Clinical trial NCT06331884
Conditions: Safety and Tolerability Healthy Volunteers
Sponsor: 4TEEN4 Pharmaceuticals GmbH
Start date: 2024-03-07; Completion date: 2024-09-04
Enrollment: 24 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Dipeptidyl peptidase 3 (DPP3) is a protease involved in the degradation of several cardiovascular mediators. During cardiogenic shock, upregulation of the vasoconstrictive molecule angiotensin II is a physiologic and potentially life-saving response aimed at maintaining adequate tissue perfusion. As circulating (c)DPP3 is able to effectively cleave angiotensin II, it may represent a novel factor contributing to hemodynamic instability during cardiogenic shock.

Recently, a cDPP3-antagonizing antibody called AK1967 (commonly referred to as Procizumab) has been developed. In animal models of cardiogenic- and septic shock, inhibition of cDPP3 by AK1967 resulted in improved cardiac function and survival. Furthermore, AK1967 has shown an excellent safety record in different preclinical studies. In the current study the safety, tolerability and pharmacokinetics/-dynamics of AK1967 will be investigated in healthy male subjects.

Detailed description:

Arms: arm 1: None arm 2: None arm 3: None arm 4: None
Interventions: intervention 1: AK1967 (Procizumab) intervention 2: Placebo
Intervention descriptions: intervention 1: DPP3 inhibition using the humanized monoclonal antibody AK1967 (Procizumab) intervention 2: Application of placebo

Protocol documents:
Prot_000.pdf: 
 
 
 
Cover Page 
 
CT-P1-001 
  
 
 
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Cover Page – CTP 
 
Title of the trial: A randomized double-blind placebo-controlled phase 1 study on the safety, 
tolerability and pharmacokinetics/-dynamics of escalating single intravenous doses of 
AK1967 (Procizumab) in healthy male volunteers 
NCT number: NCT06331884 
Document Date: 05/22/2024 
Attachments: 
- 
CTP Version 5.0 






















































































































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SAP_001.pdf: 
 
 
 
Cover Page 
 
CT-P1-001 
  
 
 
Page 1 of 1 
Confidentiality Notice: This document and the contained information are confidential and may 
not be reproduced or shared without written permission of 4TEEN4 Pharmaceuticals GmbH. 
Cover Page – SAP 
 
Title of the trial: A randomized double-blind placebo-controlled phase 1 study on the safety, 
tolerability and pharmacokinetics/-dynamics of escalating single intravenous doses of 
AK1967 (Procizumab) in healthy male volunteers 
NCT number: NCT06331884 
Document Date: 09/02/2024 
Attachments: 
- 
SAP Version 1.0 


Printed on 03.09.2024 08:09:02
QM record
Valid from:
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Version:
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QMA_01049_Clinical Statistical Analysis Plan CT-
P1-001
Status:
Released
Filing: https://fourteenfour.dhcvisioncloud.com/dhcvision/document.jsp?key=DATABASE:1001;IO:1529088
Page 1 of 1
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Clinical Statistical Analysis Plan CT-P1-
001
Signature
Date
Creation
Bourgeois Karine (Dr.)
09/02/2024 17:01:21 (UTC +1)
Review
Dirksmeyer Thies (Dr.)
09/02/2024 17:02:42 (UTC +1)
Richter Kathleen
09/02/2024 17:14:27 (UTC +1)
Uba Theo Ikenna
09/02/2024 19:06:36 (UTC +1)
Release
Bourgeois Karine (Dr.)
09/02/2024 22:04:02 (UTC +1)
Latest Changes
Version
Change Description
Valid From
(mm/dd/yyyy)
1
Initial version
09/02/2024
Older Changes
(from most new to oldest)
Version
Change Description
Valid From
(mm/dd/yyyy)
Inapplicable
Inapplicable
Inapplicable
Attachments

QMA_01049_Attachment_Clinical Statistical Analysis Plan CT-P1-001


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 Record Title: Clinical Statistical Analysis Plan CT-P1-001
  
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Statistical Analysis Plan
Record Title: Clinical Statistical Analysis Plan CT-P1-001
Contents
1
Background and scope ....................................................................................................................3
2
Study objectives ..............................................................................................................................3
3
Trial design ......................................................................................................................................4
3.1
Sample size determination / power analysis...........................................................................5
3.2
Hypotheses..............................................................................................................................5
4
Population .......................................................................................................................................5
5
Endpoints for the primary, secondary objectives and exploratory endpoints................................6
5.1
Endpoints for the primary objectives ......................................................................................6
5.2
Endpoints for the secondary objectives ..................................................................................6
5.3
Endpoints for the exploratory objectives ................................................................................7
6
General conventions .......................................................................................................................7
6.1
Treatment groups....................................................................................................................7
6.2
Layout of tables .......................................................................................................................7
6.3
General calculation rules.........................................................................................................8
6.4
Descriptive statistics..............................................................................................................10
7
Analysis sets and data review criteria ...........................................................................................10
7.1
Data sets for analysis.............................................................................................................10
7.2
Handling of missing data, drop-outs, extreme values and protocol violations .....................11
8
Data analysis..................................................................................................................................11
8.1
Statistical software ................................................................................................................11
8.2
Definitions, derived variables and handling of missing data.................................................11
Definitions of time points / time windows....................................................................................11
Derived variables...........................................................................................................................12
Handling of missing values ............................................................................................................12
8.3
Disposition.............................................................................................................................12
8.4
Demographic data, screening and baseline characteristics ..................................................12
8.5
Analysis of primary endpoints...............................................................................................13


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8.6
Analysis of secondary endpoints ...........................................................................................15
Descriptive pharmacokinetic analyses ..........................................................................................16
8.7
Additional analyses................................................................................................................18
9
Contents of statistical tables and figures ......................................................................................19


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1
Background and scope
This SAP is created for the study and protocol as stated below:
Table 1 Basic trial information.
EU-CT number
2023-507035-37-00
Trial name
Not applicable
Clinical Trial Protocol Version
Latest Version 5
All abbreviations are included in the Glossary
The following documents are attached to the QM record of this SAP:
-
None (the Data Review Plan and Report are referenced in DHC VISION)
2
Study objectives
The objective of the AK1967 Phase 1 study is to evaluate the safety and tolerability of single 
escalating doses of AK1967 in healthy male volunteers. Main trial endpoints associated with this 
objective are fully described in the study protocol. To summarize, endpoints for this safety outcome 
are defined as differences between the different AK1967 dosage groups and the placebo group in:
-
Reported number of adverse events after IMP administration
-
Kinetics of vital signs at baseline and following IMP administration
-
Local tolerability at site of IMP infusion
-
Safety laboratory parameters at baseline and following IMP administration
-
12-lead electrocardiogram (ECG) parameters at baseline and following IMP administration
There are several secondary trial objectives. A full overview is again provided in the study protocol. 
To summarize, secondary trial measurements/endpoints discussed in this document include:
-
Pharmacokinetics of the IMP (including AUC, Cmax, Terminal T1/2, clearance, VD) 
-
Pharmacodynamics (enzyme activity and concentration of DPP3) at baseline and following 
IMP administration.
-
Pharmacodynamics related to the renin-angiotensin system (blood plasma levels of renin-
angiotensin system metabolites) at baseline and following IMP administration
-
Pharmacodynamics related to catecholamines (blood plasma levels of adrenergic 
metabolites) at baseline and following IMP administration
-
Related to safety and pharmacodynamics: To determine the effects of AK1967 on 
inflammatory cytokines.


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 Record Title: Clinical Statistical Analysis Plan CT-P1-001
  
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Primary objective(s):
-
To assess the safety and tolerability of single escalating doses of AK1967 in healthy male 
volunteers.
Secondary objective(s):
-
To determine the pharmacokinetics of single escalating doses of AK1967 in healthy male 
volunteers
-
To determine the effects of AK1967 on DPP3 activity and concentration (pharmacodynamics) 
in healthy male volunteers
-
To determine the effects of AK1967 on renin-angiotensin system metabolites 
(pharmacodynamics) in healthy male volunteers.
-
To determine the effects of AK1967 on circulating adrenergic metabolite concentrations 
(pharmacodynamics) in healthy male volunteers.
-
To determine the effects of AK1967 on inflammatory cytokines, including, but not limited to 
TNF, IL-6, IL-8, IL-10.
3
Trial design
Number of centers
1 center
Randomized?
Yes
Randomization ratio
Per dose group, 8 volunteers will participate of which 6 subjects will 
receive AK1967, and 2 will receive placebo. With 3 escalating dose 
groups, a total of 6 subjects will receive placebo. In total 24 subjects will 
participate resulting in a 1:1:1:1 ratio (treatment arm A: treatment arm 
B: treatment arm C: control group)
Blinded?
Double-blind
Dosing
Intravenous infusion (single i.v. dose) over 2 hours
Placebo controlled
Yes
Treatment arms
single i.v. dose of 3 mg/kg AK1967 (treatment arm A)
single i.v. dose of 6 mg/kg AK1967 (treatment arm B)
single i.v. dose of 12 mg/kg AK1967 (treatment arm C)
single i.v. dose of Placebo (control group)
PK profile days
Pre-dose


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Day 1 (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24 hour ± 2 mins)
Day 2 (± 1 hour)
Day 3 (± 3 hours)
Day 7 (± 1 day)
Day 14 (± 2 days)
Day 21 ((± 3 days)
Day 28 (± 3 days)
Table 2 Treatment groups and sample sizes
Treatment
N
Treatment arm A: 3 mg/kg AK1967
6
Treatment arm B: 6 mg/kg AK1967
6
Treatment arm C: 12 mg/kg AK1967
6
Control group: Placebo
6
Total (randomization ratio per dose group 1:1:1:1)
24
For a flow chart of planned study procedures, please refer to the study protocol.
3.1
Sample size determination / power analysis
As the primary endpoint is safety and no comparative statistical analyses will be performed on this 
endpoint, a sample size calculation is not warranted. A generally accepted group size (for dose 
escalating phase 1 studies) of 6 subjects per dosing group will be included (in addition to the 2 
placebo-treated subjects per dosing group, also resulting in a total of 6 in the placebo-group). A 
sample size of 6 subjects per treatment group is regarded as being large enough to justify 
continuation of the trial with the next dosing group in the absence of relevant adverse events related 
to the study medication. Likewise, one or more severe or serious adverse events considered to be 
related to the study medication could be enough to discontinue the study without a statistical 
difference in (S)AEs between the groups.
3.2
Hypotheses
The null hypothesis is that there is no difference between the AK1967 group (all doses combined) 
and the Placebo group regarding the primary safety endpoint.
4
Population
The main inclusion criteria are defined as:


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-
Written informed consent to participate in this trial prior to any study-mandated procedure.
-
Male subjects aged 18 to 35 years inclusive.
-
Subjects must agree to use a reliable way of contraception with their partners from study 
entry until one month after study drug administration. 
-
BMI between 18 and 30 kg/m², with a lower limit of body weight of 50 kg and an upper limit 
of 100 kg. 
-
Considered healthy as determined by medical history, physical examination, vital signs, 12-
lead electrocardiogram, and conventional laboratory parameters.
Exclusion criteria are described in the protocol. 
5
Endpoints for the primary, secondary objectives and exploratory 
endpoints
5.1
Endpoints for the primary objectives
Safety analysis:
The endpoints for the primary objective are to determine over the 28 days study period:
-
Reported number of adverse events from baseline (just prior to the start of IMP 
administration) up until the last follow-up visit 28 days after IMP administration.
-
(Changes in) vital signs from baseline to during the first 24 hours after start of IMP 
administration, as well as during the six follow-up visits. Vital signs included are: heart rate, 
blood pressure, peripheral oxygen saturation and body temperature.
-
Local tolerability at site of i.v. infusion of the IMP.
-
Safety laboratory parameters from baseline (just prior to the start of IMP administration) up 
until the last follow-up visit 28 days after IMP administration. Laboratory parameters 
included are: Hb, Htc, leukocytes, leukocyte differential blood count, thrombocytes, sodium, 
potassium, creatinine, urea, alkaline phosphatase, ALT, AST, bilirubin, GGT, CK, CRP, PT, 
APTT, fibrinogen.
-
12-lead electrocardiogram (ECG) at baseline (screening), compared to ECGs performed 2 
hours, 9 hours and 7 days after start of IMP administration.
5.2
Endpoints for the secondary objectives
Secondary analysis:
-
Pharmacokinetics of AK1967 (including AUC, Cmax, Terminal T1/2, Cl, V) from baseline (just 
prior to the start of IMP administration) up until the last follow-up visit 28 days after IMP 
administration.
-
Pharmacodynamics (enzyme activity and concentration of DPP3) from baseline (just prior to 
start of IMP administration) up until the last follow-up visit 28 days after IMP administration.


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 Record Title: Clinical Statistical Analysis Plan CT-P1-001
  
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-
Pharmacodynamics related to the renin-angiotensin system (blood plasma levels of renin-
angiotensin system responses) at baseline (just prior to start of IMP administration) and at 2 
and 10 hours after start of IMP administration.
-
Pharmacodynamics related to catecholamines (blood plasma levels of adrenergic 
metabolites) at baseline (just prior to start of IMP administration) and at 2 and 10 hours after 
start of IMP administration.
Plasma levels of inflammatory mediators (including, but not limited to TNF, IL-6, IL-8, IL-10), 
measured from baseline (just prior to start of IMP administration) up until 24 hours after start of IMP 
administration.
Endpoints for Pharmacokinetics:
Key PK parameters, including:
-
peak plasma concentrations (Cmax)
-
time to Cmax (tmax)
-
systemic exposure (AUC)
-
volume of distribution (V)
-
systemic clearance (Cl)
-
terminal half-life (t1/2)
5.3
Endpoints for the exploratory objectives
Not applicable
6
General conventions
6.1
Treatment groups
The following treatment groups will be considered for analysis where applicable and meaningful:
-
AK1967 3 mg/kg
-
AK1967 6 mg/kg
-
AK1967 12 mg/kg
-
Placebo
6.2
Layout of tables
For numbering of tables, listings and figures, please refer to section 9. Nevertheless, in order to 
facilitate programming, this numbering might be slightly changed in the final outputs.


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Table 3 Layout of tables
Issue
Specification
Basic layout of end-of-text tables 
and listings (header, footnote)
Titles for all tables, figures, listings:
•
Study: CT-P01-001 / EU CT-2023-507035-37-00
Text (Title, footnotes, organizational 
variables 
and 
column 
headers, 
contents of table)
Case-sensitive, the first letter should be capitalized
Subject identification numbers
SUBJID (as recorded in eCRF)
Categorical Variables
Display of all possible categories (even if a category is 
not present in the data). 
In order to increase readability, large tables may also be 
presented with only the categories observed (e.g. 
frequency tables for adverse events by primary system 
organ class, preferred term and severity). 
In case a category does not occur
Display absolute frequency as 0. 
Display of units
Presentation case-sensitive and in square brackets, e.g. 
Concentration [mg/mL].
6.3
General calculation rules
Data will be listed with the observed number of decimal places. Summary statistics will be presented 
to the same number of decimal places as the observed data, apart from the means and medians (to 
one more decimal place), and standard deviations (two more decimal places). Calculated data and 
summary statistics of calculated data will be presented with an appropriate number of decimal 
places.
In case of replicate measurements (e. g. three determinations of a parameter at one planned time-
point), means of these measurements will be calculated and will be used for summary statistics 
without any prior rounding. 


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Data from unscheduled assessments will only be listed and not presented in summary statistics. 
Relative study times and dates will be calculated with respect to the start time and date of the 
administration of study drug.
Calculation of relative days from onset of adverse events:
The relative day for onset of an adverse event is calculated as day of onset minus day of 
start of treatment.
Duration of adverse events:
The duration of an adverse event (expressed in days) is calculated as stop date minus start 
date plus 1.
If parts of the start/stop date of an adverse event are missing (day, month, or year), the relative day 
for onset and/or the duration of the adverse event are not calculated. 
In order to determine if an adverse event or the worsening of an adverse event is a treatment-
emergent event, the following rules will be used as a worst-case scenario:
-
If the date (and time) of onset or change is completely known, the adverse event is 
considered as treatment-emergent, provided the day (and time) of onset or change is after 
or on the same day (and time) as first study treatment.
-
If the time of onset or change is missing, but the day, month and year is known, the adverse 
event is considered as treatment-emergent, provided the day of onset or change is after or 
on the same day as first study treatment, unless other information (e.g. stop date/time) 
suggest otherwise.
Calculation of absolute changes from baseline:
-
post-dose value - baseline value
Calculation of relative changes from baseline:
-
(post-dose value - baseline value)/baseline value
Frequencies / percentages for qualitative data:
-
Number of observations (frequency)
-
Percent
Percentages will be calculated as frequencies divided by the number of subjects in the 
population the analysis is based on (i.e. this may include missing data in the denominator).


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6.4
Descriptive statistics
Basic descriptive statistics for continuous data comprise:
-
Number of observations (N)
-
Arithmetic mean
-
Standard deviation (SD)
-
Minimum (Min)
-
Lower quartile (Q1)
-
Median
-
Upper quartile (Q3)
-
Maximum (Max)
Descriptive statistics for continuous variables assumed as log-normally distributed (e. g. PK 
parameters):
-
Number of observations (N)
-
Number of observations ≥ LLOQ (NLOQ)
-
Arithmetic mean
-
Arithmetic SD
-
Arithmetic coefficient of variation (CV)
-
Geometric mean
-
Geometric SD
-
Geometric CV (%) (gCV)
-
Q1
-
Median
-
Q3
-
Min
-
Max
7
Analysis sets and data review criteria
7.1
Data sets for analysis
A general definition of data sets for analysis will be:
Full analysis set
The full analysis set includes all randomized subjects who 
-
have received study medication 
-
have follow up data for the study primary and secondary outcomes 


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For the analysis per dose, subject that did not receive the dose per protocol should be grouped to the 
dosing group closest to the administered dose or analysed and reported separately.
Pharmacokinetic analysis set (PKS)
The pharmacokinetic analysis set includes all subjects who:
-
received the study medication
-
showed sufficient compliance concerning medication, i.e. received the full dose of study 
medication
-
have sufficient concentration PK data to calculate reliable estimates of at least one PK 
parameter
-
are without any protocol violation that would interfere with the interpretation of the PK 
data.
7.2
Handling of missing data, drop-outs, extreme values and protocol violations
For an overview of checks for identification of protocol violations and data review, please refer to the 
data review plan.
8
Data analysis
8.1
Statistical software
Statistical analysis will be performed using R statistical software, version 4.3.2 or higher 
(https://www.r-project.org/) on a Windows 7 (or higher) personal computer. The calculation of 
pharmacokinetic parameters will be performed with WinNonlin, version 8.4, Pharsight Corporation, 
Mountain View, CA. 
For each script/test performed using R, the Rmarkdown function will be used to document the 
output of the computerized system for future reference.
8.2
Definitions, derived variables and handling of missing data
Definitions of time points / time windows
Baseline will be defined per parameter and will be the value/measurement at the following 
timepoints:
Parameter for
Timepoint
Demographics
Screening
Vital signs
Baseline (before IMP administration)
ECG
Baseline (Screening)
Local laboratory assessment
Baseline (before IMP administration)
Arterial blood gas analysis
Baseline (before IMP administration)


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Parameter for
Timepoint
Biomarker (DPP3 concentration and 
activity)
Baseline (before IMP administration)
Derived variables
Extent of exposure:
Extent of exposure will be defined as total exposure in mg and calculated as:
𝑡𝑜𝑡𝑎𝑙 𝑒𝑥𝑝𝑜𝑠𝑢𝑟𝑒 [𝑚𝑔] = 20 𝑚𝑔
𝑚𝑙∗𝐼𝑀𝑃 𝑣𝑜𝑙𝑢𝑚𝑒 [𝑚𝑙] ∗𝑓𝑎𝑐𝑡𝑜𝑟
where IMP volume is the body weight adjusted IMP volume before addition of NaCl and factor equals 
1 for treatment group 12 mg/kg AK1967, 1/2 for treatment group 6 mg/kg AK1967, and 1/4 for 
treatment group 3 mg/kg AK1967. Total exposure will not be calculated for the Placebo treatment 
group.
In case of premature termination of the infusion, total exposure will additionally be multiplied by 
𝑣𝑜𝑙𝑢𝑚𝑒 𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 [𝑚𝑙]
50 𝑚𝑙
 in order to account for only partly administration of study drug.
Exposure per kg body weight will be calculated as:
𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒 𝑝𝑒𝑟 𝑘𝑔 𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 
𝑚𝑔
𝑘𝑔=  𝑡𝑜𝑡𝑎𝑙 𝑒𝑥𝑝𝑜𝑠𝑢𝑟𝑒 [𝑚𝑔]
𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 [𝑘𝑔]
Body mass index (BMI):
𝐵𝑀𝐼= 𝑊𝑒𝑖𝑔ℎ𝑡 [𝑘𝑔]
𝐻𝑒𝑖𝑔ℎ𝑡 [𝑚]²
Handling of missing values
Data from subjects who prematurely terminate the trial will be used to the maximum possible 
extent. Missing values will not be inputted.
8.3
Disposition
An overview of all subjects who entered the study, who were randomized and who completed the 
study will be provided by treatment group and overall. 
8.4
Demographic data, screening and baseline characteristics
Demographic data and baseline characteristics will be summarized for the safety analysis set. 
Screening data (prior to study participation) will be shown as listings.


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Demographic data and baseline characteristics
Summary statistics will be presented for age, weight, height, BMI, vital signs at screening and 
baseline (body temperature, heart rate, MAP, systolic and diastolic blood pressure, respiratory rate), 
Hb, Hct, leukocytes, leukocyte differential blood count, thrombocytes, sodium, potassium, creatinine, 
urea, alkaline phosphatase, ALT, AST, bilirubin, GGT, CK, CRP, PT, APTT, fibrinogen. Frequency tables 
for qualitative data (gender, age) will be provided. In case a value of one of these parameters is an 
outlier in a subject, this will be described separately. 
In order to check for a potential disbalance of baseline characteristics of the three treatment groups, 
the Kruskal-Wallis test will be performed for continuous baseline characteristics.
Categorical variables will be compared using the Chi² test for contingency tables.
8.5
Analysis of primary endpoints
The analyses of the endpoints for safety and tolerability will be performed on the full analysis set.
Reported number of Adverse Events and local tolerability
Adverse events (including assessment of local tolerability at the infusion site, which is reported in an 
AE format as well) will be recorded from the moment of study drug infusion, throughout the study 
and the follow-up visits. The same accounts for serious adverse events. (S)AEs will be categorized 
into different organ-systems based on reported symptoms. (S)AEs represent a categorical variable, 
meaning they are summarized as counts and percentages for different dosage groups. Consequently, 
no formal data distribution assessment can be performed for this datatype.
An overview of all adverse events (original term) will be generated as subject data listings, including 
onset relative to start of treatment, duration, intensity, seriousness and potential relationship to the 
study drug, action taken and outcome. The incidence of treatment-emergent adverse events as well 
as number of subjects with treatment-emergent adverse events will be summarized by potential 
relationship to the study drug (certain, probable/likely, possible, unlikely, unrelated) for all treatment 
groups. 
Vital signs
All determined vital signs (oxygenation, heart rate, systolic, diastolic -and mean arterial blood 
pressure) are registered continuously during the experimental protocol at a measurement frequency 
once per 5 seconds. Artefacts may be removed. This high-frequency data will first be averaged into 
10-minute intervals (calculating simple moving averages), as a correction measure for registration of 
spurious measurements. The exception for this approach is temperature, for which no high-
frequency measurements are performed, meaning no data transformation is required prior to 
statistical analysis. As vital signs measured during each of the follow-up visits also represent single 


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measurements, no data transformation of these measurements is required prior to statistical 
analysis. Minimum and maximum values for vital signs assessed within one day will be analyzed using 
descriptive summary statistics (including changes from baseline) by treatment group and time point 
(Day 1, Day 2, Day 3 etc.). 
Data distribution for all vital signs (both averages of high-frequency data and single measurements) 
will be evaluated using the Kolmogorov-Smirnov test and, if applicable, normal/non-normal 
distribution will be reported. All variables will be expressed both as mean ± standard deviation (SD), 
as well as medians and interquartile ranges (IQR) in tables and graphical representations of the data. 
In case a value of one of these parameters is an outlier in a subject, this will be described separately.
Safety laboratory parameters & inflammatory parameters
All safety laboratory parameters (see the study protocol for a full overview) are measured at set 
intervals during the experimental protocol, as well as during each of the studys follow-up visits. All 
safety laboratory measurements represent single timepoint measurements, meaning no data 
transformation prior to analysis needs to be performed. Complete listings for laboratory values will 
be generated. Laboratory values outside the normal range will be marked. Minimum and maximum 
values will be reported (including changes from baseline) by treatment group and time point (Day 1, 
Day 2, Day 3 etc.). In case a value of one of these parameters is an outlier in a subject, this will be 
described separately.
Data distribution for all safety laboratory parameters will be evaluated using the Kolmogorov-
Smirnov test and normal/non-normal distribution will be reported. All variables will be expressed 
both as mean ± standard deviation (SD), as well as medians and interquartile ranges (IQR) in tables 
and graphical representations of the data. In case a value of one of these parameters is an outlier in a 
subject, this will be described separately.
Results from laboratory tests will be summarized by treatment group and time point using 
descriptive summary statistics, including changes from baseline. Relative changes (for laboratory 
values) from baseline to follow-up visits (post-study drug administration minus baseline) will be 
categorized as:
-
Maximum increase from baseline: (maximum value post-study drug administration  
baseline) / baseline 
≥ 1.5, ≥ 2.0
-
Maximum decrease from baseline: (baseline - minimum value post-study drug 
administration) / baseline 
≤ 0.5


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A frequency table by treatment dose will be generated for these categorized changes.
In addition, changes from from within normal range to above normal range will be evaluated and 
categorized as:
-
changes to at least 1.5-time upper limit normal, changes to at least twice upper limit 
normal.
A frequency table by treatment will be generated for these categorized changes from normal at 
baseline.
In case a value of one of these parameters is an outlier in a subject, this will be described separately.
12-lead ECG parameters
Non-quantifiable differences in ECGs deemed to be of clinical relevance by a clinical investigator will 
be registered as an (S)AE. For differences in ECG-related (S)AEs, the same ground-rules apply as those 
listed under reported number of adverse events and local tolerability (please refer to this section). 
Frequencies of overall interpretation (normal, abnormal and clinically not significant, abnormal and 
clinically significant) will be tabulated by treatment group and time point.
For numerically quantifiable differences in ECG-parameters (e.g. QTc-time, see the study protocol for 
a full overview), data distribution will be assessed using the Kolmogorov-Smirnov test and 
normal/non-normal distribution will be reported. Individual measurement timepoints will be 
expressed as both mean ± standard deviation (SD), as well as medians and interquartile ranges (IQR). 
Extent of exposure
Total exposure [mg] and relative exposure [mg/kg] will be analyzed by descriptive summary statistics.
8.6
Analysis of secondary endpoints
Pharmacokinetic data analyses
The PK parameters will be analyzed by descriptive summary statistics. AK1967 concentration-time 
profiles for individual subjects will be analyzed with non-compartmental approaches using a 
validated commercially available software package (WinNonlin, version 8.4, Pharsight Corporation, 
Mountain View, CA). Nominal times will be used in the analysis. For data reported as BLQ and ALQ 
(below and above limit of quantification, respectively), the low and high limit of detection value will 
be used in the analysis. 
Pharmacokinetic (PK) parameters will be calculated including area under the concentrationtime 
curve (AUC), maximum concentration (Cmax), time to Cmax (Tmax), and terminal half-life (t ½). 


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The maximum drug concentration (Cmax) and time to maximum drug concentration (Tmax) will be 
determined from observed values. The area under the curve (AUC0-t) will be calculated for PCZ 
plasma concentration-time profiles using the trapezoidal method derived from non-compartmental 
analysis from time zero (i.e. start of drug infusion) to the last detectable concentration. 
Data distribution will be assessed using the Kolmogorov-Smirnov test and normal/non-normal 
distribution will be reported. Data will be displayed both tabular and graphically as means ± SD, as 
well as medians ± IQR. In case a value of one of these parameters is an outlier in a subject, this will be 
described separately.
Dose proportionality of Cmax and AUC0∞ of the different AK1967 dose groups will be calculated by 
dividing the specific PK parameter by the corresponding value in the lower dose groups and 
comparing with the corresponding fold change in dose. This is to assess which dosage groups should 
be reported as being non-dose proportional.
Pharmacokinetic parameters
The following parameters will be determined from the individual plasma concentration-time profiles 
of AK1967 using non-compartmental methods based on actual relative sampling times:
Single concentration data will be shown with the relevant number of decimal places as provided by 
the analytical lab. Derived parameters, as far as they do not present single concentrations such as 
Cmax, will be listed with an appropriate number of relevant digits (at least 3).
Exposure parameters (Cmax and AUC0-last) will also be dose-normalized (i.e. divided by dose) for the 
determination of dose-dependency.
Descriptive pharmacokinetic analyses
The PK analysis will be performed for all active treatment groups.
The individual time courses of the plasma concentrations of AK1967 will be listed and summarized 
descriptively by treatment group and time point. 
Summary statistics by time point will be calculated including only those samples which were taken 
according to the pre-defined sampling schedule, i.e.: Day 1 (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24 
hour ± 2 mins), Day 2 (± 1 hour), Day 3 (± 3 hours), Day 7 (± 1 day), Day 14 (± 2 days), Day 21 ((± 3 
days), and Day 28 (± 3 days). 
For the calculation of summary statistics, when data is reported as BLQ and ALQ (below and above 
limit of quantification, respectively), the low and high limit of detection value will be used in the 
analysis to obtain the means, standard of deviations, coefficient of variation, medians and 
interquartile ranges.


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Individual plasma concentration-time profiles will be plotted for AK1967 (up to 6 profiles in one plot) 
for each dose group considering all subjects with available PK concentration data using actual relative 
sampling times. Mean (SD), as well as median (IQR) of AK1967 plasma concentrations will be plotted 
for all active treatment groups. Individual and geometric mean concentration versus time curves will 
be plotted using both linear and semi-logarithmic scaling. 
Summary statistics will be calculated for each pharmacokinetic parameter by dose group based on 
PKs.
Pharmacodynamic data analyses (effects of AK1967 on DPP3 enzyme activity, concentration, renin-
angiotensin and adrenergic system metabolites)
-
To determine the effects of AK1967 on DPP3 activity and concentration in healthy male 
volunteers.
-
To determine the effects of AK1967 on renin-angiotensin system metabolites in healthy male 
volunteers.
-
To determine the effects of AK1967 on circulating adrenergic metabolite concentrations in 
healthy male volunteers.
-
To determine the effects of AK1967 on inflammatory cytokines, including, but not limited to TNF, 
IL-6, IL-8, IL-10.
Different biomarkers/measurements associated with AK1967s pharmacodynamic activity (see the 
study protocol for a full overview) are measured at set intervals during the experimental protocol. 
DPP3 enzyme activity and concentrations measurements are also performed during each of the 
studys follow-up visits. All pharmacodynamic biomarker measurements represent single timepoint 
measurements, meaning no data transformation prior to analysis needs to be performed. DPP3 
enzyme activity, concentration, adrenergic and renin-angiotensin system metabolite results will be 
analysed by descriptive summary statistics for the different scheduled determination time points as 
well as expressed as changes from baseline. 
Data distribution for all pharmacodynamic biomarkers will be evaluated using the Kolmogorov-
Smirnov test and normal/non-normal distribution will be reported. All variables will be expressed 
both as mean ± standard deviation (SD), as well as medians and interquartile ranges (IQR) in tables 
and graphical representations of the data. In case a value of one of these parameters is an outlier in a 
subject, this will be described separately. Between group differences in pharmacodynamic 
biomarkers will be assessed using one-way-RM-ANOVA tests (for normally distributed data), or 
Kruskal-Wallis tests (for non-normally distributed data). In case of a p-value of <0.05 for these tests 
(implying the presence of dosage groups with differences in a vital sign at a specific timepoint), 
subsequent Bonferroni or Mann-Whitney-U post-hoc tests will be performed (depending on data 
distribution), to assess which subgroups are different, and in what direction.


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8.7
Additional analyses
Pharmacodynamic data analyses (effects of AK1967 on DPP3 enzyme activity, concentration and 
renin-angiotensin metabolites)
-
To determine the effects of AK1967 on DPP3 concentration (corrected and non-corrected) in 
healthy male volunteers.
-
To determine the correlation between DPP3 concentration (corrected and non-corrected) and 
DPP3 activity.
-
To determine the effects of AK1967 on renin-angiotensin system response measured in non-
stabilized (equilibrium) plasma samples at day 7.
-
To determine the effects of AK1967 on renin-angiotensin system metabolites measured in 
stabilized (protease inhibitor) and non-stabilized (equilibrium) plasma samples over all 
timepoints.
-
To determine the correlation between renin-angiotensin system modulation measured in 
stabilized (protease inhibitor) and non-stabilized (equilibrium) plasma samples over all 
timepoints.
-
To determine the correlation between DPP3 activity and renin-angiotensin modulation measured 
in stabilized (protease inhibitor) and non-stabilized (equilibrium) plasma samples.
-
To determine the correlation between DPP3 concentration (corrected and non-corrected) and 
renin-angiotensin modulation measured in stabilized (protease inhibitor) and non-stabilized 
(equilibrium) plasma samples.
All pharmacodynamic biomarker measurements represent single timepoint measurements, meaning 
no data transformation prior to analysis needs to be performed. DPP3 enzyme activity, concentration 
(corrected and non-corrected values) and renin-angiotensin system metabolite (in stabilized and 
non-stabilized plasma) results will be analysed by descriptive summary statistics for the different 
scheduled determination time points, as well as expressed as changes from baseline. Correlations 
between pharmacodynamic parameters will be reported. 
Data distribution for all pharmacodynamic biomarkers will be evaluated using the Kolmogorov-
Smirnov test and normal/non-normal distribution will be reported. All variables will be expressed 
both as mean ± standard deviation (SD), as well as medians and interquartile ranges (IQR) in tables 
and graphical representations of the data. In case a value of one of these parameters is an outlier in a 
subject, this will be described separately. Between group differences in pharmacodynamic 
biomarkers will be assessed using one-way-RM-ANOVA tests (for normally distributed data), or 
Kruskal-Wallis tests (for non-normally distributed data). In case of a p-value of <0.05 for these tests 
(implying the presence of dosage groups with differences in a vital sign at a specific timepoint), 
subsequent Bonferroni or Mann-Whitney-U post-hoc tests will be performed (depending on data 
distribution), to assess which subgroups are different, and in what direction.


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Additional analyses related to the effects of AK1967 on safety endpoints
Between group differences in vital signs will be assessed using one-way-RM-ANOVA tests (for 
normally distributed data), or Kruskal-Wallis tests (for non-normally distributed data). In case of a p-
value of <0.05 for these tests (implying the presence of dosage groups with differences in a vital sign 
at a specific timepoint, subsequent Bonferroni or Mann-Whitney-U post-hoc tests will be performed 
(depending on data distribution), to assess which subgroups are different, and in what direction.
Between group differences over time for vital signs, safety laboratory and inflammatory parameters 
(i.e. laboratory/inflammatory parameter changes during the course of the experimental protocol) will 
be analyzed using two-way-RM-ANOVA tests (for normally distributed data), or two-way-RM-ANOVA 
tests on ranked transformed data (for non-normally distributed data). Both tests will report the 
time*group interaction term to establish significance, which will be defined as p <0.05.
9
Contents of statistical tables and figures 
14 Tables and figures referred to but not included in the text
14.1 Demographic data and general study information
Table
Table summarizing the number of subjects screened, randomized, and included in each 
analysis set 
Table
Demographic data (descriptive summary statistics)
Table
Structural comparison of baseline characteristics (continuous variables)
Table
Structural comparison of baseline characteristics (categorical variables)
14.2 Analysis of primary and secondary endpoints 
14.2.1 Analysis of primary endpoints
14.2.1.1 Vital signs
Table
Descriptive summary table of vital signs at each time point
Table
Change from baseline in vital signs at each time point per dosing group
Listing
Listing of individual subject vital signs at each time point.
Figure
Line graphs showing the mean and standard deviation of vital signs over time per dosing 
group.
Figure
Box plots of vital signs at each time point per dosing group.
14.2.1.2 Safety laboratory & Inflammatory parameters
Table
Descriptive summary statistics for laboratory parameters at each time point
Table
Change from baseline in laboratory parameters at each time point per dosing group 
Listing
Listing of individual subject laboratory parameters at each time point
Table
Descriptive summary statistics for inflammatory parameters
Listing
Listing of inflammatory parameters
14.2.1.3 ECG


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Table
Summary tables of ECG parameters
Change from baseline in ECG parameters at each time points per dosing group
Listing
Listing of individual subject ECG results at each time point
Figure
Line graphs showing the mean and standard deviation of ECG parameters over time
14.2.1.4 Extent of exposure
Table
Median dose exposure per dosing group
14.2.1.5 Adverse events
Table
Table of the number and percentage of subjects experiencing adverse events, categorized 
by severity, potential relationship to the investigational medicinal product (IMP) and 
dosing group
Table
Summary table of adverse events by system organ class and preferred term (including 
changes in vital signs and any laboratory changes)
Table
Table summarizing local tolerability at the site of i.v. infusion, including any reported 
reactions or issues
Listing
Listing of all adverse events by subject, including severity, potential relationship to IMP, 
and outcome
Figure
Bar charts or pie charts showing the distribution of adverse events by severity and 
potential relationship to IMP
14.2.2 Analysis of secondary endpoints
14.2.2.1 Pharmacokinetic data analysis
Table
Descriptive summary statistics for PK parameters
Table
Individual subject PK parameters values
Listing
Individual time courses of AK1967
Figure
Individual time courses for 3 mg/kg AK1967 (linear scale)
Figure
Individual time courses for 6 mg/kg AK1967 (linear scale)
Figure
Individual time courses for 12 mg/kg AK1967 (linear scale)
Figure
Individual time courses for 3 mg/kg AK1967 (semi-log. scale)
Figure
Individual time courses for 6 mg/kg AK1967 (semi-log. scale)
Figure
Individual time courses for 12 mg/kg AK1967 (semi-log. scale)
Figure
Geometric means of AK1967 (linear scale)
Figure
Geometric means of AK1967 (semi-log. scale)
Listing
Listing of individual subject PK data, including concentration-time data
14.2.2.2 Pharmacodynamic data analysis
Table
Summary table of enzyme activity of DPP3 at each time point
Table
Change from baseline in DPP3 activity at each time point per dosing group
Table
Summary table of enzyme concentration (corrected and non-corrected) of DPP3 at each 
time point
Table
Change from baseline in DPP3 concentration (corrected and non-corrected) at each time 
point per dosing group
Table
Summary table of blood plasma levels of renin-angiotensin system metabolites in non-
stabilized plasma


Form printed on 
Form Valid From:
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Form
 FB_00079_Statistical Analysis Plan
Form Version:
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Record Title:
 Record Title: Clinical Statistical Analysis Plan CT-P1-001
  
Filing: https://fourteenfour.dhcvisioncloud.com/dhcvision/document.jsp?key=DATABASE:1001;IO:1527897
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be reproduced or shared without written permission of 4TEEN4 Pharmaceuticals GmbH.
Table
Change from baseline in renin-angiotensin metabolites in non-stabilized plasma at each 
time point per dosing group
Table
Summary table of blood plasma levels of renin-angiotensin system metabolites in 
stabilized plasma
Table
Change from baseline in renin-angiotensin metabolites in stabilized plasma at each time 
point per dosing group
Table
Summary table of blood plasma levels of adrenergic metabolite results
Table
Change from baseline in adrenergic metabolites at each time point per dosing group
Table
Summary table of plasma levels of inflammatory mediators at each time point
Table
Change from baseline in inflammatory mediators at each time point per dosing group 
Listing
Listing of individual subject PD data for enzyme-activity, concentration (corrected and 
non-corrected) of DPP3, renin-angiotensin system metabolites (in stabilized and non-
stabilized), adrenergic metabolites, and inflammatory mediators
Figure
Line graphs showing the mean, standard deviation, median and IQR of enzyme-activity of 
DPP3 over time per dosing group
Figure
Line graphs showing DPP3 concentration (corrected and non-corrected) over time per 
dosing group
Figure
Line graphs showing the mean and standard deviation of blood plasma levels of renin-
angiotensin system metabolites in non-stabilized plasma over time per dosing group
Figure
Line graphs showing the mean and standard deviation of blood plasma levels of renin-
angiotensin system metabolites in stabilized plasma over time per dosing group
Figure
Line graphs showing the mean and standard deviation of blood plasma levels of 
adrenergic metabolites over time per dosing group
Figure
Line graphs showing the mean and standard deviation of blood plasma levels of 
inflammatory mediators over time per dosing group
14.2.3 Additional analyses
Figure
PK/PD Line graphs showing the mean, standard deviation, median and IQR of enzyme-
activity of DPP3 and AK1967 PK values over time per dosing group
Figure
PK/PD Line graphs showing DPP3 concentration (corrected and non-corrected) and 
AK1967 PK values over time per dosing group
Figure
Line graphs showing DPP3 activity-concentration (corrected and non-corrected) 
correlation over time per dosing group
Figure
PK/PD Line graphs showing the mean and standard deviation of blood plasma levels of 
renin-angiotensin system metabolites in non-stabilized plasma and AK1967 PK values 
over time per dosing group
Figure
PK/PD Line graphs showing the mean and standard deviation of blood plasma levels of 
renin-angiotensin system metabolites in stabilized plasma and AK1967 PK values over 
time per dosing group
Figure
PK/PD Line graphs showing the mean and standard deviation of blood plasma levels of 
adrenergic metabolites and PK values over time per dosing group
Figure
PK/PD Line graphs showing the mean and standard deviation of blood plasma levels of 
inflammatory mediators and AK1967 PK values over time per dosing group


Form printed on 
Form Valid From:
08/03/2024
Form
 FB_00079_Statistical Analysis Plan
Form Version:
1
Record Title:
 Record Title: Clinical Statistical Analysis Plan CT-P1-001
  
Filing: https://fourteenfour.dhcvisioncloud.com/dhcvision/document.jsp?key=DATABASE:1001;IO:1527897
Page 22 of 22
Confidentiality Notice: This document and the contained information are confidential and may not 
be reproduced or shared without written permission of 4TEEN4 Pharmaceuticals GmbH.