Clinical trial NCT03203564
Conditions: Phase I Study in Healthy Volunteers to Evaluate ECG Effect
Sponsor: Isofol Medical AB
Start date: 2017-07-04; Completion date: 2017-08-25
Enrollment: 33 participants; Allocation: RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to evaluating ECG effects, safety, tolerability and pharmacokinetics of single ascending dose of Modufolin® in healthy male volunteers

Detailed description:
An adaptive randomised, double-blind, single-centre, placebo-controlled Phase I study evaluating ECG effects, safety, tolerability and PK of single ascending doses of Modufolin® for Injection, 100 mg in healthy male volunteers. Thirty-three (33) eligible and consenting subjects will be included in 3 cohorts, 11 subjects in each cohort. Within each cohort, subjects will be randomised to receive either placebo (3 subjects) or Modufolin® for Injection, 100 mg (8 subjects). There will be 3 pre-defined ascending dose-levels. Additional dose levels may be considered if recommended by the internal Safety Review cCommittee. There will be an interval between each dose level to allow time for safety data to be analyzed and evaluated by the iSRC. The iSRC will have the choice to decide to escalate the dose as planned, reduce or increase the dose escalation step, repeat the dose, reduce the dose or terminate the study. The total study duration for the subjects will be approximately 5 weeks and there will be in total 3 visits to the clinic. Subjects will be screened for eligibility according to study-specific inclusion/exclusion criteria within 4 weeks prior to start of stud treatment (Visit 1; Screening visit). The subjects will be confined to the research clinic from the evening before dosing (Day -1) until 24 hrs post dose (Days 1 and 2). The subjects should be fasting overnight (8 hrs) before IMP/placebo administration until 4 hrs post-dose. A Follow-up Visit will be performed 5 to 10 days after dose administration of for each cohort.

Arms: arm 1: Three cohorts, 8 subjects will be randomised to Modufolin ® for injection 100 mg arm 2: Three cohorts, 3 subjects will be randomised to placebo
Interventions: intervention 1: Modufolin (arfolitixorin)
Intervention descriptions: intervention 1: Thirty-three eligible and consenting subjects will be included in 3 cohorts, 11 subjects in each cohort, Within each cohort, subjects will be randomized to receive either placebo (3 subjects) or Modufolin® for injection, 100mg (8 subjects)

Protocol documents:
Prot_001.pdf: 
Clinical Study Protocol ISO-FF-001, FINAL, 05 Dec 2016 
CONFIDENTIAL    
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Clinical Study Protocol 
EudraCT No. 
2016-004874-16 
Investigational Medicinal Product 
Modufolin® for Injection, 100 mg 
Study code 
ISO-FF-001 
Protocol Version and date 
FINAL, 05 Dec 2016 
STUDY TITLE 
An adaptive, randomized, double-blind, single-center, placebo-controlled Phase I study evaluating ECG 
effects, safety, tolerability and pharmacokinetics of single ascending doses of [6R]-5,10-Methylene 
Tetrahydrofolate (Modufolin® for Injection, 100 mg) in healthy male volunteers 
Development phase 
Phase I 
Test product and dosage 
Modufolin® for Injection, 100 mg  
(dose groups: 200, 350, 500 mg/m2) 
Comparator product 
Placebo, 0.9% NaCl sterile solution 
Sponsor Signatory 
Anders Rabbe, Chief Executive Officer 
Isofol Medical AB  
Biotech Center 
Arvid Wallgrens Backe 20, 5th level 
SE-413 46 Gothenburg, Sweden 
Phone: +46 (0)70 764 65 00 
E-mail: anders.rabbe@isofolmedical.com
Principal Investigator 
Cornelia Lif-Tiberg, MD 
CTC Clinical Trial Consultants AB 
Clinical study conduct and 
management 
CTC Clinical Trial Consultants AB 
Uppsala University Hospital, 
Entrance. 85, 2nd level  
SE-751 85 Uppsala, Sweden 


 
 
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The following amendments have been made to the Final Clinical Study Protocol version 1.0:  
Amendment No. 
Date of Amendment 
 
 
Revised protocol version (if applicable) 
 
 
 
 
 
 
 
 
 
 
  
 


 
 
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2 
STUDY SYNOPSIS 
Study Title 
An adaptive, randomized, double-blind, single-center, placebo-controlled Phase I study evaluating 
ECG effects, safety, tolerability and pharmacokinetics of single ascending doses of [6R]-5,10-
Methylene Tetrahydrofolate (Modufolin® for Injection, 100 mg) in healthy male volunteers 
Study code 
ISO-FF-001 
EudraCT No 
2016-004874-16 
Study period 
Estimated date of first subject enrolled:   Q2 2017 
Estimated date of last subject completed: Q2 2018 
Phase of development 
Phase I 
 
Coordinating Investigator  
Cornelia Lif-Tiberg, MD 
CTC Clinical Trial Consultants AB, Uppsala, Sweden 
Study design 
Single ascending dose study (SAD) 
Objectives  
Primary objective 
To evaluate effects on electrocardiogram (ECG) parameters after single ascending doses of 
Modufolin® for Injection, 100 mg in healthy male volunteers. 
Secondary objectives  
To evaluate safety, tolerability and pharmacokinetics following single ascending doses of Modufolin® 
for Injection, 100 mg in healthy male volunteers. 
Number of subjects planned 
Thirty-three (33) male subjects will be included in the study. Subjects who are prematurely 
withdrawn from the study for any reason except the occurrence of Treatment-emergent AEs (TEAEs) 
assessed as possibly or probably related to study treatment may be replaced during the course of the 
study. 
Diagnosis and main eligibility criteria 
Healthy male volunteers aged 18 to 60 years with a body surface area ≤ 2 m2. 
Methodology 
This study is an adaptive randomized, double-blind, single-center, placebo-controlled Phase I study 
evaluating ECG effects, safety, tolerability and pharmacokinetics of single ascending doses of 
Modufolin® for Injection, 100 mg in healthy male volunteers. 
At least 33 eligible and consenting subjects will be included in 3 cohorts, 11 subjects in each cohort. 
Within each cohort, subjects will be randomized to receive either placebo (3 subjects) or Modufolin® 
for Injection, 100 mg (8 subjects). There will be 3 pre-defined ascending dose-levels. Additional dose 
levels may be considered if recommended by the internal Safety Review Committee (iSRC). There 
will be an interval of at least 7 days between each dose level to allow time safety data to be analyzed 
and evaluated by the iSRC. The iSRC will have the choice to decide to escalate the dose as planned, 
reduce or increase the dose escalation step, repeat the dose, reduce the dose or terminate the study.  


 
 
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Subjects will be screened for eligibility according to study-specific inclusion/exclusion criteria within 
4 weeks prior to start of study treatment (Visit 1; Screening visit). The subjects will be confined to the 
research clinic from the evening before dosing until 24 hrs post dose (Visit 2; Days -1, 1 and 2). The 
study subjects should be fasting overnight (8 hrs) before dose administration until 4 hrs post-dose.  
A Follow-up Visit will be performed 5 to 10 days after administration of Investigational Medicinal 
Product (IMP)/placebo for each cohort. 
Investigational Medicinal Product (IMP), dosage and mode of administration 
Modufolin® for Injection, 100 mg 
The pre-defined dose levels are 200, 350 and 500 mg/m2. 
Intravenous (i.v.) bolus injection (injection time 3 min) 
Non-investigational Medicinal Product  
Placebo: 0.9% NaCl sterile solution 
Blinding 
IMP (Modufolin® for Injection, 100 mg) and Placebo are not identical in appearance and all efforts 
will be made at the clinic in order to maintain the blind. Both the IMP and the placebo will be masked 
in such a way that study subjects and study staff will remain blinded during the study. An un-blinded 
study nurse will prepare the IMP/placebo for injection and will administer the IMP/placebo to the 
study subject. The un-blinded study nurse performing the dose administration will not be involved in 
any study-specific assessments or evaluations. 
Duration of treatment 
Each subject will receive a single bolus i.v. injection of IMP or Placebo on study Day 1. 
Duration of subject involvement in the study 
The total study duration will be approximately 5 weeks and there will be in total 3 visits to the clinic 
(Screening, In-clinic period and Follow-up visit). 
Safety assessments  
The primary safety evaluation parameter is: 
x 
Change-from-baseline QTcF (∆QTcF) 
The secondary safety evaluation parameters are: 
x 
Change-from-baseline HR, PR and QRS interval (∆HR, ∆PR and ∆QRS) 
x 
Categorical outliers for QTcF, HR, PR interval, QRS interval; 
x 
Categorical analysis for T wave morphology; 
x 
Relationship between Modufolin® for Injection, 100 mg plasma concentration and ∆∆QTc 
x 
Frequency, seriousness and intensity of AEs  
x 
Physical examination 
x 
Vital signs 
x 
Safety laboratory measurements (Hematology/Clinical chemistry) 
Pharmacokinetic (PK) assessments 
Plasma PK characteristics will be determined for: 
[6R]-5,10- Methylene Tetrahydrofolate (MTHF; Modufolin® for Injection, 100 mg) 
[6S]-5-Methyl-Tetrahydrofolate (5-Methyl-THF) 


 
 
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[6S]-Tetrahydrofolate (THF) 
[6S]-5-Formyl-Tetrahydrofolic acid (5-Formyl-THF) 
The PK parameters to be measured/calculated are: 
AUC 
Area under the plasma concentration-time curve from time 0 to 
infinity. Calculated using the linear/log trapezoidal rule with 
extrapolation to infinity. 
AUClast 
Area under the plasma concentration-time curve from time 0 to 
the last time point t. 
AUCW
Area under the plasma concentration-time curve in a dosing 
interval. Calculated using the linear/log trapezoidal rule with 
extrapolation to W, if required. 
AUC%Extrap 
Percentage of AUC that is due to extrapolation from the last 
concentration to infinity. 
AUMC 
Area under the first moment curve extrapolated to infinity based 
on the last observed concentration. 
C0 
Back extrapolated concentration at time point 0 after intravenous 
administration. 
Cmax 
Maximum plasma concentration 
C(time) 
Plasma concentration at a certain time  
Css 
Steady-state drug concentration in plasma  
CL 
Total body clearance for intravenous administration 
LLOQ 
Lower Limit of Quantification 
Mean 
Arithmetic mean, otherwise specified 
MRT 
Mean Residence Time extrapolated to infinity. 
NCA 
Non-Compartmental pharmacokinetic Analysis 
PK 
Pharmacokinetics 
Tau (W) 
Dosing interval 
tmax 
Time at which maximum plasma concentration is observed. 
t1/2 
Terminal half-life 
Oz (lambda z) 
Terminal rate constant. Calculated by log-linear regression of 
concentrations versus time. 
Vz 
Volume of distribution based on the terminal phase. 
Vss 
Volume of distribution based at steady state. 
Statistical methods 
A more technical and detailed elaboration of the principal features will be presented in a separate 
Statistical Analysis Plan (SAP). 
All statistical analysis of the study will be performed using the statistical software SAS for Windows 
Version 9.4. In all calculations, zero will be substituted for concentrations below the quantification 
limit of the assay. Data collected from all subjects will be presented in data listings. Both absolute 


 
 
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values and change from baseline values for each subject will be given where applicable. All 
continuous data will be listed with the same precision as will be presented in the database. Data 
listings will be sorted by treatment, subject ID and time point. A missing value will be represented by 
an empty cell and no imputation will be made. 
Continuous data will be summarized in tables using number of subjects (n), mean, median, standard 
deviation (SD), standard error (SE), 90% confidence interval (CI; based on a t-distribution if not 
otherwise stated), minimum, and maximum by study time point. Categorical data will be summarized 
by time point using frequencies and percentages. Percentages will be rounded to the nearest tenth. 
Population counts will be used as the denominator in the calculation of percentages unless otherwise 
specified.  
Data Analysis Sets  
Per Protocol Analysis Set 
The Per Protocol Analysis Set will consist of all subjects who have been randomized and completed 
the study period without any major protocol deviations. All protocol violations will be judged as 
major or minor at the clean file meeting. 
Safety Analysis Set  
The Safety Analysis Set will include all subjects who receive at least one dose of IMP (any dose of 
Modufolin® or matching placebo) and have at least one post-dose safety assessment. 
PK Analysis Set  
The PK Analysis Set will include all subjects who have evaluable plasma concentration data for 
Modufolin® and for whom one or more of the designated PK parameters can be determined. 
QT/QTc Analysis Set  
The QT/QTc Analysis Set will include all subjects in the Safety Analysis Set with measurements at 
baseline as well as on-treatment with at least one post-dose time point with a valid ΔQTcF value. The 
QT/QTc Analysis Set will be used for the by-time point and categorical analyses in the 
electrocardiogram analysis. 
PK/QTc Analysis Set  
The PK/QTc Analysis Set will include all subjects who are in both the QT/QTc Analysis Set and PK 
Analysis Set with at least one pair of post-dose PK and QTcF data from the same time point. The 
PK/QTc Analysis Set will be used for the exposure-response analysis in the electrocardiogram 
analysis.  
Sample Size Considerations 
Assuming a 1-sided 0.05 significance level and a standard deviation of 7 milliseconds (msec) for 
ΔQTcF, a total of 33 evaluable subjects who complete Modufolin® (24 subjects) and placebo (9 
subjects), separately will be sufficient to achieve 82% power to exclude a prolongation of 10 msec or 
longer of the upper 1-sided 95% CI of the mean ΔΔQTcF, assuming that the prolongation is 3 msec at 
the geometric mean peak Modufolin® concentration. Under the same assumptions, a total of 66 
subjects (48 Modufolin® and 18 placebo) will achieve 97.5% power.  
Exposure-Response Analysis (Primary objective) 
The relationship between the plasma concentration of Modufolin® and change-from-baseline QTcF 
(∆QTcF) will be quantified using a linear mixed-effects modeling approach with ΔQTcF as the 
dependent variable, plasma concentration of Modufolin® as the covariate, treatment (active or 
placebo) and time as categorical factors, and a random intercept per subject. The degrees of freedom 
for the model estimates will be determined by the Kenward-Rogers method. From the model, the 
slope (i.e., the regression parameter for the concentration) and the treatment effect-specific intercept 


 
 
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(defined as the difference between active and placebo) will be estimated together with 2-sided 
90% CI. The estimates for the time effect will be reported with degrees of freedom and standard error 
SE.  
The geometric mean of the individual Cmax values for subjects in each of the active drug groups will 
be determined. The predicted effect and its 2-sided 90% CI for placebo-corrected change-from-
baseline QTcF (ΔΔQTcF) (i.e., the product with the slope estimate + treatment effect) at this 
geometric mean Cmax will be obtained for each Modufolin® dose separately. If the upper bound of the 
90% CI of the model predicted QTcF effect is below 10 msec at clinically relevant plasma levels of 
Modufolin® it will be concluded that Modufolin® does not cause clinically concerning QTc 
prolongation.  
By-Time point Analysis 
The analysis for QTcF will be based on a linear mixed-effects model with change-from-baseline 
QTcF (ΔQTcF) as the dependent variable, time (categorical), treatment (Modufolin® for Injection, 
100 mg and placebo), and time-by-treatment interaction as fixed effects, and baseline QTcF as a 
covariate. Subject will be included as a random effect for the intercept. Subject dosed with placebo 
will be analyzed as a pooled group. An unstructured covariance matrix will be specified for the 
repeated measures at post-dose time points for each subject. If the model with unstructured 
covariance matrix fails to converge, other covariance matrix such as autoregressive and compound 
symmetry will be considered. From this analysis, the least-squares (LS) mean and 2-sided 90% CIs 
will be calculated for the contrast “Modufolin® versus placebo” at each dose of Modufolin® for 
Injection, 100 mg and each post-dose time point, separately.  
For HR, PR, and QRS intervals, the analysis will be based on the change-from-baseline values (ΔHR, 
ΔPR, ΔQRS). The same model will be used as described for QTcF. The LS mean, SE and 90% CI 
from the statistical modeling for both change-from-baseline and placebo-corrected change-from-
baseline values will be listed in the tables and graphically displayed. 
Categorical Analyses 
The analysis results for categorical outliers, and T-wave morphology will be summarized in 
frequency tables with counts percentages for both number of subjects and number of time points. For 
categorical outliers, the number (percentage) of subjects as well as time points who had increases in 
absolute QTcF values >450 and ≤480 msec, >480 and ≤500 msec, and 500 msec, and changes from 
pre-dose baseline of >30 and ≤60 msec, and >60 msec; increase in PR from pre-dose baseline >25% 
to a PR>200 msec; increase in QRS from pre-dose baseline >25% to a QRS >120 msec; decrease in 
HR from pre-dose baseline >25% to a HR <50 bpm; and increase in HR from pre-dose baseline >25% 
to a HR >100 bpm will be determined. For T-wave morphology, the analyses will be focused on 
change from baseline (i.e., treatment-emergent changes). 
Pharmacokinetic analysis (Secondary objective) 
The PK analysis will be based on the PPAS and subcontracted by the Sponsor to PKxpert. The PK 
parameters will be calculated by non-compartmental analysis (NCA) using the software Phoenix 
WinNonlin® version 6.4 or later (Pharsight Corporation, U.S.A.).  
 
 
 


 
 
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3 
TABLE OF CONTENTS 
2 
STUDY SYNOPSIS .................................................................................................. 3 
3 
TABLE OF CONTENTS ......................................................................................... 8 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ....................... 14 
5 
IMPORTANT MEDICAL PROCEDURES TO BE FOLLOWED BY THE 
INVESTIGATOR ................................................................................................... 17 
5.1 
Medical emergencies contacts ........................................................................... 17 
5.2 
Overdose .............................................................................................................. 17 
6 
INVESTIGATOR AND STUDY ADMINISTRATIVE STRUCTURE ............ 18 
7 
INTRODUCTION .................................................................................................. 20 
7.1 
Project background ............................................................................................ 20 
7.2 
Investigational Medicinal Product .................................................................... 21 
7.2.1 
Product characteristics ...................................................................................... 21 
7.2.2 
Mechanism of action......................................................................................... 21 
7.2.3 
Non-clinical safety ............................................................................................ 21 
7.2.4 
Clinical experience ........................................................................................... 21 
7.3 
Risk/benefit assessment ...................................................................................... 22 
7.3.1 
Summary of risk management .......................................................................... 23 
8 
STUDY OBJECTIVES AND ENDPOINTS ........................................................ 24 
8.1 
Primary objective ............................................................................................... 24 
8.1.1 
Primary endpoints ............................................................................................. 24 
8.2 
Secondary objectives .......................................................................................... 24 
8.2.1 
Secondary endpoints ......................................................................................... 24 
9 
INVESTIGATIONAL PLAN ................................................................................ 25 
9.1 
Overall study design and schedule of events .................................................... 25 
9.1.1 
Dose levels ........................................................................................................ 28 
9.1.2 
Internal Safety Review Committee ................................................................... 28 
9.1.3 
Stopping criteria for dose escalation................................................................. 28 
9.2 
Rationale for study design and dose groups..................................................... 29 
10 
STUDY POPULATION ......................................................................................... 30 
10.1 
Recruitment......................................................................................................... 30 
10.2 
Screening and enrolment log ............................................................................. 30 
10.3 
Number of subjects ............................................................................................. 30 
10.4 
Inclusion criteria ................................................................................................. 30 


 
 
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10.5 
Exclusion criteria ................................................................................................ 31 
10.6 
Restrictions during the study ............................................................................ 32 
10.6.1 
General restrictions ........................................................................................... 32 
10.6.2 
Prior and concomitant therapy .......................................................................... 33 
10.7 
Criteria for subject withdrawal ........................................................................ 33 
10.7.1 
General withdrawal criteria .............................................................................. 33 
10.7.1.1 QTc withdrawal criteria ................................................................................ 33 
10.7.1.2 Liver chemistry withdrawal criteria .............................................................. 34 
10.7.2 
Procedures for discontinuation of a subject from the study ............................. 34 
10.7.1 
Subject replacement .......................................................................................... 34 
10.8 
Randomisation .................................................................................................... 34 
10.9 
Blinding ............................................................................................................... 35 
10.10 
Emergency decoding of blinded treatment during the study ......................... 35 
11 
TREATMENTS ...................................................................................................... 35 
11.1 
Identity of Investigational Medicinal Product ................................................. 35 
11.2 
Non- Investigational Medicinal Product (Placebo) ......................................... 36 
11.3 
Packaging and labelling of Investigational Medicinal Product ...................... 36 
11.4 
Conditions for storage ........................................................................................ 37 
11.5 
Dispensing and accountability ........................................................................... 38 
11.6 
Treatment administration.................................................................................. 38 
11.7 
Continuation of treatment with Investigational Medicinal Product ............. 39 
11.8 
Treatment compliance........................................................................................ 39 
11.9 
Return and destruction of Investigational Medicinal Product....................... 39 
12 
STUDY ASSESSMENTS ....................................................................................... 39 
12.1 
Recording of data ............................................................................................... 39 
12.2 
Demographics and other baseline characteristics ........................................... 39 
12.2.1 
Informed consent .............................................................................................. 39 
12.2.2 
Demographic information ................................................................................. 40 
12.2.3 
Weight and height ............................................................................................. 40 
12.2.4 
Medical/surgical history ................................................................................... 40 
12.2.5 
HIV and Hepatitis B/C...................................................................................... 40 
12.2.6 
Urine drug screen .............................................................................................. 40 
12.2.7 
Alcohol breath test ............................................................................................ 40 
12.3 
Assessments related to primary endpoint ........................................................ 41 


 
 
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12.3.1 
Cardiodynamic assessment ............................................................................... 41 
12.3.2 
Thorough QT plus ECG extraction technique .................................................. 41 
12.3.3 
High-precision QT analysis .............................................................................. 41 
12.4 
Assessments related to secondary endpoints .................................................... 42 
12.4.1 
Safety resting 12-lead ECG .............................................................................. 42 
12.4.2 
Adverse events .................................................................................................. 43 
12.4.3 
Physical examination ........................................................................................ 43 
12.4.4 
Vital signs ......................................................................................................... 43 
12.4.5 
Laboratory safety assessments .......................................................................... 43 
12.4.6 
Pharmacokinetic measurements........................................................................ 44 
12.5 
Adverse Events.................................................................................................... 44 
12.5.1 
Event definitions ............................................................................................... 44 
12.5.1.1 Adverse Event ............................................................................................... 44 
12.5.1.2 Serious Adverse Event .................................................................................. 44 
12.5.1.3 Serious Adverse Drug Reaction .................................................................... 45 
12.5.1.4 Suspected Unexpected Serious Adverse Reaction ........................................ 45 
12.5.2 
Adverse Event assessment definitions .............................................................. 45 
12.5.2.1 Assessment of severity/intensity ................................................................... 45 
12.5.2.2 Assessment of causal relationship ................................................................ 46 
12.5.2.3 Assessment of Outcome ................................................................................ 46 
12.5.3 
Collecting Adverse Events ............................................................................... 46 
12.5.4 
Recording Adverse Events ............................................................................... 47 
12.5.5 
Reporting Serious Adverse Events ................................................................... 47 
12.5.6 
Treatment and follow-up of Adverse Events .................................................... 47 
12.6 
Appropriateness of measurements .................................................................... 48 
13 
PROCEDURE FOR BIOLOGICAL SAMPLES ................................................ 48 
13.1 
Sample collection for pharmacokinetic measurement .................................... 48 
13.2 
Bioanalytical method for pharmacokinetic measurements ............................ 48 
13.3 
Volume of blood .................................................................................................. 48 
13.4 
Handling, storage and destruction of laboratory samples .............................. 49 
13.5 
Chain of custody of biological samples ............................................................. 49 
13.6 
Withdrawal of informed consent for donated biological samples.................. 49 
14 
ETHICAL AND REGULATORY REQUIREMENTS ...................................... 49 
14.1 
Ethical conduct of the study .............................................................................. 49 


 
 
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14.2 
Ethics and regulatory review ............................................................................. 49 
14.3 
Subject information and consent ...................................................................... 50 
14.4 
Subject information card ................................................................................... 50 
14.5 
Subject data protection ...................................................................................... 51 
14.6 
Changes to the approved Clinical Study Protocol ........................................... 51 
14.7 
Audits and inspections ....................................................................................... 51 
14.8 
Insurance ............................................................................................................. 51 
15 
STUDY MANAGEMENT ..................................................................................... 52 
15.1 
Training of study site personnel ........................................................................ 52 
15.2 
Clinical monitoring ............................................................................................. 52 
15.3 
Source data document ........................................................................................ 52 
15.4 
Study agreements................................................................................................ 53 
15.5 
Study time table and end of study ..................................................................... 53 
15.6 
Discontinuation of the study .............................................................................. 53 
15.7 
Reporting and publication ................................................................................. 53 
15.7.1 
Clinical Study Report ....................................................................................... 53 
15.7.2 
Confidentiality and ownership of study data .................................................... 53 
15.7.3 
Publication ........................................................................................................ 54 
15.8 
Archiving ............................................................................................................. 54 
16 
DATA MANAGEMENT ....................................................................................... 54 
16.1 
Case Report Form .............................................................................................. 54 
16.2 
Database management plan and database design ........................................... 54 
16.3 
External data ....................................................................................................... 54 
16.4 
Medical encoding ................................................................................................ 55 
16.5 
Database lock ...................................................................................................... 55 
17 
STATISTICAL METHOD AND DETERMINATION OF SAMPLE SIZE .... 56 
17.1 
General ................................................................................................................ 56 
17.2 
Determination of sample size ............................................................................. 56 
17.3 
Analysis data sets ................................................................................................ 56 
17.3.1 
Full Analysis Set ............................................................................................... 56 
17.3.2 
Per Protocol Analysis Set ................................................................................. 56 
17.3.3 
Safety Analysis Set ........................................................................................... 57 
17.3.4 
PK Analysis Set ................................................................................................ 57 
17.3.5 
QT/QTc Analysis Set ........................................................................................ 57 


 
 
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17.3.6 
PK/QTc Analysis Set ........................................................................................ 57 
17.4 
Electrocardiogram analysis ............................................................................... 57 
17.4.1 
Baseline............................................................................................................. 57 
17.4.2 
QT correction methods and parameters ............................................................ 57 
17.4.3 
Exposure-response analysis (Primary analysis) ............................................... 57 
17.4.4 
By-time point analysis: ..................................................................................... 58 
17.4.5 
Categorical analyses ......................................................................................... 59 
17.5 
Description of study population ........................................................................ 59 
17.5.1 
Demographics and baseline characteristics ...................................................... 59 
17.5.2 
Medical history and concomitant medication ................................................... 59 
17.5.3 
Treatment compliance ...................................................................................... 59 
17.6 
Analysis of primary endpoints .......................................................................... 59 
17.6.1 
Electrocardiogram............................................................................................. 59 
17.6.2 
Telemetry .......................................................................................................... 60 
17.7 
Analysis of secondary endpoints ....................................................................... 60 
17.7.1 
Physical examination ........................................................................................ 60 
17.7.2 
Vital signs ......................................................................................................... 60 
17.7.3 
Safety laboratory analyses ................................................................................ 60 
17.7.4 
Adverse Events ................................................................................................. 60 
17.7.5 
Definition and calculation of folate pharmacokinetic parameters .................... 60 
17.8 
Statistical/analytical issues ................................................................................. 62 
17.8.1 
Handling of dropouts or missing data ............................................................... 62 
17.8.2 
Interim analyses and data monitoring ............................................................... 62 
18 
APPENDICES ........................................................................................................ 63 
18.1 
Signature page .................................................................................................... 63 
18.2 
Declaration of Helsinki....................................................................................... 64 
18.3 
Summary of safety and pharmacokinetic data from Isofol Medical AB’s 
previous clinical studies in cancer patients ...................................................... 65 
19 
REFERENCES ....................................................................................................... 68 
 
 
 


 
 
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List of Tables 
Table 1  
Schedule of events (2 pages) ................................................................................ 26 
Table 2 Dose escalation and stopping rules .......................................................................... 29 
Table 3 Composition of pharmaceutical formulation............................................................ 35 
Table 4 Manufacturers of IMP .............................................................................................. 36 
Table 5 T-wave morphology categories (assessed manually) ............................................... 42 
 


 
 
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4 
  LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Abbreviation or term                   
Explanation 
5-FU 
5-Fluorouracil 
5-Methyl-THF 
[6S]-5-Methyl-Tetrahydrofolate  
5-Formyl-THF 
[6S]-5-Formyl-Tetrahydrofolate  
AUC 
Area under the plasma concentration-time curve 
from time 0 to infinity  
AUClast 
Area under the plasma concentration-time curve 
from time 0 to the last time point t 
AUCW
Area under the plasma concentration-time curve in a 
dosing interval 
AUC%Extrap 
Percentage of AUC that is due to extrapolation from 
the last concentration to infinity 
AUMC 
Area under the first moment curve extrapolated to 
infinity based on the last observed concentration 
ALT 
Alanine transaminase 
BP 
Blood Pressure 
C0 
Back extrapolated concentration at time point 0 after 
intravenous administration 
Cmax 
Maximum plasma concentration 
C(time) 
Plasma concentration at a certain time  
Css 
Steady-state drug concentration in plasma  
CA 
Competent Authority 
CI 
Confidence Interval 
CL 
Total body clearance for intravenous administration 
CRF 
Case Report Form 
CSP 
Clinical Study Protocol 
CSR 
Clinical Study Report 
CTC 
Clinical Trial Consultants AB 
CTCAE 
Common Terminology Criteria for Adverse Events 
CV 
Coefficient of Variation 
DLT 
Dose Limiting Toxicity 
DMP 
Data Monitoring Plan 
DNA 
Deoxyribonucleic acid 
ECG 
Electrocardiogram 


 
 
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EEA 
European Economic Area 
FdUMP 
5-fluoro-2’-deoxyuridine 5’-monophosphate 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
Hb 
Hemoglobin 
HDMTX 
High Dose Methotrexate 
HR 
Heart Rate 
hrs 
Hours 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation 
ICF 
Informed Consent Form 
IEC 
Independent Ethics Committee 
IMP 
Investigational Medicinal Product 
IND 
Investigational New Drug 
iSRC 
Internal Safety Review Committee 
i.v. 
Intravenous 
LLOQ 
Lower Limit of Quantification 
LLV 
Levoleucovorin 
LS 
Least-squares 
LV 
Leucovorin 
lambda z 
Terminal rate constant 
MedDRA 
Medical Dictionary for Regulatory Activities 
min 
Minute 
msec 
Millisecond 
MTHF 
[6R]-5,10-Methylene Tetrahydrofolate 
MTHF-HS 
[6R]-5,10-Methylene Tetrahydrofolate-Hemi Sulfate  
MTX 
Methotrexate 
n 
Number 
NCA 
Non-compartmental Pharmacokinetic Analysis 
PK 
Pharmacokinetic 
PQ 
The PQ interval starts at the beginning of the atrial 
contraction and ends at the beginning of the 
ventricular contraction 
PR 
The PR interval is measured from the beginning of 
the P wave to the beginning of the QRS complex 


 
 
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PT 
Preferred Term 
q2w 
every 2 weeks 
QRS 
Corresponds to ventricular depolarizations 
QT 
The QT interval is the time from start of the Q wave 
to the end of the T wave 
QTc 
Corrected QT interval 
qwk 
every week 
RR 
The R-R interval is used to calculate heart rate (time 
between beats) 
SAD 
Single Ascending Dose 
SADR 
Serious Adverse Drug Reaction 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SMD 
Study Maximum Dose 
SDV 
Source Data Verification 
SE 
Standard Error 
SOC 
System Organ Class 
SOP 
Standard Operating Procedures 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Tau (W) 
Dosing interval 
tmax 
Time at which maximum plasma concentration is 
observed 
t1/2 
Terminal half-life 
 
TEAE 
Treatment-emergent Adverse Event 
TCA 
Tricyclic antidepressants 
THC 
Tetrahydrocannabinol 
THF 
[6S]-Tetrahydrofolate 
TS 
Thymidylate Synthase 
TQT 
Thorough QT 
ULN 
Upper Limit of Normal 
Vz 
Volume of distribution based on the terminal phase 
Vss 
Volume of distribution based at steady state 
WHO 
World Health Organisation 
 


 
 
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5 
IMPORTANT MEDICAL PROCEDURES TO BE FOLLOWED BY THE 
INVESTIGATOR 
5.1 
Medical emergencies contacts 
The Principal Investigator is responsible for ensuring that procedures and expertise are 
available to handle medical emergencies during the study. A medical emergency usually 
constitutes a Serious Adverse Event (SAE) and is to be reported as such. Detailed SAE 
reporting procedures are included in Section 12.5.5. 
In the case of a medical emergency the Investigator may contact the Medical Responsible 
Person at Isofol Medical AB, Gothenburg, Sweden. 
Name 
Function in the study 
Telephone number and e-mail 
Karin Ganlöv, MD Sponsor’s Medical Officer 
Phone: +46 (0)70243 37 50 
E-mail: karin.ganlov@isofolmedical.com 
 
5.2 
Overdose 
An overdose is a dose in excess of the dose specified for each cohort in this Clinical Study 
Protocol (CSP).   
Over-dosing is not likely to occur in this study since Modufolin® for Injection, 100 mg will be 
administered by site personnel under medical surveillance. Modufolin® for Injection, 100 mg 
is the active metabolite of Leucovorin (LV) and based on historical data, an overdose of 
Modufolin® for Injection, 100 mg is unlikely to be life threatening.  However, if a subject 
would be injected with Modufolin® for Injection, 100 mg at a higher dose than the prescribed 
dose, the Principal Investigator must be informed immediately, the subject should be 
monitored, and any adverse events (AEs) should be reported. 
Overdose should be recorded as follows:  
x An overdose with associated AEs is recorded as the AE diagnosis/symptoms on the 
relevant AE modules in the Case Report Form (CRF). 
x 
An overdose without associated symptoms is only reported in the subject´s medical 
records. 
 
 


 
 
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6 
INVESTIGATOR AND STUDY ADMINISTRATIVE STRUCTURE 
Sponsor 
Sponsor’s Chief Medical Officer 
Isofol Medical AB 
Biotech Center 
Arvid Wallgrens Backe 20, 5th level 
SE-413 46 Gothenburg, Sweden 
Karin Ganlöv, MD 
Phone: +46 (0)70 243 37 50 
E-mail: karin.ganlov@isofolmedical.com 
 
Sponsor’s Chief Scientific Officer 
Anders Vedin, MD, PhD 
Phone: +46 (0)708 761 570 
E-mail: anders.vedin@isofolmedical.com 
 
 
Sponsor’s Chief Executive Officer 
Anders Rabbe 
Phone: +46 (0)707 646 500 
E-mail: anders.rabbe@isosfolmedical.com 
 
 
 
Clinical Conduct and Management 
Principal Investigator 
CTC Clinical Trial Consultants AB 
Uppsala University Hospital, 
Entrance. 85, 2nd level  
SE-751 85 Uppsala, Sweden 
Cornelia Lif-Tiberg, MD 
Phone: +46 (0)73 978 94 45 
E-mail: cornelia-lif-tiberg@ctc-ab.se 
 
Co-Investigator 
 
Fredrik Huss, MD, PhD, Associate Professor 
Phone: +46 (0)70 794 15 74 
E-mail: fredrik.huss@clinicaltrialconsultants.se 
 
 
 
 
Biostatistics 
Biostatistician 
Devicia 
Energigatan 10 B 
SE-434 37 Kungsbacka, Sweden 
Fredrik Hansson, MSc 
Phone: +46 (0)721 93 55 01 
E-mail: fredrik.hansson@devicia.se 
 
 
 
Pharmacokinetics 
PKxpert AB 
Tomtebogatan 32 
SE-113 38 Stockholm, Sweden 
 
Pharmacokineticists 
Annsofi Nihlén, PhD 
Achim Orzechowski, PhD 
Phone: +46 (0)765 698 175  
E-mail: annsofi.nihlen@pkxpert.com 
 
 
 
 
 
 
 


 
 
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iCARDIAC 
iCardiac Technologies 
150 Allens Creek Rd 
Rochester  
NY 146 18, USA 
Phone. +1 585295-7610 
Börje Darpö, MD PhD 
Chief Scientific Officer 
Phone: +46 (0)763 902 130 
E-mail: borje.darpo@icardiac.com 
 
 
 
 
Laboratory (Bioanalysis) 
Charles River Laboratories 
Edinburgh                                          
Elpinstone Research Centre 
Tranent, East Lothian,  
EH33 2NE 
Edinburgh 
United Kingdom 
 
Contact Person at Charles Rivers Labs. 
Colin Pattison, BSc  
Phone: +44(0) 1875 618066 
Fax: +44(0) 1875 614555 
E-mail: colin.pattison@crl.com 
 
 
 
 
Laboratory (Safety) 
Clinical Microbiology 
Dag Hammarskjölds väg 17 
SE-752 37 Uppsala, Sweden 
 
Laboratory (Safety) 
Clinical Chemistry 
Uppsala University Hospital, Entrance 85 
SE-751 85 Uppsala, Sweden 
 
 
 
Signatures required are provided in Appendix 18.1. 


 
 
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7 
INTRODUCTION 
Isofol Medical AB is developing novel therapeutics for unmet needs in oncology. The lead 
clinical candidate Modufolin® for Injection, 100 mg ([6R]-5,10-Methylene Tetrahydrofolate 
[MTHF-HS]) is a novel folate-based compound developed to improve the efficiency of a 
range of antimetabolite treatments used within oncology. Three different therapeutic areas of 
special interest have been identified and are included in the development program of 
Modufolin® for Injection, 100 mg: (i) rescue therapy for High Dose Methotrexate (HDMTX) 
treatments, (ii) modulation of 5-Fluorouracil (5-FU) activity via Thymidylate Synthase (TS), 
and (iii) Modulation of antifolate activity. 
7.1 
Project background 
Folate-based pharmaceuticals have been used for years as rescue agents for reduction of toxic 
plasma levels of Methotrexate (MTX) during HDMTX treatment1,2 and also in combination 
with the antimetabolite 5-FU for treatment of gastrointestinal cancers as they significantly 
improve the efficacy of 5-FU.3 The most commonly administered folate-based drug is LV.  
Isofol Medical AB has in close collaboration with its strategic R&D partner Merck & Cie, a 
leading manufacturer of reduced folates, successfully manufactured Modufolin® for Injection, 
100 mg, an endogenous folate-based compound. Modufolin® for Injection, 100 mg is 
developed to increase the efficacy and decrease the side effects of chemotherapeutic agents 
such as the antifolate drug 5-FU and the multi-targeted antifolate Pemetrexed which are used 
in the treatment of solid tumors.  
Folates are also standard of care as rescue treatment in HDMTX treatment regimens for 
osteosarcoma. The variability of the pharmacokinetics (PK) of folates is considerable and may 
impact the safety and/or efficacy of HDMTX treatments. The timing of the rescue greatly 
influences overall tumor-free survival by improving the index of normal vs. malignant cell 
rescue while still maintaining fairly tolerable toxicity profile.2,4 
Isofol Medical AB’s hypothesis is that the administration of Modufolin® for Injection, 100 mg 
will result in greater bioavailability and higher intracellular concentrations of the active 
metabolite compared to LV administration, as several metabolic steps are bypassed by 
Modufolin® for Injection, 100 mg administration.5 It is also suggested that Modufolin® for 
Injection, 100 mg may reduce potential inter- and intrapatient variability and as a 
consequence allow for safer and more predictable treatments.  
The primary objective of the current clinical study is to evaluate potential effects on 
electrocardiogram (ECG) parameters following single doses administration of Modufolin® for 
Injection, 100 mg in healthy volunteers. To allow exclusion of a small effect on the QT 
interval, serial ECG monitoring is implemented in the study, a highly precise ECG 
methodology will be used and data will be analysed using exposure response analysis. Three 
dose levels of Modufolin® for Injection, 100 mg will be studied, including two 
supratherapeutic doses, which will obviate the need for a positive control in the study.6  
This methodology promises much higher precision in gauging the cardiac safety of 
compounds in Phase I human studies. 


 
 
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7.2 
Investigational Medicinal Product  
7.2.1 Product characteristics 
The drug product, Modufolin® for Injection, 100 mg, is a lyophilized powder (100 mg 
Modufolin® per vial, calculated as free acid) for administration after reconstitution with 
10 mL of water for intravenous (i.v.) injection. Modufolin® for Injection, 100 mg is not 
authorised for sale in any country. 
7.2.2 Mechanism of action 
The active drug substance in Modufolin® for Injection, 100 mg, ([6R]-5,10-Methylene 
Tetrahydrofolic acid hemisulfate [MTHF-HS]), is a stable formulation of the naturally 
occurring diastereoisomer of MTHF, the endogenous co-substrate of thymidylate synthase 
(TS) which is involved in the enzymatic pathway of nucleotide substrates necessary for DNA 
synthesis.5 
In contrast to other available folates such as LV and Levoeucovorin (LLV), Modufolin® for 
Injection, 100 mg does not require enzymatic metabolic activation. MTHF is the key 
metabolite of LV and may be directly involved in the formation of 5-fluoro-2'-deoxyuridine-
5'-monophosphate-thymidylate synthase (FdUMP-TS) ternary complex. The hypothesis is 
that patients which are not capable of metabolizing LV could benefit from treatment with 
Modufolin® for Injection, 100 mg.  
7.2.3 Non-clinical safety 
Modufolin® for Injection, 100 mg has been evaluated in preclinical Good Laboratory Practice 
(GLP) rat and dog studies. In summary, twice daily i.v. administration of Modufolin® for 
Injection, 100 mg for up to 28 days is well tolerated in rats at doses up to 100 mg/kg/day and 
in beagle dogs at doses up to 50 mg/kg/day. The active pharmaceutical ingredient of 
Modufolin® for Injection, 100 mg has also been evaluated in various non-GLP mouse models 
in combination with different anti-metabolites such as Pemetrexed and 5-FU. No safety issues 
were raised in the non-clinical safety studies. 
7.2.4 Clinical experience 
Completed clinical studies 
ISO-CC-002 was the first phase I/II non-Investigational New Drug (IND) clinical study with 
Modufolin® for Injection, 100 mg in patients diagnosed with operable colorectal cancer which 
was performed at Sahlgrenska University Hospital in Gothenburg, Sweden. In this clinical 
study, 60 or 200 mg/m2 of Modufolin® or LLV was administered as single doses prior to 
surgery with the objective of comparing PK/pharmacodynamic profiles in plasma, tumors, 
and tumor adjacent mucosa. Thirty-two (32) patients were included in this study, of whom 16 
were exposed to Modufolin® for Injection, 100 mg. None of the 24 AEs reported in the study 
were assessed as related to study treatment.  
ISO-MC-091 was an extended feasibility phase I/II non-IND clinical study of Modufolin® for 
Injection, 100 mg and Pemetrexed single agent, given as neoadjuvant treatment in patients 
with resectable rectal cancer. 
Twenty-four (24) patients with operable rectal cancer received Modufolin® for Injection, 


 
 
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100 mg at doses of 10, 50, 100, or 500 mg/m2 qwk for 10 weeks in combination with 
Pemetrexed as part of a neoadjuvant setting for evaluation of the feasibility and activity of the 
treatment.7,8 The safety data revealed that 20 of 24 patients had at least one AE that was 
judged to be related to the study drugs. Eleven SAEs were reported by 5 patients. One SAE 
(fever) was assessed as an SAR related to the study drug Pemetrexed.  
 
On-going clinical studies 
ISO-MTX-003 is an open-label, multicenter, phase I/II non-IND clinical study designed to 
identify the Modufolin® for Injection, 100 mg dose with the most favorable safety prospect 
and confirmed ability to mitigate HDMTX induced toxicity during treatment of osteosarcoma 
patients. The study was designed to (i) investigate the safety of Modufolin® rescue therapy, 
(ii) identify a recommended dose for further study, (iii) and evaluate the feasibility of 
Modufolin® as rescue therapy for HDMTX treatment.  
ISO-CC-005 is an open-label, multi-site, phase I/II non-IND clinical dose cohort study which 
will investigate the tolerability of Modufolin® for Injection, 100 mg in combination with a 
fixed dose of 5-FU alone or together with a fixed dose of Oxaliplatin or Irinotecan in patients 
with stage IV colorectal cancer. Toxicity will be evaluated for 4 dose levels (30, 60, 120 and 
240 mg/m2) in 5 treatment arms, see treatment regimen below: 
Modufolin® for Injection, 100 mg: 
Arm 1: 
30, 60, 120 and 240 mg/m2.  
Arm 2-3: 
30 and 60 mg/m2 i.v. bolus injection, twice q2w (i.e., on Day 1 and Day 2 of 
each chemotherapy cycle)  
Arm 4: 
60, 120 and 240 mg/m2 i.v. bolus injection, Day 1 only of each chemotherapy 
cycle). Enrolment in next dose cohort if the lower dose shows favorable 
tolerability. 
Arm 5:  
Selected Phase 2 dose from Arm 4. 
Chemotherapies (4 cycles over 8 weeks): 
Arm 1:  
5-FU (500 mg/m2) alone 
Arm 2:  
5-FU (500 mg/m2) and Oxaliplatin (85 mg/m2)  
Arm 3:  
5-FU (500 mg/m2) and Irinotecan (180 mg/m2) 
Arm 4:  
5-FU (400 mg/m2+2400 mg/m2) and Oxaliplatin (85 mg/m2) 
Arm 5:  
5-FU (400 mg/m2+2400 mg/m2) and Oxaliplatin (85 mg/m2) + bevacizumab 
(5 mg/kg) 
For further details on completed and on-going clinical studies, refer to the Investigator’s 
Brochure (IB).  
A summary of safety and PK data from Isofol Medical AB’s previous clinical studies on 
Modufolin® for Injection, 100 mg in cancer patients is provided in Appendix 18.3.  
7.3 
Risk/benefit assessment 
Modufolin® for Injection, 100 mg is a stable formulation of the naturally occurring 
diastereoisomer of MTHF, the endogenous co-substrate of thymidylate synthase which is 
involved in the enzymatic pathway of nucleotide substrates necessary for DNA synthesis. 


 
 
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Based on available data to date, the occurrence of any adverse reactions due to Modufolin® 
for Injection, 100 mg alone is not expected. Modufolin® for Injection, 100 mg has been safely 
administered to 72 patients at doses between 7.5 and 500 mg/m2 with no serious side-effects 
specifically related to Modufolin® for Injection, 100 mg alone. There is no evidence that 
Modufolin® for Injection, 100 mg may be of safety concern for patients, either after 
administration of repeated doses or after administration of single high doses. No SAEs 
assessed as possibly related to Modufolin® for Injection, 100 mg alone have been reported.  
Consequently, all SAEs assessed as having a possible relationship to Modufolin® for 
Injection, 100 mg should be reported as Suspected Unexpected Serious Adverse Reactions 
(SUSARs). 
A review of the safety data from the non-IND clinical studies ISO-MC-091, ISO-CC-002, 
ISO-MTX-003 and ISO-CC-005 identified fatigue and nausea as the most common AEs 
assessed as possibly related to study treatment. However, these events were no more frequent 
than could be expected for the same cytotoxic agents in the presence of other folates.  
7.3.1 Summary of risk management 
As the healthy volunteers in this study will have no medical benefit from participation, their 
safety and well-being is of utmost importance.  
The subjects will however remain at the research clinic for 24 hrs after the administration of 
the IMP/placbeo and will be closely monitored by medical staff. A final safety check will take 
place at a Follow-up visit scheduled 5 to 10 days after dose administration.   
The Principal Investigator at the research clinic will ascertain that adequate facilities and 
procedures are available to handle emergency situations should they occur during the study. 
The medical staffs at CTC Clinical Trial Consultants AB (hereafter referred to as CTC) have 
extended experience from early Phase I studies and there are adequate procedures in place to 
handle unexpected and expected adverse reactions in the study subjects.  
There can be risks related to medical devices used in the study e.g., indwelling venous 
catheters, but they are devices used in routine medical care and the risk is considered low and 
ethically justifiable. Study specific evaluations and sampling procedures like blood-pressure 
measurements using a blood pressure cuff and frequent blood-sampling, can cause transient 
discomfort but the risk is deemed to be low and ethically justifiable.  
The use and frequency of the above mentioned risk factors have been kept at a low level that 
still will meet the scientific and medical goals for the study and at the same time not expose 
the healthy volunteers participating in the study for risks that would not be ethically 
justifiable. 


 
 
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8 
STUDY OBJECTIVES AND ENDPOINTS 
8.1 
Primary objective 
To evaluate effects on ECG parameters after single ascending doses of Modufolin® for 
Injection, 100 mg in healthy male volunteers  
8.1.1 Primary endpoints 
The primary evaluation parameter is: 
x Change-from-baseline QTcF (∆QTcF) 
8.2 
Secondary objectives  
To evaluate safety, tolerability and PK following single ascending doses of Modufolin® for 
Injection, 100 mg in healthy male volunteers 
8.2.1 Secondary endpoints 
The secondary evaluation parameters are: 
x 
Change-from-baseline heart rate, PR and QRS interval (∆HR, ∆PR and ∆QRS) 
x Categorical outliers for QTcF, HR, PR interval, QRS interval; 
x Categorical analysis for T wave morphology; 
x Relationship between Modufolin® plasma concentration and ∆∆QTc 
x 
Frequency, seriousness and intensity of AEs  
x 
Physical examination 
x 
Vital signs 
x 
Safety laboratory measurements  
x 
Plasma PK characteristics of Modufolin® for Injection, 100 mg and its 
metabolites:  
[6R]-5,10- Methylene Tetrahydrofolate (MTHF; Modufolin® for Injection, 
100 mg) 
[6S]-5-Methyl-Tetrahydrofolate (5-Methyl-THF) 
[6S]-Tetrahydrofolate (THF) 
[6S]-5-Formyl-Tetrahydrofolic acid (5-Formyl-THF) 
The PK parameters to be evaluated are listed in Section 17.7.5. 


 
 
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9 
INVESTIGATIONAL PLAN 
9.1 
Overall study design and schedule of events 
An adaptive randomised, double-blind, single-centre, placebo-controlled Phase I study 
evaluating ECG effects, safety, tolerability and PK of single ascending doses of Modufolin® 
for Injection, 100 mg in healthy male volunteers. 
Thirty-three (33) eligible and consenting subjects will be included in 3 cohorts, 11 subjects in 
each cohort. Within each cohort, subjects will be randomised to receive either placebo 
(3 subjects) or Modufolin® for Injection, 100 mg (8 subjects).  
There will be 3 pre-defined ascending dose-levels. Additional dose levels may be considered 
if recommended by the internal Safety Review cCommittee (iSRC; see Section 9.1.2). There 
will be an interval of at least 7 days between each dose level to allow time for safety data to 
be analyzed and evaluated by the iSRC. The iSRC will have the choice to decide to escalate 
the dose as planned, reduce or increase the dose escalation step, repeat the dose, reduce the 
dose or terminate the study.  
The total study duration for the subjects will be approximately 5 weeks and there will be in 
total 3 visits to the clinic. Subjects will be screened for eligibility according to study-specific 
inclusion/exclusion criteria (see Sections 10.4 and 10.5) within 4 weeks prior to start of study 
treatment (Visit 1; Screening visit). The subjects will be confined to the research clinic from 
the evening before dosing (Day -1) until 24 hrs post dose (Days 1 and 2). The subjects should 
be fasting overnight (8 hrs) before IMP/placebo administration until 4 hrs post-dose. A 
Follow-up Visit will be performed 5 to 10 days after dose administration of for each cohort. 
The timing of all study assessments is shown in the Schedule of events provided in Table 1. 
Further details about the randomization procedures are presented in Section 10.8, procedures 
for maintaining the blind in Section 10.9 and treatment administration in Section 11.6.


 
                                    
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Table 1  
Schedule of events (2 pages) 
Visit number 
1 
2 
3 
 
Sreening 
In-clinic period 
FU 
Day 
-28 to -1 
-1 
1 
2 
6 to 11 
Time points 
- 
 
-2 h 
-45 
min 
-30 
min 
-15 
min 
0 
5 
min 
15 
min 
30 
min 
1 h 
2 h 
3 h  
4 h 
5 h 
6 h 
8 h 
12 h 
24 h 
- 
Time windows (min) 
- 
- 
- 
±2 
±2 
±2 
 
±2 
±2 
±2 
±5 
±5 
±5 
±5 
±5 
±5 
±5 
±5 
±60 
- 
Informed consent 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical/surgical history 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Weight 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs (BP and pulse) 
x 
 
 
 
 
x 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
x 
Hematology, clinical 
chemistry 
x 
 
x1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
Urine analysis (dip stick) 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV, hepatitis B and C 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drugs of abuse 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Alcohol screen 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12-lead safety ECG2 
x 
 
x2 
 
 
 
 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
x 


 
 
 
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Visit number 
1 
2 
3 
 
Sreening 
In-clinic period 
FU 
Day 
-28 to -1 
-1 
1 
2 
6 to 11 
Time points 
- 
 
-2 h 
-45 
min 
-30 
min 
-15 
min 
0 
5 
min 
15 
min 
30 
min 
1 h 
2 h 
3 h  
4 h 
5 h 
6 h 
8 h 
12 h 
24 h 
- 
Time windows (min) 
- 
- 
- 
±2 
±2 
±2 
 
±2 
±2 
±2 
±5 
±5 
±5 
±5 
±5 
±5 
±5 
±5 
±60 
- 
Holter ECG 
(Cardiodynamic)3 
 
 
x3 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Randomisation 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IMP/placebo administration 
 
 
 
 
 
 
x4 
 
 
 
 
 
 
 
 
 
 
 
 
 
PK blood sampling5 
 
 
 
 
 
x 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
AE reporting 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Prior and concomitant 
medication 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
FU=Follow-up Visit; min=minutes; h=hour; BP=Blood Pressure; ECG=Electrocardiogram; IMP=Investigational Medicinal Product; PK=Pharmacokinetic; AE=Adverse Event 
1 Within 1 hr prior to dose administration. 
2 Safety ECGs will be printed on-site and interpreted by the Investigator. Pre-dose safety ECGs in triplicate will be extracted within 1 hr prior to dose administration. 
3 Continuous12-lead ECG recording (Holter ECG) will start 2 hrs prior to dose administration on Day 1. Holter ECGs will be extracted at 3 time points before dosing (-45, -30 and -15 min). 
Subjects will be supinely resting for at least 10 min prior to time points for ECG recordings. When time points for ECG recordings coincide with other assessments, procedures should be 
carried out in the following order: (1) Holter ECG, (2) safety ECG, (3) vital sign, (4) PK blood sampling and (5) safety blood sampling.   
4 Subjects should be fasting overnight (8 hrs) before IMP/placebo administration and until 4 hrs post-dose. 
5 Time 0 = end of IMP/placebo infusion. For further information on the PK blood sampling procedures, refer to Section 13.1. 


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9.1.1 Dose levels 
The proposed dose levels are 200, 350 and 500 mg/m2 Modufolin® for Injection, 100 mg. 
Each dose will be administered as a single bolus i.v. injection. 
Depending on results obtained during the study, the number of dose levels, the actual dose, 
frequency of dosing and planned sampling times might have to be adjusted. These 
adjustments will be documented in non-substantial amendments to the CSP. 
The study maximum dose (SMD) will not exceed 500 mg/m2 administered as single bolus 
i.v. injection.  
9.1.2 Internal Safety Review Committee  
The iSCR will consist of:  
x The Principal Investigator or delegate 
x A Sponsor Medical representative or delegate 
Additional internal or external experts may be consulted by the iSCR if needed. 
After each completed dose level, the iSCR will evaluate all available safety data. The decision 
may be to give the next intended dose, a greater or smaller dose increment than the intended 
dose, a repeated dose, a lower dose or to stop dosing. 
A dose escalation beyond 500 mg/m2 will not be mandated by the iSRC unless authorized by 
the regulatory authority in a substantial amendment to this CSP. 
The randomization code may be broken by the iSRC during the assessment process (partial 
un-blinding) but only for subjects with severe AEs (≥grade 3) to enable their decision on 
continued dosing of further cohorts or to stop the dose escalation. The medical staff and the 
subjects will remain blinded for the treatments (active drug or placebo) to be administered in 
the subsequent dose groups/cohorts in order to minimize bias. 
The decision of the iSRC on the next dose level will be taken in consensus between the iSRC 
members and documented appropriately in a safety evaluation form provided by CTC. 
9.1.3 Stopping criteria for dose escalation 
The Principal Investigator and the iSRC will follow the recommendations and grading system 
of Common Terminology Criteria for Adverse Events (CTCAE) v4.039 (see Section 12.5.2.1) 
but also take into account the recommendations published by M Sibille et al. 201010 which is 
an adaptation to FIH studies of the grading systems previously proposed by National Cancer 
Institute (NCI),9  World Health Organization (WHO),11 National Institutes of Health (NIH)12 
and Food & Drug Administration (FDA).13 The grade, the frequency of AEs and the blinding 
will be taken into account.  
At the individual level, basically, a Grade 2 AE is at minimum a safety alert leading to caution 
and closer assessment of safety in other subjects. An escalation to Grade 3 applies if rapid 
worsening, concomitant findings, clinical symptoms and signs occur. A Grade 3 AE always 
supports stopping the suffering subject from continuing participation in the study.  
At the dose group level, the algorithm supporting the stopping rules to be strictly followed is 
presented in Table 2. An un-blinding, limited to suffering subjects, will be applied. A 33% 


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frequency of AEs or findings (Grade 3 or 2, as explained above) of the same types will trigger 
a stop of dose escalation. However, depending on the type and potential risk of the AE, any 
modulation can be decided, for example, for safety concerns, a lower frequency level. 
Following the iSRC risk evaluation of AEs (Grade ≥3) it may be decided to, for the next dose 
level to either repeat the current dose level, lower the dose or stop dosing. 
 
Table 2 
Dose escalation and stopping rules 
If 0/111 subjects have Dose 
Limiting Toxicity (DLT) 
Escalate to the next higher 
dose level 
No un-blinding 
If 1/8 subjects on active 
drug has DLT 
Repeat the current dose level 
or escalate to the next higher 
dose level 
Partial un-blinding (of subjects 
with DLT) 
If ≥2/8 subjects on active 
drug have DLT 
Stop further dosing 
Partial un-blinding (of subjects 
with DLT) 
1 Control subjects included 
9.2 
Rationale for study design and dose groups 
A Single Ascending Dose (SAD) design was chosen for the evaluation of the cardiac safety of 
Modufolin® for Injection, 100 mg. The adaptive study design allows for flexible dose 
escalation and involves careful monitoring of the subject’s well-being. The study will also 
provide important PK data to support the design of further studies, both in healthy volunteers 
and in patients. The time points for PK blood sampling were selected based on data obtained 
from previous clinical studies (see summary of PK data in Appendix 18.3). After dose 
administration, the time points for extraction of ECG data are paired with PK blood sampling.  
A placebo control will be used to establish the frequency and magnitude of changes in 
endpoints that may occur in the absence of active treatment.  
Randomization will be used to minimize bias in the assignment of subjects to dose groups and 
to increase the likelihood that known and unknown subject attributes (e.g., demographic and 
baseline characteristics) are evenly balanced across treatment groups.  
Blinded treatment will be used to reduce potential bias during data collection and evaluation 
of endpoints. 
The primary objective of the current clinical study is to evaluate potential effects on ECG 
parameters following single doses administration of Modufolin® for Injection, 100 mg in 
healthy volunteers. To allow exclusion of a small effect on the QT interval, serial ECG 
monitoring is implemented in the study, a highly precise ECG methodology will be used and 
data will be analysed using exposure response analysis.  
Three dose levels of Modufolin® for Injection, 100 mg will be studied (200, 350 and 500 
mg/m2), including two supratherapeutic doses, which will obviate the need for a positive 
control in the study.6 Based on current data the expected clinical dosing regimen in future 
clinical trials in metastatic colorectal cancer patients (lead indication) is 120 mg/m2 
Modufolin® for Injection, 100 mg. Doses of Modufolin® up to 500 mg/m2/day have been 
administered to patients in a previous non-IND clinical study with no safety concerns 
(ISO-MC-091).  


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10 STUDY POPULATION  
10.1 Recruitment 
The subjects will be recruited from a database of healthy volunteers at CTC and from 
advertising in media including social media. 
10.2 Screening and enrolment log 
A screening number will be allocated to each subject undergoing screening. The Investigator 
will keep records of all subjects screened and included. The reason for screen failure should be 
stated for all subjects screened but not included. The reason for withdrawal should be stated for 
all subjects included but not completed.  
If a subject cannot receive the planned dose of IMP/placebo within 28 days after screening (i.e., 
the time interval between signing informed consent until dose administration, the subject should 
be rescreened before proceeding in the study). 
10.3 Number of subjects 
Thirty-three (33) male subjects will be included in the study.  
To account for potential drop-outs and/or additional cohorts depending on PK data and 
recommendations from the iSRC, additional male subjects might be included in the study (see 
Section 9.1.2). 
For replacements of subjects discontinuing the study, see Section 10.7.1.  
10.4 Inclusion criteria 
For inclusion in the study, subjects must fulfil the following criteria:  
1. Willing and able to provide a written informed consent for participation in the study.  
2. Healthy male subject aged 18-60 years inclusive. 
3. Body Mass Index (BMI) ≥ 18 and ≤ 30 kg/m2 and weight at least 50 kg and no more 
than 100 kg at screening and body surface area ≤ 2 m2. 
4. Clinically normal medical history, physical findings, vital signs, ECG and laboratory 
values at the time of screening, as judged by the Investigator.  
5. Willing to use condom and highly effective contraceptive methods with a failure rate 
of < 1% to prevent pregnancy1 and drug exposure to a partner and refrain from 
donating sperm from the date of dosing until 3 months after dosing of the 
IMP/placebo. 
                                                 
1 Combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation 
(oral, intravaginal, transdermal), progestogen-only hormonal contraception associated with inhibition of ovulation 
(oral, injectable, implantable), intrauterine device (IUD), intrauterine hormone-releasing system (IUS), bilateral 
tubal occlusion, vasectomized partner/vasectomy, sexual abstinence. 


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10.5 Exclusion criteria 
Subjects must not enter the study if any of the following exclusion criteria are fulfilled: 
1. History of any clinically significant disease or disorder which, in the opinion of the 
Investigator, may either put the subject at risk because of participation in the study, or 
influence the results or the subject’s ability to participate in the study. 
2. Any clinically significant illness, medical/surgical procedure or trauma within four 
weeks of the first administration of IMP/placebo. 
3. Any planned major surgery within the duration of the study. 
4. Any positive result on screening for serum hepatitis B surface antigen, hepatitis C 
antibody and Human Immunodeficiency Virus (HIV). 
5. After 10 minutes (min) supine rest at the time of screening, any vital signs values 
outside the following ranges:  
x Systolic BP > 150 mm Hg 
x Diastolic BP > 90 mm Hg 
x Pulse < 40 or > 85 beats per min 
 
6. Prolonged QTcF (>450 ms), cardiac arrhythmias or any clinically significant 
abnormalities in the resting ECG at the time of screening, as judged by the Investigator. 
7. History of severe allergy/hypersensitivity or on-going allergy/hypersensitivity, as 
judged by the Investigator, or history of hypersensitivity to drugs with a similar 
chemical structure or class to Modufolin® (i.e., folate derivatives). 
8. Regular use of any prescribed or non-prescribed medication including antacids, 
analgesics, herbal remedies, vitamins and minerals within two weeks prior to the 
administration of IMP/placebo, except occasional intake of paracetamol (maximum 
2000 mg/day; and not exceeding 3 000 mg/week), at the discretion of the Investigator 
and nasal decongestants without cortisone or antihistamine for a maximum of 10 days, 
at the discretion of the Investigator. 
9. Regular use of any prescribed or non-prescribed medication which could influence 
folate and vitamin B12 status within 30 days prior to the administration of IMP/placebo. 
10. Administration of another new chemical entity (defined as a compound which has not 
been approved for marketing) or has participated in any other clinical study that 
included drug treatment with less than three months between administration of last dose 
and first dose of IMP/placebo in this study. Subjects consented and screened but not 
dosed in previous phase I studies are not excluded. 
11. Current smokers or users of nicotine products. Irregular use of nicotine (e.g., smoking, 
snuffing, chewing tobacco) less than three times per week is allowed before screening 
visit. 
12. Positive screen for drugs of abuse or alcohol at screening or on admission to the unit 
prior to administration of the IMP/placebo.  
13. Current or history of alcohol abuse and/or use of anabolic steroids or drugs of abuse. 


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14. Intake of xanthine and/or taurine containing energy drinks within two days prior to 
screening. 
15. Plasma donation within one month of screening or blood donation (or corresponding 
blood loss) during the three months prior to dosing.   
16. Investigator considers the subject unlikely to comply with study procedures, restrictions 
and requirements. 
10.6 Restrictions during the study 
10.6.1 General restrictions 
x Contraception Requirements: The male volunteers are expected to use condom and 
other effective contraceptive methods with a failure rate of < 1% to prevent pregnancy2 
and drug exposure of a partner and refrain from donating sperm from the date of dosing 
until 3 months after last dosing of the IMP/placebo. 
x Meals and Dietary Restrictions: Study subjects will receive standardized meals during 
study days at the clinic. No breakfast will be served before dose administration. Lunch 
will be served 4 hrs post-dose, a snack at 6 hrs post-dose and dinner 9 hrs post-dose. 
Water is allowed ad libitum at the clinic. 
x Fasting: The subjects should be fasting overnight (8 hrs) before IMP/placebo 
administration until 4 hrs post-dose. 
x Alcohol: Consumption of alcohol is not allowed within 48 hrs prior to all clinic visits 
until after the Follow-up Visit. 
x Coffee: Consumption of up to five cups of coffee per day will be allowed during the 
study.  
x Xanthine or taurine containing products/beverages: Energy drinks (e.g., Redbull) are not 
allowed during the study. 
x Nicotine: Smoking or use of nicotine-containing products is not allowed during the 
study. 
x Grapefruit and grapefruit containing products: Consumption of grapefruit and/or 
grapefruit containing products is not allowed during the study. 
x Exercise: The subjects must refrain from strenuous exercise (defined as greater than 
70% of the maximal pulse rate for one hour or more) during the study. 
x Blood donation: The subjects must not donate blood or plasma during the study until 
three months after the final medical examination at the Follow-up Visit. 
                                                 
2 Combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation 
(oral, intravaginal, transdermal), progestogen-only hormonal contraception associated with inhibition of ovulation 
(oral, injectable, implantable), intrauterine device (IUD), intrauterine hormone-releasing system (IUS), bilateral 
tubal occlusion, vasectomised partner, sexual abstinence. 


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10.6.2 Prior and concomitant therapy 
 
In general, no concomitant medications or therapies, including herbal remedies, vitamin 
supplements and over-the-counter (OTC) products, will be allowed during participation in the 
study.  
In particular, intake of medications which could influence folate and vitamin B12 status is 
prohibited during the study. 
However, the following will be allowed: 
x Paracetamol in doses up to 2000 mg/day for a maximum of 3 consecutive days. If this 
amount of paracetamol is not sufficient for treatment of the subject, withdrawal should 
be considered. 
x Nasal decongestants without cortisone or antihistamine for a maximum of 10 days. 
Other medications considered necessary for the subject’s safety and well-being, may be given 
at the discretion of the Investigator during the residential period. Following consultation with 
the Sponsor, the Investigator will determine whether or not the subject should continue in the 
study. 
10.7 Criteria for subject withdrawal 
10.7.1 General withdrawal criteria  
Subjects are free to discontinue their participation in the study at any time and for whatever 
reason without affecting their right to an appropriate follow-up investigation or their future 
care. If possible, the reason for withdrawal of consent should be documented. 
Subjects may be discontinued from the study at any time at the discretion of the Investigator 
for any of the following reasons:  
x Severe non-compliance to study protocol procedures, as judged by the Investigator 
and/or Sponsor. 
x Subject is lost to follow-up. 
x Significant AEs posing a risk for the subject, as judged by the Investigator and/or 
Sponsor. 
x Withdrawal of informed consent to the use of biological samples. 
10.7.1.1 QTc withdrawal criteria 
A subject meeting the criteria below will be withdrawn from the study. The same QT 
correction formula will be used to determine discontinuation throughout the study. 
x 
QTcF > 500 millisecond (msec)  
x 
Change from baseline:  QTc >60 msec to QTc > 480 msec 
Withdrawal decisions will be based on an average QTc value of triplicate ECGs. If an ECG 
demonstrated a prolonged QT interval, two more ECGs will be obtained over a brief period 
and the averaged QTc values of the three ECGs used to determine whether the subject should 
be discontinued from the study. 


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10.7.1.2 Liver chemistry withdrawal criteria 
Liver chemistry threshold stopping criteria have been designed to assure subject safety and to 
evaluate liver event etiology.14  
Study treatment will be stopped if any of the following liver chemistry stopping criteria is 
met: 
x Alanine transaminase (ALT) 3 x Upper Limit of Normal (ULN) and total bilirubin ≥ 
2xULN (>35% direct bilirubin); or ALT 3xULN and INR > 1.5) 
 
NOTE: serum bilirubin fractionation should be performed if testing is available. If 
fractionation is unavailable, urinary bilirubin is to be measured via dipstick (a 
measurement of direct bilirubin, which would suggest liver injury). 
x ALT 5xULN. 
x ALT 3xULN if associated with symptoms (new or worsening) believed to be related to 
hepatitis (such as fatigue, nausea, vomiting, right upper quadrant pain or tenderness or 
jaundice) or believed to be related to hypersensitivity (such as fever, rash or 
eosinophilia). 
 
Subjects with ALT 3xULN and < 5xULN and bilirubin < 2xULN, who do not exhibit 
hepatitis symptoms or rash, will be allowed to continue study treatment as long as they are 
monitored weekly for four weeks.  
10.7.2 Procedures for discontinuation of a subject from the study 
A subject who prematurely discontinues participation in the study will always be asked about 
the reason(s) for discontinuation and the presence of any AEs. If possible, they will be seen 
by the Investigator and assessed according to the procedures scheduled for the follow-up visit. 
Any ongoing AEs will be followed as described in Section 12.5.6. 
10.7.1 Subject replacement 
Subjects who are prematurely withdrawn from the study for any reason except the occurrence 
of Treatment-emergent AEs (TEAEs) assessed as possibly or probably related to study 
treatment may be replaced during the course of the study. 
10.8 Randomisation 
Subjects in each cohort will be randomized to receive either placebo (3 subjects) or active 
treatment (8 subjects). 
The randomization list will be generated by CTC or delegate and provided to the packing 
company. The original randomization list will be kept in a sealed envelope by the randomizer. 
Sealed treatment code envelopes will be kept by CTC and the Sponsor.  


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10.9 Blinding 
This is a double-blinded study and the allocation of treatments will not be disclosed until 
clean file has been declared and the database has been locked.  
The IMP and the placebo are not identical in appearance and all efforts will be made at the 
clinic in order to maintain the blind. Both the IMP and the placebo will be masked in such a 
way that study subjects and study staff will remain blinded during the study. An un-blinded 
study nurse will prepare the IMP/placebo and administer the solutions to the study subject. 
The un-blinded study nurse will not be involved in any study-specific assessments or 
evaluations. 
10.10 Emergency decoding of blinded treatment during the study 
The treatment code may only be broken by the study medical staff in case of emergency when 
knowledge of the treatment received is necessary for the proper medical management of the 
subject. The code breaking procedure should be carefully documented in the CRF. 
The randomization code may be broken by the iSRC during the assessment process (partial 
un-blinding) but only for subjects with severe AEs (≥grade 3) to enable their decision on 
continued dosing of further cohorts or to stop the dose escalation. The medical staff and the 
subjects will still be blinded for the treatments (active drug or placebo) to be administered in 
the subsequent cohorts in order to minimize bias (see Section 9.2). 
11 TREATMENTS 
11.1 Identity of Investigational Medicinal Product  
Test product (Modufolin® for Injection, 100 mg) 
Pharmaceutical formulation 
Modufolin® for Injection, 100 mg is formulated as a lyophylized white to light yellowish or 
light beige powder containing 100 mg Modufolin® per vial (calculated as free acid, for 
composition see Table 3). After reconstitution with 10 mL sterile water, reconstituted 
Modufolin® Injection, 100 mg is a clear yellowish fluid ready for intravenous (i.v.) use as 
bolus injection (final concentration of 10 mg/mL). The resulting solution for injection is 
isotonic and has a pH of 8.5.  
Table 3 
Composition of pharmaceutical formulation 
Component 
Amount/vial 
Function 
Sodium hydroxide 
0.3 g 
For pH adjustment 
Trisodium citrate dihydrate 
0.2 g 
Buffer (to guarantee a defined stable pH 
after reconstitution in water) 
Modufolin® drug substance 
0.1 g 
Active pharmaceutical ingredient 
 


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11.2 Non- Investigational Medicinal Product (Placebo) 
The placebo in this study will be 0.9% NaCl sterile solution.  
11.3 Packaging and labelling of Investigational Medicinal Product 
The drug product, Modufolin® for Injection, 100 mg is manufactured, released and tested in 
compliance with Good Manufacturing Practice (GMP) by the following companies as 
summarised in Table 4. 
Table 4 
Manufacturers of IMP 
Name 
Address 
Function 
Cobra Biopharma Matfors AB 
(previously Unitech Biopharma 
AB) 
Storjordenvägen 2 
SE-864 31 Matfors, Sweden 
Manufacture 
Batch release 
Merck & Cie 
Im Laternenacker 5 
8200 Schaffhausen 
Switzerland 
Release testing 
Unimedic AB 
Storjordenvägen 2 
SE-864 31 Matfors, Sweden 
Release testing (particulate 
matter) 
Pharmacontrol MQL AB 
Virdings Allé 2 
SE-754 50 Uppsala Sweden 
Release testing (sterility) 
 
 
Primary packaging (Modufolin® for Injection, 100 mg): 
Lyophilized powder:  
10 mL type 1 glass vials with lyophilization stopper (20 mm, 
siliconized, Helvoet FM157.  
 
Secondary packaging (Modufolin® for Injection, 100 mg): 
Each box will contain 10 x 10 mL glass vials. 
 
Labels will comply with applicable current Good Manufacturing Practice (GMP) 
requirements.15 


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The following labels will be used for Study ISO-FF-001: 
 
Primary packaging- Label to be attached to vial 
 
For clinical trial                          
Study No. ISO-FF-001 
Caution: New Drug--Limited by Federal law to 
investigational use 
Modufolin® for Injection, 100 mg 
Batch No: XXXX 
Store at 2 - 8°C (refrigerator) 
Principal investigator: Cornelia Lif-Tiberg, MD 
Sponsor: Isofol Medical AB, Phone:  +46 (0)702 43 37 50 
 
 
Secondary packaging- Label to be attached to each box containing 10 vials 
 
For clinical trial                            
Study No. ISO-FF-001 
Principal investigator: Cornelia Lif-Tiberg, MD 
Caution: New Drug--Limited by Federal law to 
investigational use 
Modufolin® for Injection, 100 mg  
10x100mg  
To be dissolved with Water for Injection according to 
separate preparation description 
Batch No: XXXX 
Store at 2 - 8°C (refrigerator) 
 
Expiry date: YYYY-
MM-DD 
Sponsor: Isofol Medical AB,  
Arvid Wallgrens Backe 20  
Gothenburg, Sweden 413 46  
Phone:  +46 (0)702 43 37 50 
 
11.4 Conditions for storage 
Modufolin® for Injection, 100 mg vials are stored at refrigerated storage conditions 2-8 °C.  
Stability data demonstrate that the drug product is stable when stored at 2-8 °C for up to 
48 months. The current shelf life is 42 months.  


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Stability data on the solution for injection (the dissolved powder) show that the reconstituted 
IMP is stable for up to 2 hrs at room temperature after reconstitution.   
11.5 Dispensing and accountability 
CTC AB and the Investigator will maintain a Drug Dispensing Log detailing the dates and 
quantities of study medication received, dispensed to and used by each subject and study 
medication returned or destroyed at the end of the study. Any discrepancies between 
dispensed and returned IMP must be explained and documented. Products deliberately and/or 
accidentally destroyed by the Investigator/ pharmacy or the subject must be accounted for. 
11.6 Treatment administration 
The pre-defined dose levels are 200, 350 and 500 mg/m2 of Modufolin® for Injection, 100 mg. 
There will be 3 cohorts, 11 subjects in each cohort. Subjects will be assigned to a cohort in a 
consecutive manner. In each cohort, 8 subjects will receive Modufolin® for Injection, 100 mg 
and 3 subjects will receive placebo. The IMP/placebo will be administered as a single dose on 
Study Day 1. The IMP/placebo will be administered in the morning and the time will be 
recorded in the CRF. The study subjects should be fasting overnight (8 hrs) before 
IMP/placebo administration until 4 hrs post-dose. There will be an interval of at least 7 days 
between each cohort to allow time for the iSRC to evaluate safety data before proceeding to 
the next dose level. 
Route of administration 
Intravenous bolus injection (injection time 3 min). 
 
Preparation and handling of IMP solution (Modufolin® for Injection, 100 mg): 
1. The preparation of the solution for injection will be done by trained personnel, for 
example a site pharmacist or a registered nurse. There will be 2 un-blinded persons 
working together, one person will handle the IMP and perform the reconstitution of 
the drug product while the other person will supervise the process. The process will 
also be recorded on video tape. 
2. The reconstitution of each vial of IMP powder in 10 mL of water for injection should 
be done using aseptic technique. 
3. Gently swirl each vial until the powder is completely dissolved. The resulting solution 
is clear and ranges in color from colorless to yellow. 
4. The solution should be inspected visually for particulate matter and discoloration prior 
to administration. If particles are present, do not administer. 
5. The reconstituted solution can be kept at room temperature. 
6. Administration should preferably be done directly after reconstitution and must be 
done within 2 hrs after reconstitution. 
7. If administration cannot take place for whatever reason, the vial should be handled in 
accordance with the study-specific instructions on IMP disposal. 


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11.7 Continuation of treatment with Investigational Medicinal Product 
This is a Phase I study in healthy volunteers who will have no medical benefit from the 
treatment and thus there will be no treatment with Modufolin® for Injection, 100 mg after end 
of study participation. 
11.8 Treatment compliance 
All study products will be administered at the research clinic under medical supervision to 
ensure compliance. 
11.9 Return and destruction of Investigational Medicinal Product 
Any unused IMP will be returned to the Sponsor or the Hospital Pharmacy for destruction. 
Empty containers will be destroyed at the study site. The Monitor will perform final IMP 
accountability reconciliation at the study end to verify that all unused IMP is adequately 
destroyed/returned and documented. 
12 STUDY ASSESSMENTS 
The study assessments are described in the sections below and the timing of these assessments 
are detailed in the Schedule of events in in Section 9.1 (Table 1). 
12.1 Recording of data 
The Principal Investigator will provide the Sponsor with all data produced during the study 
from the scheduled study assessments. He/she ensures the accuracy, completeness, legibility, 
and timeliness of the data reported to Sponsor in the CRF and in all required reports. 
When time points for ECG recordings coincide with other assessments, procedures should be 
carried out in the following order:  
1. Holter ECG 
2. Safety ECG 
3. Vital sign 
4. PK blood sampling  
5. Safety blood sampling.   
The actual PK blood sampling time should always be recorded in the CRF and will be used in 
the calculation of the PK parameters. Pre-dose assessments may be performed up to 60 min 
prior to dosing if not specified in the Schedule of events in Section 9.1 (Table 1). 
12.2 Demographics and other baseline characteristics 
12.2.1 Informed consent 
Signed informed consent must be obtained before any screening procedures are initiated. The 
informed consent procedure is further described in Section 14.3. 


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12.2.2 Demographic information 
The following demographic data will be recorded: gender, age, and ethnic origin. 
12.2.3 Weight and height 
Weight and height will be measured without shoes. BMI will be calculated from the height 
and weight recorded and rounded to the nearest whole number. 
12.2.4 Medical/surgical history 
Medical/surgical history will be obtained by interview in order to verify that the eligibility 
criteria are met. 
12.2.5 HIV and Hepatitis B/C 
Subjects will be tested for HIV and Hepatitis B/C prior to inclusion into the study in order to 
protect personnel handling the blood samples. 
12.2.6 Urine drug screen 
Urine will be screened for drugs of abuse at screening and prior to dose administration using 
the AlereTM Drug Screen Test Panel. Additional random tests can be performed during the 
study. 
The following substances will be included in the screen panel:  
 
 
Amphetamine 
Methadone 
Barbiturates 
Methamphetamine 
Benzodiazepines 
Methylenedioxymethamphetamine (MDMA) 
Buprenorphine 
Morphine 
Clonazepam 
Opiate 
Cocaine 
Oxycodone 
Fentanyl 
Phencyclidine 
Ketamine 
Propoxyphene 
Marijuana (Tetrahydrocannabinol [THC]) 
Tramadol 
 
Tricyclic antidepressants (TCA) 
12.2.7 Alcohol breath test 
An alcohol breath test will be performed at screening and prior to dose administration. 
Additional random tests can be performed during the study. 


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12.3 Assessments related to primary endpoint  
12.3.1 Cardiodynamic assessment 
Twelve-lead ECGs will be extracted from continuous recordings (Holter recordings) prior to 
and serially after dosing with Modufolin® for Injection, 100 mg at time points as shown in the 
Schedule of events (Table 1; Section 9.1). Holter ECGs will be extracted at 3 time points 
before dosing. Subjects will be supinely resting for at least 10 min prior to time points for 
ECG recordings.  
The 12-lead Holter and ECG equipment will be supplied and supported by iCardiac 
Technologies, Inc.   
All ECG data will be collected using a Global Instrumentation (Manlius, NY, USA) M12R 
ECG continuous 12 lead digital recorder. The continuous 12-lead digital ECG data will be 
stored onto SD memory cards. ECGs to be used in the analyses will be selected by pre-
determined time points as defined in the in the Schedule of events (Table 1; Section 9.1) and 
will be read centrally by iCardiac Technologies, Inc. 
The following principals will be followed in iCardiac’s core laboratory: 
x ECG analysts are blinded to the subject, visit and treatment allocation. 
x Baseline and on-treatment ECGs for a particular subject will be over-read on the same 
lead and will be analyzed by the same reader. 
x The primary analysis lead is lead II. If lead II is not analyzable, then primary lead of 
analysis will be changed to another lead for the entire subject data set. 
The following is a brief description of ECG analysis methods utilized by iCardiac’ core 
laboratory. 
12.3.2 Thorough QT plus ECG extraction technique 
Ten 14-second digital 12-lead ECG tracings will be extracted from the continuous Holter 
recordings using the ‘Thorough QT (TQT) Plus method’, a computer-assisted and statistical 
process utilized by iCardiac Technologies. The method enables extraction of ECGs with the 
lowest HR variability and noise within the protocol-specified extraction time window (e.g., 
the HR and QT changes from beat-to-beat in the range of <10%). At each protocol-specified 
time point, 10 ECG replicates will be extracted from a 5-min “ECG window” (typically, the 
last 5 min of the 15-min period when the subject is maintained in a supine or semi-recumbent 
quiet position). 
12.3.3 High-precision QT analysis 
High-precision QT analysis will be performed on all analyzable (non-artifact) beats in the 
10 ECG replicates.  Statistical quality control procedures are used to review and assess all 
beats and identify “high” and “low” confidence beats using several criteria, including: 
x QT or QTc values exceeding or below certain thresholds (biologically unlikely). 
x RR values exceeding or below certain thresholds (biologically unlikely). 
x Rapid changes in QT, QTc or RR from beat to beat. 


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Measurements of all primary ECG parameters (QT, QTc, RR) in all recorded beats of all 
replicates that are deemed “high confidence” is performed using COMPAS software. All low 
confidence beats are reviewed manually and adjudicated using pass-fail criteria. The final QC 
assessment is performed by a cardiologist. The beats found acceptable by manual review are 
included in the analysis. The median QT, QTc, and RR value from each extracted replicate is 
calculated, and then the mean of all available medians from a nominal time point is used as 
the subject’s reportable value at that time point. 
Categorical T-wave morphology analysis and the measurement of PR and QRS intervals will 
be performed manually in 3 of the 10 ECG replicates at each time point (Table 5). Each 
fiducial point (onset of P-wave, onset of Q-wave, offset of S-wave, and offset of T-wave) is 
electronically marked. 
In addition to the T-wave categorical analysis, the presence of abnormal U-waves is noted. 
Table 5 
T-wave morphology categories (assessed manually) 
Category 
Description 
Normal T-wave 
Any T-wave not meeting any criterion below 
Flat T-waves 
T amplitude < 1 mm (either positive or negative) including flat 
isoelectric line 
Notched T-wave (+) 
Presence of notch(es) of at least 0.05 mV amplitude on ascending or 
descending arm of the positive T-wave 
Biphasic 
T-wave that contains a second component with an opposite phase that 
is at least 0.1 mV deep (both positive and negative/positive and 
polyphasic T-waves included) 
Normal T-wave (-) 
T amplitude that is negative, without biphasic T-wave or notches 
Notched T-wave (-) 
Presence of notch(es) of at least 0.05 mV amplitude on descending or 
ascending arm of the negative T-wave 
12.4 Assessments related to secondary endpoints 
12.4.1 Safety resting 12-lead ECG 
Single 12-lead safety ECGs will be recorded in supine position after 10 min of rest using the 
site's ECG machine or printouts from the GI Holter device. HR and PQ/PR, QRS, QT and 
QTcF intervals will be recorded.  
Safety ECG recordings will be performed at baseline in triplicate, after dose administration (at 
time points in the vicinity of Tmax) and before discharge from the unit.  
Safety ECGs will be reviewed and interpreted on-site by the investigator.  


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12.4.2 Adverse events  
The frequency, seriousness and intensity of AEs will be collected and reported as described in 
detail in Section 12.5. 
12.4.3 Physical examination 
A complete physical examination will include assessments of the head, eyes, ears, nose, 
throat, skin, thyroid, neurological, lungs, cardiovascular, abdomen (liver and spleen), lymph 
nodes and extremities. 
12.4.4 Vital signs 
Systolic and diastolic blood pressure (BP) and pulse will be measured in supine position after 
10 min of rest.  
12.4.5 Laboratory safety assessments 
Blood samples for analysis of clinical chemistry, hematology and coagulation parameters will 
be collected through an indwelling venous catheter and sent to the certified clinical chemistry 
laboratory at Uppsala University Hospital and analyzed by routine analytical methods.  
Urine analysis will be performed at the research clinic using dip sticks. 
The following safety laboratory parameters will be assessed at time-points defined in 
Section 9.1: 
Clinical Chemistry 
Hematology 
Alanine aminotransferase (ALT) 
Hematocrit 
Alkaline phosphatase (ALP) 
Hemoglobin (Hb) 
Albumin 
Platelet count 
Aspartate aminotransferase (AST) 
Red blood cell (RBC) count 
Bilirubin (total and conjugated) 
 
Calcium 
White blood cell (WBC) count with differential 
count 
Chloride 
Creatinine 
 
Magnesium 
Urinalysis (dip stick) 
Phosphorous 
Glucose 
Potassium 
Erythrocytes 
Sodium 
Nitrite 
Urea nitrogen 
Protein 
Uric acid 
Specific gravity 
 
pH 


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12.4.6 Pharmacokinetic measurements 
Blood sampling for PK measurements of MTHF and its metabolites will be performed as 
described in Section 13.1 (Sample collection for PK measurement) and 
Section 13.2 (Bioanalytical method for PK measurements).  
The specific PK parameters to be evaluated are listed in Section 17.7.5. 
12.5 Adverse Events  
The Principal Investigator is responsible for ensuring that all medical staff involved in the 
study is familiar with the content of this section and the content of the CTC Standard 
Operating Procedures (SOPs) regarding emergencies and Phase I studies.  
12.5.1 Event definitions  
12.5.1.1 Adverse Event  
An AE is any untoward medical occurrence in a clinical study subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An AE can therefore be any unfavorable and unintended sign (including clinically 
significant abnormal values from relevant tests, such as clinical safety laboratory tests, 
ECGs, vital signs), symptom, or disease temporally associated with the use of an IMP, 
regardless of whether it is considered related to the IMP. 
A baseline event is any AE in a clinical study subject that occurs after he signed the Informed 
Consent Form (ICF) up until the first administration of IMP/placebo. 
A treatment emergent AE (TEAE) is any AE not present prior to the initiation of IMP/placebo 
administration or any event already present that worsens in either intensity or frequency 
following exposure to the IMP/placebo. 
12.5.1.2 Serious Adverse Event  
An SAE is any AE that: 
x results in death 
x is life-threatening (this refers to an event in which the subject was at risk of death at 
the time of the event; it does not refer to an event that hypothetically might have 
caused death had it been more severe) 
x requires inpatient hospitalization or prolongation of existing hospitalization 
x results in persistent or significant disability/incapacity 
x is a congenital anomaly/birth defect 
x is medically important (this refers to an event that may not be immediately life-
threatening or result in death or hospitalization, but may jeopardize the subject or 
may require intervention to prevent any of the SAEs defined above) 
 


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Examples of medically important events are intensive treatment in an emergency room for 
allergic bronchospasm or blood dyscrasias, convulsions that do not result in hospitalization, 
development of drug dependency, and drug abuse. 
Planned hospitalizations or surgical interventions for a condition that existed before the 
subject signed the ICF and that did not change in intensity are not SAEs. 
If there is any doubt as to whether an AE meets the definition of an SAE, a conservative 
viewpoint must be taken, and the AE must be reported as an SAE. 
12.5.1.3 Serious Adverse Drug Reaction  
The term Serious Adverse Drug Reaction (SADR) is to be used whenever either the 
Investigator or Sponsor or designee assessed the SAE as possibly or probably related to the 
IMP. 
12.5.1.4 Suspected Unexpected Serious Adverse Reaction  
A Serious Unexpected Serious Adverse Reaction (SUSAR) is any SADR whose nature or 
intensity is not consistent with the current version of the IB. 
12.5.2 Adverse Event assessment definitions  
12.5.2.1 Assessment of severity/intensity 
The grading of the severity/intensity of AEs will follow the CTCAE v4.03.9 Grade refers to the 
severity of the AE. The CTCAE displays Grades 1 through 5 with unique clinical descriptions 
of severity for each AE based on this general guideline. 
The Investigator must assess the severity/intensity of an AE using the following definitions, 
and record it on the Adverse Event Form in the CRF: 
Grade 1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
Grade 2 
Moderate; minimal, local or non-invasive intervention indicated; limiting 
age-appropriate instrumental Activities of Daily Living (ADL)*. 
Grade 3 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; 
limiting self- care ADL**. 
Grade 4 
Life-threatening consequences; urgent intervention indicated. 
Grade 5 
Death related to AE. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
**Self- care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 


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12.5.2.2 Assessment of causal relationship 
The Investigator must assess the causal relationship between a TEAE and the IMP using 
the definitions below and record it on the Adverse Event Form in the CRF as well as on the 
Serious Adverse Event Report Form, if applicable: 
x Probable – the AE has a strong temporal relationship to the IMP or recurs on re-
challenge, and another etiology is unlikely or significantly less likely 
x Possible – the AE has a suggestive temporal relationship to the IMP, and an 
alternative etiology is equally or less likely 
x Not related – the AE has no temporal relationship to the IMP or is due to 
underlying/concurrent illness or effect of another drug (that is, there is no 
causal relationship between the IMP and the AE). 
 
An AE is considered causally related to the use of the IMP when the causality assessment is 
probable or possible. 
For a baseline event, a causality assessment is not relevant. 
12.5.2.3 Assessment of Outcome 
The Investigator must assess the outcome of an AE using the definitions below and record 
it on the Adverse Event Form in the CRF: 
x Recovered – the subject has recovered completely, and no symptoms remain. 
x Recovering – the subject’s condition is improving, but symptoms still remain. 
x Recovered with sequelae – the subject has recovered, but some symptoms 
remain (for example, the subject had a stroke and is functioning normally, but 
has some motor impairment). 
x Not recovered – the subject’s condition has not improved and the symptoms are 
unchanged (for example, an atrial fibrillation has become chronic). 
x Death 
12.5.3 Collecting Adverse Events 
AEs (including baseline events) identified using any of the following methods will be 
recorded: 
x AEs spontaneously reported by the subject 
x AEs observed by the Investigator or medical personnel 
x AEs elicited based on non-leading questions from the Investigator or medical 
personnel 
Collection of baseline events starts after the subject signs the ICF and continues until the first 
administration of IMP/placebo. 
TEAE collection starts with administration of the IMP/placebo and continues until the last 
follow-up assessment. Any AE with start date on the day of first IMP/placebo administration 
must be recorded with start time. 


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At the Follow-up Visit, information on new AEs or SAEs, if any, and stop dates for AEs 
recorded and on-going during the dosing period must be recorded. 
12.5.4 Recording Adverse Events 
AEs (including baseline events) must be recorded on an Adverse Event Form in the CRF. 
The Investigator must provide information on the AE, preferably with a diagnosis or at 
least with signs and symptoms; start and stop dates, start and stop time; intensity; causal 
relationship to IMP/placebo; action taken, and outcome. 
If the AE is serious, this must be indicated in the CRF. Furthermore, the Investigator must 
fill out the Serious Adverse Event Report Form and report the SAE to the Sponsor as 
described in Section 12.5.5. 
AEs, including out-of-range clinically significant clinical safety laboratory values, must be 
recorded individually, except when considered manifestations of the same medical condition 
or disease state; in such cases, they must be recorded under a single diagnosis. 
If the severity/intensity of an AE increases a new Adverse Event Form must be completed in 
the CRF. 
12.5.5 Reporting Serious Adverse Events 
The Investigator must report SAEs to the Sponsor immediately (within 24 hrs) after 
becoming aware of them, by contacting: 
Karin Ganlöv, MD 
Isofol Medical AB 
Arvid Wallgrens Backe 20 
SE-413 46 Gothenburg, Sweden 
Telephone (mobile): +46 (0)702 43 37 50 
E-mail: karin.ganlov@isofolmedical.com 
The same information must also be sent to the CTC SAE email inbox: sae@ctc-ab.se. 
To report SAEs, the Serious Adverse Event Report Form for clinical studies provided must be 
used. The first report should contain as much information as possible, and if more information 
about the subject’s condition becomes available a follow-up report must be submitted with the 
additional information using the same procedure as for the initial report. 
The Sponsor or a delegate will assume responsibility for reporting SAEs to the Competent 
Authorities (CAs) in accordance with local regulations.  
The Sponsor is responsible for informing the Investigators concerned of relevant information 
about SUSARs that could adversely affect the safety of subjects.  
12.5.6 Treatment and follow-up of Adverse Events 
Subjects with AEs that occur during the study must be treated according to daily clinical 
practice at the discretion of the Investigator. 
AEs must be followed up until resolution or until the last Follow-up visit, whichever comes 
first. At the Follow-up Visit, information on new AEs, if any, and stop dates for previously 


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reported AEs must be recorded. AEs on-going at the last Final-up visit may be followed up 
until assessed as stable or until resolution as judged by the Investigator. 
It is the responsibility of the Investigator to follow up on all SAEs until the subject has 
recovered, stabilized, or recovered with sequelae, and to report to the Sponsor all relevant new 
information using the same procedures and timelines as those for the initial report. Relevant 
information includes discharge summaries, autopsy reports, and medical consultation. 
SAEs spontaneously reported by a subject to the Investigator within 30 days after the last 
follow-up assessment must be handled in the same manner as SAEs occurring during the 
study. These SAEs will be reported to the Sponsor. 
12.6 Appropriateness of measurements 
Standardized methods for measurements of safety, tolerability and PK will be used. 
13 PROCEDURE FOR BIOLOGICAL SAMPLES 
13.1 Sample collection for pharmacokinetic measurement 
Blood samples for PK measurements will be drawn by venipuncture or an indwelling venous 
catheter (opposite arm from drug administration) into chilled a-Monovette® EDTA K3EDTA 
2.6 mL tubes at time points specified in the Schedule of events (Table 1; Section 9.1). 
The time points for PK blood sampling will start from end time of injection post dosing.  
The exact sampling time will be recorded in the CRF and used for the PK calculations. 
After blood collection, the vacutainer tubes will be inverted and centrifuged within 40 min at 
3000 rpm for 10 min at approximately 4o C according to local standard procedures. The 
separated plasma is transferred into two aliquots of 0.3 mL each in microtubes 
(polypropylene) and immediately placed at -80° C.  
The plasma samples will be shipped to Charles River Laboratories (UK) for determination of 
MTHF, 5-Formyl-THF, 5-Methyl-THF and THF. 
13.2 Bioanalytical method for pharmacokinetic measurements 
The bioanalytical method for the determination of levels of MTHF, 5-Formyl-THF, 5-Methyl-
THF and THF will be described in a separate bioanalytical laboratory manual. 
13.3 Volume of blood 
During the ambulatory visits, blood samples will be taken by venipuncture and during the stay 
in the research clinic through an indwelling intravenous catheter. 
The anticipated volume of blood samples collected during the study from each subject will 
not exceed 450 mL per subject (i.e., less than the volume drawn during a regular blood 
donation).  


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13.4 Handling, storage and destruction of laboratory samples 
All biological samples will be registered in a tissue-bank at CTC (893).  
Any remains from the safety laboratory samples will be disposed of after analyses.  
The samples for analyses of PK will be stored at -80°C until analyzed. The samples will be 
disposed of after the Clinical Study Report (CSR) has been finalized. 
13.5 Chain of custody of biological samples 
A full chain of custody is maintained for all samples throughout their lifecycle.  
CTC keeps full traceability of collected biological samples from the subjects while in storage 
at the research clinic until shipment and keeps documentation of receipt of arrival.  
The sample receiver (the analytical laboratory) keeps full traceability of the samples while in 
their storage and during use until used or disposed of.  
The Sponsor keeps oversight of the entire life cycle through internal procedures, monitoring 
of study sites and auditing of external laboratory providers.  
13.6 Withdrawal of informed consent for donated biological samples 
If a subject withdraws consent to the use of biological samples donated, the samples will be 
disposed of /destroyed, if not already analyzed and documented.  
The Principal Investigator will ensure that:  
x Subject withdrawal of informed consent is notified immediately to Sponsor.  
x Biological samples from the subject, if stored at the research clinic, are immediately 
identified, disposed of/destroyed and the action is documented.  
The Sponsor has to ensure that the laboratory(ies) holding the samples is/are informed about 
the withdrawn consent immediately and that samples are disposed of/destroyed or returned to 
the research clinic and the action is documented. 
14 ETHICAL AND REGULATORY REQUIREMENTS 
14.1 Ethical conduct of the study 
The study will be performed in accordance with ethical principles that have their origin in the 
Declaration of Helsinki and are consistent with International Conference on Harmonisation 
(ICH)/Good Clinical Practice (GCP) E6 (R1), EU Clinical Trials Directive, and applicable 
local regulatory requirements. 
A link to the Declaration of Helsinki is included in Appendix 18.2. 
14.2 Ethics and regulatory review 
The Principal Investigator is responsible for submission of the CSP, the patient information 
and ICF, any other written information to be provided to the subjects and any advertisements 


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used for recruitment of subjects to applicable Independent Ethics Committee (IEC) for 
approval.  
The Sponsor is responsible for submission of study documents to the applicable CA to local 
regulatory requirements. 
Approval must be obtained in writing from both IEC and CA before the first subject can be 
recruited. 
The Sponsor will provide the CA, IEC and Principal Investigators with safety updates/reports 
according to local requirements. Progress reports and notifications of SUSARs will be 
provided to the IEC according to local regulations and guidelines. 
14.3 Subject information and consent 
It is the responsibility of the Investigator or an authorized associate to give each potential 
study subject adequate verbal and written information before any study specific assessments 
are performed. 
The information will include the objectives and the procedures of the study as well as any 
risks or inconvenience involved. It will be emphasized that participation in the study is 
voluntary and that the subject may withdraw from participation at any time and for any 
reason, without any prejudice. All subjects will be given the opportunity to ask questions 
about the study and will be given sufficient time to consider participation before signing the 
ICF. 
Before performing any study-related procedures the ICF must be signed and personally dated 
by the subject (or their legally acceptable representative and/or witness, as applicable) and by 
the Investigator. A copy of the subject information including the signed ICF will be provided 
to the subject. 
Documentation of the discussion and the date of informed consent must be recorded in the 
source documentation and in the CRF. The subject information sheet and the signed ICF 
should be filed by the Investigator for possible future audits and/or inspections.  
The final approved version of the subject information and ICF must not be changed without 
approval from the Sponsor and the applicable IEC.  
14.4 Subject information card  
The subject will be provided with a Subject Information Card including the following 
information:  
x That he/she is participating in a clinical study 
x Subject study ID  
x That he is treated with the IMP/placebo 
x The name and phone number of the Investigator  
x Name and address of the Sponsor 


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14.5 Subject data protection 
The ICF includes information that data will be recorded, collected and processed and may be 
transferred to European Economic Area (EEA) or non-EEA countries. In accordance with the 
European Union Data Protection Directive (95/46/EC), the data will not identify any persons 
taking part in the study. 
The potential study subject (or the subject’s legally acceptable representative and/or witness, 
as applicable) should be informed that by signing the ICF he approves that authorized 
representatives from Sponsor and CTC, the concerned IEC and CA have direct access to 
his/her medical records for verification of clinical study procedures. This agreement is to be 
substantiated in a separate document, according to local requirements. 
The subject has the right to request access to his/her personal data and the right to request 
rectification of any data that is not correct and/or complete.  
The Investigator must file a Subject Identification List which includes sufficient information 
to link records, i.e., the CRF and clinical records. This list should be preserved for possible 
future inspections/audits but should not be made available to the Sponsor except for 
monitoring or auditing purposes. 
14.6 Changes to the approved Clinical Study Protocol  
Any proposed change to the approved Final CSP (including appendices) will be documented 
in a written and numbered Clinical Protocol Amendment. All amendments including 
substantial changes to the protocol must be approved by the appropriate IEC and/or CA 
before implementation according to applicable regulations. 
14.7 Audits and inspections 
Authorized representatives of Sponsor, a CA, or an IEC may perform audits or inspections at 
the research clinic, including Source Data Verification (SDV). The purpose of an audit or 
inspection is to systematically and independently examine all study-related activities and 
documents, to determine whether these activities were conducted, and data were recorded, 
analyzed, and accurately reported according to the protocol, ICH-GCP guidelines and any 
applicable regulatory requirements. The Investigator will contact the Sponsor immediately if 
contacted by a CA about an inspection at the center. 
14.8 Insurance 
Subjects will be covered under Sponsor’s liability insurance policy through the Swedish 
Pharmaceutical insurance (Läkemedelsförsäkringen). Subjects will also be covered under 
Isofol Medical AB’s liability insurance policy through “IF Skadeförsäkring AB”. The 
certificates of insurance and an information leaflet containing essential information about the 
insurance coverage can be provided upon request. The participating subjects are also 
protected in accordance with national regulations, as applicable. CTC has a company 
insurance covering services performed by CTC. 


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15 STUDY MANAGEMENT 
15.1 Training of study site personnel 
Before enrolment of the first study subject a Sponsor representative or delegate will perform a 
study initiation visit at the research clinic. The requirements of the CSP and related 
documents will be reviewed and discussed and the investigational staff will be trained in any 
study specific procedures and system(s) utilized. 
It is the responsibility of the Investigator to ensure that all personnel involved in the study are 
fully informed of all relevant aspects of the study, and have a detailed knowledge of and 
training in the procedures that are to be executed by them. Any new information of relevance 
to the performance of this study must be forwarded to the staff involved in a timely manner. 
The Investigator will keep a list of all personnel involved in the study together with their 
function and study related duties delegated. A Curriculum Vitae will be available for all staff 
delegated study-specific duties. 
15.2 Clinical monitoring  
The study site will be periodically visited by a Monitor from an independent group at times 
agreed on by the Investigator and the Monitor. At the time of each monitoring visit, the 
function of the Monitor is to:  
x provide information and support to the investigational team. 
x confirm that facilities and resources remain acceptable. 
x confirm that the investigational team is adhering to the CSP, applicable SOPs, 
guidelines, manuals and regulatory requirements.  
x verify that data are being accurately and timely recorded in the CRFs and that IMP 
accountability checks are being performed. 
x verify that data in the CRF are consistent with the clinical records (SDV) in accordance 
with the Monitoring Plan.  
x verify that the correct informed consent procedure has been adhered to for participating 
subjects.  
x ensure that withdrawal of informed consent to the use of the subject’s biological 
samples will be reported and biological samples are identified and disposed 
of/destructed accordingly, and that this action is documented and reported to the 
subject. 
x verify that AEs are recorded and reported in a timely manner and according to the CSP. 
When the study has been completed and all queries have been resolved and the database has 
been locked, the Monitor will perform a close-out visit. 
15.3  Source data document 
A separate Source Data Verification List will be generated for each site before start of 
enrolment, specifying the location of the source of derived information appearing in the CRF. 


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This document must be signed by the Principal Investigator and the Monitor to confirm 
agreement before start of recruitment. 
The paper CRF is considered source data when data is entered directly into the CRF. 
The Investigator should guarantee access to source documents to the Monitor, CAs and the 
IECs, if required. 
15.4 Study agreements 
The Principal Investigator must comply with all the terms, conditions, and obligations of the 
Clinical Study Agreement for this study. 
Agreements between Sponsor and CTC must be in place before any study-related procedures 
can take place, or subjects be enrolled. 
15.5 Study time table and end of study 
The end of the clinical part of the study is defined as the last visit of the last subject 
participating in the study. 
The study is expected to start in Quarter 2, 2017 and to be completed by Quarter 2, 2018.  
15.6 Discontinuation of the study 
The Sponsor reserves the right to discontinue the study at any time, but intends only to 
exercise this right for valid scientific or administrative reasons. 
After such a decision, the Investigator must inform all participating subjects and perform 
relevant assessments, preferably according to the scheme for the final assessments. All 
delivered and unused study products and other study materials must be returned and all CRFs 
completed as far as possible. 
15.7 Reporting and publication 
15.7.1 Clinical Study Report  
A summarizing report must be submitted to the applicable CA and IEC within 12 months after 
completion of the study (in accordance with LVFS 2011:19, Chapter 9). 
A CSR, in compliance with ICH E3; Structure and content of Clinical Study Reports, 
describing the conduct of the study, the statistical analysis performed and the results obtained, 
will be prepared by CTC. The CSR will be reviewed and approved by, as a minimum, the 
Principal Investigator, the Statistician and the Sponsor. 
15.7.2 Confidentiality and ownership of study data 
Any confidential information relating to the IMP or the study, including any data and results 
from the study, will be the exclusive property of the Sponsor. The Investigator and any other 
persons involved in the study will protect the confidentiality of this proprietary information 
belonging to the Sponsor.   


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15.7.3 Publication 
The results from this study will be submitted for publication at the discretion of the Sponsor.  
15.8 Archiving  
The Principal Investigator is responsible for maintaining essential documents, (as defined in 
ICH E6 GCP, Section 8) for 10 years after finalization of the CSR. This includes any original 
source documents related to the study, the Subject Identification List (providing the sole link 
between named subject source records and anonymous CRF data), the original signed ICFs 
and detailed records of disposition of IMP.  
It is the responsibility of the Sponsor to inform the Investigator/institution as to when these 
documents no longer need to be retained. 
The Sponsor will archive the Study Master File (SMF) in accordance with ICH E6 GCP, 
Section 8 and applicable regulatory requirements. 
16 DATA MANAGEMENT 
16.1 Case Report Form  
Data will be collected in paper CRFs specifically designed for this study. The Investigator or 
an authorized person will record subject data in the CRF in a precise and accurate manner. 
Abbreviations should not be used. The Investigator is responsible for the data entered and will 
sign off the CRF at each visit and at the end of the study. The data should be recorded as soon 
as they are generated. CRF entries must be made with an archive resistant pen. Any correction 
should be marked by a strike-through and the correct information should be written next to the 
error. All corrections must be signed and dated. Correction fluid must not be used. Only 
persons authorized by the Investigator are allowed to make entries to the CRF. 
16.2 Database management plan and database design 
Detailed information on data management will be described in a study-specific Data 
Management Plan (DMP). The person entering data into the database is not allowed to 
attempt any personal interpretation or to make any decisions on the data other than 
self-evident corrections as listed in the study-specific Data Entry Instructions or Data 
Handling Report. Single data entry type will be applied.  
Data validation/data cleaning procedures are designed to assure validity and accuracy of 
clinical data. These procedures consist of manual reviewing during data entry and 
computerized edit checks and queries for identifying data values that are outside the allowed 
range, protocol violations, incomplete or inconsistent. The Data Validation Plan specifies the 
checks that are to be performed on subject data for the study. All study-specific and standard 
data validation programming will be tested in a separate testing environment prior to use on 
production data. 
16.3 External data 


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External data may be received in electronic format or paper printout. Key variables are 
defined in order to uniquely identify each sample record. File and data formats are agreed 
with the external data provider. Any electronically transferred data must contain origin, date 
created, date sent and number of records at minimum. 
The continuous 12-lead digital ECG data will be stored onto Secure Digital memory cards.   
16.4 Medical encoding 
Medical encoding will be performed by trained personnel at CTC. Adverse events and 
medical history verbatim terms are encoded using the Medical Dictionary of Regulatory 
Activities (MedDRA), latest version available when approving the DMP.  
Prior and concomitant medications will be coded according to the WHO Anatomic 
Therapeutic Chemical classification system.  
All coding will be approved by Sponsor. 
16.5 Database lock 
When all data have been entered, discrepancies solved and all reconciliation with the SAE 
database is complete, the database will be locked and the data will be analyzed and reported in 
a CSR. 
 
 


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17 STATISTICAL METHOD AND DETERMINATION OF SAMPLE SIZE 
The principal features of the statistical analysis to be performed are described in this section. 
A more technical and detailed elaboration of the principal features will be presented in a 
separate Statistical Analysis Plan (SAP). 
17.1 General 
All statistical analysis of the study will be performed using the statistical software SAS for 
Windows Version 9.4. In all calculations, zero will be substituted for concentrations below 
the quantification limit of the assay. Data collected from all subjects will be presented in data 
listings. Both absolute values and change from baseline values for each subject will be given 
where applicable. All continuous data will be listed with the same precision as will be 
presented in the database. Data listings will be sorted by treatment, subject ID and time point. 
A missing value will be represented by an empty cell and no imputation will be made. 
Continuous data will be summarized in tables using number of subjects (n), mean, median, 
standard deviation (SD), standard error (SE), 90% confidence interval (CI; based on a t-
distribution if not otherwise stated), minimum, and maximum by study time point.  
Categorical data will be summarized by time point using frequencies and percentages.  
Percentages will be rounded to the nearest tenth. Population counts will be used as the 
denominator in the calculation of percentages unless otherwise specified.  
17.2 Determination of sample size 
Assuming a 1-sided 0.05 significance level and a standard deviation of 7 msec for ΔQTcF, a 
total of 33 evaluable subjects who complete Modufolin® (24 subjects) and placebo (9 
subjects), separately will be sufficient to achieve 82% power to exclude a prolongation of 
10 msec or longer of the upper 1-sided 95% CI of the mean ΔΔQTcF, assuming that the 
prolongation is 3 msec at the geometric mean peak Modufolin® concentration. Under the 
same assumptions, a total of 66 subjects (48 Modufolin® and 18 placebo) will achieve 97.5% 
power.  
Note that this calculation is conservative, since it does not take into account any gain in 
precision due to the use of all data of each subject with the help of a linear mixed effects 
model. 
17.3 Analysis data sets 
17.3.1 Full Analysis Set  
The Full Analysis Set will consist of all subjects who have been randomized and received one 
dose of IMP/placebo. 
17.3.2 Per Protocol Analysis Set 
The Per Protocol Analysis Set will consist of all subjects who have been randomized and 
completed the study period without any major protocol deviations. All protocol violations will 
be judged as major or minor at the clean file meeting. 


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17.3.3 Safety Analysis Set  
The Safety Analysis Set will include all subjects who receive at least one dose of IMP/placebo 
and have at least one post-dose safety assessment. 
17.3.4 PK Analysis Set  
The PK Analysis Set will include all subjects who have evaluable plasma concentration data 
for Modufolin® and for whom one or more of the designated PK parameters can be 
determined. 
17.3.5 QT/QTc Analysis Set  
The QT/QTc Analysis Set will include all subjects in the Safety Analysis Set with 
measurements at baseline as well as on-treatment with at least one post-dose time point with a 
valid ΔQTcF value. The QT/QTc Analysis Set will be used for the by-time point and 
categorical analyses in the electrocardiogram analysis. 
17.3.6 PK/QTc Analysis Set  
The PK/QTc Analysis Set will include all subjects who are in both the QT/QTc Analysis Set 
and PK Analysis Set with at least one pair of post-dose PK and QTcF data from the same time 
point. The PK/QTc Analysis Set will be used for the exposure-response analysis in the 
electrocardiogram analysis.  
17.4 Electrocardiogram analysis 
17.4.1 Baseline  
For all ECG parameters, baseline is defined as the average of the measured QTc intervals 
from the 3 pre-dose time points (45, 30, and 15 min pre-dose) on Day 1.   
17.4.2 QT correction methods and parameters 
The QT and RR value for each beat will be used for HR correction. 
Replicate ECGs will be extracted in up to 10 replicates from each nominal time point pre-
specified in the protocol. The median value from each extracted replicate will be calculated, 
and then the mean of all available medians (minimum 3 medians) from a nominal time point 
will be used as the subject’s reportable value at that time point.    
QTcF:  
The Fridericia's correction QTcF is defined as: QTcF = QT/RR1/3. 
17.4.3 Exposure-response analysis (Primary analysis)  
The relationship between the plasma concentration of Modufolin® and change-from-baseline 
QTcF (∆QTcF) will be quantified using a linear mixed-effects modeling approach with 
ΔQTcF as the dependent variable, plasma concentration of Modufolin® as the covariate, 
treatment (active or placebo) and time as categorical factors, and a random intercept per 
subject.  The degrees of freedom for the model estimates will be determined by the Kenward-
Rogers method. From the model, the slope (i.e., the regression parameter for the 


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concentration) and the treatment effect-specific intercept (defined as the difference between 
active and placebo) will be estimated together with 2-sided 90% CI. The estimates for the 
time effect will be reported with degrees of freedom and SE.  
The geometric mean of the individual Cmax values for subjects in each of the active drug 
groups will be determined. The predicted effect and its 2-sided 90% CI for placebo-corrected 
change-from-baseline QTcF (ΔΔQTcF) (i.e., the product with the slope estimate + treatment 
effect) at this geometric mean Cmax will be obtained for each Modufolin® dose separately. If 
the upper bound of the 90% CI of the model predicted QTcF effect is below 10 msec at 
clinically relevant plasma levels of Modufolin®, it will be concluded that Modufolin® does not 
cause clinically concerning QTc prolongation.  
The plot of the observed median-quantile Modufolin® concentrations and associated mean 
ΔΔQTcF (90% CI) adjusted for diurnal effects together with the regression line presenting the 
predicted ΔΔQTcF (90% CI) (as described in the publication by Tornoe et al.16 will be used to 
evaluate the adequacy of the model fit to the assumption of linearity and the impact on 
quantifying the exposure response relationship. Additional exploratory analyses (via graphical 
displays and/or model fitting) will include accounting for a delayed effect (hysteresis) and the 
justification for the choice of pharmacodynamic model (linear versus nonlinear) as follows. 
Investigation of hysteresis: 
If a QTc effect (∆∆QTcF) exceeding 10 msec cannot be excluded in the by-timepoint analysis 
of the two highest dose groups, hysteresis, i.e., difference in peak QT response and Cmax, will 
be explored through visual inspection of overlaid PK and ΔΔQTc time curves and through so 
called hysteresis loops for each dose of Modufolin® for Injection, 100 mg  
Appropriateness of a linear model: 
To assess the appropriateness of a linear model, normal QQ-plots for the residuals and plots 
of weighted residuals vs. concentration and vs. fitted values will be produced. The scatter plot 
of residuals vs concentration by Loess (i.e., locally weighted scatterplot smoothing as 
described in the publication by Cleveland17) fitting will be also produced with an optimal 
smoothing parameter selected by the Akaike information criterion with a correction (AICC).18 
In addition, a model with a quadratic term in concentration will be fitted and the quadratic 
term will be tested on the two-sided 5% level. If there is an indication that a linear model is 
inappropriate, additional models will be fitted, in particular: 
x An E-max model; 
x A log-linear model where the plasma concentration C is replaced by log(C/C0), C0 is 
the limit of quantification of the assay used to determine C and all values below C0 are 
replaced by C0 (i.e., log[C0/C0] = 0). 
The Exposure-Response analysis will then be repeated for the model found to best 
accommodate the nonlinearity detected. 
17.4.4 By-time point analysis:  
The analysis for QTcF will be based on a linear mixed-effects model with change-from-
baseline QTcF (ΔQTcF) as the dependent variable, time (categorical), treatment (Modufolin® 
for Injection, 100 mg and placebo), and time-by-treatment interaction as fixed effects, and 
baseline QTcF as a covariate. Subject will be included as a random effect for the intercept. 
Subject dosed with placebo will be analyzed as a pooled group. An unstructured covariance 


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matrix will be specified for the repeated measures at post-dose time points for each subject. If 
the model with unstructured covariance matrix fails to converge, other covariance matrix such 
as autoregressive and compound symmetry will be considered. From this analysis, the least-
squares (LS) mean and 2-sided 90 % CIs will be calculated for the contrast “Modufolin® 
versus placebo” at each dose of Modufolin® and each post-dose time point, separately.  
For HR, PR, and QRS intervals, the analysis will be based on the change-from-baseline values 
(ΔHR, ΔPR, ΔQRS). The same model will be used as described for QTcF. The LS mean, SE 
and 90% CI from the statistical modeling for both change-from-baseline and placebo-
corrected change-from-baseline values will be listed in the tables and graphically displayed. 
17.4.5 Categorical analyses 
The analysis results for categorical outliers, and T-wave morphology will be summarized in 
frequency tables with counts percentages for both number of subjects and number of time 
points. For categorical outliers, the number (percentage) of subjects as well as time points 
who had increases in absolute QTcF values >450 and ≤480 msec, >480 and ≤500 msec, and 
500 msec, and changes from pre-dose baseline of >30 and ≤60 msec, and >60 msec; increase 
in PR from pre-dose baseline >25% to a PR>200 msec; increase in QRS from pre-dose 
baseline >25% to a QRS >120 msec; decrease in HR from pre-dose baseline >25% to a 
HR <50 bpm; and increase in HR from pre-dose baseline >25% to a HR >100 bpm will be 
determined. For T-wave morphology, the analyses will be focused on change from baseline 
(i.e., treatment-emergent changes). 
17.5 Description of study population 
17.5.1 Demographics and baseline characteristics 
Descriptive statistics for demographics, weight and height will be presented by treatment. 
17.5.2 Medical history and concomitant medication 
Medical/surgical history and prior/concomitant medications will be presented by treatment 
using descriptive statistics and listings. 
17.5.3 Treatment compliance 
The number of subjects treated in each treatment period and their individual dose will be 
tabulated. 
17.6 Analysis of primary endpoints 
17.6.1 Electrocardiogram  
All ECG data will be listed for each subject and summarized as the vital signs parameters. In 
addition, ECGs will be categorized as ”normal”, ”abnormal, not clinically significant”, or 


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”abnormal, clinically significant” (as judged by the Investigator) and summarized by 
treatment and period using frequency tables.  
17.6.2 Telemetry 
Ambulatory ECG telemetry will be used for cardiac surveillance up to 24 hrs after dose 
administration. 
17.7 Analysis of secondary endpoints 
17.7.1 
Physical examination 
Abnormal findings will be specified and presented by subject and summarized by treatment 
and period. 
17.7.2 Vital signs 
Vital signs (systolic/diastolic blood pressure and pulse will be summarized by treatment and 
period using descriptive statistics.  
17.7.3 Safety laboratory analyses 
Safety laboratory data will be presented by individual time courses for each parameter and 
subject and summarized by treatment and period. 
17.7.4 Adverse Events  
All AE data will be fully listed by Investigator terms and MedDRA Preferred Term (PT). AE 
data will be summarized by System Organ Class (SOC) and PT.  
17.7.5 Definition and calculation of folate pharmacokinetic parameters 
The PK analysis will be based on the PPAS and subcontracted by the Sponsor to PKxpert. 
The PK parameters will be calculated by non-compartmental pharmacokinetic analysis (NCA) 
using the software Phoenix WinNonlin® version 6.4 or later (Pharsight Corporation, U.S.A.).  
The following non-compartmental PK parameters will be assessed for [6R]-5,10-MTHF, 
[6S]-5-Methyl-THF, [6S]-THF and [6S]-5-Formyl-THF, if data permit: 
AUC 
Area under the plasma concentration-time curve from time 0 to 
infinity. Calculated using the linear/log trapezoidal rule with 
extrapolation to infinity. 
 
The log trapezoidal rule is used from time tmax to the last time point t: 


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Extrapolation to infinity is performed using: 
 
AUClast 
Area under the plasma concentration-time curve from time 0 to the 
last time point t. 
AUCW
Area under the plasma concentration-time curve in a dosing interval. 
Calculated using the linear/log trapezoidal rule with extrapolation to 
W, if required 
AUC%Extrap 
Percentage of AUC that is due to extrapolation from the last 
concentration to infinity: 
 
AUMC 
Area under the first moment curve extrapolated to infinity based on 
the last observed concentration. 
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C0 
Back extrapolated concentration at time point 0 after intravenous 
administration 
Cmax 
Maximum plasma concentration 
C(time) 
Plasma concentration at a certain time  
Css 
Steady-state drug concentration in plasma 
CL 
Total body clearance for intravenous administration. 
AUC
Dose
CL  
 
LLOQ 
Lower Limit of Quantification 
Mean 
Arithmetic mean, otherwise specified 
MRT 
Mean Residence Time extrapolated to infinity. 
AUC
AUMC
MRT  
 
 
NCA 
Non-Compartmental pharmacokinetic Analysis 
PK 
Pharmacokinetics 
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Tau (W) 
Dosing interval 
tmax 
Time at which maximum plasma concentration is observed. 
t1/2 
Terminal half-life: 
 
Oz (lambda z) 
Terminal rate constant. Calculated by log-linear regression of 
concentrations versus time 
Vz 
Volume of distribution based on the terminal phase. 
z
AUC
Dose
Vz
O
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Vss 
Volume of distribution based at steady state. 
CL
MRT
Vss
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Descriptive statistics for the PK parameters will be presented by treatment group with number 
of measurements, arithmetic mean, SD, Coefficient of variation (CV), median, minimum, 
maximum, geometric mean, geometric CV%. 
17.8 Statistical/analytical issues 
17.8.1 Handling of dropouts or missing data 
For information, refer to the SAP. 
17.8.2 Interim analyses and data monitoring 
Not applicable. 
z
t
O
2
ln
½  




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18.2 Declaration of Helsinki 
http://www.up.ac.za/media/shared/Legacy/sitefiles/file/45/2875/declarationofhelsinki_fortaleza_brazil
2013.pdf 


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18.3 Summary of safety and pharmacokinetic data from Isofol Medical AB’s previous 
clinical studies in cancer patients 
Therapeutic dose 
The ongoing clinical study ISO-CC-005 is investigating 60, 120 and 240 
mg/m2. Based on current data the expected clinical dosing regimen in 
future clinical trials in metastatic colorectal cancer (lead indication) 
patients is 120 mg/m2 Modufolin®. 
Maximum tolerated dose 
Doses of Modufolin® up to 500 mg/m2/day has been administered to 
patients in study ISO-MC-091. 
From animal studies: 
In dogs, the 28-day toxicology studies indicate that doses of 200 mg/m2 
Modufolin® for Injection, 100 mg may be administered twice daily (i.e., 
total daily dose of 400 mg/m2) resulting in safety ratios between dogs 
and humans above 10. Looking at the safety exposure margins for rats, 
the safety factors would still be above 10 even if 200 mg/m2 doses 
were administered up to 6-8 times daily (i.e., total daily doses of 
1,200-1,800 mg/m2). 
Principal adverse events 
Modufolin® alone is not anticipated to induce any AEs or DLTs. 
Modufolin® is a biomodulator of the 5-FU anticancer effect in colorectal 
cancer or as a rescue treatment after High Dose Methotrexate treatment. 
In the ISO-CC-005 study in metastatic CRC patients (Modufolin® 
administered together with drugs such as 5-FU, irinotecan and 
oxaliplatin): 
Most common AEs: nausea (25), diarrhea (24), fatigue (24) and vomiting 
(13). 
Main DLT AE: Neutropenia (8) 
Maximum dose tested 
Single Dose 
In the ISO-CC-002 study, colorectal cancer 
patients have been exposed to single Modufolin® 
doses of 60 or 200 mg/m2. 
In the ISO-MC-091 study, rectal cancer patients 
have been exposed to single and multiple doses 
ranging between 10 and 500 mg/m2/day. 
Multiple Dose 
In the ISO-MC-091 study, rectal cancer patients 
have been exposed to multiple doses ranging 
between 10 and 500 mg/m2/day. 
Exposures Achieved at 
Maximum Tested Dose 
Single Dose 
Cycle 1 Day 15, 500 mg/m2 n=6, (ISO-MC-
091) 
Modufolin® drug substance ([6R]-MTHF) 
(parent) 
C(10min) 23300 ±6680 ug/L 
AUC(0-4h) 13600 ±3430 h*ug/L 
[6S]-5-tetrahydrofolate ([6S]-THF) Cycle 1 Day 
15, 500 mg/m2 (ISO-MC-091) 
C(10min) 30200 ±5780 ug/L 
AUC(0-4h) 66700±12900 h*ug/L 
[6S]-5-methyl-tetrahydrofolate 
([6S]-methyl-THF)  
C(10min) 1600 ±400 ug/L 
AUC(0-4h) 17400 ±3120 h*ug/L 
 


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Multiple Dose 
Cycle 3 Day 15, 500 mg/m2 (ISO-MC-091) 
Modufolin® drug substance ([6R]-MTHF) 
(parent)  
C(10min) 24200 ±2410 ug/L 
AUC(0-4h) 14900 ±1450 h*ug/L 
[6S]-5-Tetrahydrofolate ([6S]-THF) 
C(10min) 31600 ±6330 ug/L 
AUC(0-4h) 70800±12500 h*ug/L 
[6S]-5-Methyl-Tetrahydrofolate 
([6S]-Methyl-THF)  
C(10min) 1400 ±420 ug/L 
AUC(0-4h) 14900 ±4150 h*ug/L 
Range of linear PK 
60 and 200 mg/m2 (ISO-CC-002) and 500 mg/m2 (ISO-MC-091) 
AUClast: Average values: 2270 h*ug/L at 60 mg/m2 (tlast = 1-1.5 h, 
AUC0-2h could not be calculated) 
AUC0-2h: Average values: 7730 h*ug/L at 200 mg/m2 and 13200 h*ug/L 
at 500 mg/m2.  
Thus, dose proportional increase was seen comparing 60 to 200 mg/m2, 
and a minor sub-proportional increase to dose increase was seen, 
comparing up to 500 mg/m2. It should be emphasized that 60 and 200 
mg/m2 was in one study with colon cancer patients (ISO-CC-002) and 
500 mg/m2 was in another study with rectal cancer (ISO-MC-091). 
Accumulation at steady 
state 
x Modufolin® drug substance ([6R]-MTHF) (parent, active 
metabolite), no accumulation after once daily dosing up to 500 
mg/m2/day. 
Metabolites 
Most relevant other metabolites:  
[6S]-5-Tetrahydrofolate ([6S]-THF)  and 
([6S]-5-Methyl-Tetrahydrofolate ([6S]-Methyl-THF) 
Both metabolites can serve as precursors to MTHF, but are not 
active in itself 
Absorption 
Absolute/Relative 
Bioavailability 
N/A, IV administration 
Tmax 
x Median (range) for parent 
N/A, IV administration 
x Median (range) for metabolites 
[6S]-5-tetrahydrofolate ([6S]-THF): The 
concentration of the metabolite, THF, after IV 
administration of Modufolin® increased rather 
rapidly, tmax ranged between 5-15 min at 60 
mg/m2 and 15-45 min at 200 mg/m2.  
[6S]-5-Methyl-Tetrahydrofolate 
([6S]-Methyl-THF):  
tmax ranged between 1.5-4 h at 60 mg/m2 and 
3-6 h at 200 mg/m2  
Distribution 
Vd 
The distribution volume (Vss) of MTHF was in 
the range of 9.2-22.7 L in the 60 mg/m2 dose 
group and 9.4-29.3 L in the 200 mg/m2 dose 
group.  


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% bound 
Not performed 
Elimination 
Route 
x Primary route; percent dose eliminated 
IV. Not studied, but it is anticipated that most of 
the dose will entering the endogenous folate 
metabolism. 
x Other routes 
N/A 
Terminal t½   
x Mean (± SD) for parent 
The elimination half-life calculated for two 
patients in the high dose level (200 mg/m2, 
measurable concentrations until 6 h) were 1.5-
1.8 h, but the major part of the exposure of the 
parent compound was eliminated faster, a multi-
compartmental elimination curve was seen for 
Modufolin® drug substance ([6R]-MTHF) .  
x Mean (± SD) for metabolites 
[6S]-5-Tetrahydrofolate ([6S]-THF): one phase 
elimination curve with an elimination half-life 
less than 1.5 h  
[6S]-5-Methyl-Tetrahydrofolate 
([6S]-Methyl-THF): The concentration of 
methyl THF could be measured until 6 - 24 h. 
The PK of methyl THF for two patients at 200 
mg/m2 could be described by a one phase 
elimination curve with an elimination half-life of 
4.3-14.5 h  
CL 
The clearance (CL) of methylene THF, parent,  
was in the range of 31-60 L/h 
Intrinsic Factors 
Age 
Currently no information available (popPK).  
Sex 
Currently no information available (popPK).  
Race 
Currently no information available (only 
caucasians studied hitherto). 
Hepatic & Renal 
Impairment 
Currently no information available. 
Extrinsic Factors 
Drug interactions 
Not performed 
Food Effects 
Intravenous administration / No food effects 
expected 
Expected High Clinical 
Exposure Scenario 
In the other investigated indication with the treatment of Modufolin® 
(Osteosarcoma), the drug candidate is used as rescue after High dose 
methotrexate administration. In scenarios of extreme Methotrexate 
levels, according to the Children’s Oncology Group, doses of folic acids 
(e.g.Leucovorin/Calcium folinate) might be administered up to 
maximum 1500 mg q6h until Methotrexate levels are <0.1 µM. This 
corresponds to a maximum dosage of 750 mg q6h Modufolin®. 
(Modufolin® is the active metabolite of Leucovorin/Calcium folinate, 
hence half the dosage is needed) 


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5. Danenberg PV et al. Folates as adjuvants to anticancer agents: Chemical rationale and 
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7. Wettergren Y et al. (2015). A pharmacokinetic and pharmacodynamic investigation of 
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8. Gustavsson B et al. (2015). Phase 1 dose de-escalation trial of the endogenous folate 
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9. National Cancer Institute, Cancer Therapy Evaluation Program. Common terminology 
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10. Sibille et al. (2010). A safety grading scale to support dose escalation and define 
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11. WHO (1981). Recommendations for grading of acute and subacute toxicity: reporting 
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SAP_000.pdf: