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DOI: 10.1055/a-2594-1472
Cost-Effective Synthesis of Bule-OH: An Alternative to Bulevirtide for Hepatitis B and D Research
Funding None.
Abstract
Bulevirtide is a hepatitis B virus/hepatitis D virus (HDV) entry inhibitor that has been commercially available in Europe. It is manufactured via solid-phase synthesis of 47 amino acids with N-terminal myristoylation on MBHA resin. This production method presents significant environmental and economic challenges that limit the accessibility for patients. This study aims to develop a more sustainable and cost-efficient alternative through the rational design of Bule-OH, a Bulevirtide analogue featuring C-terminal carboxyl modification, rather than Bulevirtide's C-terminal amide. In this work, Bule-OH is successfully synthesized by combining fermentation expression with chemical modification techniques to reduce production costs. Bule-OH has a carboxylic acid group at the C-terminus, whereas Bulevirtide has an amide. We compared the pharmacological profiles of Bule-OH and Bulevirtide by determining their functional performance, including activity and enzymatic stability; and biophysical properties, including stability and self-association assays, followed by their pharmacokinetic evaluation in hNTCP (human sodium taurocholate cotransporting polypeptide) mice model of HDV infection. The results showed that the pharmacokinetic characteristics of Bule-OH and Bulevirtide in the target organ, the liver, were similar, whereas Bule-OH had a stronger liver targeting ability. These findings suggest that Bule-OH has the potential to be an economical and efficient alternative to Bulevirtide.
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]).
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]).
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.
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.
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.
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).
Conflict of Interest
None declared.
Ethical Approval
The in vivo pharmacokinetic experiments were reviewed and approved by the Ethical Committee of Experimental Animals at the Chia Tai Tianqing Pharmaceutical Group.
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- 22 Jiang H, Chen W, Wang J, Zhang R. Selective N-terminal modification of peptides and proteins: recent progresses and applications. Chin Chem Lett 2022; 33 (01) 80-88
- 23 Zeng Z, Tan R, Chen S. et al. Di-PEGylated insulin: a long-acting insulin conjugate with superior safety in reducing hypoglycemic events. Acta Pharm Sin B 2024; 14 (06) 2761-2772
- 24 Di L. Strategic approaches to optimizing peptide ADME properties. AAPS J 2015; 17 (01) 134-143
Address for correspondence
Publication History
Received: 17 February 2025
Accepted: 24 April 2025
Article published online:
19 May 2025
© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
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