Conditions: Ischemic Stroke
Keywords: Acute Ischemic Stroke Fibrinolysis Fortelyzin
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 336
Healthy volunteers: no

Brief summary:
The aim of the study is to determine if single-bolus recombinant nonimmunogenic staphylokinase is effective and save thrombolytic agent in patients with ischemic stroke in comparison to alteplase.

Detailed description:
Experimental Drug Profile. The active substance of Fortelyzin is Forteplase. It's recombinant protein which contains aminoacid sequence of staphylokinase. It is single chain molecula, consists of 138 aminoacids, weight 15.5 kDa. When staphylokinase is added to human plasma containing a fibrin clot, it preferentially reacts with plasmin at the clot surface, forming a plasmin-staphylokinase complex. This complex activates plasminogen trapped in the thrombus. The plasmin-staphylokinase complex and plasmin bound to fibrin are protected from inhibition by alpha2-antiplasmin. Once liberated from the clot (or generated in plasma), however, they are rapidly inhibited by alpha2-antiplasmin. This selectivity of action confines the process of plasminogen activation to the thrombus, preventing excessive plasmin generation, alpha2-antiplasmin depletion, and fibrinogen degradation in plasma. In rabbits anti forteplase antibodies are not produced. It was achieved by replacement of amino acids in immunogenic epitop of molecule staphylokinase. Blood fibrinogen decrease after i.v. injection of Fortelyzin less 10% within first 24 hours. Angiographic data suggests that restoration of coronary blood flow appears in up to 80% of patients with STEMI after i.v. injection of Fortelyzin.

Main goals of the study are to prove an efficacy of the single-bolus intravenous injection of recombinant nonimmunogenic staphylokinase (Fortelyzin) in comparison with bolus infusion alteplase(Actilyse) in patients with ischemic stroke.

To prove a safety and to assess possible adverse events in the single-bolus intravenous injection of recombinant nonimmunogenic staphylokinase (Fortelyzin) in comparison with bolus infusion alteplase (Actilyse) in patients with ischemic stroke.

Study Design. All eligible patients will be randomized in two equal groups for administration recombinant nonimmunogenic staphylokinase (Fortelyzin) or alteplase (Actilyse) by using "envelope method" of randomization. It is an open-lable study. Each of agents will be administered no longer then 4,5 hours from symptoms onset. Comparative agent will be administered as prescribed in its instructions. All patients will be examination for 90 days

Arms:
arm 1: Lyophilizate for solution making for intravenous injection, 5 mg (745000 ME). 10 mg of drug reconstituted in 10 ml of 0.9% solution of NaCl given as single i.v. bolus over 5 - 10 seconds arm 2: Intravenous alteplase 0.9 mg/kg (10% bolus and 90% as IV infusion over 1 hour, maximum 90 mg)

Interventions: intervention 1: Recombinant staphylokinase intervention 2: Alteplase
Intervention descriptions: intervention 1: 10 mg of drug reconstituted in 10 ml of 0.9% solution of NaCl given as single i.v. bolus over 5 - 10 seconds intervention 2: Intravenous alteplase 0.9 mg/kg (10% bolus and 90% as IV infusion over 1 hour, maximum 90 mg)

Protocol document:
Prot_SAP_000.pdf: 
------------------------------------------------------------------------------------------------------------------------------------------------ 
------------------------------------------------------------------------------------------------------------------------------------------------ 
 
 
 
CLINICAL TRIAL PROTOCOL 
 
 
Multicenter open-label randomized comparative trial of the efficacy and safety of 
single bolus injection of Fortelyzin® (Supergene, LLC) and bolus-infusion of 
Actilyse® (Boehringer Ingelheim Pharma GmbH) in patients with ischemic stroke 
 
 
 
Study drug: 
Fortelyzin® 
(substance Forteplase®) 
Trial Phase: 
III b 
Trial design: 
Multicenter, open-label, randomized, 
comparative, non-inferiority trial of efficacy 
and safety 
Sponsor: 
The Russian Academy of Sciences,  
Supergene, LLC, Russia 
Version:  
1.0, approved by the Ministry of Health of 
the Russian Federation as of July 15, 2016  
 
 
 
 
 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 2 of 67 
INFORMATION ABOUT THE PRINCIPAL INVESTIGATOR 
The National Investigator will coordinate the research activities of all research centers 
participating in the multicenter clinical trial. 
 
GUSEV EVGENY IVANOVICH 
Pirogov Russian National Research Medical University of the Russian Ministry 
of Health 
Head of the Department of Neurology, Neurosurgery and Medical Genetics of the 
Faculty of Medicine, Honored Scientist of the Russian Federation, Academician of the 
Russian Academy of Sciences, MD, Professor 
e-mail: egusev2339@gmail.com 
mob. phone: +7 906 796-89-06 
 
I read all the pages of the Clinical Trial Protocol «Multicenter open-label randomized 
comparative trial of the efficacy and safety of single bolus injection of Fortelyzin® 
(Supergene, LLC) and bolus-infusion of Actilyse® (Boehringer Ingelheim Pharma 
GmbH) in patients with ischemic stroke», and I agree that it contains all the information 
necessary to conduct this trial. I will conduct the trial as described in this Protocol and 
comply with all terms and conditions specified therein. I confirm that I will conduct 
the trial in accordance with the current legislation, the Declaration of Helsinki, and the 
principles of Good Clinical Practice approved at the International Conference on 
Harmonization (ICH GCP). I also guarantee that the co-investigator(s) and the other 
subjects involved will conduct the trial in accordance with these documents. 
 
 
 
Principal Investigator’s signature: 
Signature:   
 
 
 Date:   
/ 
 
 
/ 
 
 
 dd/mm/yyyy 
 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 3 of 67 
CONTACT INFORMATION 
Ethics Committee of the Russian Ministry of Health: 
3 Rakhmanovsky Lane, Moscow, 127994, Russian Federation 
tel: +7 (495) 625-41-90 
 
 
Sponsors: 
Russian Academy of Sciences 
14 Solyanka Street, Moscow, 109240 
tel./fax: +7 (495) 698-56-50 
 
Supergene, LLC  
Luzhnetskaya nab., 6, building 1, office 301, 119270, Moscow 
tel./fax: +7 (495) 287-98-07 
 
Contact information of the Project Principal Investigator: 
GUSEV EVGENY IVANOVICH 
phone: +7 906 796-89-06 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 4 of 67 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ............................................................................................................................................. 5 
PROTOCOL SUMMARY .................................................................................................................................................. 7 
1. BACKGRAUND FOR THE TRIAL ............................................................................................................................ 14 
2. GENERAL INFORMATION ABOUT THE STUDY DRUG ..................................................................................... 16 
3. POTENTIAL BENEFITS AND RISKS TO PARTICIPANTS .................................................................................... 33 
4. REGULATORY FRAMEWORK AND ETHICAL ASPECTS OF THE STUDY ...................................................... 34 
4.1. ETHICS COMMITTEE ................................................................................................................................................. 35 
4.2. PATIENT INFORMATION SHEET ................................................................................................................................ 36 
5. TRIAL AIM .................................................................................................................................................................. 37 
6. TRIAL OBJECTIVES .................................................................................................................................................. 37 
7. TRIAL POPULATION ................................................................................................................................................. 38 
7.1. INCLUSION CRITERIA ................................................................................................................................................ 38 
7.2. EXCLUSION CRITERIA............................................................................................................................................... 38 
7.3. TRIAL COMPLETION AND POST-TRIAL THERAPY ....................................................................................................... 40 
7.4. CRITERIA FOR EARLY TERMINATION OF TRIAL PARTICIPANTS .................................................................................. 41 
7.5. CRITERIA FOR EARLY TERMINATION OF THE TRIAL .................................................................................................. 42 
8. DESIGN AND PLAN OF RESEARCH ACTIVITIES ................................................................................................ 43 
9. TRIAL PROCEDURES ................................................................................................................................................ 46 
9.1. DESCRIPTION OF SOME RESEARCH PROCEDURES ...................................................................................................... 50 
9.1.1. Anamnesis ..................................................................................................................................................... 50 
9.1.2. Collection of information about concomitant and previous therapy ............................................................. 51 
9.1.3. Physical examination ..................................................................................................................................... 51 
9.1.4. Vital signs ...................................................................................................................................................... 51 
9.1.5. Electrocardiography ...................................................................................................................................... 51 
9.1.6. Complete and biochemical blood analysis .................................................................................................... 51 
9.1.7. Common urine analysis ................................................................................................................................. 52 
10. THERAPY DURING THE TRIAL ............................................................................................................................ 53 
PREVIOUS TREATMENT ................................................................................................................................................... 55 
PERMITTED CONCOMITANT THERAPY ............................................................................................................................. 55 
PROHIBITED CONCOMITANT THERAPY ............................................................................................................................ 56 
11. EFFICACY ASSESMENT ......................................................................................................................................... 56 
12. SAFETY ASSESSMENT ........................................................................................................................................... 56 
15. CONFIDENTIALITY................................................................................................................................................. 62 
16. PARTICIPATION IN THE TRIAL AND COMPENSATION .................................................................................. 62 
17. CALCULATION OF SAMPLE SIZE ........................................................................................................................ 63 
18. STATISTICAL ANALYSIS OF RESEARCH MATERIALS ................................................................................... 65 
19. MONITORING AND AUDIT .................................................................................................................................... 65 
20. REPORTING .............................................................................................................................................................. 66 
21. PUBLICATIONS ........................................................................................................................................................ 67 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 5 of 67 
LIST OF ABBREVIATIONS 
AG 
– angiography 
AIS 
– acute ischemic stroke 
BP 
– blood pressure 
ALAT 
– alanine aminotransferase  
AST 
– aspartate aminotransferase  
APPT 
– activated partial thromboplastin time 
RSC 
– Russian Cardiology Society  
WHO 
– World Health Organization 
DBP 
– diastolic blood pressure  
DWI 
– diffusion-weighted image  
DS MAH 
– Duplex scanning of major arteries of the head  
IPIC 
– Individual Patient Identification Code 
CRF 
– Case Report Form 
К+ 
– blood serum potassium 
CT 
– computed tomography 
CPK 
– creatine phosphokinase 
MP 
– medicinal product 
INR 
– international normalized ratio 
MRI 
– magnetic resonance imaging 
mRS 
– modified Rankin scale  
UAR 
– unforeseen adverse reaction 
AE 
– adverse event 
AMI 
– acute myocardial infarction  
ADCC 
– acute disorders of cerebral circulation  
SBP 
– systolic blood pressure  
SH 
– subarachnoid hemorrhage 
MA 
– middlecerebral artery 
SAE 
– serious adverse event  


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 6 of 67 
SAK 
– staphylokinase  
TPA 
– tissue plasminogen activator 
TLT 
– thrombolytic therapy 
HDL-C 
– high-density lipoprotein cholesterol 
RR 
– respiratory rate  
HR 
– heart rate 
EC 
– Ethics Committee 
ECG 
– electrocardiography  
BI 
– Barthel Index for activities of daily living 
ECASS 
– European Cooperative Acute Stroke Study  
Hb 
– hemoglobin  
Ht 
– hematocrit  
ICH 
– International Conference on Harmonisation 
GCP 
– Good Clinical Practice  
GUSTO 
– Global Use of Strategies to Open Occluded Coronary Arteries  
Na+ 
– Sodium in blood  
NIHSS 
– National Institutes of Health Stroke Scale 
NYHA 
– New York Heart Association  
TIMI 
– Thrombolysis in Myocardial Infarction 
TFC 
– TIMI Frame Count  
TOAST 
– Trial of ORG 10172 in Acute Stroke Treatment  
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 7 of 67 
PROTOCOL SUMMARY 
Official Title: 
Multicenter open-label randomized comparative trial of the efficacy 
and safety of single-bolus intravenous injection of Fortelyzin® 
(manufactured by Supergene, LLC) and bolus infusion of Actilyse® 
(manufactured by Boehringer Ingelheim Pharma GmbH) in patients 
with ischemic stroke 
Short title: 
FRIDA 
Trial phase: 
III b 
Trial aim: 
Assessment of the efficacy and safety of single-bolus intravenous 
injection of Fortelyzin® in comparison with bolus infusion of 
Actilyse® in patients with ischemic stroke 
Trial objectives: 
• To assess the efficacy of single-bolus intravenous injection of 
Fortelyzin® in comparison with bolus infusion of Actilyse® in 
patients with ischemic stroke  
• To assess the safety and possible adverse events of single-bolus 
intravenous injection of Fortelyzin® in comparison with bolus-
infusion of Actilyse® in patients with ischemic stroke  
Trial design: 
A multicenter, open-label, randomized, comparative, parallel-group 
non-inferiority trial of efficacy and safety. In clinical centers, 
patients will be randomized using the "envelope method" into two 
equal groups of 168 patients each (a total of 336 people, including 
10 % of those who may have dropped out) and assigned to receive 
either Fortelyzin® or Actilyse®. The drugs will be administered no 
later than 4,5 hours after ischemic stroke onset and after signing the 
informed consent. Fortelyzin® will be given as single i.v. bolus over 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 8 of 67 
5-10 seconds. Actilyse® will be administered in accordance with the 
instruction for use. 
Patients will be monitored for 90 days: 1-2 days in the Neurological 
Intensive Care Unit and the Department of Neurology in the 
remaining days before discharge (on average 14 days), with an 
outpatient visit on the 90th day. 
Enrollment of patients into the trial will be conducted on a 
competitive basis. 
Trial duration: 
The trial completion date is the 90th day after the final trial patient was 
included in the trial. All patients will be examined for 90 days 
Trial population: Men and women aged 18 years and older with a diagnosis of 
ischemic stroke no later than 4.5 hours after the onset of symptoms. 
Sample size: 
The total number of patients included in the trial – 336 people, 
including 10 % of possible dropouts 
Study drug: 
Fortelyzin®, registration number – 001941 as of December 18, 2012. 
Pharmaceutical form: Lyophilizate for preparation of a solution for 
intravenous administration, 5 mg (745,000 IU), supplied with a 
solvent 
Composition: 
Active substance: Forteplase® (recombinant protein containing the 
amino acid sequence of staphylokinase) 5 mg (745,000 IU). 
Excipients:  
 
 
L-arginine - 15,0 mg, 
 
 
 
 
 
L-histidine – 2,0 mg, 
 
 
 
 
 
glycine – 30,0 mg, 
 
 
 
 
 
povidone-17: 20,0 mg, 
 
 
 
 
 
polysorbate-20: 0,4 mg. 
Solvent: 5 mL ampule of sodium chloride 0.9%, solution 
for injection 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 9 of 67 
Inclusion 
criteria: 
• Men and women aged 18-80 (inclusive) years old. 
• Verified diagnosis of ischemic stroke (neuroimaging (CT/MRI) 
signs of ischemic stroke, from 5 to 25 points on the NIHSS scale).  
• Duration of symptoms of ischemic stroke is not more than 
4.5 hours before starting thrombolytic therapy. 
• The patient's consent to use reliable contraceptive methods 
throughout the trial and within 3 weeks after: 
– women who have a negative pregnancy test and use the following 
means of contraception: intrauterine devices, oral contraceptives, a 
contraceptive patch, long-acting injectable contraceptives, a 
double-barrier method of contraception. Women who are not at 
risk of pregnancy for health-related reasons (documented 
conditions: hysterectomy, tubal ligation, infertility, menopause for 
more than 1 year) may also be involved in the trial);  
– men who use barrier contraception. Men who unable to fertilize 
(documented conditions: vasectomy, infertility) may also be 
involved in the trial. 
• A participant provided signed and dated informed consent of 
participation in the trial. 
Exclusion 
criteria: 
• The time of the onset of the first symptoms is more than 4.5 hours 
from the onset of the disease or the time of the onset of the first 
symptoms of a stroke is not known (for example, the development 
of a stroke during sleeping - the so-called "night stroke") 
• Increased sensitivity to alteplase, gentamicin (residual traces 
from the production process). 
• Systolic blood pressure greater than 185 mm Hg or diastolic 
blood pressure greater than 110 mm Hg or the need for 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 10 of 67 
intravenous administration of drugs to reduce the blood pressure 
to these boundaries. 
• Neuroimaging (CT/MRI) signs of intracranial hemorrhage, brain 
tumors, arteriovenous malformation, brain abscess, aneurysm of 
cerebral vessels. 
• Surgery on the brain or spinal cord. 
• Suspicion of subarachnoid hemorrhage. 
• Signs of severe stroke: clinical signs (stroke scale NIH> 25), 
neuroimaging (according to CT of the brain and/or MRI of the 
brain in the DWI, the ischemia focuses on the territory of more 
than 1/3 of the MA pool). 
• Simultaneous administration of oral anticoagulants, for example, 
warfarin with INR> 1.3. 
• The use of direct anticoagulants (heparin, heparinoids) in the 48 
hours preceding the stroke with APTT values above the norm.  
• Prior stroke or severe head injury within 3 months. 
•  Significant regression of neurological symptoms during the 
observation of the patient. 
• Mild neurological symptoms (NIH <4 points).  
• Hemorrhagic stroke or stroke unspecified in medical history. 
• Strokes of any genesis in the history of a patient with diabetes 
mellitus. 
• Gastrointestinal bleeding or bleeding from the genitourinary 
system in the last 3 weeks. Confirmed exacerbations of gastric 
ulcer and duodenal ulcer during the last 3 months. 
• Extensive bleeding, present or during the previous 6 months. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 11 of 67 
• Severe liver disease, including liver failure, cirrhosis, portal 
hypertension (with varicose veins of the esophagus), active 
hepatitis. 
• Acute pancreatitis. 
• Bacterial endocarditis, pericarditis. 
• Aneurysms of arteries, malformations of arteries and veins. 
Suspicion of exfoliating aortic aneurysm. 
• Neoplasms with an increased risk of bleeding. 
• Large operations or severe injuries within the last 14 days, minor 
surgery or invasive manipulations in the last 10 days. 
• Puncture of uncompensated arteries and veins during the last 7 
days. 
• Prolonged or traumatic cardiopulmonary resuscitation (more 
than 2 min). 
• Pregnancy, obstetrics, 10 days after birth. 
• The number of platelets is less than 100,000/μL. 
• Blood glucose less than 2.8 mmol/l or more than 22.5 mmol/l. 
• Hemorrhagic diathesis, including renal and hepatic insufficiency. 
• Data on bleeding or acute trauma at the time of examination. 
• Seizures in the onset of the disease, if there is an uncertainty that 
the seizure is a clinical manifestation of ischemic stroke with a 
postictal residual deficiency. 
 
Trial procedures: • collection of anamnesis, physical examination, measurement of 
vital signs (respiratory rate, heart rate, blood pressure) 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 12 of 67 
• assessment of neurological status according to generally accepted 
methods with the trial of cerebral, meningeal, and focal 
symptoms 
• assessment of neurological status using NIHSS, which allows to 
quantify the severity of neurological deficits 
• Brain CT or MRI performed within 40 minutes of the patient's 
admission to the hospital 
• angiography of cerebral vessels (if necessary) 
• duplex scanning of the major arteries of the head (if necessary) 
• venous blood sampling for complete blood count (with platelet 
count) and biochemical blood analysis (with glucose level 
determination), coagulogram (INR, APTT, fibrinogen). The 
results of the tests should be obtained within 20 minutes from the 
moment the patient admitted to the hospital 
• common urine analysis 
Efficacy criteria:  • The primary efficacy endpoint is a good functional recovery on 
day 90 (modified Rankin scale, mRS, 0-1 score).  
• Secondary combined efficacy endpoint on day 90 – mRS (0-1) + 
NIHSS (0-1 score) + Barthel Index (BI) - 95 points or higher. 
• Additional efficacy criterion – NIHSS score after 24 hours and 
after 90 days. 
Safety criteria: 
• Safety endpoint - overall mortality on day 90. 
• Safety endpoint - hemorrhagic transformation (all cases). 
• Safety endpoint - symptomatic hemorrhagic transformation (as 
defined by ECASS III). 
• The number of SAE and AE by organs and systems. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 13 of 67 
Finally: 
Each research center prepares a report, which is signed by the 
Principal Investigator and approved by the head of the medical 
institution, where the center is based. The submission deadline is up 
to 1 month after the completion of the trial. The statistical processing 
is carried out by an independent statistical expert according to the 
Case Report Form (CRF). 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 14 of 67 
1. BACKGRAUND FOR THE TRIAL  
Ischemic stroke is one of the leading causes of death and disability worldwide. 
In 2004, the World Health Organization declared stroke a global epidemic. The annual 
mortality rate from stroke in Russia is one of the highest in the world (175 per 100,000 
people). The early 30-day mortality after stroke is 34.6%, and about half of the cases 
die within a year. Stroke is the leading cause of disability in the population. According 
to the National Stroke Registry, 31 % of stroke survivors require assistance, and 20% 
are not able to walk on their own. Only 8% people stroke after can return to their 
previous work. Stroke imposes special obligations on the family members of the 
patient, significantly reducing their labor potential. 
International experience shows that the decline in mortality from cardiovascular 
diseases is achieved by a coordinated set of measures, including raising public 
awareness about the risk factors and prevention of cardiovascular diseases, the 
introduction of effective prevention programs, and improvement of the medical care 
system in stroke. 
New approaches to the treatment of ischemic stroke include the use of modern 
and highly effective brain substance reperfusion techniques in the first hours after a 
stroke. These methods are aimed at restoring blood flow in the damaged vessel, which 
allows to prevent the development of irreversible damage to the brain substance or 
reduce its amount, i.e. minimize the severity of residual neurological deficit. 
According to the Decrees of the RF Government as of December 27, 2007, No. 
1012 and No. 186 as of March 2, 2009, Russia has begun the implementation of 
measures aimed at improving medical care for cardiovascular patients. Regional 
Vascular Centers and Primary Vascular Departments have established units for the 
treatment of patients with acute disorders of cerebral circulation (ADCC). The 
procedure for providing medical care to patients with ADCC is developed and 
approved by the orders of the RF Ministry of Health and Social Development of July 
6, 2009, No. 389n and No. 44n of February 2, 2010. Within the framework of these 
measures, modern methods of diagnosis, treatment, rehabilitation, and secondary 
prevention of stroke are being introduced in the departments for the treatment of 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 15 of 67 
patients with ADCC, including thrombolytic therapy, which is aimed at restoring blood 
flow in the damaged vessel. 
The safety and efficacy of systemic thrombolysis therapy using Actilyse® in 
patients with ischemic stroke have been proven in a number of large randomized 
placebo-controlled clinical trials (NINDS, ECASS I, II, III, ATLANTIS), as well as the 
SITS-MOST and SITS-ISTR registers. 
In addition, a high frequency of hemorrhagic transformation (6-23.8%) and a 
high 3-month mortality rate (18.2%) were noted in some studies with alteplase, which, 
according to the researchers, is associated with an initially high neurological deficit. 
In this regard, the search for new thrombolytic drugs that can improve the 
efficacy and safety of thrombolytic therapy is an important scientific and economic 
task. 
 
 
 
 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 16 of 67 
2. GENERAL INFORMATION ABOUT THE STUDY DRUG 
Brand name: Fortelyzin®, valid registration number - 001941 as of December 18, 
2012. 
Chemical name: recombinant protein, which contains the amino acid sequence of 
staphylokinase. 
Pharmacology and pharmacokinetics 
Fortelyzin® refers to the so-called group of fibrin-selective thrombolytics. 
 
The active substance of Fortelyzin® is Forteplase, which is a single-chain 
molecule consisting of 138 amino acids with a molecular weight of 15.5 kDa.  
 
Forteplase® is an original recombinant protein obtained using genetically 
engineered E. coli and containing the amino acid sequence of modified (non-
immunogenic) staphylokinase. 
In contrast to the native staphylokinase, 3 amino acids in the immunodominant 
epitope were replaced in the Forteplase® molecule. 
Forteplase® is not an enzyme and activates plasminogen forming a 
stoichiometric complex at a 1:1 ratio. 
Lysine-11 of the N-terminal site of plasminogen and glutamine-46, lysine-50, 
glutamine-65, and asparagine-69 of the C-terminal site of the drug play a key role in 
the interaction of Forteplase® and plasminogen. 
 
Along with this, the N-end of the substance acts as a "moving arm" that captures 
the plasmin molecule and, as a result, a ternary complex composing of plasminogen- 
Forteplase®-plasmin, is formed. 
 
Depending on different conditions, plasminogen can take three different 
conformations: α, β, and γ. 
Plasminogen was determined to have a closed α-conformation in the 
bloodstream; a semi-open β-conformation occurs when plasminogen is bound to intact 
fibrin and a fully open γ-conformation occurs when plasminogen is bound to partially 
degraded fibrin. 
 
The conversion of plasminogen to plasmin by plasminogen activators requires 
an open γ-conformation. Forteplase® interacts only with plasminogen in the γ-


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 17 of 67 
conformation and does not bind to plasminogen in the closed form, i.e., which is in the 
circulation. 
The mechanism of Forteplase® fibrinolytic action involves its initial binding to 
plasmin generated on the fibrin clot with subsequent activation of γ-plasminogen. 
Fortelyzin®, compared to the classic plasminogen activator Actilyse®, is the only 
thrombolytic that forms a ternary complex: plasmin-Forteplase-plasminogen activator.  
 
The second mechanism of Forteplase® fibrin selectivity results from the 
difference in the inhibition rate of the plasmin-Forteplase® complex in the bloodstream 
and on the surface of fibrin, i.e. on the clot. This complex is neutralized by α2-
antiplasmin in blood plasma 100 times faster than on the fibrin surface. 
 
Fibrin selectivity of Fortelyzin® increases the therapeutic efficacy and prevents 
the activation of circulating plasminogen, and may also increase the safety of 
thrombolytic therapy by minimizing exposure to circulating blood fibrinogen. 
 
The thrombolytic fibrin selectivity also causes its dose-dependent effect. The 
dose of the moderately selective thrombolytic - Actilyse® is 100 mg for AMI patients 
and 90 mg for patients with ischemic stroke, while the dose of the fibrin-selective 
thrombolytic- Fortelyzin® - is 15 mg for AMI and 10 mg for ischemic stroke. 
 
Also, Fortelyzin® does not cause the formation of neutralizing anti-
staphylokinase antibodies in the blood. In rare cases, the transient formation of anti-
drug antibodies (in low titers) may be observed. 
 
After intravenous administration of Fortelyzin®, there is a slight decrease in blood 
fibrinogen, less than 10 %, within 1 day after administration. 
Fortelyzin® has a short half-life. The main organ for excretion of the drug (75%) 
is the liver. 
 
Pharmacokinetics of a single bolus injection of Fortelyzin® at a dose of 15 mg 
for AMI patients showed a short half-life (t1/2 α) of 5.7 minutes and t1/2 β in the so-
called terminal phase of 30 minutes. 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 18 of 67 
Pharmaceutical form: Lyophilizate for preparation of a solution for intravenous 
administration. 
One vial of Fortelyzin® contains: 
Active substance: Forteplase® (a recombinant protein containing the amino acid 
sequence of staphylokinase), 5 mg (745,000 IU). 
Excipients: L-arginine - 15,0 mg, L-histidine - 2,0 mg, glycine - 30,0 mg, povidone -
17:20,0 mg, polysorbate -20:0,4 mg. 
Solvent: 5mL ampule of sodium chloride 0.9% solution for injection. 
 
 
The results of Fortelyzin® preclinical studies  
 
Preclinical study of toxicological properties was conducted in the Russian 
Cardiology Research Complex of the Ministry of Health of the Russian Federation 
(Director – Academician Chazov E.I.) in accordance with the requirements set out in 
the "Manual on Experimental (Preclinical) Study of New Pharmacological Substances" 
(Moscow, 2005) of the Federal Service for Surveillance in Healthcare and Social 
Development of the Russian Federation. 
 
 
A single intraperitoneal administration of Fortelyzin® in the range of tested 
doses of 1250-28750 mg/kg to BALB/c mice did not cause any signs of intoxication 
and death of the animals. The highest dose tested on mice -28750 mg/kg - was more 
than 216,000 times higher than the highest therapeutic dose for humans (10 mg/person 
or 0.133 mg/kg). 
 
The toxicity study of Fortelyzin® in subchronic experiments, when the drug was 
administered intravenously to rabbits for 2 weeks at doses of 2.66 mg/kg (substance) 
and 1.33 mg/kg (dosage form), did not show the damaging effect of the drug on the 
main organs and body systems of experimental animals. The doses tested were 20 and 
10 times higher than the highest therapeutic dose recommended for humans. It was 
found that Fortelyzin® does not have mutagenic properties. 
 
Also, a 2-week intravenous administration of Fortelyzin® to experimental 
animals did not establish any local irritant effect of the drug.  


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 19 of 67 
 
At the tested dose of 2.66 mg/kg, Fortelyzin® did not cause any deformities and 
embryo malformations and did not affect the reproductive function of the animals. 
 
Fortelyzin®, at the doses and sensitization schemes tested, did not reveal any 
allergizing effect and had no effect on the humoral and cellular immunity, which 
indicates the absence of immunotoxicity of the drug. 
 
According to the results of the preclinical study, Fortelyzin® was recommended 
for clinical trials as a fibrinolytic agent – plasminogen activator (Pharmacotherapeutic 
Group of the State Registration of Medicinal Products). 
 
Fortelyzin® clinical trial according to the Protocol "An open-label, prospective study 
to evaluate the safety, tolerability, and efficacy of Fortelyzin® (a recombinant protein 
containing the amino acid sequence of staphylokinase) vs. Actilyse® in patients with 
acute myocardial infarction (phase I-II)", the authorization of the Federal Service for 
Supervision in Healthcare (hereinafter–“Roszdravnadzor”) No. 400 dated 01.10.2009, 
showed the following: 
 
– a bolus and bolus+infusion i.v. injection of Fortelyzin® at a dose of 10 mg in 
comparison with Actilyse® in patients with acute myocardial infarction with ST-
segment elevation (STEMI) showed comparable tolerability and safety of the study 
drug. 
 
– a bolus and bolus+infusion i.v. injection of Fortelyzin® at a dose of 10 mg in 
comparison with Actilyse® in patients with acute myocardial infarction with ST-
segment elevation (STEMI) showed the efficacy of the study drug comparable to that 
of Actilyse®; 
 
– it is recommended to increase the dose of Fortelyzin® to 15 mg, administered 
according to the two schemes: the first scheme: 10 mg bolus + 5 mg bolus with an 
interval of 30 minutes; the second one: 10 mg bolus + 5 mg infusion for 30 minutes. 
 
The further study assessed a bolus and bolus-infusion of Fortelyzin® at a dose 
of 15 mg vs. Actilyse® according to the Protocol "An open-label, prospective study of 
tolerability and efficacy of Fortelyzin® vs. Actilyse® in patients with acute myocardial 
infarction (phase III)", the authorization of Roszdravnadzor No. 196 dated 28.04.2010. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 20 of 67 
 
A total of 90 people were screened in 6 clinics, and 41 patients in the Fortelyzin® 
study group and 13 patients in the Actilyse® study group were included in the study. 
 
In the Fortelyzin® group, a combination of bolus + infusion was administered 
to 21 patients, and 20 patients received a double bolus dose. 
 
Assessment of the tolerability of the study drug revealed no serious adverse 
events (SAE) or hemorrhagic stroke in any of the groups observed. There was one 
adverse event in the form of "no-reflow" syndrome in the Fortelyzin® group and one 
bleeding in the Fortelyzin® group (considered as not related to the drug administration) 
and in the Actilyse® group (related to the drug administration). 
 
There were no allergic reactions, nausea, and vomiting after administration of 
the drugs in both groups of patients. The maximum drop in blood fibrinogen on the 
first day after administration of Actilyse® was 38 %, and after administration of 
Fortelyzin® - only 7 %. The difference is statistically significant. 
 
There was no decrease in blood pressure, increased pulse, and heart rate in both 
groups of patients. 
 
Thus, the clinical study of bolus and bolus+infusion administration of 
Fortelyzin® showed their comparable tolerability with Actilyse® in STEMI patients. 
 
Direct and indirect methods were used to assess the blood flow through the 
coronary arteries. 
 
Coronary angiography was performed as a direct method (within 180 minutes 
after administration of Fortelyzin® or Actilyse®). Assessment of coronary blood flow 
was carried out according to the TIMI criteria. Evaluation of the TIMI frame count 
(TFC), the so-called aggregated frame-by-frame count, was carried out. 
 
As an indirect method, the control of the QRST complex dynamics of the 
electrocardiogram was performed. When coronary blood flow was restored, there was 
a rapid decrease in the ST segment in the leads in which it was elevated, and the 
formation of negative ("coronary") T-waves were observed. 
At the patient’s first visit, the following parameters were evaluated: a reduction 
of > 50% of the initial ST segment in the lead, where its elevation was maximal 3 
hours after the administration of drugs; the rate of a reduction of the initial ST 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 21 of 67 
segment in the lead, where its elevation was maximal after 90 and 180 minutes after 
drug administration in % (30%, 50% and 70% of the initial value); the blood flow 
through the coronary arteries according to the results of coronary angiography (TIMI 
and TFC scales). 
Table 1. Clinical Efficacy Assessment of Fortelyzin® and Actilyse® 
Parameters 
Fortelyzin® 
Actilyse® 
 
Double bolus 
(n = 20) 
bolus + infusion 
(n = 21) 
Total 
(n = 41) 
Total 
(n = 13) 
Clinical signs of 
reperfusion 
17 
19 
36 
11 
ECG signs of 
reperfusion 
16 
19 
35 
11 
 
Table 1 demonstrates that the clinical efficacy expressed in the absence of signs 
of angina was observed in 36 patients (88%) in the Fortelyzin® group, with 35 of them 
(85%) having electrocardiographic (ECG) signs of reperfusion. In the Actilyse® group, 
11 out of 13 patients (85%) also had clinical and ECG signs of reperfusion. 
Table 2. Angiographic assessment of the efficacy of Fortelyzin® and Actilyse® 
Parameters 
Fortelyzin® 
Actilyse® 
Double bolus 
 (n = 20) 
bolus + infusion 
 (n = 21) 
Total 
 (n = 41) 
Total 
 (n = 13) 
TIMI 0  
3 
2 
5 
1 
 
1  
1 
1 
2 
2 
 
2  
6 (30%) 
6 (29%) 
12 (29%) 
6 (46%) 
 
3  
10 (50%) 
12 (57%) 
22 (54%) 
4 (31%) 
TIMI 2 + 3 
16 (80%) 
18 (86%) 
34 (83%) 
10 (77%) 
 
 
Table 2 shows that after bolus administration of Fortelyzin®, a complete 
reperfusion of blood flow (TIMI 3) is observed in 10 patients (50%), incomplete 
reperfusion of blood flow (TIMI 2) in 6 patients (30%), in total – in 16 patients (80 %). 
 
After bolus infusion of Fortelyzin®, the reperfusion of blood flow according to 
TIMI 3 criteria was observed in 12 patients (57%); according to TIMI 2 criteria – in 
6 patients (29%); in total, in 18 patients (86%). The blood flow did not restore in 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 22 of 67 
4 patients (20 %) after bolus injection of Fortelyzin® and in 3 patients (14%) after 
bolus infusion of the drug. 
In total, in the Fortelyzin® group (n = 41), the blood flow was restored in 
34 patients (83%), in the Actilyze® group – in 10 patients (77 %). It should be noted 
that according to TIMI criteria 3, a higher degree of blood flow reperfusion is observed 
in the Fortelyzin® group (54%) compared with the Actilyse® group (31%). 
 
Thus, the results of the study show the comparable efficacy of the drugs both in 
bolus and bolus-infusion administration. 
Fortelyzin® was recommended for medical use, including its bolus 
administration at the prehospital stage by emergency medical teams. 
 
According to the results of preclinical and phase I-II and III clinical trials, 
Fortelyzin® is registered as a medicinal product for medical use: registration certificate 
LP-001941 as of December 18, 2012. 
 
The project of creating an innovative domestic drug Fortelyzin® involved the 
participation of leading medical and scientific centers: Lomonosov Moscow State 
University (MSU), I. M. Sechenov First Moscow State Medical University, RUDN 
University (the Peoples' Friendship University of Russia), Tomsk Research Institute of 
Cardiology SB Russian Academy of Medical Sciences and others and took more than 
12 years. 
 
When developing the Fortelyzin® substance and pharmaceutical form, the 
requirements of the Russian, British, European, and American Pharmacopoeias were 
taken into account. 
 
The production of Fortelyzin® is carried out in compliance with the current 
regulations and GMP rules in the Russian Federation (License No. 11035-LS-P of 
February 11, 2011). 
 
Monitoring the efficacy and safety of Fortelyzin®. Continuous monitoring of the 
efficacy and safety of Fortelyzin® is carried out and covers 1,531 STEMI patients for 
the period from June 2013 to June 2015. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 23 of 67 
The average age of STEMI patients who received Fortelyzin® was 65 years, 322 
people were older than 75 years old (of the total number of patients, 61% are men). 
According to the localization of myocardial infarction, the patients were distributed 
almost equally (Table 3). 
Table 3. Main characteristics of patients treated with Fortelyzin® 
Number of 
patients 
Sex, 
m/f 
Average 
age, Min-
max 
(years old) 
Number of 
patients 
> 75 years old 
Localization of infarction 
Average 
door-to-
needle time 
Min-max 
(min) 
anterior  
posterior  
1531 
924 / 607 
65 
32-89 
322 
789 
742 
176 
40-720 
 
 
The time from the onset of symptoms of myocardial infarction to the 
administration of Fortelyzin® was 176 min., which is 10% less than in 40 regions of 
the Russian Federation implementing the Vascular Program, but 50% higher than in 
the studies carried out by Tomsk Research Institute of Cardiology and almost 2 times 
higher than in the STREAM trial.  
 
 
ECG evidence of myocardial reperfusion was observed in 1,148 patients out of 
1,531, which was 75%. Angiography confirmed coronary reperfusion in 141 out of 179 
patients (79%), and TIMI 3 was observed in 104 out of 179 patients, which was 58%. 
 
 
Hemorrhagic stroke occurred in 7 out of 1,531 patients (0.46%), with 2 patients 
older than 75 years. The 30-day mortality rate was 5.3%. Major non-cerebral bleeding 
was observed in 3.7%, of which: 1.6% required blood transfusion; minor bleeding from 
the puncture sites was noted in 10.7% of patients (Table 4).  
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 24 of 67 
Table 4. Evaluation of the efficacy and safety of Fortelyzin® 
Signs of reperfusion 
(reduction in the ST 
elevation by 50 %) 
(N = 1531) 
TIMI 
(N = 177) 
intracranial hemorrhage 
Death within 30 days 
Major non-cerebral 
bleeding 
Bleeding required blood 
transfusion 
Minor bleeding 
3 
2 
3 + 2 
1148 
74,9 % 
104 
58 
% 
37 
21 % 
141 
79 % 
7 
0,46 % 
among them 
2 > 75 years old 
81 
5,3 % 
56 
3,7 % 
25 
1,6 % 
165 
10,7 % 
 
 
These data on the efficacy and safety of Fortelyzin®, based on the monitoring 
of 1,531 STEMI patients, indicate the results comparable to thrombolytics Actilyse® 
and Metalyse®. 
 
Since 2015, the original drug Fortelyzin® (a recombinant protein containing the 
amino acid sequence of staphylokinase) has been included by the RF Government in 
the updated list of vital and essential medications for medical use. 
 
Background for the dose of Fortelyzin® in patients with ischemic stroke  
To find a safe and effective dose of Fortelyzin® for AIS patients, in vitro and 
in vivo experiments were performed. 
At the first stage, the in vitro studies of molecular mechanisms of fibrinolysis of 
the fibrin-selective thrombolytics used in the treatment of AIS patients were carried 
out to compare Actilyse® and Purolase with Fortelyzin® used in the treatment of AMI. 
The trial was performed in cooperation with the laboratory of fibrinolysis of the 
MSU Chemistry Department (the head of the laboratory prof. R. B. Aisina, the head of 
the Department of Chemical Enzymology, the member of the Russian Academy of 
Sciences (RAS) - S. D. Varfolomeev). 
The study of the drug amidolytic activity revealed that Fortelyzin® is more 
fibrin-selective thrombolytic than Actilyse® and Purolase, which determined its dose: 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 25 of 67 
10 times less than Actilyse® dose and 5 times less than Purolase. It was also found that 
Fortelyzin® does not bind to fibrinogen placed in a 0.9% NaCl solution in vitro, 
compared to Actilyse® and Purolase, which reduce fibrinogen in the NaCl solution by 
50% of its initial value. 
Thus, the experimental studies performed in vitro to compare Fortelyzin® with 
Actilyse® and Purolase used in IS patients confirmed the principal possibility for 
Fortelyzin® application both in AMI patients and in patients with AIS.  
Thus, it was necessary to make a comparison of these three drugs on AIS models 
in animals, the choice of which was associated with their different species specificity. 
It is known that staphylokinase is fibrin-selective in humans, primates, pigs, and 
rabbits, neutral in rats, and fibrin-nonselective in dogs, which caused massive 
hemorrhages and animal death in experimental studies. 
Therefore, it was decided to make an experimental AIS model in rabbits. 
The creation of such model was a separate scientific task performed jointly with 
the Laboratory of Drug Toxicology of the Russian Cardiology Research 
Complex (RCRPC) of the RF Ministry of Health (the head of the laboratory, prof. E. 
V. Arzamassev, Director – Academician E. I. Chazov). The creation of a rabbit model 
of acute ischemic stroke is described in detail in the Investigator's Brochure. 
Evaluation of thrombolytic efficacy involved the determination of the degree of 
lysis of the embolus inserted in the right middle-cerebral artery of the rabbit, the size 
of cerebral infarction before and after injection of thrombolytics, and neurological 
status of the rabbit according to the modified Bederson scale (MBS) (Bederson JB, et 
al. Rat middle cerebral artery occlusion: evolution of the model and development of a 
neurologic examination. Stroke. 1986;17:472-476.) before and after thrombolysis: 
A grade of 0 indicates no neurological damage;  
A grade of 1 indicates minor to moderate neurological damage; 
A grade of 2 indicates severe neurological damage, including hemiparesis, 
persistent sopor/stupor (Persistent Vegetative State (PVS), and circadian behavior; 
A grade of 3 indicates death. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 26 of 67 
Safety assessment of the thrombolytic use in AIS rabbits involved the 
determination of the number of deaths, hemorrhagic transformation, bleeding, and the 
concentration of blood fibrinogen. 
Based on the results of the in vitro studies of Fortelyzin®, Actilyse® and 
Purolase, the following doses of the drugs were chosen for their administration to AIS 
rabbits: Fortelyzin® - 0.133 mg/kg, Actilyse® - 1.33 mg/kg, and 0,665 mg/kg of 
Purolase, which, based on the rabbit weight equal on average to 3 kg, is 0.4 mg of 
Fortelyzin®, 4 mg of Actilyse® and 2 mg of Purolase. 
These doses correspond to 10 mg of Fortelyzin®, 100 mg of Actilyse®, and 
8 mln/IU of Purolase in humans.  
Fortelyzin® at a dose of 0.4 mg was injected intravenously as a bolus 
(5 seconds), and 4 mg of Actilyse® and 2 mg of Purolase were injected intravenously 
for 30 minutes. 
Efficacy evaluation revealed a 50% reduction in the size of the embolus inserted 
into the right middle cerebral artery of rabbits when using each of the thrombolytics.  
The pathomorphological study of the brain of AIS rabbits showed the greatest 
reduction in the size of brain infarction when using Fortelyzin® – 530 mm3, Actilyse® 
– 450 mm3, Purolase – 400 mm3 from the initial 1250 mm3 (the difference between the 
drugs was not statistically significant). 
Injection of Fortelyzin® reduced the neurological damage by 0.91 points of 
MBS, Actilyse® – 0.65 points of MBS, and Purolase by 0.58 points of MBS from the 
initial 2.3 MBS scores in each group of the thrombolytics. 
Application of Fortelyzin® revealed a high correlation between the reduction in 
the size of brain infarction and a degree of reduction in the neurological symptoms 
according to MBS (r = 0.67, p< 0.001). 
Safety assessment of the doses used - Fortelyzin® 0.4 mg, Actilyze® – 4 mg, 
and Purolase® 2 mg revealed no deaths, hemorrhagic transformations and bleeding in 
AIS rabbits. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 27 of 67 
When applying 0.4 mg of Fortelyzin®, the reduction of fibrinogen was 15% 
from the baseline, when applying Actilyse® and Purolase® – 40% and 52%, 
respectively. 
At the next stage, the efficacy and safety of the double doses of Fortelyzin® 
(0.8 mg), Actilyse® (8.0 mg), and Purolase® (4.0 mg) in thrombolysis in rabbits with 
ischemic stroke were evaluated. The double doses studied correspond to 20 mg of 
Fortelyzin®, 200 mg of Actilyse®, and 16 mln/IU of Purolase® in humans. 
Evaluation of the efficacy of double doses of the studied drugs in AIS rabbits 
has shown that such doses do not increase the area of lysis of the embolus, decrease 
the volume of brain infarction and reduce the amount of neurological damage 
according to the modified Bederson scale (MBS). 
However, the double doses of Actilyse® and Purolase® led to a catastrophic 
drop in blood fibrinogen of more than 90%, causing the massive cerebral hemorrhages 
and death of animals. 
The data obtained correspond to the literature data on the application of 
immunogenic staphylokinase in rabbits with ischemic stroke in comparison with 
Actilyse® (S. Vanderschueren, et al. Intravenous Thrombolysis With Recombinant 
Staphylokinase Versus tissue-type Plasminogen Activator in a Rabbit Embolic Stroke 
Model. Stroke. 1997; 28:1783-1788). 
Therefore, the study of the two doses of Fortelyzin® - 0.4 mg and 0.8 mg - in 
rabbits with ischemic stroke, corresponding to 10 mg and 20 mg of Fortelyzin® in 
humans, showed their equal effectiveness and greater safety of a dose of 0.4 mg. 
In connection with the above, a 10 mg dose of Fortelyzin® was selected for the 
treatment of patients with ischemic stroke. 
First, such choice is due to the fact that the dose of 10 mg equivalent to 0.4 mg 
of Fortelyzin® in AIS rabbits has shown its high safety and comparable effectiveness 
with the doses of Actilyse® (4 mg) and Purolase (2 mg) used in ischemic stroke. 
Secondly, the 10 mg dose of Fortelyzin® has also shown high efficacy and safety 
in the clinical trials (phase I and II) (page 18 of this protocol) of Fortelyzin® in 
comparison with Actilyse® (100 mg) in AMI patients. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 28 of 67 
Indications 
 
Ischemic stroke in men and women aged 18 and older 
 
After 80 years with caution 
 
The time from the onset of stroke symptoms is less than 4.5 hours. 
 
Contraindications 
 
Cerebral  
1. Neuroimaging (CT, MRI) signs of intracranial hemorrhage, brain tumors 
2. Hemorrhagic stroke or stroke of an unspecified nature in the anamnesis 
3. Rapid improvement or mild symptoms by the time of the beginning of TLT 
4. Signs of a severe stroke: clinical [score on the National Institutes of Health Stroke 
Scale (NIHSS) > 25), neuroimaging (based on CT of the brain and/or MRI of the 
brain in the diffusion-weighted imaging (DWI) mode, the focus of ischemia extends 
to the territory of more than 1/3 of the CMA pool) 
5. Seizures in the onset of the disease, if there is no certainty that the seizure is a 
clinical manifestation of ischemic stroke with a postictal residual deficiency 
6. Previous stroke or severe traumatic brain injury within 3 months 
7. Stroke of any genesis in the history combined with diabetes mellitus 
8. Suspected subarachnoid hemorrhage (SAH) 
9. Surgical intervention on the brain or spinal cord in the anamnesis 
 
Cerebral and somatic  
10. Arterial aneurysms, defects in the development of arteries or veins 
11. Tumors with a high risk of bleeding 
 
Somatic  
12. Hypersensitivity to any component of the drug 
13. Hemorrhagic diathesis 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 29 of 67 
14. Arterial hypertension greater than 185/105 mm Hg or the need for intensive 
reduction below these figures 
15. Bacterial endocarditis, pericarditis 
16. Gastrointestinal bleeding or bleeding from the genitourinary system in the last 3 
weeks. Confirmed exacerbations of gastric ulcer and duodenal ulcer during the last 3 
months. 
17. Liver failure (cirrhosis, active hepatitis, portal hypertension) 
18. Acute pancreatitis 
19. Present bleeding or extensive bleeding in the last 6 months 
20. Extensive surgery, trauma, delivery, puncture of non-compressible vessels, 
cardiopulmonary resuscitation within the last 10 days 
21. Pregnancy. Before the study, women of childbearing age will be asked to have a 
urine pregnancy test (determination of chorionic gonadotropin (HCG) in the urine). 
22. Data on bleeding or acute injury at the time of examination. 
 
Laboratory  
23. Administration of warfarin and INR > 1,3 
24. Use of heparin for 48 hours with increased aPTT 
25. Thrombocytopenia less than 100,000/µl 
26. Glycemia less than 2.8 and more than 22.5 mmol/l 
 
Posology and method of administration 
For Intravenous Use Only! 
 
Fortelyzin® is administered intravenously as early as possible from the onset of 
clinical symptoms, but no later than 4.5 hours from its onset. The content of each of 
the two vials of 5 mg (745,000 IU) is diluted in 5 ml of isotonic sodium chloride 
solution. Fortelyzin® administered intravenously as a bolus dose of 10 mg for 5-10 s. 
The solution should be prepared immediately before use and should not be stored. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 30 of 67 
According to clinical guidelines, when conducting thrombolytic therapy with 
Fortelyzin® in the intensive care unit, it is necessary to ensure that the following vital 
signs are monitored for at least 24 hours: 
* blood pressure; 
* heart rate; 
* frequency of respiratory movements; 
* body temperature; 
* oxygen saturation. 
 
During thrombolytic therapy and within 24 h after its completion, it is necessary 
to monitor the dynamics of neurological status. 
 
Drug-Drug Interactions 
Fertilyzin® should not be administered concurrently with other medications. If 
severe bleeding occurs (especially from non-compressible vessels), the administration 
of the thrombolytic should be stopped, and fresh frozen plasma should be administered. 
Due to the possible development of arterial hypertension observed during 
thrombolysis with Actilyse®, according to clinical guidelines antihypertensive drugs 
can be used in dosage forms for intravenous administration: 
* angiotensin-converting enzyme inhibitors -enalaprilate (Enap R); however, 
simultaneous use of angiotensin-converting enzyme inhibitors with alteplase increases 
the risk of angioedema; 
* nitrates – isosorbite dinitrate (isoket), nitroglycerin; however, the frequent 
side effect of nitrates is headache, which can mask the development of intracranial 
hemorrhage; 
* vasodilators – sodium nitroprusside; 
* ganglioblockers-azamethonium bromide (Pentamine); 
* alpha-blockers-urapidil (Ebrantil), proxodolol (Albetor); 
* calcium channel blockers - nimodipine (Nimotop). 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 31 of 67 
Side effect/Adverse events 
 
Bleeding of varying severity is possible. Bleeding events associated with 
thrombolytic therapy can be divided into two large groups: 
– external bleeding (usually occurred at the sites of blood vessel punctures); 
– internal bleeding in any part or cavity of the body. 
 
The possibility of allergic reactions is not excluded. 
 
As a rule, therapeutic doses of Fortelyzin® do not lead to arterial hypotension, 
but the possibility of its occurrence cannot be excluded. 
Experience of using Fortelyzin® in AMI patients with simultaneous use of 
anticoagulant and dual antithrombotic therapy showed a minimal percentage of 
hemorrhagic stroke (0.46 %) or major bleeding requiring blood transfusion (1.6%). 
 
However, due to the short half-life of Fortelyzin® and its minimal effect on the 
blood coagulation system (drop in fibrinogen is less than 10 %), no clotting factor 
replacement is required. 
 
In the case of bleeding, accompanied by a decrease in hemoglobin by more than 
3 g/dl, the use of blood components transfusion is required. 
 
Bleeding that occurs within the first 24 hours after Fortelyzin® administration 
can be regarded as associated with drug use. 
 
Hemorrhagic transformation of the brain lesion is symptomatic if its 
development leads to an increase in the total NIHSS score by 4 points or more. In most 
cases of intracerebral hemorrhages, asymptomatic hemorrhagic transformation is 
registered after thrombolytic therapy. It is detected by CT or MRI and often 
accompanied by clinical improvement and is evidence of reperfusion. 
If intracerebral hemorrhage is suspected, neuroimaging (brain CT or MRI) must 
be performed. If necessary, the patient should be consulted by a neurosurgeon. 
 
Overdose (intoxication) 
 
Adverse drug effects from overdose can result in hemorrhagic complications. 
 
In the case of local bleeding – it can be stopped without stopping the drug 
administration. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 32 of 67 
 
If life-threatening bleeding occurs, Fortelyzin® administration should be 
stopped and fresh frozen plasma or whole blood should be prescribed. If necessary, to 
neutralize the effect of Fortelyzin®, an anti-fibrinolytic agent can be administered, for 
example, aminocaproic acid, or tranexamic acid. 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 33 of 67 
3. POTENTIAL BENEFITS AND RISKS TO PARTICIPANTS  
 
The expected benefit of using the drug is to restore normal blood circulation in 
the ischemic areas of the brain, which will normalize brain functions and avoid further 
disability of patients.  
 
The possible undesirable effects of Fortelyzin® is the likelihood of bleeding. 
Local bleeding (injection sites, oral cavity), as a rule, does not require additional 
treatment. 
 
Internal bleeding may be caused by latent forms of peptic ulcer, esophageal 
erosion, bleeding from hemorrhoidal veins, esophageal veins, etc. Thus, careful history 
taking with special attention to the above-mentioned diseases and a clear selection of 
patients taking into account all contraindications allows, in most cases, to minimize the 
risk of bleeding.  
During thrombolytic therapy in patients with ischemic stroke, hemorrhagic 
transformation of the focus is possible. However, it is believed that the benefit of using 
drugs of this group in patients with thrombosis significantly exceeds the risk of 
bleeding. 
 
During the study, all adverse events will be carefully controlled. If they appear, 
all available data will be evaluated and appropriate measures will be taken by the staff 
of the research centers. 
 
 All participants in this clinical study are insured. If a patient is harmed in the 
trial and this harm is a direct result of the action of the study drug and/or medical 
manipulations used in accordance with the Study Protocol, the Insurance Company 
undertakes to fully reimburse the participant all expenses for the necessary medical 
care. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 34 of 67 
4. REGULATORY FRAMEWORK AND ETHICAL ASPECTS OF THE 
STUDY 
The clinical trial is conducted in accordance with the guidelines of the 
International Conference on Harmonization Good Clinical Practice (ICH GCP), the 
ethical principles set out in the World Medical Association Declaration of Helsinki 
"Recommendations guiding physicians in biomedical 
research 
involving 
human subjects" (1964-2008), the Directive 2001/20/EC of the European Parliament 
and the requirements of the legislation of the Russian Federation: 
 
• Federal Law No. 61-FZ "On Circulation of Medicines" (as amended of October 
11, 2010); 
• Federal Law No. 323-FZ of November 21, 2011 “On the fundamentals of 
health protection in the Russian Federation” (as amended of July 2, 2013); 
• ICH Harmonized Tripartite Guidelines for Good Clinical Practice, 1996  
• Rules for quality clinical trials in the Russian Federation (OST 42-511-99); 
• Rules of clinical practice in the Russian Federation (2003); 
• National Standard of the Russian Federation GOST R 52379-2005 "Good 
Clinical Practice" (approved by the order of the Federal Technical Regulation 
and Metrology Agency (Rosstandart) No. 232-ST of September 27, 2005); 
• RF Government Regulation of September 13, 2010, No. 714 “On the approval 
of model rules for Compulsory Insurance of Life and Health of a patient involved 
in clinical trials of a medicinal product” (as amended by the RF Government No. 
393 of May 18, 2011);  
• Order of the Ministry of Health and Social Development of the Russian 
Federation No. 774 of August 31, 2010 “On Ethics Board”; 
• Guideline for quality clinical trials of medicinal products. Edited by A. N. 
Mironov, Moscow, 2012; 
• RF Government Resolution No. 1012 of December 27, 2007, and No. 186 of 
March 2, 2009; 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 35 of 67 
• Order of the Ministry of Health and Social Development of the Russian 
Federation No. 389n of July 6, 2009 “On Approval of Medical Care Procedure 
to patients with Acute disorders of Cerebral Circulation”; 
• Order of the Ministry of Health of the Russian Federation No. 928n of November 
15, 2012 “On Approval of Medical Care Procedure to patients with Acute 
disorders of Cerebral Circulation”; 
• Russian clinical guidelines for thrombolytic therapy in ischemic stroke, 
developed by the Russian Society of Neurologists and the National Stroke 
Association (Principal Investigator – Academician of RAS E. I. Gusev and 
corresponding member of RAS V. I. Skvortsova), 2012; 
• Methodological guide "Thrombolytic therapy in ischemic stroke", of the 
Ministry of Health and Social Development of the Russian Federation, edited by 
a corresponding member of RAS V. I. Skvortsova, Moscow, 2010; 
• Recommendations for thrombolytic therapy in patients with ischemic stroke. 
Moscow, 2014; 
• Guidelines for the Early Management of Patients with Acute Ischemic Stroke: 
A Guideline for Healthcare Professionals from the American Heart 
Association/American Stroke Association. Stroke, 2013; 44; 870-947.  
The trial will be initiated in all research centers only after receiving the written 
permission to conduct the study, approval of the Ethics Committee, and receiving 
signatures on the clinical trial protocol of each of the parties involved in the study. 
Researchers will be timely familiarized with the materials of the study before it 
begins. The qualification of researchers will meet the requirements necessary to 
conduct high-quality clinical trials. 
The selection of prospective study participants is made on a volunteers basis. 
 
4.1. Ethics Committee 
The materials of the clinical trial (including the protocol, Information Sheet, and 
materials provided to the patient) will be approved by the Ethics Committee of the 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 36 of 67 
Ministry of Health of the Russian Federation before the start of the study. All 
amendments to the Protocol and updated versions of the Patient Information Sheet will 
also be submitted to the Ethics Committee. 
The clinical trial will be conducted in accordance with the Protocol approved by 
the Ethics Committee. 
4.2. Patient Information Sheet 
 
Informed consent will be obtained from all prospective study participants before 
any research procedures are initiated. Prior to this, the Investigator must fully inform a 
patient about all aspects of clinical research, including its purposes, study procedures, 
expected risks and benefits of participation, and voluntariness of participation. 
The patient must be provided with written information about the study 
(Information Sheet), approved by the Ethics Committee. The patient's information 
sheet will contain all information about the planned clinical trial, as well as the terms 
of confidentiality and the use of the patient’s data. The rights, safety, and well-being 
of the participants will take priority and prevail over the interests of science and society. 
If necessary, information about the procedure for obtaining informed consent from 
patients/volunteers can be submitted to the Ethics Committee. 
Prior to the patient's participation in the clinical trial, two (2) copies of the 
Informed Consent Form with the patient's name must be signed and personally dated 
by the patient or the patient's legal representative, or by a person who conducted the 
informed consent discussion. One copy of signed and dated written Informed Consent 
will be provided to the patient, and the other will be kept by the Investigator. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 37 of 67 
The information obtained during the study that may reveal the patient's identity 
will be kept secret and can only be disclosed within the limits established by law. 
 
5. TRIAL AIM 
 
Assessment of the efficacy and safety of single-bolus intravenous injection of 
Fortelyzin® in comparison with bolus infusion of Actilyse® in patients with ischemic 
stroke. 
6. TRIAL OBJECTIVES 
• To assess the efficacy of single-bolus intravenous injection of Fortelyzin® in 
comparison with bolus infusion of Actilyse® in patients with ischemic stroke.  
• To assess the safety and possible adverse events of single-bolus intravenous injection 
of Fortelyzin® in comparison with bolus infusion of Actilyse® in patients with 
ischemic stroke. 
 
When the patient's condition does not allow him/her to express his/her will and 
there is no legal representative and the need for treatment is urgent, the question of 
medical intervention in the interest of the patient and his/her inclusion in the study is 
resolved by a council of physicians, and if it is not possible – by his/her attending (duty) 
physician with following notification of the Principal Investigator, officials of the 
medical institution and the legal representative. 
The Patient Information Sheet and Informed Consent Form must be reviewed 
and approved by the Ethics Committee. The final version of the Study Protocol, 
Individual Patient Registration Form, information for the patient, and Informed 
Consent Form must be approved by the Russian Ministry of Health and the Local 
Ethics Committee (if any) at the Research Center.  
All subsequent amendments to the above-mentioned documents will also be 
submitted for approval to these institutions. In addition, these same institutions will be 
informed about all adverse events associated or possibly related to the use of the study 
drug. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 38 of 67 
7. TRIAL POPULATION 
Description of the trial population 
 
This trial is planned to involve adult patients of both sexes aged 18 years and 
older with a verified diagnosis of ischemic stroke. 
7.1. Inclusion criteria  
• Men and women aged 18-80 (inclusive) years old. 
• Verified diagnosis of ischemic stroke (neuroimaging (CT/MRI) signs of 
ischemic stroke, from 5 to 25 points on the NIHSS scale).  
• Duration of symptoms of ischemic stroke is not more than 4.5 hours before 
starting thrombolytic therapy. 
• The patient's consent to use reliable contraceptive methods throughout the 
trial and within 3 weeks after: 
– women who have a negative pregnancy test and use the following means of 
contraception: intrauterine devices, oral contraceptives, a contraceptive patch, long-
acting injectable contraceptives, a double-barrier method of contraception. Women 
who are not at risk of pregnancy for health-related reasons (documented conditions: 
hysterectomy, tubal ligation, infertility, menopause for more than 1 year) may also 
be involved in the trial.);  
– men who use barrier contraception. Men who unable to fertilize (documented 
conditions: vasectomy, infertility) may also be involved in the trial.  
• A participant provided signed and dated informed consent of participation in 
the trial. 
7.2. Exclusion criteria  
• The time of the onset of the first symptoms is more than 4.5 hours from the 
onset of the disease or the time of the onset of the first symptoms of a stroke 
is not known (for example, the development of a stroke during sleep - the so-
called "night stroke"). 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 39 of 67 
• Increased sensitivity to alteplase, gentamicin (residual traces from the 
production process). 
• Systolic blood pressure greater than 185 mm Hg or diastolic blood pressure 
greater than 110 mm Hg or the need for intravenous administration of drugs 
to reduce the blood pressure to these boundaries. 
• Neuroimaging (CT, MRI) signs of intracranial hemorrhage, brain tumors, 
arteriovenous malformation, brain abscess, aneurysm of cerebral vessels. 
• Surgery on the brain or spinal cord. 
• Suspicion of subarachnoid hemorrhage. 
• Signs of severe stroke: clinical signs (stroke scale NIH> 25), neuroimaging 
(according to CT of the brain and/or MRI of the brain in the DWI, the 
ischemia focuses on the territory of more than 1/3 of the MA pool). 
• Simultaneous administration of oral anticoagulants, for example, warfarin 
with INR> 1.3. 
• The use of direct anticoagulants (heparin, heparinoids) in the 48 hours 
preceding the stroke with APTT values above the norm.  
• Prior stroke or severe head injury within 3 months. 
• Significant regression of neurological symptoms during the observation of 
the patient 
• Light neurological symptoms (NIH <4 points) 
• Hemorrhagic stroke or stroke, unspecified in history. 
• Strokes of any genesis in the history of a patient with diabetes mellitus. 
• Gastrointestinal bleeding or bleeding from the genitourinary system in the 
last 3 weeks. Confirmed exacerbations of gastric ulcer and duodenal ulcer 
during the last 3 months. 
• Extensive bleeding, present or within the previous 6 months. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 40 of 67 
• Severe liver disease, including liver failure, cirrhosis, portal hypertension 
(with varicose veins of the esophagus), active hepatitis. 
• Acute pancreatitis. 
• Bacterial endocarditis, pericarditis. 
• Aneurysms of arteries, malformations of arteries and veins. Suspicion of 
exfoliating aortic aneurysm. 
• Neoplasms with an increased risk of bleeding. 
• Large operations or severe injuries within the last 14 days, minor surgery or 
invasive manipulation in the last 10 days. 
• Puncture of uncompensated arteries and veins during the last 7 days. 
• Prolonged or traumatic cardiopulmonary resuscitation (more than 2 min). 
• Pregnancy, obstetrics, 10 days after delivery.  
• The number of platelets is less than 100,000/μL. 
• Blood glucose less than 2.8 mmol/l or more than 22,5 mmol/l. 
• Hemorrhagic diathesis, including renal and hepatic insufficiency. 
• Data on bleeding or acute trauma at the time of examination. 
• Seizures in the onset of the disease, if there is no certainty that the seizure is a 
clinical manifestation of ischemic stroke with a postictal residual deficiency. 
7.3. Trial completion and post-trial therapy 
 
The trial for a patient will be considered completed after the 6th visit on the 90th 
day after thrombolysis.  


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 41 of 67 
7.4. Criteria for early termination of trial participants 
Follow-up should be continued until the patient's condition is restored; until the 
diagnosis of an adverse event or a change in the safety parameter is stabilized or 
established chronically; as long as there are clinical indications for follow-up, or the 
duration of follow-up is determined by an attending physician-investigator. 
 
A patient should be withdrawn from the trial immediately after any of the 
following situations occur: 
• a patient or his/her legal representative or attending physician sends a request 
to exclude the patient from the trial, based on the withdrawal of Informed 
Consent (the patient's unwillingness to continue participation in the trial); 
• pregnancy; 
• significant violation of the Protocol procedures;  
The reason for each unscheduled termination should be noted in the Case Report 
Form (CRF) in the section "Trial Completion". Follow-up will be performed for all 
patients who have dropped out of the trial due to adverse events or other safety 
parameters.  
Follow-up will also be performed for all patients who have dropped out of the trial 
for other reasons, but also experience adverse events or have other safety parameters 
that might lead to discontinuation of therapy. In the case of early termination, the 
treatment of the underlying disease continues in accordance with recommendations and 
health standards.  
In the case of pregnancy, every effort should be made to fully monitor the course 
of pregnancy until the moment of delivery, and child growth. 
 
Randomized patients who did not complete the trial for reasons including those 
related to the trial drug will be included in the analysis of efficacy and safety data. 
Replacement of the removed randomized patients with newly included ones is not 
provided. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 42 of 67 
7.5. Criteria for early termination of the trial 
 
7.6. Exclusion of Research center  
The trial conducted at a separate clinical center may be terminated if the Sponsor, 
Investigator, Regulatory Authorities, or the Ethics Committee of the clinical center 
deem it necessary for any reason. 
 
 
 
The trial may be completed prematurely: 
• if the trial reveals serious adverse events associated with the use of the trial drug and 
makes its further use unacceptable from an ethical point of view; 
• when new information is received that indicates a high risk for the trial participants; 
• in the case of circumstances considered as force majeure events; 
• at the request of the Federal Regulatory Authorities and at the decision of the Sponsor. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 43 of 67 
8. DESIGN AND PLAN OF RESEARCH ACTIVITIES 
The trial will include 336 patients with ischemic stroke. The patients will be 
monitored for 90 days: 1-2 days in the Neurological Intensive Care Unit and the 
Department of Neurology in the remaining days before discharge (on average 14 days), 
with an outpatient visit on the 90th day. 
Before the trial, all patients will undergo the following procedures: collection of 
thorough medical history, measurement of vital signs, physical examination, 
assessment of neurological status, CT/MRI scans of the brain, duplex scanning, and 
angiography of the brain vessels (if necessary), ECG, blood and urine samples for 
laboratory tests: 
*complete blood count: hemoglobin, red blood cells, hematocrit, leukocyte formula: 
neutrophils, eosinophils, basophils, monocytes, lymphocytes, platelets, ESR; 
*biochemical blood parameters: total protein, creatinine, urea, AST, ALT, CK, total 
bilirubin, glucose, sodium, potassium, cholesterol, HDL-C, triglycerides; 
*coagulogram: fibrinogen, activated partial thromboplastin time (aPTT), INR, 
prothrombin; 
The current trial is a multicenter, open-label, randomized, parallel-group, phase 
III b non-inferiority trial to compare the efficacy and safety of the trial drug. 
Start of research, initiation of centers: « 
 
»  
 
 20 
  
Enrollment of patients: «  
»  
 
 20 
  
Result processing and reporting: « 
 
»  
 
 20 
  
The enrollment period may be extended to total patient enrollment if a research 
center fails to enroll the planned number of patients. The end date of the trial is 
considered to be the end date of the trial in all patients involved. 
 
In each research center, the subject of the trial is assigned a serial number that 
corresponds to the sequence of patient inclusion in the trial. The serial number is 
reflected in the Case Report Form (CRF). The list of patient’s identification is stored in 
the trial file. Patients are included in the trial only after signing the Informed Consent 
Form. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 44 of 67 
*common urine analysis: color, transparency, reaction, density, glucose, protein, 
bilirubin/urobilinogen, white blood cells, red blood cells. 
After enrollment, patients will be randomized using the "envelope method" into 
two equal groups of 168 patients each (a total of 336 people, including 10% who may 
have dropped out) and assigned to receive either Fortelyzin® or Actilyse®. 
Fortelyzin® will be given as single i.v. bolus over 5 - 10 seconds. Actilyse® will be 
administered following the instruction for use. 
To compare the efficacy and safety of Fortelyzin® vs Actilyse®, patients will 
be monitored for 90 days after administration of the drug. During this period, patients 
will have 6 visits (Table 5). 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 45 of 67 
Table 5. Plan of the trial 
 
V 1 
V 2 
V 3 
V 4 
V 5 
V 6 
 
0 
1 day 
2 days 
7 days 14 days 90 days 
Signing Informed consent  
+ 
– 
– 
– 
– 
– 
Demographic data 
+ 
– 
– 
– 
– 
– 
Previous treatment 
+ 
– 
– 
– 
– 
– 
CT/MRI SCAN 
+ 
+ 
– 
+ 
– 
– 
ASPECTS 
+ 
+ 
– 
+ 
– 
– 
Dynamics of neurological status 
– 
+ 
+ 
– 
– 
– 
Changes by organs and systems 
– 
+ 
+ 
+ 
+ 
– 
ТОAST criteria 
– 
– 
+ 
– 
– 
– 
Medical history 
+ 
– 
– 
– 
– 
– 
Measurement of vital signs  
+ 
+ 
+ 
+ 
+ 
+ 
Physical examination 
+ 
– 
– 
– 
– 
– 
Concomitant diseases 
+ 
– 
– 
– 
– 
+ 
NIHSS 
+ 
+ 
+ 
+ 
+ 
+ 
Bartel 
– 
– 
– 
+ 
+ 
+ 
Rankin 
+ 
– 
+ 
+ 
+ 
+ 
Meningeal symptoms 
+ 
+ 
+ 
+ 
+ 
– 
ECG 
+ 
– 
– 
+ 
+ 
– 
Duplex scanning of the major arteries of the 
head (if necessary) 
+ 
– 
– 
+ 
– 
– 
Coagulogram 
+ 
+ 
+ 
+ 
+ 
– 
Complete blood count 
+ 
– 
+ 
+ 
+ 
– 
Biochemical blood analysis 
+ 
– 
– 
+ 
+ 
– 
Blood sampling for Pharmacokinetic analysis  
– 
+ 
– 
– 
– 
– 
Common urine analysis 
+ 
– 
+ 
+ 
+ 
– 
Inclusion/Exclusion criteria  
+ 
– 
– 
– 
– 
– 
Angiography of cerebral vessels (if necessary) 
+ 
– 
– 
– 
– 
– 
Administration of Fortelyzin® or Actilyse® 
– 
+ 
– 
– 
– 
– 
Change in concomitant therapy 
– 
+ 
+ 
+ 
+ 
+ 
Registration of AE  
– 
+ 
+ 
+ 
+ 
+ 
Bleeding assessment 
– 
+ 
+ 
+ 
+ 
– 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 46 of 67 
9. TRIAL PROCEDURES 
Visit 1 (Start of the trial) 
Prior to administration of the trial drug, all patients will undergo the following 
procedures:  
• determination of inclusion/non-inclusion criteria 
• signing of Informed consent  
• collection of demographic data (date of birth, sex) 
• collection of medical history (duration of the underlying disease, previous 
therapy, allergological anamnesis, etc.) 
• physical examination  
• evaluation of changes by organs and systems 
• assessment of neurological status using the NIHSS and modified Rankin scales 
(mRS) 
• Duplex scanning of the major arteries of the head (if necessary) 
• CT/MRI examination of the brain 
• assessment of CT changes according to ASPECTS (Alberta Stroke Program 
Early CT Score) 
• angiography of the cerebral vessels (if necessary) 
• blood sampling for hematological and biochemical analyses 
• coagulogram 
• common urine analysis 
• ECG 
• assessment of the patient's compliance with the inclusion/exclusion criteria 
• randomization  
Assignment of individual patient identification code (IPIC) 
The Investigator must ensure the anonymity of the trial subjects. The full names of 
all patients participated in the trial are strictly confidential. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 47 of 67 
After signing the Informed Consent Form, each patient will be assigned an 
Individual Patient Identification Code (IPIC) in accordance with the current legislation 
(Table 6). 
Table 6. Individual Patient Identification Code 
Permission of the Ministry of 
Health of the Russian 
Federation No. 
Permission's date of issue, 
DD/MM/YYYY/ 
Serial number of the medical 
organization specified in the 
Permission 
Patient's initials 
Patient's date of birth, 
DD/MM/YYYY/ 
A unique number assigned to 
the patient by the Investigator, 
consisting of letters and 
numbers (the name of the 
protocol and the screening 
number under which the 
patient was included in the 
trial) 
 
 
 
 
 
 
 
The individual patient identification code (IPIC) is established by the Insurant 
based on the patient data provided by the Investigator. The individual patient 
identification code is reported by the Insurant to the Investigator for inclusion in his 
medical documentation. The IPIC will be reflected in the primary documentation, Case 
Report Form, and Patient Information Sheet with the informed consent form. The IPIC 
will be transmitted by the Code Registry by the Insurant to the Insurer. 
 
The individual identification code assigned to the patient is not subject to change. 
The Investigator should maintain a separate Identification Registry containing 
the individual numbers, surnames, dates of birth, addresses, phone numbers, and 
medical records (if available). Only the initials, dates of birth, and unique patient 
numbers that are part of the Individual Patient Identification Code will be used in the 
report and trial documentation. 
The randomization plan will be known by the Principal Investigator and 
Regulatory Authorities. 
 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 48 of 67 
Visit 2. 
• Administration of Fortelyzin® or Actilyse®. 
• On Visit 2, the changes in neurological status will be recorded, as well as 
registration and evaluation of bleeding and adverse events based on patient 
complaints, data from neurological, physical, and laboratory-instrumental 
studies. 
Table 7. Monitoring of parameters at the moment of thrombolysis 
Trial parameter 
Frequency of examination 
BP 
Every 15 minutes to 2 hours after the start of 
thrombolysis 
Vital signs 
Every 30 minutes for 6 hours 
NIHSS 
Every 15 minutes for 1 hour 
Meningeal symptoms  
Every 15 minutes for 1 hour 
Table 8. Monitoring of parameters during the first day (24h) after thrombolysis 
Trial parameter 
Frequency of examination  
BP 
Every hour until the end of the day (within 24 hours) 
Vital signs 
Every 30 minutes to 6 hours, then every hour until the 
end of the day 
Meningeal symptoms  
Every hour for 24 hours 
NIHSS 
Every hour for 24 hours 
 
• CT/MRI 24 hours after TLT or if the patient's condition worsens 
• assessment of CT changes according to ASPECTS  
• registration of adverse events (AE) 
• assessment of bleeding 
All adverse events identified should be recorded in the Case Report Form (CRF). 
The Investigator should report the adverse events to Roszdravnadzor within the next 
24 hours. The assessment of bleeding is carried out according to the corresponding 
scales (see Safety assessment). Monitoring of coagulogram parameters (fibrinogen) 
will be carried out within 24 h from the time of Fortelyzin® or Actilyse® 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 49 of 67 
administration. CT/MRI examination of the brain will be repeated 24 hours after the 
thrombolysis or if the patient's condition worsens. 
During the visit, patients will receive treatment in accordance with the Russian 
Clinical Guidelines for Thrombolytic Therapy in Ischemic Stroke, 2015. 
Visit 3. 
• verification of the stroke subtype according to TOAST criteria 
• measurement of vital signs 
• physical examination 
• assessment of neurological status scales scores 
• coagulogram 
• complete blood count 
• common urine analysis 
Visit 4. 
• measurement of vital signs 
• physical examination 
• assessment of neurological status scales scores 
• CT/MRI scans 
• assessment of CT changes according to ASPECTS  
• ECG 
• ultrasound examination of brain vessels (if necessary) 
• coagulogram 
• complete blood count 
• biochemical analysis of blood 
• common urine analysis. 
Visit 5. 
• measurement of vital signs 
• physical examination 
• assessment of neurological status scales scores 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 50 of 67 
• ECG 
• coagulogram 
• complete blood count 
• biochemical analysis of blood 
• common urine analysis. 
 
During visits 2-5, information about adverse events and bleeding will be 
collected and recorded into the Case Report Form. The data on adverse events will be 
shared with the Sponsor. In addition, information about changes in concomitant 
therapy will also be recorded. 
Visit 6. 
• measurement of vital signs 
• collection of information about SAE and AE, as well as recording the Bartel 
index scores, Rankin and NIHSS scale scores 
• collection of information about concomitant diseases. 
Unscheduled visits 
 
Unscheduled visits may be made at the discretion of the Investigator or at the 
initiative of the patient. For all unscheduled visits, the corresponding CRF page 
should be filled out.  
9.1. Description of some research procedures 
9.1.1. Anamnesis  
 
Collection of the anamnesis of the studied disease will be carried out during the 
screening, as well as information on significant past or current concomitant diseases 
(for example, allergic reactions, congenital and acquired heart and vascular diseases, 
other serious diseases, organ transplantation, mental disorders). 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 51 of 67 
9.1.2. Collection of information about concomitant and previous therapy 
 
All medications taken within 30 days prior to screening must be registered in the 
primary documentation. Separately, it will be clarified whether the patient took any 
prohibited drugs that prevent his/her inclusion in the trial. 
9.1.3. Physical examination  
 
Physical examination should be performed by the research physician. It includes 
(at a minimum) assessment of the patient’s general condition and the following body 
systems: lymph nodes, mouth, and pharynx, lungs, cardiovascular system, abdominal 
cavity, extremities, musculoskeletal system, nervous system, and skin. 
 
All changes considered clinically significant should be reported as an anamnesis 
before the study drug administration, and after the patient received the study drug, all 
changes should be reported as adverse events. 
9.1.4. Vital signs 
 
Vital signs include blood pressure (BP), heart rate (HR), and respiratory rate 
(RR), which must be measured and recorded by the staff of the research center. 
Measurements of blood pressure and heart rate should be taken at the same body 
position throughout the trial. 
9.1.5. Electrocardiography 
 
Electrocardiography (ECG) is performed in accordance with normal clinical 
practice. A prerequisite is the registration of all 12 standard leads. 
ECG interpretation is performed by an authorized researcher. It is necessary to 
indicate the date of ECG registration and provide a general conclusion. ECG records 
should be kept in the patient's medical records.  
9.1.6. Complete and biochemical blood analysis  
 
The tests are carried out in accordance with the methodology adopted in the 
clinical center laboratory. The following parameters will be measured: 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 52 of 67 
– complete blood count (number of red blood cells, white blood cells, platelets, 
hemoglobin, hematocrit, leukocyte formula, ESR) 
– biochemical blood parameters (glucose, ALT, AST, bilirubin, creatinine, total 
protein, sodium, potassium, CK, urea, cholesterol, HDL-C, triglycerides) 
– coagulogram (fibrinogen, INR, APTT, prothrombin index) 
In the case of clinically significant abnormalities, these phenomena are recorded 
as a concomitant disease (during the screening) or as an adverse event (during the trial).  
9.1.7. Common urine analysis 
 
To perform the analysis, approximately 50 ml of the first-morning portion of 
urine is taken, and the analysis is carried out according to local practice. The following 
parameters are measured:  
 – General characteristics (color, transparency, pH, density, protein, glucose, 
bilirubin/urobilinogen). 
– Microscopy of urinary sediment (red blood cells, white blood cells, epithelium, 
cylinders, bacteria, salts). 
 
In the case of clinically significant abnormalities, these phenomena are recorded 
as a concomitant disease (during the screening) or as an adverse event (during the trial). 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 53 of 67 
10. THERAPY DURING THE TRIAL 
Study drug: Fortelyzin® 
Manufacturer: Supergene, LLC, Russia 
Drug description 
Pharmaceutical form: lyophilizate for preparation of a solution for intravenous 
administration, supplied with a solvent 
Detailed description: 
Lyophilizate: tablet-like freeze-dried mass of white or almost white color. 
Solvent: colorless transparent liquid. 
Reconstituted solution: colorless, transparent or slightly opalescent liquid.  
Composition: 
Active substance: Forteplase® (recombinant protein containing the amino acid sequence 
of staphylokinase) 5 mg (745,000 IU). 
Excipients: L-arginine - 15,0 mg, L-histidine - 2,0 mg, glycine - 30,0 mg, povidone -
17:20,0 mg, polysorbate -20:0,4 mg. 
Solvent: 5mL ampule of sodium chloride 0.9% solution for injection 
Dosage regimen: Fortelyzin® will be assigned at a dose of 10 mg (1,490,000 IU).  
 
Route of administration: intravenously in the form of a single bolus injection for 5-
10 sec. The solution of Fortelyzin® is prepared immediately before administration: the 
content of a 5 mg vial (745,000 IU) is diluted in 5 ml of 0.9% sodium chloride solution. 
Non-storable! 
Labeling: «Product for clinical trial only».  
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 54 of 67 
Active Comparator: Actilyse® 
Manufacturer: Boehringer Ingelheim Pharma GmbH & Co. KG  
Drug description 
Pharmaceutical form: lyophilizate for preparation of a solution for infusion supplied 
with a solvent. 
Detailed description: 
Lyophilizate: freeze-dried mass of white or light yellow color. 
Solvent: colorless transparent liquid 
Composition: 
Active substance: Alteplase 50,0 mg 
Excipients: L-arginine: 1742.0 mg, 
Phosphoric acid, 85 %: up to pH 7.2 ± 0.2, 
Polysorbate 80: 3.5-5.0 mg. 
Note 
The excess is 3.5 % of the nominal amount of the active ingredient and excipients in 
the vial. 
Residual traces: gentamicin (used in the production process). 
Each vial of solvent contains: 
Water for injection: 50 ml 
1 ml of the solution after dilution contains 1 mg of alteplase. 
Dosage regimen: 
 
Actilyse ® is administered in accordance with the instructions for use in 
ischemic stroke at a dose of 0.9 mg/kg, maximum 90 mg, if a patient's weight exceeds 
55 kg: 
• 10% of the dose is administered intravenously via a syringe for one minute 
(bolus); 
• the main dose of the drug (90% of the dose) is administered within 60 minutes 
(using a syringe dispenser / infusomat); 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 55 of 67 
• during the infusion of the main dose, it is allowed to stop and continue the 
administration of Actilyse®, but the total duration of drug administration cannot 
exceed one hour after bolus (10% of the dose). 
• Labeling: «Product for clinical trial only». 
Previous treatment 
 
Every effort should be made to find out what treatment the patient received 
within 30 days before administration of the study drug. The names of the drugs, the 
start and end dates of their use should be registered in the CRF. 
Permitted concomitant therapy 
 
In the first 24 hours after thrombolysis, the use of drugs to lower blood pressure 
is allowed: angiotensin-converting enzyme inhibitors and angiotensin receptor 
antagonists. If blood pressure drops during thrombolysis, the use of vasopressors 
(dobutamine, dopamine, and norepinephrine) is allowed. 
 
In the case of allergic reactions during thrombolysis, the use of glucocorticoids 
and antihistamines is allowed. 
If severe bleeding (especially from non-compressible vessels) occurs, the 
thrombolytic should be stopped and administration of fresh frozen plasma or fresh 
blood should be performed. 
 
After 24 hours after thrombolysis, the use of antiplatelet agents – ASA, 
clopidogrel, ticlopidine, ticagrelor, anticoagulants: unfractionated or low-molecular-
weight heparin, warfarin, dabigatran, bivalirudin, apixaban, rivaroxaban, fondaparinux 
is allowed. These drugs are permitted after thrombolysis only after CT/MRI 
examination to exclude intracranial hemorrhages (symptomatic and asymptomatic) and 
hematomas (Clinical recommendations for thrombolytic therapy in ischemic stroke, 
2015). 
 
In the absence of hemorrhagic transformation, the use of ASA is allowed during 
the entire follow-up period (90 days) after thrombolysis. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 56 of 67 
Prohibited concomitant therapy 
 
In this trial, the use of antiplatelet agents (ASA, clopidogrel, ticagrelor, 
ticlopidine), anticoagulants (NPH, NMG, fondaparinux), oral anticoagulants 
(phenylin, syncumar), vitamin K inhibitor (warfarin), direct inhibitors of thrombin and 
blood clotting Factor X (dabigatran, bivalirudin, apixaban, rivaroxaban), glycoprotein 
IIb/IIIa receptor blockers, drugs that affect the rheological properties of blood 
(agapurin, vinpocetine) is prohibited within 24 hours after thrombolysis. 
11. EFFICACY ASSESMENT 
 
This trial involves a comparison group. Evidence of Fortelyzin® efficacy will 
be compared with the results of the application of Actilyse®. 
Criteria for the efficacy evaluation of thrombolytic therapy 
• The primary efficacy endpoint is a good functional recovery on day 90 (modified 
Rankin scale (mRS), 0-1 score).  
• Secondary combined efficacy endpoint on day 90 – mRS (0-1) + NIHSS (0-1 score) 
+ Barthel Index (BI) - 95 points or higher. 
• Additional efficacy criterion – NIHSS score after 24 hours and after 90 days. 
12. SAFETY ASSESSMENT 
• Safety endpoint - overall mortality on day 90. 
• Safety endpoint - hemorrhagic transformation (all cases). 
• Safety endpoint - symptomatic hemorrhagic transformation (as defined by ECASS 
III). 
• The number of SAE and AE by organs and systems. 
 
The assessment of safety is based on the number and severity of adverse events 
that may occur in the course of the trial. The drug is considered as well tolerated if no 
severe adverse events are registered during the trial (fatal outcome or life-threatening 
conditions of the patient), which, according to the Investigator, may be associated with 
the application of the drug studied. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 57 of 67 
Adverse events 
 
Adverse event (AE) is any unfavorable medical occurrence in a patient or clinical 
trial subject administered a pharmaceutical product, which may not have a causal 
relationship with its use. Thus, an adverse event can be any adverse symptom 
(including deviation of the laboratory value from the norm), a complaint, or a disease, 
the time of occurrence of which does not exclude a causal relationship with the use of 
the medicinal product, regardless of the presence or absence of such communication. 
 
Information about adverse events should be collected at each visit by 
interviewing the patient, as well as during physical examination and evaluation of 
laboratory and instrumental data. All adverse events that occur during the trial should 
be evaluated and documented. 
When registering an adverse event, the following information should be reflected, 
if possible: 
• the exact time of AE occurrence и разрешения  
• the nature of the adverse event 
• severity (mild, moderate, severe) 
• causal relationship with the study drug 
• whether the adverse event is serious  
• actions taken (whether AE required any therapeutic measures)  
• outcome 
Adverse Event Grading: 
• Mild: the presence of signs or symptoms that do not affect daily activities. 
• Moderate: event has sufficient intensity to interfere with daily activities. 
• Severe: inability to work or perform daily activities. 
 
The connection between the development of an adverse event and the use of 
the study drug is determined by the WHO scale: 
• Certain: Clinical manifestations of unforeseen adverse reaction (UAR), laboratory 
findings deviations that occur during the period of drug administration, can not be 
explained by the presence of existing diseases and the influence of other factors. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 58 of 67 
Manifestations of UAR regress after the drug discontinuation and occur again after 
repeated administration. 
• Probable. Clinical manifestations of UAR, laboratory findings deviations are 
associated with the time of drug administration, unlikely to be related to 
concomitant diseases or other factors, and which regress with drug withdrawal. 
Response to the drug re-challenge is unknown. 
• Possible. Clinical manifestations of UAR, laboratory findings deviations 
associated with the time of drug administration, but they can be explained by the 
presence of concomitant diseases or other drug administration and the influence of 
chemical compounds. Information on drug withdrawal response is unclear. 
• Questionable. Clinical manifestations of UAR, changes in laboratory findings 
occur in the absence of a clear temporal association with the product 
administration; other factors are present (medicinal products, diseases, chemicals), 
which may be the cause of their occurrence.  
• Unlikely. Clinical manifestations of UAR, laboratory findings deviations related 
to UAR, are difficult to assess. Additional data are needed for assessment, or the 
data are currently being analyzed. 
• Unclassified. Reports on suspected UAR cannot be assessed, due to insufficient 
information, or it is contradictory. 
 
In the case of adverse events, the choice of measures (follow-up, non-drug or 
drug therapy, hospitalization, and/or prolongation of existing hospitalization) is made 
by the physician-investigator based on the patient’s interests to create optimal 
conditions and provide the patient with needed medical care. 
 
Patients are monitored in the center until the full completion of any adverse event 
that occurred during the trial, and in the case of the development of a chronic disease - 
until the condition is stabilized. Adverse events in the form of changes in laboratory 
findings are observed until the normalization of the values or until the diagnosis of the 
chronic disease that caused such changes. 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 59 of 67 
Serious adverse event (SAE)  
A serious adverse event (SAE) is defined as any adverse event (AE) that at any dose 
results in: 
* death and/or disability; 
* development of a life-threatening condition; 
* prolongation of existing hospitalization; 
* development of congenital anomalies or malignant tumors.  
 
In addition, any severe bleedings will be considered as SAEs in this trial:  
• "major bleeding" according to the TIMI classification 
• "severe or life-threatening bleeding" according to the GUSTO classification 
• symptomatic intracerebral hemorrhage (SICH) was defined as any hemorrhage with 
neurologic deterioration, as indicated by an NIHSS score for 4 points and more than 
its value at baseline or the lowest one at 7 days after thrombolysis or any 
hemorrhage leading to death and had been identified as a predominant cause of 
neurologic deterioration (definition ECASS III) 
Adverse events that do not fall into these categories should not be regarded as 
serious. 
Any SAE, regardless of the causal relationship with the drug under trial, must be 
reported to Roszdravnadzor no later than 24 hours after the Investigator became aware 
of the SAE. 
 
All paragraphs in the appropriate SAE report form must be filled in. Information 
about the SAE should be included in the Case Report Form. 
Bleedings 
All bleeding events should be registered in the patient's CRF. Each bleeding is 
classified according to the GUSTO and TIMI scales. Bleeding that is classified 
according to TIMI as "major" or "severe or life-threatening " – according to the 
GUSTO scale are considered as SAE in this trial. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 60 of 67 
Safety assessment is carried out based on the number and severity of AEs and SAEs 
detected during the trial. 
If a serious adverse event occurs, the Investigator must: 
− provide (if necessary) appropriate qualified medical care, including laboratory 
and instrumental studies; 
− inform Roszdravnadzor within 24 hours about the occurrence of SAE and send 
(fax, e-mail) a Report form on a serious adverse event; 
− indicate all adverse events in the patient's CRF; 
− continue to monitor the patient’s condition until the SAE is fully completed. 
In addition, if there is major or moderate bleeding, its localization is indicated:  
− gastrointestinal tract; 
− urogenital tract; 
− retroperitoneal space; 
− central nervous system;  
− parenchymal organs. 
In minor bleeding occurs, the immediate cause of its occurrence and/or localization 
is indicated: 
− puncture; 
− damage to blood vessels; 
− bleeding from the gums. 
 
 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 61 of 67 
13. CASE REPORT FORM 
Primary data means all the information contained in the original medical records 
and their certified copies, describing the results of clinical observations, examinations, 
and other activities, allowing to restage the trial and assess it. Primary data is included 
in the primary documentation (originals or certified copies thereof). 
Data entered in the CRF will be confirmed by other primary documents. 
For the tests performed in the central laboratory, it is necessary to attach a copy 
of the analysis results to the CRF. 
All information obtained during the trial about the patient's condition, data from 
laboratory and instrumental studies, and adverse events are recorded in the CRF. 
Erroneous records are crossed out with a single line so that it can be read. Correct 
records are made nearby. All records and corrections are dated and signed by the 
physician-investigator.  
The original documents should be available for periodic review. The original 
CRFs are transferred to the Sponsor upon completion of the trial.  
14. DRUG STORAGE AND ACCOUNTING  
 
The study drug will be properly labelled. The label will indicate the name and 
dose of the drug, expiration date, storage conditions. The drug package will have an 
inscription: ”For clinical trial use only”. It is unacceptable to use the drug in patients 
not included in this trial. 
The drug is delivered to a research center on demand and stored in a dry place at 
a temperature of 2-25 °C. Only Investigators have access to the drug. The packaging 
of the used drug must be preserved and returned to the Sponsor. 
 
The Regulatory Authorities will require accounting for all the necessary drugs 
received by each research center. The following information should be entered into the 
register: the date of issue, date of administration, dose, and patient to whom the drug 
was administered. When using each dose of the drug, the corresponding record is made 
in the patient's medical history and in the patient's CRF (date and time, batch number 
of the drug, and the initials of the person who prepared the drug for administration). 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 62 of 67 
The patient's number, date, and time of administration should also be indicated on the 
drug package. 
 
Drug accounting will be controlled by the Monitor during visits to the research 
center. 
 
At the end or termination of the trial, the form for accounting the trial product 
should be completed and verified; all discrepancies should be clarified, and their cause 
should be indicated in the documentation. According to the requirements of the 
Regulatory Authorities, all unused research materials must be returned to the Sponsor 
at the end of the trial. It is allowed to dispose of empty vials in the research center, but 
only after the use of the drug has been checked by the Monitor. 
15. CONFIDENTIALITY 
 
Confidentiality of a patient’s personal data is protected by the current laws and 
legislation. The names and surnames of all patients participating in the trial are strictly 
confidential. Participants are identified by their initials and the individual number 
assigned. 
 
The surname and other personal information will not be included in the reports 
and publications related to this trial. Direct access to the medical records will be 
available to the trial Monitor, the audit, and representatives of the control bodies when 
verifying the authenticity of the information obtained during the trial. 
16. PARTICIPATION IN THE TRIAL AND COMPENSATION 
 
No payments and/or compensation are provided for participation in this trial. 
The trial drug, medical examinations, laboratory, and instrumental studies are free of 
charge for all participants. The patient has the right to information about the results of 
medical examinations.  
 
In accordance with Article 44 of the Federal Law of April 12, 2010, No. 61-FZ 
"On the Circulation of Medicines", all patients involved in a clinical trial are subject to 
compulsory life and health insurance. In the case of harm to the patient's health as a 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 63 of 67 
result of the clinical trial, the patient is obliged to provide the Insurer with an individual 
identification code for receiving the insurance premium. 
17. CALCULATION OF SAMPLE SIZE 
No multicenter randomized clinical trials of thrombolytic therapy in ischemic 
stroke have been conducted in the Russian Federation before. Available scientific 
literature is related to the pilot application of Actilyse® in a limited number of AIS 
patients (10-16 people) (V. I. Skvortsova et al., 2006, N. A. Shamalov, 2015). Thus, 
the data of the SITS-MOST international register and the results of randomized 
multicenter clinical trials of Actilyse® in comparison with placebo in patients with 
ischemic stroke were used as the basis for the sample size justification. 
The calculation of the number required was carried out taking into account the 
capacity of 80 % and planning of a non-inferiority study in parallel groups 
[V. I. Sergienko, I.B. Bondareva "Mathematical statistics in clinical trials"; GEOTAR-
Media, Moscow, 2006]. The null hypothesis (H0) was that the thrombolytic therapy 
with Fortelyzin® is worse than the standard thrombolytic therapy with Actilyse®. This 
means that frequency of the primary efficacy criterion in the comparison group 
(PActilyse) is higher than in the group of the study drug (РFortelyzin), taking into account 
the “no worse” boundary (δ): 
H0: РFortelyzin – PActilyse ≤ -δ  
The alternative hypothesis (HA) is that Fortelyzin® therapy is no less effective 
than the standard thrombolytic therapy with Actilyse: 
HA: РFortelyzin – PActilyse > -δ  
The calculation of the sample size for the “no worse" hypothesis, the primary criterion 
representing the proportion of respondents, and even 1:1 distribution in therapy groups 
is carried out according to the following formula [Chow S-Ch., Shao J., Wang H., 
"Sample Size Calculations in Clinical Research", 2003]: 
𝑛= (𝑧1−𝛼+ 𝑧1−𝛽)2
(𝛿−𝛥)2
[𝑝1(1 −𝑝1) + 𝑝2(1 −𝑝2)] 
where 𝑛 – the number of patients in each of the groups compared; 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 64 of 67 
𝑝1 – the proportion of respondents (reaching the primary efficacy criterion) in the 
group of the study drug; 
𝑝2 – the proportion of respondents in the comparison group with Actilyse®; 
𝛥= 𝑝1 −𝑝2 – the expected difference between the proportions of respondents in the 
groups of the study drug and the comparison drug; 
𝛿 – “no worse” boundary 
α and β – probabilities of type I and II errors, respectively; 
Z1-α and Z1-β – critical values of the standard normal distribution.  
The limit of clinical significance was chosen to be 16.9% and was calculated as 
follows.  
The frequency of reaching the primary efficacy criterion in the thrombolytic 
therapy of ischemic stroke using Actilyse®, namely, the percentage of good functional 
recovery 3 months after thrombolysis (MRS – 0-1 score) according to the International 
Register (SITS-MOST) is 54.8 % (total number of patients is 6 483) (Wahlgren N et 
al., SITS-MOST investigators. Multivariable analysis of outcome predictors and 
adjustment of main outcome results to baseline data profile in randomized controlled 
trials; Safe Implementation of Thrombolysis in Stroke Monitoring Study (SITS-
MOST). Stroke 2008; 39: 3316-3322).  
The frequency of reaching the primary efficacy criterion when using placebo – 
the percentage of good functional recovery (MRS-0-1 score) in patients with ischemic 
stroke 3 months after thrombolysis, is on average 37.9%. This value was obtained 
according to the ECASS II, Atlantis, and ECASS III studies, where the favorable effect 
when using placebo was 36.6%, 32 %, and 45.2%, respectively (Hacke W et al, for the 
Second European-Australasian Acute Stroke Study Investigators. Randomized double-
blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in 
acute ischaemic stroke (ECASS II. Lancet 1998:352:1245-1251),  
(Clark W.M., et al., for the Atlantis Study Investigators. for the Second 
European-Australasian Acute Stroke Study Investigators. Recombinant tissue-type 
plasminogen activator for ischemic stroke 3 to 5 hours after symptom onset. The 
Atlantis Study: A Randomized Controlled Trial. JAMA 1999;282:2019—26),  
(Hacke W, et al. "Thrombolysis with Alteplase 3 to 4.5 Hours after Acute 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 65 of 67 
Ischemic Stroke". ECASS III The New England Journal of Medicine. 2008. 
359(13):1317-1329).  
Thus, the difference between a good clinical recovery in patients with AIS with 
the use of Actilyse® and placebo is 54,8% – 37,9% = 16,9%. Thus, the “no worse " 
boundary was set as 16%. 
Assuming equal proportions of reaching the primary efficacy criterion in the 
study drug group and the comparison drug group of 54.8% (Δ = 0), with the established 
“no worse” boundary of 16% and demonstration at the bilateral significance level of 
5%, the minimum number of patients included in the trial is estimated as 152 people in 
each therapeutic group to maintain 80% of the comparison power. Taking into account 
the possible 10% dropout, it is recommended to increase the sample size to 336 patients 
(168 patients in each group). 
18. STATISTICAL ANALYSIS OF RESEARCH MATERIALS 
 
For Statistical Analysis R 3.5.1 package will be used. 
Normally distributed variables will be described as mean (SD), ordinal variables 
as median (IQR), and categorical variables as number and percentage per category. For 
calculation of significant differences between proportions, we shall use the χ2 method 
and for medians the Mann-Whitney U test (with a two-sided 5% level of significance). 
Ninety-five percent confidence intervals will be calculated for quantitative data. 
The primary outcome and other continuous variables will be analyzed with a 
linear regression model. Binary variables will be tested using logistic regression 
models adjusted for the same stratification variables, p-values for the difference 
between treatment groups will be extracted from these models. 
19. MONITORING AND AUDIT 
 
Monitoring and audit will be conducted by Roszdravnadzor in accordance with 
the requirements of the GCP. At the same time, Investigators should provide direct 
access to the center's documentation and medical documentation. 
 
In order to ensure that all the requirements of the Protocol are met, monitoring 
visits will be carried out periodically during the trial. A research center may also be 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 66 of 67 
subject to inspections by other Regulatory Authorities. The primary medical 
documentation will be checked to verify its compliance with the data entered in the 
CRF. 
 
National Regulatory Authorities may conduct audits during and after the trial. 
 
The audit may include but is not limited to, monitoring of the correct storage and 
delivery of the study drug, availability of necessary documentation, compliance with 
procedures of signing the Informed Consent, medical records management, compliance 
with the Protocol of the trial as a whole, and comparison of data reflected in the CRFs 
with the information included in the primary documentation. 
 
Prior to the start of the trial, the Monitor should conduct an initiating visit to the 
research center to ensure that all materials (CRF, study drug, etc.) have been received 
by the center in proper condition, and to train the Investigator and the center's staff to 
comply with the procedures and requirements of the Protocol. 
 
Monitoring of the research center can be carried out through visits and telephone 
contacts. The Monitor of clinical research (Alexey A. Nikonov, Ph.D., Professor of the 
Department of Neurology, Neurosurgery and Medical Genetics of the Medical Faculty 
of the Pirogov Russian National Research Medical University of the RF Ministry of 
Health, mob. phone: +7 916 141-95-48) will perform monitoring visits in accordance 
with a pre-determined schedule and with sufficient frequency to assess the rate of 
patient’s inclusion, verification of data entered in the CRF in comparison with the data 
of the primary documentation, monitor the compliance with the Trial Protocol and the 
study drug handling. 
After the completion of participation of all patients, the Monitor will conduct a final 
visit.  
20. REPORTING 
 
Each research center compiles a report, which is signed by the Principal 
Investigator and approved by the head of the medical institution on the basis of which 
the center is located. 


CLINICAL TRIAL PROTOCOL 
------------------------------------------------------------------------------------------------------------------------------------------------ 
Page 67 of 67 
 
The statistical processing is carried out by an independent statistical expert on 
the basis of the CRF. 
 
The deadline for report submission is up to 1 month after the completion of the 
trial. 
 
The final report will contain clinical and statistical reports, as well as appendixes 
with individual data and statistical analysis. 
 
The suggested structure of the report: the purpose of the trial, methods (including 
any deviations from the Trial Protocol), evaluation of the results, discussion of adverse 
events, and their interpretation. 
 
21. PUBLICATIONS 
 
After the end of the trial, the data will be summarized and prepared for 
publication. 
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
 
Principal Investigator’s Signature 
Signature Date