Synthesis and preclinical pharmacokinetic study of DHA-10, a novel potential antifungal pogostone analogue

Article abstract of DOI:10.1111/jphp.12750

Objectives: The emergence of fungal disease calls for the urgency of development of novel drug. In this study, we developed a novel pogostone analogue, DHA-10 and investigated its preclinical pharmacokinetics, tissue distribution, excretion and protein binding rate in rats. Methods: DHA-10 was synthesized with dehydroacetic acid (DHA) as the starting material and the structure confirmed by NMR and HRMS. The LC-MS/MS was applied to quantitative analysis of DHA-10 concentrations in the biological samples. Key finding: DHA-10 was eliminated rapidly in rat plasma with half-lives of 3.39 ± 0.5, 3.24 ± 0.32 and 3.80 ± 0.40 h after single oral doses of 70, 140 and 280 mg/kg, respectively, and showed linear pharmacokinetic within the examined dosage range. The oral bioavailability of DHA-10 was 69.09 ± 3.9%. DHA-10 distributed widely in tissues with highest tissue concentration was found in small intestine at 2.5 h postdose, followed by the stomach, liver and uterus. Approximately, 1.50 ± 0.26% and 9.12 ± 2.53% of parent drug was excreted via the urine and faeces within 48 h, respectively; 1.45 ± 0.12% was excreted into the bile up to 36 h after a single oral administration of 140 mg/kg. Binding rate of DHA-10 with plasma protein was about 78.80 ± 1.75% in a concentration-independent manner. Conclusions: DHA-10 was successfully synthesized and characterized. The preclinical pharmacokinetics study in rats supported the further development of this new antifungal candidate compound.

Full text of DOI:10.1111/jphp.12750

Research Paper
Synthesis and preclinical pharmacokinetic study of DHA-10,
a novel potential antifungal pogostone analogue
Ming Liua , Yanfen Zhoub, Jianwei Zhuc, Bo Mab, Zheng Fangb and Qi Zhangb
aSchool of Life Science & Pharmaceutical Engineering, Nanjing Tech University, bSchool of Pharmaceutical Sciences, Nanjing Tech University, and
cInstitute of Advanced Materials (IAM), Nanjing Tech University, Nanjing, China
Keywords
Abstract
pogostone analogue; synthesis;
pharmacokinetics; tissue distribution;
excretion
Objectives The emergence of fungal disease calls for the urgency of development
of novel drug. In this study, we developed a novel pogostone analogue, DHA-10
and investigated its preclinical pharmacokinetics, tissue distribution, excretion
and protein binding rate in rats.
Correspondence
Qi Zhang and Zheng Fang, School of
Pharmaceutical Sciences, Nanjing Tech
University, Nanjing 210009, China.
E-mails: nancyzhang03@hotmail.com and
fzcpu@njtech.edu.cn
Methods DHA-10 was synthesized with dehydroacetic acid (DHA) as the start-
ing material and the structure confirmed by NMR and HRMS. The LC-MS/MS
was applied to quantitative analysis of DHA-10 concentrations in the biological
samples.
Key finding DHA-10 was eliminated rapidly in rat plasma with half-lives of
3.39 Æ 0.5, 3.24 Æ 0.32 and 3.80 Æ 0.40 h after single oral doses of 70, 140 and
280 mg/kg, respectively, and showed linear pharmacokinetic within the examined
dosage range. The oral bioavailability of DHA-10 was 69.09 Æ 3.9%. DHA-10
distributed widely in tissues with highest tissue concentration was found in small
intestine at 2.5 h postdose, followed by the stomach, liver and uterus. Approxi-
mately, 1.50 Æ 0.26% and 9.12 Æ 2.53% of parent drug was excreted via the
urine and faeces within 48 h, respectively; 1.45 Æ 0.12% was excreted into the
bile up to 36 h after a single oral administration of 140 mg/kg. Binding rate of
DHA-10 with plasma protein was about 78.80 Æ 1.75% in a concentration-inde-
pendent manner.
Received October 23, 2016
Accepted April 18, 2017
doi: 10.1111/jphp.12750
Conclusions DHA-10 was successfully synthesized and characterized. The pre-
clinical pharmacokinetics study in rats supported the further development of this
new antifungal candidate compound.
antifungal drugs (polyenes, azoles, echinocandins, allylami-
nes and DNA analogues etc.) are available for candidiasis
Introduction
The fungi have emerged as major causes of human disease,
ranging from superficial infections of the mucosal surfaces
or skin to systemic infections.[1,2] The invasive infections
(IFIs) are an increasingly important cause of morbidity and
mortality in immunocompromised patients, especially can-
cer patients with chemotherapy-induced neutropenia,
transplant recipients receiving immunosuppressive therapy
and human immunodeficiency virus (HIV)-infected
patients.[3–7]
treatment[10] and the application of these drugs are greatly
limited by their toxicities, side effects, drug interactions,
drug delivery routes and the emergences of drug resis-
tance.[11] Multiple adverse factors have stimulated research
directed towards the discovery of novel chemical entities
with alternative mode of action to treat fungal infections
and prevent drug resistance. Pogostone (PO, C12H16O4)
has been investigated for its significant antimicrobial activ-
ity against a large number of clinically relevant fungal and
(myco) bacterial strains, and showed anti-Candida activity
both in vitro and in vivo.[12,13] Unlike major antifungal
drugs in current clinical use (azole, polyenes, echinocandins
bacteria),[10] PO belongs to a different chemical structure
Candida species are the most common opportunistic
fungal pathogens in humans, with Candida albicans being
the most prevalent microbial population in mucosal and
systemic infections.[8,9] So far, only a few classes of
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
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A promising 1-hydroxypyridone antifungal compound
Ming Liu et al.
family of pyranones. But the oil–water partition coefficient
(Log P) of pogostone was not ideal and affected its biologi-
cal transport in the body. Therefore, a novel pogostone
analogue was synthesized which retained the biological
actions 2-pyrone ring of the pogostone parent molecule
with improved water solubility and better pharmacological
properties by modifying the structure of green food grade
antibacterial agent (DHA). The pharmacodynamic study of
the cooperative group indicated that DHA-10 [3-(2-diethy-
lamino acetyl-4-hydroxy-6-2H-pyran-2-one hydrochloride]
has good effects on Candida albicans, Candida glabra, Can-
dida tropicalis and Candida famala.
in CH3CN (40 ml), stirring at room temperature for 0.5 h.
Diethylamine (0.37 g, 5 mmol) was then added dropwise
to the liquid followed by potassium carbonate (0.82 g,
6 mmol), stirring at room temperature for 5 h. After the
reaction, the resultant mixture was filtered and solvent was
removed under vacuum. The residue was then dissolved in
ethyl acetate and washed with saturated aqueous NaCl. The
organic layer was dried over anhydrous sodium sulphate
and solvent removed under vacuum. The crude product
was purified by flash chromatography on silica gel, gradient
elution with ethyl acetate in petroleum ether to obtain the
yellow product. The product obtained was then dissolved
in CH2Cl2, stirring at À10 °C and hydrogen chloride solu-
tion was added. After the reaction, the desired product was
recrystallized by ethyl acetate. Due to the poor solubility of
DHA-10 in organic solvents, it is hard to characterize
DHA-10 by nuclear magnetic resonance. Therefore, the free
base form of DHA-10 was characterized with nuclear mag-
netic resonance shown in Appendix S2.
With the increasing significance of DHA-10 as a poten-
tial antimycotic agent, it is important to investigate the in-
vivo process of DHA-10. An HPLC-MS/MS method was
validated and applied to the study of the pharmacokinetics,
distribution, excretion and plasma protein binding rate of
DHA-10 in rats. To date, this is the first report on the syn-
thesis and pharmacokinetics of novel pogostone analogues
(DHA-10). The results could be used for the analysis of effi-
cacy, and toxicity, more importantly guidance for lead opti-
mization thus played a critical role in the development of
potential pyrone antifungal drugs.
Animals
The Sprague-Dawley rats (male and female, weight
200 Æ 20 g) were obtained from the Animal Center of
Nanjing Medical University (Nanjing, China; certificate no.
SCXK2009-001), were used in this study. All rats were accli-
matized for one week under controlled environmental con-
ditions (temperature was 23–25 °C; humidity was 50%
with a 12 h-dark–light cycle). Before administration, rats
were fasted for 12 h with free access to water. The experi-
mental protocols were approved by the Animal Ethics
Committee of Nanjing Tech University.
Materials and Methods
Chemicals and reagents
DHA-10
[3-(2-diethylamino
acetyl-4-hydroxy-6-2H-
pyran-2-one hydrochloride] was synthesized, purified and
characterized in the Pharmaceutical Sciences College of
Nanjing Tech University, (Nanjing, China). HPLC-grade
acetonitrile and methanol were purchased from Merck
(Darmstadt, Germany). Formic acid of LC-MS grade was
obtained from Sigma (St. Louis, MO, USA). Water was
purified by employing a Milli-Q water purification system
(Millipore, Billerica, USA). All other reagents were at ana-
lytical grade.
Experimental design
Pharmacokinetic study
Twenty-four Sprague-Dawley rats (12 males and 12
females) were divided into four groups randomly (six
rats/per group, three males and three females). DHA-10
was dissolved in normal saline. The first three groups of
rats received a single oral (i.g.) administration of DHA-
10 at 70, 140 and 280 mg/kg, respectively, and blood
samples (200 ll) were collected into heparinized tubes at
predose, 0.25, 0.75, 1.5, 2.0, 2.5, 2.75, 3.0, 3.5, 4, 6, 8, 12
and 24 h postdose. The fourth group rats were given
intravenous (i.v.) administration via lateral tail vein at
the dose of 140 mg/kg and blood samples (200 ll) were
collected into heparinized tubes at predose, 0.033, 0.083,
0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8,12 and 24 h postdose.
All samples were centrifuged at 5315 9 g for 10 min to
obtain the plasma that were separated and kept at
À80 °C until analysis.
Synthesis and characterization of DHA-10
The synthesis route used to synthesize the pogostone ana-
logues (DHA-10) is outlined in Figure 1. Firstly, a mixture
of dehydroacetic acid (1.681 g, 10 mmol), 5, 5-Dibromo-
barbituric acid (2.859 g, 10 mmol), p-toluenesulfonic acid
(0.172 g, 1 mmol) and AlCl3 (0.067 g, 0.5 mmol) in
CH3CN (20 ml) was stirred under reflux for 15 h. After the
reaction, the temperature was cooled down and solvent
removed under vacuum. The residue was then dissolved in
dichloromethane and washed with H2O. The organic layer
was dried over anhydrous sodium sulphate and solvent
removed under vacuum to obtain the product. Secondly,
the bromide intermediate (1.225 g, 5 mmol) was dissolved
2
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Ming Liu et al.
A promising 1-hydroxypyridone antifungal compound
Figure 1 The synthesis route of DHA-10, where a and b are the first and second step reaction products.
Tissue distribution study
obtain a final concentration of 6, 48 and 120 lg/ml
(n = 3). The mixture of DHA-10 and plasma was incu-
bated at 37 °C for 2 h to allow equilibration. Then a 100-
ll aliquot was removed to the centrifugal filter unit for
the total concentration analysis in each sample, including
free and bound DHA-10 concentrations. The remaining
volume using filters to ultrafiltration. Centrifugation was
carried out at 12 000g for 45 min. 400 ll phosphoric acid
buffer solution (PBS, PH 7.4) of DHA-10 at a concentra-
tion of 6, 48 and 120 lg/ml (n = 3) was processed with
the same plasma ultrafiltration method to evaluate the
adsorption of drug and membrane. Using the instrument
LC-MS/MS for the determination of total drug concentra-
tion (A) and the free drug concentration (B), then accord-
ing to the formula F = (A À B)/A calculated plasma
protein binding rate.
Eighteen rats were divided into three groups (n = 6 per
group, three males and three females) randomly. All rats
were oral administered single dose of 140 mg/kg DHA-10.
At 0.5, 2.5 and 12.0 h postdose, one group rats were sacri-
ficed, samples of heart, liver, spleen, lung, kidney, stomach,
intestine, brain, gonadal (testis, uterus and ovary) were har-
vested, rinsed in normal saline and blotted dry with filter
paper. Tissue homogenized was obtained by homogeniza-
tion in normal saline (1 ml/0.2 g tissue) and stored at
À80 °C until analysis.
Excretion study
After single-dose oral administration of DHA-10 at
140 mg/kg, six rats (half male and half female) were indi-
vidually placed in stainless-steel metabolic cages with ad li-
bitum access to food and water, allowing separate
collection of urine and faeces. Urine samples were collected
at intervals of 0–2, 2–4, 4–8, 8–12, 12–24, 24–36 and
36–48 h postdose. Faeces samples were collected at 0–12,
12–24, 24–36 and 36–48 h. The volume of urine for each
collection period was recorded, respectively. The faeces
were individually dried at 50 °C dry oven for 24 h, weighed
and recorded, and then homogenized in normal saline to
prepare the suspension (200 mg/ml; w/v) before storing at
À80 °C.
Sample processing
In this study, different samples were extracted by different
methods. In detail, an aliquot of 100 ll of biological
samples (plasma, urine, bile) were spiked with 10 ll IS
(2 lg/ml) solution. Then, 400 ll ethyl acetate for plasma
or 400 ll methanol for urine and bile was added and vor-
texed for 5 min immediately. The mixture solutions were
cold centrifuged at 10600 9 g for 10 min maintained at
4 °C temperature. Subsequently, 300 ll of supernatant
layer was transferred into a new 1.5-ml Eppendorf tube
(EP) and evaporated to dryness in vacuum concentrator.
Residues were reconstituted in 100 ll mobile phase sol-
vents (acetonitrile: water, 55 : 45, v/v) by vortexing for
5 min and centrifuged. 80 ll supernatant was transferred
to a fresh vial and 5 ll was injected into LC-MS/MS analy-
sis system for analysis.
To study the bile excretion, another six rats (half male
and half female) were anesthetized by subcutaneous injec-
tion of 2% pentobarbital sodium solution (w/v). A cannula
was inserted into the bile duct of each animal. Blank bile
was collected before DHA-10 was administered. After oral
administration of DHA-10 at the dose of 140 mg/kg, bile
samples were collected at 0–2, 2–4, 4–6, 6–8, 8–10, 10–12,
12–24 and 24–36 h postdose and volume recorded before
stored at À80 °C.
Each weighed tissue sample was homogenized in normal
saline (1 ml/0.2 g tissue, w/v). 100 ll of tissue homogenate
was spiked with 10 ll of IS solution (2 lg/ml) and 400 ll
of ethyl acetate solution, and then vortex-mixed for 5 min.
Subsequent steps were in accordance with above-men-
tioned plasma sample preparation.
Plasma protein binding study
Ultrafiltration method was applied to the determination of
plasma protein binding (PPB) rate. 25 ll DHA-10 work-
ing solution was added to 475 ll fresh rat plasma to
Faeces were dried at 50 °C dry oven for 24 h, individu-
ally weighed and recorded, and then homogenized in nor-
mal saline to prepare the suspension (200 mg/ml, w/v).
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
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A promising 1-hydroxypyridone antifungal compound
Ming Liu et al.
Subsequent steps were in accordance with above-men-
tioned plasma sample preparation.
1 White solid; m.p. = 118–119 °C. 1HNMR (CDCl3,
400 MHz) d = 15.40(s, 1H), 5.95(s, 1H), 4.64(s, 2H),
2.25(s, 3H); HRMS (ESI) m/z calcd for C8H7O4Br
[M + H]+246.9528, found 246.9486.
Analytical methodology
2
1HNMR (CDCl3, 400 MHz) d = 12.05(s, 1H), 6.43(s,
1H), 4.70(s, 2H), 3.57(q, 2H, J = 7.1 Hz), 3.49(q, 2H,
J = 7.2 Hz), 2.52(s, 3H), 1.34(t, 3H), 1.28(t, 3H,
J = 7.2 Hz); HRMS (ESI) m/z calcd for C12H17NO4
[M + H]+240.123, found 240.1244.
DHA-10 concentrations in the plasma, tissue, faeces, urine
and bile were quantified by LC-MS/MS and the analytical
method was shown in Appendix S1. The separation was
achieved on a Kromasil 100-5C18 (150 mm 9 2.1 mm,
5 lm) column with MS detection operated in positive
MRM mode at m/z 240.0 ? 222.0 and m/z 199.0 ? 125.2
for DHA-10 and IS, respectively. The assay was linear over
the concentration range (r2 > 0.995) with the LLOQ of
0.010 lg/ml. The intra- and interday precisions (R.S.D.)
were ≤11.67%, whereas accuracy ranged from À13.96% to
14.66%. The extraction recovery, stability and matrix effect
were within the acceptable limits.[14]
Plasma pharmacokinetic study
Mean plasma concentration-time profiles and log of plasma
concentration-time curves of DHA-10 after single oral and
intravenous administration were shown in Figure 2. The
major pharmacokinetic parameters were calculated by a
noncompartmental model based on the statistical moment
and listed in Table 1. The terminal elimination half-life
Data analysis
(T1/2
)
of three oral dose groups was 3.39 Æ 0.58,
SPSS 19.0 (SPSS Inc., Chicago, USA) and Origin Pro 8.6
(Origin Lab Co., Northampton, USA) software were used for
various calculations. The pharmacokinetic parameters and
the concentration-time curves were analysed through a non-
compartmental method by DAS 2.0 Software (Mathematical
Pharmacology Professional Committee of China, Shanghai,
China). Levels of statistical significance were assessed using
the Student’s t-test. P < 0.05 was considered as statistical sig-
nificance. All the data were expressed as the mean Æ SD.
3.24 Æ 0.32 and 3.80 Æ 0.40, respectively, and the mean
residence time (MRT) ranging from 5.66 to 6.96 h. Total
plasma clearance CL (0.37 Æ 0.045, 0.32 Æ 0.019 and
0.34 Æ 0.048 l/h per kg) was not different across the inves-
tigated dosage range, respectively. The mean plasma Cmax
and AUC0–∞ values of the three doses indicated an appar-
ent dose-proportionality and correlation coefficient (r) is
0.995 and 0.998, respectively. As for the other parameters
including Tmax, MRT, CL, Vd and T1/2, there were no sig-
nificant differences in the three dosages (P > 0.05). All
these results supported the linear plasma pharmacokinetics
of DHA-10 among the investigated dose range in rats (70–
280 mg/kg). The oral bioavailability (F) of DHA-10 at the
dose of 140 mg/kg was estimated to be 69.09 Æ 3.9%.
Results
Synthesis and characterization
DHA-10 was synthesized and its spectral data given below:
Figure 2 Mean plasma concentration-time curves of DHA-10 and log of plasma concentration-time curves after a single oral administration at a
dose of 70 140 280 mg/kg (n = 6).
4
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Ming Liu et al.
A promising 1-hydroxypyridone antifungal compound
Table 1 Pharmacokinetic parameters of DHA-10 after a single-dose administration in rats (n = 6)
Oral
Intravenous
140 mg/kg
Parameter
70 mg/kg
140 mg/kg
280 mg/kg
Cmax (lg/l)
40.53 Æ 4.88
2.25 Æ 0.27
79.79 Æ 1.77
2.42 Æ 0.38
134.72 Æ 8.30
2.21 Æ 0.33
283.91 Æ 52.78
–
Tmax (h)
T1/2z (h)
3.39 Æ 0.58
3.24 Æ 0.324
436.214 Æ 24.63
440.357 Æ 24.96
0.32 Æ 0.019
1.49 Æ 0.17
3.80 Æ 0.40
3.63 Æ 0.15
631.40 Æ 90.23
637.40 Æ 92.14
0.223 Æ 0.031
1.17 Æ 0.17
4.72 Æ 0.44
4.94 Æ 0.48
AUC0–t (lg/l 9 h)
AUC0–∞ (lg/l 9 h)
CLz/F (l/h per kg)
Vz/F (l/kg)
192.43 Æ 24.53
194.32 Æ 25.77
0.37 Æ 0.045
1.776 Æ 0.32
5.44 Æ 0.30
820.89 Æ 112.48
834.45 Æ 118.34
0.34 Æ 0.048
1.86 Æ 0.204
6.59 Æ 0.27
MRT0–t (h)
6.34 Æ 0.32
MRT0–∞ (h)
5.658 Æ 0.40
6.55 Æ 0.42
6.96 Æ 0.27
Figure 3 The distribution of DHA-10 in rats after oral administration
of 140 mg/kg at 0.5, 2.5 and 12.0 h.
Tissue distribution study
The concentration of DHA-10 (Figure 3) was detected in
heart, liver, spleen, lung, kidney, stomach, small intestine,
brain, fat, muscle and gonadal (testis, uterus and ovary)
samples. The results indicated that after oral administra-
tion, the DHA-10 underwent a wide distribution in rat tis-
sues within the time course examined. The highest tissue
concentration was found in small intestine at 2.5 h, fol-
lowed by the stomach, liver and uterus with very little
DHA-10 found in brain. DHA-10 concentration decreased
obviously at 12 h postdose.
Figure 4 Urinary, faecal and biliary cumulative excretion of DHA-10
in rats after single-dose administration of 140 mg/kg.
Excretion study
after a single oral administration, 1.50 Æ 0.26% of DHA-
10 was excreted as original form in urine within 48 h and
9.12 Æ 2.53% of DHA-10 was excreted as unchanged form
Excretion of DHA-10 in rat urine, faeces and bile was pre-
sented in Figure 4 and Table 2. The result indicated that
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
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A promising 1-hydroxypyridone antifungal compound
Ming Liu et al.
in faeces within 48 h period. The amount of DHA-10 in
faeces reached to 8.48 Æ 2.71% of the dose within 24 h,
and then increased slowly over time. Biliary excretion
within 36 h was about 1.45 Æ 0.12%.
protein binding were designed and carried out based on the
LC-MS/MS. method which proved to be sensitive, rapid
and reproductive and suitable to the pharmacokinetic study
in rats.
Pharmacokinetics of DHA-10 showed linear kinetic
character within the dose range studied. T1/2 of DHA-10
was 3.15–3.83 h, which was longer than that of PO, 53.31–
51.96 min,[13] suggesting that DHA-10 eliminated more
slowly. The highest tissue concentration was found in small
intestine and the second in liver, which implied that the
small intestine might be the main absorption site of the
drug and liver played an important role in the biotransfor-
mation of DHA-10. High concentrations of DHA-10 were
also found in uterus and ovary, which indicated that these
two tissues could be the therapeutic targets. This discovery
was obviously meaningful considering that gynecological
diseases caused by fungal infections were common in
women. DHA-10 was detectable in testis, suggesting that
DHA-10 could get through the blood-testis barrier and
have effects on testis infected by fungus. The amount of
DHA-10 decreased to a very low level at 12 h postdosing in
all of the examined tissues.
Plasma protein binding study
The plasma protein binding rates of DHA-10 were mea-
sured to be 77.26 Æ 1.72, 80.04 Æ 0.92, 79.11 Æ 0.61% for
three different concentrations, respectively. The average
plasma protein binding rate was 78.80 Æ 1.75%. There was
no significant differences at three different levels of DHA-
10 in rats (P > 0.05), indicating that plasma protein bind-
ing to DHA-10 had no concentration dependence.
Discussion
Pogostone is one of the secondary metabolites from
Pogostemon cablin (Blanco) Benth (Lamiaceae), serving as
the effective component of the antimicrobial activity. Previ-
ous report showed that PO may develop as a serious novel
antifungal compounds with proper structure modifica-
tion.[15] We have synthesized a series of PO analogues and
evaluated their antimicrobial activities, of which DHA-10
showed the most antifungal potential. Here, the synthesis
and pharmacokinetic properties of DHA-10 were investi-
gated for the first time.
Excretion results showed that up to 48 h, the total
unchanged DHA-10 excreted in urine and faeces was only
10.62 Æ 0.25% of the oral dose, suggesting that most of
DHA-10 may excrete as metabolite forms.
The drug in bound form to plasma protein is not avail-
able for interaction with its biological targets such as
enzyme, receptor transporter, etc. The ratio of free and
The preclinical pharmacokinetic study including the
plasma profiles, tissue distribution, excretion and plasma
Table 2 Urine, faeces and biliary cumulative excretion of DHA-10 in rats during each time interval after single-dose administration of 140 mg/kg
(n = 6)
Matrix
Urine
Time (h)
Excretion amount (lg, mean Æ SD)
Excretion accumulation (lg, mean Æ SD)
Excretion accumulation (% of oral dose)
0–2
2–4
57.27 Æ 15.10
73.83 Æ 34.28
85.41 Æ 32.14
143.44 Æ 62.55
49.36 Æ 51.70
10.31 Æ 6.87
57.27 Æ 15.10
131.10 Æ 21.86
216.51 Æ 51.49
359.95 Æ 88.63
412.18 Æ 74.05
424.91 Æ 74.75
425.13 Æ 74.67
800.47 Æ 285.61
2374.64 Æ 759.13
2553.83 Æ 708.05
2554.38 Æ 708.00
24.70 Æ 8.26
0.205 Æ 0.054
0.468 Æ 0.078
0.773 Æ 0.184
1.286 Æ 0.317
1.462 Æ 0.264
1.499 Æ 0.267
1.500 Æ 0.262
2.86 Æ 1.02
4–8
8–12
12–24
24–36
36–48
0–12
12–24
24–36
36–48
0–2
0.22 Æ 0.12
Faeces
Bile
800.47 Æ 285.61
1574.17 Æ 772.77
179.19 Æ 138.95
0.56 Æ 0.28
8.48 Æ 2.71
9.12 Æ 2.53
9.12 Æ 2.53
24.70 Æ 8.26
0.078 Æ 0.030
0.27 Æ 0.083
0.49 Æ 0.091
0.67 Æ 0.094
0.81 Æ 0.096
0.94 Æ 0.099
1.43 Æ 0.12
2–4
54.04 Æ 17.33
62.53 Æ 12.07
49.68 Æ 11.89
38.78 Æ 5.42
78.75 Æ 23.28
4–6
141.28 Æ 25.50
190.96 Æ 26.34
229.74 Æ 26.89
265.69 Æ 27.63
404.08 Æ 34.02
405.00 Æ 34.01
6–8
8–10
10–12
12–24
24–36
35.95 Æ 14.22
138.39 Æ 20.43
0.91 Æ 0.12
1.45 Æ 0.12
6
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Ming Liu et al.
A promising 1-hydroxypyridone antifungal compound
plasma protein bound drugs will have an impact on phar-
macokinetics, pharmacodynamics and safety margins of the
compound. The rates of DHA-10 binding to plasma pro-
tein were less than 80% in a concentration-independent
manner has a certain reference value to guide the dosage of
administration.
(1.50 Æ 0.26%) and faeces (9.12 Æ 2.53%). The infor-
mation from the synthesis and pharmacokinetic of
DHA-10 supported further drug design optimization in
the development of both current and future series’ as
potential antifungal agents for the effective treatment of
invasive fungal infection diseases.
Conclusions
Declarations
In summary, this is the first report for the synthesis of
DHA-10 and studies of the preclinical pharmaceutics,
tissue distribution, excretions and plasma protein bind-
ing of DHA-10 in rats. The results showed linear dispo-
sition of DHA-10 at the examined dose range, with
Cmax and AUC0–∞ being proportional to the adminis-
tered dosages in rats. The oral bioavailability of the drug
was calculated to approximately 69.09 Æ 3.9%. In tissue
distribution studies, the highest level in small intestine,
followed by the liver suggests that DHA-10 was mainly
absorbed in the small intestine and metabolism mainly
in the liver. Very little DHA-10 crossed the blood–brain
barrier (BBB). Only 10.62 Æ 0.25% of the administered
DHA-10 was excreted as the unchanged form from urine
Conflict and interest
All Authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by National Science and Technol-
ogy Major Projects for ‘Major New Drugs Innovation and
Development’ (No. 2013ZX09103001-004) and ‘Specialized
Research Fund for the Clinical Medicine of Jiangsu Pro-
vince’ (No. SBL201330086). The authors would like to
thank William Gu from the University of Auckland for
improving the standard of written English of the manu-
script.
patient. Curr Infect Dis Rep 2006; 8: 13. Li Y-C et al. Anti-Candida albicans
References
434–441.
activity and pharmacokinetics of
pogostone isolated from Pogostemonis
Herba. Phytomedicine 2012; 20: 77–83.
8. Pfaller MA, Diekema DJ. Rare and
emerging opportunistic fungal patho-
1. Odds FC. Molecular phylogenetics
and epidemiology of Candida albi-
cans. Future Microbiol 2010; 5: 67–79.
2. Pfaller MA, Diekema DJ. Epidemiol-
ogy of invasive candidiasis: a persis-
tent public health problem. Clin
Microbiol Rev 2007; 20: 133–163.
gens: concern for resistance beyond Can- 14. US Food and Drug Administration.
dida albicans and Aspergillus fumigatus. J
Clin Microbiol 2004; 42: 4419–4431.
Guidance for Industry: Bioanalytical
Method Validation. Washington: US
Department of Health and Human
Services, 2001.
9. Walsh TJ et al. Infections due to
emerging and uncommon medically
important fungal pathogens. Clin 15. Yi Y-Y et al. Synthesis and antimicro-
3. Richardson M, Lass-Flo€rl C. Changing
epidemiology of systemic fungal infec-
tions. Clin Microbiol Infect 2008; 14
(Suppl. 4): 5–24.
Microbiol Infect 2004; 10 (Suppl. 1):
48–66.
bial evaluation of pogostone and its
analogues. Fitoterapia 2013; 84:
135–139.
10. Sanglard
D et al. Antifungal drug
4. Fridkin S, Jarvis W. Epidemiology of
nosocomial fungal infections. Clin
Microbiol Rev 1996; 9: 499–511.
resistance mechanisms in fungal
pathogens from the perspective of
transcriptional gene regulation. FEMS
Yeast Res 2009; 9: 1029–1050.
Supporting Information
Additional Supporting Information
may be found in the online version of
this article:
5. Groll A et al. Trends in the post-
mortem epidemiology of invasive fun-
gal infections at a university hospital.
J Infect 1996; 33: 23–32.
11. Enoch DA et al. Invasive fungal infec-
tions: a review of epidemiology and
management options. J Med Microbiol
2006; 55: 809–818.
Appendix S1. HPLC-MS/MS method
for the determination of DHA-10 in
rat plasma, tissue, faeces, urine and
bile.
6. Pfaller MA. Epidemiological typing
methods for mycosis. Clin Infect Dis
1992; 14: 4–10.
12. Chena H et al. LC-MS/MS determina-
tion of pogostone in rat plasma and
its application in pharmacokinetic
studies. Biomed Chromatogr 2013; 27:
1092–1099.
Appendix S2. The characterization
of DHA-10.
7. Upton A, Marr AK. Emergence of
opportunistic mould infections in the
hematopoietic stem cell transplant
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