Introduction
The Copaifera genus (Leguminosae), commonly known as “Copaíbas”, “Copaibeiras”, or “Copaívas”,
encompasses approximately 70 species of large trees that are widely distributed in
Brazil, primarily in the northern region, with a particular emphasis on the states
of Amazonas, Pará, and Roraima [1], [2], [3]. The oleoresins extracted from the trunk of these plants have long been utilized
in traditional Brazilian medicine, demonstrating various beneficial properties such
as anti-inflammatory, antimicrobial, analgesic, wound healing, antitumor, purgative,
and antiparasitic activities [1], [2], [3]. Moreover, these oleoresins hold significant pharmacological value and are commercially
sold as crude oil to serve as raw material for producing cosmetics [1], [3], [4].
These oleoresins exhibit variable viscosity and color and comprise volatile sesquiterpenes
and nonvolatile acid diterpenes [1], [5]. Despite notable variations in the chemical composition among Brazilian Copaifera species, sesquiterpene compounds such as δ-cadinene, α-cadinol, α-cubebene, β-elemene, α-copaene, α-humulene, β-caryophyllene, and caryophyllene oxide, as well as kaurane, clerodane, and labdane
diterpenes, have been identified in all examined species [1], [6], [7].
Among the various Copaifera oleoresins found in Brazil, the oleoresin from Copaifera duckei Dwyer is the most prominent representative of Amazonian “Copaíbas”. This oleoresin
comprises approximately 72% nonvolatile components, including the diterpenes ent-polyalthic acid and dihydro-ent-agathic acid [6], [7] ([Fig. 1]).
Fig. 1 Chemical structures of the major nonvolatile constituents of Copaifera duckei oil resin: ent-polyalthic acid (1) and dihydro-ent-agathic acid (2).
In Brazilian folk medicine, the recommended oral dosage is 3 – 5 drops dispersed in
warm water or honey, taken up to three times daily to treat internal ailments [8], [9]. Despite the oral administration of these oleoresins, there is a lack of information
regarding their pharmacokinetic studies. Consequently, we have developed and validated
an analytical method to determine the pharmacokinetic profiles of ent-polyalthic and dihydro-ent-agathic acids in rat plasma samples following a single oral dose of C. duckei oleoresin administration.
Results and Discussion
The mass spectra in the mobile phase and the chromatograms of plasma samples of the
ent-polyalthic and dihydro-ent-agathic acids and the internal standard (IS) warfarin are depicted in [Figs. 2] and [3]. The matrix effect, linearity, precision, accuracy, and stability are shown in [Table 1]. The carryover tests of the ent-polyalthic and dihydro-ent-agathic acids and the IS warfarin are shown in [Fig. 3]. Chromatographic peak areas of the blank plasma samples were lower than 20% of the
lower limit quality control (LLQC) areas, as shown in [Fig. 3].
Fig. 2 Full scan mass spectrum of Copaifera duckei oil resin, dihydro-ent-agathic, ent-polyalthic acids, and warfarin sodium (internal standard) diluted in the mobile phase.
Fig. 3 Chromatograms of the lower limit quality control (LLQC), low-quality control (LQC),
and high-quality control (HQC) samples and of blank plasma samples immediately following
HQC sample analysis of ent-polyalthic and dihydro-ent-agathic acids.
Table 1 Validation parameters of the method of analysis of ent-polyalthic and dihydro-ent-agathic acids in rat plasma samples.
|
NMF = normalised matrix factor, CV = coefficient of variation, RSE = relative standard
error, LLQC = lower limit of quantification, LQC = low-quality control, MQC = medium
quality control, HQC = high-quality control
|
|
Parameter
|
ent-Polyalthic acid
|
Dihydro-ent-agathic acid
|
|
Matrix effect
Mean NMF (CV%)
LQC
HQC
|
0.40 (8.61%)
0.39 (4.74%)
|
0.94 (5.83%)
0.51 (6.06%)
|
|
Linearity range (µg/mL)
Regression equation
Correlation coefficient (r)
|
0.57 – 114.74
y = 0.422 126 × x − 0.226 519
0.993 176
|
0.09 – 18.85
y = 3.16 888 × x − 0.146 839
0.996 044
|
|
Stability
|
CV%
|
RSE%
|
CV%
|
RSE%
|
|
Shor-term (25 °C, 6 h)
|
|
LQC
|
(n = 3) 4.03
|
− 6.26
|
(n = 4) 3.51
|
− 3.71
|
|
HQC
|
(n = 4) 3.78
|
− 10.14
|
(n = 4) 2.60
|
− 7.58
|
|
Post-processing (12 °C, 10 h)
|
|
LQC
|
(n = 3) 3.00
|
− 12.65
|
(n = 3) 5.73
|
− 5.77
|
|
HQC
|
(n = 4) 6.81
|
5.84
|
(n = 5) 2.03
|
2.36
|
|
Freeze/thaw cycles (25 °C, 6 h)
|
|
LQC
|
(n = 5) 1.52
|
4.59
|
(n = 5) 4.09
|
− 5.30
|
|
HQC
|
(n = 5) 3.03
|
10.82
|
(n = 5) 4.37
|
− 6.26
|
|
Precision and accuracy
|
CV%
|
RSE%
|
CV%
|
RSE%
|
|
Intra-assay
|
|
LLQC
|
(n = 7) 2.61
|
− 0.20
|
(n = 7) 5.02
|
3.34
|
|
LQC
|
(n = 7) 3.53
|
8.57
|
(n = 7) 5.17
|
− 5.60
|
|
MQC
|
(n = 7) 6.30
|
4.81
|
(n = 7) 3.29
|
− 4.89
|
|
HCQ
|
(n = 7) 4.05
|
− 4.24
|
(n = 7) 2.27
|
− 2.53
|
|
Inter-assays (3 assays)
|
|
LLQC
|
(n = 22) 8.57
|
0.10
|
(n = 22) 5.98
|
8.80
|
|
LQC
|
(n = 17) 9.61
|
− 1.46
|
(n = 20) 6.45
|
− 1.06
|
|
MQC
|
(n = 22) 6.48
|
0.47
|
(n = 22) 7.07
|
− 2.24
|
|
HCQ
|
(n = 19) 7.30
|
3.69
|
(n = 21) 3.20
|
− 5.01
|
Both ent-polyalthic and dihydro-ent-agathic acid pharmacokinetics were characterized as a bicompartmental structural
model with first-order absorption and elimination. Plasma concentrations versus time
curves of ent-polyalthic and dihydro-ent-agathic acids are presented in [Fig. 4]. The typical values of the parameters and interindividual variability (IIV) are
presented in [Table 2] (θ
i =
θ
TV × eη
, where θ
i is the parameter value of an individual animal, θ
TV is the population parameter typical value, and η is a random variable with mean zero and variance ω
2).
Fig. 4 Observed plasma concentrations over time of ent-polyalthic acid and dihydro-ent-agathic acids in 44 Wistar rats following a 200-mg/kg Copaifera duckei oil resin dose administrated by gavage (91.79 mg/kg of ent-polyalthic acid and 18.08 mg/kg of dihydro-ent-agathic acid).
Table 2 Estimates of the population pharmacokinetic model of ent-polyalthic acid and dihydro-ent-agathic acid in rats following oral administration (gavage) of a 200-mg/kg dose of
Copaifera duckei oleoresin (91.79 mg/kg of ent-polyalthic acid and 18.08 mg/kg of dihydro-ent-agathic
acid).
|
Parameter
|
ent-Polyalthic acid
|
Dihydro-ent-agathic acid
|
|
Estimates
|
Bootstrap (n = 1000)
|
Estimates
|
Bootstrap (n = 1000)
|
|
Typical value (θ)
RSE
|
IIV (ω
2)
RSE
|
Typical value median
PI (2.5 – 97.5%)
|
IIV median
PI (2.5 – 97.5%)
|
Typical value (θ)
RSE
|
IIV (ω
2)
RSE
|
Typical value median
PI (2.5 – 97.5%)
|
IIV median
PI (2.5 – 97.5%)
|
|
CL/F: apparent clearance. Vc/F: apparent central volume of distribution; Q: intercompartmental
clearance; Vp/F: apparent peripheral volume of distribution; IIV: interindividual
variability. RSE%: residual standard error; PI: percentile interval range; SD: standard
deviation. Cmax: maximum concentration. Tmax: time to achieve Cmax. t
1/2: half-life. Relative F: relative bioavailability between ent-polyalthic/dihydro-ent-agathic acids
|
|
Ka
(h−1)
|
0.47
12.16%
|
–
|
0.47
(0.37 – 0.90)
|
–
|
0.28
12.9%
|
–
|
0.28
(0.22 – 0.67)
|
–
|
|
CL/F
(L/h)
|
0.15
13.91%
|
0.25
44.68%
|
0.15
(0.099 – 0.19)
|
0.26
(0.05 – 0.75)
|
0.04
9.3%
|
0.329
26.1%
|
0.04
(0.03 – 0.05)
|
0.31
(0.16 – 0.54)
|
|
Vc/F
(L)
|
0.04
20.17%
|
–
|
0.04
(0.03 – 0.09)
|
–
|
0.01
27.6%
|
|
0.01
(0.01 – 0.04)
|
|
|
Q/F
(L/h)
|
0.13
11.91%
|
–
|
0.14
(0.10 – 0.21)
|
–
|
0.02
30.1%
|
|
0.02
(0.01 – 0.05)
|
|
|
Vp/F
(L)
|
2.48
30.36%
|
0.98
37.09%
|
2.50
(1.33 – 4.66)
|
0.98
(0.26 – 2.05)
|
0.18
14.0%
|
–
|
0.19
(0.14 – 0.31)
|
–
|
|
σ
2
|
0.15
21.26%
|
–
|
0.14
(0.09 – 0.20)
|
–
|
0.17
15.6%
|
–
|
0.16
(0.11 – 0.22)
|
–
|
|
|
Median
PI (25 – 75%)
|
Mean
(SD)
|
|
|
Median
PI (25 – 75%)
|
Mean
(SD)
|
|
|
|
Tmax
(h)
|
0.50
(0.45 – 0.75)
|
0.64
(0.36)
|
|
|
0.75
(0.50 – 1.50)
|
1.26
(0.87)
|
|
|
|
Cmax
(µg/mL)
|
41.63
(33.25 – 50.91)
|
42.75
(13.22)
|
|
|
22.65
(16.97 – 29.36)
|
22.03
(9.80)
|
|
|
|
t
1/2
(h)
|
11.60
(7.85 – 15.15)
|
11.60
(7.14)
|
|
|
3.49
(2.52 – 4.78)
|
3.49
(2.17)
|
|
|
|
AUC0–48
(μg · h/mL)
|
130.74
(96.43 – 176.64)
|
144.89
(131.16)
|
|
|
103.00
(69.84 – 150.67)
|
118.76
(68.62)
|
|
|
|
Relative F
|
|
|
0.245
|
|
|
|
|
The residual variability of both acids was described by a proportional residual error
model. Therefore, the concentration estimates of each time (j) and animal (i) was
Yij = F
ij
+ F
ij
× εij
where Yij is the observed concentration, Fij is the concentration estimate, and ε
ij is a random variable with mean zero and variance σ
2 ([Table 2]).
The goodness of fit plot (GOF) and visual predictive check (VPC) of ent-polyaltic and dihydro-ent-agatic acids ([Figs. 5] and [6]) indicated a good fit of the predicted plasma concentrations to the observed data,
and the bootstraps ([Table 2]) indicate acceptable bias and good accuracy of the fixed and random effects estimates.
In addition, the bootstrap had only 11.1% minimization failures.
Fig. 5 Goodness of fit plot of ent-polyaltic and dihydro-ent-agatic acids. Observed concentrations over population and individual predictions
(left). Conditional weighted residuals (CWRES) over population predictions and time
(right). Red line: trend line; dashed lines: identity line and two and half times
the identity (left plots); 2, 0, and − 2 CWRES (right plots).
Fig. 6 Visual predictive check (VPC) of ent-polyaltic and dihydro-ent-agatic acids in rat plasma over time. Dots: observed plasma concentrations. Lines:
5th, 50th, and 95th percentiles of observed concentrations. Shaded areas: 5th, 50th,
and 95th percentiles of the simulated concentrations (n = 1000).
The influence of animal weight and age on the pharmacokinetic parameters of ent-polyaltic
and dihydro-ent-agatic acids were explored however due to the uniformity of weight
and age values among the animals no effect was identified.
To our knowledge, this is the first method for analyzing ent-polyaltic and dihydro-ent-agatic acids in the plasma of rats by LC-MS/MS. The method presented a wide linearity
range and low lower limit of quantification (LLOQ) (0.57 – 114.74 and 0.09 – 18.85 µg/mL
plasma, respectively), which makes it suitable to be applied to preclinical pharmacokinetic
studies.
We could not analyze the ent-polyalthic and dihydro-ent-agathic acids by multiple reactions monitoring (MRM). Despite several fragmentation
tests with different ionization energies and argon flux, their ionized molecules did
not generate fragment ions appreciably. A similar difficulty was also described by
Gasparetto et al. [10] in the analysis of kaurenoic acid, a diterpene contained in Guaco.
The sample preparation method did not present a significant matrix effect for standard
or lipemic plasma samples ([Table 1]). However, the matrix effect for hemolyzed plasma was higher than 15%, and the hemolyzed
plasma samples should be disregarded.
The method is selective for ent-polyalthic and dihydro-ent-agathic acids at concentrations ≥ the LLQC. The chromatogramsʼ peak areas of interferents
were lower than 20% compared to the chromatogramsʼ peak analytes at LLQC concentrations
([Fig. 3]).
The analytical method showed good intra- and inter-assay precision and accuracy, with
the coefficient of variation (CV) and relative standard error (RSE) of quality controls
lower than 15%. Similarly, the stability of the freeze/thaw cycles, post-processing
in an auto-injector, and short-term on a bench had a CV and RSE lower than 15%.
The C. duckei oleoresin sample was prepared at 20 mg/mL in saline with Cremophor 10% to furnish
a homogeneous suspension able to provide the dose of 200 mg of oleoresin per kilogram
of rat in a volume not exceeding 10 mL/kg of rat, as described by the Good Practices
for the Administration of Substances and Blood Collection [11]. Blood samples in volumes lower than 400 µL were collected from the caudal vein,
not exceeding three samples per animal. The volume collected was less than 10% of
the animalʼs total blood volume, making volume replacement unnecessary, according
to the Good Practice Guide for the Administration of Blood Substances and Collections,
which would cause changes in pharmacokinetic parameters [11].
This is the first report on the population pharmacokinetics of ent-polyalthic and dihydro-ent-agathic acids. With few reports on the bioavailability of natural products and limited
population pharmacokinetic studies on natural products, finding similar data for comparison
is difficult.
ent-Polyalthic and dihydro-ent-agathic acids have similar lipophilicity (logP = 4.9 and 4.7, respectively) and polar
surface area (50.4 and 74.6 Å2, respectively) [12]. However, the apparent clearance (CL/F), central apparent volume of distribution
(Vc/F), and peripheral apparent volume of distribution (Vp/F) of ent-polyalthic acid are approximately 3.75-, 4.00-, and 13.78-folds higher than the dihydro-ent-agathic ones, and the relative bioavailability between ent-polyalthic and dihydro-ent-agathic acids is 0.245 ([Table 2]).
Due to the Brazilian populationʼs extensive use of copaibaʼs oleoresin, further studies
are needed to establish safe and effective doses for humans. Furtado et al. [13] reported no genotoxic activity at doses up to 2000 mg of oleoresin/kg of animal
for six different Copaifera species (C. duckei, Copaifera multijuga, Copaifera paupera, Copaifera pubiflora, Copaifera
reticulata, and Copaifera trapezifolia). However, they observed cytotoxic activity of C. duckei oleoresin on Chinese hamster fibroblast cells at a concentration of 9.8 µg/mL. Castro-e-Silva
et al. [14] reported the oral treatment of rats with 600 mg oleoresin/kg/day for 7 days and
after partial hepatectomy. They also observed a decrease in hepatocellular proliferation
and mitochondrial breathing in the liver. Noteworthy is that in the present study,
the C. duckei oleoresin sample was prepared at 20 mg/mL in saline with
Cremophor 10% to furnish a homogeneous suspension able to provide the dose of 200 mg
of oleoresin per kilogram of rat in a volume not exceeding 10 mL/kg of rat, as described
by the Good Practices for the Administration of Substances and Blood Collection.
Finally, it is possible to conclude that the developed LC-MS/MS analytical method
is reliable for the pharmacokinetic studies of Copaifera oleoresin ent-polyalthic
and dihydro-ent-agathic acid diterpenes. The CL/F, Vc/F, and Vp/F of ent-polyalthic acid are higher than that of dihydro-ent-agathic. It is also essential to observe that the literature reports that diterpenes
with an oxidized furan ring, such as teucrin A, are known liver toxic compounds [15]. All these facts indicate the need for further toxicological and clinical studies
to better understand the safety aspects of using Copaifera oleoresin.
Material and Methods
Plant materials and chemicals
C. duckei Dwyer oleoresin was collected in Belém (Pará State, Brazil) at coordinates S01°06.933′,
O48°19.781′. A voucher specimen (NID:96/2012) was obtained and deposited in the Herbarium
of the Brazilian Agricultural Research Corporation (Embrapa Eastern Amazon). The Botanist
Silvane Tavares Rodrigues identified the specimen (voucher number 175 206). ent-Polyalthic acid and dihydro-ent-agathic acid ([Fig. 1]) were isolated and identified through nuclear magnetic resonance analysis by our
research group from the same oleoresin used in this study [6], [16]. Their purity was also evaluated by integrating the area under the signals corresponding
to the compounds of interest in the 1H NMR spectrum.
The IS was warfarin sodium, purchased from FURP. The following HPLC grade reagents
were used: Milli Q Plus purified water (Millipore), methanol (Merck), acetonitrile
(Sigma-Aldrich), methyl tert-butyl ether (Fischer Scientific), and isopropanol (J. T. Baker). Formic acid and
glacial acetic acid (J. T. Baker), both of analytical grade, were employed as mobile
phase modifiers.
Development and validation of an analytical method for ent-polyalthic acid and dihydro-ent-agathic acid in rat plasma
LC-MS/MS analysis
Chromatographic analysis was conducted using an Alliance e2695 Waters system (Waters
Corp.). A LiChrospher 100 CN (5 µm) column with a LiChroCART 125 – 4 mm (Merck) pre-column
was utilized and maintained at a temperature of 27 °C. The mobile phase consisted
of a mixture of water, acetonitrile, isopropanol, and formic acid (64.8 : 20 : 15 : 0.2 v/v/v/v)
with a 0.7 mL/min flow rate. Mass spectrometry detection was performed using a Quattro
Micro Liquid Chromatograph triple quadrupole (Micromass) equipped with an electrospray
ionization (ESI) source. The ent-polyalthic acid and dihydro-ent-agathic acid were detected in the single ion recording (SIR) mode, while warfarin
(IS) was detected in the MRM mode. The ESI source was set to the negative mode. The
parameters were configured as follows: capillary voltage − 3.00 kV, source and desolvation
temperatures of 125 and 400 °C, respectively, cone and desolvation gas flow (N2) were set at 50 and 800 L/h,
respectively, and argon was employed as the collision gas at a rate of 0.15 mL/min.
The cone voltage, collision energies, and ion transitions for each analyte were optimized
according to [Table 3]. Data acquisition and sample quantifications were performed using MassLynx 4.1 version
(Waters).
Table 3 Precusor/product ion pairs and parameters for multiple reactions monitoring of ent-polyalthic acid, dihydro-ent-agathic acid, and the internal standard (IS) warfarin.
|
Compound
|
Retention time (min)
|
MS mode
|
Transitions
(precursor > product)
|
Cone voltage (V)
|
Collision energy (eV)
|
|
ent-Polyalthic acid
|
11.18
|
SIR
|
315
|
50
|
2
|
|
Dihydro-ent-agathic acid
|
5.72
|
SIR
|
335
|
50
|
2
|
|
IS
|
5.06
|
MRM
|
307.1 > 161.5
|
30
|
20
|
Preparation of standard solutions
Stock solutions of ent-polyalthic (5 mg/mL) acid, dihydro-ent-agathic acid (1 mg/mL), and warfarin sodium (IS, 5 mg/mL) were prepared separately
in methanol and kept at − 20 °C. The calibration standards were prepared by successive
dilutions in methanol to obtain concentrations of 1.15, 2.29, 3.44, 4.59, 6.88, 9.18,
22.95, 45.90, 91.79, 114.74, 137.69, 183.58, and 229.48 µg of ent-polyalthic acid/mL methanol and 0.19, 0.38, 0.57, 0.75, 1.13, 1.51, 3.77, 7.54, 15.08,
18.85, 22.62, 30.16 e 37.70 µg of dihydro-ent-agathic acid/mL methanol. Warfarin sodium (IS) solution was further diluted in methanol
to obtain a 5 µg/mL concentration.
Sample preparation
Fifty microliters of plasma sample (or blank plasma for calibration curves) were added
to 2000 µL microtubes (Axygen Scientific) to begin the sample preparation. Subsequently,
25 µL of IS solution (5 µg/mL), 50 µL of 0.75 M acetic acid, and 25 µL of methanol
(or 25 µL of each standard solution for calibration curves) were added. The microtubes
were then vortexed for 30 s using a vortex mixer (Phoenix Luferco, model AP56). After
mixing, 500 µL of the extraction solution (methyl tert-butyl ether : isopropanol, 4 : 1 v/v) were added to the microtubes. Another 30-s
vortex mixing was performed, followed by centrifugation for 10 min at 21 500 g, 4 °C (Himac CF8DL). The resulting supernatants were carefully transferred to other
clean microtubes and subjected to evaporation until dryness using a vacuum concentrator.
The dried residues were then reconstituted in 100 µL of the mobile phase and mixed
for 30 s. The processed samples were stored in the automatic injector at
12 °C, and 60 µL of each sample were injected into the LC-MS/MS system. For the construction
of calibration curves, peak area ratios (analyte/IS) were plotted against plasma concentrations.
The concentration ranges were 0.57 to 114.74 µg/mL and 0.09 to 18.85 µg/mL for ent-polyalthic acid and dihydro-ent-agathic acid, respectively.
Method validation
The analytical method was validated according to FDA and EMA guidelines [17]. The quality control samples were prepared at the plasma concentrations shown in
[Table 4].
Table 4 Quality control concentrations of ent-polyalthic and dihydro-ent-agathic acids in rat plasma.
|
Quality control sample
|
ent-Polyalthic acid (µg/mL)
|
Dihydro-ent-agathic acid (µg/mL)
|
|
LLQC: lower limit concentrations quality control, LQC: lower concentration quality
control, MQC: medium concentration quality control, HQC: higher quality control concentrations,
DQC: dilution quality control
|
|
LLQC
|
0.57
|
0.09
|
|
LQC
|
1.72
|
0.28
|
|
MQC
|
57.37
|
9.42
|
|
HQC
|
91.79
|
15.08
|
|
DQC
|
114.74
|
18.85
|
The matrix effect was assessed using eight 50 µL aliquots of blank plasma obtained
from different rats, including two lipemic samples, two hemolyzed samples, and four
standard samples. The blank plasma extracts were then spiked with standard solutions
at concentrations corresponding to the high-quality control (HQC) and low-quality
control (LQC), along with the addition of the IS solution. Additionally, exact standard
solutions in methanol spiked with the IS solution were analyzed. The matrix factor
normalized with the IS (NMF) was calculated by dividing the peak area ratios of analyte/IS
from the post-extracted plasma samples by the peak area ratios of analyte/IS from
the neat solutions. The matrix effect was determined by calculating the CV of all
obtained MFs.
Selectivity was evaluated using blank plasma from eight different sources, including
four standard samples, two lipemic samples, and two hemolyzed samples. The resulting
chromatograms were compared to LLQC concentration samples.
Linearity was assessed through triplicate calibration curves, including a blank and
zero samples. The carryover effect was evaluated by consecutively injecting three
blank samples, followed by injecting the sample at the HQC concentration.
Precision and accuracy (intraday and inter-day) were evaluated by performing seven
replicates of LLQC, LQC, medium quality control (MQC), and HQC of ent-polyalthic and dihydro-ent-agathic acids in a single analytical run (intra-assay) and in three different analytical
runs (inter-assay). The precision and accuracy results are expressed as the CV and
RSE.
Stability tests in rat plasma were conducted using four replicates of LQC and HQC
samples. For the freeze/thaw stability evaluation, the LQC and HQC replicates were
subjected to three cycles of freezing at − 70 °C for 24 h, followed by thawing at
room temperature and freezing again at − 70 °C for 24 h. The samples were then analyzed
at the end of the three cycles. Short-term stability was assessed by keeping the LQC
and HQC samples at room temperature for 1 h before preparation and analysis. Post-processing
stability was evaluated by storing the processed LQC and HQC samples in the automatic
injector at 12 °C for 24 h before analysis. The stability results are expressed as
the CV and RSE.
Population pharmacokinetics of ent-polyalthic acid and dihydro-ent-agathic acid in rats
Animal experiments
Male Wistar rats (260 ± 30 g, n = 44) were housed in metabolic cages under controlled
temperature (25 ± 1 °C), relative humidity (40 – 70%), a 12-h light-dark cycle, and
with free access to food and water. On December 9, 2015, the Ethics Committee of Universidade
de Franca (CEUA) approved the experimental protocol on the Use of Animals under protocol
number 057/15.
After 12 h fasting, 20 mg/mL C. duckei oleoresin, dissolved in physiological saline
solution with 1.0% Cremophor, were orally administered by gavage at a dose of 200 mg/kg
(91.79 mg/kg of ent-polyalthic acid and 18.08 mg/kg of dihydro-ent-agathic acid) to
a total of 44 rats. Serial blood samples of 200 µL (3 to 4 samples per animal) were
collected from tail vein at 5, 15, 30, 45 min, and 1, 1.5, 2, 3, 4, 6, 8, 12, 16,
18, 20, 24, 30, 36, and 48 h (n = 8 for each sampling time) after oleoresin administration
and transferred to tubes containing heparin as an anticoagulant (Liquemine 5000 IU;
Roche). After centrifugation (10 min, 9560 g, 4 °C), the plasma samples were stored at − 70 °C until analysis.
Population pharmacokinetic models
Population pharmacokinetics models of ent-polyalthic and dihydro-ent-agathic acids in rats were evaluated by nonlinear mixed-effects modelling conducted
in NONMEN software, version 7.4.3 (ICON Development Solutions), with compiler GNU
Fortran 4.6 (Free Software Foundation, Inc.) and interface PsN, version 4.9.0 (Perlspeaks-NONMEM)
[18]. R version 3.6.1 (R Foundation for Statistical Computing) was used to reorganize
the dataset, statistical summaries, and graphics.
The estimations were conducted based on the first-order conditional estimation with
the interaction method (FOCE-I). The model-building criteria included (1) successful
minimization, (2) reduced relative standard error and shrinkage values of estimates,
(3) number of significant digits, (4) successful covariance step, (5) correlation
between model parameters, and (6) acceptable gradients at the last iteration [19], [20].
All fixed and random effects were introduced into the model according to a stepwise
procedure exploring mono- or bicompartmental pharmacokinetics with first order and
elimination and different absorption models (first-order, lag-time, or transit compartment
models). The residual variability was explored by additive, proportional, or proportional
combined with additive error models. The IIV was explored in all parameters assuming
a log-normal distribution.
Comparison between hierarchical models was based on graphic and statistical methods
that included (1) reduction of the objective function value (OFV) and AIC (Akaike
information criteria), (2) values of relative standard error and shrinkage, (3) GOF
that included plots of the predicted population (PRED) and individual (IPRED) concentrations
versus the observed concentrations and the conditional weighted residuals (CWRES)
versus population predicted concentrations and time [20], [21].
A stepwise forward inclusion/backward elimination procedure was used for covariate
selection, according to the concept that the difference in − 2 log likelihood between
two models is approximately χ2 distributed, with degrees of freedom equal to the difference in the number of parameters
between the hierarchical models [22]. The covariates were introduced one by one and retained if a decrease in OFV of
at least 3.84 units (p < 0.05) was observed. During the backward elimination procedure,
an increase in OFV of at least 7.8 units (p < 0.005) was used as a criterion for a
significant effect.
The predictive performance of ent-polyalthic and dihydro-ent-agathic acid pharmacokinetic models was assessed via graphical and statistical methods,
including VPCs [20], [21], [23] and bootstrapping [24], in addition to graphical evaluation of the GOF. VPCs were obtained from 1000 simulations
per animal of the ent-polyalthic and dihydro-ent-agathic acid plasma concentrations from 0 to 48 h. Bootstrap analysis identified
bias, stability, and precision of the estimates obtained with the model and was performed
with 1000 new datasets generated by resampling individuals (with replacement) from
the original dataset.
The area under the plasma concentration from 0 to 48 h (AUC0 – 48) of ent- polyalthic and dihydro-ent-agathic acids was calculated by the trapezoidal rule in R version 3.6.1 from the
modelʼs individual predicted concentrations over time. Then, the relative bioavailability
between ent-polyalthic/dihydro-ent-agathic acids of each animal was accessed through the equation:
F = AUC0 – 48 of ent-polyalthic acid × dose of dihydro-ent-agathic acid
AUC0 – 48 of dihydro-ent-agathic acid × dose of ent-polyalthic acid
Contributorsʼ Statement
Data collection: F. A. Aguila, Nardotto G. H. B., Oliveira, L. C.; design of the study:
F. A. Aguila, Bastos, J. K., Veneziani R. C. S., Nardotto G. H. B., Oliveira, L. C.,
Rocha, A., Lanchote, V. L., Ambrosio S.R; statistical analysis: F. A. Aguila, Nardotto
G. H. B., Oliveira, L. C., Rocha, A., Lanchote, V. L., Ambrosio S.R; analysis and
interpretation of the data: F. A. Aguila, Bastos, J. K., Veneziani R. C. S., Nardotto
G. H. B., Oliveira, L. C., Rocha, A., Lanchote, V. L., Ambrosio S.R; drafting the
manuscript: Bastos, J. K., Veneziani R. C. S., Rocha, A., Lanchote, V. L., Ambrosio
S.R; critical revision of the manuscript: Bastos, J. K., Veneziani R. C. S., Lanchote,
V. L., Ambrosio S.R, Ambrosio S. R.
