Bioactivatable Pseudotripeptidization of Cyclic Dipeptides to Increase the Affinity toward Oligopeptide Transporter 1 for Enhanced Oral Absorption: An Application to Cyclo(l -Hyp- l -Ser) (JBP485)

Article abstract of DOI:10.1021/acs.jmedchem.9b00358

The cyclic dipeptides generally present lower affinity toward intestinal oligopeptide transporter 1 (PEPT1) than the linear dipeptides. JBP485 (cyclo(l-Hyp-l-Ser)) is a low-affinity substrate of PEPT1 with poor oral bioavailability. However, JBP923 (l-Hyp-l-Ser) is a high-affinity substrate of PEPT1 with high oral absorption. We hypothesize that the bioactivatable pseudo-tripeptidization prodrug strategy is promising to increase the affinity of cyclic dipeptides toward PEPT1. To test our hypothesis, we design five amino acid ester prodrugs of JBP485. Compared with JBP485, the optimal prodrug (JBP485-3-CH₂-O-valine, J3V) demonstrates improved affinity of PEPT1, oral bioavailability in rats and beagle dogs. Moreover, J3V can dose-dependently protect against liver injury. Additionally, J3V is stable in the gastrointestinal tract, beneficial to the PEPT1-mediated membrane transport, and is bioactivated in the enterocytes and hepatic cells, essential to elicit its bioactivity. In summary, the bioactivatable pseudo-tripeptidization strategy shows potential in increasing affinity of PEPT1 to enhance oral bioavailability of cyclic dipeptides.

Full text of DOI:10.1021/acs.jmedchem.9b00358

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Article
Bioactivatable pseudo-tripeptidization of cyclic dipeptides to
increase the affinity toward oligopeptide transporter 1 for enhanced
oral absorption: an application to cyclo(L-Hyp-L-Ser) (JBP485)
Qikun Jiang, Jiangnan Zhang, Peiyue Tong, Yan Gao, Yiqin Lv, Changyuan Wang,
Meiling Luo, Mengchi Sun, Jian Wang, Yao Feng, Linlin Cao, Gang Wang, Yang Wang,
Qiming Kan, Tianhong Zhang, Yongjun Wang, Kexin Liu, Jin Sun, and Zhonggui He
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00358 • Publication Date (Web): 08 Aug 2019
Downloaded from pubs.acs.org on August 10, 2019
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Bioactivatable pseudo-tripeptidization of cyclic dipeptides to increase the affinity
toward oligopeptide transporter 1 for enhanced oral absorption: an application
to cyclo(L-Hyp-L-Ser) (JBP485)
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a
Qikun Jiang,a Jiangnan Zhang,f Peiyue Tong,a Yan Gao,a Yiqin Lv, Changyuan
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Wang,f Meiling Luo,a Mengchi Sun,a Jian Wang,b Yao Feng,b Linlin Cao,c Gang
Wang,d Yang Wang,d Qiming Kan,e Tianhong Zhang,a Yongjun Wang,a Kexin Liu,f
Jin Sun,*,a Zhonggui He*,a
a
Department of Pharmaceutics, Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
b
Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang
Pharmaceutical University, Ministry of Education, Shenyang 110016, China
c Department of Pharmaceutics, The Second Hospital of Dalian Medical University,
Dalian 116023, China
d Department of Pharmaceutics, Guang Xi University of Chinese Medicine, Guangxi
530001, China
e
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang
110016, China
f Department of Clinical Pharmacology, Dalian Medical University, Dalian 116044,
China
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ABSTRACT
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The cyclic dipeptides generally present lower affinity toward intestinal oligopeptide
transporter 1 (PEPT1) than the linear dipeptides. JBP485 (cyclo(L-Hyp-L-Ser)) is a
low-affinity substrate of PEPT1 with poor oral bioavailability. However, JBP923
(L-Hyp-L-Ser) is a high-affinity substrate of PEPT1 with high oral absorption. We
hypothesize that the bioactivatable pseudo-tripeptidization prodrug strategy is
promising to increase the affinity of cyclic dipeptides toward PEPT1. To test our
hypothesis, we design five amino acid ester prodrugs of JBP485. Compared with
JBP485, the optimal prodrug (JBP485-3-CH2-O-valine, J3V) demonstrates improved
affinity of PEPT1, oral bioavailability in rats and beagle dogs. Moreover, J3V can
dose-dependently protect against liver injury. Additionally, J3V is stable in the
gastrointestinal tract, beneficial to the PEPT1-medited membrane transport, and is
bioactivated in the enterocytes and hepatic cells, essential to elicit its bioactivity. In
summary, the bioactivatable pseudo-tripeptidization strategy shows potential in
increasing affinity of PEPT1 to enhance oral bioavailability of cyclic dipeptides.
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KEYWORDS: bioactivatable pseudo-tripeptidization, cyclic dipeptides, linear
dipeptides, JBP485
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INTRODUCTION
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Cyclic dipeptides are heterocyclic compounds comprising of two amino acid
residues linked to a central diketopiperazine (DPK) ring structure, and have various
biological activities such as anti-inflammatory1, anti-microbial2, anti-diabetic activity3.
However, it had been reported that the intestinal permeability of some cyclic
dipeptides was lower than that of its counterpart, the linear dipeptides4, 5. Most linear
dipeptides could be actively transported by oligopeptide transporter 1 (PEPT1,
SLC15A1), which is highly expressed in the apical membrane of enterocytes6,
whereas the cyclic dipeptides generally present lower affinity toward PEPT1 than the
linear dipeptides7, even not recognised by PEPT18, 9. The reason for low affinity of
PEPT1 for cyclic dipeptides is that the key pharmacophores of linear dipeptides
responsible for interacting with PEPT1 were masked after cyclization, such as
C-terminal carboxylic or N-terminal amino group of linear dipeptides6, 10. Several
formulation approaches have been attempted previously to increase the bioavailability
of cyclic dipeptides, including the preparation of PLGA-based nanoparticles11 or
emulsions12. However, these strategies have some limitations, such as complex
preparation process13,low drug loading14 and unfavourable stability15. Until now, no
prodrug approach has been adopted to improve the oral bioavailability of cyclic
dipeptides.
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In recent years, the understanding of membrane transporters in the intestine has
prompted a novel transporter-targeted prodrug approach to overcome the poor
membrane permeability16. Since PEPT1 could mediate the transport of dipeptides,
tripeptides and various of peptidomimetics, the broad substrate specificity of PEPT1
makes it an attractive target for designing oral prodrugs10. The PEPT1-mediated
prodrug approach is effective in increasing intestinal permeability and oral absorption,
using amino acids as targeting promoieties17. Most amino acid prodrugs are classified
as amide- and ester-type prodrugs, in which α-carboxylic or α-amine group of amino
acids is conjugated with functional groups of the parent compounds (hydroxyl, amine,
and carboxyl)18, respectively. The amino acid ester prodrugs are preferred for
designing PEPT1-targeted prodrugs, because they show reasonably good stability
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over the gastrointestinal pH range and simultaneously maintain a rapid rate of
enzymatic-mediated bioactivation. For examples, valganciclovir, the valine ester
prodrugs of poorly absorbed nucleoside analogue antiviral drug-ganciclovir, exhibited
good oral absorption19. This PEPT1-mediated prodrug approach was also successfully
applied to other poorly absorbed nucleosides by our group to increase oral
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absorption20-22
.
Inspired by the above findings, we hypothesize that the pseudo-tripeptidization
strategy, amino acid ester prodrugs of cyclic dipeptides, could increase the binding
affinity of PEPT1. Our assumption is stated as follows: firstly, since the PEPT1
recognition is influenced by peptide size, the amino acid esters of cyclic dipeptides,
the size of which is similar to tripeptides, would be accommodated by the binding
pocket of PEPT1; secondly, the N-terminal amino group of amino acids introduced
into the prodrugs would facilitate interaction with PEPT1. Furthermore, according to
the chemical/enzymatic stability of amino acid ester prodrugs, we proposed that the
high chemical stability would reduce the conversion of the amino acid ester prodrugs
of cyclic dipeptides prior to absorption in the gastrointestinal (GI) tract, whereas
bioactivation by multiple enzymes in the enterocytes and hepatic cells could allow for
quick and quantitative conversion to the active parent molecule.
JBP485 (cyclo(L-Hyp-L-Ser), Figure 1A) and JBP923 (L-Hyp-L-Ser, Figure 1B)
are firstly isolated from hydrolysate of human placenta (Laennec®) and used to treat
anti-hepatitis and anti-aging23, 24. The activity of JBP485 is notably higher than that of
JBP923 at the same dose after intravenous administration25. However, JBP485 has a
weaker potency after oral administration, due to the low oral bioavailability (about
20%) for JBP485, but JBP923 is nearly 100% absorbed25, 26. It had been reported that
JBP485 and JBP923 were actively transported by PEPT1. However, the PEPT1
affinity of JBP923 was higher than that of JBP485, with inhibition constant (Ki) for
JBP485 and JBP923 in relation to the uptake of Gly-Sar being 36.0 and 2.5 mM,
respectively5. The low binding affinity of JBP485 toward PEPT1 was responsible for
low oral bioavailability of JBP485.
Herein, we put forward the bioactivatable pseudo-tripeptidization strategy to
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increase the PEPT1 affinity of JBP485. We had synthesized five amino acid ester
prodrugs of JBP485 (Figure 1(C-G)). These prodrugs were screened regarding the
chemical/metabolic stability study, cellular permeation in the Caco-2 cell monolayers
and oral pharmacokinetic study in rats and beagle dogs. These results demonstrated
that the optimal prodrug (JBP485-3-CH2-O-Valine, J3V) was stable in the
gastrointestinal tract of rats and then was bioactivated by multiple activating enzymes
in the intestine and liver of rats. J3V exhibited significantly high bioavailability
compared to JBP485 after oral administration in rats and beagle dogs, due to the high
PEPT1 affinity of J3V.
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RESULTS AND DISCUSSION
Design and synthesis of JBP485 and its prodrugs
To increase the binding affinity of PEPT1 for JBP485, five amino acid prodrugs
(JBP485-3-CH2-O-Valine
(J3V),
(J3I),
JBP485-3-CH2-O-Leucine
(J3L),
(J3P),
JBP485-3-CH2-O-Isoleucine
JBP485-3-CH2-O-Phenylalanine
JBP485-7-O-Valine (J7V)) were synthesized according to the synthetic procedures
(Figure 2). Firstly, four prodrugs (J3V, J3L, J3I, J3P) were synthesized by
conjugating the low steric hindrance 3-methyl-hydroxyl of JBP485 with the series of
the carboxyl groups of amino acids (L-valine, L-leucine, L-isoleucine,
L-phenylalanine) to investigate the effects of amino acid promoieties on the PEPT1
affinity; secondly, compared to J3V, J7V was designed to study the steric hindrance
effect of ester bond on the affinity for PEPT1. JBP485 and its prodrugs were fully
characterized by HPLC, H1 NMR, and HRMS. Detailed synthetic procedures, yields,
and HPLC purity were given in the experimental sections.
Stability Study
Chemical stability
To target effectively the PEPT1 transporter in the apical side of enterocytes, the
prodrugs should be stable in the GI tract prior to intestinal membrane transport. The
chemical stability was examined over the gastrointestinal pH range (0.1M HCl, pH
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4.5 acetate buffer, pH 5.8 phosphate buffer, pH 6.8 phosphate buffer and pH 7.2
phosphate buffer). As shown in Table 1, JBP485 was less susceptible to different pH
changes, demonstrating that JBP485 was stable in the GI tract. The stability for all
prodrugs increased with decreasing pH and followed the rank order of
J3I>J7V>J3V>J3P=J3L. In addition, the t1/2 values in the simulated gastric/intestinal
fluids were similar to those in 0.1 M HCl and pH 6.8 phosphate buffer, demonstrating
that all prodrugs were not hydrolyzed by pepsin and trypsin in the GI tract. The t1/2
values of J3V, J7V and J3I was 7.9-20.0 h in the simulated gastric/intestinal fluids,
indicating that these prodrugs could ensure sufficient stability during the transport
process in the intestine. However, J3P and J3L could be instable in the intestine
because of short t1/2 values in the simulated intestinal fluids.
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Metabolic stability
Except for chemical stability, metabolic stability of prodrugs was investigated in
rat jejunum and liver homogenates and plasma (Table 1). The metabolic stability
study provided the bioactivation performances of prodrugs to JBP485 in these
metabolic sites after oral administration. The t1/2 was >20 h for JBP485 in jejunum
and liver homogenate and plasma, thus excluding the possibilities that low
bioavailability of JBP485 was mainly due to instability in these metabolic sites. This
was also consistent with the previous results that the first-pass hepatic metabolism of
JBP485 could be neglected because its bioavailability after port vein injection was
85% 26. Additionally, the rank order in metabolic stability of all prodrugs was the
same as those in chemical stability. The t1/2 in tissue homogenates and plasma were
3-35-fold shorter than that in pH 7.4 phosphate buffer, indicating the
enzyme-catalyzed hydrolysis for all prodrugs in tissues. Moreover, the t1/2 values of
all prodrugs in the jejunum and liver homogenates were shorter than that in the
plasma, implying that the primary bioactivation sites of prodrugs might be the liver
and intestine.
Caco-2 Membrane Permeability
The permeability of JBP485 and its prodrugs across Caco-2 cell monolayers was
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shown in Figure 3A. The amino acid prodrugs (J3V, J3I, J7V) showed 2.4-11.6-fold
increase in permeability compared to JBP485, while J3L and J3P didn’t increase the
permeability. J3V showed the highest permeability and was nearly similar to JBP923,
a known substrate of PEPT1 with up to 100% oral bioavailability in rats.
Consequently, J3V was chosen for further study to investigate the single-pass
intestinal perfusion study, cellular uptake study, oral pharmacokinetics and
antihepatitis activity.
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Correlation of lipophilicity and permeability
The pH-dependent distribution coefficient (log D), predicted by Discovery
Studio 3.5 Client software, was the principal parameter to estimate lipophilicity of
chemical compound. Figure 3B showed the correlation between Caco-2 membrane
permeability and lipophilicity. JBP923 had the lowest log D value and JBP485 and its
prodrugs had similar log D values. However, lipophilicity was not correlated with the
high Caco-2 membrane permeability of JBP923 and J3V, indicating that a different
permeation pathway than passive diffusion is responsible for the high permeability of
JBP923 and J3V.
Single-pass intestinal perfusion (SPIP) study in rats
The effective permeability (Peff) of JBP485, J3V and JBP923 were determined
using the in situ single-pass intestinal perfusion system in rats. As shown in Figure 3C,
JBP485 had low permeability in the jejunum, consistent well with the low
permeability in Caco-2 cell monolayers. However, the permeability of J3V was
similar to JBP923, and was 6.03-fold higher than JBP485. To investigate whether the
increased intestinal permeability of J3V was due to improved affinity toward PEPT1,
an inhibition study was performed by co-perfusing three compounds (JBP485,
JBP923, J3V) with Gly-Sar (the well-known and non-metabolized PEPT1 substrate),
respectively. The Peff values of JBP485, J3V and JBP923 in the presence of Gly-Sar
were reduced by 2.5-, 6.8-, 7.3-fold, respectively, demonstrating that JBP485, J3V
and JBP923 were the typical substrates of PEPT1 and the affinity of J3V was higher
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than that of JBP485.
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Cellular uptake mechanism of JBP485 and J3V
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The SPIP study strongly suggested that JBP485, J3V and JBP923 were PEPT1
substrates and the PEPT1 affinity of J3V was the highest. To further verify these
findings, the cellular uptake mechanism of J3V and JBP485 in the Caco-2 cells was
investigated, with JBP923 as a positive control. The PEPT1 transporter is driven by
an inwardly-directed proton gradient which catalyzes the co-transport of the substrates.
As shown in Figure 4A, J3V and JBP923 showed an approximate 1.7-2.5-fold
increase in cellular uptake compared to JBP485 at pH 6.0. However, the cellular
uptake amounts of JBP485, J3V and JBP923 were significantly decreased when
changing pH from 6.0 to 7.4 or co-perfusing with 50 mM Gly-Sar at pH 6.0 (Figure
4A-4B), respectively. These results were consistent with the results of SPIP study,
further confirming that these compounds were substrates of PEPT1.
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Since other transporters, such as OATP-B (SLC 21A9), OCT1 (SLC 22A1) and
OCT3 (SLC 22A3), could be involved in the peptides transport through small intestine
wall, the inhibition kinetic assay of OATP-B and OCTs (OCT1 and OCT3) substrates
in Caco-2 cells was also evaluated in order to confirm whether JBP485 and J3V had
interacted with these transporters. Figure 4C indicated that J3V and JBP485 showed
no significant difference in cellular uptake in the presence or absence of captopril
(substrates of OATP-B) and metformin (substrates of OCT1-3), demonstrating that
JBP485 and J3V were not considered to be the substrates of OATP-B and OCTs.
We further compared the PEPT1 affinity of JBP485, J3V and JBP923, using the
saturable transport and Gly-Sar inhibition kinetic assays in Caco-2 cells. Kinetic
analysis showed that the Km value of J3V was roughly 3.0-fold lower than that of
JBP485, which compared favorably to JBP923 (Figure 5A-5C, Table 2). Additionally,
the IC50 values for JBP485, J3V and JBP923 to inhibit Gly-Sar uptake in Caco-2 cells
were listed from the curves (Figure 5D, Table 2). The rank order of IC50 values of
these compounds against the Gly-Sar uptake was similar to the rank order of Km
values of the saturable kinetics. The J3V and JBP923 showed the highest inhibition
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effects on Gly-Sar uptake, with IC50 values being 2.9, 1.76 and 18.2 mM, respectively,
which demonstrated that J3V and JBP923 showed 6.3-10.3-fold improvement in
affinity over JBP485.
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Molecular docking
The crystal structure of the hPEPT1 protein has not been resolved, and so we
chose a homology modeling of hPEPT1. To date, bacterial peptide transporters have
been proven to be valid and reliable model system contributing to understand the
molecular basis of peptide recognition within hPEPT1 transporters. We selected the
PEPTso with high degree of sequence conservation within the transmembrane (TM)
region (35% identity) as PEPT1 proteins. The Glu595 and Arg34 were the two
important conserved residues on the substrate-binding sites of PEPTso27, 28. The
interaction profiles of the homology model with these ligands (JBP485, J3V and
JBP923) were illustrated in Figure 6. These ligands interacted with the key
substrate-binding site residues (Glu595, Arg34) of PEPTso, demonstrating that these
ligands could be recognised via the PEPTso. However, around 1.3-fold and 1.5-fold
increase in binding energy scores obtained from -4.8 up to -6.4 and -7.2 kcal/mol for
JBP485, J3V and JBP923, respectively. These indicated that stronger binding of J3V
and JBP923 with PEPTso would favor its transportation in comparison with JBP485.
These findings were consistent with the above results of cellular permeability and
uptake study. This discrepancy for the PEPT1 affinity of these ligands was due to the
difference of bond strength between these ligands (JBP485, J3V and JBP923) and
binding site residue (Glu595) of PEPTso. The 7-hydroxy of JBP485 formed the
hydrogen bond with the negative charge of Glu595. However, the amino of JBP923
and J3V could make ion-dipole interaction with Glu595. It is well known that the
energies of hydrogen bond (2-8 kcal/mol) is smaller than that of ion−dipole
interaction (>100 kcal/mol)29, implying that J3V and JBP923 would presumably be
more stably interacting with Glu595 of PEPTso as compared to JBP485. These results
were consistent with our hypothesis that the pseudo-tripeptides would be
accommodated by the binding pocket of PEPT1 and the N-terminal amino group of
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amino acid introduced into the pseudo-tripeptide could interact highly with PEPT1.
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Pharmacokinetic study
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The PEPT1 seems to be highly conserved, in the aspect of sequence, tissue
distribution, localization and function, between mammalian species. The PEPT1 gene
sequences of rat (NM_057121.1) and beagle dog (NM_001003036) has >80%
similarity to those of human (NM_005073)30. Similar intestinal distribution of PEPT1
and similar function in transporting its substrates in SD rats, beagle dogs and humans
were observed as well30-32. So we performed the pharmacokinetic (PK) study to assess
the absorptive rate, extent and mechanism of JBP485 and its prodrugs in rats and
beagle dogs.
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Pharmacokinetic profile of oral JBP485 and its prodrugs in rats
The oral bioavailability of prodrugs (J3V, J3I, J3L, J3P, J7V) compared to
JBP485 was tested in SD rats following oral administration. Figure 7A summarized
the plasma concentration-time profiles and Table 3 listed the pharmacokinetic
parameters. The PEPT1 could allow for fast and efficient uptake of its substrates due
to a high-capacity system, indicating small Tmax and high AUC0-t and Cmax for the
high affinity substrates. The Tmax of J3V, J3I, J7V was 1.4-2.2-fold lower than that of
JBP485. Also, the AUC0-t and the Cmax of J3V, J3I and J7V were approximately
1.5-2.4-fold and 1.5-4.1-fold greater than that of JBP485, respectively. J3V exhibited
the highest bioavailability among these prodrugs, because L-valine might have the
preferred combination of chain length and branch at the β-C of the amino acid, which
facilitated the binding with PEPT1. However, J3P and J3L exhibited 2.4-2.8-fold
decrease in oral bioavailability compared to JBP485 because of low affinity of PEPT1
and instability in the gastrointestinal tract.
To further evaluate the role of PEPT1 in the oral absorption of J3V in vivo, we
conducted competitive inhibition study involving co-administration with Gly-Sar. The
absolute bioavailability of J3V decreased from 50.1% to 27.1% in the presence of
Gly-Sar and the inhibition ratio was 45.8% (Figure 8A, Table 4). The above results
showed the competitive inhibition of Gly-Sar on the PEPT1-mediated transport of
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J3V did occur in vivo, further indicating that the high bioavailability of J3V was made
by enhanced affinity to PEPT1.
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It had also been mentioned that JBP485 could interact with other influx
transporters or regulate their expression, eg. PEPT2 (SLC 15A2)33, OCT2 (SLC 22A2)
and OAT1 (SLC 22A6), OAT3 (SLC 22A8)34. Considering the complex
circumstances, it was very important to exclude the possibilities that the changes of
PK parameters of JBP485 and J3V in rats were related to other transporters expect for
PEPT1. The reasons were listed as followings: 1) OCT235, 36, PEPT29, 37, OATs
(OAT1, OAT3)35, 38, highly expressed in kidney but not in intestine, represented the
renal reabsorption and secretory pathway for substrates in kidney. Additionally, Liu
had reported that JBP485 alone could not up-regulate the expressions of OCT2, which
could be irrespective of substrate induction34. Furthermore, valaciclovir was also
substrates of PEPTs (PEPT1 and PEPT2) and OATs39, 40, but the oral bioavailability
of valaciclovir was significantly enhanced by targeting effectively the PEPT1
transporter in the apical side of enterocytes, not other transporters41. Consequently, we
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believed that the oral absorption of JBP485 and J3V through the intestinal wall was
not related to its recognition as a substrate by OCT2, PEPT2 and OATs; 2) Although
the expression of OATP-B, OCTs (OCT1 and OCT3) transporters is observed in
intestine, the substrates are commonly characterized as the large hydrophobic anions
for OATPs35, 42 and the organic cations for OCTs36. Additionally, JBP485 and J3V
were not the substrates proved by the inhibition kinetic assays (captopril for OATP-B
and metformin for OCTs) in Figure 4C.
Even if PEPT1 is a high-capacity transporter, it is significant to determine
whether PEPT1 shows capacity-limited absorption at high dose. The J3V and JBP485
were orally administered to the rats at doses of 12 and 100 mg/kg and the plasma
concentrations of JBP485 were shown in Figure 8C-8D and Table 4. There was a
proportional increase of Cmax and AUC0-t with dosing increase of J3V (Figure 8D),
suggesting that PEPT1-mediated transport of J3V was not saturated at high dose in
rats. However, the corresponding AUC0-t and Cmax of JBP485 did not increase
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proportionally with dose over the 10-fold range after orally administration of JBP485
at the dose of 12 and 100 mg/kg, demonstrating that saturation of the plasma
concentration occurred for oral administration of JBP485. Consequently, we can draw
conclusions that the capacity absorption process at the high dose has been observed
for the high-affinity substrates of PEPT1 and the pseudo-tripeptidization strategy
could improve maximum transport capacity of PEPT1 for cyclic dipeptides.
Bioactivation sites study of J3V
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A core issue in developing successful prodrugs is balancing the need for stability
prior to absorption in the gastrointestinal tract and easily conversion to parent drug
after absorption. To investigate the in vivo bioconversion sites of J3V, portal vein and
systemic plasma concentrations of J3V and JBP485 were determined. According to
the chemical stability study, we assumed that J3V could pass through the apical
membrane of intestinal epithelia primarily in intact form. J3V was prone to be
metabolized in the intestinal epithelium cells, with AUC0-120min value in the portal
plasma for JBP485 and J3V of 119.2 nM*min/mL and 24.93 nM*min/mL, suggesting
that the majority bioactivation of J3V did occur in the enterocytes. The underlying
reason was that there were some hydrolytic enzymes contributing to bioconversion of
J3V, agreeing well with the instability of J3V in the intestine homogenates. In the
systemic circulation, only JBP485 could be detected in the systematic plasma,
suggesting the liver was the second-role bioactivation site. (Figure 8B, Table 5).
Pharmacokinetic profile of oral JBP485 and J3V in Beagle dogs
To evaluate the pharmacokinetic study of JBP485 and J3V in beagle dogs, J3V
and JBP485 were both orally administered to male beagle dogs. Figure 7B
summarized the plasma concentration-time profiles and Table 3 listed the
pharmacokinetic parameters. Compared to JBP485, J3V showed a 3.2-fold higher
Cmax (85.9 nM/mL for J3V versus 26.65 nM/mL for JBP485) and 2.6-fold higher
AUC0−t (233.25 nM*h/mL for J3V versus 89.15 nM*h/mL for JBP485) and 2.6-fold
lower Tmax (0.58 h for J3V versus 1.5 h for JBP485). J3V exhibited the higher
bioavailability and shorter Tmax in comparison with JBP485 after oral administration
in beagle dogs, consistent with the pharmacokinetic study in rats. Moreover, no J3V
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in the systematic plasma was observed, implying rapid enzymatic activation in the
intestinal epithelium cells, liver and plasma.
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Anti-hepatitis activities in vivo
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Although J3V could increase oral bioavailability in comparison with JBP485, it
is important to show that J3V also could maintain the pharmacological effect of
JPB485 or even perform better. We investigated the protective effect of JBP485 and
J3V on alpha-naphthylisothiocyanate (ANIT)-induced liver injury in SD rats, with
ursodeoxycholate sodium (UDCA) as a positive control. To preferably observe the
hepatoprotective effect of JBP485 and J3V, we selected three doses of JBP485 (6.25,
12.5 and 25 mg/kg) and four doses of J3V (3.125, 6.25, 12.5 and 25 mg/kg) for oral
administration and two doses of JBP485 (6.25, 12.5 mg/kg) for intraperitoneal
administration in accordance with the results of pharmacokinetics. As shown in
Figure 9A, the serum total bilirubin (TBIL), an important indicator of cholestasis, was
13.0-fold higher in the ANIT-treated rats than in the control rats. The high level of
serum TBIL in ANIT-treated rats was significantly reduced when rats were orally
administered with UDCA (100 mg/kg), different dose of JBP485 and J3V and were
intraperitoneally injected with different dose of JBP485. Additionally, JBP485 and
J3V could decrease T-BIL in a dose-dependent manner. As expected, high-dose oral
J3V (25 mg/kg) and intraperitoneal JBP485 (12.5 mg/kg) exhibited similar and
excellent performance, which was also validated by similar oral absolute
bioavailability in the pharmacokinetic study. Figure 9B-9C showed that the varying
tendencies of serum alanine aminotransferase (ALT) and aspartate aminotransferase
(AST), another biochemical indicator of hepatic cell damage, were similar to that of
T-BIL.
To further evaluate the liver histological changes, the liver sections were stained
by H&E. The mononuclear and polymorphonuclear of hepatocytes were shrunk and
the cytoplasmic swelling was accompanied with massive necrosis in ANIT-treated
rats by microscopic examination (Figure 10B). The arrows indicated that the
ANIT-induced hepatocellular edema and necrosis were alleviated in rats after
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treatment of UDCA, JBP485 and J3V, separately (Figure 10C-10L), which was
consistent with the results of serology study. These results suggested that J3V could
maintain remarkable hepatoprotective effects of JBP485 on ANIT-induced liver
injury or even perform better.
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CONCLUSIONS
In this study, we develop the bioactivatable pseudo-tripeptidization strategy of
cyclic dipeptides to increase the binding affinity towards PEPT1, leading to enhanced
intestinal permeability accompanied by better stability of prodrugs in the GI tract and
enzymes-mediated bioactivation to the bioactive parent compound in the intestinal
epitheliums and hepatic cells. Our findings disclosed that the bioactivatable
pseudo-tripeptidization strategy of cyclic dipeptides could be adopted to improve oral
absorption of cyclic dipeptides with no/low affinity toward PEPT1, which could
provide more possibilities in clinical translation for cyclic dipeptides with high
bioactivity.
EXPERIMENTAL SECTIONS
General Information
JBP923 (purity: 98.8%) were purchased from Zhejiang Ontores Biotech Co., Ltd.
(Zhejiang, Hangzhou, China); L-Ser-L-Hyp-L-Gly was supplied from Zhejiang
taihepna biotechnologies Co., Ltd; cyclo(L-Pro-Gly) (purity: 98.3%) were obtained
from Anhui Research Peptide Biotechnology Co., Ltd. HBSS buffers and HEPES
buffers were brought from Dalian Meilun Biotech Co., Ltd. (Liaoning, Dalian, China);
The ter-butyloxycarbonyl (Boc) protected amino acids (Boc-L-Valine,
Boc-L-Isoleucine, Boc-L-Phenylalanine, Boc-L-Leucine) were obtained from GL
Biochem
Co.,
Ltd
(China,
(DMAP),
Shanghai);
Carbodiimide
(EDCI),
(NMM),
4-Dimethylamino-pyridine
N-methylmorpholine
1-hydroxybenzotriazole (HOBT), and dicyclohexylcarbodiimide (DCC) were
purchased from Kaile Chemicals (Shanghai,China); Glycylsarcosine (Gly-Sar) was
purchased from Sigma-aldrich (St. Louis, MO). All other chemicals used were
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Synthesis of JBP485 and PEPT1-targeted prodrugs of JBP485
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All compounds were analyzed by nuclear magnetic resonance(1H NMR) and
high resolution mass spectrometry (HRMS). The purity of JBP485 and
PEPT1-targeted prodrugs was ≥ 95% as determined by high-performance liquid
chromatography (HPLC).
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Synthesis of JBP485
The synthesized procedure was shown in Figure 2A. The Cmpd1 (1g, 3.63 mol)
was dissolved in 10 mL sodium acetate buffer (10 mM, pH 4.5) at 90 °C for 2 h. The
mixture was purified on HPLC to obtain Cmpd2. The prepared mixture was frozen
and lyophilized for 12 h to afford JPB485. The compound was obtained in 42% yield
as a white solid: 1H NMR (600 MHz, DMSO-d6): δ 7.82 (s, 1H), 5.12 (d, J = 3.0 Hz,
1H), 4.70 (t, J= 5.8 Hz, 1H), 4.41-4.31 (m, 1H), 4.29 (d, J= 4.1 Hz, 1H), 4.07 (d, J =
4.8 Hz, 1H), 3.78-3.61 (m, 2H), 3.56 (dd, J =12.6, 4.7 Hz, 1H), 3.20 (d, J = 12.6 Hz,
1H), 2.05 (dd, J = 13.0, 6.3 Hz, 1H), 1.87 (ddd, J = 12.9, 11.3, 4.4 Hz, 1H); HRMS
(ESI): [M +H], calculated 201.08698, found 201.08695.
Synthesis of JBP485-3-CH2-O-Amino Acid (AA)
The synthesized procedure was shown in Figure 2B. A mixture of Cmpd3 (10 g,
24.4 mmol), Cmpd4 (3.48 ml, 29.33 mmol) and sodium carbonate (33.1 g, 29.3 mmol)
was dissolved in DMF and stirred at room temperature (r.t.) for 12 h. The solvent was
evaporated. The residues were dissolved in DCM and were washed by 5% H3PO4,
H2O, NaHCO3, brine and dried over by Na2SO4 (anhydrous) and were filtered and
concentrated under the vacuum to afford Cmpd5. The Cmpd5 (13.8 g, 24.4 mmol)
o
was charged with 25% DEA/THF (140 ml) solution and cooled down to -5 C with
the protection of nitrogen. The solution was stirred under r.t. for 1 h. The reaction
mixture was then concentrated under the vacuum and purified by column
chromatography to afford Cmpd6. A mixture of Cmpd6, Cmpd7, EDCI, HOBt and
NMM was fully dissolved in DCM and stirred for 2 h. The reaction mixture was then
washed by 5% H3PO4, H2O, NaHCO3 (saturated solution), brine and dried over by
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Na2SO4 (anhydrous). It was filtered and concentrated under the vacuum to afford oily
product. It was purified by column chromatography (DCM/MeOH=1000/1 to 700/1)
to afford Cmpd8. The Cmpd8 (6.2 g, 10.55 mmol) and 25% DEA/THF (50 mL)
9
o
solution were mixed and stirred at -5 C for 1 h. The reaction mixture was then
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concentrated under the high vacuum to afford Cmpd9. A mixture of Cmpd9 (660 mg,
1.1 mmol), Cmpd10a (245 mg, 1.1 mmol) or Cmpd10b (285 mg, 1.1 mmol) or
Cmpd10c (256 mg, 1.1 mmol) or Cmpd10d (256 mg, 1.1 mmol), DCC and DMAP
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was fully dissolved in DCM and then cooled down to -5 C with the protection of
nitrogen. The reaction mixture was stirring under r.t. for 12 h. The reaction mixture
was filtered and concentrated under the vacuum to afford Cmpd11a-11d. 8mL of the
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cleavage cocktail (TFA/Tis/H2O=95/2.5/2.5) was cooled down to -5 C and then
Cmpd11a (800mg), Cmpd11b (928 mg), Cmpd11c (837 mg), Cmpd11d (837 mg)
were added, respectively. The reaction mixture was stirred under r.t. for 1 h. The
mixture was then precipitated into ether and was washed by ether thrice. The white
precipitation formed was collected through centrifugation at 3500 rpm for 10 min and
was purified on pre-HPLC to obtain Cmpd12a-12d. The Cmpd12a-12d were frozen
and lyophilized for 12 h, respectively.
JBP485-3-CH2-O-Val (J3V, 12a): The title compound was obtained in 25%
1
yield as a white solid: H NMR (600 MHz, DMSO-d6): δ 8.40 (s, 3H), 8.27 (s, 1H),
4.55-4.47 (m, 2H), 4.43 (ddd, J = 11.5, 6.2, 1.6 Hz, 1H), 4.39 (dq, J = 8.8, 4.8 Hz,
1H), 4.32 (t, J = 4.5 Hz, 1H), 3.57 (dd, J = 12.6, 4.6 Hz, 1H), 3.24 (d, J = 12.6 Hz,
1H), 2.17 – 2.05 (m, 2H), 1.85 (ddd, J = 12.8, 11.4, 4.3 Hz, 1H), 0.95 (dd, J = 9.3, 7.0
Hz, 6H); HRMS (ESI): [M+H], calculated 300.15540, found 300.15510.
JBP485-3-CH2-O-Phe (J3P, 12b): The title compound was obtained in 18%
1
yield as a white solid: H NMR (600 MHz, DMSO-d6): δ 8.41 (s, 3H), 8.28 (s, 1H),
7.34 (dd, J = 8.0, 6.6 Hz, 2H), 7.31-7.22 (m, 3H), 4.54-4.41 (m, 3H), 4.40-4.26 (m,
3H), 3.53 (dd, J = 12.6, 4.6 Hz, 1H), 3.24 (d, J = 12.5 Hz, 1H), 3.14 (dd, J = 14.4, 6.3
Hz, 1H), 3.07 (dd, J = 14.4, 6.3 Hz, 1H), 2.08 (dd, J = 13.1, 6.3 Hz, 1H), 1.90 (ddd, J
= 12.8, 11.2, 4.3 Hz, 1H); HRMS (ESI): [M+H], calculated 348.15540, found
348.15499.
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JBP485-3-CH2-O-Leu (J3L, 12c): The title compound was obtained in 15%
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yield as a white solid: H NMR (600 MHz, DMSO-d6): δ 8.31 (s, 2H), 8.27 (s, 1H),
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5.21 (s, 1H), 4.49 (dt, J = 8.9, 3.3 Hz, 2H), 4.43 (ddd, J = 11.5, 6.2, 1.6 Hz, 1H), 4.36
(dd, J = 11.8, 5.8 Hz, 1H), 4.32 (t, J = 4.5 Hz, 1H), 3.95 (t, J = 7.2 Hz, 1H), 3.56 (dd,
J = 12.6, 4.6 Hz, 1H), 3.25 (d, J = 12.6 Hz, 1H), 2.09 (dd, J = 13.0, 6.3 Hz, 1H), 1.85
(ddd, J = 12.7, 11.3, 4.3 Hz, 1H), 1.79-1.70 (m, J = 6.6 Hz, 1H), 1.64 (dt, J = 14.1, 7.2
Hz, 1H), 1.53 (dt, J = 14.2, 7.3 Hz, 1H), 0.86 (dd, J = 6.5, 5.3 Hz, 6H); HRMS (ESI):
[M +H], calculated 314.17105, found 314.17094.
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JBP485-3-CH2-O-Ile (J3I, 12d): The title compound was obtained in 22% yield
as a white solid: 1H NMR (600 MHz, DMSO-d6): δ 8.34 (s, 3H), 8.26 (s, 1H), 5.199 (s,
1H), 4.55-4.46 (m, 2H), 4.43 (ddd, J = 11.5, 6.2, 1.7 Hz, 1H), 4.39 (dd, J = 10.9, 3.9
Hz, 1H), 4.32 (s, 1H), 3.99 (d, J = 3.6 Hz, 1H), 3.56 (dd, J = 12.6, 4.7 Hz, 1H), 3.24
(d, J = 12.6 Hz, 1H), 2.09 (dd, J = 12.9, 6.2 Hz, 1H), 1.90-1.67 (m, 2H), 1.50-1.33 (m,
1H), 1.33-1.10 (m, 1H), 0.90 (d, J = 6.9 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H); HRMS
(ESI): [M +H], calculated 314.17105, found 314.17098.
Synthesis of JBP485-7-O-Val (J7V, 19)
The synthesized procedure was shown in Figure 2C. A mixture of Cmpd13 (10 g,
26.1 mmol), Cmpd14 (4.71 g, 26.1 mmol), NMM, EDCI and HOBt was dissolved in
DMF and stirred under r.t. for 2 h. The solvent was evaporated. The residues were
dissolved in DCM and washed by 5% H3PO4, H2O, NaHCO3, brine and dried over by
Na2SO4 (anhydrous). It was filtered and concentrated under the vacuum to afford
oiley products. It was purified by column chromatography on a silica gel
(DCM/MeOH=50/1 to 20/1) to afford Cmpd15. The Cmpd15 (6.8 g, 13.32 mmol)
o
was charged with 25% DEA/THF solution and cooled down to -5 C with the
protection of nitrogen. The solution was stirred under r.t. to afford Cmpd16 and was
concentrated under the vacuum. A mixture of Cmpd16 (2 g, 7.8 mmol), Cmpd17
(1.69 g, 7.8 mmol), DCC, DMAP was fully dissolved in DCM and stirred under r.t.
for 12 h. The reaction mixture was filtered and concentrated under the vacuum to
afford Cmpd18. 8 mL of cleavage cocktail (TFA/Tis/H2O=95/2.5/2.5) was cooled
down to -5oC and then Cmpd18 (800 mg) was added. The reaction mixture was stirred
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under r.t. for 1 h. The mixture was then precipitated into ether and was washed by
ether thrice. The white precipitation formed was collected through centrifugation at
3500 rpm for 10 min and was purified on pre-HPLC to obtain Cmpd19. The Cmpd19
was frozen and lyophilized for 12 h. The compound was obtained in 20% yield as a
white solid: 1H NMR (600 MHz, DMSO-d6): δ 8.37 (s, 3H), 8.00 (s, 1H), 5.42 (t, J =
4.9 Hz, 1H), 4.78 (s, 1H), 4.41 (ddd, J = 11.5, 6.2, 1.7 Hz, 1H), 4.09 (td, J = 4.3, 1.6
Hz, 1H), 3.91 (d, J = 4.8 Hz, 1H), 3.87 (dd, J = 13.7, 5.1 Hz, 1H), 3.73 (d, J = 4.3 Hz,
2H), 3.43 (d, J = 13.7 Hz, 1H), 2.25 (dd, J = 13.8, 6.3 Hz, 1H), 2.22-2.13 (m, 2H),
0.99 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H); HRMS (ESI): [M +H], calculated
300.15540, found 300.15517.
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Stability Study
Chemical stability
The chemical stability of JBP485 and its prodrugs was studied in buffers with
various pHs (0.1 M HCl, pH 4.5 acetate buffer, pH 5.8 phosphate buffer, pH 6.8
phosphate buffer and pH 7.2 phosphate buffer) and simulated gastric fluids (SGF, pH
1.2) and simulated intestinal fluids (SIF, pH 6.8). Above solutions were prepared
according to methods described in the Chinese Pharmacopoeia (2015 edition).
JBP485 and its prodrugs were dissolved in buffers and simulated gastric/intestinal
fluids to obtain the solutions with a concentration of 5 mM. 200 μL samples were
taken at different time points (0, 0.25, 0.5, 1, 2, 4, 8, 12, 20 h) and analyzed by HPLC.
Metabolic stability
The metabolic stability study in rats was conducted in accordance with protocols
approved by the Animal Care and Use Committee at Shenyang Pharmaceutical
University. The jejunum and liver were excised from euthanized 6-week-old male
Sprague-Dawley (SD) rats (weighing 200−250 g, no strains of animals) from
Shenyang Pharmaceutical University and were washed with ice-cold PBS buffer at
pH 7.4. The tissues which added to ice-cold PBS buffer at pH 7.4 were then
homogenized by ultrasonic homogenizer and centrifuged at 3500 rpm for 10 min. The
supernatant was collected and the protein content was obtained by the Bio-Rad DC
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Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA). The plasma was obtained
from the rat jugular vein after centrifugation at 13000 rpm for 10 min. JBP485 and its
prodrugs (5 mM) were incubated with liver (200 μg/mL of protein), intestinal
homogenates (200 μg/mL of protein) and plasma at 37 °C. Samples (200mL) were
taken at specific time points (0, 10, 20, 30, 60, 90, 120 min) and quenched with 600
μL of ice-cold acetonitrile, and then centrifuged at 13000 rpm (4 ℃) for 10 min. The
supernatant was analyzed using HPLC.
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Apparent first-order degradation kinetics and rate constants were measured by
using rates of hydrolysis. The percentage of remaining pordrugs was plotted against
corresponding time points, the half-life (t1/2) obtained from linear regression of
pseudo-first-order kinetics. The relation between the rate constants (k) and slopes of
the plots is explained by the following equation:
k=2.303×slope (log C vs time)
The t1/2 is then calculated by the following equation:
Caco-2 membrane permeability
Caco-2 cells were seeded on 12-well transwell inserts (0.4 μm pore size, area
1.12 cm2, Corning, NY) at a density of 1.0×104 cells/cm2 and cultured in MEM (10%
FBS, 1 mM sodium pyruvate, 1% non-essential amino acids and 1% L-glutamine;
Invitrogen, Carlsbad, CA). Caco-2 Cells were cultured in 5% carbon dioxide and 90%
humidity at 37 °C. Monolayers with transepithelial electrical resistance (TEER)
values >300 Ω/cm2 were used for the study. 0.5 mL of pH 6.0 Hank’s balanced salt
solution (HBSS) and 1.5 mL of pH 7.4 HBSS buffer were applied to the apical (AP)
layer and the basolateral (BL) layer, respectively. The plates were maintained at 37 °C
for 20 min. The AP layer medium was aspirated and replaced with 0.5mL of JBP485
and its prodrugs (0.5 mM) solution in pH 6.0 HBSS buffer and the BL layer medium
was replaced with 1.5 mL of pH 7.4 HBSS buffers. 200 μL of samples from BL layer
medium were collected at 5, 15, 30, 45 and 60 min for evaluating absorptive transport
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and were replaced with HBSS buffers at pH 6.0. Each experiment was repeated three
times. Drug concentrations were determined by the UPLC-MS/MS method and the
apparent permeability coefficient (Papp) was calculated using the following formula.
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where A is the surface area of the monolayer (cm2), C is the initial concentration of
JBP485 and its prodrugs (μmol) in the donor solution and dQ/dt is the steady-state
flux (μmol/s).
Single-pass intestinal perfusion (SPIP) study in rats
According to the ethical guidelines for the care and use of laboratory animals of
Shenyang Pharmaceutical University, animal experiments were conducted. The
6-week-old male Sprague-Dawley rats (SD rats, weighing 200−250 g, no strains of
animals) were bought from Shenyang pharmaceutical University and were fasted for
12 h before the perfusion experiment. Briefly, SD rats were anesthetized with
urethane (1.5 g/kg) by intraperitoneal injection and then fixed on an operation table at
37 °C. The abdomen was opened by a midline incision of 3-4 cm and about 10 cm of
jejuna was carefully exposed. After the jejuna were cleared with saline solution (37
°C), one end was connected to a peristaltic pump (BT100, Changzhou Purui Precision
Pump Co., Ltd., China) and the other end was connected to a collecting vial. The
peristaltic pump rate was 0.2 mL/min. The HEPES buffer at pH 6.0 containing 5 mM
of JBP485, JBP923 and J3V was used and the outflow samples were collected at 15,
30, 45, 60, 90, 120 min after the steady state at the rate of 0.2 mL/min. The length and
radius of the jejuna were accurately measured at the end of the experiment. Samples
(200 μL) were quenched with 600 μL of ice-cold acetonitrile and centrifuged at 13000
rpm (4 ℃) for 10 min. The supernatant was analyzed using HPLC. The effective
permeability (Peff) was determined using the following equation:
where Q is the perfusion buffer flow rate (0.2 mL/min) of the inlet solution, Cout/Cin is
the ratio of the outlet and the inlet concentrations (μmol/mL) of tested compounds,
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Vout/Vin is the ratio of the outlet and inlet volume (the density of perfusates is
determined as 1 kg/L), R and L are the radius and length of the intestinal segment
(cm), respectively.
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Cellular Uptake Study
Caco-2 cells were seeded at a density of 1×104 cells/cm2 in 24-well plates. The
concentration- and proton-dependent transport of JBP485, J3V and JBP923 were
made to validate the cellular uptake mechanism. The concentration-dependent uptake
of JBP485, J3V and JBP923, ranging from 0.2 to 5 mM, was determined in Caco-2
cells with 10 min incubation at 37 ℃. The Michaelis-Menten constant (Km) was
calculated by Eadie–Hofstee plot. The Caco-2 cells were incubated with JBP485, J3V
and JBP923 (0.5 mM) in the culture medium of either pH 6.0 or pH 7.4 HBSS buffer
with 10 min for proton-dependent uptake. After incubation, cells were washed three
times with ice-cold PBS at pH 7.4 to stop uptake. The cells were then homogenized in
0.3 mL water and 20 μL of suspension was used to determine protein content by the
Bio-Rad DC Protein Assay. The remaining cell homogenization was centrifuged at
13000 rpm for 10 min, and then the supernatant of samples was collected stored at -80
°C until analysis by UPLC/MS/MS.
An inhibition assay was conducted using 0.5 mM Gly-Sar as a probe substrate to
assess if the uptake of Gly-Sar was inhibited by JBP485, J3V and JBP923 (0.5 to 50
mM). The half-maximal inhibitory concentration (IC50) values were obtained by
Nonlinear data fitting (Graph Pad Prism V5.0). In addition, 50 mM Gly-Sar was
assessed to determine their inhibitory on 0.5 mM JBP485, J3V and JBP923 uptake
mediated by PEPT1. Furthermore, the inhibition study was also evaluated whether 50
mM captopril (the substrates for OATP-B) and metformin (the substrates for OCT1-3)
could inhibit 0.5 mM JBP485 and J3V uptake.
Molecular docking
The amino acid sequence of human PEPT1 (hPEPT1) is solute carrier having
708 amino acids. According to the two-dimensional (2D) structure, ligands (JBP485,
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J3V and JBP923) were built into three-dimensions using DS 3.0. We built homology
models of PEPT1 based on prokaryotic PEPTSo structures (PDB code: 4UVM,
sequence identities of 35%) by automatic model of SWISS-MODEL server. All
molecular docking experiments were performed with AutoDock Vina software. The
other docking parameters were set to the default values. In order to improve the
confidence level of the computer simulation, results with less than 2 Å
root-mean-square deviations (RMSD) were accepted. The optimized parameters of
simulations were as follows: as described previously43. Discovery Studio 4.0
Visualizer was used to display the interaction models between PEPT1 and ligands.
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Pharmacokinetics study
Pharmacokinetic profile of oral JBP485 and its prodrugs in rats
All pharmacokinetic study in rats was conducted in accordance with protocols
approved by the Animal Care and Use Committee at Shenyang Pharmaceutical
University. The 5-week-old male SD rats (weighing 200−250 g, no strains of animals)
were obtained from Shenyang Pharmaceutical University and were maintained on a
12 h light−dark cycle with access to food and water. The rats were fasted but had free
access to water for 12 h before experiments. Six groups of SD rats (n=6, technical
replicates) were orally treated with JBP485, J3V, J3I, J3L, J3P and J7V at a JBP485
dose of 12 mg/kg. To calculate the absolute bioavailability, JBP485 was also given
intravenously to rats at a dose of 6 mg/kg. J3V (12 mg/kg, calculate as JBP485) was
administrated to rats alone or with Gly-Sar (100 mg/kg) to verify whether J3V was the
substrate of PEPT1 or not. In addition, J3V (12 and 100 mg/kg, calculated as JBP485)
and JBP485 (12 and 100 mg/kg) were orally administered to rats to investigate the
dose effects. Blood samples (200 μL) were withdrawn from the jugular vein at 0.083,
0.167, 0.333, 0.5, 1, 2, 4, 6, 8, 10 h and then centrifuged (13000 rpm, 4 °C for 10 min),
respectively. The supernatant of samples was stored at -80 °C until UPLC-MS/MS
analysis. The absolute bioavailability of JBP485 and its prodrugs was calculated using
the following equation.
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where F is the absolute oral bioavailability, AUCpo and AUCiv are the area from time
zero to the last time point under the plasma concentration-time curves of oral and
intravenous administration, respectively. Dpo and Div are the oral and intravenous
administration doses, respectively.
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Bioactivation sites study of J3V
The systemic and hepatic portal pharmacokinetics of J3V was evaluated to
investigate the bioactivation sites in rats after an oral administration of J3V (12 mg/kg,
calculated as JBP485). Portal vein and jugular samples were withdrawn
simultaneously at 15, 30, 60, 120 min and then centrifuged (13000 rpm, 4 °C for 10
min), respectively. The supernatant of samples was stored at -80 °C until bioanalysis.
Pharmacokinetic study in Beagle dogs
The pharmacokinetic study in beagle was conducted in accordance with the
guidelines recommended by the Animal Care and Use Committee at Shenyang
Pharmaceutical University. The 2-year-old male beagle dogs (weighing 8-10 kg, no
strains of animals) obtained from Shenyang Pharmaceutical University were orally
administered with JBP485 (12 mg/kg) and J3V (12 mg/kg, calculated as JBP485) in
aqueous solution. Blood samples (1 mL) were collected from the jugular vein via
direct vein puncture at 0.167, 0.25, 0.33, 0.5, 1, 2, 4, 6, 8 h and placed into sodium
heparin tubes. Blood samples were centrifuged at 13000 rpm for 10 min at 4 °C and
collected in tubes and stored at -80 °C until bioanalysis.
Anti-hepatitis activities in vivo
The 6-weeks-old male SD rats (weighing 200-250 g, no strains of animals) were
purchased from Shenyang Pharmaceutical University. All pharmacokinetic study in
rats was conducted in accordance with protocols approved by the Animal Care and
Use Committee at Shenyang Pharmaceutical University. The rats were fasted but had
free access to water for 12 h before experiments. 1-Naphthyl isothiocyanate (ANIT)
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was dissolved in olive oil and compounds (JBP485 and J3V) were dissolved in water
and ursodeoxycholate sodium (UDCA) was suspended in 5% sodium
carboxymethylcellulose (CMC-Na) solution. The animals were randomly divided into
12 groups with 6 rats in each group as following: Group 1: The rats served as normal
control and received water at 30 min before and 8, 22, 32 and 46 h after olive oil was
intraperitoneally (i.p) injected; Group 2: the rats were i.p injected at the dose of 50
mg/kg ANIT to induce cholestasis; Group 3: UDCA was orally administered to the
ANIT-treated rats at doses of 100 mg/kg at 30 min before and 8, 22, 32 and 46 h after
ANIT treatment; Group 4-6: JBP485 was orally administered to the ANIT-treated rats
at doses of 6.25, 12.5 and 25 mg/kg in the same manner as described in Group 3;
Group 7-8: JBP485 was i.p injected to the ANIT-treated rats at doses of 6.25, 12.5
mg/kg in the same manner as described in Group 3; Group 9-12: J3V was orally
administered to the ANIT-treated rats at doses of 3.125, 6.25, 12.5 and 25 mg/kg in
the same manner as described in Group 3. Serum was collected 48 h after ANIT
treatment for determining hepatic biochemical index. For serum collection, rats were
anesthetized with chloral hydrate and blood was collected from abdominal aorta.
Blood was then left on the coagulation-promoting tubes and centrifuged for 5 min at
3500 rpm to obtain the supernatant as serum. The total bilirubin concentration (T-BIL)
and the activity of liver specific cytosolic enzymes (ALT and AST) in the rat serum
were determined using the appropriate assay kits (Nanjing Jiancheng Pure Chemical
Industries). Moreover, the cut liver was immersed in 4% triformol solutions and
stained with hematoxylin–eosin (H&E) for morphological examination.
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Analytical methods
Pre-HPLC Analysis JBP485 and its prodrugs were purified by preparative high
performance liquid chromatography (pre-HPLC) (Hitachi Technologies, Japan) that
consist of a Hitachi L-2130 pump, Hitachi L-2420 UV detector. Separation was
achieved using a C18 column (250 mm×20 mm, 5 μm) maintained at 30 °C with a
mobile phase consisting of methol:water (5:95, v/v) for JBP485 and
methol:water:hydrochloric acid (5:95:0.1, v/v/v) for all prodrugs.
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HPLC Analysis The samples from purity study of JBP485 and its prodrugs were
analyzed by Hitachi HPLC instrument (Hitachi Technologies, Japan) that consist of a
Hitachi 1110 pump, Hitachi 1210 auto sampler, and Hitachi 1410 UV detector.
Separation was achieved using a C18 column (25mm×4.6mm, 5μm) maintained at 30
°C with a mobile phase consisting of methol:water (5:95, v/v) for JBP485 and
acetonitrile:water:trifluoroacetic acid (5:95:0.1, v/v/v) for all prodrugs. The flow rate
was 1.0 mL/min and the UV detection were carried out at 205 nm.
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The samples from stability study and SPIP study of JBP485 and its prodrugs
were analyzed by WATERS e2695 HPLC instrument (WATERS Technologies, CA,
USA) that consist of a WATERS e2695 pump, WATERS e2695 autosampler, and
WATERS 2489 UV detector. Separation was achieved using a C18 column
(25mm×4.6mm, 5μm) maintained at 30 °C with a mobile phase consisting of
acetonitrile:water:trifluoroacetic acid (5:95:0.1, v/v/v). The flow rate was 1.0 mL/min
and the UV detection were carried out at 205 nm.
UPLC-MS-MS Analysis Cell samples from monolayer permeability study and
cellular uptake study and plasma samples from pharmacokinetic study were analyzed
using ultra performance liquid chromatography–dual mass spectrometry
(UPLC–MS/MS) method performed on ACQUITY UPLCTM system (Waters Corp.,
Milford, MA). Prior to analysis, frozen samples were thawed on ice. The samples (50
μL) were processed using a protein precipitation method by addition of 50 μL of
internal standard (cyclic (L-Pro-L-Gly), 0.1 μM) dissolved in acetonitrile and 100 μL
of acetonitrile, followed by vortex mixing for 30 s and then centrifugation at 13000
rpm for 10 min at 4 °C. A 150 μL aliquot of supernatant was dried by nitrogen and the
dried samples were reconstituted by solutions containing 50 μL of acetonitrile and 50
μL of water. A volume of 10 μL was injected into the liquid chromatography
instrument for quantitative analysis. The separations were carried out on the C18
column (50 mm×2.1 mm, 1.7 μm; Waters Corp.) with mobile phase composed of
methanol/water/acetic acid (5/95/0.1, v/v/v). In the MS/MS detector, the cone voltage
was maintained at 35 V, collision energy was at 18 eV and argon collision gas
pressure was 4.0×10−3 mbar. Data acquisition was by multiple reactions monitoring in
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positive ion electrospray ionization mass spectrometry. The compounds were
analyzed by multiple reaction monitoring of the transitions of m/z 200.1→86.2 for
JBP485, m/z 300.3→72.1 for J3V, m/z 314.3→72.1 for J3I, m/z 314.3→72.1 for J3L,
m/z 348.3→72.1 for J3P, m/z 300.3→72.1 for J7V, m/z 219.2→86.0 for JBP923, and
155.2→70.0 for internal standard, respectively.
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Statistical analysis
All the quantitative data were expressed as their mean±S.D. (standard deviation),
and the statistical analysis was made using the ANOVA test. The differences were
considered to be statistically significant at p< 0.05
SUPPLEMENT INFORMATION
Additional supporting documents are available, including 1H NMR, HRMS
spectra and HPLC chromatography of JBP485 and its prodrugs (J3V, J3L, J3P, J3I,
J7V) and the 3D pdb files for docking of JBP485, JBP923 and J3V inside the binding
site of hPEPT1
.
AUTHOR INFORMATION
Corresponding Authors
* Zhonggui He. E-mail address: hezhonggui@vip.163.com.
* Jin Sun. E-mail address: sunjin66@21cn.com.
ORCID
Zhonggui He: 0000-0002-5907-3875
Jin Sun: 0000-0001-7385-9017
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
This work was financially supported by the National Nature Science Foundation
of China (No. 81773656, 81573497, U1608283) and Key Projects of Technology
Bureau in Shenyang (No18400408) and the Key Projects of Liaoning Province
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Department of Education (No. 2017LZD03).
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ABBREVIATIONS USED
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PEPT1, oligopeptide transporter 1; JBP485, cyclo(L-Hyp-L-Ser); JBP923,
L-Hyp-L-Ser; J3V, JBP485-3-CH2-O-Valine; J3L, JBP485-3-CH2-O-Leucine; J3I,
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JBP485-3-CH2-O-Isoleucine;
JBP485-7-O-Valine; Boc-, ter-butyloxycarbonyl-; EDCI, Carbodiimide; DMAP,
4-Dimethylamino-pyridine; NMM, N-methylmorpholine; HOBT,
J3P,
JBP485-3-CH2-O-Phenylalanine;
J7V,
1-hydroxybenzotriazole; DMF, dimethylformamide; DCM, dichloromethane; DCC,
dicyclohexylcarbodiimide; r.t., room temperature; Gly-Sar: glycylsarcosine; DPK,
diketopiperazine; GI, gastrointestinal; Ki, inhibition constant; HPLC,
high-performance liquid chromatography; NMR, nuclear magnetic resonance; MS:
mass spectrometry; UPLC-MS/MS, ultra performance liquid chromatography-dual
mass spectrometry; t1/2, half-time; FBS, fetal bovine serum; IC50, half-maximal
inhibitory concentration; TEER, transepithelial electrical resistance; Papp, apparent
permeability coefficient; HBSS, Hank’s balanced salt solution; AP: apical; BL:
basolateral; SPIP: single-pass intestinal perfusion; Km, michaelis constant; i.v.,
intravenous; 2D structure, two-dimensional structure; SD rats: Sprague-Dawley rats;
PDB, Protein Data Bank; S.D., standard deviation.
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