WHAT THIS STUDY ADDS
- This
study revealed both the safety and pharmacokinetics of the improved
formulation of a novel drug S-ORT in the healthy volunteers for the
first time.
- Overall, the good safety and appropriate pharmacokinetics of S-ORT in
humans can provide a rationale for the safe and reasonable basis for
the application of this formulation in phase II clinical programs.
1. Introduction
Oxiracetam (ORT), whose chemical name is 4-hydroxy-2-oxo-1-pyrrolidine
acetamide, is the β-hydroxy derivative of Piracetam. It has been
clinically used to treat various cognitive dysfunctions, improve
learning ability, as well as promote and enhance memory[1-6]. Through the interaction with glutamate
receptors, ORT can selectively act on the cerebral cortex and
hippocampus to protect, activate or promote the recovery of nerve cell
functions. However, the drug itself does not display any direct vascular
and central excitatory effects, and the impact of ORT on learning and
memory has been reported to be long-lasting one[7-8]. In addition, this product has been reported
to exert a positive protective effects on craniocerebral trauma,
cerebrovascular injury, and mild to a moderate multiple cerebral
infarction dementia [9]. ORT molecule contains an
asymmetric carbon atom at the 4th position of the
pyrrole ring, which can exist in the two isomer forms: S-ORT and R-ORT[10], and the chemical structural formulas of
these two isomers has been shown in Fig. 1.
Chirality is essential for various biological activities, and most of
the active compounds are chiral molecules [11].
The chemical and physical properties of chiral molecules are basically
the same, while the biological activity, mechanism of action and
toxicity of one form of the enantiomer drug may differ drastically[12-13]. ORT, as a ”racetam” nootropic drug, can
exhibit significant stereospecificity, and subtle differences in
structure that can lead to significant changes in its pharmacological
activities [14]. Previous studies[15] have found that the effects of S-ORT was
significantly better than that of the R-ORT, when inducing and enhancing
the long-term potentiation effect of rat hippocampal slices.
Additionally, in the presence of glutamate, S-ORT could also stimulate
Ca2+ to be taken up into the cerebellum in a
dose-dependent manner, while R-ORT was found to be ineffective[16]. Recent studies [17-18]have shown that compared with R-ORT, S-ORT is the major active
ingredient of ORT, which can reduce cognitive impairment caused by
chronic cerebral hypoperfusion in the rats. The data also suggested that
only S-ORT administration was beneficial for treatment. Therefore, the
development of one single isomer of S-ORT could help to significantly
reduce drug dose, decrease toxicity, improve efficacy, and provide a
more precise relationship about toxicology/toxicokinetics and
pharmacokinetic/pharmacodynamic [19].
Although the use of S-ORT alone may have several advantages as
elaborated above, only racemic ORT is in clinical use currently, and
there are no reports related to the clinical application of S-ORT.
Therefore, this study for the first time explored the PK characteristics
of S-ORT used as an injection in healthy volunteers, analyzed the PK of
single and multiple-dose injection, and also compared the PK difference
of the two enantiomers. At the same time, the safety and tolerance of
this product was also carefully evaluated, thereby providing a safe and
reasonable basis for the clinical use of this formulation in phase II
clinical programs.
2. Methods
2.1 Study design
The study was a randomized, open, single-arm phase I clinical trial and
was divided into three parts (Fig. 2): Part 1 (single-dose
administration), the subjects were randomly divided into three different
groups with 10 people in each group, and they were injected with 2.0 g,
4.0 g, and 8.0 g S-ORT by intravenous infusion, respectively. Each
subject only received one dosage of the drug, and after obtaining the
results of clinical observations and laboratory examinations of the
previous dosage, if no unacceptable AE was found, the next dose was
administered. For the 4.0 g group, the urine was also collected to
calculate the cumulative excretion rate. In part 2 (comparison of
chirality PK), 12 subjects were randomly divided into two groups, one of
which was injected with 3.0 g S-ORT by intravenous infusion during the
first period, thereafter, 6.0 g racemic ORT was injected after a 7-day
washout period. For the other group, the subjects were first injected
with 6.0 g racemic ORT, and then subsequently injected with 3.0 g S-ORT.
In part 3 (multiple-dose administration), 10 subjects were intravenously
instilled with 4.0 g S-ORT once a day consecutively for 7 days. The
ratio of male to female in each group was 1:1.
The volunteers who were screened and qualified for inclusion in the
various groups were monitored by the clinicians during their
participation in the clinical trial. The trial site was located in the
Phase I clinical research ward of Yijishan Hospital of Wannan Medical
College. During the trial, subjects were advised to avoid strenuous
activities, and abstain from smoking, drinking alcohol or other caffeine
and carbonated (e.g., tea, chocolate, coke) drinks. The physical and
laboratory examination results of the subjects were recorded before and
after the test (e.g., blood routine, urine routine, stool routine, blood
biochemistry and related inspection).
2.2 Participants
The study was approved by the former State Food and Drug Administration
(SFDA), and the batch number was 2013L02096. The trial strictly abided
by the rules specified in “Helsinki Declaration” and “Quality Control
of Clinical Trial of Drugs” as issued by SFDA. The research protocol
and its various amendments, the investigator’s manual, the informed
consent and the subject recruitment materials were carefully reviewed
and approved by the ethics committee of Yijishan Hospital of Wannan
Medical College. The eligibility criteria were as follows: Healthy male
and female volunteers with age of 18-40, and with body mass index
ranging from 19-24 were included in this study. Thereafter, based on the
medical history, screening tests (e.g., electrocardiogram, blood
routine, blood biochemistry, urine routine, serum virology examination
and physical examination), qualified volunteers were included in the
trial. However, pregnant and lactating women, people with a history of
drug dependence or mental illness in the past two years, volunteers who
have participated in other drug clinical trials 3 months before the test
were excluded from this study. Besides, those who were found allergic to
the drugs and food, and positive for anti-HIV, anti-HCV, HBsAg tests
were also excluded.
2.3 Drugs
S-ORT for injection packed in 1 g/bottle (batch number: 20140401), was
produced by Nanjing Youke Pharmaceutical Co., Ltd., and provided
by Nanjing Youke Biomedical
Research Co., Ltd. ORT for injection was 1 g/bottle (batch number:
262131043), produced by Shiyao Group Ouyi Pharmaceutical Co., Ltd., and
was provided by Nanjing Youke Biomedical Research Co., Ltd.
2.4 Safety assessments
For Part 1 and Part 2, the blood pressure, pulse, respiration, and
temperature of subjects were measured at 1, 2, 4, 6, 8, 12, 24, 48 h
before and after the drug administration. The complaints and positive
signs of the subjects were also observed and recorded. In Part 3, vital
signs were measured at 1, 2, 4, 6, 8, 12 h before and after
administration from day 1 to day 6. At day 7, the time points of vital
signs detection were similar to Part 1 tests. The complaints and
positive signs of the subjects were again observed and recorded. In
addition, examinations of all the
subjects, including physical examinations, blood routine, blood
biochemistry, urine routine, stool routine and occult blood test,
coagulation function, electrocardiogram and other examinations were
performed upon entering and leaving the group. During the entire study
period, the occurrence of AEs was carefully recorded, and the severity,
duration, results, and possible relationship with drug administration
were evaluated. All the safety data was properly analyzed by detailed
statistical analysis.
2.5 Blood and urine sampling
For Part 1 and Part 2, 4 mL venous blood was carefully collected before
administration (0 h) and after administration 0.17, 0.33, 0.50, 0.75,
1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24.0 h. For part1 4.0 g
dose group, the urine samples of the subjects were collected before
administration (0 h), and after administration at different time
intervals 0-4, 4-8, 8-12, 12-24, 24-36 and 36-48 h. Thereafter, the
amount of urine at each time was recorded. 5-10 mL urine was collected
and stored at -70℃ for further sample analysis. For part 3, 0.4 mL
venous blood was collected on day 1 and day 7 before administration (0
h) and after administration at various time points such as 0.17, 0.33,
0.50, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24 h. 4 mL
blood samples of trough concentration were collected on day 5 and day 6
before administration. All the collected blood samples were placed in
the heparinized centrifuge tubes, centrifuged at 3500 rpm for 5 minutes,
and the upper layer of plasma was frozen and stored at -70 ℃ for sample
analysis.
2.6 Bioanalytical methods
Plasma and urine sample in part1 and part3Instruments: API3200 mass spectrometer (Applied Biosystems
Sciex, USA), Analyst 1.4.2 data processing software;Chromatographic conditions: Lichrospher C18(4.6mm×150mm, 5μm) chromatographic column; Mobile phase:
acetonitrile-water phase (The aqueous phase contained 10
mmol∙L-1 ammonium acetate and 0.1% formic acid)
(85:15). Mass spectrometry conditions: electrospray ionization
source (ESI), positive ion detection mode. The compound parameters for
S-ORT: parent ion: m/z 159.1, daughter ion: m/z 114.0; Piracetam (IS):
parent ion: m/z143.0, daughter ion: m/z126.0. Plasma sample
processing: plasma samples were treated by precipitating the protein
with acetonitrile, and analyzed by HPLC-MS/MS. Urine sample
processing: urine samples was diluted with methanol after being treated
by precipitating the protein with methanol and then analyzed by
HPLC-MS/MS.
Plasma sample in part2 Instrument : Waters high
performance liquid chromatography (HPLC), Waters Empower processing
software; Chromatographic conditions :
CHIRALPAK® ID (4.6mm×250mm, 5μm) chromatographic
column; Mobile phase: n-hexane-anhydrous ethanol (anhydrous ethanol
contained 0.1 % trifluoroacetic acid) (82:18); UV detection wavelength:
210 nm. Plasma sample processing:after the plasma samples were
precipitated protein by acetonitrile, the entire supernatant was blown
dry with nitrogen, dissolved in 300 μL mobile phase and then transferred
into sample bottle for HPLC analysis.
For part 1 and part 3, the linearity of plasma concentration measurement
was found to be 0.5-400 μg∙mL-1, and the urine
concentration was found in the range of 20-2000
μg∙mL-1. For part 2, the linearity of plasma
concentration measurement was 5-400 μg∙mL-1. The
samples that exceeded the limitation of linearity were diluted and then
further analyzed. The analysis method was carried out very carefully,
exactly met the requirements of the verification guidelines for
biological sample analysis as issued by FDA, and all verification
parameters were noted to be within the acceptable range.
2.7 Pharmacokinetic data analysis
The blood concentration of S-ORT at different time points was analyzed
by DAS2.1 software (version 2.1, China) to calculate the PK parameters
of non-compartmental model. The maximum plasma concentration
(Cmax) and the time to reach Cmax(Tmax) was obtained directly from the observed plasma
concentrations. The elimination rate constant ke was
obtained from the terminal slope of the semi-logarithmic drug
concentration-time curve. The elimination half-life time
T1/2 was calculated with ln2/ke.
AUC0-t was determined by the linear trapezoidal method
from the area under the curve within the time from administration to the
lowest blood concentration that could be detected.
AUC0-∞ was the area under the curve from administration
to extrapolation to infinity. In the formula:
AUC0-∞=AUC0-t+Ct/ke,
Ct was the last measurable concentration. Clearance rate
CLz = D/AUC0-∞ (D was dose).
Vz = CL/ke. AUCss, the
AUC at steady state, was equal to AUC0-τ (multiple
doses), which was calculated by the trapezoidal method within the dosing
interval τ. At steady state, trough concentration
Css,min and peak concentration Css,maxwas directly obtained from the measured plasma concentration-time data.
The average steady-state plasma concentration of Cav was
found to be equal to AUCss/τ. The fluctuation
coefficient
DF=(Cmax-Cmin)/Cav ×
100%. The accumulation index Rac was calculated based
on the ratio of the measured value of AUCss on the 7th
day in relation to the measured value of AUC0-24h on the
1st day, and the formula used was Rac=
AUCss/AUC0-24h (single dose). The urine
concentration data was used to determine the cumulative excretion rate
of the drug within the last 48 hours.
2.8 Statistics
The descriptive statistical analysis was performed on the number of
selected subjects, dropouts and removals, demographic statistics and
other baseline characteristics. The safety analysis used descriptive
statistical methods to analyze the degree of AE, the relationship
between duration and drug administration, and outcomes were analyzed on
a case-by-case basis. The incidence rate, number and frequency of AE and
other adverse reactions were also carefully calculated. The main PK
difference between the three doses of a single administration was
analyzed by one-way analysis of variance, and the dose-proportion
relationship about the main PK parameters was analyzed by Power Model[20-21]. The model formula used was: ln (PK
parameter) = α+β*ln (Dose), and the PK parameters included
Cmax and AUC, α was the intercept, β was the slope. The
linear relationship was evaluated according to the 90% confidence
interval (CI) of the slope β. If the CI value included 1, the
relationship of non-linear dose-proportion was considered as not
significant, while the linear dose-proportion relationship could be
established. If the CI was entirely within the judgment interval, the PK
parameter and dose were found to display a linear PK characteristic. If
the CI was noted to be entirely outside the judgment interval, within
the given dose range, the PK parameter and dose were not considered to
display a linear PK characteristic. If the CI overlapped with the
judgment interval, a defined conclusion could not be given within the
administered dose range. The PK parameters correlation between Part1 and
Part3 and the PK difference between male and female were analyzed using
group t -test. The paired t- test was used to analyze the PK
parameters of S-ORT after two periods of intravenous infusion with 3.0 g
S-ORT and 6.0 g racemic ORT. The sample size of the study was the
standard conventional sample size used for evaluating the safety and PK
characteristics in the phase I clinical study, and was not calculated
based on statistical power.
3. Results
3.1 Subject disposition and demographics
The 52 subjects included in the study completed the study as planned,
and no cases dropped in or were eliminated during the study. There was
no significant difference noted in the baseline value of each treatment
group. The demographical and baseline characteristics of the study
subjects have been shown in Table 1.
3.2 Safety
At the end of each dose group, physical examination, vital signs,
electrocardiogram and laboratory test indicators (blood routine, urine
routine, blood biochemistry, blood coagulation function, etc.) of
subjects did not show significant clinical changes. 4 subjects (3 in
Part 1 4.0 g group, 1 in Part 1 8.0 g group) showed abnormal laboratory
test indicators, which was further analyzed by the doctor and found to
have no clinical significance. The 4 subjects came to review on time and
confirmed that they were back to normal state. 1 subject in the Part1
8.0 g group showed abnormal urine test, which was related to menstrual
cycle, and returned to normal after 4 days. During the trial, 1 mild AE
occurred, that is, 1 subject in Part 3 had abdominal distension and
discomfort on the 4th day of the trial, which was
naturally resolved without any medication or other treatments. The study
doctor concluded that the AE might be related to the drug. During the
study, no other SAE occurred in any other group of the subjects.
3.3 Pharmacokinetics
The PK parameters of each dose group of part1 have been shown in Table
2. One-way analysis of variance was used to analyze the PK parameters
between the groups, and the results showed that the main PK parameters,
such as T1/2 and Vz, between the dose
groups were not statistically significant (p>0.05).
However, Cmax, AUC0-t, and
AUC0-∞ increased upon increasing the drug dose, and the
difference between groups was statistically significant
(p<0.05). The average drug concentration-time curve of the
three dose groups in part 1 has been shown in Fig. 3. From the
log-averaged drug concentration-time curve, it could be clearly seen
that the elimination rates of the three dose groups were basically the
same. The linear regression was performed with dose as abscissa, and
with Cmax, AUC0-t, and
AUC0-∞ as ordinates. After spearman correlation
analysis, the correlation coefficient of Cmax-dose was
0.940, P<0.001. The correlation coefficient of
AUC0-t-dose was noted to be 0.941, P<0.001.
The correlation coefficient of AUC0-∞-dose was observed
to be 0.937, P<0.001. The results showed that
Cmax, AUC0-t, and AUC0-∞increased with increasing dose, and had a linear correlation with dose.
The power model was used to evaluate the linearity of PK parameters and
dose. Cmax, AUC0-t and
AUC0-∞ were fitted to the dose by the power model
formula ln (PK)=α+β*ln (Dose), and the β values (90% CI) were 0.837
(0.733-0.942), 0.822 (0.710-0.934), and 0.810 (0.696-0.923),
respectively. For Cmax, AUC0-t and
AUC0-∞, 90% CI of β value did not contain 1, indicating
that in the 2.0-8.0 g dose interval, Cmax,
AUC0-t and AUC0-∞ of S-ORT increased
upon increasing dose, whereas Cmax/dose,
AUC0-t/dose, and AUC0-∞/dose showed a
significant decreasing trend.
Among them, urine of 4.0 g group was collected. Urinary cumulative
excretion rate at 48 h after S-ORT administration was 59.99%±5.41%.
The urinary excretion rate data and profile has been shown in Fig. 4.
The results clearly indicated that most of the S-ORT was excreted
through the urine, and the proportion was approximately 60%.
The trough concentrations before blood sampling from day 5 to day 7, and
24 h after administration has been shown in Table 3. As depicted in the
table, the trough concentrations were almost similar, and analyzed using
analysis of variance. The results showed that there was no statistically
significant difference (P>0.05) and indicated that a steady
state can be reached with a dose once a day for 5 days.
For 4.0 g multiple-dose group, the
PK data on 1st day and 7th day after
administration was compared, and paired t- test was used to
analyze the PK parameters. As shown in Table 4, the main PK parameters,
such as T1/2, Cmax (Css
-max), AUC0-t (AUCss), and
AUC0-∞ displayed no significant difference between day 1
and day 7 (P>0.05). The average drug concentration-time
curve of the 4.0g multiple-dose group on day 1 and day 7 has been shown
in Fig.5. From the Fig.5, it can be clearly noted that the drug
concentration-time curves of single and multiple doses were basically
the same when attaining a steady state. In addition, upon comparing the
logarithmic scale drug concentration-time curves, their eliminations
were found to be basically the same. The accumulation index
Rac was calculated based on the ratio of the measured
value of AUCss after attaining a steady state on the day
7 to the calculated value of AUC0-24h on day 1, and the
result was noted to be 1.05(0.08). The accumulation index was
1.08(0.06), which was calculated with the formula Rac=1/
(1-e-ke*τ). Both of them were found to be basically
the same and approximately close to 1, thereby indicating that S-ORT
could not cause significant accumulation in body with a once-a-day
dosing regimen.
To analyze the effects of gender on PK, the various PK parameters
(Cmax, AUC0-t, AUC0-∞,
T1/2, Tmax, Vz,
CLz) of Part1 and Part3 were subjected to a group based
on genders for t- test, and the results have been shown in Table
5. The results indicated that Cmax,
AUC0-t, AUC0-∞, and CLzin the 2.0 g dose group of part1, Vz of part3 on day 1,
and AUCss, AUC0-∞, CLzof part3 on day 7 showed significant differences between the
genders (P<0.05). The
gender differences in the PK parameters of the remaining dose groups can
be ignored (P>0.05).
The paired t -test was used to analyze the PK difference of S-ORT
after intravenous infusion of 3.0
g S-ORT and 6.0g racemic ORT. The results have been shown in Table 6.
The drug concentration-time curves of S-ORT and R-ORT after intravenous
instillation of 6.0 g ORT were almost identical, as shown in Fig. 6. The
results suggested that the two kinds of ORT isomers in the body had no
obvious interactions and conversions.
4. Discussion
ORT has been used safely in the past, with only few reports related to
major adverse reactions [1,22-23], and patients
may develop anxiety, skin itching, rash, nausea, stomach pain, etc.,
which can subside by itself after discontinuing the drug. The active
ingredient of this product is the S-ORT, which is an improved drug with
relatively low risk, and without any reports of obvious dose-related AE
occurring in the study. During the trial, 1 subject in Part 3 showed
abdominal distension and discomfort on day 4. Interestingly, previous
reports have indicated that administration of ORT racemates could cause
nausea, vomiting, stomach upset and other adverse reactions[24]. Therefore, it was hypothesized that the
correlation between AE and the drug was ”possibly related”, but a
detailed analysis may be required. The severity of AE was mild, and SAE
did not occur during this study.
To establish a chiral separation method for ORT racemates, several
reversed-phase and normal-phase chromatography systems have been
carefully evaluated. When a reversed-phase system was used, the ORT
enantiomers could be properly separated at the baseline. As a
consequence, the normal phase system of Chiralpak ® ID
chiral column for chromatographic separation was used. However, the
mobile phase in the normal phase system can be associated with major
disadvantages such as potential explosion hazard and poor ionization in
the mass spectrometry ion source [25-26].
Therefore, the chiral separation was performed on Waters HPLC.
Currently, there are no specific requirements for the sample size to
conduct the safety and PK study in the phase I clinical trials of drugs.
The sample size of this study is the regular sample size employed in a
phase I clinical study for evaluating safety and PK, and is not
calculated based on the statistical power. However, it is considered
more optimal if the number of subjects can answer their potential
research purposes.
The variance analysis model of dose-proportion correlation analysis
(Table 2) showed that the ratios of the mean exposures of three dose
groups (low, middle and high) were 1:2.07:3.17 (Cmax),
1:1.96:3.08 (AUC0-t), and 1:1.91 :2.99
(AUC0-∞), respectively. The values of
Cmax/dose, AUC0-t/dose, and
AUC0-∞/dose in 8.0 g dose group were observed to be
relatively lower. This study applied the power model to analyze the
does-proportion relationship, for Cmax,
AUC0-t and AUC0-∞, 90% CI of β values
was found in the left of 1. The CI and the judgment interval (calculated
according to the bioequivalence criterion 0.80-1.25) overlapped
substantially, and the defined conclusion could not be reached within
the given dose range, which might be due to the parallel experimental
design, small sample size in each dose group (only 10 cases in each
group), and individual differences in the subjects enrolled for this
study.
In Part 2, after dose correction, Cmax,
AUC0-t, AUC0-∞ were basically consistent
with the results in part1, but T1/2 and
Vz were found to be significantly lower. In the
statistical moment model, T1/2 was calculated by the
elimination rate at the end of the drug concentration-time curve, and
the last time point of sampling appeared to have a greater effect on the
calculation results. However, due to the limitation of detection
sensitivity (the LLOQ was observed to be 5 μg/mL in part 2). Moreover,
the blood concentrations at 12 h and 24 h after administration in part2
were observed to be lower than LLOQ, and the end sampling time for PK
parameter calculation in this group varied from 8 h to 10 h, which might
explain that both T1/2 and Vz were noted
to be relatively lower.
The prototype of this product was found to be mainly excreted through
urine (excretion rate was about 60%). However, the metabolism of this
product in the body has not been extensively analyzed, and the follow-up
study will further explore both the metabolism and the mass balance of
S-ORT in accordance with the proper requirements.
In conclusion, this was the first phaseⅠclinical study to explore both
the safety , tolerance and pharmacokinetics characteristics of the
improved novel drug of S-ORT in the healthy Chinese volunteers. No
safety concerns were identified with the single-dose intravenous
infusion in a range of 2.0-8.0 g and multiple-dose administrations of
4.0 g for 7 consecutive days. These findings could provide valuable
information for the further investigation of S-ORT injection in phase II
clinical studies.
Acknowledgement
The authors wish to thank all subjects who participated in the studies,
and internal and external project teams for their services.
Author Contributions
Clinical study design: H.T.X., J.S., J.S., Y.J.
Clinical study conduct: D.H.L., S.H., Y.W.J.
Pharmacokinetic study: D.H.L., H.S., M.D., X.H.L., B.Y.
Drafting manuscript: D.H.L., H.S.
Editing manuscript: D.H.L., H.S., H.T.X., J.S.
Conflict of Interest Statement
Y.J. and J.S. are employees of the study sponsor, Nanjing Youke
Biomedical Co. LTD. D.H.L, H.S., Y.W.J., M.D., X.H.L., B.Y., J.S.,
H.T.X. are employees of Anhui Provincial Center for Drug Clinical
Evaluation. Haitang Xie was Principal Investigator of the clinical
studies.
Funding Information
The clinical studies were funded by Nanjing Youke Biomedical Co. LTD.
Data Availability Statement
Research data are not shared.
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Tables
Table 1. Demographics and baseline characteristics (mean (SD))
of subjects in part 1, part 2, and part 3