specific to the antiplatelets and thrombolytic agents, currently available drugs approved by Food and Drug Administration (FDA) have several limitations, such as resistance (to clopidogrel), and parenteral-only dosage forms (of glycoprotein IIb/IIIa inhibitors, such as abciximab, tirofiban, lamifiban, eptifibatide). Even though these agents seem highly effective in diabetes and are recommended in diabetic patients, such limitations have made them unsuitable for long-term prevention in a lifelong disease, such as diabetes. This drives the development of a more effective antiplatelet treatment regimen, particularly for prevention of atherothrombotic events in diabetic patients with PAD, in whom the risk for developing CV events are much higher and the prevalence are rising.

In the last few years, DLBS1033, a bioactive fraction derived from Lumbricus rubellus earthworm, have been developed, possesses both antithrombosis as well as thrombolytic activities.28 Based on its pharmacological activity, safety profile27,28,29,30,31,32,33 as well as its simple administration (i.e. oral preparation), DLBS1033 can be considered as a new potential agent that is hypothetically beneficial for secondary prevention of cardiovascular events, particularly in diabetes mellitus patients. Besides, the thrombolytic activity of DLBS1033 may have a significant role in reducing other potential risk factors related to haemostasis parameters, such as fibrinogen and D-dimer levels.

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Page : 19 of 69 3. OBJECTIVES

The objectives of this study are:

1. To investigate clinical efficacy of DLBS1033 in combination with aspirin compared to aspirin alone, for the improvement of ankle-brachial index (ABI) as well as other hemostasis parameters in diabetic patients with peripheral arterial disease.

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4. STUDY DESIGN 4.1 Overview

This is a prospective, randomized, double-blind, double-dummy, and controlled study of DLBS1033, over a total of twelve weeks of therapy. There are 96 subjects (48 subjects per arm) planned to complete the study. To anticipate a withdrawal rate of about 15%, a total of 114 subjects are required to be enrolled in the study.

Subjects will be screened consecutively and eligible subjects will be randomized to receive aspirin tablet 80 mg once daily and either the investigational product, DLBS1033 tablet at a dose of 490 mg three times daily, or its placebo. Clinical and laboratory

examinations to evaluate the investigational drug’s efficacy and safety will be performed

at baseline and at the interval of six weeks over the twelve week-course of therapy, while drug dispensing and accountability will be carried out at interval of 3 weeks.

Diabetic subjects who have been being under therapy with aspirin can directly start with study treatment. For those who are currently not under therapy with aspirin, though, i.e. newly-diagnosed diabetic subjects (with PAD) who have never been treated with aspirin, or those who have been being treated with antiplatelet(s) other than aspirin, there will be a run-in period during which they will receive (or be switched to) aspirin treatment, without any other antiplatelets, for two weeks. After then, they will receive study medication. The schematic figure of the study design is shown in Figure 1 (Page 21).

All subjects will also be instructed to follow a lifestyle modification,40 i.e. cease cigarette smoking (for smokers), follow a healthy diet enriched with protein but restricted in saturated fat, cholesterol and salt, and have routine exercise (i.e. aerobic and weight-bearing exercise for at least 4 to 5 times @ 30 minutes, per week) during their participation in the study. There will be no special treatment or control performed by the Investigators concerning the implementation of this lifestyle modification, thus it becomes essential for the Investigators to provide an earnest education and information as well as the reminder to the patient about this matter.

Before its commencement, the study protocol must obtain approval from the Ethics Committee of Medical Faculty, University of Udayana, Bali. Informed consent will be obtained from each clinical trial subject or their legal guardian. Eligible clinical trial subject will receive study medication and be closely monitored by the Investigator during their participation in the study to evaluate the outcome.

4.2 Design and Methods

The study procedures and methods are described schematically in Figure 1 (Page 21). Eligible subjects are randomly allocated into two treatment groups as follow:

1. Treatment group I (DLBS1033)

:  DLBS1033 enteric-coated tablets @ 490 mg, three times daily, for 12 weeks

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2. Treatment group II (Control)

:  Placebo DLBS1033 enteric-coated tablets, three times daily, for 12 weeks

 Aspirin enteric-coated tablet 80 mg, once daily, for 12 weeks

The study medication is provided in double-blind, double-dummy preparations. Subjects take the study medication everyday over twelve weeks of study period and are instructed to come to the clinic for follow-up visits at interval of six weeks.

Figure 1. Scheme of Study Design.

Lipid profile: LDL, HDL, TC, TG Hematology

DLBS1033 enteric-coated tablets 490 mg tid Aspirin enteric-coated tablets 80 mg od

Group 2 (n = 57)

Placebo DLBS1033 tablets tid Aspirin enteric-coated tablets 80 mg od ±24 hours

Diabetes profile: HbA1c, FPG, 2h-PG Lipid profile: LDL, HDL, TC, TG diabetes subjects with PAD who have never been on aspirin therapy or those who have been being treated with antiplatelet(s) other than aspirin.

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4.3 Efficacy Endpoints

4.3.1 Primary Efficacy Endpoint

The primary efficacy endpoint will be: change (augmentation) of resting ankle-brachial index (ABI) from baseline to the end of study (week 12th).

4.3.2 Secondary Efficacy Endpoints

The secondary efficacy endpoints are:

 Change of resting ABI from baseline to week 6th of treatment.

 Change of hs-CRP from baseline to week 6th and 12th of treatment.

 Change of thromboxane B2 (TXB2) from baseline to week 6th and 12th of treatment.

 Change of fibrinogen level from baseline to week 6th and 12th of treatment.

 Change of d-dimer level from baseline to week 6th and 12th of treatment.

4.3.3 Other Endpoints

Other endpoints evaluated which are not directly associated with the efficacy of the investigational product :

 Change of HbA1c from baseline to the end of study (week 12th).

 Change of fasting plasma glucose (FPG) level from baseline to the end of study (week 12th).

 Change of 2-hour post prandial glucose (2-hour PG) level from baseline to the end of study (week 12th).

 Change of lipid profile (LDL, HDL, Total Cholesterol, and Triglyceride levels) from baseline to the end of study (week 12th).

4.4 Safety Endpoints

Unless specified time of measurement is mentioned, all safety parameters will be measured at baseline, week 6th, and week 12th of treatment:

 Vital signs: blood pressure, heart rate, respiratory rate.

 Routine hematology: Hb, Ht, RBC, WBC, differential of WBC, platelet count.

 Liver function:

o_{Serum ALT (SGPT)}

o_{Serum AST (SGOT)}

o_{Alkaline phosphatase}

Liver function will be examined at baseline and at the end of study (week 12th).

 Renal function:

o_{Serum creatinine}

o_{Blood Urea Nitrogen (BUN)}

Renal function will be examined at baseline and at the end of study (week 12th).

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Page : 23 of 69 o_{Prothrombin Time (PT)}

o_{Activated Partial Thromboplastin Time (aPTT).}

 Adverse event will be observed and carefully evaluated along the course of the study.

4.5 Rationale for Study Design

A parallel, randomized, double-blind, double-dummy, and controlled study was considered as the best design to evaluate the efficacy and safety of a new oral fibrinolytic agent, DLBS1033, in combination with aspirin in diabetic patients with PAD. Random allocation will expose every study subject to an equal opportunity to receive either the investigational product (DLBS1033) or its placebo, on top of aspirin treatment. A double-blind design will minimize any possible bias in evaluating the result of the trial. Placebo or dummy tablets of DLBS1033 will be used in this study in order to maintain the double-blinding procedure.

In this study, aspirin alone was chosen as the active control. This is based on the rationale that in line with the investigational drug, DLBS1033, aspirin also exerts its action through the hemostasis system (i.e. as an antiplatelet), and is recommended for prevention of ischemic events in diabetic patients who are at increased CVD risk.20,23,24

To date, we do not have any clinical data yet, comparing the inhibitory effect of DLBS1033 on platelet aggregation to that of aspirin in healthy volunteers. Therefore it is ethically and scientifically considerable to administer the new agent on top of aspirin as the currently recommended standard regimen. Further, such a combination is also expected to produce a synergistic effect as both agents have different mechanism of action in improving vascular system thus preventing cardiovascular events.

In order to set a matching or comparable baseline hemostasis condition in all subjects, before they receive study medication, newly-diagnosed diabetic subjects (with PAD) who have never been on aspirin therapy will be enrolled in a two-week run-in period during which they will be given aspirin; while those who have been being treated with other antiplatelet(s) than aspirin, will be switched to aspirin treatment during the run-in period. Such an aspirin-treated run-in period is set for a period of two weeks based on the average lifespan of a platelet, which is normally 7–10 days.

A three-week interval of follow-up visit was determined to minimize the chance of losing study subjects along the way during the study, as well as to control the compliance of study subjects with the study regimens and procedure. A twelve-week treatment period was regarded appropriate and adequate to examine the augmentation of ankle-brachial index (ABI) as well as all the secondary endpoints after the medication by the investigational products.

In this study, hs-CRP, TXB2, fibrinogen, and d-dimer as risk factors of PAD will also be

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and thrombolytic/fibrinolytic activities. Those parameters have been known to have direct or indirect correlation with increasing risk of PAD in diabetes patients.8,9,10,11,12

Meanwhile, safety evaluation of DLBS1033 will also be evaluated in this study, including the evaluation on the vital signs, cardiac, liver and renal function, hematology, hemostasis parameters, as well as other clinical adverse events occurred during the study period, either observed by the investigator or reported by the subjects.

4.6 Rationale for Dosage Regimen

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Page : 25 of 69 5. STUDY SITE AND POPULATION

5.1 Study Site, Principal Investigator and Team

Principal Investigator of this study will be Prof. Dr. dr. Ketut Suastika, SpPD-KEMD. In conducting the study, the principal investigator will be assisted by a study coordinator: dr. Pande Made Oka Arimas, S.Ked. and several co-investigators, as follows: dr. Wira Gotera, SpPD, dr. A. A. Gede Budhi Tresna, SpPD, dr. I Made Pande Dwipayana, SpPD, and dr. Made Ratna Saraswati, SpPD.

This will be a single-center-study with the study site at Department of Internal Medicine, Faculty of Medicine, University of Udayana/Sanglah Hospital, situated at Jl. Diponegoro, Denpasar, Bali.

Clinical laboratory assigned for this study will be Prodia Laboratory Denpasar, situated at Jl. Diponegoro No. 192, Denpasar 80114, Indonesia.

5.2 Number of Patients to be Studied

Planned number of enrolled subjects: 114 subjects (57 subjects in each group) Anticipated withdrawal rate during the study period: 15%

Planned number of complete subjects: 96 subjects (48 subjects in each group)

5.3 Patient Replacement

There will be no patient replacement in the study for every withdrawn subject.

5.4 Nature of Population

Study population will be diabetes patients with PAD with resting ABI of 0.41–0.90 who attend the study site.

5.5 Subject Eligibility 5.5.1 Inclusion Criteria

1. Signed informed consent before any trial related activities. 2. Male or female subjects of 40 – 65 years of age.

3. Diagnosis of diabetes mellitus defined as HbA1c level of  6.5% (for newly diagnosed diabetes) or based on medical history.

4. Presence of peripheral arterial disease with resting ankle-brachial index (ABI) of 0.41–0.90 inclusive (i.e. mild to severe PAD, without critical limb ischemia).2,16,39 (Note: the reference leg should be determined at this point. Henceforth, ABI improvement will be based on measurement on the same leg as reference).

5. Able to take oral medication.

6. Patients or patients’ legally acceptable representatives are able and willing to record adverse events in a diary.

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5.5.2 Exclusion Criteria