Key words favipiravir - pharmacokinetics -
in vitro In vivo correlation (IVIVC) - ethnic effect.
Introduction
Viruses, unlike bacteria and other free-living microbes, cannot grow in culture.
Viruses must reproduce in a living cell. A virus includes genetic information in the
form of DNA or RNA [1 ]. These genetic
materials are surrounded by a protein coat called a capsid or an outer lipid
envelope [2 ]. A broad variety of diseases are
caused by viruses which may be simple acute disorders or life threatening infections
[3 ]. One of these highly contagious and
pathogenic viruses is SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)
which have appeared in late 2019 caused a pandemic of high mortality respiratory
disease, named COVID-19 (coronavirus disease 2019) [4 ]. This necessitates fast invention of vaccines and drugs. There are
major platforms for vaccine developments as nucleic acid (DNA and RNA), live
attenuated or inactivated virus, subunit (protein, recombinant proteins or
polysaccharide) and viral vectors (replicating or non-replicating) approaches [5 ]. These vaccines may vary in their
effectiveness at reducing the incidence and severity of SARS-CoV-2 infection [6 ]. Despite the effectiveness of vaccines, no
particular drugs have been approved for COVID-19 as of yet [7 ]. Many drugs are demonstrating their
antiviral activity against SARS-CoV-2 by inhibiting the fusion process during viral
entry into the host cells as baricitinib [8 ]
and umifenovir [9 ]. Protease inhibitor drug
like lopinavir is considered a potential candidate for COVID-19 treatment especially
in combination with ritonavir [10 ]. Remdesivir
is a nucleotide analogue prodrug that disturbs viral replication of majority of the
single stranded RNA viruses like coronaviruses [11 ]. Following this family, there is favipiravir, an oral RNA-dependent
RNA polymerase (RdRp) inhibitor which already approved for treatment of influenza in
Japan [12 ]. Favipiravir could be administered
in a safe therapeutic dose to give effective concentration against the SARS-CoV-2
infection [13 ]. It has hydrophobic
macromolecular structures of low solubility. It is slightly soluble at pH 2.0 to 5.5
and sparingly soluble at pH 5.5 to 6.1. Its log P=0.8 [14 ]. For formulation development, its in-vitro
dissolution and the efficiency with which a medication is released from the dosage
form should be established [15 ]. Stability
testing for favipiravir in its pharmaceutical formulation was investigated by
Marzouk et al. [16 ] Determination of
favipiravir in human plasma for bioequivalence studies was reported. [17 ]
[18 ]
Favipiravir pharmacokinetic parameters were estimated in Japanese healthy volunteers
and Cpmax occurred within 2 hours and it is eliminated mainly via
aldehyde oxidase metabolism and partially by xanthine oxidase [19 ], with an elimination half-life of
2–5.5 hours. Patients in the United States had a plasma
concentration levels that was roughly 50 percent lower than those in Japan for the
same dose [20 ]. Due to pharmacokinetic
concerns and varying efficacy outcomes [21 ],
our study is needed to calculate the pharmacokinetic parameters in healthy Egyptian
volunteers to identify how to dose patients treated with favipiravir. Also to
establishing in vitro in vivo correlation (IVIVC) to correlate in vitro drug data to
in vivo performance to waive regulatory requirements for the evidence of in vivo
bioavailability and to set reliable specifications for in vitro dissolution
method.
Materials And Methods
Materials
Favipiravir was kindly supplied by Optrix Laboratories private Limited, India.
Lamivudine, used as an internal standard (IS), was bought from LGC GmbH,
Germany. Potassium dihydrogen phosphate, sodium dihydrogen phosphate, ortho
phosphoric acid, acetic acid, ammonium formate and formic acid were purchased
from Scharlau, Spain. Methanol and acetonitrile (HPLC grade), Sigma Aldrich,
Germany.
Pharmaceutical product
Avigan 200 mg Tablets, manufactured by Toyama Chemical Co., Ltd.,
Japan.
Analytical methodology
For in-vitro dissolution testing: HPLC Alliance/e2695S separation module,
Waters LC system equipped with photodiode array detector (2998 PDA), (USA) was
used. Separation was achieved on Inertsil C18
(250×4.6 mm, 5 µm) column using acetonitrile:
10 mM phosphate buffer (pH 3) (40:60, v/v) as a mobile phase
that was pumped at a flow rate of 1 mL/min. with UV detection at
320 nm.
For in-vivo testing: Waters Acquity UPLC H Class-Xevo TQD system (USA) was used
for assay of favipiravir in human plasma. It was equipped with electrospray
ionization. Chromatographic separation of analytes was carried out on Acquity
UPLC HSS C18 (100×2.1 mm, 1.8 μm) column
using methanol-10 mM ammonium formate+0.1% formic acid in
gradient mode as a mobile phase at a flow rate of
0.35 mL/minute. Other source dependent parameters were cone gas
flow, 50 L/hr; desolvation gas flow, 800 L/hr;
capillary voltage, 2.41 kV, source temperature, 120°C;
desolvation temperature, 550°C. The optimum values for compound
dependent parameters like cone voltage and collision energy were set at
33 V and 15 eV for FAV and 20 V and 15 eV for
IS, respectively. Detection of the ions was performed in the multiple-reaction
monitoring (MRM) mode, by monitoring the transition pairs (precursor to product
ion) of m/z 156 to m/z 113 for FAV, m/z 230 to
m/z 112 for IS. Mass Lynx software version 4.1 was used to control all
parameters of UPLC and MS.
In-vitro dissolution assessment
In vitro dissolution studies were carried out for Avigan 200 mg tablets
using USP Apparatus 2 - Rotating Paddle in three different dissolution media,
namely, pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8. All tests
were done in triplicate.
All dissolution media were kept at 37 º C±0.5
º C at 50 rpm. Samples of 5 mL were
withdrawn from each dissolution medium at time intervals 5, 10, 15, 20 and
30 minutes and were replaced by 5 mL of fresh dissolution medium
to keep the volume constant. Membrane filters of 0.45 µm were
used to filter the withdrawn samples and the first part of the filtrate was
discarded. One mL of the filtrate of each sample was placed in 10 mL
volumetric flask and completed to volume with mobile phase (filtered degassed
mixture of phosphate buffer (pH 3): acetonitrile (60: 40, v/v). A volume
of 20 µL of each sample was injected into HPLC-UV apparatus
using a validated method of analysis to detect the percent dissolved of
favipiravir at 320 nm.
Study subjects and study design
Twenty-seven male healthy Egyptian volunteers were recruited to participate in
this study. Sample size was calculated by SAS software. Demographic data
including age, height, weight and body mass index and physical examinations and
vital signs were examined. All laboratory tests including biochemical,
serological and urine analyses were carried out. One tablet was swallowed by
each volunteer under fasting condition for 10 hours pre-dose. This
clinical study was done in accordance with the Guideline for Good Clinical
Practice of the International Conference on Harmonization [22 ] and the principles of the World Medical
Association’s Declaration of Helsinki [23 ] and was authorized by the ethics committee of Advanced Research
Center (ARC), Egypt. Each volunteer signed an informed consent form written in
lay language to understand his role and rights in this study and the possible
risks [24 ].
Blood sampling
Blood samples were obtained from the volunteers at 0.00 (pre-dose), 0.25, 0.50,
0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3, 3.5, 4, 5, 6, 8 and
10 hours after dose administration. The collected blood samples were
centrifuged at 3500 rpm for 10 minutes and then transferred
directly into 5-mL plastic tubes. The plasma samples were stored at the study
site in an ultra-deep freezer at -80 º C until
measurement.
Pharmacokinetic analysis
The pharmacokinetic parameters (The maximum concentration in plasma
(Cpmax ), time to reach it (tmax ), the area under the
curve (AUC0-t and AUC0-∞ ) and the terminal
elimination half-life (t1/2 ) were calculated by
non-compartmental analysis using Phoenix WinNonlin software (version 8.1;
Certara USA, Princeton, NJ, USA). The AUC0–t was calculated
by the linear trapezoidal rule. The elimination rate constant (k) was estimated
from the slope of the terminal elimination phase of the plasma
concentration-time curve and hence the elimination half-life
(t1/2 ) was calculated by using the formula:
t1/2= 0.693/k. The
AUC0-∞ was calculated using the following formula:
AUC0-∞ =AUC0–t+ Cp*/k
Where Cp* is the last measured concentration.
In vitro in vivo correlation (IVIVC)
The cumulative percent dissolved of favipiravir at 5, 15 and 30 minutes
were correlated with partial areas under the curves of the in-vivo plasma
concentration – time curves namely, AUC0–0.5 ,
AUC0–0.75 and AUC0–1 till reaching the
peak (Cpmax ). This procedure was done for each dissolution medium (pH
1.2, pH 4.5 and pH 6.8). The % dissolved was depicted on x-axis
(independent variable) and the calculated partial areas were depicted on y-axis.
The coefficient of determination (r2 ) and the slope were calculated
by linear regression analysis using Microsoft Excel software to develop level C
correlation [25 ].
Statistical evaluation
The Student t-test was used to determine the significance difference between the
means of two sets of data for in vitro dissolution data. Microsoft Excel
365 was used to perform such a calculation. z-test is used to compare
between 2 sample means [26 ]. These
calculations were done applying special syntax using SPSS software ver. 16.0
(IBM, Armonk, NY, USA).
Results
Chromatographic conditions and analytical method validation
A simple LC method was developed and validated to separate favipiravir from
dissolution medium, additives and/or excipients. Linearity was tested
over a concentration range of 2–28 µg/mL with a
linear regression equation of: Y=58797 X+3081 and
r=0.9998. Where, Y is peak area, X is concentration
(µg/mL), and r is coefficient of correlation. Quantitation of
favipiravir in human plasma samples was achieved by applying a previously
validated UPLC–MS/MS method. The analytical method was validated
in terms of specificity and selectivity, linearity, precision and accuracy,
recovery, matrix effect, dilution accuracy and stability [18 ].
In Vitro Dissolution of Favipiravir
The individual dissolution profiles of favipiravir at three different dissolution
media are presented in [Fig. 1 ]. It was
observed that>97% was dissolved after 5 minutes in all
dissolution media and ~ 100% was dissolved after
30 minutes in both acetate buffer pH 4.5 and phosphate buffer pH 6.8
while 97.19% was dissolved after 30 minutes in pH 1.2.
Statistical analysis by one-way ANOVA showed that there is a significant
difference between the dissolution results of the acid medium pH 1.2 and acetate
buffer medium pH 4.5 (p=0.0368) and there is a significant difference
between the dissolution results of the acid medium pH 1.2 and phosphate buffer
medium pH 6.8 (p=0.0017). It was found that there is also a significant
difference between the dissolution profile of acetate buffer medium pH 4.5 and
phosphate buffer medium pH 6.8 (p=0.0105). The mean dissolution time
(MDT) was calculated to be 2.13, 2.80 and 3.16 minutes for acid medium,
acetate buffer medium and phosphate buffer respectively.
Fig. 1 In vitro dissolution of favipiravir in different
dissolution media (pH 1.2, pH 4.5 and pH 6.8).
Demographic data
Thirty male subjects were screened but 27 subjects completed the clinical study.
All physical examinations and vital signs including blood pressure, pulse and
body temperature showed normal results. All lab results were in normal range.
The mean age of the volunteers was 33.3±10.14 years (20–53
years), mean weight was 73.31±11.44 kg
(49–92 kg), mean height was 172.76±5.41 cm
(165.0–187.5 cm) and mean body mass index was 24.61±3.99
(16.37–31.11).
Pharmacokinetic evaluation
Plasma samples of human volunteers were analyzed by a validated LC-MS/MS method
[18 ]. The average plasma
concentration-time profiles following a single oral dosing of 200 mg
Avigan tablets to 27 healthy Egyptian volunteers is presented in [Fig. 2 ]. The estimated median time
required to achieve the highest concentration in plasma (Tmax ) was
0.75 h and ranged from 0.5 to 2.5 h. The calculated mean maximum
plasma concentration (Cpmax )±Standard deviation (SD) of
favipiravir was 5966.45±1767.26 ng/mL
(3714.66–10793.00 ng/mL). The mean area under the curve
(AUC0-t ) ± SD was calculated to be
12577.14±4967.23 ng.h/mL
(7787.76–29892.55 ng.h/mL) while the mean
AUC0-∞ ±SD was assessed to equal
13325.54±5049.05 ng.h/mL
(8136.41–30642.65 ng.h/mL) and the ratio of
AUC0-t to AUC0-∞ was calculated to be
94.38%. The volunteers showed elimination half-life
(t1/2 ) ranged from 0.87 h to 2.34 h with
an estimated average value of 1.25±0.29 h. It was found that the
estimated pharmacokinetic parameters Cpmax and AUC of favipiravir
were higher in healthy adult Japanese than in American subjects as published in
the Review Report by “Pharmaceuticals and Medical Devices Agency: Avigan
(favipiravir)” [27 ]. The outcomes
of our study were compared to these pharmacokinetic parameters and tabulated in
[Table 1 ].
Fig. 2 Plasma concentration of Favipiravir after oral
administration of one tablet of Avigan® 200 mg Tablets
to 27 healthy Egyptian volunteers.
Table 1 Pharmacokinetic parameters of single oral dose of
400 mg of favipiravir in healthy adult Japanese and American
subjects Vs. Pharmacokinetic parameters of single oral dose of
200 mg in healthy adult Egyptian volunteers.
Pharmacokinetic parameter
Japanese subjects* (400 mg)
American subjects* (400 mg)
Egyptian subjects (200 mg)
Median T
max
, h
0.5
0.6
0.75
Cp
max
, ng/mL
(CV)**
16590 (6.0)
12170 (20.4)
5966.45 (29.62)
AUC
0-∞
, ng.h/mL
(CV)**
39410.00 (16.0)
26740.00 (18.2)
13325.54 (37.89)
t
1/2
,
h±SD***
1.60±0.20
1.40±0.10
1.25±0.29
*Data was retrieved from reference 27.;
** CV is the coefficient of variation.;
*** SD is the standard deviation.
In vitro in vivo correlation
Linear regression models were used to correlate mean AUC0-t values
(in vivo data) with the percent dissolved at different time points
(in vitro data). Understanding the in vitro properties of possible
formulations that could predict their in vivo performance is essential in
pharmaceutical product development [28 ].
The level C IVIVC of AUC0–0.5 , AUC0–0.75
and AUC0–1 values of favipiravir for the various %
dissolved at different dissolution media pH 1.2, pH 4.5 and pH 6.8 at 5, 15 and
30 minutes are shown in [Fig.
3a–c ]. The calculated coefficients of determination
(r2 ) were 0.8798, 0.8926 and 0.9501 for dissolution media pH 1.2,
pH 4.5 and pH 6.8 respectively.
Fig. 3 Level C IVIVC models for favipiravir for mean AUCs vs.
percent dissolved at different dissolution media (a ) pH 1.2,
(b ) pH 4.5 and (c ) pH 6.8.
Discussion
Most people infected with SARS-CoV-2 virus will suffer from mild to moderate
respiratory infection symptoms and recover without the need for additional therapy.
Some, though, will get very ill and require medical treatment. Many antiviral drugs
and monoclonal antibodies were authorized for COVID-19 in persons who are more prone
to get severely ill. The efficacy of favipiravir against several viral infections
has emerged as possible therapy for COVID-19 [29 ]. The pharmacokinetic profile of favipiravir is quite complex [30 ]. Inconsistency in the efficacy outcomes of
favipiravir treatment in many clinical studies can be attributed to a variety of
factors, including research design, demographic, and ethnicity [31 ]. These findings urge to investigate the
pharmacokinetic characteristics of favipiravir in various ethnic groups. Our work
focuses on the examination of Pk profile in healthy Egyptian volunteers and compares
the results of Pk parameters with published data in other ethnic groups. After
completing the screening phase and exhibiting normal vital signs, blood and urine
tests, twenty-seven male healthy Egyptian participants were chosen for the
study.
The pharmacokinetic results revealed considerable similarities between American and
Egyptian volunteers if the values are corrected with respect to the dose. z-test was
used to demonstrates how far one sample data point is far from another known sample
mean using either a known population standard deviation or a sample standard
deviation [32 ]
[33 ]. It was found that there is no significant difference between
Cpmax of Egyptian and American volunteers (p=0.620), while
there is significant difference between Egyptian and Japanese volunteers
(p=0.0001). It was also observed that there is no significant difference
between AUC0-∞ of Egyptian and American volunteers
(p=0.924), while there is significant difference between Egyptian and
Japanese volunteers (p=0.0001). In another study to evaluate the
bioequivalence of two favipiravir oral tablet formulations (200 mg) in
Caucasian adult males under fasting conditions, the observed Cpmax of the
reference product was 5002.171±1231.177 ng/mL and
AUC0-∞ was
10152.115±2507.694 ng.h/mL. Tmax was
0.75 h while t1/2 was 1.319 h [34 ]. The results showed substantial comparisons
between Caucasian and Egyptian volunteers. According to Michael G. and Marc H.,
favipiravir has a highly complex pharmacokinetic profile [21 ]. Physiologic condition can also influence
medication pharmacokinetic characteristics and dosage recommendations [35 ]. Due to limited number of participants,
more clinical pharmacokinetic studies in different ethnic groups are required to
adjust dosing of favipiravir in different patients.
In vitro dissolution is a critical test used in formulation development to
evaluate a drug’s characteristics. It is often used as a predictor of in
vivo performance and is routinely used in quality control to evaluate the
performance of solid dosage forms and to examine the batch-to-batch variation.
There is no official monograph for in vitro dissolution of favipiravir. As a
result, the purpose of this research is to develop an optimum in vitro
dissolution test for favipiravir IR tablets. A novel HPLC method was developed and
validated for in vitro assay of favipiravir. It was realized that there is a
considerable disparity in the dissolution profiles of all dissolution media.
The Level C IVIVC was created by correlating certain Pk parameter as
AUC0-t to the percentage of drug dissolved in three distinct
dissolving media at various times. Because the results showed a better fit [36 ], phosphate buffer medium (pH 6.8) is
recommended as the optimal dissolution medium for level C IVIVC. These findings were
consistent with results obtained by Göktuğ et al. [37 ] who concluded that 900 mL of
phosphate buffer (pH 6.8) maintained at 37.0±0.5°C is
considered as proper dissolution medium for favipiravir tablets if using USP
(apparatus II) at 50 rpm for 30 min. In vitro-in vivo correlations
(IVIVCs) are generally practiced for modified-release (MR) formulations.
Consequently, there are few publications regarding Level C IVIVC for immediate
release (IR) preparations which might be helpful in product development and setting
dissolution specifications [38 ]. Filippos
Kesisoglou et al. demonstrated level C correlation for the disintegration versus
Cpmax of suvorexant immediate release tablets formulated using solid
dispersion methodology. They came to the conclusion that disintegration time might
be used as a substitute for dissolution in the establishment of IVIVC [39 ]. Dissolution data at certain times versus
in vitro mean dissolution time, were used to create highly predictive level C IVIVC
models for IR metformin tablets [40 ].
Cpmax vs. numerous in vitro dissolution parameters were successfully
used to attain level C IVIVC for carbamazepine IR products [41 ]
[42 ].
Conclusion
This study was performed on healthy Egyptian volunteers to assess the probable ethnic
or regional variations in pharmacokinetics of favipiravir. It was concluded that
there are similarities in the pharmacokinetic profile of favipiravir in healthy
Egyptian, Caucasian, and American volunteers but there is a considerable difference
between Egyptian and Japanese volunteers. In the future, clinical pharmacokinetic
studies will be conducted on COVID-19 Egyptian patients to compare their
pharmacokinetic parameters with other ethnic groups. Also, in this study, level C
IVIVC model was implemented to select the recommended dissolution medium for
favipiravir IR tablets as crucial preference for drug dissolution testing.
Data availability statement
The data are available as preprint on Research Square.
