Keywords
Bulevirtide - Bule-OH - production costs - pharmacokinetic
Introduction
Hepatitis B virus (HBV) infection is a significant global public health issue and
is the third leading cause of mortality globally causing an estimated 800,000 deaths
per year.[1] In clinical therapy, first-line pharmaceuticals typically encompass nucleoside analogs
and PEGylated interferon (PEG-IFN). However, the former is prone to induce drug resistance,
whereas the latter can lead to side effects reminiscent of influenza-like symptoms
and myalgia.[2]
[3] Therefore, there is a pressing clinical necessity for the development of antiviral
therapies against HBV that employ innovative mechanisms of action.[4] In 2012, researchers identified sodium taurocholate cotransporting polypeptide (NTCP)
as a receptor that facilitates HBV entry into hepatocytes.[5] Inhibiting HBV entry can prevent the infection of uninfected cells, and reinfection
of already infected cells, and produces superior therapeutic outcomes when used in
conjunction with other anti-HBV drugs.[6] Targeting the viral entry process is an efficacious strategy to prevent postexposure
infections, halt mother-to-child transmission, and avoid infections associated with
organ transplantation.[7]
Bulevirtide developed by the German biotechnology firm MYR GmbH is an entry inhibitor
that targets NTCP.[8] Bulevirtide is derived from the PreS1 domain of the large surface antigen protein
of HBV spanning amino acid residues 2 to 48, and competitively binds to the NTCP,
thereby preventing HBV entry into cells.[9] Bulevirtide is now commercially available in Europe and is administered through
once-daily subcutaneous injections at a dosage of 2 mg.[10] Clinical evidence indicates that Bulevirtide is well-tolerated and exhibits significant
therapeutic efficacy.[11]
Bulevirtide is prepared from the synthesis of 47 natural amino acids, followed by
the N-terminal myristoylation using MBHA resin.[12] However, the solid-phase synthesis is not only costly and time-consuming but also
necessitates the use of excess hazardous reagents and solvents.[13] Reports indicate that the solid-phase synthesis of 1 kg of 19-peptide generates
13,063 kg of waste material, which contradicts the principles of green chemistry and
incurs additional costs for the treatment of pollutants.[14] In 2022, the Food and Drug Administration declined to approve Bulevirtide for marketing
in the United States, citing concerns over its manufacturing and distribution processes.
Furthermore, hepatitis D virus (HDV) and HBV infections require long-term medication,
imposing a significant burden on patients in terms of extensive treatment durations
and exorbitant drug costs.[15] In summary, there is a pressing need to develop a more cost-effective and environmentally
friendly method for the preparation of Bulevirtide to meet the clinical demands for
anti-HBV infection treatment.[16]
To reduce Bulevirtide's production costs, recombinant fermentation technology was
employed to successfully obtain a fragment comprising 47 natural amino acids. Given
that recombinant expression is limited to natural amino acids, the N-terminal myristoylation
of Bulevirtide cannot be obtained through fermentation.[17] However, a selective N-terminal myristoylation method can be developed by modulating
the solvent composition and pH levels.[18] Leveraging both fermentation expression and chemical modification techniques, we
have successfully synthesized a Bulevirtide analog, designated as Bule-OH. Bule-OH
is characterized by a carboxylic acid functional group at the C-terminus, whereas
Bulevirtide possesses an amide structure. Preliminary cost analysis under controlled
laboratory conditions suggests that Bule-OH production may offer significant cost
advantages compared with conventional Bulevirtide synthesis methods. These initial
findings require further validation at the production scale, suggesting a potential
for substantial process economics improvement in optimized systems. Furthermore, we
conducted an exhaustive comparative analysis of the biological activity, stability,
self-association propensity, and enzymatic stability of Bule-OH and Bulevirtide, and
assessed their pharmacokinetic profiles in an NTCP mouse model. The study revealed
that Bule-OH and Bulevirtide exhibit similar pharmacokinetic characteristics in the
liver, with Bule-OH demonstrating a more pronounced hepatic targeting capability.
These findings suggest that Bule-OH holds the potential to serve as a cost-effective
alternative to Bulevirtide.
Materials and Methods
Cell Lines and Reagents
All materials and chemicals were commercially available. The Huh-7D-NTCP cell line
was provided by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Purchase source of
reagents for N-terminal myristoylation is described in the [Supporting Information] (available in the online version).
Hepatitis B Virus Infection Inhibition Test
Huh-7D-NTCP cells were plated at a density of 2 × 105/mL (100 μL/well) and maintained in a Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 2.5% dimethyl sulfoxide (DMSO), on the second day, 5% fluorescein isothiocyanate
was added and incubation was continued for 24 hours.
Infectious HBV particles were obtained from HepAD38 (tet-off) cell line supernatants.
Briefly, cells were maintained in DMEM (Sigma-Aldrich, St. Louis, Missouri, United
States)/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1%
penicillin–streptomycin. Viral supernatants were collected at 72 hours postconfluence,
filtered through 0.45-μm membranes, and concentrated by polyethylene glycol (PEG8000)
precipitation. Viral titers were determined by quantitative PCR using HBV-specific
primers (forward: 5′-GTGTCTGCGGCGTTTTATCA-3′; reverse: 5′-GACAAACGGGCAACATACCTT-3′),
with working concentrations normalized to 100 multiplicity of infection (MOI) for
infection experiments.
The HBV virus derived from the supernatant of HepAD38 cells was diluted with an infection
dose of 100 MOI. The dilution medium was DMEM with 2.5% DMSO and 4% PEG8000 (Sigma-Aldrich,
St. Louis, Missouri, United States). Eight different concentrations of compounds of
1000, 200, 40, 8, 1.6, 0.32, 0.064, and 0.0128 nmol/L were prepared using the same
dilution medium. The infection-blocking group was added with different concentrations
of compounds (100 μL/well) 30 minutes before the addition of diluted virus (100 μL/well).
The contents were gently pipetted 4 to 6 times and then centrifuged at 1,000 rpm for
30 minutes at 25°C. The cells were incubated for 16 to 24 hours, followed by the removal
of the infection solution, washing with phosphate buffer saline (PBS) three times,
and the addition of DMEM containing 2.5% DMSO and 5% FBS. On the 11th day, the cell
supernatant was collected, and the hepatitis B surface antigen level in the cell supernatant
was measured by determining absorbance values using a microplate reader (SpectraMaxi3x,
Molecular Devices, San Jose, California, United States), and the response values of
Bulevirtide and its analogs at different concentrations were calculated using GraphPad
Prism 8.01 software after linear fitting of the standard curve between concentration
and signal value.
Bulevirtide Synthesis
MBHA resins with a loading capacity of 0.25 to 0.6 mmol/g were used for Bulevirtide
Synthesis. A detailed methodology is provided in the [Supporting Information] (available in the online version).
Expression and Purification of 1–47
A recombinant fusion sequence of SUMO and 1–47, namely: SUMO-(1–47), was engineered
by gene fusion. The 1–47 fusion sequence is derived from Bulevirtide at positions
2–48, but its C-terminus is a carboxyl group rather than an amide. 1–47 constructs
were expressed in Escherichia
coli and subsequently purified via reversed-phase C4 chromatography. The detailed methodology
is provided in the [Supporting Information] (available in the online version).
N-terminal Myristylation of 1–47
The 1–47 was dissolved in PBS (50 mmol/L K2HPO4, pH 6.5) to a final concentration of 5 mg/mL; then, a 50% volume of acetonitrile
(ACN) was added. The myristoylated monosuccinimide ester ethanol solution was added
in a feed ratio of 1:1. The reaction proceeded for 2 hours and was stopped by adjusting
the pH to 4.0 using 0.1 mol/L HCl. The reaction mixture was diluted to a concentration
of ACN constituted less than 10% of the solution, filtered and purified using reverse-phase
high-performance liquid chromatography (RP-HPLC) (Waters 600E-2487, Milford, Massachusetts,
United States) on C4 columns (Kromasil C4, 10 μm, 10 × 250 mm, 300 Å; AKZO NOBEL,
Sweden) with ACN/trifluoroacetic acid (TFA) as the mobile phase. The purity and identification
of the products were determined using HPLC (DIONEX UltiMate 3000, Thermo Fisher, Waltham,
Massachusetts, United States) and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS;
Waters Acquity ultra-performance liquid chromatography [UPLC]-Xevo G2-XS-QTof, Milford,
Massachusetts, United States).
High-Performance Liquid Chromatography Analysis
HPLC analysis was performed on a Kromasil C4 column (4.6 × 150 mm, 3.5 μm, 100 Å)
with a flow rate of 1 mL/min. Detection wavelengths were set at 215 and 280 nm, with
an injection volume of 20 μL. The mobile phase consisted of (A) 0.1% TFA in water
and (B) 0.1% TFA in ACN.
Liquid Chromatography–Tandem Mass Spectrometry Analysis
LC–MS/MS analysis was conducted using an Acquity UPLC system coupled with a QDA mass
detector. The QDA parameters were as follows: electrospray ionization in positive
mode, capillary voltage of 0.8 kV, desolvation gas temperature of 600°C, and ion source
temperature of 120°C.
Plasma Stability of Bule-OH and Bulevirtide
Bule-OH and Bulevirtide were added to human plasma (600 μL, Sigma-Aldrich) to a final
concentration of 50 μg/mL, respectively, and incubated at 37°C with continuous temperature-controlled
shaking. Aliquots of 100 μL were collected at 0, 0.5, 1, 2, 4, 8, 10, 12, 16, 20,
and 24 hours. To each sample, 200 μL of precipitant solution (consisting of ACN and
methanol in a 3:2 ratio, with 0.1% glacial acetic acid) was added. The mixture was
vortexed and centrifuged at 18,000 g for 7 minutes at 4°C. The supernatant (200 μL)
was taken, of which 20 μL was subjected to LC–MS/MS analysis.
Gastrointestinal Enzyme Stability of Bule-OH
Bule-OH and Bulevirtide were diluted in PBS (pH 6.8) to a final concentration of 250 μg/mL,
of which 500 μL was taken and an equal volume of trypsin (parenzyme) or chymotrypsin
(each at a concentration of 2 U/mL) was added to a final concentration of 125 μg/mL
of the sample. The sample was incubated in a water bath at 37°C and at intervals of
0, 10, 20, 30, 45, and 60 minutes, 80 μL of the sample was taken and 40 μL of 1 mol/L
hydrochloric acid was added to quench the reaction, followed by HPLC analysis.
Blue-OH and Bulevirtide were diluted to a final concentration of 100 μg/mL using PBS
with pH values being 7.4, 5.3, and 2.6, respectively. A 500 μL of the above solution
was added to an equal volume of gastric protease (pepsin) at a concentration of 7
U/mL, to a final concentration of 50 μg/mL. The sample was incubated at 37°C in a
water bath, and at 0, 10, 20, 30, 45, and 60 minutes, 200 μL of the sample was taken
and added to 800 μL of methanol to quench the reaction, followed by HPLC analysis.
Determination of Self-Association Tendency of Bulevirtide and Bule-OH
Bule-OH and Bulevirtide were dissolved in PBS to reach a concentration of 1 mg/mL.
The solutions were shaken at a speed of 100 rpm using a shaking incubator (Shihping
SPH-2112D, Shanghai, China) at 37°C. Absorbance at 340 nm was measured at designated
time points, and the entire procedure lasted for 85 hours. The preparation and packaging
of the solutions must be conducted under sterile conditions. Given the self-association
propensity of glucagon-like peptide-1 7–37 (GLP-1 (7–37), MedChemExpress), this peptide
served as a positive control in our comparative assessment of the self-association
behaviors of Bule-OH and Bulevirtide.
Pharmacokinetic Studies
Male human sodium taurocholate cotransporting polypeptide (hNTCP) mice were purchased
from the Pharmacological Evaluation Institute of Chia Tai Tianqing Pharmaceutical
Group. Prior to administration, the experimental animal underwent a 12-hour fasting
period, followed by a 4-hour feeding period and access to water ad libitum. The mice
were randomized into groups (n = 3), and each group was given a single subcutaneous dose of Bulevirtide and Bule-OH
(463 nmol/kg). Blood was collected at 0.5, 1, 2, 4, 6, 8, 10, 24, 30, 48, and 72 hours'
postinjection. Blood samples were collected in ethylene diamine tetraacetic acid-coated
test tubes, kept on ice, and centrifuged at 1,200 × g for 15 minutes at 4°C. Plasma
was transferred to micronic tubes and stored at −80°C until further analysis.
Male hNTCP mice were randomized into groups (n = 3), and each group was given a single subcutaneous dose of Bulevirtide and Bule-OH
(463 nmol/kg). Liver tissue was collected at 1, 8, 24, and 72 hours' postinjection.
The liver was extracted and washed 2 to 3 times with physiological saline to eliminate
any blood and eliminate surface connective tissue fat. The samples were then cut into
small pieces and repeatedly washed with physiological saline until there was no blood.
A small amount of physiological saline was added, and the liver was homogenized using
a tissue grinder or homogenizer.
Introduce 100 μL of plasma or liver tissue homogenate. Then, 50 μL of internal standard
solution and 150 μL of precipitant (solvent: ACN: methanol [3:2] [containing 0.1%
glacial acetic acid]) were added. The mixture was vortexed and centrifuged at 18,000 g
at 4°C for 7 minutes. A 200 μL of the supernatant was collected, of which 20 μL aliquot
was subjected to LC–MS/MS analysis. Pharmacokinetic parameters were calculated using
a two-compartment model with Drug and Statistics software (DAS, ver. 2.0; Mathematical
Pharmacology Professional Committee of China, Shanghai, China).
Statistical Analysis
Prism software version 8.01 (GraphPad, La Jolla, California, United States) was used
for nonlinear regression and statistical analyses. The half-maximal inhibitory concentration
(IC50) was determined by fitting a sigmoidal dose–response inhibition “log (inhibitor)
versus response–variable slope (four parameters)”. Graphs depict the average ± standard
deviation of at least three independent experiments. All normally distributed data
were compared using Student's t-test or one-way analysis of variance with post hoc analysis (Games–Howell's procedure)
as appropriate. p-Values of <0.05 were considered statistically significant.
Results
Design, Expression, and Purification of 1–47
Sequence 1–47 represents the native amino acid sequence of Bulevirtide, as depicted
in [Fig. 1A], which outlines the expression and purification process ([Supplementary Figs. S6] and [S7], [Supporting Information], available in the online version). We engineered an E. coli strain to incorporate a small ubiquitin-like modifier (SUMO) fusion tag ([Fig. 1B]). Electrophoresis analysis (SDS-PAGE) indicated that the intermediate protein in
the lysate exhibited a band at 15 kDa during fermentation, aligning with the predicted
molecular weight ([Fig. 1C]). The band's intensity increased with extended fermentation times, signifying the
accumulation of the intermediate protein. Postenzymatic cleavage, the 1–47 peptide,
owing to its small size, was not visible on the gel; however, the SUMO tag was evident
at 11 kDa, corresponding to the expected molecular weight, thus confirming the cleavage's
success ([Fig. 1D]). The 1–47 peptide eluted directly through the Q XL chromatography, whereas the
SUMO tag bound to the column, facilitating their separation ([Fig. 1E]). Despite the SUMO tag's removal, the sample contained impurities ([Fig. 1F]). Employing reverse purification ([Fig. 1G]), we achieved a peptide purity of over 95% ([Fig. 1H]).
Fig. 1 Expression and purification of peptide 1–47. (A) The preparation route for 1–47. (B) Schematic representation of the expression vector for SUMO-1–47. (C) Electrophoretic analysis showing the expression profile of SUMO-1–47 across the
fermentation period. (D) Electrophoretic analysis showing the protein expression after SUMO-1–47' enzymatic
digestion and purification. (E) The chromatographic profile of Q XL purification. (F) Monitoring of the Q XL elution process using RP-HPLC with UV absorption chromatography
at 215 nm. (G) RP-HPLC conditions. (H) The chromatographic peak of purified 1–47 was obtained using RP-HPLC with UV detection
at 215 nm. RP-HPLC, reverse-phase high-performance liquid chromatography.
Bule-OH Preparation Through N-terminal Modification of the 1–47 Sequence with Myristoylation
Bule-OH was obtained by adding a myristoyl modification at the N-terminus ([Fig. 2A]). The N-terminal modification process of 1–47 was optimized, and the specific results
are shown in [Table 1]. Initially, since 1–47 is soluble in water, whereas myristoyl succinimidyl ester
is not, we added ACN to facilitate the dissolution of both. Subsequently, we found
that the modification efficiency was highest under conditions of pH 6.5. By reducing
the substrate ratio, we achieved a high N-terminal modification efficiency of 1–47
with approximately 36.87% yield and 32.19% recovery ([Fig. 2B]). The specific procedure involved dripping a solution of myristoyl succinimidyl
ester in ethanol into a buffered solution of 1–47 under conditions of pH 6.5 and 50%
ACN for a reaction time of 1 hour, followed by adjusting the pH to 2 with 0.1 mol/L
hydrochloric acid to terminate the reaction, thus achieving N-terminal myristoyl modification.
The purity of Bule-OH exceeds 95% by an RP-HPLC analysis ([Fig. 2D]). Secondary mass spectrometry analysis indicated that the sequence of Bule-OH is
consistent with Bulevirtide ([Fig. 2C]), and high-resolution mass spectroscopy (HRMS) results showed that its actual molecular
weight matches the theoretical value ([Supplementary Fig. S9], [Supporting Information], available in the online version). Since 1–47 is obtained through fermentation expression,
its C-terminus is a carboxylic acid, which is different from the amide at the C-terminus
of Bulevirtide. The analysis of the N-terminal fragment ion peaks in the secondary
mass spectrum indicated that the myristic acid modification occurred at the N-terminus
([Fig. 2E]).
Fig. 2 Structural characterization and purification analysis of Bule-OH. (A) The schematic representation of the Bule-OH sequence. (B) Monitoring of the N-terminal modification of 1–47 with myristic acid using RP-HPLC
with UV absorption chromatography at 215 nm. (C) Sequence information of Bule-OH characterized by MS/MS spectrometry. (D) The chromatographic peak of purified Bule-OH was obtained using RP-HPLC with UV
detection at 215 nm. (E) MS/MS spectrometry-based analysis of the ion peak of the N-terminal fragment of
Bule-OH. MS/MS, tandem mass spectrometry; RP-HPLC, reverse-phase high-performance
liquid chromatography.
Table 1
N-terminal modification efficiency of 1–47 under various modification conditions
|
Entry
|
Buffer
|
Feed ratio
|
Residual amount (%)
|
Yield (%)
|
|
1
|
PBS (pH 4.0, 50 mmol/L)
|
1:10
|
100
|
NA
|
|
2
|
PBS (pH 5.0, 50 mmol/L)
|
1:10
|
100
|
NA
|
|
3
|
PBS (pH 6.0, 50 mmol/L)
|
1:10
|
12.21
|
11.23
|
|
4
|
PBS (pH 6.5, 50 mmol/L)
|
1:10
|
10.84
|
16.95
|
|
5
|
PBS (pH 7.0, 50 mmol/L)
|
1:10
|
3.19
|
10.35
|
|
6
|
PBS (pH 7.5, 50 mmol/L)
|
1:10
|
1.72
|
7.46
|
|
7
|
PBS (pH 8.0, 50 mmol/L)
|
1:10
|
NA
|
4.51
|
|
8
|
PBS (pH 8.5, 50 mmol/L)
|
1:10
|
NA
|
2.43
|
|
9
|
PBS (pH 9.0, 50 mmol/L)
|
1:10
|
NA
|
1.08
|
|
10
|
PBS (pH 9.5, 50 mmol/L)
|
1:10
|
NA
|
NA
|
|
11
|
PBS (pH 6.5, 50 mmol/L)
|
1:5
|
7.69
|
25.88
|
|
12
|
PBS (pH 6.5, 50 mmol/L)
|
1:4
|
10.54
|
29.09
|
|
13
|
PBS (pH 6.5, 50 mmol/L)
|
1:3
|
12.63
|
30.90
|
|
14
|
PBS (pH 6.5, 50 mmol/L)
|
1:2
|
26.50
|
36.35
|
|
15
|
PBS (pH 6.5, 50 mmol/L)
|
1:1
|
32.19
|
36.87
|
|
16
|
PBS (pH 6.5, 50 mmol/L)
|
1:0.8
|
35.24
|
31.27
|
|
17
|
PBS (pH 6.5, 50 mmol/L)
|
1:0.6
|
38.42
|
29.68
|
Abbreviations: NA, not applicable; PBS, phosphate buffer saline.
Bulevirtide was synthesized using standard solid-phase peptide synthesis with the
9-fluorenyl methyl chloroformate (Fmoc) strategy, as depicted in [Fig. 3A]. The crude peptides, postcleavage, were subjected to purification on a C4 column
using RP-HPLC with a mobile phase of ACN/acid glacial, yielding a sample with a purity
exceeding 95%, as shown in [Fig. 3B]. The purity and characteristics of Bulevirtide were determined by RP-HPLC and HRMS
([Supplementary Figs. S1]–[S5], [Supporting Information], available in the online version). Furthermore, as illustrated in [Fig. 3C], the preparation cost of Bule-OH has been reduced to approximately 1/26th that of
Bulevirtide.
Fig. 3 Synthesis, purification, and cost analysis of Bulevirtide. (A) Bulevirtide preparation route. (B) The chromatographic peak of Bulevirtide was obtained using an RP-HPLC with UV detection
at 215 nm. (C) The cost comparison was derived from our internal accounting of raw material expenses
and labor hours invested in the synthesis and purification processes to obtain 5 g
batches of each compound with >95% purity (USD/5 g basis).
Fig. 4 Comparative evaluation of the antiviral activity, solubility, and stability profiles
of Bulevirtide and Bule-OH. (A) Dose–response curves of Bulevirtide and Bule-OH based on blocking HBV infection.
(B) Anti-HBV activity. (C) Change in solution transmittance of Bule-OH, Bulevirtide, and GLP-1 (7–37) at pH
7.4 and 37°C under continuous agitation. (D) Plasma stability of Bule-OH and Bulevirtide. (E) Stability comparison between Bule-OH and Bulevirtide in parenzyme (1 U/mL). (F) Stability comparison between Bule-OH and Bulevirtide in chymotrypsin (1 U/mL). (G) Percentage remaining of intact Bule-OH and calculated t
1/2 upon incubation with pepsin (3.5 U/mL). (H) Percentage remaining of intact Bulevirtide and calculated t
1/2 upon incubation with pepsin (3.5 U/mL). HBV, hepatitis B virus.
Fig. 5 Comparative pharmacokinetic profiles of Bulevirtide and Bule-OH following their subcutaneous
administration in hNTCP mice. (A) Blood concentration curve. (B) Liver tissue drug concentration curve. (C) Liver blood ratio of Bulevirtide and Bule-OH. hNTCP, human sodium taurocholate cotransporting
polypeptide.
In vitro Bioactivity of Purified Bulevirtide Analogs
The anti-HBV viral activity assay was conducted using Huh-7D-NTCP cells. As depicted
in [Fig. 4A], [B], Bule-OH exhibited a reduction in activity by approximately 50% relative to Bulevirtide,
yet its maximum inhibition rate remained unchanged. This reduction in Bule-OH's activity
is attributed to the substitution of an amide with a carboxyl group at the C-terminus.
Despite this, Bule-OH retains potent anti-HBV activity, warranting further development
as a potential oral analog.
Plasma Stability and Association Tendency
Self-association increases the volume of peptide molecules, adversely affects solubility,
and may impair both biological activity and stability. A thermal aggregation assay
was performed under continuous agitation to assess the changes in the transmittance
rate of Bule-OH, Bulevirtide, and GLP-1 (7–37). As illustrated in [Fig. 4C], the transmittance of the GLP-1 (7–37) solution increased by over 15% within 6 hours,
whereas the transmittance of the Bule-OH and Bulevirtide solutions increased by less
than 5% within 85 hours. These findings suggest that Bule-OH and Bulevirtide exhibit
a lower tendency to self-association.
The C-terminal amide group of peptides is crucial for their stability. As shown in
[Fig. 4D], Bulevirtide maintains its stability, with 85% of the compound remaining after 24 hours.
In contrast, Bule-OH, which features a carboxyl group in place of the C-terminal amide,
exhibits reduced plasma stability. These findings suggest that the C-terminal amide
group significantly contributes to Bulevirtide's plasma stability.
Enzymatic Stability Studies
The C-terminal amide of peptides plays a significant role in their enzymatic stability.
We assessed the enzymatic stability of Bulevirtide and Bule-OH in the presence of
parenzyme, chymotrypsin, and pepsin. Our data show that Bule-OH degrades more rapidly
in parenzyme compared with Bulevirtide ([Fig. 4E], all p < 0.001). Similarly, Bule-OH exhibits reduced stability in chymotrypsin relative
to Bulevirtide ([Fig. 4F], p < 0.01). The C-terminal amide structure prevents degradation by gastrointestinal
enzymes. Pepsin is inactive at pH 7.4 and is incapable of degrading the peptides.
Conversely, as the pH decreases, pepsin's activity increases, resulting in a shorter
half-life of the peptides. At equivalent pH levels, Bulevirtide exhibits a longer
half-life than Bule-OH ([Fig. 4G], [H]), suggesting that it is more stable against pepsin.
Pharmacokinetic Study of Human Sodium Taurocholate Cotransporting Polypeptide Mice
following Subcutaneous Injection
The in vivo pharmacokinetics of Bule-OH and Bulevirtide were evaluated in hNTCP mice following
subcutaneous injection of 463 nmol/kg. The corresponding plasma concentration-time
profiles are depicted in [Fig. 5A], with pharmacokinetic parameters detailed in [Table 2]. The analysis revealed that the pharmacokinetics of both compounds, specifically
463 nmol/kg doses of Bulevirtide and Bule-OH, adhered to a two-compartment model postinjection.
Bulevirtide achieved a C
max of 1,740 ng/mL at 2 hours, whereas Bule-OH reached a C
max of 4,928 ng/mL within 1 hour after injection. The half-life of Bule-OH is 7.5 times
that of Bulevirtide. Bulevirtide is below the detection limit (1 ng/mL) at 10 hours,
whereas Bule-OH can still be detected at 48 hours. Moreover, Bule-OH demonstrated
a significantly higher plasma exposure, approximately 16.5-fold that of Bulevirtide.
Table 2
Pharmacokinetic parameters of Bulevirtide and Bule-OH in plasma
|
Analyst
|
AUClast (h•ng/mL)
|
C
max (ng/mL)
|
T
max
(h)
|
HL_Lambda_z
(h)
|
AUC 1–10
(h•ng/mL)
|
|
Bule-OH
|
13,863
|
4,928
|
1
|
7.38
|
113,188
|
|
Bulevirtide
|
6,850
|
1,740
|
2
|
0.98
|
6,850
|
Abbreviations: AUClast, the area under the curve from time zero to the last measurable
concentration; C
max, maximum plasma concentration observed; T
max, time to reach maximum observed concentration; HL_Lambda_z, half-life associated
with the terminal elimination rate constant (λz); AUC1–10, the area under the concentration-time
curve from hour 1 to hour 10.
Bulevirtide targets the liver, and our study evaluated drug concentrations in the
liver as depicted in [Fig. 5B] and detailed in [Table 3]. Bule-OH and Bulevirtide both achieve their C
max at 8 hours, with similar C
max values being observed. The liver tissue exposure is comparable for each compound.
The liver-to-plasma concentration ratio was determined by correlating drug concentrations
in liver tissue with those in plasma at equivalent time points. [Fig. 5C] shows that the liver-to-plasma ratio for Bule-OH exceeds that of Bulevirtide at
all measured time points, suggesting that Bule-OH has a more pronounced liver-targeting
capability.
Table 3
Pharmacokinetic parameters of Bulevirtide and Bule-OH in liver tissue
|
Analyst
|
AUClast (h•ng/mL)
|
C
max (ng/mL)
|
T
max
(h)
|
AUC8-72
(h•ng/mL)
|
Maximum liver–blood ratio
|
|
Bule-OH
|
2,305,284
|
70,000
|
8
|
1,922,152
|
11,889
|
|
Bulevirtide
|
1,832,720
|
65,333
|
8
|
1,832,720
|
2,965
|
Abbreviations: AUClast, the area under the curve from time zero to the last measurable
concentration; C
max, maximum plasma concentration observed; T
max, time to reach maximum observed concentration; AUC8–72, the area under the concentration-time
curve from hour 8 to hour 72.
Discussion
Bulevirtide is a drug for the treatment of HBV and HDV infections, which significantly
diminishes viral load and improves patients' quality of life. Nonetheless, its high
manufacturing costs and extended treatment durations impose a considerable financial
strain on patients, particularly in developing nations with a high prevalence of HBV.[19] In this study, we explored a Bulevirtide analog, Bule-OH, by a combined fermentation–chemical
synthesis. A comparative cost evaluation under laboratory conditions demonstrated
that Bule-OH required significantly reduced production expenditure relative to standard
Bulevirtide manufacturing ([Fig. 3C]).
Bulevirtide has been separated into two components: a sequence of 47 natural amino
acids and an N-terminal myristoyl group. We generated a fusion protein, SUMO-1–47,
by engineering E. coli and inducing expression through fermentation. The SUMO tag improved the solubility
of the fusion sequence, thereby enhancing protein expression and stability.[20] After removal of the SUMO tag using Ubl-specific protease 1, we purified the 1–47
fragment to high purity via anion-exchange chromatography followed by RP-HPLC.
In our investigation of N-terminal modification strategies for the 1–47, we identified
potential interference from the lysine residues at positions 37 and 45. Therefore,
we sought a selective N-terminal modification method. Since the N-terminus is typically
exposed to the solvent, the solvent composition was adjusted. A mixed solvent of 50%
ACN and water was chosen, which effectively dissolved myristoyl succinimidyl ester
and 1–47, and improved the N-terminal modification efficiency.[21] Considering that the pKa value of the N-terminal amine (∼7–8) is lower than that
of the ε-lysine amine (pKa of about 10), we controlled the pH value to selectively
modify the N-terminus.[22] Experiments indicated that the highest modification efficiency was achieved at pH
6.5. We optimized the amount of activated ester and found that 1 equiv. of activated
ester increased the reaction efficiency while reducing the Lys-ε-amine modification
by-products. Ultimately, we developed an N-terminal modification method that achieved
a modification efficiency of 36.87% for 1–47. Through RP-HPLC purification, Bule-OH
was obtained with a purity exceeding 97% and a purification yield of 92%, and 30.25%
1–47 was able to be recovered. However, since the C-terminus of Bule-OH is a carboxylic
acid, whereas the C-terminus of Bulevirtide is an amide, we are concerned that this
difference might affect its properties.
We subsequently compared Bule-OH with Bulevirtide across several characteristics:
antiviral activity, propensity for self-association, plasma stability, and resistance
to enzymatic degradation. The anti-HBV activity of Bule-OH was found to be half that
of Bulevirtide, suggesting that the N-terminal amide group enhances antiviral efficacy.
Both compounds demonstrated a low self-association propensity, which is beneficial
for pharmaceutical development.[23] Additionally, the stability of Bule-OH and Bulevirtide in plasma, as well as in
the presence of various enzymes, was evaluated. The stability of Bule-OH is slightly
reduced compared with Bulevirtide, but still satisfactory. Bule-OH has a carboxyl
group at the C-terminus, whereas Bulevirtide has an amide group at the C-terminus,
which contributes to the stability of the peptide.[24]
We performed a comparative analysis of two compounds in hNTCP mice. Our data revealed
that Bule-OH has a higher clearance rate than Bulevirtide, aligning with in vitro stability observations. Bule-OH also showed enhanced plasma exposure and reached
C
max more rapidly, suggesting a quicker absorption profile. However, both compounds exhibited
similar hepatic distribution patterns and pharmacokinetic characteristics. Notably,
Bule-OH had a higher liver-to-blood ratio, underscoring its superior hepatic targeting.
Comparative pharmacokinetic analysis in hNTCP mice indicated that although Bule-OH
mirrored Bulevirtide, its liver targeting was superior. Collectively, these results
imply that Bule-OH could serve as a cost-effective substitute for Bulevirtide.
Conclusion
Bulevirtide has demonstrated significant efficacy in reducing viral load and improving
the quality of life for patients with HBV and HDV infections. However, its high manufacturing
costs and prolonged treatment durations pose substantial financial challenges, particularly
in developing countries with high HBV prevalence. Our laboratory-scale cost comparison
revealed that the integrated fermentation–chemical production of Bule-OH was significantly
more economical than traditional Bulevirtide synthesis methods. The innovative approach
of using a SUMO tag to enhance protein expression and stability, followed by selective
N-terminal modification, resulted in Bule-OH with high purity and satisfactory stability,
albeit with slightly reduced antiviral activity compared with Bulevirtide. Despite
this, Bule-OH exhibited superior hepatic targeting and comparable pharmacokinetic
properties. However, further research is needed to fully understand its pharmacodynamic
and toxicological profiles, and continued optimization of the production process could
enhance its economic benefits, potentially expanding access to treat HBV and HDV infections
globally.
Supporting Information
Purchase source of the reagents used in this work; the synthesis purification, and
characterization of Bulevirtide; the protein expression and purification of the 1–47;
N-terminal myristoylation of 1–47; as well as HPLC spectrum and HRMS of Bulevirtide
([Supplementary Figs. S1] and [S2], available in the online version); schematic diagram depicting the enzymatic cleavage
of Bulevirtide by V8 ([Supplementary Fig. S3], available in the online version), Bulevirtide peptide map ([Supplementary Fig. S4], available in the online version); HRMS of enzymatic digestion fragment ([Supplementary Fig. S5], available in the online version); HPLC spectrum and HRMS of 1–47 ([Supplementary Figs. S6] and [S7], available in the online version), and HPLC spectrum and HRMS of Bule-OH ([Supplementary Figs. S8] and [S9], available in the online version), are included in the [Supporting Information] (available in the online version).
