Comparison of the Pharmacokinetics (PK) and Pharmacodynamics (PD) Biosimilarity of Proposed Biosimilar Rapid-Acting Insulin Aspart (I004) and NovoLog After Single-Dose Subcutaneous Administration to Healthy Volunteers: A Single-Center Randomized, Double-blinded, Two-Treatment, Two-period, Two-sequence Crossover, Hyperinsulinemia-Euglycemic Clamp
Phase: PHASE2
Conditions: Pharmacokinetics, Pharmacodynamics
Sponsor: Amphastar Pharmaceuticals, Inc.

Study Protocol 
Protocol No.: API-I004-CL-B 
Title: Comparison of the Pharmacokinetics (PK) and Pharmacodynamics (PD) Biosimilarity of Proposed 
Biosimilar Rapid-Acting Insulin Aspart (I004) and NovoLog® after Single-Dose Subcutaneous 
Administration to Healthy Volunteers 
Version: 1.1 
Date: 05 August 2021 
NCT05539872 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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SYNOPSIS 
General information 
(1) Protocol No.         API-I004-CL-B  
(2) Title: 
 
Comparison of the Pharmacokinetics (PK) and Pharmacodynamics (PD) 
Biosimilarity of Proposed Biosimilar Rapid-Acting Insulin Aspart (I004) 
and NovoLog® after Single-Dose Subcutaneous Administration to 
Healthy Volunteers  
(3) Subtitle:  
A Single-Center Randomized, Double-blinded, Two-treatment, Two-
period, Two-sequence Crossover, Hyperinsulinemia-Euglycemic Clamp 
Study   
(4)Proposed Drug: Biosimilar Insulin Aspart Solution (I004, to-be-marketed formulation) 
100U/mL (
) 
(5) Comparator:  
NovoLog®, 100U/mL (
) 
(6) Delivery Path:      Subcutaneous (SC) injection             
(7) Sponsor: 
 
Amphastar Pharmaceuticals, Inc. 
(8) Objectives: 
To determine PK/PD biosimilarity between the I004 and NovoLog® 
through assessment of in vivo PK and PD in healthy adult volunteers. 
(9) Phase:                Phase II/III (Pivotal) 
 
 
 
I. Study Design 
 
This study is a randomized, double-blinded, two-treatment, two-period, two-sequence crossover 
pivotal Biosimilar study in sixty (60) healthy volunteers (male and female, age between 18 and 
65 years old).  The purpose of this study is to establish the PK and PD biosimilarity of proposed 
biosimilar I004 and the US-approved NovoLog®. The I004 will use the to-be-marketed 
formulation. 
 
The study includes one (1) screening visit, two (2) study visits (separated by at least 7 days) and a 
follow-up visit. In each study visit, a single injection will be administered before undergoing an 
euglycemic clamp that lasts for 12 hours. Adverse events will be documented. Study participation 
is expected to last up to 10 weeks. However, Screening visit window, Treatment visit window, and 
Follow-up visit window could be extended to accommodate state-issued Stay at Home orders due 
to the COVID-19 pandemic. 
 
1) Screening Visit: Subject will sign informed consent and be assigned a screening ID, as SI4-B-
mm, where SI4-B represents “Screening for I004 Study B”, and mm is screening sequential 
number. Each subject will be evaluated per inclusion/exclusion criteria. Measurements of:  
 
(1) 
physical examination (PE); 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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(2) 
vital signs; 
(3) 
ECG;  
(4) 
urinalysis (UA);  
(5) 
urine drug screen; 
(6) 
alcohol (urine or breathalyzer);  
(7) 
pregnancy test (serum for screening and urine test on day 1; females of childbearing 
potential); 
(8) 
fasting blood tests (hematology, clinical chemistry including fasting serum glucose, 
TSH, HIV, Hepatitis B-Antigen and C-Antibody tests)  
 
shall be conducted. Qualified subjects will be scheduled for study visits. 
 
2) Two (2) Study Visits:  
 
After satisfying the enrollment criteria, each subject will be assigned a unique study subject ID as 
I4-B-nn, I4-B represents study for “I004 Study B”, and nn is the sequential number. The subject 
will be randomized into one of the two treatment sequences, T-R (I004 in the 1st visit and 
NovoLog® in the 2nd visit) or R-T (NovoLog® in the 1st visit, and I004 in the 2nd visit), respectively.  
A washout period of at least 7 days will be scheduled between the two study visits. At each study 
visit, the subject is given a single dose SC injection of either I004 or NovoLog® which is prepared 
per the randomization code.   
 
Participants will be asked to be restrained from alcohol or caffeine-containing food or drinks for 
24h and strenuous activity for the 72h period prior to coming in for study visits. Due to COVID-
19 pandemic, subjects will be admitted to the clinical research unit on the day (day 1) of each 
treatment.  A standard dinner will be given and finished before 8pm. The participants will take at 
least 10 hour fast before dosing. 
 
Twenty-eight (28) serial PK blood samples after treatment and during the clamp period (12 hours) 
will be collected for serum insulin aspart, C-peptide, and endogenous human insulin 
measurements. No food will be allowed during the PK/PD sampling period (12 hours).  Water 
intake and infusion will be preferably controlled at the minimal level during the clamp study. 
 
Baseline vital signs and ECG will be performed at each study visit. On clamp days, both vital signs 
and ECG will be measured at baseline which is 15 min (± 5 min) prior to dosing, at 5 (± 3 min) 
minutes, 60 min (± 10 min), 180 min (± 20 min) and 720 min (±30 min) post-dose. 
 
(3) Follow-up Evaluation  
 
All treated subjects will be scheduled for a follow up visit after the study visit-2 (or ET visit). 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Subjects will be asked if they have experienced any adverse event since the last study visit. An 
F/U evaluation will be performed, which includes: reviews of vital signs, ECG, fasting clinical lab 
tests (hematology, clinical chemistry, and Urinalysis), and physical examination (PE). 
 
II. Subject 
 
A sufficient number of healthy volunteers will be enrolled to obtain sixty (n=60) evaluable 
subjects. All subjects will be included in or excluded from the study based on the following 
inclusion/exclusion criteria.  
 
Inclusion Criteria:  
 
Candidates will be qualified only if they meet all of the following criteria: 
 
(1) Upon review, agree to participate and sign informed consent. 
(2) Healthy male and female subjects ≥ 18 to ≤ 65 years of age. 
(3) Body mass index (BMI) ≥ 18.5 to ≤ 29.9 kg/m2. 
(4) Weight ≥ 50 kg. 
(5) Fasting plasma glucose of < 100 mg/dL (5.5 mmol/L) measured with YSI at 
site; one repeat test is allowed. 
(6) HbA1c < 5.7 %. 
(7) Non-smoker for ≥ 3 months prior to Screening. 
(8) 
Female candidates must be >1 year post-menopausal, surgically sterile, or 
practicing a clinically acceptable form of birth control and confirmed by 
negative serum pregnancy test at Screening. 
 
Exclusion Criteria: Subjects will be excluded for any of the following reasons: 
 
(1) History of diabetes mellitus. 
(2) Resting blood pressure (BP) > 140/90 mmHg or < 90/60 mmHg. Subjects BP 
may be re-checked. 
(3) Participation in an investigational drug/device study within 30 days or 5 half-
lives within the last dose of any study drug, whichever is longer. 
(4) History of any serious adverse reaction or hypersensitivity to any of 
the investigational product components. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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(5) Have significant history of or current cardiovascular, respiratory, hepatic, 
renal, gastrointestinal, endocrine, hematological, or neurological disorders or 
abnormalities, or other major systemic disease that, according to the 
investigator, would unduly risk the subject’s safety or may impact the conduct 
of the study. 
(6) Subject shows evidence of significant active neuropsychiatric disease, including 
taking prescription medication for such diseases (including anti-depressant 
/anti-anxiety medication). 
(7) Presence of clinically significant physical, laboratory, or ECG findings at 
Screening that, in the opinion of the Investigator, may interfere with any 
aspect of study conduct or interpretation of results, or may present a safety 
issue to that particular subject (laboratory results may be re-checked once on 
a separate day per Investigator discretion). 
(8) Long QT syndrome or family history of long QT syndrome or corrected 
QT interval (QTcF) > 450 ms in men, > 470 ms in women at Screening. 
(9) Liver function test results of AST and/or ALT ≥ 2.5 upper normal limit (ULN). 
(10) Subject has a history of syncope. 
(11) History of any major surgery within 6 months. 
(12) History of any active infection, other than mild viral illness within 30 days 
prior to dosing. 
(13) History of blood clots (e.g., deep vein thrombosis or embolism) or a 
frequent appearance in 1st degree relatives as judged by the Investigator. 
(14) Known history or positive test of hepatitis B surface antigen (HBsAg), hepatitis 
C antibody (HCV Ab), or human immunodeficiency virus type 1 (HIV-1) or 2 
(HIV-2) antibody. 
(15) History of alcohol abuse as judged by the Investigator within approximately 1 
year. Average weekly alcohol intake > 21 units/week (males) and > 14 
units/week (females) or are unwilling to stop alcohol consumption from 24 
hours prior to each dosing until discharged from the clinical research unit 
(CRU). Positive alcohol test at Screening. (One unit of alcohol equals about 250 
mL of beer or lager, one glass of wine, or 20 mL of spirits). 
(16) History of illicit drug abuse, including marijuana, within approximately 1 year 
or evidence of current use as judged by the Investigator. Positive drug test at 
Screening. 
(17) Donation or loss of > 500 mL of blood within 56 days. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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(18) Chronic use of or over-the-counter or prescription medication within 7 or 14 days 
prior to dosing (apart from vitamin/mineral supplements, occasional 
paracetamol, or birth control methods [Desogestrel is not allowed]). 
(19) Unable to comply with the safety monitoring requirements of this clinical study 
or is considered by the investigator to be an unsuitable candidate for the study. 
 
III.Dosing Regimens  
 
Qualified subjects will participate in two (2) study visits separated by at least 7 days. These 
subjects will be treated at each study visit as described in Table A below by a qualified 
healthcare professional per the randomized sequences. 
 
Table A.  Treatments and Dose for I004-CL-B Study 
 
 
Double-blinded study medications will be dispensed in identical appearing syringes using aseptic 
technique by the designated site personnel. I004 or NovoLog will be drawn with 0.3mL insulin 
syringes that have a 0.01 mL (one unit on insulin syringe) graduation. 
 
A Biostator, a 510(k)-cleared device that provides continuous assay of blood glucose 
measurements and minute-by-minute adaptations of glucose infusion rates (GIR), will be set up 
with infusion lines for blood glucose management and then the insulin aspart will be administered 
while subjects are under observation in the clinical research by qualified staff. The investigational 
product will be administered (at time-point zero) via subcutaneous injection into the abdominal 
wall into the peri-umbilical area using a standardized skin-fold technique. An area of skin 
approximately 5 cm lateral to the umbilicus on either side of the abdomen will be cleaned and used 
for the insertion of the needle. The needle will be promptly inserted at an approximately 45°degree 
angle into a lifted skin fold. The insulin aspart will be administered, and the needle should remain 
in the skin for at least 20 seconds after administration to reduce the possibility of leakage. The 
Treatment
Arm-T
Arm-R
Study Drug Name
I004
NovoLog 
Manufacturer
API and Scource
Insulin Aspart 
Insulin Aspart 
Dosage & Strength
Sterile Solution 100 U/mL
Sterile Solution 100 U/mL
No. of Subjects
Dose Regimen
0.2 IU/kg
0.2 IU/kg
Delivery Path
Subcutaneous injection
Subcutaneous injection
60 healthy volunteers participate in                                 
two (2) crossover treatment sequences, T-R or R-T.

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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needle will be very slowly withdrawn or may be held for a few seconds before it is fully withdrawn 
to minimize leakage of insulin. The injection site will be marked with a pen.  
 
IV. 
PK/PD Blood Sampling  
 
(1) 
PK Sampling  
 
PK Sampling Procedure 
 
A catheter will be inserted into one of the antecubital region veins for blood sampling. 
Alternatively, collection from a vein from the hand or forearm for easier access or via venipuncture 
is also acceptable. PK blood draws should use the opposite arm of the glucose infusion/clamp. 
Preferably, the arm with better venous access will be used for PK samplings, and the opposite arm 
for the glucose infusion/clamp. In case of extraordinary situations, such as IV infiltration etc., the 
PK samples could be collected from the arm of the clamp, for safety reason. 
 
After each blood sample collection, a catheter flush will be performed to avoid catheter clot 
formation and prevent interference between samples. 
 
At each PK sampling point, the first 1 mL of blood will be discarded for waste (utilizing a 
disposable tube), and blood samples (~6 mL per sample), will be collected in a serum separation 
tube and left to stand at room temperature for 30 - 60 minutes. Each sample will be centrifuged at 
2-8°C, 1,000-1,300 g for 20 minutes. 
 
Isolated serum will be distributed into two (2) 2.0 mL cryo vials, approximately 1.5 mL in the first 
aliquot and the remainder in the second aliquot. The serum samples will be frozen on dry ice and 
then immediately (within 60 minutes following serum isolation) stored in a freezer at -20°C or 
lower until analysis. 
 
PK Sampling Schedules and Time Window  
 
PK sampling: a total of twenty-eight (28) blood samples for each study visit will be collected. The 
blood sampling schedule and allowed time window during each study visit are listed in Table B. 
 
Sample ID  
The sample ID for PK samples (before centrifugation) is defined as    
      
                                                            “I4-B-nn-V-XX:” 
 
where, I4-B-nn is the subject ID; V is the study visit code, V=1 or 2; and XX is the two-digit time-

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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point sequential number of the sample: 01, 02, 03, …28 insulin as listed in Table B. 
 
Table B PK Blood Sampling Schedule (6 mL per sample) 
 
 
The sample ID for PK samples (after centrifugation) is defined as   
 
                                                           “I4-B-nn-V-XX-k” 
 
where k is the aliquot number (A or B). The sample ID labels will be printed in blue colored fonts 
with label size for the tube is 1/2”×1¾” (Avery label 5667), as demonstrated below: 
 
                 
 
Sample Transportation and Analysis 
 
Prior to sample transportation, all samples should be stored in a box and must be organized in 
sequential order by subject and separated by aliquot. The chain of custody, temperature control 
and related documents for sample transportation must be recorded. During transportation of PK 
samples from the site to Amphastar, the temperature should be below -20˚C. The transportation 
between the study site and the test laboratory must be completed within 48 hrs. The chain of 
custody, elapsed time for transport, temperature control and related documents for sample transfer 
must be recorded and documented. All serum samples will be stored at -50 ºC or lower in the 
Seq. #
Time 
window
Sample 
No.,"XX"
#
Time 
window
Sample 
No.,"XX"
1
-60 min
01
15
75 min
±3 min
15
2
-30 min
02
16
80 min
16
3
0 min
03
17
90 min
17
4
5 min
04
18
105 min
18
5
10 min
05
19
120 min
19
6
20 min
06
20
150 min
20
7
25 min
07
21
180 min
21
8
30 min
08
22
210 min
22
9
40 min
09
23
4 h
23
10
50 min
10
24
5 h
24
11
55 min
11
25
6 h
25
12
60 min 
12
26
8 h
26
13
65 min 
13
27
10 h
27
14
70 min 
14
28
12 h
28
Total
28 (168mL)
Scheduling
Scheduling
±3 min
±20 min
± 5 min
±1 min
±2 min
±5 min
±10 min
I4-B-nn-V-XX-k 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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laboratory. Serum samples will be analyzed for the concentrations of (i) insulin aspart, (ii) C-
peptide, and (iii) endogenous human insulin with a validated method of ultra-high pressure liquid 
chromatography/high resolution mass spectrum (UPLC/HRMS) by R & D Department of 
Amphastar under GMP, please refer to Section 7. 15 for details.  
 
(2) PD Glucose Clamp  
 
On the study visit, following ≥ 10 hour overnight fast, a 12-hour euglycemic clamp will be 
initiated.  
 
At least 1 hour prior to the clamp, three venous catheters will be inserted into peripheral veins to 
connect subjects to the Biostator, to allow for the sampling of glucose, insulin and or other 
analytes, and to continuously infuse glucose 20%.  
 
The determination of blood glucose concentrations will start at least 60 minutes prior to dosing. 
The mean of three blood glucose (BG) readings from -30 to -10 minutes (i.e., -30, -20 and -10 
minutes) will be used to determine the individual subject’s fasting blood glucose level for each 
dosing visit. The individual blood glucose clamp target level will be determined for each subject 
by the mean fasting glucose level minus 5 mg/dL. 
 
If differences between initial fasting blood glucose values (-30, -20, -10 min) exceed the limit of 
10 mg/dL, subjects will need to be rescheduled. Additionally, subjects will not be dosed if mean 
fasting plasma glucose is < 75 mg/dL due to proximity to overt hypoglycemia or > 99 mg/dL. 
Subjects may be re-scheduled once to come back to the site on a later day and may be dosed if the 
mean fasting plasma glucose is ≥ 75 mg/dL or ≤ 99 mg/dL prior to dosing. 
 
The study drug will be administered at approximately 08:00 (t=0) by qualified staff via SC 
injection as described in section 6.4.  
 
Following the study drug administration, the clamp procedure will start. Onset of insulin action 
will be identified by a ≥ 5mg/dL decline of BASELINE level and start of the glucose infusion rate 
(GIR). After the onset of insulin action is identified, an infusion of glucose 20% will then be 
utilized as needed to maintain target level of blood glucose concentration. 
 
The GIR will be based on the rates suggested by a published clamp algorithm. The clamp operator 
can adapt the glucose infusion rate manually as deemed appropriate. The GIR required to keep 
blood glucose at target level (±10%) will be recorded every one minute over 12 hours and data 
will be used to calculate PD parameters.  
 
The subjects will remain fasting in a supine or semi-supine position during the entire glucose 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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clamp. The subjects will be allowed to sip water throughout the clamp but will otherwise remain 
fasting. 
 
V. Study Site and Investigators 
 
The study will be performed at  
 
 
Chula Vista, CA, 91911 
USA 
 
The detailed information about the study site can be found on FDA Form 1572, including: Name, 
address, and statement of qualifications of each principal investigator; name of each sub-
investigator working under the supervision of the investigator; name and address of the research 
facilities to be used; name and address of each reviewing IRB per US 21 CFR 312.23 section 
6(iii)b. 
 
Principal Investigators (PI) responsible for the conduct of this study will be board certified 
physicians who are familiar with the study medications and trained to handle any untoward events 
related to medications.  
The PI is responsible for the global aspects of the research study. For safety monitoring, a study 
physician or healthcare professional, who is certified for Advanced Cardiac Life Support, will be 
available at the study site at the time of study drug administration and during the entire time of PK 
blood sampling period. The Institutional Review Board (IRB) acknowledges one PI per study site. 
Qualifications for the PI and/or Co-Investigator(s) are listed on the FDA Form 1572. 
 
VI. Study Evaluation 
 
1) Pharmacokinetics (PK) Endpoints for Insulin Aspart (IA) 
 
(1) Primary PK Endpoints for IA 
(i) CIAmax, defined as the maximum serum IA concentration;  
(ii) AUCIA(0-12h), defined as area under the curve (AUC) of serum IA concentrations for 
time 0 to 12 hours; and 
 
(2) Secondary PK Endpoints for IA 
 
(i) AUCIA(0-∞) of Insulin Aspart,  
(ii) AUCIA(0-2h),  
(iii) tmax for IA (tIAmax),  
(iv) Apparent clearance (CL/F),  

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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(v) Apparent volume of distribution (Vz/F), and  
(vi) Half-life (t1/2). 
 
2) Pharmacokinetics (PK) Endpoints for Human Insulin (HI) 
 
(1) Primary PK Endpoints for HI 
(i) CHImax, defined as the maximum serum HI concentration;  
(ii) AUCHI(0-12h), defined as area under the curve (AUC) of HI concentrations for time 0 
to 12 hours; and 
 
(2) Secondary PK Endpoints for HI: tmax for HI (tHImax) 
 
3) Pharmacodynamics (PD) endpoints  
 
The PD parameters are used to measure the study drug action over time as measured by the 
hyperglycemic-euglycaemic clamp procedure. During the clamp procedure, blood glucose 
concentrations are held constant after the administration of NovoLog® by adjusting the 
exogenous glucose injection rate (GIR). GIR, denoted as G, is defined as infusion rate of 
glucose administered intravenously needed to maintain target blood glucose level. GIR data 
will be reported as mg/kg/min.  
 
(1) Primary PD Endpoints 
 
(i) Gmax: Maximum GIR, defined as maximum infusion rate of glucose administered 
intravenously needed to maintain target blood glucose level.  
(ii) AUCG(0-12h) for GIR: defined as area under the curve (AUC) of GIR, i.e. the total 
amount of glucose infused over the duration of the clamp procedure.  
 
Both Gmax and AUCG of GIR are calculated based on smoothed GIR curves. 
 
(2) Secondary PD Endpoints 
 
(i) 
AUCGA(0-12h) for GA (or GA), where GA stands for “GIR for IA” to reflect the PD 
effect of only IA.  
(ii) 
GAmax 
(iii) 
AUCG(0-last), 
(iv) 
AUCG(0-2h), 
(v) 
Value of last measurable GIR, Glast 
(vi) 
Time until maximum glucose infusion rate is reached, tGmax 
(vii) Time of start of GIR post-dose, tGonset 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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(viii) Time of last measurable GIR, tGlast 
(ix) 
t-G50%early, defined as the time to 50% maximal GIR before tGmax 
(x) 
t-G50%late, defined as the time to 50% maximal GIR after tGmax 
 
4) Statistical Analysis 
 
The ratio (T to R) of geometric means and 90% confidence intervals of the ratio of geometric 
means for primary and secondary endpoints are to be estimated and evaluated. 
 
VII. Safety Evaluation 
 
(1) Changes in vital signs and ECG from baselines will be analyzed at designated post-dosing time 
points for two treatments, respectively. 
 
(2) The results of laboratory tests, including hematology, clinical chemistry and UA, will be 
assessed. Comparisons between data at Screening and that at the follow-up visit (F/U) will be 
performed. Unscheduled visit for safety laboratory testing is allowed and at the discretion of 
PI during the COVID-19 pandemic. 
 
(3) Adverse events (AEs): The occurrence of AEs will be compared for the two treatments.  Two 
AE of special interests: hypoglycemia and injection site reaction will be closed monitored and 
elevated.  
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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TABLE OF CONTENTS 
 
1.0 
INTRODUCTION .......................................................................................................................................... 19 
1.1 
Background ................................................................................................................................................ 19 
1.2 
Rationale for the Proposed Study ............................................................................................................... 19 
1.3 
Summary of Pre-Clinical /Clinical Studies ................................................................................................ 20 
1.4 
Rationale for Treatment and Dose ............................................................................................................. 20 
2.0 
STUDY OBJECTIVES .................................................................................................................................. 21 
2.1 
Primary Objectives ..................................................................................................................................... 21 
2.2 
Secondary Objectives ................................................................................................................................. 21 
2.3 
Safety Objectives ....................................................................................................................................... 22 
2.4 
Study Endpoints ......................................................................................................................................... 22 
3.0 
INVESTIGATORS AND STUDY SITES ..................................................................................................... 24 
3.1 
Investigational Site ..................................................................................................................................... 24 
3.2 
Principal Investigator(s) ............................................................................................................................. 24 
3.3 
Laboratory .................................................................................................................................................. 24 
4.0 
STUDY DESIGN AND DESCRIPTION ....................................................................................................... 25 
4.1 
Study Design .............................................................................................................................................. 25 
4.2 
Study Description ....................................................................................................................................... 25 
4.3 
Rationale for Study Design and Endpoints ................................................................................................ 28 
4.3.1 
Criteria for Early Termination of the Study ........................................................................................... 29 
4.3.2 
Criteria for Early Termination of Individual Subjects ........................................................................... 30 
5.0 
STUDY POPULATION ................................................................................................................................. 31 
5.1 
Inclusion Criteria ........................................................................................................................................ 31 
5.2 
Exclusion Criteria ...................................................................................................................................... 31 
5.3 
Prohibited Medications .............................................................................................................................. 33 
5.4 
Check-in Criteria on Day 1 ........................................................................................................................ 33 
6.0 
STUDY MATERIALS ................................................................................................................................... 35 
6.1 
Investigational Products ............................................................................................................................. 35 
6.2 
Packaging, and Labeling of Investigational Products ................................................................................ 35 
6.3 
Storage and Drug Accountability of Investigational Products ................................................................... 35 
6.4 
Dose Regimen ............................................................................................................................................ 36 
6.5 
Overdose .................................................................................................................................................... 37 
6.6 
Randomization and Blinding ...................................................................................................................... 37 
6.7 
Breaking of Blinded Code .......................................................................................................................... 38 
6.8 
Auxiliary Supply ........................................................................................................................................ 39 
7.0 
STUDY PROCEDURE .................................................................................................................................. 40 
7.1 
Informed Consent ....................................................................................................................................... 40 
7.2 
Screening .................................................................................................................................................... 40 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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7.3 
Demographics and Medical History ........................................................................................................... 40 
7.4 
Physical Examination ................................................................................................................................. 41 
7.5 
Height, Weight, and BMI ........................................................................................................................... 41 
7.6 
Vital Signs .................................................................................................................................................. 41 
7.7 
Concomitant Illness and Therapy ............................................................................................................... 41 
7.8 
Procedures for Clinical Laboratory Samples .............................................................................................. 42 
7.9 
Contraception ............................................................................................................................................. 44 
7.10 
Pregnancy .............................................................................................................................................. 44 
7.11 
ECG Procedure ...................................................................................................................................... 45 
7.12 
Fasting Plasma Glucose ......................................................................................................................... 45 
7.13 
Standardized Meals ................................................................................................................................ 45 
7.14 
Euglycemic Glucose Clamp ................................................................................................................... 46 
7.15 
Pharmacokinetic Assessments and Schedule ......................................................................................... 48 
7.16 
Pharmacodynamic Assessments and Schedule ...................................................................................... 51 
7.17 
Tolerability Assessments ....................................................................................................................... 51 
7.18 
Blood Volume ........................................................................................................................................ 52 
8.0 
ADVERSE EVENTS ..................................................................................................................................... 53 
8.1 
Definitions .................................................................................................................................................. 53 
8.1.1 
Adverse Event (AE) ............................................................................................................................... 53 
8.1.2 
Treatment Emergent Adverse Event (TEAE) ........................................................................................ 53 
8.1.3 
Clinical Laboratory Event ...................................................................................................................... 53 
8.1.4 
Unexpected Adverse Event/ Unexpected Suspected Adverse Reaction ................................................ 53 
8.1.5 
Serious Adverse Event (SAE)/ Serious Suspected Adverse Reaction ................................................... 54 
8.1.6 
Life-Threatening Adverse Event/Life-Threatening Suspected Adverse Reaction ................................. 54 
8.1.7 
Severity of AEs ...................................................................................................................................... 54 
8.1.8 
Relationship to Study Treatment ........................................................................................................... 55 
8.2 
Procedures .................................................................................................................................................. 55 
8.2.1 
Collection and Recording of AEs .......................................................................................................... 55 
8.2.2 
Collection and Reporting of SAEs ......................................................................................................... 56 
8.3 
Anticipated AEs ......................................................................................................................................... 56 
8.3.1 
Drug Related AEs .................................................................................................................................. 57 
8.3.2 
Procedure Related AEs .......................................................................................................................... 58 
8.4 
Follow-up of AEs and SAEs ...................................................................................................................... 58 
Safety Reporting to IRBs or IECs, and Regulatory Authorities is described below. ........................................... 59 
9.0 
DATA HANDLING AND MANAGEMENT ................................................................................................ 60 
9.1. 
Data Quality Assurance and Monitoring .................................................................................................... 60 
9.2. 
Electronic Case Report Forms (eCRFs) ..................................................................................................... 60 
9.3. 
Clinical Trial Drug Accountability Data .................................................................................................... 60 
9.4. 
Trial Documents ......................................................................................................................................... 61 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
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9.5. 
Reporting and Publication .......................................................................................................................... 61 
9.6. 
Archive ....................................................................................................................................................... 61 
10.0 
STATISTICAL METHODS ........................................................................................................................... 62 
10.1 Analysis Plan ................................................................................................................................................... 62 
10.1.1  Populations .............................................................................................................................................. 62 
10.1.2 
Analysis of Demographics and Other Baseline Characteristics ........................................................ 62 
10.1.3 
Analysis of the PK Endpoints ............................................................................................................ 62 
10.1.4 
Analysis of the PD Endpoints ............................................................................................................ 63 
10.1.5 
Safety Analysis and Endpoints .......................................................................................................... 63 
10.2 
Interim Analysis ......................................................................................................................................... 63 
10.3 
Determination of Sample Size.................................................................................................................... 63 
11.0 
QUALITY CONTROL AND QUALITY ASSURANCE .............................................................................. 64 
11.1 
Monitoring ................................................................................................................................................. 64 
11.2 
Protocol Deviations .................................................................................................................................... 64 
12.0 
ETHICAL ASPECTS OF THE STUDY ........................................................................................................ 65 
12.1 
Institutional Review Board and/or Independent Ethics Committee ........................................................... 65 
12.2 
Regulatory Authorities ............................................................................................................................... 65 
12.3 
Responsibilities of the Investigator ............................................................................................................ 66 
12.4 
Informed Consent ....................................................................................................................................... 66 
12.5 
Subject Confidentiality ............................................................................................................................... 67 
12.6 
Publication, Disclosure, and Clinical Study Registration Policy ............................................................... 67 
12.7 
Insurance and Compensation for Injury ..................................................................................................... 68 
13.0 
REFERENCES ............................................................................................................................................... 69 
APPENDIX A ............................................................................................................................................................. 72 
APPENDIX B .............................................................................................................................................................. 73 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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LIST OF TABLES 
 
Table 6-1 Storage Conditions........................................................................................................35 
 
Table 6-2 Sample Treatment Allocation Scheme..........................................................................37 
 
Table 7-1 Clinical Laboratory Assessments..................................................................................41 
 
Table 7-2 PK Blood Sampling Schedule.......................................................................................48 
 
 
 
 
 
 
 
 
 
 
LIST OF FIGURES 
 
Figure 4-1 Study Design Schematic..............................................................................................23 
  
Figure 7-1 MSIA Technology Workflow......................................................................................51 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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LIST OF ABBREVIATIONS 
 
AE 
 
 
 
 
 
Adverse event  
ALT 
 
 
 
 
 
Alanine aminotransferase 
 
ANOVA 
 
 
 
 
Analysis of variance  
AST 
 
 
 
 
 
Aspartate aminotransferase 
 
AUC  
 
 
 
 
Area under the concentration-time curve 
AUCGIR 
 
 
 
 
Area under the glucose infusion rate-time curve 
BG 
 
 
 
 
 
Blood glucose  
BMI 
 
 
 
 
 
Body mass index 
 
BW 
 
 
 
 
 
Body weight  
CDM  
 
 
 
 
Clinical data management 
 
CI 
 
 
 
 
 
Confidence interval 
 
CL/F  
 
 
 
 
Apparent clearance 
 
Cmax  
 
 
 
 
Maximum concentration 
 
CRF 
 
 
 
 
 
Case report form 
 
CRO  
 
 
 
 
Contract Research Organization 
CSR 
 
 
 
 
 
Clinical Study Report  
CV 
 
 
 
 
 
Coefficient of variation 
 
DMP  
 
 
 
 
Data management plan 
 
DNA  
 
 
 
 
Deoxyribonucleic acid 
 
ECG  
 
 
 
 
Electrocardiogram 
 
Ecrf 
 
 
 
 
 
Electronic CRF 
 
ELISA  
 
 
 
 
Enzyme linked immunosorbent assay 
FDA  
 
 
 
 
Food and Drug Administration 
 
FPG 
 
 
 
 
 
Fasting plasma glucose 
 
FSFV  
 
 
 
 
First subject first visit  
GA 
 
 
 
 
 
Glucose infusion rate (GIR) for Insulin Aspart (IA) 
GCP  
 
 
 
 
Good Clinical Practice 
 
GIR 
 
 
 
 
 
Glucose infusion rate  
HbA1C 
 
 
 
 
Glycosylated hemoglobin 
 
HbsAg  
 
 
 
 
Hepatitis B surface antigen 
 
HCV  
 
 
 
 
Hepatitis C virus 
 
HI 
 
 
 
 
 
Human Insulin 
HIV 
 
 
 
 
 
Human immunodeficiency virus 
IA 
 
 
 
 
 
Insulin Aspart 
IP 
 
 
 
 
 
Investigational Product 
IB 
 
 
 
 
 
Investigator’s brochure 
 
ICH 
 
 
 
 
 
International Conference on Harmonization 
IEC 
 
 
 
 
 
Independent Ethics Committee 
 
INR 
 
 
 
 
 
International normalized ratio 
IRB 
 
 
 
 
 
Institutional Review Board 
 
IV 
 
 
 
 
 
Intravenous 
 
Kg 
 
 
 
 
 
Kilogram 
 
Lb 
 
 
 
 
 
Pound  
LC/MS/MS  
 
 
 
 
Liquid Chromatography with tandem mass 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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spectrometry 
LLOQ  
 
 
 
 
Lower Limit of Quantification 
LS mean 
 
 
 
 
Least square mean 
 
MSIA  
 
 
 
 
Mass Spectrometric Immunoassay 
MSIA‐HR/MS 
Mass spectrometric immunoassay with high 
resolution and accurate mass detection 
Mcg 
 
 
 
 
 
Microgram 
 
MedDRA 
 
 
 
 
Medical Dictionary for Regulatory Activities 
Mg 
 
 
 
 
 
Milligram 
 
m/z 
 
 
 
 
 
mass to charge ratio 
Nmol  
 
 
 
 
Nanomol 
 
OTC  
 
 
 
 
Over the counter 
 
PD 
 
 
 
 
 
Pharmacodynamics 
 
PE 
 
 
 
 
 
Physical examination  
PI 
 
 
 
 
 
Principal Investigator  
PK 
 
 
 
 
 
Pharmacokinetics 
 
PP 
 
 
 
 
 
Per-Protocol  
rDNA  
 
 
 
 
Recombinant deoxyribonucleic acid 
SAE 
 
 
 
 
 
Serious adverse event  
SAP 
 
 
 
 
 
Statistical analysis plan 
 
SC 
 
 
 
 
 
Subcutaneous  
SOE 
 
 
 
 
 
Schedule of events 
 
SUSAR 
 
 
 
 
Suspected unexpected serious adverse reaction 
t½ 
 
 
 
 
 
Terminal insulin half-life 
 
TEAE  
 
 
 
 
Treatment emergent adverse event 
TG 
 
 
 
 
 
Triglycerides  
tGIRmax 
 
 
 
 
Time to maximum glucose infusion rate 
Tmax  
 
 
 
 
Time to maximum serum insulin concentration (in 
 
 
 
 
 
 
concentration time curve) 
 
TSH 
 
 
 
 
 
Thyroid-stimulating hormone  
ULN  
 
 
 
 
Upper limit of normal  
US 
 
 
 
 
 
United States  
USP 
 
 
 
 
 
Unites States Pharmacopeia  
Vz/F  
 
 
 
 
Apparent volume of distribution at terminal phase 
WHO  
 
 
 
 
World Health Organization 
 
YSI 
 
 
 
 
 
YSI 2300 STAT Glucose Analyzer 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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1.0 INTRODUCTION 
 
1.1 Background 
 
In 2015, diabetes affected 30.3 million Americans or 9.4% of the total US population, including 
7.2 million people who were undiagnosed.1 Recently, there have been dramatic yearly increases 
in the incidence of diabetes, and should this upward trend continue, the total prevalence of 
diagnosed and undiagnosed diabetes in the US adult population is projected to increase to as much 
as 33% by the year 2050.2 
 
The results of the Diabetes Control and Complication Trial (DCCT) demonstrated that tight 
glycemic control can prevent or decrease the microvascular complications of diabetes, such as 
retinopathy, nephropathy, neuropathy as well as reduce cardiovascular– related death.3 To 
optimize blood glucose control in patients, intensive insulin regimens are used, although current 
replacement insulin formulations only approximate physiologic insulin secretion, resulting in high 
postprandial blood glucose peaks.4 
 
Another disadvantage of intensive insulin regimens is the risk of hypoglycemia. The development 
of rapid-acting insulins with accelerated absorption has improved the postprandial glucose 
excursions and allowed for more precise dosing.5,6,7 
 
The development of rapid-acting insulin analogs with fast absorption has reduced postprandial 
glucose excursions and has allowed greater flexibility in insulin therapy as well as a more 
physiological insulin replacement with more precise dosing regimens.8 
 
Both I004 and NovoLog® are insulin Aspart [rDNA origin] injection, rapid-acting human insulin 
analogs used to lower blood glucose. I004 and NovoLog are homologous with regular human 
insulin, with the exception of a single substitution of the amino acid proline by aspartic acid in 
position B28, and is produced by recombinant DNA technology. The insulin formulation is 
available in a concentration corresponding to 100 units/mL (U-100). 
 
Please refer to the Investigator's Brochure /Package Insert9 for further information on NovoLog®. 
 
1.2 Rationale for the Proposed Study 
 
This study will assess PK and PD data that will be necessary for a future biosimilar approval. 
Therefore, the comparison of rapid-acting insulin I004 and Novolog with regard to the AUCIA (0-
12h), area under the serum insulin profile for the drug, and AUCGIR (0-12h), area under the glucose 
infusion rate as primary endpoints, will be performed.  
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Safety, cardiac safety (ECG), and tolerability after SC administration will also be determined. 
 
1.3 Summary of Pre-Clinical /Clinical Studies 
 
Information regarding characterization, structure and properties of Insulin aspart [rDNA origin] 
are provided in the Novolog Investigator’s Brochure (IB). 
 
For data of in vitro and in vivo pharmacologic and toxicologic studies and clinical studies, 
reference is made to 
(Novolog, 
). The sponsor refers to the 
information and FDA safety determinations in the approved product labeling for these products. 
 
Novolog will be commercially procured from the US market and will be used as the investigational 
product and comparator in this study. It will be administered at the same dose and in the same 
fashion, consistent with the approved product information. 
 
1.4 Rationale for Treatment and Dose 
 
The dose of 0.2 units/kg body weight has been chosen for this study in order to provide a robust 
dose-response relationship relevant for healthy subjects. In addition, the dose used is representative 
of doses in healthy subjects and is within the dose range used in other clinical glucose clamp 
studies conducted to date, where no safety concerns have been observed at this dose level. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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2.0 
STUDY OBJECTIVES 
 
2.1 Primary Objectives 
 
To assess and compare the PK profile of I004 and Novolog by: 
(i) Maximum insulin concentration, CIAmax 
(ii) Area under the insulin concentration-time curve, AUCIA (0-12h) 
 
To assess and compare the PD profile of I004 and Novolog by: 
(i) Maximum glucose infusion rate, Gmax 
(ii) Area under the curve for the glucose infusion rate (GIR)-time curve from 
administration to end of clamp time, AUCG (0-12h) 
 
2.2 Secondary Objectives 
 
To assess and compare the PK profile of I004 and Novolog by: 
(i) 
Partial AUCIA, eg, AUCIA (0-2h) 
(ii) 
Area under the insulin concentration curve extrapolated to infinite time, 
AUCIA(0-∞) 
(iii) 
Time until Cmax is reached, tIAmax 
(iv) 
Apparent clearance, CL/F 
(v) 
Apparent volume of distribution, Vz/F 
(vi) 
Apparent terminal half-life, t1/2 
 
To assess and compare the PD profile of I004 and Novolog by: 
(i) 
Total and partial AUC of GIR, eg, AUCG(0-last), AUCGIR(0-2h) 
(ii) 
Value of last measurable GIR, Glast 
(iii) 
Time until maximum glucose infusion rate is reached, tGmax 
(iv) 
Time of start of GIR post-dose, tGonset 
(v) 
Time of last measurable GIR, tGlast 
(vi) 
t-G50%early, defined as the time to 50% maximal GIR before tGmax 
(vii) t-G50%late, defined as the time to 50% maximal GIR after tGmax 
 
To assess the confounding effect of endogenous human insulin: 
 
(i) PK profile of endogenous human insulin  
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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2.3 Safety Objectives 
 
To assess safety and tolerability of I004 and Novolog by: 
(i) 
Incidence of adverse events (including hypoglycemia) 
(ii) 
Clinical findings on physical examination 
(iii) 
Clinical laboratory parameters (hematology, serum chemistry and urinalysis) 
(iv) 
Vital signs (blood pressure, temperature, respiratory rate, and heart rate) 
measurements 
(v) 
12-lead ECG parameters 
(vi) 
Injection site reaction 
 
 
2.4 Study Endpoints 
 
1) Pharmacokinetic (PK) Endpoints for Insulin Aspart (IA) 
 
(1) Primary PK Endpoints for IA 
(i) CIAmax, defined as the maximum serum IA concentration;  
(ii) AUCIA(0-12h), defined as area under the curve (AUC) of serum IA concentrations for 
time 0 to 12 hours; and 
 
(2) Secondary PK Endpoints for IA 
(i) AUCIA(0-∞) of Insulin Aspart,  
(ii) AUCIA(0-2h),  
(iii) tmax for IA (tIAmax),  
(iv) Apparent clearance (CL/F),  
(v) Apparent volume of distribution (Vz/F), and  
(vi) Half-life (t1/2). 
 
2) Pharmacokinetic (PK) Endpoints for Human Insulin (HI) 
 
(1) Primary PK Endpoints for HI 
(i) CHImax, defined as the maximum serum HI concentration;  
(ii) AUCHI(0-12h), defined as area under the curve (AUC) of HI concentrations for time 0 
to 12 hours; and 
 
(2) Secondary PK Endpoints for HI: tmax for HI (tHImax) 
 
3) Pharmacodynamic (PD) endpoints  
 
The PD parameters are used to measure the study drug action over time as measured by the 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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hyperglycemic-euglycaemic clamp procedure. During the clamp procedure, blood glucose 
concentrations are held constant after the administration of NovoLog® by adjusting the 
exogenous glucose injection rate (GIR). GIR, denoted as G, is defined as infusion rate of 
glucose administered intravenously needed to maintain target blood glucose level. GIR data 
will be reported as mg/kg/min.  
 
(1) Primary PD Endpoints 
 
(i) Gmax: Maximum GIR, defined as maximum infusion rate of glucose administered 
intravenously needed to maintain target blood glucose level.  
(ii) AUCG(0-12h) for GIR: defined as area under the curve (AUC) of GIR, i.e. the total 
amount of glucose infused over the duration of the clamp procedure.  
 
Both Gmax and AUCG of GIR are calculated based on smoothed GIR curves. 
 
(3) Secondary PD Endpoints 
 
(i) 
AUCGA(0-12h) for GA (or GA), where GA stands for “GIR for IA” to reflect the PD 
effect of only IA.  
(ii) 
GAmax 
(iii) 
AUCG(0-last), 
(iv) 
AUCG(0-2h), 
(v) 
Value of last measurable GIR, Glast 
(vi) 
Time until maximum glucose infusion rate is reached, tGmax 
(vii) Time of start of GIR post-dose, tGonset 
(viii) Time of last measurable GIR, tGlast 
(ix) 
t-G50%early, defined as the time to 50% maximal GIR before tGmax 
(x) 
t-G50%late, defined as the time to 50% maximal GIR after tGmax 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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3.0 
INVESTIGATORS AND STUDY SITES 
 
3.1 Investigational Site 
 
The study will be performed at  
 
 
 
Chula Vista, CA, 91911 
USA 
 
The detailed information about the study site can be found on FDA Form 1572, including: Name, 
address, and statement of qualifications of each principal investigator; name of each sub-investigator 
working under the supervision of the investigator; name and address of the research facilities to be 
used; name and address of each reviewing IRB per US 21 CFR 312.23 section 6(iii)b. 
 
3.2 Principal Investigator(s)  
 
Principal Investigators (PI) responsible for the conduct of this study will be board certified 
physicians who are familiar with the study medications and trained to handle any untoward events 
related to medications.  
 
The PI is responsible for the global aspects of the research study. For safety monitoring, a study 
physician or healthcare professional, who is certified for Advanced Cardiac Life Support, will be 
available at the study site at the time of study drug administration and during the entire time of PK 
blood sampling period. The Institutional Review Board (IRB) acknowledges one PI per study site. 
Qualifications for the PI and Co-Investigator(s) are listed on the FDA Form 1572. 
 
3.3 Laboratory 
 
Samples of blood for evaluation of hematology and chemistry (metabolic panels) will be analyzed 
by the clinical laboratory with certification from a recognized accreditation agency (e.g., College of 
American Pathology or Clinical Laboratory Improvement Amendments certification). Refer to 
Table 7-1 for a list of parameters analyzed, and the laboratory manual for details regarding specimen 
sample collection, processing, and shipment procedures. PK samples will be processed and analyzed 
by R&D Department, Amphastar Pharmaceuticals, Inc. 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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4.0 
STUDY DESIGN AND DESCRIPTION 
 
4.1 Study Design 
 
This is a pivotal, phase II/III, single dose, double-blind, randomized, two-treatment, two-period, 
crossover, euglycemic glucose clamp study to assess the safety, pharmacokinetic and 
pharmacodynamic profiles of insulin I004 and Novolog in healthy subjects. Each period will include 
a 12-hour clamp and blood sampling procedure. 
 
At least sixty (60) eligible subjects will be randomized to receive a total of two administrations of 
study drug on two separate dosing visits. Each subject will receive one administration of either 0.2 
units/kg I004 or 0.2 units/kg Novolog. Replacement of dropouts and withdrawals will take place in 
order to ensure at least 60 subjects complete the study. 
 
Each subject will undergo a Screening Visit, followed by two treatment periods, each consisting of 
a one-day in-house treatment period, with one euglycemic clamp procedure, for a total of two 
glucose clamp procedures per subject. The Wash-out Period between treatment periods will be at 
least 7 days. A Follow-up Visit will take place after the last dose of study drug in treatment period 
2. The original duration of participation in this study, including Screening, Treatment and Follow-
up will approximately be up to 10 weeks, as shown in Figure 4-1. However, Screening visit window, 
Treatment visit window, and Follow-up visit window could be extended to accommodate state-
issued Stay at Home orders due to the COVID-19 pandemic.  
 
Figure 4-1 Study Design Schematic 
 
 
 
Due to the COVID-19 pandemic, original Screening visit window (≤30 days), Treatment visit window (7-21 days), and Follow-up 
visit window (1-14 days) could be extended to accommodate state-issued Stay at Home orders.  
 
4.2 Study Description 
 
Screening Visit: 
 
Before the Screening takes place, potential subjects for the study will be provided with written and 
oral information about the study and the procedures involved. Subjects have to sign the IRB/IEC 
informed consent form (ICF) prior to entering the study. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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The Screening Visit will be performed prior to the first dosing, in order to identify eligible subjects 
for the study. All assessments performed at the Screening visit are stated in the SOE (Appendix A) 
and will be recorded in the electronic case report form (eCRF). See section 7.1.2 and 9.2 for further 
details. 
 
Treatment Period 1: 
 
All eligible subjects will check in to the clinic in the morning of Day 1 for a one-day in-house period, 
to standardize meals and ensure adequate fasting time prior to the clamp procedure. Check–in 
assessments will be performed to ensure subject meets the check-in requirements. 
 
Subjects will be randomized to one of two treatment sequences (TR or RT), where treatments will 
consist of single SC study drug administrations. Please see section 6.6 for further details. 
 
On Day 1, following ≥ 10 hour overnight fast, a 12-hour euglycemic clamp will be initiated. EMEA 
(European Medicines Agency) recommends typical clamp duration to be 8 to 10 hours for rapid-
acting insulins.16 The 12-hour duration of the clamp study follows the recommendations from the 
EMEA and takes into account the known duration of action of the US-approved Novolog, with a 
half-life 81 minutes.9 
 
At least 1 hour prior to the clamp, three venous catheters will be inserted into peripheral veins to 
connect subjects to the Biostator, to allow for the sampling of glucose, insulin and or other analytes, 
and to continuously infuse glucose 20%.  
 
The determination of blood glucose concentrations will start at least 60 minutes prior to dosing. The 
mean of three blood glucose (BG) readings from -30 to -10 minutes (i.e., -30, -20 and -10 minutes) 
will be used to determine the individual subject’s fasting blood glucose level for each dosing visit. 
The individual blood glucose clamp target level will be determined for each subject by the mean 
fasting glucose level minus 5 mg/dL. 
 
If differences between initial fasting blood glucose values (-30, -20, -10 min) exceed the limit of 10 
mg/dL, subjects will need to be rescheduled. Additionally, subjects will not be dosed if mean fasting 
plasma glucose is < 75 mg/dL due to proximity to overt hypoglycemia or > 99 mg/dL. Subjects may 
be re-scheduled once to come back to the site on a later day and may be dosed if the mean fasting 
plasma glucose is ≥ 75 mg/dL or ≤ 99 mg/dL prior to dosing. 
 
The study drug will be administered at approximately 08:00 (t=0) by qualified staff via SC injection 
as described in section 6.4. 
 
Following the study drug administration, the clamp procedure will start. Onset of insulin action will 
be identified by a ≥ 5mg/dL decline of BASELINE level and start of the GIR. After the onset of 
insulin action is identified, an infusion of glucose 20% will then be utilized as needed to maintain 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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target level glucose concentration. 
 
The GIR will be based on the rates suggested by a published clamp algorithm. The clamp operator 
can adapt the glucose infusion rate manually as deemed appropriate. The GIR required to keep blood 
glucose at target level (±10%) will be recorded over 12 hours and data will be used to calculate PD 
parameters. For details on the clamp procedure please see section 7. 14. 
 
The subjects will remain fasting in a supine or semi-supine position during the entire glucose clamp. 
The subjects will be allowed to sip water throughout the clamp but will otherwise remain fasting. 
 
After the end of the clamp procedure, subjects will be disconnected from the Biostator. 
 
Post-dose procedures, such as vital signs, ECG and injections site assessment will be performed 
during/after the clamp procedure as stated in the SOE (Appendix A). 
 
Blood glucose will be measured to ensure that plasma glucose levels are stable and thereafter the 
subjects will receive a meal. Subjects will stay in the clinic throughout the treatment period until the 
glucose clamp has been completed and after deemed safe by the Investigator. 
 
Wash-out period: 
 
Subjects will be scheduled to come in for the next treatment period after a wash-out period of at 
least 7 days. 
 
Treatment Periods 2: 
 
All subjects will check in to the clinic in the morning of Day 1 for the second of the in-house 
treatment period. All procedures and the clamp will follow the same procedures as stated for the 
first treatment period, except for the change of the study drug. 
 
Body weight from Day 1 of Treatment Period 1 under fasting condition will be used to calculate the 
drug dose for Treatment Period 2. 
 
Follow-up Visit: 
 
A Follow-up Visit will be performed after the last dosing to perform final safety procedures. The 
End of Study Form must be completed. Please see SOE (Appendix A) for details. 
 
Sampling and Assessment: 
 
Time points for study procedures and sample collection are specified in section 7.1. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Safety assessments will occur throughout the duration of the study, including monitoring of adverse 
events (AEs), clinical laboratory tests (chemistry, hematology and urinalysis), vital signs 
measurements (blood pressure, heart rate, respiration rate, and aural temperature), 12-lead 
electrocardiograms (ECGs) and physical examinations. 
Sampling collections for PK, PD and glucose measurements will be performed as specified in 
Appendix B. Sample collection for PK and PD parameter will be collected under fasting condition 
for 12 hours. 
 
Blood samples for the analysis of C-peptide and endogenous human insulin will be collected with 
every PK sample. The standardized measurements of the Biostator will be verified on a regular basis 
using the YSI reference device. 
 
 
4.3 Rationale for Study Design and Endpoints 
 
A single dose cross-over design is chosen in order to allow each subject to act as his/her own control 
in order to reduce variability. 
 
A minimum 7-day wash-out period before and between the two treatment periods is introduced to 
ensure a sufficient wash-out from previous dosing, in order to avoid any carry-over effects. 
 
Randomization is used to avoid bias introduced through an association between trial drug allocation 
order and subject characteristics. 
 
Blinding of participants and personnel is introduced in order to avoid performance bias. 
 
Due to the variability in fasting blood glucose concentrations in subjects with type 1 diabetes, which 
might affect the metabolic response to the study drug, healthy subjects have been chosen. A study 
in healthy subjects is likely to produce less PK variability compared with that in patients with 
potentially confounding factors such as underlying and/or concomitant disease and concomitant 
medications. C-peptide and endogenous human insulin in parallel to the insulin aspart concentration 
is measured during the study, to detect potential changes in endogenous insulin secretion and enable 
a correction for endogenous insulin secretion, if necessary. 
 
Enrollment criteria will favor a generally insulin-sensitive population and diseases/conditions and 
medications associated with insulin resistance have been excluded. 
 
The sample size of 60 subjects is chosen as it represents a typical number for a biosimilar study. 
The use of the euglycemic glucose clamp method is chosen to assess PD in a standardized fashion 
and for safety reasons to avoid hypoglycemia. 
 
Glucose levels below approximately 60 mg/dL must be avoided because they result in the 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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stimulation of counter regulatory hormones (epinephrine, glucagon, cortisol, growth hormone) to 
increase blood glucose concentrations and lead to a rapid and pronounced worsening of insulin 
sensitivity, thus influencing the estimated time-action profile of the investigated insulin preparation. 
Therefore, blood glucose (BG) inclusion criterion prior to the clamp is limited to a fasting plasma 
glucose value ≥ 75 mg/dL (for the mean of the -30 ≤ to -10 min glucose measurements), as the target 
clamp level is 5 mg/dL below the mean fasting BG values. The chosen target level of mean fasting 
glucose minus 5 mg/dL will ensure that endogenous insulin secretion is sufficiently suppressed and 
will not interfere with PK and PD assessments. 
 
For the study design, the FDA and EU guidance on the development of similar biological medicinal 
products and prior studies, evaluating the pharmacology of rapid-acting insulins, have been taken 
into consideration.10,11,12 
 
Safety is further emphasized by the design of the study: The clamp procedures in this study will be 
conducted at a specialized early phase research center, where the subjects will be under supervision 
of physicians, research nurses and additional medical staff experienced in investigating new insulins, 
incretins or other metabolic compounds. Following the glucose clamp procedures, subjects may be 
released from the investigational site or may be observed for longer, if deemed appropriate and 
needed by the Investigator to monitor for the risk of hypoglycemic episodes. 
 
4.3.1 Criteria for Early Termination of the Study 
 
The study will be completed as planned unless one or more of the following criteria are satisfied 
that require temporary suspension or early termination of the study. 
 
• New information or other evaluation regarding the safety or efficacy of the study 
medication that indicates a change in the known risk/benefit profile for the compound, 
such that the risk/benefit is no longer acceptable for subjects participating in the study. 
 
• Significant violation of Good Clinical Practice (GCP) that compromises the ability to 
achieve the primary study objectives or compromises subject safety. 
 
• Failure to meet expected enrollment goals. 
 
• Administrative Reasons. 
 
In the event that the Sponsor, an institutional review board (IRB)/ independent ethics committee 
(IEC), or regulatory authority elects to terminate or suspend the study or the participation of an 
investigational site, a study-specific procedure for early termination or suspension will be provided 
by the Sponsor; the procedure will be followed by applicable investigational sites during the course 
of termination or study suspension. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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4.3.2 Criteria for Early Termination of Individual Subjects 
 
Subjects may withdraw their consent to participate in the study at any time. 
 
If a subject withdraws consent, the date and reason for consent withdrawal should be documented. 
Subjects will be encouraged to remain in the clinic for safety assessments until the Investigator 
deems that it is safe for the subject to be discharged. Subject data will be included in the analysis up 
to the date of the consent withdrawal. 
 
• 
AE or SAE that requires discontinuation at the discretion of the Investigator 
 
• 
Protocol violation: If protocol violation or concurrent illness occurs, which, in the 
clinical judgment of the Investigator or after discussion with the Sponsor, may 
invalidate the study by interfering pharmacokinetically or pharmacodynamically 
with the investigational products, the subject will be withdrawn by the Investigator. 
 
• 
Lost to follow-up. The subject did not return to the clinic and attempts to contact the 
subject were unsuccessful. Attempts to contact the subject must be documented. 
 
• 
Voluntary withdrawal of consent (mandatory removal from study). 
 
• 
Discretion of Investigator (document reason on CRF). 
 
• 
Subject becomes pregnant or begins breastfeeding (mandatory). 
 
• 
Study discontinuation by Sponsor. 
 
Wherever possible, the tests and evaluations, including those listed for the Follow-up Visit should 
be performed for all subjects who discontinue prior to the completion of the study. 
 
In the event the Investigator determines to terminate a subject participation in the Clinical Study, 
the Investigator must notify the Sponsor of such decision and rationale immediately in writing. In 
all cases, the appropriate IRB/IEC and other applicable regulatory authorities shall be informed. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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5.0 
STUDY POPULATION 
 
5.1 Inclusion Criteria 
 
Subjects must meet all of the following criteria at the Screening Visit to be considered eligible to 
participate in the study: 
 
(1) 
Upon review, agree to participate and sign informed consent. 
(2) 
Healthy male and female subjects ≥ 18 to ≤ 65 years of age. 
(3) 
Body mass index (BMI) ≥ 18.5 to ≤ 29.9 kg/m2. 
(4) 
Weight ≥ 50 kg. 
(5) 
Fasting plasma glucose of < 100 mg/dL (5.5 mmol/L) measured with YSI at site; one 
repeat test is allowed. 
(6) 
HbA1c < 5.7 %. 
(7) 
Non-smoker for ≥ 3 months prior to Screening. 
(8) 
Female candidates must be >1 year post-menopausal, surgically sterile, or practicing 
a clinically acceptable form of birth control and confirmed by negative serum 
pregnancy test at Screening. 
 
5.2 Exclusion Criteria 
 
Subjects who meet any of the following criteria at the Screening Visit will be excluded from 
participating in the study: 
 
(1) 
History of diabetes mellitus. 
(2) 
Resting blood pressure (BP) > 140/90 mmHg or < 90/60 mmHg. Subjects BP may 
be re-checked. 
(3) 
Participation in an investigational drug/device study within 30 days or 5 half-lives 
within the last dose of any study drug, whichever is longer. 
(4) 
History of any serious adverse reaction or hypersensitivity to any of the 
investigational product components. 
(5) 
Have significant history of or current cardiovascular, respiratory, hepatic, renal, 
gastrointestinal, 
endocrine, 
hematological, 
or 
neurological 
disorders 
or 
abnormalities, or other major systemic disease that, according to the investigator, 
would unduly risk the subject’s safety or may impact the conduct of the study. 
(6) 
Subject shows evidence of significant active neuropsychiatric disease, including 
taking prescription medication for such diseases (including anti-depressant /anti-

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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anxiety medication). 
 
(7) 
Presence of clinically significant physical, laboratory, or ECG findings at Screening 
that, in the opinion of the Investigator, may interfere with any aspect of study conduct 
or interpretation of results, or may present a safety issue to that particular subject 
(laboratory results may be re-checked once on a separate day per Investigator 
discretion). 
(8) 
Long QT syndrome or family history of long QT syndrome or corrected QT interval 
(QTcF) > 450 ms in men, > 470 ms in women at Screening. 
(9) 
Liver function test results of AST and/or ALT ≥ 2.5 upper normal limit (ULN). 
(10) 
Subject has a history of syncope. 
(11) 
History of any major surgery within 6 months. 
(12) 
History of any active infection, other than mild viral illness within 30 days prior to 
dosing. 
(13) 
History of blood clots (eg, deep vein thrombosis or embolism) or a frequent 
appearance in 1st degree relatives as judged by the Investigator. 
(14) 
Known history or positive test of hepatitis B surface antigen (HBsAg), hepatitis C 
antibody (HCV Ab), or human immunodeficiency virus type 1 (HIV-1) or 2 (HIV-2) 
antibody. 
(15) 
History of alcohol abuse as judged by the Investigator within approximately 1 year. 
Average weekly alcohol intake > 21 units/week (males) and > 14 units/week 
(females) or are unwilling to stop alcohol consumption from 24 hours prior to each 
dosing until discharged from the clinical research unit (CRU). Positive alcohol test 
at Screening. (One unit of alcohol equals about 250 mL of beer or lager, one glass of 
wine, or 20 mL of spirits). 
(16) 
History of illicit drug abuse, including marijuana, within approximately 1 year or 
evidence of current use as judged by the Investigator. Positive drug test at Screening. 
(17) 
Donation or loss of > 500 mL of blood within 56 days. 
(18) 
Chronic use of or over-the-counter or prescription medication within 7 or 14 days 
prior to dosing (apart from vitamin/mineral supplements, occasional paracetamol, or 
birth control methods [Desogestrel is not allowed]). 
(19) 
Unable to comply with the safety monitoring requirements of this clinical study or is 
considered by the investigator to be an unsuitable candidate for the study. 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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5.3 Prohibited Medications 
 
Use of prescription or nonprescription drugs is prohibited from 7-14 days prior to dosing. 
 
Vitamins, mineral supplements, occasional paracetamol and /or birth control methods are allowed 
after discretion of the Investigator. Desogestrel is not allowed. 
 
5.4 Check-in Criteria on Day 1 
 
Subjects will be educated about the factors that may influence insulin sensitivity, such as refraining 
from strenuous exercise within 72 hours prior to the injection of the investigational product and 
during the in-house period, avoidance of alcohol and herbal supplements within 24 hours prior to 
dosing, caffeinated drinks (caffeine containing food and beverages within 24 hours prior to dosing), 
smoking or medication and illness/infection. 
 
Treatment Period 1 
 
At check-in for Treatment Period 1, subjects will not be allowed to check in if they meet any of the 
following criteria and the treatment period may be rescheduled one time with a time window of 1-
14 days. 
 
(1) 
Positive alcohol breath test. 
(2) 
Positive urine drug screen test. 
(3) 
Positive urine pregnancy test in female subjects. If sample is positive, blood sample 
will be sent to local lab for confirmation. A negative test result is mandatory prior to 
dosing. 
(4) 
Any medical condition that could interfere with glucose metabolism, as judged by 
the Investigator. 
(5) 
Any use of medicine (prescribed or over-the-counter) other than any allowed 
concomitant medications within the last 7-14 days prior to dosing. 
(6) 
Strenuous exercise within the last 72 hours prior to injection of the investigational 
product. 
(7) 
Consumption of caffeine containing food and beverages within 24 hours prior to 
dosing. 
 
On Day 1, subjects will not be allowed to be dosed if they meet the following criterion and the 
treatment period may be rescheduled one time with a time window of 1-14 days. 
 
(1) 
Subjects whose differences between initial fasting blood glucose values (-30, -20, -

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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10 min) determined by the YSI exceed the limit of 10 mg/dL or whose mean FPG is 
< 75 mg/dl or > 99 mg/dL will be rescheduled. 
 
Treatment Period 2 
 
At check-in for Treatment Period 2, subjects will not be allowed to check in if they meet any of the 
following criteria and the treatment period may be rescheduled one time (1-14 days later). The 
Sponsor will be notified about the rescheduling in a timely manner. 
 
(1) 
Positive alcohol breath test. 
(2) 
Positive urine drug screen test. 
(3) 
Positive urine pregnancy test in female subjects. If sample is positive, blood sample 
will be sent to local lab for confirmation. A negative test result is mandatory prior to 
dosing. 
(4) 
Any medical condition that could interfere with glucose metabolism, as judged by 
the Investigator. 
(5) 
Any use of medicine (prescribed or over-the-counter) other than any allowed 
concomitant medications within the last 7-14 days prior to dosing. 
(6) 
Strenuous exercise within the last 72 hours prior to injection of the investigational 
product. 
(7) 
Consumption of caffeine containing food and beverages within 24 hours prior to 
dosing. 
(8) 
Weight change of ≥ 5% compared to the weight for the first dosing. 
 
On Day 1, subjects will not be allowed to be dosed if they meet the following criterion and the 
treatment period may be rescheduled one time with a time window of 1-14 days. 
 
(1) 
Subjects whose differences between initial fasting blood glucose values (-30, -20, -
10 min) determined by the YSI exceed the limit of 10 mg/dL or whose mean FPG is 
< 75 mg/dl or > 99 mg/dL will be rescheduled. 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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I004 or Novolog must be stored securely in a refrigerator at 2 ℃ to 8 ℃ (36 °F to 46 °F), and should 
not be exposed to excessive heat, direct sunlight and never be frozen. It must be allowed to come to 
room temperature on the morning of dosing. 
 
The study staff is required to document the receipt, dispensing, and return/destruction of IP and 
supplies provided by or on behalf of the Sponsor. The Study Site must return all used and unused 
vials of I004 or Novolog to the Sponsor or designee or destroy them upon request. Drug supplies 
will be counted and reconciled at the site before being returned to the Sponsor or designee or being 
destroyed. 
 
The quantity of reserve samples of study drugs under test will be sufficient to meet the retention 
sample requirements per 21 CFR 320.38 (b) and (c), and 320.63 19, 20, 21. The investigator and 
designated dispenser are responsible to carry out the on-site drug retained activities in accordance 
to this protocol, 21 CFR 320.38 and 320.63.  
 
All samples will be stored in an area with controlled access. The retention samples will be stored for 
five (5) years or longer following product approval. 
 
The Investigator or Investigator’s authorized staff must ensure the availability of proper storage 
conditions. The temperature of all study drugs will be monitored over 24 hours a day, 7 days a week 
(24/7). In case of incorrect storage, the Sponsor and monitor must be contacted without delay. 
 
No study drugs may be dispensed to any person not enrolled in the study. 
 
6.4 Dose Regimen 
 
I004 or Novolog will be drawn from vials by an unblinded staff with commercially available, 
appropriate insulin syringes (eg, Becton and Dickinson products) using standard sterile methods. 
The purpose of using insulin syringe dosing is to maintain blinding and consistency of dosing 
methodology across treatments. 
The dose will be 0.2 units/kg at each dosing (Day 1/ Treatment Period 1 and Day 1/ Treatment 
Period 2), based on the body weight measured at Day 1 of Treatment Period 1 under fasting 
condition. 
 
Dose calculation: 
 
To calculate the amount of insulin for each subject, the body weight in kilograms (kg) measured on 
Day 1 of Treatment Period 1 under fasting condition will be determined (rounded to two decimal 
places) and the amount of insulin calculated will be rounded up or down to the nearest whole unit. 
 
I004 or Novolog dose = 0.2 units/kg x _ _. _ _ kg = _ _ units. Doses will be rounded to nearest 1 
unit (0.01ml). 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Example: A subject with a body weight of 74.90 kg will receive 15 U insulin (74.9 × 0.2 = 14.98, 
which is rounded up to 15) 
 
Standard rounding rules (0.49 unit round down, 0.50 unit round up) will be used. IP will be drawn 
with insulin syringes that have a 0.01 mL (one unit on insulin syringe) gradation. 
 
The IP will be administered while subjects are under observation in the clinical research by qualified 
staff. The investigational product will be administered (at time-point zero) via subcutaneous 
injection into the abdominal wall into the peri-umbilical area using a standardized skin-fold 
technique. An area of skin approximately 5 cm lateral to the umbilicus on either side of the abdomen 
will be cleaned and used for the insertion of the needle. The needle will be promptly inserted at an 
approximately 45°degree angle into a lifted skin fold. The IP will be administered, and the needle 
should remain in the skin for at least 20 seconds after administration to reduce the possibility of 
leakage. The needle will be very slowly withdrawn or may be held for a few seconds before it is 
fully withdrawn to minimize leakage of IP. The injection site will be marked with a pen. 
 
Every new injection should be administered in a new location, rotating around the umbilicus of the 
abdomen. The time of each dose will be recorded in the source documents and on the eCRFs. 
 
For detailed instructions please see Novolog Product Insert.9 
 
6.5 Overdose 
 
If a study medication error occurs, it should be documented as Protocol Deviation. A brief 
description should be provided in the deviation, including whether the subject was symptomatic (list 
symptoms) or asymptomatic, and the event accidental or intentional. 
 
Dosing details should be captured on the Electronic Case Report Form. If the subject takes a dose 
of Study Drug that exceeds protocol specifications and the subject is symptomatic, then the 
symptom(s) should be documented as an AE and be reported. 
 
Should an overdose occur, the Investigator or designee should contact the Sponsor within 24 hours. 
6.6 Randomization and Blinding 
 
Subjects will be randomly assigned to one of two treatment sequences (TR or RT), where treatments 
will consist of single SC IP administrations: 
 
• Treatment T: 0.2 units/kg I004; Solution for subcutaneous injection, 100 units/mL 
 
• Treatment R: 0.2 units/kg Novolog; Solution for subcutaneous injection, 100 units/mL 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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 after consulting with the PI and, if possible, with the Sponsor. 
Drug identification information is to be unblinded only if this is required for subjects’ safety. 
 
In the event that an unblinding has occurred, the circumstances around the unblinding (e.g., date, 
time and reason) must be documented and the clinical monitor notified as soon as possible. Only the 
Principal Investigator or delegate and the respective subject’s code will be unblinded. Study site 
personnel and Sponsor personnel directly associated with the conduct of the study will not be 
unblinded. 
 
If the study site needs to unblind a subject, the sponsor will, if possible, be contacted prior to 
breaking the blinding. In all cases, the Study Monitor must be notified within 24 hours after 
emergency unblinding. 
 
6.8 Auxiliary Supply 
 
will supply laboratory material necessary for PK sampling, all the safety hematology, 
biochemistry, and urinalyses (incl. pregnancy test) in collaboration with the specific laboratory. 
 
 will also provide all necessary material needed for the glucose clamp procedure. 
Glucose, sodium chloride solutions, heparin, syringes, and needles used in the clamp will be 
provided by 
. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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7.0 
STUDY PROCEDURE 
 
7.1 
Informed Consent 
 
Written informed consent will be obtained from each subject prior to performing any study-specific 
evaluations. The informed consent document is subject to review and approval by the Sponsor and 
will be approved by a qualified IRB. Only the most recently IRB-approved informed consent 
document must be used to consent prospective study subjects. The Investigator or a person 
designated by the Investigator and under the Investigator’s responsibility, will fully inform the 
potential study subject of all pertinent aspects of the clinical study, including written information 
given approval/favorable opinion by the IRB/IEC. They will have the opportunity to ask questions. 
If the subjects wish to participate in the study, they must sign and date an ICF. 
 
7.2 Screening 
 
Subjects who have signed the informed consent, will be assigned a Screening ID, as 
 
“SI4-B-mm”, where S stands for Screening, I4-B represents “I004 Study B”, and mm is the 
Screening sequential number. 
 
If subjects are fasting (only water for ≥10 hours), all Screening assessments may be done on the 
same day. If subjects are not fasting, they will be invited to return for a second Screening visit to 
complete any missing Screening procedures (e.g. laboratory assessments). 
 
Investigators must account for all subjects who sign informed consent forms. The Investigator will 
keep a Subject Screening Log at the site. 
 
Subjects who have screen failed may be allowed to re-screen once at the discretion of the 
Investigator. A new Screening number will be assigned. 
 
Subjects who meet all inclusion criteria and none of the exclusion criteria are eligible for this study. 
Subjects will be invited for the in-house treatment periods. They will be instructed on factors 
influencing insulin sensitivity such as refraining from strenuous exercise for three days prior to 
dosing, avoidance of alcohol and herbal supplements, caffeinated meals and drinks, smoking or 
medication and illness/infection as stated in section 5.4. 
 
 
7.3 Demographics and Medical History 
 
Demographic information and medical history, including smoking status, and medication history 
will be obtained at Screening. 
The history of prior insulin exposure or use will be documented.  

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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7.4 Physical Examination 
 
The baseline physical examination will consist of the following body systems: (1) eyes; (2) ears, 
nose, throat, neck, thyroid; (3) cardiovascular system; (4) respiratory system; (5) gastrointestinal 
system, mouth; (6) dermatologic system; (7) extremities; (8) musculoskeletal system; (9) central and 
peripheral nervous system and (10) lymph nodes. 
 
A physical exam will be performed by an Investigator (or a qualified physician at the investigational 
site) at time points indicated in the SOE (Appendix A). An additional physical examination may be 
performed in case the subjects have symptoms or at the discretion of the Investigator. 
 
7.5 Height, Weight, and BMI 
 
Height (without shoes) will be measured once, during the Screening Visit. 
 
Weight (without shoes) will be measured fasting, in the morning, with light clothing and post void 
at time points indicated in the SOE (Appendix A). 
 
BMI (kg/m2) will be calculated from height and weight. 
 
The body weight at Day 1, Treatment Period 1 under fasting condition will be used to calculate all 
further drug dose for the following clamp experiment. Therefore, the body weight will be recorded 
as stated in the SOE (Appendix A) and should stay within the range of 5 % throughout the study. If 
body weight is outside the range, subject will be rescheduled. 
 
7.6 
Vital Signs 
 
Vital signs will include body temperature (aural), supine blood pressure (after 5 minutes resting), 
respiration rate and pulse rate (after 5 minutes resting). Vital sign measurements will be performed 
at days indicated in the SOE (Appendix A). 
 
On clamp days, vital signs will be measured at baseline which is 15 min (± 5 min) prior to dosing, 
at 5 (± 3 min) minutes, 60 min (± 10 min), 180 min (± 20 min) and 720 min (±30 min) post-dose. 
 
Additional measures will be performed as deemed necessary by the Investigator. 
 
7.7 
Concomitant Illness and Therapy 
 
Concomitant therapy is any medication given in addition to the investigational product (including 
over-the-counter medications, herbal medications, and vitamin supplements) administered between 
Screening and follow-up. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Concomitant illness is any significant medical condition or disease that is present at study start 
(signing of informed consent). This includes clinically significant laboratory, electrocardiogram 
(ECG), or physical examination abnormalities noted at Screening examination. 
 
Details of all concomitant illnesses and therapies must be recorded at study entry/Screening and 
must be recorded on the subject’s CRF. Any changes in concomitant medication must be recorded 
at each visit. If the change influences the subject’s eligibility to continue in the study, the Sponsor 
must be informed. The information collected for each concomitant medication includes, at a 
minimum, start date, stop date or continuing, and indication. 
 
AEs related to administration of these therapies or procedures must also be documented on the 
appropriate eCRF. 
 
7.8 
Procedures for Clinical Laboratory Samples 
 
All samples will be collected in accordance with acceptable laboratory procedures. 
Laboratory samples will be taken as described in the SOE (Appendix A). 
 
Table 7-1 Clinical Laboratory Assessments 
Hematology 
Serum Chemistry 
Urinalysis 
CBC with differential: 
Hepatic function panel: 
Routine urinalysis with 
Hematocrit 
Alanine aminotransferase 
microscopic examination on 
Hemoglobin 
(ALT/SGPT) 
positives(a): 
mean corpuscular volume 
albumin, serum 
Color 
(MCV) 
alkaline phosphatase, serum 
appearance, 
mean corpuscular hemoglobin 
aspartate aminotransferase 
specific gravity 
(MCH) 
(AST/SGOT) 
pH 
mean corpuscular hemoglobin 
bilirubin, direct 
protein 
concentration (MCHC) 
bilirubin, total 
glucose 
red cell distribution width 
protein, total, serum 
ketones 
(RDW) 
occult blood 
percentage and absolute 
Renal function panel: 
leukocyte esterase 
differential counts 
Albumin, serum 
Nitrite 
platelet count 
BUN 
bilirubin 
red cell count (RBC) 
BUN: creatinine ratio 
urobilinogen 
white blood cell count (WBC) 
calcium, serum 
carbon dioxide, total 
chloride, serum 
creatinine, serum 
glucose, plasma (c) 
phosphorus, serum 
potassium, serum 
sodium, serum 
Additional parameters: 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Lactic acid dehydrogenase 
(LDH) 
Gamma-Glutamyl Transferase 
Magnesium 
Uric Acid 
Free fatty acid (FFA) 
Diagnostic Screening 
Serum/Plasma/Whole Blood 
Urine 
Breath 
HBsAg 
Drug Screen Profile 
Alcohol breath test at timepoints 
Anti-HCV 
Urine drug screen, including 
stated in the SOE at the 
TSH (b) 
marijuana, at timepoints stated 
investigational site 
PTT 
in the SOE via commercial kit at 
INR 
the investigational site. 
HbA1c 
Anti-HIV-1 
Anti-HIV-2 
 
 
 
Urine pregnancy testing via 
Female Subjects Only 
commercial kit at site. 
human chorionic gonadotropin 
(hCG) as stated in SOE 
 
follicle-stimulating hormone 
(FSH) test for postmenopausal 
 
women (defined as amenorrheic 
female subjects < 60 years of age 
and not surgically sterile) at 
Screening. 
 
 
(a) 
These tests are done on all routine urinalysis and if protein, leukocyte, occult blood, and nitrites are all 
negative, microscopic examination is not performed; just the above parameters are reported. 
(b) 
In the event of abnormal TSH, Free T3/T4 may be collected at Investigator discretion. 
 
(c) 
Fasting Plasma Glucose at Screening in-house, test via YSI 
 
The designated laboratory will perform all necessary laboratory tests listed above. The results of 
laboratory tests will be sent to the Investigator or designee, who is responsible for reviewing these 
results. All laboratory safety data will be faxed or transferred electronically. 
 
Laboratory reports must be signed and dated by the Investigator or designee indicating that the report 
has been reviewed and any abnormalities have been assessed for clinical significance. 
 
All clinically significant laboratory abnormalities detected after Screening must be recorded as an 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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AE. A clinically significant laboratory abnormality may be verified by retesting and may be 
followed upon discretion of the Investigator. 
 
Unscheduled visit for safety laboratory testing is allowed and at the discretion of PI during the 
COVID-19 pandemic. 
 
7.9 
Contraception 
 
Females must be non-pregnant and non-lactating, or either surgically sterile (e.g., bilateral tubal 
ligation, hysterectomy, bilateral salpingectomy, bilateral oophorectomy) or post-menopausal for 
>12 months. The site will make an effort to retrieve medical records to document the sterility, 
however, the absence of records will not exclude the subject. In the event that medical records cannot 
be obtained, serum pregnancy testing will be conducted at Screening, and serum and urine 
pregnancy testing will be conducted throughout the study. Postmenopausal status will be confirmed 
through testing of FSH levels outside the normal range (as specified by responsible lab) at Screening 
for amenorrheic female subjects <60 years of age. 
 
Female subjects must use an adequate method of contraception, defined as adequate hormonal 
contraception to have started at least 7 days prior to the Screening visit, or an intra-uterine device to 
have been in place for at least 2 months prior to the Screening visit, and throughout the study 
(including washout intervals between treatments periods). 
 
During the course of the study, regular human chorionic gonadotropin (hCG) pregnancy tests will 
be performed for sterilized women. In addition to a negative serum hCG pregnancy test at Screening, 
subjects also must have a negative urine hCG pregnancy test at check-in to in-house periods and at 
the follow-up visit. 
 
7.10 Pregnancy 
 
Women known to be of childbearing potential are allowed to participate in the study. In the event a 
subject becomes pregnant during the study, she should be withdrawn, and the study drug should be 
immediately discontinued. However, the subject will be encouraged to complete the post-treatment 
follow-up portion of the study to the extent that study procedures do not interfere with the pregnancy. 
 
If the pregnancy occurs at any time during the study and the 30 days of last dose of active study 
medication, the pregnancy should be reported immediately to the Sponsor, using a pregnancy 
notification form. 
 
Study subjects will give consent on enrollment that the Investigator will report any pregnancy during 
the study to the Sponsor and that they will be asked to provide information about her pregnancy, 
delivery, and the health of her infant until age one month. Payment for all aspects of obstetrical care, 
child, or related care will be the subject’s responsibility. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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All reported pregnancies will be followed up to final outcome, using the pregnancy and pregnancy 
follow-up forms. The outcome, including any premature termination, will be reported to the 
Sponsor. An evaluation after the birth of the child may also be conducted. 
 
Pregnancy complications must be recorded as adverse event(s). If the infant has a congenital 
anomaly/birth defect this must be reported and followed up as a serious adverse event. 
 
7.11 ECG Procedure 
 
A standard 12-lead ECG will be recorded after 5 minutes in a supine position. The Investigator (or 
designee) will interpret the ECG’s by use of an electronic measurement using the following 
categories: within normal limits, abnormal but not clinically significant, or abnormal with clinical 
significance. ECG’s are performed according to the SOE (Appendix A). The following parameters 
will be recorded from the subject’s ECG trace: heart rate, QT interval, PR interval, QRS interval, 
RR interval, and QTc (corrected) using the Fridericia correction (QTcF = QT ÷ cube root of the R-
R interval [where R-R is the duration of the entire cardiac cycle]). 
 
When ECGs are to be collected at the same time point as a blood collection, ECGs should be 
collected first to avoid any artificially increased heart rates due to the blood collection. 
 
On clamp days, ECGs will be performed at baseline which is 15 min (±5 min) prior to dosing, at 5 
min (±3 min), 60 min (±10 min), 180 min (± 20 min) and 720 min (±30 min) post-dose. 
 
ECGs will be performed at days indicated in the SOE (Appendix A). 
 
In some cases, it may be appropriate to repeat abnormal ECGs. If a machine-read QTc value is 
prolonged, repeat measurements may not be necessary if the Investigator’s interpretation determines 
that the QTc value is in the acceptable range. 
 
7.12 Fasting Plasma Glucose 
 
Fasting plasma glucose will be measured during the in-clinic period with an YSI 2300 STAT glucose 
analyzer at time points stated in the SOE (Appendix A). 
 
7.13 Standardized Meals 
 
At each period, the importance of maintaining the body weight within a 5% range throughout the 
study will be explained. The counseling for each subject will be individualized based on their actual 
measured body weight. Additionally, subjects will be counseled to maintain their normal diet and 
exercise regimen. They will be instructed not to start any new diets, supplements, or exercise 
programs during the study. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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During in-house period(s), subjects will receive standardized meals. Meal will be provided after the 
clamp procedure has been terminated. 
 
7.14 Euglycemic Glucose Clamp 
 
7.14.1 Biostator® Overview 
 
The Biostator Automated Glucose Analyzer (MTB Medizintechnik, Amstetten, Germany) is a 
510(k)-cleared device that automates the glucose clamp technique providing continuous assay of 
blood glucose measurements and minute-by-minute adaptations of glucose infusion rates (GIR). 
 
In the clamp scenario, the Biostator automatically calculates and infuses the appropriate amount of 
Dextrose 20%, based on the actual measured blood glucose and its deviation from target level. The 
Biostator analyzer, using an internal algorithm, makes minute by minute adjustments to GIR, as 
necessary, to keep the blood glucose concentration at the target level. Adjustments are done per 
current blood glucose measurement and the variability observed during the last five readings. 
 
7.14.2 Glucose Clamp Set-Up 
 
The Biostator is set up with infusion lines for blood glucose management. One 0.9% sodium chloride 
(NaCl) and two Dextrose 20% (D20) IV infusion lines are connected via an infusion module. All 
three lines are linked together using three-way valves which are then connected to the subject 
through an IV catheter on the contralateral arm (contralateral and ipsilateral are defined as relative 
to Biostator). A second IV line is inserted (generally the dorsal hand vein, the lateral wrist vein of 
ipsilateral arm or any peripheral arm vein) and setup through a different Biostator module using a 
Double Lumen Catheter (DLC). One lumen is used for a continuous blood sample draw at an 
estimated rate of 2 mL/hr; while the other lumen, is used to infuse a low concentration of heparinized 
saline solution to the catheter tip where it is then mixed with the blood drawn. This mix avoids 
sample clotting within the tubing. The DLC vein site is covered by a heating pad for arterialization 
of venous blood flow. The Biostator system programming and operation is done per 
 
standard operating procedures. An external D20% IV line is connected to the infusion lines. This 
infusion is manually operated by the technicians running the clamp in coordination with the medical 
staff and is used (e.g., in case the GIR delivered by the Biostator is insufficient). 
 
As a background validation with a second methodology for safety, the standardized measurements 
of the Biostator will be verified on a regular basis using the YSI reference (YSI 2300 STAT or 
equivalent YSI Glucose Analyzer; Yellow Springs Instruments, Ohio, USA) device at 
approximately every 30 minutes. 
 
Please see Appendix B for a detailed Clamp Sampling Schedule. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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7.14.3 Glucose Clamp Procedure 
 
During the study, subjects will undergo two euglycemic clamp procedures, one per treatment period. 
All clamp procedures will be performed identically. 
 
Subjects will be connected to the Biostator through peripheral arm veins as described in section 
7.14.2. The clamp procedure will follow instructions described in section 4.2. 
 
The Biostator will be turned on and programmed. The body weight in kilograms used for Biostator 
programming on both clamps, corresponds to the weight measurement obtained on Day 1 of 
Treatment Period 1. At least one hour before dosing (t= - 60 min), the Biostator will start recording 
data. During this pre-dose monitoring period, glucose readings are obtained and analyzed with a 
YSI 2300 STAT or equivalent YSI Glucose Analyzer (Yellow Springs Instruments, Yellow Springs, 
Ohio, USA) at the following time points relative to the start of the trial drug infusion: -60, -30, -20 
and -10 minutes. The average of blood glucose values prior to dosing (-30 min, -20 min, -10 min) 
correspond to each subject’s individual BASELINE glycemic level. The glucose CLAMP TARGET 
level is determined by subtracting 5 mg/dL from the BASELINE glycemic level (Target level = 
BASELINE - 5 mg/dL). Subjects whose differences between initial fasting BG values (-30, -20, -10 
min) exceed the limit of 10 mg/dL will be rescheduled to another day. Additionally, subjects will 
not be dosed if mean fasting plasma glucose is < 75 mg/dL due to proximity to overt hypoglycemia 
or > 99 mg/dL. Subjects may be re-scheduled once to come back to the site on a later day and may 
be dosed if the mean fasting plasma glucose is ≥ 75 mg/dL or ≤ 99 mg/dL prior to dosing. 
 
At t= 0-min, the investigational product will be administered per randomization schedule. 
 
After dosing, the onset of insulin action will be identified by a ≥ 5mg/dL decline of glucose 
concentration from the baseline level. After the onset of insulin action is identified, glucose infusion 
will then be utilized as needed to maintain the target level (± 10%) using a variable infusion of 
D20%. This GIR is calculated and infused by the Biostator. The glucose infusion is continuously 
diluted with saline solution at an approximate rate of 30 ml/hour to avoid the remote possibility of 
localized pain or venous thrombosis as complications. In case a high GIR is necessary, a part of the 
glucose infusion will be given by an external infusion pump. The infusion rate provided by the 
external pump will then be added to the infusion rate of the Biostator. 
 
The subjects will remain fasting and in a supine or semi supine position during the entire glucose 
clamp. The subjects are allowed to sip water throughout the trial and will otherwise remain fasting. 
 
7.14.4 Glucose Clamp Quality Parameter 
 
The quality of the clamp data will be reviewed on a regular basis by the PI and the clamp supervisors 
at regularly scheduled Data Review Meetings. The following parameters are included in the review: 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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• Clamp coefficient of variation (CV): Coefficient of variation of device BG measurements 
calculated by the following formula: 
 
(SD of Device BG / Mean Device BG) * 100 
 
• Clamp deviation from the target (DFT): Mean difference (mg/dL) between device and target 
BG level represented as a percentage, calculated as follows: 
 
[Mean (device BG – target level) / target level] * 100 
 
• Accuracy: Mean difference of all paired BG measurements of device vs reference method 
represented as a percentage. 
 
Calculations for CV and DFT will be based on each clamp from the minute that the glucose infusion 
rate is initiated (Start of GIR) to the minute that the glucose infusion rate is no longer required (End 
of GIR) or at the end of the clamp at the 12-hour mark, whichever appears first. Note that an End of 
GIR is not necessarily an end of the clamp. 
 
In case a subject’s GIR requirement is intermittent or has interrupted periods during the clamp, the 
PI will review the clamp data and determine how the clamp quality will be calculated. 
 
Generally, overall mean clamp CV and DFT for the clamps should be less than 10%. If the calculated 
CV or DFT for an individual clamp is ≥ 12%, the clamp data for that clamp should be considered 
for exclusion from the final data analysis and the clamp should be repeated (if possible), or the 
subject should be replaced, following discussion between the Investigator and the Sponsor. If the 
accuracy calculation is > 12% but the CV and DFT are < 12%, it will be at the discretion of the 
Principal Investigator to determine if the clamp data quality is acceptable. 
Clinical Data 
Management Team and the Sponsor will also be consulted. 
 
 
7.15 Pharmacokinetic Assessments and Schedule 
 
Blood for PK analysis of the IP, the human insulin analog, will be collected at the time points 
indicated in the SOE (Appendix A) and follow the PK blood sampling schedule below. 
 
At each sampling time point, 1.0 mL of blood will be discarded for waste (utilizing a throw away 
tube). Thereafter, one 6.0 mL whole blood sample per scheduled time point will be collected to 
provide a minimum of 1.5 mL of serum for PK measurements and 1.5 mL of serum as a secondary 
back-up sample. Instructions for sample processing and shipment are provided below. 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Table 7-2 PK Blood Sampling Schedule (6 mL per sample) 
 
 
PK Sample Processing and Shipment Instructions are described below: 
 
(1) 
Sampling Procedure 
 
A catheter will be inserted into one of the antecubital region veins for blood sampling. Alternatively, 
collection from a vein from the hand or forearm for easier access or via venipuncture is also 
acceptable. PK blood draws should use the opposite arm of the glucose infusion/clamp. Preferably, 
the arm with better venous access will be used for PK samplings, and the opposite arm for the 
glucose infusion/clamp. In case of extraordinary situations, such as IV infiltration etc, the PK 
samples could be collected from the arm of the clamp, for safety reason. 
 
After each blood sample collection, a catheter flush will be performed to avoid catheter clot 
formation and prevent interference between samples. 
 
At each PK sampling point, the first 1 mL of blood will be discarded for waste (utilizing a disposable 
tube), and blood samples (~6 mL per sample), will be collected in a serum separation tube and left 
to stand at room temperature for 30 - 60 minutes. Each sample will be centrifuged at 2-8°C, 1,000-
1,300 g for 20 minutes. 
 
Isolated serum will be distributed into two (2) 2.0 mL cryo vials, approximately 1.5 mL in the first 
aliquot and the remainder in the second aliquot. The serum samples will be frozen on dry ice and 
then immediately (within 60 minutes following serum isolation) stored in a freezer at -20°C or lower 
Seq. #
Time 
window
Sample 
No.,"XX"
#
Time 
window
Sample 
No.,"XX"
1
-60 min
01
15
75 min
±3 min
15
2
-30 min
02
16
80 min
16
3
0 min
03
17
90 min
17
4
5 min
04
18
105 min
18
5
10 min
05
19
120 min
19
6
20 min
06
20
150 min
20
7
25 min
07
21
180 min
21
8
30 min
08
22
210 min
22
9
40 min
09
23
4 h
23
10
50 min
10
24
5 h
24
11
55 min
11
25
6 h
25
12
60 min 
12
26
8 h
26
13
65 min 
13
27
10 h
27
14
70 min 
14
28
12 h
28
Total
28 (168mL)
Scheduling
Scheduling
±3 min
±20 min
± 5 min
±1 min
±2 min
±5 min
±10 min

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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until analysis. 
 
(2) 
PK Sample ID 
 
The sample ID for PK blood samples (before centrifugation) is defined as: 
 
“I4-B-nn-v-XX” 
 
where I4-B represents study for “I004 Study B”, nn is the subject ID, v is the treatment period/visit 
number and XX is the two-digit time-point sequential number of the sample: 01, 02, 03, …28 as 
listed in Table 7-2. 
 
The sample ID for PK samples (after centrifugation) is defined as “I4-B-nn-v-XX-k” and k is the 
aliquot number (A or B). The PK sample ID labels, with the size of “1/2” × 1¾” (Avery label 5667), 
should be printed in black font as demonstrated below: 
 
“I4-B-nn-XX-k” 
 
(3) 
Sample Transportation 
 
Prior to sample transportation, all samples should be stored in a box and must be organized in 
sequential order by subject and separated by aliquot. The chain of custody, temperature control and 
related documents for sample transportation must be recorded. During transportation of PK samples 
from the site to Amphastar, the temperature should be below -20ºC. 
 
(4) 
Bioanalytical Methods of Insulin Aspart 
 
An automated, high‐throughput method for targeted quantification of Insulin Aspart (I004) in human 
serum includes (i)  mass spectrometric immunoassay (MSIA) for immune-enrichment which is 
based on the Thermo Scientific™ MSIA™ platform and (ii) analysis by UPLC with high resolution 
mass spectrometer (UPLC/HRMS) will be performed.  
 
MSIA™ platform approaches based on immune-enrichment prior to the mass spectrometric 
determination, using  the insulin antibodies immobilized on a solid support first to capture the 
molecule of interest from the matrix, then subsequent washing cycles are used to remove 
contaminants and finally the analyte is eluted prior to its analysis by UPLC/HRMS.  
 
The HR-MS detection provides significant advantages over enzyme linked immunosorbent assay 
(ELISA) methods because the selectivity of MS allows detection of co-eluting analogs. Full-scan 
MS data could be analyzed due to the low noise and selectivity of the MSIA extraction technology. 
Co-eluting insulin analogs were easily separated and identified based on the accurate m/z values of 
each precursor’s charge state and corresponding isotopes. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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The results of the method feasibility study indicated the method is accurate with the LLOQ of 50 
pg/mL or 0.05 ng/mL with a good linearity over the quantitation range. 
 
(5) 
Bioanalytical Methods of Endogenous Insulin 
 
Amphastar developed sensitive and specific UPLC/HR-MS method to detect the endogenous human 
insulin. 
 
Figure 7-1 MSIA Technology Workflow 
 
 
 
7.16 Pharmacodynamic Assessments and Schedule 
 
The pharmacodynamic parameter GIR will be determined from the concentration-time data for all 
evaluable subjects. 
 
Serum C-peptide and endogenous human insulin will be analyzed out of the PK sample at all PK 
sampling timepoints as stated in the PK sampling schedule. Instructions for PK sample processing 
and shipment are provided in section 7.15. 
 
Please see Table 7-2 for PK Blood Sampling Schedule. 
 
7.17 Tolerability Assessments 
 
After injection of the study drug, the injection site will be marked with a pen. Assessment of study 
drug injection site will be performed at 15 (±3) min, at 60 (±10) min and at 720 (±30) min after 
dosing. 
 
The local reaction from the injection site, the insertion site, and the adhesive will be evaluated 
quantitatively using a Draize scale or similar scale by qualified study staff. If an injection site 
reaction like pain on palpation, itching, erythema, edema, induration is observed, it must be recorded 
as an AE and then will be evaluated using the following scale: 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Erythema will be evaluated as follows: 
 
• 0 – No erythema 
• 1 – Very slight erythema (barely perceptible) 
• 2 – Well-defined erythema 
• 3 – Moderate to severe erythema 
• 4 – Severe erythema (beet redness) to slight eschar formations (injuries in depth) 
 
Edema will be evaluated as follows: 
 
• 0 – No edema 
• 1 – Very slight edema (barely perceptible) 
• 2 – Slight edema (edges of area well defined by definite raising) 
• 3 – Moderate edema (raised approximately 1 mm) 
• 4 – Severe edema (raised more than 1 mm and extending beyond the area of exposure) 
 
The diameter of the affected area will be measured with a paper measurement in cm and the 
condition of the injection site will be recorded. Digital photography will be used to document all 
positive injection site reactions. In case of clinically significant injection site reactions, subjects may 
undergo a dermatological consultation. 
 
7.18 
Blood Volume 
 
Total blood sampling volume for subjects will approximately be 390 mL. 
 
Even in case of further unexpected blood sampling, extension of blood sampling period or necessary 
retesting, sampling volume will not exceed 420 mL. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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8.0 
ADVERSE EVENTS 
 
8.1 Definitions 
 
8.1.1 Adverse Event (AE) 
 
An AE is any undesirable and unintended medical event occurring to a subject in a clinical study, 
whether or not related to the study products. This includes events from the first study related activity 
after the subject has signed the informed consent and until post treatment follow-up period as defined 
in the protocol. The following should not be recorded as AEs, if recorded as medical 
history/concomitant illness on the eCRF at Screening: 
 
• Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened from the first study related activity after the subject has signed the informed 
consent 
 
• Pre-existing conditions found as a result of Screening procedures 
 
• Pre-existing events that has not worsened in intensity or frequency from baseline 
 
8.1.2 Treatment Emergent Adverse Event (TEAE) 
 
A treatment-emergent AE (TEAE) is defined as any clinically significant event not present before 
exposure to study drug or any event already present that worsens in either intensity or frequency 
after exposure to study drug. 
 
8.1.3 Clinical Laboratory Event 
 
A clinical laboratory AE is any clinically significant laboratory abnormality that suggests a disease 
and/or organ toxicity and is of a severity, which requires active management, (i.e. change of dose, 
discontinuation of study product, more frequent follow-up or diagnostic investigation). 
 
A laboratory re-test and/or continued monitoring of an abnormal value is not considered an 
intervention. In addition, repeated or additional noninvasive testing for verification, evaluation or 
monitoring of an abnormality is not considered an intervention. 
 
8.1.4 Unexpected Adverse Event/ Unexpected Suspected Adverse Reaction 
 
An unexpected AE/unexpected suspected adverse reaction is an AE or suspected adverse reaction 
that is not listed in the Investigator Brochure or Protocol, as occurring with a class of drugs or as 
anticipated from the pharmacological properties of the drug, but are not specifically mentioned as 
occurring with the particular drug under investigation, or is not listed at the specificity or severity 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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that has been observed; or, if an Investigator Brochure is not required or available, is not consistent 
with the risk information described in the general investigational plan or elsewhere in the current 
application. 
 
8.1.5 Serious Adverse Event (SAE)/ Serious Suspected Adverse Reaction  
 
An SAE is defined as any untoward medical occurrence that at any dose: 
 
(1) 
Results in death. 
(2) 
Is life threatening. 
• The term “life threatening” 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 if it were more severe. 
 
(3) 
Requires inpatient hospitalization or prolongation of existing hospitalization. 
Hospitalization is defined as an admission of greater than 24 hours to a medical 
facility and does not always qualify as an AE. 
(4) 
Results in persistent or significant disability/incapacity. 
(5) 
Leads to a congenital anomaly/birth defect. 
 
(6) 
Is an important medical event that satisfies any of the following: 
 
• May require intervention to prevent items 1 through 5 above. 
 
• May expose the subject to danger, even though the event is not immediately life 
threatening or fatal or does not result in hospitalization. 
 
8.1.6 Life-Threatening Adverse Event/Life-Threatening Suspected Adverse Reaction 
 
A life-threatening AE/life-threatening suspected adverse reaction, in the view of either the 
Investigator or Sponsor, places the patient or suspect at immediate risk of death. It does not include 
an adverse reaction that, had it occurred in a more severe form, might have caused death. 
 
The determination of whether an AE is life threatening can be based on the opinion of the 
Investigator or Sponsor. If either, the Sponsor or investigator believes that the event is serious or life 
threatening, the event must be considered serious and evaluated by the Sponsor for expedited 
reporting (21 CFR 312.32(a) and 312.32(c)(1)). 
 
8.1.7 Severity of AEs 
 
The different categories of intensity (severity) are characterized as follows: 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Mild:  
 
The event is transient, easily tolerated by the subject and does not 
affect the subject’s daily activities. 
 
Moderate: 
 
The event causes the subject discomfort and interrupts the subject’s 
usual daily activities. 
 
Severe: 
 
The event is incapacitating and causes considerable interference with 
                                    the subject’s usual activities. 
 
 
8.1.8 Relationship to Study Treatment 
 
Very Likely Related: There is clear evidence that the event is related to the use of the IP. 
The AE follows a reasonable temporal sequence from 
administration of a drug (including the course after withdrawal of 
the drug), or for which possible involvement of the drug can be 
argued. 
 
Probably Related: 
The AE follows a reasonable temporal sequence from the IP 
administration, and there is a high degree of certainty that the AE 
is related to study drug administration (eg, it represents a known 
reaction to the IP or other drugs in its class or is predicted by the 
known pharmacological properties of the drug). 
Unknown: 
The event follows a reasonable temporal sequence from the IP 
administration, and cannot be reasonably explained by the 
subject’s clinical state or other causes (eg, disease under study, 
concurrent diseases, or concomitant medications). 
Unlikely Related: 
An event for which an alternative explanation is more likely (eg, 
concomitant medications or ongoing medical conditions) or the 
temporal relationship to IP administration and/or exposure suggests 
that a causal relationship is unlikely (for reporting purposes, 
Unlikely will be grouped together with Not Related) 
Definitely Not 
Related: 
An AE that does not follow a reasonable temporal sequence from 
administration of IP and/or that can reasonably be explained by 
other factors, such as underlying diseases, complications, 
concomitant drugs and concurrent treatments. 
 
8.2 Procedures 
 
8.2.1 Collection and Recording of AEs 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Collection of all AEs (serious AEs and non-serious AEs) will commence from the time the subject 
signs the informed consent to participate in the study until the post-treatment follow-up visit. At 
each study visit, the Investigator will assess whether any subjective AEs have occurred. In order to 
avoid bias in eliciting AEs, a non-specific question, such as “How have you been feeling since your 
last visit?” may be asked. Subjects may report AEs occurring at any other time during the study. 
 
All subjects experiencing AEs, whether considered associated with the use of the study medication 
or not, must be monitored and given appropriate medical treatment at the discretion and judgement 
of the Investigator until the symptoms subside and any clinically relevant changes in laboratory 
values have returned to baseline or until there is a satisfactory explanation for the changes observed. 
All AEs will be documented in the AE page of the eCRF, whether or not the Investigator concludes 
that the event is related to the drug treatment. The event term, start and stop dates, severity, action 
taken with study drug and outcome, will be documented, along with the Investigator’s opinion of 
the causal relationship between the event and the IP. 
 
8.2.2 Collection and Reporting of SAEs 
 
When an SAE occur, it should be reported according to the following procedure: 
 
An SAE form must be completed immediately or within 24 hours of first onset or notification of the 
event. The information should be completed as fully as possible but contain, at a minimum a short 
description of the event and the reason why the event is categorized as serious, subject identification 
number, Investigator’s name, name of the study medication and a causality assessment. 
 
In the interest of subject safety, and in order to fulfill regulatory requirements, all SAEs (regardless 
of their relationship to study drug) should be reported to the Sponsor or a designated qualified vendor 
within 24 hours of the study site’s first knowledge of the event. 
 
The collection of SAEs will begin after the subject signs the informed consent form and stop at the 
end of the subject’s follow-up period. An Initial Serious Adverse Event Form should be completed, 
and a copy should be provided to the Sponsor or designated qualified vendor. 
 
All serious and unexpected adverse event reporting will adhere to 21 CFR 312.32 for IND drugs and 
21 CFR 314.80 for marketed drugs (15-day alerts). The Institutional Review Board(IRB) and all 
Investigators will be notified of the alert reports per FDA regulations. 
 
 
8.3 Anticipated AEs 
 
An anticipated AE is defined as any adverse events described in the Product Information or this 
protocol. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Normal precautions taken for a human study, including the provision of emergency equipment, will 
be taken during this study. Qualified and well-trained physicians and medical staff will instruct the 
subjects. Insulin or the preservatives in the insulin products may give rise to allergic reactions. 
 
8.3.1 Drug Related AEs 
 
8.3.1.1 Hypoglycemia 
 
There is a small risk of severe hypoglycemia (requiring assistance of the study staff) when an 
exogenous insulin analog is administered. The glucose clamp will be performed under continuous 
surveillance and frequent monitoring of blood glucose levels as specified. Since there is 
simultaneous infusion of glucose, the occurrence of severe hypoglycemia is unlikely. If a blood 
glucose value ≤ 50 mg/dL is recorded and confirmed by YSI, or the subject has neurogenic 
symptoms (palpitations, tremor, hunger, sweats) or symptoms of neuroglycopenia (confusion, 
lethargy, agitation, reduced level of consciousness) at a BG ≤ 56 mg/dL, the subject must be treated 
at the discretion of the Investigator to prevent or alleviate neuroglycopenic symptoms. Treatment 
may include ingestion of carbohydrates (e.g. apple juice), iv glucose bolus injections, iv glucose 
infusions or other methods as required per Investigator. Subjects will be closely monitored, and it 
will be a clinical decision made by the Investigator to move from oral to iv glucose bolus, to iv 
glucose infusion. Severe hypoglycemia (as defined by the ADA Workgroup13) should be treated at 
the Investigator’s discretion according to best available medical practice. As a safety precaution, a 
glucose solution will be available near the subject. An intravenous cannula will be placed, flushed 
with 2.0 mL sterile saline and maintained in a prominent forearm vein throughout the post-dose PK 
sampling period to enable prompt reliable venous access should iv glucose be required to treat 
hypoglycemia. 
 
Hypoglycemic events will be recorded as adverse events, if symptoms develop and BG is ≤56 mg/dl, 
or if blood glucose measured is ≤ 50 mg/dL, even if no symptoms are present. The subjects will only 
be discharged from the clinic, if deemed safe by the Investigator. 
 
8.3.1.2 Hypokalemia 
 
Insulin products can cause a shift in potassium from extracellular to intracellular space, possibly 
leading to hypokalemia that, if left untreated, may cause respiratory paralysis, ventricular 
arrhythmia, and death. 
 
8.3.1.3 Hypersensitivity and Allergic Reactions 
 
• Local redness, lipodystrophy, swelling and itching at the injection site after I004 or 
Novolog injection. 
 
• Local reaction and generalized myalgia due to metacresol, an excipient in I004 or 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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Novolog. 
 
• Severe, life-threatening, generalized allergy, including anaphylaxis due to the insulin 
product, including whole body rash, dyspnea, wheezing, hypotension, tachycardia or 
diaphoresis. 
 
• Vasovagal response, bruising, tenderness and rarely infection due to venous line. 
 
8.3.2 Procedure Related AEs 
 
8.3.2.1 Catheter Placement 
 
Study procedures involve the placement of a catheter which may lead to allergic reaction, redness, 
swelling, bruising, pain, bleeding or infection at catheter insertion site. 
 
8.3.2.2 Adhesive tape 
 
The study procedures also involved the use of adhesive to secure the placement of medical 
equipment. The adhesive may cause an allergic reaction, redness, swelling or itching when in contact 
with the skin. 
 
8.3.2.3 Blood draw 
 
Subjects will participate in several blood draws throughout the course of the study which have the 
potential to cause a venous line-vasovagal response, bruising, tenderness, and rarely infection. 
 
8.4 Follow-up of AEs and SAEs 
 
All AEs should be followed up and subjects will be rendered appropriate medical care and treatment 
at the discretion of the Investigator until resolution or until the Investigator and Sponsor concludes 
that “further follow-up is not necessary”. If the AE has not resolved by the post-treatment follow-
up visit, the stop date will be recorded as “ongoing.” 
 
All SAEs should be followed up until resolution or permanent outcome of the event or until the 
Investigator and Sponsor judge that further follow-up is not necessary. 
If information is not available at the time of the first report and becomes available at a later date, the 
Investigator should complete a follow-up SAE form at the earliest possible or provide other written 
documentation and fax it immediately within 24 hours of receipt of information to the Sponsor or 
designee. Copies of any relevant data from the hospital notes (e.g., ECGs, laboratory tests, discharge 
summary, postmortem results) should be sent accordingly. 
 
All other non-serious AEs must be followed until the outcome of the event is “recovering” (for 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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chronic conditions), or “recovered”, or until the end of the post-treatment follow-up stated in the 
protocol, whichever comes first, and until all queries related to these AE’s have been resolved. 
 
Safety Reporting to IRBs or IECs, and Regulatory Authorities is described below. 
 
The Sponsor or designated qualified vendor will be responsible for reporting all suspected 
unexpected serious adverse reactions (SUSARs) and any other applicable SAEs to regulatory 
authorities, Investigators and IRBs or IECs, as applicable, in accordance with national regulations 
in the countries where the study is conducted. 
 
Relative to the first awareness of the event by/or further provision to the Sponsor or Sponsor’s 
designee, SUSARs will be submitted within 7 days for fatal and life-threatening events and 15 days 
for other SUSARs, unless otherwise required by national regulations. 
 
The Sponsor or designated qualified vendor will also prepare an expedited report for other safety 
issues where these might materially alter the current benefit-risk assessment of an investigational 
medicinal product or that would be sufficient to consider changes in the investigational medicinal 
products administration or in the overall conduct of the study. The investigational site will forward 
a copy of all expedited reports to his or her IRB or IEC in accordance with national regulations. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
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9.0 
DATA HANDLING AND MANAGEMENT 
 
9.1. Data Quality Assurance and Monitoring 
 
The trial will be monitored according to current Amphastar SOP.  
 
The site monitor will conduct monitoring of the investigational activities for the purpose of ensuring 
subject safety and study compliance to applicable regulatory guidelines.  The Investigator will 
permit Amphastar authorized monitors to access the subject source documents, eCRFs, clinical 
supplies dispensing and storage area and study documentation as frequently as necessary and agrees 
to assist the site monitors with their activities.  The study site will make the eCRFs available, provide 
missing or corrected data and PI will sign the eCRFs. Personal or subject identifying information 
will be treated as confidential and will NOT be publicly accessible.  
The trial information may be reviewed by regulatory authorities or independent QA auditors.  The 
study site may be inspected during or after completing the study. The Investigators agree to allow 
inspectors from regulatory agencies to have access to all trial records, including subject source 
documents. By participating in this study, the Investigator agrees to these requirements and will 
assist the inspectors in their duties. 
 
9.2. Electronic Case Report Forms (eCRFs) 
 
Electronic Data Capture (EDC) system will be used for recording of eCRFs for the conduct of the 
study by Amphastar, eCRF will be used for the purpose of capturing data for all consented 
participants in the study. The 24 hour clock will be used for all time entries. 
 
Subject source documents are the study subject records maintained at the study site. In some cases, 
the source documents may be the hospital’s or the physician’s clinical records. Therefore, the 
information collected on the eCRFs must match those records. Amphastar personnel will review all 
eCRFs to ensure the data is verifiable and accurate. At all times, the PI has final responsibility for 
the accuracy and authenticity of all clinical and laboratory data entered on the eCRFs. The eCRFs 
will be completed as soon as possible after the data become available. 
 
9.3. Clinical Trial Drug Accountability Data 
 
The Study Site Research Pharmacist or designated dispenser acknowledges that the drug supplies 
are investigational and, as such, must be handled in accordance with the protocol and labeling. The 
Research Pharmacist or designated personnel will maintain adequate records showing the receipt, 
dispensing, return, or other disposition of the investigational study material, including the date, 
quantity, lot or code number, and identification of subjects (number, initials) who received or 
returned study drug.  
 
The Research Pharmacist or designated dispenser and Amphastar’s unblinded site monitor will 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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review study supply records, including dispensing and accountability records, at monitoring visits.  
 
It is the responsibility of the investigator and research pharmacist/designated dispenser to ensure 
that, when the study is completed or discontinued, unused supplies of study materials will be 
returned or disposed of, as directed by Amphastar’s site monitors or CRA. Unused study 
medications must be available for verification by Amphastar’s site monitors or CRA. 
 
 
9.4. Trial Documents 
 
The trial documents, including the protocol signature page signed by the investigators, Investigators’ 
curriculum vitae, IRB approval notice and IRB member list, approved Informed Consent Form, 
laboratory verification and institutional accreditation, will be submitted to Amphastar 
Pharmaceuticals, Inc. 
 
9.5. Reporting and Publication 
 
The clinical trial report will be prepared according to the Amphastar Standard Operating Procedure 
for Clinical Trial Reporting.  
 
9.6. Archive 
 
The Investigator will retain a copy of all study documents, including reports to the IRB/Ethics 
Committee and to the sponsor, in accordance with the FDA and local regulations.  The FDA 
regulations and/or ICH guidelines state that the Investigator must maintain study documents for a 
set period of time. The Investigator will arrange for the retention of the Subject Identification for at 
least 15 years after the Final Study Report has been signed. All other documents should be kept for 
the maximum period of time permitted by the institution. 
  
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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10.0 
STATISTICAL METHODS 
 
10.1 Analysis Plan 
A statistical analysis plan (SAP) will be prepared. 
 
10.1.1  Populations 
 
(1) Population for Safety Evaluation – Treated Population  
 
The safety evaluation will be conducted for the treated population, namely all subjects who 
received at least one dose of study drug 
 
(2)  Population for Pharmacokinetic (PK) Evaluation 
 
The PK evaluation population will include all subjects with sufficient evaluable PK data appropriate 
for the evaluation of interest (with no major protocol deviations or violations thought to significantly 
affect the PK of the drug). 
 
(3) Population for Pharmacodynamic (PD) Evaluation 
 
The PD evaluation population will include all subjects with sufficient evaluable PD data appropriate 
for the evaluation of interest (with no major protocol deviations or violations thought to significantly 
affect the PD of the drug). 
 
10.1.2 Analysis of Demographics and Other Baseline Characteristics 
 
Demographic and baseline characteristics will be summarized for all subjects overall and by 
treatment. Summary statistics (e.g., number of subjects, mean, median, standard deviation, and 
range) will be generated for continuous variables (e.g., age and weight) and the number and 
percentage of subjects within each category will be presented for categorical variables. 
 
10.1.3 Analysis of the PK Endpoints 
 
The analyses of the PK endpoints will assess and characterize the difference of the insulin I004 and 
Novolog in regard to the primary and secondary objective and endpoints. 
 
The PK endpoints will be derived from the individual serum insulin I004 and Novolog profiles. 
 
Insulin AUCIA(0-12h) and CIAmax are the primary PK endpoints of interest. Partial AUCs, tIAmax, etc, 
are of secondary interest as listed in Section 2.4. 
 
PK assessments will be analyzed using descriptive and comparative statistical methods. 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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10.1.4 
Analysis of the PD Endpoints 
 
The analyses of the PD endpoints will assess and characterize the difference of the insulin I004 and 
Novolog in regard to the primary and secondary objective and endpoints. 
 
The PD endpoints will be derived from the GIR parameter. 
 
GIR parameter AUCG(0-12h) and Gmax are the primary PK endpoints of interest. Other GIR profiles, 
such as tGmax, are of secondary interest as listed in Section 2.4. 
 
PD assessments will be analyzed using descriptive and comparative statistical methods. 
 
10.1.5 
Safety Analysis and Endpoints 
 
Safety and tolerability of the study drugs will be assessed by collection and review of adverse events, 
tolerability, laboratory parameters, physical examination, vital signs, and ECG parameters 
throughout the duration of the study. Safety analysis will involve examination of the descriptive 
statistics and individual subject listings for any effects of study treatment on clinical tolerability and 
safety. 
 
AEs will be summarized using the treated population. 
 
All AEs will be coded using MedDRA. Data will be summarized using preferred term and primary 
system organ class. 
 
Adverse event summaries will include the overall incidence (by system organ class and preferred 
term), events by maximum intensity, event by relationship to study treatment, events leading to 
discontinuation of study drug, and serious adverse events. Physical exam, vital signs, ECG, and 
laboratory parameters (hematology, chemistry, and urinalysis) will be summarized descriptively by 
treatment. 
 
10.2 Interim Analysis 
 
No interim analysis is planned. 
 
10.3 Determination of Sample Size 
 
 
 
 
the minimal sample size is estimated as 60 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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11.0 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
11.1 Monitoring 
 
The study will be monitored by the sponsor’s monitor or a qualified designee. 
 
Monitoring visits to the study site will be made periodically during the study to ensure that all aspects 
of the protocol and GCP are followed, CRFs are completed correctly, and drug accountability is 
monitored. The Monitor will visit the study site at least once before First Subject First Visit (FSFV) 
(Initiation Visit), at least once during the clinical part of the study, and at least once after Last Subject 
Last Visit (LSLV). Furthermore, the Monitor must be available for discussions by telephone. 
 
The Monitor must be given direct access to source documents, such as original documents, data, and 
records. Direct access includes permission to examine, analyze, verify any record(s) and report(s) 
that are important to evaluation of the clinical study. The study will be monitored to verify integrity 
and validity of the data. Monitoring will follow a Monitoring Plan. 
 
Additionally, QC monitoring of the clinical study for protocol and GCP compliance will be 
conducted periodically by qualified staff of 
 
 
11.2 Protocol Deviations 
 
The Investigator should not deviate from the protocol, except where necessary to eliminate an 
immediate hazard to study subjects. Should other circumstances arise that will require deviation 
from protocol-specified procedures, unless there is an emergency or immediate need, the 
Investigator should contact the medical monitor and Sponsor to review and discuss the implications 
of the deviation and determine the appropriate course of action. Any deviation must be documented, 
stating the reason and date, the action taken, and the impact for the subject and/or the study. The 
documentation must be kept in the Investigator’s Study File and the Sponsor’s Study Master File. 
 
 
 
 
 
 
 
 
 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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12.0 
ETHICAL ASPECTS OF THE STUDY 
 
This study will be conducted in accordance with the Protocol, the International Conference on 
Harmonization (ICH), Guideline for Good Clinical Practice: Consolidated Guidance (E6) and 
applicable regulatory requirements including clinical research guidelines established by the Basic 
Principles defined in the U.S. 21 CFR Parts 50, 56, and 312 and the principles enunciated in the 
Declaration of Helsinki (revised version Fortaleza 2013).14,15 
 
12.1 Institutional Review Board and/or Independent Ethics Committee 
 
Prior to commencement of the study, the protocol, any amendments, subject information/informed 
consent form, any other written information to be provided to the subject, subject recruitment 
procedures, information about payments and compensation available to subjects if not mentioned in 
the subject information, the Investigator’s current CV and/or other documentation evidencing 
qualifications, and other documents as required by the local Independent Ethics Committee 
(IEC)/Institutional Review Board (IRB) should be submitted. Written approval/favorable opinion 
must be obtained from IEC/IRB prior to commencement of the clinical study start. 
 
The Investigator shall provide to Sponsor or its designee a copy of the written and dated 
approval/favorable opinion by the applicable IRB/IEC. 
 
During the study, the Investigator must promptly report the following to the IEC/IRB: Updates to 
IB, unexpected SAEs where a causal relationship cannot be ruled out, substantial amendments to 
the protocol, non-substantial amendments, deviations to the protocol implemented to eliminate 
immediate hazards to the subjects, new information that may affect adversely the safety of the 
subjects or the conduct of the study (including new risk/benefit analysis in case it will have an impact 
on the planned follow-up of the subjects), annually written summaries of the study status and other 
documents as required by the local IEC/IRB. 
 
Substantial amendments must not be implemented before approval/favorable opinion, unless 
necessary to eliminate hazards to the subjects. 
 
The Investigator must maintain an accurate and complete record of all submissions made to the 
IEC/IRB. The records should be filed in the Investigator’s Study File and copies must be provided 
to the Sponsor. 
 
12.2 Regulatory Authorities 
 
Regulatory Authorities will receive the Protocol, Amendments to the Protocol, and reports on SAEs 
and the Integrated Clinical Study Report (CSR) according to FDA regulations. 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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12.3 Responsibilities of the Investigator 
 
The Investigator will conduct this clinical study in compliance with all applicable national, state, 
local or regional laws and regulatory requirements of the countries in which the clinical study is 
performed. The Investigator will align his or her conduct in accordance with the “Responsibilities 
of the Investigator”. All study procedures and assessments may be performed by qualified designees 
per Investigator’s discretion, such as medical doctors (MD), physician assistants (PA) and /or nurse 
practitioners (NP). 
 
In compliance with the Clinical Study Protocol, Good Clinical Practice and applicable regulatory 
requirements, the Investigator must permit auditing by or on the behalf of the Sponsor and inspection 
by applicable regulatory authorities. The Investigator agrees to allow the auditors/inspectors to have 
direct access to his/her study records for review, being understood that these personnel are bound 
by professional secrecy and as such will not disclose any personal identity or personal medical 
information. The Investigator will make every effort to help with the performance of the audits and 
inspections, giving access to all necessary facilities, data, and documents. As soon as the Investigator 
is notified of a future inspection by the authorities, he or she will inform the Sponsor or its designee 
and authorize the Sponsor or its designee to participate in this inspection. Any results and 
information arising from the inspections by the regulatory authorities will be immediately 
communicated to the Sponsor. The Investigator shall take appropriate measures required by the 
Sponsor to take corrective actions for all problems found during the audit or inspections. 
 
Clinical research studies sponsored by the Sponsor are subject to ICH GCP and all the applicable 
local laws and regulations. The responsibilities imposed on Investigators by the FDA are 
summarized in the “Statement of Investigators” (Form FDA 1572), which must be completed and 
signed before the Investigator may participate in this study. 
 
12.4 Informed Consent 
 
Once signed, the original informed consent form, subject authorization form (if applicable), and 
subject information sheet (if applicable) will be stored in the Investigator’s site file. The Investigator 
must document the date the subject signs the informed consent in the subject’s medical record. 
Copies of the signed informed consent form, the signed subject authorization form (if applicable), 
and subject information sheet (if applicable) shall be given to the subject. 
 
All revised informed consent forms must be reviewed and signed by relevant subjects or the relevant 
subject’s legally acceptable representative in the same manner as the original informed consent. The 
date the revised consent was obtained should be recorded in the subject’s medical record, and the 
subject should receive a copy of the revised informed consent form. 
 
 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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12.5 Subject Confidentiality 
 
The Sponsor and designees affirm and uphold the principle of the subject’s right to protection against 
invasion of privacy. Subject records will be kept private except when ordered by law. The following 
individuals will have access to study subject records: Principal Investigator and designees, study 
Sponsor, monitors and auditors, the FDA, other government offices and the IRB. 
 
Throughout this study, a subject’s source data will only be linked to the Sponsor’s clinical study 
database or documentation via a unique identification number. As permitted by all applicable laws 
and regulations, limited subject attributes, such as sex, age, or date of birth, and subject initials may 
be used to verify the subject and accuracy of the subject’s unique identification number. 
 
The Investigator must agree to permit the Sponsor’s monitor or designee’s monitor, representatives 
from any regulatory authority, the Sponsor’s designated auditors, and the appropriate IRBs and IECs 
to review the subject’s source data or documents, including, but not limited to, laboratory test result 
reports, ECG reports, admission and discharge summaries for hospital admissions occurring during 
a subject’s study participation, and autopsy reports. Access to a subject’s original medical records 
requires the specific authorization of the subject as part of the informed consent process. The 
confidentiality of the verified data and the protection of the subjects must be respected during these 
inspections. 
 
Copies of any subject source documents that are provided to the Sponsor must have certain 
personally identifiable information removed. 
 
12.6 Publication, Disclosure, and Clinical Study Registration Policy 
 
The Investigator will provide the Sponsor with truthful, accurate and complete test results and all 
data derived from the study. During the study, only the Sponsor may make study information 
available to other study Investigators or to regulatory agencies, except as required by law or 
regulation. Except as otherwise allowable in the clinical study site agreement, any public disclosure 
(including publicly accessible websites) related to the protocol or study results, other than study 
recruitment materials and/or advertisements, is the sole responsibility of the Sponsor. 
 
 or its designee will be responsible for preparing the Clinical Study Report. When all data 
has been fully analyzed, the Sponsor will communicate the results of the Clinical Study to the 
Investigator(s). 
 
The Investigator or qualified designee agrees to use this information only and strictly in connection 
with this Clinical Study and must not use it for other purposes without the prior written permission 
from the Sponsor. Prior to any publication, the Sponsor must be given the opportunity to review and 
comment upon any manuscript, poster, or paper that contains data derived or generated from this 
study in order to be aware of all written and oral presentations of the data and does not imply any 

 
 
Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                             Version 1.1 (08/05/2021) 
 
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editorial review or restriction of the contents of the presentation or use. 
 
12.7 Insurance and Compensation for Injury 
 
The Sponsor shall carry applicable insurance in the types and amounts necessary to cover its 
obligations herein in accordance with local laws and requirements and/or guidelines for conducting 
clinical studies in any country, unless others have shown negligence. The Sponsor renounces 
liability in the event of negligence or any other liability by the clinics or doctors conducting 
experiments or by persons for whom the said clinic or doctors are responsible. The Sponsor accepts 
liability in accordance with all applicable regulations per the Code of Federal Regulations (CFR) 
and all other applicable federal or state regulations. 
 
Each subject in the study must be insured in accordance with the regulations applicable to the site 
where the subject is participating. 
 
If a subject becomes ill or injured due to an adverse event directly resulting from use of the study 
drug or a study procedure in the course of their participation in this Clinical Study, medical treatment 
will be provided. Sponsor will pay the costs of such treatment. 

Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                              Version 1.1 (08/05/2021) 
 
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13.0 
REFERENCES 
 
1. 
Centers for Disease Control and Prevention. National Diabetes Statistics Report, 2017. 
http://www.diabetes.org/assets/pdfs/basics/cdc-statistics-report-2017.pdf 
 
2. 
Boyle JP, Thompson TJ, Gregg EW, et al. Projection of the year 2050 burden of diabetes 
in the US adult population: dynamic modeling of incidence, mortality, and prediabetes 
prevalence. Popul Health Metr. 2010;8(1):29. 
 
3. 
The Diabetes Control and Complications Trial Research Group. The effect of intensive 
treatment of diabetes on the development and progression of long-term complications in 
insulin-dependent diabetes mellitus. N Engl J Med. 1993;329:977-86. 
 
4. 
Pieber TR et al. Evaluation of a structured outpatient education program for intensive 
insulin therapy. Diabetes Care 1995;18:625-30. 
 
5. 
Lindholm A et al. Improved postprandial glycemic control with insulin aspart: a 
randomized double-blind cross-over trial in type I diabetes. Diabetes Care 1999;22:801-5. 
 
6. 
Gale EAM, the UK Trial Group: A randomized controlled trial comparing insulin lispro 
with human soluble insulin in patients with type I diabetes on intensified insulin therapy. 
Diabetic Med 2000;17:209-14. 
 
7. 
Kotsanos JG et al. Health-related quality-of life results from multinational clinical trials of 
insulin lispro: assessing benefits for a new diabetes therapy. Diabetes Care 1997;20:948-
58. 
 
8. 
Kang S et al. Subcutaneous absorption explained by insulin’s physiochemical properties: 
evidence from absorption studies of soluble human insulin and insulin analogues in 
humans. Diabetes Care 1980;14:942-48 
 
9. 
Product information NovoLog®, revised 03/2021: 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/020986s093lbl.pdf 
 
10. 
EMA: Annex to guideline on similar biological medicinal products containing 
biotechnology- derived proteins as an active substance. Guideline on non-clinical and 
clinical development of similar biological medicinal products containing recombinant 
human insulin and insulin analogues The European Agency for the evaluation of Medicinal 
Products. EMA/CHMP/BMWP/32775/2005_Rev. 1 

Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
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11. 
EMA: Guideline on similar biological products containing biotechnology-derived proteins 
as active substance: Quality issues. EMA /CHMP/BWP/49348/2005 
 
12. 
US Food and Drug Administration. United States Code of Federal Regulation (CFR), Part 
320: Bioavailability and Bioequivalence Requirements. Federal Regulations] [Title 21, 
Volume 5] [Revised as of April 1, 2019] [CITE: 21CFR320]. 
 
13. 
ADA Workgroup Report on Hypoglycemia. Diabetes Care 2013; 36:1384-95. 
 
14. 
World Medical Association. Declaration of Helsinki. Ethical Principles for Medical 
Research Involving Human Subjects. 52nd WMA General Assembly, Edinburgh, 
Scotland, October 2000. Last amended with Note of Clarification on Paragraph 29 by the 
WMA General Assembly, Washington 2002.and Note of clarification on paragraph 30 by 
the WMA General assembly, Tokyo 2004 
 
15. 
International Conference on Harmonization. ICH Harmonized Tripartite Guideline. Good 
Clinical Practice. 01-May-1996 
 
16. 
EMA: Guideline on non-clinical and clinical development of similar biological medicinal 
products 
containing 
recombinant 
human 
insulin 
and 
insulin 
analogues. 
EMEA/CHMP/BMWP/32775/2005_Rev. 1 
 
17. 
Shankar G, Arkin S, Cocea L, et al.: Assessment and reporting of the clinical 
immunogenicity of therapeutic proteins and peptides—harmonized terminology and 
tactical recommendations. AAPS J 2014;16: 658–673. 
 
18. 
FDA Guidance for Industry: Assay Development and Validation for Immunogenicity 
Testing of Therapeutic Protein Products. April 2016. 
 
19. 
Guidance for Industry: Handling and Retention of BA and BE Testing Samples and 
Retention (May 2004) 
 
20. 
Guidance for Industry: Compliance Policy for the Quantity of Bioavailability and 
Bioequivalence Samples Retained Under 21 CFR 320.38 (c) (August 2020) 
 
21. 
Questions and Answers on Biosimilar Development and the BPCI Act Guidance for 
Industry: Guidance for Industry (December 2018) 
 
 

Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                              Version 1.1 (08/05/2021) 
 
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Protocol Signature Page 
 
I confirm that I have read this protocol, I understand it, and I will work according to this protocol 
and to the ethical principles stated in the ICH Good Clinical Practice guideline and adhere to the 
core principles of the Declaration of Helsinki, or the applicable local laws and regulations of the 
study site for which I am responsible, whichever provides the greater protection of the individual. 
I will abide by the publication rules set forth in my agreement with Amphastar Pharmaceuticals, 
Inc. I will accept the monitor's inspection and overseeing of the study. I will promptly submit the 
protocol to the applicable Institutional Review Board/Ethical Committee for approval. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Investigator  
 
 
 
 
Date 
 
 
 
 
 
 
 
 
 
  
Investigator Name (Print) 
 
 
 
 
 
 
 
 
 
Title 
 
 
 
 
 
 
 
 
 
Name of Facility 
 
 
 
 
 
 
 
 
 
Street Address 
 
 
 
 
 
 
 
 
 
City, State  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Representative of  
 
 
 
             Date 
Amphastar Pharmaceuticals, Inc. 
 
 
 

Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                              Version 1.1 (08/05/2021) 
 
72 
 
 
 
 
APPENDIX A 
Schedule of Events (SOE) 
 
1To be performed after second dosing or at early termination 
 
2On each Day 1, vital signs to be measured 15 (± 5) min pre-clamp/pre-dose and at 5(± 3) min, 60 (± 10) 
min, 180 (± 20) min and 720 (± 30) min post-dose. 
 
3On each day 1, ECG 15 (± 5) min pre-clamp/pre-dose and at 5 (± 3) min, 60 (± 10) min, 180 (± 20) min and 
720 (± 30) min post-dose. 
 
4Assessment of reaction at 15 min, 60 min and 720 min post-dose. 
 
5 Unscheduled visits for safety lab testing are allowed during COVID-19 pandemic. 
ASSESSMENT
SCREEN
IN-HOUSE 
PERIOD 1
WASH-
OUT
IN-HOUSE 
PERIOD 2
FOLLOW-
UP1
IP administration
X
X
Informed consent
X
Assess eligibility criteria
X
Demography
X
Medical history
X
History of prior Insulin exposure/use
X
Sequestered in clinic
X
X
Physical examination (PE)
X
X
Hematology, serum chemistry & urinalysis5
X
X
HbA1c
X
Coagulation
X
TSH
X
Hep B, Hep C, HIV
X
FSH if postmenopausal
X
Pregnancy test(serum)
X
Pregnancy test(urine)
X
X
X
Urine drug screen & alcohol breath test
X
X
X
X
Weight
X
X
X
Height, BMI
X
Vital signs
X
X2
X2
X
12-lead ECG
X
X3
X3
X
Standardized meals
X
X
Record concomitant medications
X
X
X
X
Record AEs
X
X
X
X
Assess check-in criteria
X
X
Randomization
X
Injection site assessment
X4
X4
Clamp procedure
X
X
PK/PD assessments
X
X
Blood glucose (YSI)
X
X
X

Amphastar Pharmaceuticals, Inc.                                                                                                   I004 Clinical Study B 
Insulin Aspart Solution (I004)                                                                                          Protocol No.  API-I004-CL-B 
IND 140867                                                                                                                              Version 1.1 (08/05/2021) 
 
74 
 
 
 
Disconnect from Biostator 
1Actual starting time is approximate but nominal timing should be followed in any case. 
2Collection of blood samples for pharmacokinetic assessment at the correct time point relative to dosing is of primary 
importance.