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\begin{document}

\title{A Phase I, single and continuous administration study of Safety,
tolerability and pharmacokinetics of neorudin, a noval recombinant
anticoagulant protein, in healthy subjects}



\author[1]{Yu-bin Liu}%
\author[2]{Mei-xia Wang}%
\author[1]{Xiao-na Dong}%
\author[3]{Jia He}%
\author[1]{Lin Zhang}%
\author[1]{Ying Zhou}%
\author[3]{Xia Xia}%
\author[1]{Gui-fang Dou}%
\author[1]{Chu-tse Wu}%
\author[1]{Ji-de Jin}%
\affil[1]{Beijing Institute of Radiation Medicine}%
\affil[2]{Beijing You’an Hospital, Capital Medical University}%
\affil[3]{Beijing SH Biotechnology Co., Ltd.}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
AIMS: The aims of the study were to evaluate the tolerability, safety
and pharmacokinetics of single and continuous administration of
recombinant neorudin (EPR-hirudin, EH) by intravenous injection in
healthy subjects, and to provide a safe dosage range for phase II
clinical research. METHODS: A single and continuous administration dose
phase I clinical study was conducted. Forty-four subjects were received
EH as single-dose of 0.2-2.0 mg/kg by intravenous bolus plus drip;
Eighteen healthy subjects were randomly divided into 3 dose groups
(0.15-0.45 mg/kg/h) with 6 cases in each group in the continuous
administration trial. RESULTS: Single or continuous doses of neorudin
were generally well tolerated in healthy adult subjects. There were no
serious adverse events (SAEs), and all adverse events (AEs) were mild to
moderate. No subjects withdrew from the trial due to adverse events.
There were no clinically relevant changes in physical examination,
clinical chemistry, urinalysis or vital signs. The incidence of adverse
events was not significantly related to the dose and systemic exposure.
After the single-dose and continuous administration, the serum EH
concentration reached a peak at 0.083h,the exposure increased with the
increase of the administered dose with the mean half-life (T1/2) ranging
from 1.7 to 2.5h, the clearance (Cl) ranging from 123.9 to179.7 mL/h/kg,
and the apparent volume of distribution (Vd) ranging from 402.7 to 615.2
mL/kg. CONCLUSIONS: The safety, tolerability and pharmacokinetics
characteristics of EH can be used to guide rational drug dosing and
choose therapeutic regimens in subsequent clinical studies.%
\end{abstract}%



\sloppy


\textbf{A Phase I, single and continuous administration study of Safety,
tolerability and pharmacokinetics of neorudin, a noval recombinant
anticoagulant protein, in healthysubjects}

\textbf{Liu Yu-Bin1 Wang Mei-Xia2 Dong Xiao-Na1 He-Jia3 Zhang Lin1 Zhou
Ying1 Xia-Xia3 Dou Gui-Fang1 Wu Chu-tse1* Jin Ji-De1*}

1 Beijing Institute of Radiation Medicine,Beijing 100850,China

2 Beijing You'an Hospital, Capital Medical University, Beijing
100069,China

3 Beijing SH Biotechnology Co., Ltd., Beijing 100093,China

* Corresponding author,Wu Chu-tse E--mail:\emph{wuct@bmi.ac.cn} ; Jin
Ji-De E--mail:\emph{jinjide505@163. com}

Keywords:Clinical Trials, Patient safety, Drug metabolism,
Anticoagulants

\textbf{AIMS:} The aims of the study were to evaluate the tolerability,
safety and pharmacokinetics of single and continuous administration of
recombinant neorudin (EPR-hirudin, EH) by intravenous injection in
healthy subjects, and to provide a safe dosage range for phase II
clinical research.

\textbf{METHODS:} A single and continuous administration dose phase I
clinical study was conducted. Forty-four subjects were received EH as
single-dose of 0.2-2.0 mg/kg by intravenous bolus plus drip; Eighteen
healthy subjects were randomly divided into 3 dose groups (0.15-0.45
mg/kg/h) with 6 cases in each group in the continuous administration
trial.

\textbf{RESULTS:} Single or continuous doses of neorudin were generally
well tolerated in healthy adult subjects. There were no serious adverse
events (SAEs), and all adverse events (AEs) were mild to moderate. No
subjects withdrew from the trial due to adverse events. There were no
clinically relevant changes in physical examination, clinical chemistry,
urinalysis or vital signs. The incidence of adverse events was not
significantly related to the dose and systemic exposure. After the
single-dose and continuous administration, the serum EH concentration
reached a peak at 0.083h,the exposure increased with the increase of
the administered dose with the mean half-life (T\textsubscript{1/2})
ranging from 1.7 to 2.5h, the clearance (Cl) ranging from 123.9 to179.7
mL/h/kg, and the apparent volume of distribution (Vd) ranging from 402.7
to 615.2 mL/kg.

\textbf{CONCLUSIONS} \textbf{:}The safety, tolerability and
pharmacokinetics characteristics of EH can be used to guide rational
drug dosing and choose therapeutic regimens in subsequent clinical
studies.

\textbf{WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT?}

Animal studies have suggested that neorudin has a significant
anticoagulant activity and low bleeding feature, as well as detectible
pharmacokinetic properties.

\textbf{WHAT THIS STUDY ADDS?}

This first study in humans provided some further important reference
information about the tolerability, safety and pharmacokinetics of EH in
healthy subjects for next clinical investigation.

\textbf{1 Introduction}

With growing populations and longer life expectancy, we will see an
increase in the prevalence of thrombus formation.\textsuperscript{1}
Thrombotic events can be potentially life threatening and may prolong
the length of hospital stay and result in chronic
disability\textsuperscript{2}. Thrombosis is the most common underlying
pathology of the three major cardiovascular disorders: ischemic heart
disease (acute coronary syndrome, ACS), stroke, and venous
thromboembolism (VTE).\textsuperscript{3} ACS, the acute manifestation
of ischemic heart disease, resulting from coronary arterial thrombus
formation, remains a major cause of morbidity and mortality
worldwide.\textsuperscript{4}VTE, primarily including pulmonary embolism
and deep venous thrombosis, affects an estimated 750,000 people in the
United States each year and causes more than 100,000 deaths annually.
15.4\% of people with thromboembolism died within 90 days after the
index diagnosis.\textsuperscript{5,6}

Low-molecular heparin and low-dose unfractionated heparin can prevent
deep vein thrombosis of the lower
extremities.\textsuperscript{7,8}Unlike heparin, hirudin is an
antithrombotic substance produced by the salivary glands of the
medicinal leech Hirudo medicinalis which can directly act on thrombin
and effectively inhibit both free and bound thrombin. In some animal
models of deep vein injury, hirudin is a more effective antithrombotic
drug than heparin.\textsuperscript{9,10}However, treatment with hirudin
increases the risk of systemic bleeding, which is its main adverse
effect.\textsuperscript{11,12}

Neorudin (EPR-hirudin, EH) is a targeted hirudin variant 2-Lys47(HV2)
fusion protein, which is composed of 68 amino acids, with a theoretical
molecular weight of 7284 Da.\textsuperscript{13-15} EH was developed as
a prodrug of HV2 by introducing EPR (Glu-Pro-Arg), which is recognized
and cleaved by FXIa, into the N-terminal of HV2.\textsuperscript{16} EH
exerts antithrombotic effects by releasing its active metabolite, HV2,
at the thrombus site via FXIa-mediated cleavage of EPR, resulting in
direct inhibition of thrombin. However, when intact, EH does not display
anticoagulant activity. The construction and mechanism of EH determined
that it not only effectively inhibited thrombus formation but also
reduced the risk of bleeding by increasing the specificity and
efficiency of hirudin.\textsuperscript{17,18}

Pre-clinical studies\textsuperscript{19} have shown that EH was
effective and safe in the treatment of thrombosis in rat models of
thrombus formation. Compared with low-molecular-weight heparin and
hirudin, the bleeding side effects of EH were lower when the
antithrombotic effects were similar. After rhesus monkeys were given EH
1.0, 3.0 and 6.0 mg/kg by intravenous bolus and drip, the
half-life(T\textsubscript{1/2}) of EH were 1.2\selectlanguage{ngerman}±0.6, 1.3±0.3 and
1.0±0.1h, and Tmax was 0.05±0.00 h. The clinical indication to be used
of EH was the prevention and treatment of arteriovenous thrombosis.

The purpose of this first-in-human study was to assess the elementary
safety and pharmaco- kinetics of EH in healthy volunteers, and this
study was registered at chinadrugtrials.org.cn (Clinical Trial Registry
number: CTR20160444) {[}PRC CFDA. China drug trials. http://
www.chinadrugtrials.org.cn/eap/clinicaltrials.searchlist?keywords=CTR20160444.
(Accessed 15 March 2020){]}.

\textbf{2 Methods}

\textbf{2.1 Subjects}

Healthy, nonsmoking men or women aged 18--45 years with a body mass
index of 19--26 kg /m\textsuperscript{2} (Weight\selectlanguage{english}[?]50kg) were eligible for
inclusion in the study. The health status was determined by a prestudy
medical history, physical examination and clinical laboratory
evaluations. Women who were nursing or pregnant and subjects who were
infected with human immunodeficiency virus, hepatitis B, hepatitis C
viruses or syphilis were excluded from the study. The subjects were not
allowed to consume cigarette, alcohol or grapefruit-containing products
to screening and during admission and follow-up. The subjects who have
blood donation history or significant blood loss in the past 3 months
(\selectlanguage{english}[?]400ml) were excluded from the study.

\textbf{2.2 Design}

A Phase I, single and continuous administration study of safety,
tolerability and pharmacokinetics of EH by intravenous injection in
healthy subjects was conducted. The study, sponsored by Beijing SH
Biotechnology Co., Ltd. and Beijing Institute of Radiation Medicine, was
performed at the Phase I Unit of Beijing You'an Hospital, Capital
Medical University. Both studies were approved by applicable
institutional review boards/ethics committees and conducted in
accordance with country regulations, the International Conference on
Harmonisation Good Clinical Practice guidelines, and the principles of
the Declaration of Helsinki. Written informed consent was obtained from
all subjects.

\textbf{2.2.1 Determination of the EH dose escalation range}

According to the ''Guidelines for Estimating the Maximum Recommended
Starting Dose of Drugs for the First Clinical Trial of Healthy Adult
Volunteers'' promulgated by the CENTER FOR DRUG EVALUATION and the No
Observed Adverse Effect Level (NOAEL) calculation results of preclinical
animal trials, it was known that the most suitable NOAEL of EH for
long-term toxicity testing of cynomolgus monkeys was 1.3 mg/kg. The
recommended clinical dose of REFLUDAN,\textsuperscript{20}a similar drug
from Bayer, approved by the FDA on March 6, 1998, for the treatment of
HIT (Heparin-Induced Thrombocytopenia) was 0.4 mg/kg intravenous bolus
first, and then 0.15 mg/kg/h of continuous intravenous drip for 2-7
days. Preclinical studies of EH showed that 0.4mg/kg of hirudin was
equivalent to 0.416mg/kg EH, and the pharmacodynamic dose range of EH
animal test was about 0.06\textasciitilde{}2.13mg/kg. The preclinical
mouse and cynomolgus acute toxicity test of this drug showed that its
Maximum Tolerated Dose (MTD) was much higher than the planned clinical
effective dose. It was safe and reasonable to initially consider
0.2\textasciitilde{}2.0mg/kg as the dose increase range of this clinical
trial.

\textbf{2.2.2 Single dose regimen}

In the single-dose trial, 4 subjects were enrolled in the preliminary
trial, and 40 subjects were enrolled in the formal trial. In the
preliminary trial, considering that the test drug was first used in
humans, in order to protect the safety of the subjects to the utmost
extent, at the beginning of the preliminary trial (test drug 0.2mg/kg),
1 healthy subject was enrolled and observed for 7 days to confirm the
safety of EH\selectlanguage{english},then the remaining 3 subjects were re-entered and observed
for 7 days. In the formal trial, the randomized, placebo-controlled,
dose-escalating study was divided into 5 dose group, 8 cases per group
(Test drug: placebo=6:2), and the order of increasing dose was: 0.4,
0.8, 1.2, 1.6, 2.0 mg/kg per administration. The research center
determined the drug grouping information of the subjects according to
the random system. The single administration process was shown in Figure
1.

\textbf{2.2.3 Continuous administration}

There were 3 dose open groups for continuous administration, with 6
cases in each group, and the time of continuous administration was 24
hours. After the administration, the subjects were discharged after 2
days of hospitalization, and the trial ended after the 28th day. In each
dose group, the dose of intravenous bolus (bolus 30 seconds or more) was
0.4 mg/kg, and the increasing range of maintenance dose was 0.15, 0.30
and 0.45 mg/kg/h. One subject in each group was first enrolled, and the
remaining subjects were then enrolled after observing that there was no
safety problem at least 48 hours after the end of the administration. It
was confirmed that there was no safety problem after the last subject in
the previous dose group had completed the observation to at least the
7th day, then the subjects were entered for the next dose group.
Supervising and guiding the process of drug clinical trials was
performed by establishing a data and safety monitoring committee. The
continuous administration process was shown in Figure 2.

\textbf{2.3 Safety assessment}

The following clinical safety assessments were included in the study: a
physical examination, the vital signs (height, weight, blood pressure,
heart rate, axillary temperature and respiratory rate), clinical
laboratory tests (full blood count, urine routine, blood chemistry,
coagulation function, stool routine \& occult blood, 12-lead ECG cardiac
monitoring, chest X-ray examination) and recording of the adverse
events. Adverse experiences were monitored throughout the study. The
investigators evaluated all the clinical adverse events in terms of the
intensity (mild, moderate or severe), duration, severity, outcome and
relationship to the study drug.

\textbf{2.4 Pharmacokinetic assessments}

\textbf{2.4.1 Pharmacokinetic study of single dose}

Forty-four subjects at 6 dose levels of 0.2-2.0 mg/kg participated in
the pharmacokinetic assessment of a single dose. Serum was taken within
5 min before administration, 5min, 15min, 30min after the start of
administration, 5 min, 20 min, 40 min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h,
12h, 24h after the end of the administration to test the blood drug
concentration.

\textbf{2.4.2 Pharmacokinetic study of continuous administration dose}

Eighteen subjects in the 3 dose groups of 0.15, 0.30 and 0.45mg/kg/h
participated in the pharmacokinetics analysis of continuous
administration dose. Serum was taken within 5 min before administration,
5 min, 30 min, 4h, 8h, 12h and 24h after the start of administration, 5
min, 20 min, 40 min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 24h after the
end of the administration to test the blood drug concentration. In the
0.30 mg/kg/h dose group, plasma was also taken at 5 min, 8 h, 24 h and 5
min after the end of administration to test the blood drug
concentration, and urine was collected before administration, 0-4h,
4-8h, 8-12h, 12-16h, 16-20h, 20-24h after administration start, 0-4h,
4-8h, 8-12h, 12-16h, 16-20h, 20-24h, 24-32h, 32-40h, 40-48h after the
end of the administration to test the urine drug concentration.

\textbf{2.5 Data and statistical analysis}

The treatment-emergent adverse events (TEAE) and adverse reactions in
each dose group were summarized according to the System Organ Class and
the Preferred Term. TEAE means an adverse event that occurred at or
after the first medication to 28 days (inclusive) after the last
medication. The severity of adverse events was classified using NCI
CTCAE 4.03.

For safety data such as laboratory, vital signs, electrocardiogram,
physical examination, etc., baseline data, post-dose data, and change
from baseline were summarized according to each follow-up and each dose
group; Regarding whether the test results were normal or not and whether
they were clinically meaningful as classified data, a Shift Table was
used to describe the change from baseline to each follow-up after
administration.

Using the blood/urine PK concentration analysis set, the blood/urine
concentration of each planned time point was descriptively summarized,
and for each dose group, the concentration-time curve of each subject
and the average concentration-time curve of each group was drawn.

The pharmacokinetic data were processed and graphed using computer
programs Excel and origin Pro 8.0. The pharmacokinetic parameters were
calculated using the non-compartmental model (NCA) of Phoenix 64
(WinNonlin6.3). Medical history and adverse events were coded using
MedDRA and past and combined medications were coded using WHO Drug
Dictionary.

Statistical analysis was respectively performed for the single-dose and
continuous administration by the SAS(r) version 9.4 software (SAS
Institute Inc., Cary, NC, USA) and a P-value of 0.05 was considered
statistically significant.

\textbf{3 RESULTS}

\textbf{3.1 Subject characteristics}

In single-dose trial, 44 subjects met the selection criteria were
received medication to obtain safety data and were included in the
safety analysis set. In the continuous administration trial, 18 subjects
met the selection criteria were received medication to obtain safety
data and were included in the safety analysis set. Summary demographic
information about the 62 subjects who completed the study was summarized
in Table1. Healthy, non-smoking men or women average aged 27.4--30.9
years with a body mass index of 21.4--23.81~kg/m\textsuperscript{2} were
eligible for inclusion in the single-dose trial. Healthy, non-smoking
men or women average aged 29.7\textasciitilde{}30.5 years with a body
mass index of 22.65-23.30~kg/m\textsuperscript{2} were eligible for
inclusion in the continuous administration dose.

\textbf{3.2 Safety Results}

The adverse events (AEs) recorded during the entire study were mild to
moderate and well controlled, and most were recovered without treatment.
The incidence of adverse events had no statistically difference (P =
0.62) between the test (20 of 34 subjects) and placebo groups (5 of 10
subjects) in the single-dose study. In total, 51 AEs were reported from
20 of the 34 subjects in test group and fourteen of these were
considered to be related to the EH by the investigator (Table 2). The
incidence of AEs was notably higher in the continuous administration
dose study than in the single-dose study. 68 AEs were reported from 17
of the 18 subjects in the continuous administration dose study, and 58
out of the 68 reported AEs were considered by the investigator to be
related to EH (Table 3). Hypohemoglobinemia was the main AEs recorded in
the single-dose study, while the increase of D-dimer, reticulocyte and
activated partial thromboplastin time (APTT) were the main AEs reported
in the continuous administration study. However, no treatment or
dose-related trend in the incidence of AEs was observed. There were no
serious adverse events (SAEs) reported, no subjects were withdrawn due
to AEs, and there were no AEs leading to death.

\textbf{3.3 Pharmacokinetics}

The serum or urine samples from 62 subjects participated in the
single-dose or the continuous administration dose study were collected
for pharmacokinetics analysis using ultraperformance liquid
chromatography/tandem mass spectrometry method.\textsuperscript{21,22}
However, the data from the ten subjects participated in the placebo
group were not detected and then were ignored.

\textbf{3.3.1 Pharmacokinetics after~single-dose trial}

The mean serum concentration--time data and PK parameters of EH were
shown in Table 4 and Figure 3. Following 0.083h infusion, the serum
concentration of EH reached a peak at the end of infusion, and the
exposure increased with the increase of the dose. The
half-life(T\textsubscript{1/2}) of each dose group changed in the range
of 1.7\textasciitilde{}2.5~h. The Clearance (Cl) of each dose group
changed in the range of 123.9\textasciitilde{}179.7 mL/h/kg. The
apparent volume of distribution (Vd) changed in the range of
402.7\textasciitilde{}615.2mL/kg.

The mean serum concentration of EH gradually increased with increasing
dose, and the characteristics of serum concentration-time data in each
dose group were similar. With the increase of the administered dose, the
exposure of hirudin, the active metabolite of EH, in the serum also
gradually increased. The trend of EH plasma concentration was similar to
hirudin, basically showing a linear law. After 7h of EH administration,
the serum hirudin concentration of each dose group was too low to be
detected. The ratio of the area under curve of the hirudin to the EH was
about 2.4-6.3\%(Table 5).

\textbf{3.3.2 Pharmacokinetics after continuous administration trial}

The mean serum concentration--time data and PK parameters of EH were
summarized shown in Table 4 and Figure 4. The mean serum concentration
gradually increased with increasing dose, and the characteristics of
serum concentration--time data in each dose group were similar. With the
increase of the administered dose, the exposure of hirudin in the serum
gradually increased and the trend of EH plasma concentration was similar
to hirudin, basically showing a linear relationship. The hirudin in each
dose group was below the lower limit of quantification after 28h of
continuous administration, and the EH in each dose group was below the
lower limit of quantification after 36h of continuous administration.
The 0.45 mg/kg/h group had no significant accumulation after continuous
administration, and the accumulation ratio AR\_Cmax was less than 1.25.
At the doses of 0.15mg/kg/h and 0.30mg/kg/h, the cumulative excretion
rates of EH in urine from the start of administration to 48h after the
end of administration were 4.6\% and 9.9\% respectively and the
cumulative excretion rates of hirudin were 0.6\% and 1.2\%(Table 6). The
total cumulative excretion rates of EH plus hirudin were 5.2\% and
11.0\% respectively, suggesting some other metabolites were produced
from EH.

\textbf{4 Discussion}

\textbf{4.1 Safety}

This trial was a first-in-human trial conducted in healthy volunteers to
explore the safety, tolerability, and pharmacokinetics of EH. The action
mechanism and structure determine that neorudin gestates the high safe
and low bleeding charaterestics which have already been proved in
preclinical studies. In this study, the safety and tolerance of EH was
also reconfirmed in healthy subjects within a single-dose of 0.2-2 mg/kg
and a continuous 24-hour administration of 0.15-0.45 mg/kg/h. Although
the level of hirudin in vivo increased with the increase of EH dose, the
level of hirudin was still very low, and almost no bleeding events
occurred. The adverse events were mostly reported in the clinical study
or application of hirudin, including the adverse reactions related to
bleeding and abnormal coagulation tests, such as hypohemoglobin and
reticulocyte count increase,\textsuperscript{23-25} and were soon
recovered after symptomatic treatment or without treatment. The
antithrombotic mechanism of EH was that the nitrogen EPR short peptide
of EH cleaved through FXIa and/or FXa, and the active metabolite hirudin
was released at the microthrombus site, which inhibited activity of
thrombin and the formation of new thrombus. Normally, EH had no
antithrombotic activity due of low FXIa, and antithrombotic activity
could only be produced when the coagulation system was
activated.\textsuperscript{17} This mechanism not only effectively
inhibited thrombosis, but also significantly reduced bleeding side
effects.

Activated partial thromboplastin time (APTT) was an important parameter
to measure the therapeutic effect of hirudin and to change the
therapeutic dose. Previous experiments had shown that hirudin
significantly prolonged APTT, PT and TT, and the duration of APTT was
positively correlated with the plasma concentration of
hirudin.\textsuperscript{26,27} In this study, the prolongation of APTT
was only found in the single-dose trial of 0.2mg group (1 case) and
continuous administration of 0.30mg/kg/h (2 cases) and 0.45mg/kg/h (2
cases). The APTT increase of the case in 0.2mg group was occurred on the
8th day after administration when over 7 half-life elapsed, therefore it
was not related to the study drug. The prolongation of APTT in the
continuous administration all occurred on the day of injection, which
could be related to the EH. However, they were all mild and recovered on
the same day or the next day without any treatment. The slight
prolongation of APTT suggested that a little of hirudin was released
with the increase of EH loading, but the hirudin level in healthy
subjects remained at a low level after continuous administration,
indicating that EH was generally safe in healthy adults.

Furthermore, fibrin D-dimer increase and fibrinogen decrease were found
in several cases during the study. Many
studies\textsuperscript{28-30}indicated~that levels of D-dimer were
typically elevated with acute VTE. However, D-dimer levels may also
increase in a variety of nonthrombotic disorders such as lipemia,
hyperbilirubinemia, and hemolysis. In hospitalized and other acutely ill
patients commonly affected, D-dimer testing had less utility because of
the high frequency of false-positive results. So the meaning of D-dimer
increase in this study need to be further investigated in the next
clinical study. The decrease of fibrinogen in this study was hard to
explained with the mechanisms associated with a reduction in fibrinogen
concentration: hemodilution, consumption, and degradation. However, the
of fibrinogen decrease can be demonstrated by evaluation of D-dimer, the
other fibrin degradation product\textsuperscript{31-33}.

\textbf{4.2 Pharmacokinetics}

EH showed predictable pharmacokinetics after administered intravenously,
with exposures increasing proportionally with dose. The pharmacokinetics
parameters of EH in healthy volunteers were almost consistent with those
found in preclinical study of monkeys and rats.

The concentration of EH in serum reached the peak within 5 minutes after
the end of injection, with exposures (AUC and Cmax) increasing
proportionally with doses between 0.2\textasciitilde{}2.0mg/kg in
single-dose and 0.15\textasciitilde{}0.45mg/kg/h (24h) in continuous
administration. After intravenous administration, the EH was mainly
distributed in the extracellular fluid with an apparent distribution
volume of 402.7-615.2 ml/kg, which was independent of the dosage.

Preclinical animal studies suggested that renal excretion was a major
excretion route of EH, and the excretion in urine accounted for 65.9\%
of the total dose by isotope labeling of \textsuperscript{131}I.
However, in this clinical study, the cumulative excretion rate in urine
of prototype drugs plus hirudin only was less ten percent which
indicated that EH produced other metabolites beside hirudin to excrete
in urine. Indeed, excepting for EH and hirudin, the two major measurable
metabolites in human urine after intravenous administration of EH we
found that nine and ten metabolites were truncated at the C-terminal of
EH and hirudin, respectively. This caused a successive reduction in
amino acids at the C-terminus, which suggested the EH was metabolised to
produce hirudin and other metabolites and that hirudin was metabolised
to corresponding metabolites by the kidney\textsuperscript{17}. These
results suggested that metabolic pathway of EH in vivo was similar to
that of hirudin.\textsuperscript{18} Moreover, the pharmacokinetic
parameters and excretion of EH were also similar with
hirudin.\textsuperscript{20}

The pharmacokinetic parameters of hirudin and recombinant hirudin had
been characterised in healthy volunteers.\textsuperscript{34} Native
hirudin and recombinant hirudin undergo little, if any, hepatic
metabolism. In the studies of hirudin, more than 70\% of the drug was
excreted unchanged in the urine within the first hour after intravenous
administration, and 95\% was eliminated after 5
hours.\textsuperscript{25} After a single intravenous injection in
healthy volunteers, both native hirudin and recombinant hirudin have
rapid distribution phases. The a half-life(T\textsubscript{1/2}) in
studies with healthy volunteers ranged from 0.15 to 1.24 hours. The
maximum plasma concentration (Cmax) was 0.6 to 1.0 mg/L. The volume of
distribution (Vd) of native hirudin and recombinant hirudin ranged from
8.9 to 17.2L.\textsuperscript{34-36} These results suggested that EH was
similar with hirudin in pharmacokinetic parameters.

The ratio of area under the curve of hirudin to EH was 4.0 +- 1.2\%,
indicating that a small amount of EH was cleaved into hirudin, resulting
in the high safety and low bleeding potential of EH. In normal
organisms, the levels of activated FXa and FXIa were
low\textsuperscript{37} in that EH mainly existed in intact form, and
its active metabolite was very low. However, the relationship between
the activity level of hirudin and EH under in vivo hypercoagulable state
was not clear, and further study was needed to establish the dose-effect
relationship of EH in patients.

Interestingly, no hirudin was detected in the plasma samples of the 0.30
mg/kg/h group of continuous administration, while there was no
significant difference in the concentration of EH between plasma and
serum, which indicated that the hirudin in the serum may come from the
process of blood coagulation in vitro accompanying the activated
coagulate factors.

Considering the small sample size, the clinical therapeutic effect of EH
should be confirmed by a future phase II--III study.

\textbf{4.3 Limitations}

There were a few limitations to the conclusions that could be made from
this study. Because subjects were healthy, the pharmacokinetic data
collected represented the best case and did not include variability due
to patient covariates. As the study was performed in a small number of
subjects, conclusions related to safety can only be made for common
adverse events, but not rare adverse events.

\textbf{5 Conclusions}

The results of this study showed that the EH had good tolerance and
safety in Chinese healthy subjects, but the safety and pharmacodynamics
of this drug need to be further studied in thrombotic patients. No
dose-limiting toxicity occurred, no deaths, no serious adverse events
and serious adverse reactions, no adverse events and adverse reactions
leading to withdrawal, and most adverse reactions returned to normal
without intervention. In summary, the tolerance and PK characteristics
of EH support that phase II clinical trials of this drug is performed
and the recommended doses of EH are: 4-11.2mg/kg, and the recommended
way of administration is first intravenous bolus injection of 0.4mg/kg,
and then the dose is maintained at 0.15-0.45mg/kg/h, intravenous drip.

ACKNOWLEDGEMENTS

This study was partially supported by the grant from the Chinese
National Major Projects for New Drug Discovery (No.2018ZX09301010-001).

COMPETING INTERESTS

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

CONTRIBUTORS

Conception and design: Wu Chu-tse and Jin Ji-De

Operation of clinical experiments/acquisition of data: Liu Yu-Bin, Wang
Mei-Xia, He Jia, Zhang Lin, Zhou Ying, Xia Xia

Analysis of pharmacodynamics: Dong Xiao-Na and Dou Gui-Fang

Analysis and interpretation of data: All authors.

Drafting the manuscript or revising it critically for important
intellectual content: all authors.

DATA AVAILABILITY STATEMENT

The clinical data that support the findings of this study are available
from the corresponding author, in agreement with Beijing Institute of
Radiation Medicine, upon reasonable request.

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