Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified

Aug 3, 2018 - ... (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemo-enz...
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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified Atrial Natriuretic Peptide Derivatives Synthesized Using Chemo-enzymatic Synthesis Approaches Mitsuhiro Iwamoto,* Takahiro Yamaguchi, Yukiko Sekiguchi, Shohei Oishi, Takeshi Shiiki, Masako Soma, Kensuke Nakamura, Makoto Yoshida, Hiroyuki Chaya, Yutaka Mori, Ryuki Miyauchi, Jun Hasegawa, Takahiro Nagayama, and Takeshi Honda Downloaded via EASTERN KENTUCKY UNIV on August 4, 2018 at 05:13:35 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Sinagawa-ku, Tokyo 140-8710, Japan S Supporting Information *

ABSTRACT: Atrial natriuretic peptide (ANP) exerts beneficial pharmacological effects in the treatment of various cardiovascular disorders, such as acute congestive heart failure (ADHF). However, the clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemoenzymatic glyco-engineering using EndoS D233Q/Q303L. The most potent derivative SG-ANP-Fc conjugate extended the half-life to 14.9 d and the duration of blood pressure lowering effect to over 28 d. This new biologic modality provides an opportunity to develop outpatient therapy after ADHF.

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Fc-peptide conjugates (ANP-Fc conjugates).10 Such modifications are expected to protect ANP from degradation by plasma enzymes, reduce excretion via the kidney, and enable recycling via FcRn in ANP-HSA and ANP-Fc conjugates. Pellerin et al. reported an ANP-HSA conjugate with a half-life of 15 h in the human plasma.9 Furthermore, Mezo et al. demonstrated ANPFc conjugates with a half-life of 5−6 h.10 However, effectively long half-life that can enable outpatient therapy via weekly or monthly administration has not been reported, possibly owing to the degradation and inactivation of ANP moiety by plasma enzymes. In our search for ANP moieties that are tolerant to plasma enzymes, we identified SG-ANP (3), a glyco-modified ANP, with biantennary complex-type oligosaccharide (SG) [(NeuAc-GalGlcNAc-Man)2-Man-GlcNAc-GlcNAc-] (Figure 1). It exhibited

INTRODUCTION Atrial natriuretic peptide (ANP), one of the natriuretic peptides, was isolated from the human atrium and identified as a 28-amino acid peptide.1 Atrial natriuretic peptide activates guanylate cyclase A (GC-A), a natriuretic peptide receptor A (NPR-A), to increase the intracellular concentration of cyclic guanosine monophosphate (cGMP), resulting in multiple favorable cardiovascular effects, such as vasodilation, diuresis, inhibition of cell growth and sympathetic activity, and lowering of venous return.2 In Japan, an α-human ANP, carperitide, has been used as a first-line drug for acute decompensated heart failure (ADHF) over many years. Oishi et al. reported its potential application in the treatment of chronic heart failure, using a natriuretic peptide derivative.6 Furthermore, a novel therapeutic potential of ANP in cancer treatment has been reported by Nojiri et al., who demonstrated that the recurrence of lung cancer after surgery was significantly lower in ANPtreated patients than in the patients who were not treated with ANP;3 this is because of the activation of GC-A by ANP.4 However, the current clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min).5 Thus, the development of a long-acting GC-A activator as an ANP derivative will broaden the clinical use of ANP, including outpatient therapy (which will improve the QOL of patients), heart failure treatment, and metastasis control. Various attempts have been made to prepare a modified ANP with prolonged half-life, including synthetic and semisynthetic methods, such as elongation of sequence of peptide (mANP)7 and slow release of peptide from reversible PEG-peptide conjugate,8 albumin-peptide conjugates (ANP-HSA conjugates),9 and © XXXX American Chemical Society

Figure 1. Structure of sialyl glycan (SG).

strong GC-A activity and resistance against neprilysin (NEP) degradation in rat. We also developed a novel approach to Received: June 17, 2018 Revised: July 19, 2018

A

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. Preparation of glycosylated ANP derivatives. A. Preparation of 3. Conditions: (a) TSTU, DIPEA, DMF/H2O. B. Preparation of 5, 6, and 7. Conditions: (b) Fmoc-PEG(12)COOH, TSTU, DIPEA, DMF/H2O; (c) piperidine, DMF/H2O; (d) Fmoc-PEG(12)COOH, TSTU, DIPEA, DMF/ H2O; (e) piperidine, DMF/H2O; (f) Dibenzocyclooctyne-N-hydroxysuccinimidyl ester, DIPEA, DMF/H2O. Conditions: (g) Fmoc-(SG-)Asn, HATU, DIPEA, DMF/H2O; (h) piperidine, DMF/H2O; (i) Fmoc-PEG(12)2-COOH, TSTU, DIPEA, DMF/H2O; (j) piperidine, DMF/H2O; (k) Dibenzocyclooctyne-N-hydroxysuccinimidyl ester, DIPEA, DMF/H2O.

azido-mAb and antibody-drug conjugate (ADC) using the endoglycosidase Endo-S and its mutant.13−15 Additionally, Parson et al. reported the preparation of another type of azidomAb under optimal synthetic conditions using EndoS D233Q.16 In the preparation of ANP-mAb and ANP-Fc conjugates, we used the improved enzyme mutant EndoS D233Q/Q303L, which has higher transglycosylation efficiency compared with that of EndoS D233Q. Furthermore, it reduces the required amount of oxazoline derivative, a glycan donor for transglycosylation.17 The preparation method of the ANP-mAb conjugate (12) has been summarized in Figure 3. Monoclonal antibody (9) and immunoglobulin Fc fragment (Fc) (14) with heterogeneous glycoforms were obtained using the FreeStyle 293 expression system (Thermo Scientific), a widely used protein expression system derived from human embryonic kidney cells. Monoclonal antibody (9) was converted to GlcNAc-mAb (10) using EndoS. As reported by Tang,15 the complete removal of Endo-S after this reaction, through a 2-step purification process using the rProteinA and CHT columns, is critical for the reproducibility of the subsequent reaction, possibly because of its high affinity to IgG. GlcNAc-mAb (10) was then converted to azido-mAb (11) in the presence of azido-tagged oxazoline (8) using the Endo-S mutant. With Endo-S D233Q single mutant, oxazoline should be added intermittently (20 × 5 equiv at 5 min interval) at a pH of 6.5 to avoid the nonsite-specific glycation of mAb due to the high reactivity of oxazoline.16 To avoid such a complicated operation, we used Endo-S D233Q/Q303L, which demonstrated a high transglycosylation efficiency (>90%) with a low concentration of oxazoline (10 equiv) (8) for mAb. As expected, the non-site-specific glycation of 11 was not detected (see Figures S1 and S2). Azido-mAb (11) was then converted to the ANP-mAb conjugate (12) via click reaction in

prepare a novel biologic modality, the glyco-modified ANP-Fc conjugate, with long pharmacokinetic (PK) and pharmacodynamic (PD) profiles in rat and monkey.

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RESULTS AND DISCUSSION Design and Synthesis of ANP Derivatives. We focused on the modification of ANP with the oligosaccharide block, a highly soluble moiety, to avoid the deterioration of physicochemical properties of ANP. The homogeneous human-type oligosaccharide (SG), shown in Figure 1, was used in this study because of its safety to mammals and its commercial availability. The concise synthetic method for 3 has been summarized in Figure 2A. Compound 2 was prepared from sialylglycopeptide (SGP) and 2-acetamido-2-deoxy-β-D-glucopyranosyloxyacetic acid (GlcNAc tag) using Endo M N175Q,11,12 which acts as a transglycosidase to transfer glycan from SG-Peptide to GlcNAc-tag. Compound 2 was then treated with 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) to form N-hydroxysuccinimide ester (NHS-ester) in DMF, followed by the addition of ANP (1) in DMF/H2O to obtain 3. Further, to load ANP or SG-ANP on carrier proteins, other ANP derivatives were prepared as shown in Figure 2B. The coupling of ANP (1) and Fmoc-PEG(12)-COOH, followed by the deprotection of Fmoc group resulted in 4. The coupling and deprotection of Fmoc group was repeated, followed by the introduction of strain-activated alkyne (DBCO) derivative to obtain 5. The coupling of ANP (1) and Fmoc-(SG-)Asn, followed by the deprotection of Fmoc group resulted in 6. The above-mentioned steps were followed to also obtain 7. Design and Synthesis of ANP-mAb/ANP-Fc Conjugates. Endoglycosidase-catalyzed transglycosylation is an attractive strategy to produce a homogeneous mAb in terms of efficiency. Originally, Huang et al. reported the production of precursor B

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 3. Preparation of ANP-mAb/ANP-Fc conjugates. Step 1 is a hydrolysis reaction using EndoS. Conditions: (a) EndoS, buffer (pH 6.0). Step 2 is a transglycosylation reaction using an EndoS mutant with oxazoline (8). Conditions: (b) EndoS D233Q/Q303L, buffer (pH 6.0). Step 3 is a conjugation reaction by the click reaction. Conditions: (c) DMSO/buffer (pH 5.5).

of ANP. The EC50 values of SG- or SG-Asn-modified compounds (6, 13, and 17) were higher than that of ANP, possibly because of the steric hindrance to receptor binding. Although compounds 13 and 17 have four molecules of ANP as opposed to one molecule in compound 6, their EC50 values were higher than that of compound 6. It can be speculated that further modification or branching at the critical position affected receptor binding and effectively canceled out the valence. Notably, compound 13 had substantially high EC50 value, even compared to compound 17, which only differed in the presence or absence of two Fab-arms of antibody. It can be inferred that the presence of two mobile arms of antibody provides additional hindrance to receptor binding. In Vivo Assay of Glyco-Modified ANP. To evaluate the effect of glycosylation of ANP (1) on PK and PD, the plasma concentration of test peptides and the concentration of cGMP over time were investigated in rat after the intravenous injection of SG-ANP (3) and ANP. SG-ANP (3) exhibited higher plasma concentration and sustained PK profile, whereas ANP (1) showed very low plasma concentration at the first time point after administration. The plasma concentrations after 15 min were determined to be less than the limit of detection (Figure 4A). Consistently, the elevation of plasma cGMP level by SG-ANP (3) was more potent and lasted longer than that by ANP (Figure 4B). To elucidate the mechanism of acquired sustainability, we evaluated the effect of inhibitor of NEP (NEPi), a major metabolic enzyme of ANP.20,21 The coadministration of ANP and NEPi enhanced plasma cGMP level and improved the PD profile compared with the administration of ANP alone, suggesting the importance of NEP in improving the PD profile of ANP. However, ANP was rapidly eliminated from the plasma, and the plasma cGMP level decreased to less than one-seventh of the Cmax at 180 min after transient elevation even in coadministration of ANP and NEPi (Figure 4A and 4B). This reflects the insufficient improvement of PK profile by NEP inhibition alone (Figure 4A). In contrast, SG-ANP (3) exhibited the same sustained PK and PD profiles regardless of

the presence of ANP derivative (4) by strain-promoted azide− alkyne cycloaddition (SPAAC).18,19 The SG-ANP-mAb conjugate (13) and SG-ANP-Fc conjugate (17) were prepared following a similar method (Figure 3). The final product contained a fraction of 17-like compound without glycan on one of the two protomers, likely derived from an impurity of Fc (14) that lacked the core GlcNAc in only one protomer (Figures S5, S6, and S7). In Vitro hGC-A Agonistic Activity. To test whether the N-terminal modification of ANP can affect the pharmacological activity, we assessed the cGMP productive activity in human GC-A (hGC-A) expressing cells (Table 1). All the derivatives Table 1. In Vitro EC50 Values and the Relative Value of in Vitro Activities of ANP Derivativesa Peptide/ compound ID ANP(1) 3 6 12 13 17

Theoretical valence of ANP 1 1 4 4 4

Emax (relative to hANP response) 1.02 1.02 1.02 1.00 0.99 0.99

± ± ± ± ± ±

0.01 0.0027 0.01 0.03 0.02 0.01

EC50 (pM)

(n)

± ± ± ± ± ±

(16) (4) (4) (4) (4) (4)

22.4 45.5 119.2 20.8 454.5 147.8

1.7 0.9 4.4 4.8 38.5 7.6

a

CHO cells stably expressing human GC-A were incubated with the indicated concentration of peptides for 15 min. The concentration of cGMP was measured by the HTRF-based assay. The data are presented as the mean ± standard error of the mean. The EC50 values of hGC-A expressing cells were averaged.

enhanced cGMP production in a concentration-dependent manner, exhibiting maximum efficacy comparable with that of ANP. On the contrary, the EC50 value was affected by each modification; the compounds 3 and 12 presented EC50 values comparable with that of ANP (1). While compound 3 showed 2-fold increase in EC50 value, compound 12 equipped with four molecules of ANP showed EC50 value almost equal to that C

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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value (around 80 pmol/mL) after 48 h and sustained the concentration of cGMP at >70% of the maximum for the next 5 days. These results indicate that 13 has substantially longer GC-A activating profile when compared with that of 12, and the effect is due to the presence of oligosaccharide connected to the proximity of the N-terminus of ANP. Pharmacokinetic Profile of the ANP-mAb/ANP-Fc Conjugate. To evaluate the PK profile, two different ligand binding assays (LBA) were adopted to detect the protein. The deviation of ANP/Fc (to detect intact ANP on Fc) from Fc/Fc (to detect Fc part) reflects the degradation of the ANP peptide portion on the mAb- or Fc-conjugate. The PK profile of ANP mAb (12) and SG-ANP-mAb conjugate (13) after their intravenous administration in rat is summarized at Figure 6. We

Figure 4. Plasma concentrations of SG-ANP or ANP (A) and cGMP (B) after bolus injection of SG-ANP (3) or ANP (1) at a concentration of 100 nmol/kg (0.55 mg/kg or 0.31 mg/kg, respectively) in the presence or absence of NEPi. The data are presented as mean ± standard deviation (n = 3). The error bars that are not visible are smaller than the size of the symbols.

coadministration of NEPi (Figure 4A and 4B). While acquired sustainability is partly attributed to the acquisition of NEPtolerance by glyco-modification, presumably due to steric hindrance,22 significant increase in plasma concentrations and sustained PK and PD profiles cannot be fully explained by the acquisition of NEP-tolerance. These could partially be attributed to the additional protection against other plasma enzymes, a marginal increase in size, and/or other changes in the overall characteristic of the molecule, all of which might contribute to the clearance of the molecule. In Vivo Assay of ANP-mAb Conjugates. We speculated that the N-terminal glyco-modification of ANP might also extend the GC-A activating profile in the mAb-conjugate form. Therefore, the cGMP producing effect of the ANP-mAb conjugate (12) was compared with that of the SG-ANP-mAb conjugate (13) after their subcutaneous administration in rat (Figure 5).

Figure 6. Plasma concentrations of ANP-mAb conjugate or SG-ANPmAb after intravenous administration of ANP-mAb conjugate (12) or SG-ANP-mAb conjugate (13) at a concentration of 10 nmol/kg (1.70 or 1.79 mg/kg, respectively) in rats. The data are presented as mean ± standard deviation (n = 3). The error bars that are not visible are smaller than the size of the symbols.

focused on the intravenous administration of ANP-mAb conjugates in order to understand the potential of our lead compounds, which can be compared with that of the prior examples. Interestingly, the plasma concentration of 13 measured by both ANP/Fc and Fc/Fc methods closely overlapped for 7 d, whereas that of 12 exhibited a large deviation after 2 d. A similar deviation in the PK profile of ANP-Fc conjugates after intravenous administration at a dose of 0.5 mg/kg in Wistar rats has been reported by Mezo et al.10 They reported that the ANP peptide portion on the ANP-Fc conjugate was partially degraded by plasma enzymes in vivo, whereas the ANP peptide portion on 12 was partially degraded, most likely by plasma enzymes, and that of the SG-ANP-mAb conjugate (13) was evidently protected by the ANP-proximal oligosaccharide, significantly improving the PK profile. To predict the potential of the lead compounds for outpatient therapy in nonrodent model, we measured the plasma concentration of SG-ANP-mAb conjugate (13) and SG-ANPFc conjugate (17) after their subcutaneous administration in monkey (n = 4) (Figure 7). Interestingly, the half-life of 17 (14.9 days) was longer than that of 13 (7.1 days) despite the lower molecular weight. As the long half-life of the antibody is mainly due to the FcRn-catalyzed recycling,23 we speculated that the difference in the affinities toward monkey FcRn might

Figure 5. Plasma concentrations of cGMP after subcutaneous administration of ANP-mAb conjugate (12) or SG-ANP-mAb conjugate (13) at a concentration of 100 nmol/kg (17 or 18 mg/kg, respectively) in rats. The data are presented as mean ± standard error of the mean (n = 3). The error bars that are not visible are smaller than the size of the symbols.

The administration of 12 gradually elevated the concentration of cGMP to a maximum value (around 160 pmol/mL) after 24 h and sustained the concentration of cGMP at >30% of the maximum for the next 6 days. On the contrary, 13 exhibited a slower increase in the concentration of cGMP to a maximum D

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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17 toward monkey FcRn, both 13 and 17 are likely to have long half-lives in human, similar to that observed in monkeys. Pharmacodynamic Profile of 17. The duration of the hypotensive effect of the SG-ANP-Fc conjugate (17) was evaluated in monkey. Using a telemetry system, we continuously measured the systolic blood pressure (SBP) for 24 h on each indicated day after the subcutaneous administration of 17 (Figure 8). Delta SBP on Day −2 (Figure 8A) shows the baseline SBP fluctuation before the administration of 17. As shown in Figure 8A, the subcutaneous administration of 17 gradually decreased the SBP by approximately 20 mmHg over a period of ≥4 h and sustained the lower level throughout the day (Day 0). The SBP level was almost equally sustained from Days 0 to 1 (Figure 8B) and then gradually recovered over a course of 4 weeks (Figure 8B−E). Finally, the SBP reverted to the baseline after the fifth week (Figure 8F). SBP decreased significantly in response to SG-ANP-Fc conjugate (17) until Day 28 compared to Day 2 (Figure 8G). Thus, SG-ANP-Fc conjugate (17) demonstrated long-lasting BP-lowering effect, which was sustained for over a month, owing to its long PK profile.

Figure 7. Plasma concentrations of SG-ANP-mAb conjugate or SGANP-Fc conjugate after subcutaneous administration of SG-ANPmAb conjugate (13) or SG-ANP-Fc conjugate (17) at a concentration of 10 nmol/kg (1.79 or 0.83 mg/kg, respectively) in rats. The data are presented as mean ± standard deviation (n = 4). The error bars that are not visible are smaller than the size of the symbols.

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CONCLUSIONS We found that the N-terminal modification of ANP with oligosaccharide block (SG) results in NEP tolerance while maintaining the in vitro activity, thus improving the stability and retention of ANP in vivo. Furthermore, the robust ANP

be the reason for the difference in the PK profiles. As speculated, the Kd value of 17 (7.4 × 10−7 M) was lower than that of 13 (2.2 × 10−6 M). As the affinity toward human FcRn of 17 (7.4 × 10−7 M) and 13 (1.7 × 10−6 M) was similar to that of

Figure 8. Sustained blood pressure lowering effect of the SG-ANP-Fc conjugate in cynomolgus monkey. (A−F) Systolic blood pressure (SBP) monitored for 24 h on each indicated day by radiotelemetry, and the 1 h averaged values relative to that at −2 h on Day 0 (87.3 ± 2.4 mmHg) are plotted. The monkeys were maintained under a 12:12 h light/dark cycle; the black bars along the x-axis represent the dark cycle. (A) Subcutaneous injection of SG-ANP-Fc conjugate, compound 17, at a concentration of 10 nmol/kg (0.83 mg/kg) at time 0 on Day 0 (arrow) lowered the SBP level on Day 0 (gray circle) compared to that on Day −2 (open triangle). (B−F) Gradual recovery of SBP to the baseline is observed during the following days: Day 1 (B, gray box), Day 7 (C, black triangle), Day 14 (D, striped box), Day 21 (E, open circle), Day 28 (E, open diamond), Day 36 (F, gray triangle), and Day 43 (F, black circle). (G) Area under the curve of delta SBP from 2 to 24 h on each day is shown. Statistical differences versus Day −2 are assessed by Dunnett’s test except for Day 0 in which the data at some time points were not available. * P < 0.01. The data are presented as mean ± standard error of the mean (n = 4). E

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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(GE Healthcare) and hydroxyapatite chromatography, CHT Type I (Bio-Rad), and buffer-exchange with 50 mM phosphate buffer (pH 6.0), yielding 472 mg of 10. ESI-MS: calculated for the heavy chain of 10 (-Lys, pyrGlu), M = 50166.6, found (m/z), 50165.3 (deconvolution data); calculated for the light chain of 10, M = 23292.9, found (m/z), 23292.0 (deconvolution data). azido-mAb (11). A solution of 10 (211 mg) and 8 (33.8 mg, 10 equiv) was incubated with Endo-S D233Q/Q303L (4.2 mg) at 30 °C in 13.2 mL of 50 mM Phosphate buffer (pH 6.0) for 3.5 h. The mixture was immediately subjected to affinity chromatography, HiTrap rProteinA FF (GE Healthcare) and hydroxyapatite chromatography, CHT Type I (Bio-Rad), and buffer-exchange with 20 mM phosphate buffer (pH 6.0). Another reaction batch was prepared similarly and combined after purification to obtain 390 mg of 11. ESI-MS: calculated for the heavy chain of 11 (-Lys, pyrGlu), M = 52569.9 found (m/z), 52569.4 (deconvolution data); calculated for the light chain of 11, M = 23292.9 found (m/z), 23292.1 (deconvolution data). ANP-mAb Conjugate (12). A solution of 11 (50.0 mg) and 4 (12.9 mg, 8 equiv) was incubated at 30 °C in 4.0 mL of 20 mM phosphate buffer (pH 6.0) and 1.0 mL of DMSO for 16 h. The mixture was immediately subjected to NAP25 column chromatography (GE Healthcare) for buffer-exchange with 20 mM phosphate buffer (pH 6.0) and purified by affinity chromatography, HiTrap rProteinA FF chromatography (GE Healthcare), and buffer-exchange with 10 mM acetate buffer (pH 5.5) containing 5% sorbitol. The filter sterilization of the solution through Millex-GV (0.22 μm, PVDF, Merck Millipore) yielded 41.5 mg of 12. ESI-MS: calculated for the heavy chain of 12 (-Lys, pyr-Glu), M = 61708.2 found (m/z), 61706.4 (deconvolution data); calculated for the light chain of 12, M = 23292.9 found (m/z), 23291.7 (deconvolution data). SG-ANP-mAb Conjugate (13). A solution of 11 (113 mg) and 7 (43.1 mg, 8 equiv) was incubated at 30 °C in 8.0 mL of 20 mM phosphate buffer (pH 6.0) and 2.0 mL of DMSO for 16 h. The mixture was immediately subjected to NAP25 column chromatography (GE Healthcare) for buffer-exchange with 20 mM phosphate buffer (pH 6.0) and purified by affinity chromatography, HiTrap rProteinA FF chromatography (GE Healthcare), and buffer-exchange with 10 mM acetate buffer (pH 5.5) containing 5% sorbitol. Another reaction batch was prepared similarly and combined after purification, followed by the filter sterilization of the solution through Millex-GV (0.22 μm, PVDF, Merck Millipore) to yield 220 mg of ANPmAb-conjugate (13). ESI-MS: calculated for the heavy chain of 13, M = 66348.4 found (m/z), 66347.5 (deconvolution data); calculated for the light chain of 13, M = 23292.9 found (m/z), 23291.9 (deconvolution data). GlcNAc-Fc (15). A solution of 14 (260 mg) was incubated with Endo-S (3.8 mg) at 37 °C in 21.9 mL of 50 mM phosphate buffer (pH 6.0) for 2 h. The mixture was immediately subjected to affinity chromatography, HiTrap rProteinA FF (GE Healthcare) and hydroxyapatite chromatography, CHT Type I (Bio-Rad), and buffer-exchange with 50 mM phosphate buffer (pH 6.0), to yield 254 mg of 15. ESI-MS: calculated for the chain of 15 (-Lys), M = 25287.7; found 25286.8 (deconvolution data) azido-Fc (16). A solution of 15 (37.5 mg) and 8 (30.2 mg, 17 equiv) was incubated with Endo-S D233Q/Q303L (2.2 mg) at 30 °C in 7.3 mL of 50 mM phosphate buffer (pH 6.0) for 3.5 h. The mixture was immediately subjected to affinity chromatography, HiTrap rProteinA FF (GE Healthcare) and hydroxyapatite

derivative SG-ANP (3) elevated the cGMP level for a longer duration than that with ANP alone, and it was comparable with that of ANP coadministered with NEPi. Importantly, we have generated a novel biologic modality SG-ANP-Fc-conjugate (17), which exhibited blood pressure lowering effects for over 28 d in monkeys. To the best of our knowledge, 17 is the longest acting ANP among the chemically modified ANP derivatives ever reported, with a PK profile that could enable outpatient therapy via weekly or monthly administration of compound, which was previously not possible with conjugate molecules reported by other research groups.7−10 Our approach is distinct in that ANP is conjugated to FcRn binder and further glyco-modified to address the vulnerability to plasma enzymes, such as NEP. The ANP derivatives described herein will be investigated in the future for outpatient therapy.

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EXPERIMENTAL PROCEDURES Compound Characterization. The Supporting Information provides details of the identity (by high-resolution mass spectrometer) and purity (by HPLC or Experion Pro260 assay) of each ANP derivative. Each ANP derivative reported in the present study was purified by column chromatography before analyzing their identity and purity. SG-ANP (3). To a solution of 2 (790 mg) in DMF (18 mL), a solution of TSTU (104 mg) in DMF (2 mL) was added. Subsequently, DIPEA (241 μL) was added, and the mixture was stirred at room temperature for 60 min and used in the following reaction. ANP (1000 mg) was dissolved in 12 mL of DMF and 3.2 mL of distilled water. To this solution, 241 μL of DIPEA and 20 mL of solution containing active ester in DMF were added; the mixture was stirred for 1 h. After the reaction, 32 mL of MeCN was added, and the precipitate was collected by filtration. After washing with 30 mL of DMF/MeCN solution (1:1) and 100 mL of MeCN, the obtained solid matter was dried under reduced pressure. The product obtained was separated and purified by reverse-phase HPLC (Inertsil ODS-3; GL Sciences Inc.) using 0.1% aqueous acetic acid solution and 0.1% solution of acetic acid in MeCN as eluents and lyophilized to obtain SG-ANP (3) (1056 mg). ESI-MS: Calcd for C213H341N51O102S3: [M + 5H]5+ 1069.9, found 1069.9; [M + 4H]4+ 1337.1, found 1337.1; [M + 3H]3+ 1782.5, found 1782.4. H2N-PEG(12)-ANP (4). Compound 4 was synthesized according to the general procedure. ESI-MS: Calcd for C154H256 N46O52S3: [M + 6H]6+ 614.4, found 614.3; [M + 5H]5+ 737.0, found 737.0; [M + 4H]4+ 921.1, found 921.0. DBCO-PEG(12)2-ANP (5). Compound 5 was synthesized according to the general procedure. ESI-MS: Calcd for C200H322 N48O67S3: [M + 6H]6+ 762.2, found 762.0; [M + 5H]5+ 914.4, found 914.3; [M + 4H]4+ 1142.8, found 1142.6. (SG-)Asn-ANP (6). Compound 6 was synthesized according to the general procedure. ESI- MS: Calcd for C215H345N53O102S3: [M + 6H]6+ 901.1, found 900.9; [M + 5H]5+ 1081.1, found 1081.1; [M + 4H]4+ 1351.1, found 1350.8; [M + 3H]3+ 1801.2, found 1801.1. DBCO-PEG(12)2 -(SG-)Asn-ANP (7). Compound 7 was synthesized according to the general procedure. ESI-MS: Calcd for C288H464N56O130S3: [M + 7H]7+ 984.9, found 984.9; [M + 6H]6+ 1148.9, found 1148.7; [M + 5H]5+ 1378.4, found 1378.4. GlcNAc-mAb (10). A solution of 9 (490 mg) was incubated with Endo-S (2.5 mg) at 37 °C in 21.3 mL of 50 mM phosphate buffer (pH 6.0) for 3 h. The mixture was immediately subjected to affinity chromatography, HiTrap rProteinA FF F

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

analysis line, including the column. The InertSustain Bio C18 HP (2.1 mm i.d. × 50 mm, 3 μm, GL Sciences, Tokyo, Japan) was used as the analytical column. SG-ANP (3) or ANP (1) was used as the internal standard to analyze ANP (1) or SG-ANP (3), respectively. The plasma samples were diluted with 0.5% trifluoroacetic acid (TFA) containing the internal standard and loaded onto the InterSep 96WP C2 100 mg (GL Sciences, Tokyo, Japan) previously conditioned with 1 mL of acetonitrile, followed by 2 mL of 0.5% TFA. Subsequently, the plate was washed with 2 mL of 0.5% TFA and the analytes were eluted with 0.5 mL of 0.5% TFA−acetonitrile (1:1, v/v). Two hundred microliters of the eluate was injected into the PAC-LC/MS/MS system. Pharmacokinetics Measurement of 12, 13, and 17. Compounds 12 and 13 were administered subcutaneously at a dose of 100 nmol/kg to male Sprague−Dawley rats (Charles River, Yokohama, Japan). Compounds 13 and 17 were administered subcutaneously at a dose of 10 nmol/kg to male Cynomolgus monkeys. The blood sample was collected in the CAPIJECT Na2EDTA blood collection tube (Terumo Medical Corporation, Somerset, NJ, USA). The plasma was immediately separated by centrifugation and stored at −80 °C until further analysis by the ANP/Fc or Fc/Fc ligand binding assay (LBA). The PK parameters were calculated using Phoenix WinNonlin 6.3 (Certara, Princeton, NJ, USA). ANP/Fc Ligand Binding Assay. The form of ANP/Fc was determined using the Bioaffy 200 CD (Gyros Protein Technologies, Uppsala, Sweden) by Gyrolab xP workstation (Gyros Protein Technologies, Uppsala, Sweden). The ANP antibody [23/1] (GeneTex Inc., Irvine, CA, USA) was biotinylated and diluted to a concentration of 700 nM in PBS containing 0.1% Tween-20 (PBST) and used as the capture reagent. Goat AntiHuman IgG, Monkey ads-Alexa Fluor 647 (SouthernBiotech, Birmingham, AL, USA) was diluted to a concentration of 10 nM in Rexxip F buffer (Gyros Protein Technologies, Uppsala, Sweden) and used as the detection reagent. The plasma samples were diluted with the Rexxip HN Buffer (Gyros Protein Technologies, Uppsala, Sweden), resulting in a minimum required dilution (MRD) of 10-fold before being loaded onto CD. Data acquisition at the 5% photomultiplier tube (PMT) level was used. Regression was performed using a Gyrolab Evaluator (version 3.3.9.175, Gyros Protein Technologies, Uppsala, Sweden) with 4-parameter logistic fit without blank and with weight response. Fc/Fc Ligand Binding Assay. The form of Fc/Fc was determined using the Bioaffy 200 CD by Gyrolab xP workstation. Goat Anti-Human IgG, Monkey ads-Biotin (SouthernBiotech) was diluted to a concentration of 700 nM in PBST and used as the capture reagent. Goat Anti-Human IgG, Monkey ads-Alexa Fluor 647 was diluted to a concentration of 10 nM in the Rexxip F buffer and used as the detection reagent. The plasma samples were diluted with the Rexxip HN Buffer, resulting in a MRD of 10-fold before being loaded onto CD. Data acquisition at the 5% PMT level was used. Regression was performed by Gyrolab Evaluator with 4-parameter logistic fit without blank and with weight response. FcRn Surface Plasmon Resonance. The recombinant His-tagged human or cynomolgus FcRn expressed in FreeStyle 293-F cells were purified by HisTrap excel Ni-affinity column chromatography (GE Healthcare) and were gel-filtrated using the Superdex 200 16/60 column (GE Healthcare) in PBS buffer. The binding affinity of ANP conjugates was measured at 25 °C using the Biacore 4000 instrument (GE Healthcare).

chromatography, CHT Type I (Bio-Rad), and buffer-exchange with 10 mM acetate buffer (pH 5.5) containing 5% sorbitol. Another reaction batch was prepared similarly and combined after purification to obtain 67.9 mg of 16. ESI-MS: calculated for the chain of 16(-Lys), M = 27691.0; found 27690.4 (deconvolution data). SG-ANP-Fc Conjugate (17). A solution of 16 (30.0 mg) and 7 (27.4 mg, 7 equiv) was incubated at 30 °C in 6.0 mL of 10 mM acetate buffer (pH 5.5) containing 5% sorbitol and 1.5 mL of DMSO for 16 h. The mixture was immediately subjected to NAP25 column chromatography (GE Healthcare) and buffer-exchange with 20 mM phosphate buffer (pH 6.0) and purified with affinity chromatography, HiTrap rProteinA FF chromatography (GE Healthcare), and buffer-exchange with 10 mM acetate buffer (pH 5.5) containing 5% sorbitol. Another reaction batch was prepared similarly and combined after purification, followed by filter sterilization of the solution through Millex-GV (0.22 μm, PVDF, Merck Millipore) to obtain 61.9 mg of 17. ESI-MS: calculated for the chain of 17 (-Lys), M = 41469.4; found 41469.1 (deconvolution data). Animals. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, eighth edition, updated by the U.S. National Research Council Committee in 2011. In Vitro Human GC-A/cGMP Assay. In vitro human GC-A (hGC-A) agonistic activity was measured in accordance with a previous report.6 Briefly, CHO cells stably expressing hGC-A were seeded in a 384-well plate at a concentration of 4 × 103 cells/well and cultured for 1 d under 5% CO2 at 37 °C. After the removal of culture medium, the cells were incubated with 1.6 mM 1-methyl-3-isobutylxanthine (Merck Millipore, Billerica, MA, USA) for 10 min, followed by incubation for 15 min with ANP or test compounds. The cells were lysed and the concentration of cGMP in the lysate was determined using a cGMP assay kit (Cisbio, Codolet, France). The EC50 value was calculated with the 4-parameter curve fitting tool (SigmaPlot 12.0, Systat Software). The relative agonistic activity of ANP was calculated as the ratio of the maximum response relative to the response of 1 nM ANP, which was designated as 1.0 in each experiment. In Vivo Plasma cGMP Measurement. The plasma cGMP concentration was evaluated using 8-week-old male SD rats under isoflurane (2%) anesthesia, as described previously.6 Compound 1 or 3 at a concentration of 100 nmol/kg was administrated via bolus intravenous injection immediately after the intravenous infusion of saline or phosphoramidon (Santa Cruz Biotechnology, Dallas, TX, USA), a NEP inhibitor, at a concentration of 825 nmol/kg/min for 2 min. This was followed by continuous administration at a concentration of 165 nmol/kg/min until the end of the experiment, as previously reported by Hashimoto et al.20 The concentration of cGMP in the Na2EDTA plasma at each time point was measured using an EIA kit (GE Healthcare, Little Chalfont, UK). ANP (1) and SG-ANP (3) LC/MS/MS Assay. ANP (1) and SG-ANP (3) were determined by a peptide adsorptioncontrolled liquid chromatography-tandem mass spectrometric (PAC-LC/MS/MS) method24 using the Nexera X2 and LCMS-8060 System (Shimadzu, Kyoto, Japan) equipped with a 500 μL sample loop. Both mobile phases A (50% acetic acid) and B (methanol) were used to elute the analytes, whereas the mobile phase C (water) was used to wash the G

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

DMF,N,N-dimethylformamide; DMSO,dimethyl sulfoxide; DTT,dithiothreitol; Fc,fragment crystallizable; FcRn,Fc Receptor-neonate; Fmoc,9-fluorenylmethyloxycarbonyl; GC-A,guanylate cyclase-A; HSA,human serum albumin; mAb,monoclonal antibody; NEP,neprilysin; SBP,systolic blood pressure; SG,sialyl glycan; SGP,sialyl glycopeptide; SPAAC,strain-promoted azide− alkyne cycloaddition; TSTU,O-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

Approximately 20 response units of human or cynomolgus FcRn were captured by anti-b2m antibody (Abcam) immobilized on the CM5 sensor chip (GE Healthcare). Compounds 13 and 17 were prepared at concentrations of 3000−15 nM by 2-fold serial dilution in running buffer (10 mM sodium phosphate (pH 5.8), 0.05% polysorbate 20) and injected for 5 min over the captured FcRn in the sample flow cell and the anti-b2m antibody in the reference flow cell. The anti-b2m antibody immobilized sensor chip was regenerated using 3 M NaCl in 10 mM glycine-HCl (pH 1.5) for 1 min. The Kd was obtained by fitting a plot of response at equilibrium against the concentration using the Biacore 4000 Evaluation Software (GE Healthcare).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00427. Materials, general method, HPLC spectra of intermediates and final compounds, raw and deconvoluted MS data of intermediates and final compounds, and electropherogram of intermediates. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-3-3492-3131. Fax: +81-3-5436-8578. ORCID

Mitsuhiro Iwamoto: 0000-0002-0212-2322 Author Contributions

T.H. and T.N. conceived the idea of long-acting GC-A activator and supervised the study. M.I., T.Y., S.O., and T.S. designed the study and discussed the experimental data. M.I., Y.S., and T.Y. designed and synthesized the compounds. S.O. carried out the biology experiments. T.S., M.S., and M.Y. performed the analytic experiments (PK, PD, and FcRn). H.C. and Y.M. contributed to the design of linker and preparation of compounds. K.N., R.M., and J.H. contributed to the design and selection of the carrier protein. M.I., T.Y., and K.N. wrote the manuscript with all authors providing input. Funding

All authors were employed by Daiichisankyo Co. Ltd. at the time of this study. The authors received no external funding. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Inazu T. and Dr. Tomabechi Y. from Tokai University, Japan, for the technical advice and helpful discussions on glyco-remodeling using EndoM. We thank the members of Asubio Pharma Co., Ltd. for helpful discussions on ANP as a GC-A activator. We also thank the members of the Protein Production Research Group, Daiichi Sankyo RD Novare Co., Ltd. for the production of enzymes, mAb, and Fc used in the present study, and the members of the Natural Product Research Group, Daiichi Sankyo RD Novare Co., Ltd. for the MS analysis of ANP derivatives and for supplying glycan materials.

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ABBREVIATIONS ADHF,acute decompensated heart failure; ANP,atrial natriuretic peptide; CHO,Chinese hamster ovary; DBCO,dibenzylcyclooctyne; DIPEA,N,N-diisopropylethylamine; H

DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.bioconjchem.8b00427 Bioconjugate Chem. XXXX, XXX, XXX−XXX