A novel polyethylene glycol (PEG)‐drug conjugate of Venetoclax, a Bcl‐2 inhibitor, for treatment of acute myeloid leukemia (AML)

Abstract Background Venetoclax (VTX) is an anticancer drug. It is a selective Bcl‐2 inhibitor that is clinically used for the treatment of patients with lymphomas and leukemias. Treatment with VTX, however, is accompanied by severe adverse events such as tumor lysis syndrome and neutropenia, because VTX readily binds to serum proteins, which results in poor pharmacokinetics and poor tumor tissue concentration. To avoid such adverse events, VTX is administered using a daily or weekly ramp‐up schedule that is cumbersome in clinical situations. Aims To overcome these shortcomings, we prepared a novel polyethylene glycol (PEG)‐drug conjugate of VTX (PEG‐VTX) and evaluated its cytotoxic effects on acute myeloid leukemia (AML) both in vitro and in vivo. Methods and results VTX and 4‐armed PEG derivatives were covalently attached through an amide bond linker. In a series of in vitro studies, PEG‐VTX selectively induced potent growth inhibition of MV4‐11 human AML cells via the inducement of Bcl‐2‐mediated apoptosis. PEG‐VTX had the effect of free VTX, presumably due to the protease‐mediated release of VTX from the conjugates. In in vivo studies with AML tumor‐xenograft mice models, intravenous PEG‐VTX promoted sufficient tumor growth suppression. Compared with a regimen of oral free VTX, the intravenous regimen in those studies used a VTX dosage that was 15–30 times smaller for an OCI‐AML‐2 xenograft model and a dosing regimen that was less frequent for an MV4‐11 xenograft model. The most important development, however, was the absence of weight loss related to severe side effects throughout the treatments. An increase in water solubility and the resultant hydrodynamic size of VTX via PEGylation improved the pharmacokinetics of VTX by avoiding protein interactions and lessening the extravasation from blood. The result was an increase in tumor accumulation and a decrease in the nonspecific distribution of VTX. Conclusion The results of this study suggest that PEG‐VTX could be an alternative therapeutic option for the safe and effective treatment of patients with AML.


| INTRODUCTION
Apoptosis, or programmed cell death, is a pivotal process that removes damaged and/or unnecessary cells in the human body. In the environment of a tumor, however, defects can occur at any point along apoptotic signaling pathways and can lead to oncogenesis, tumor proliferation and metastasis, and chemoresistance. 1 The B-cell leukemia/lymphoma 2 (Bcl-2) protein was the first apoptotic regulator identified as an agent that blocks the mitochondrial apoptotic pathway 2-4 by abrogating the oligomerization of pro-apoptotic proteins, such as Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer 1 (Bak). 5 Several clinical reports have described how the Bcl-2 gene is overexpressed in 84% of patients with acute myeloid leukemia (AML) at the time of diagnosis and in almost all (95%) patients who experience a relapse. 6 Overexpression of the Bcl-2 gene is associated with poor prognosis, a low complete remission (CR) rate, a short survival time, and chemoresistance. 7,8 Accordingly, inhibition of the Bcl-2-triggered suppression of apoptosis is expected to be a promising therapeutic approach for patients with AML via inducing an effective programming of the death of cancer cells.
To date, several anticancer agents have been developed to target Bcl-2-family proteins. Obatoclax (GX15-070) is a broad inhibitor of Bcl-2 family proteins such as Bcl-2, Bcl-xL, Bcl-w, and Mcl-1, 9 and is currently undergoing a phase I study for the treatment of patients with chronic lymphocytic leukemia (CLL). 10 Although Obatoclax has produced modest clinical outcomes, it seems to cause undesired severe neurological events. 10 Navitoclax (ABT-263) is the first orally bioavailable inhibitor against Bcl-2-family proteins, including Bcl-2, Bcl-xL, and Bcl-w. 11,12 In clinical trials, Navitoclax has produced good therapeutic outcomes for both patients with lymphoid malignancies 13 and with CLL, 14 but it caused thrombocytopenia, which is a severe undesired side effect that is frequently observed in Bcl-xL. Therefore, Navitoclax was precluded from further clinical trials. In order to avoid such severe side effects due to Bcl-2 nonselective inhibition, Venetoclax (VTX; ABT-199), a selective Bcl-2 inhibitor, has been developed. 15 In clinical trials, VTX has produced substantial therapeutic outcomes in patients with lymphomas and was finally approved and marketed as VENCLEXTA. 16 Nevertheless, VTX continued to cause several adverse effects, such as tumor lysis syndrome (TLS) and neutropenia. 17 VTX is highly bound to serum proteins (>99.9%) in blood circulation, 16 which causes poor pharmacokinetics of VTX and subsequent severer adverse events. In the clinical trials of VTX, laboratory TLS, which is defined as an increase in uric acid, potassium, phosphorus, or calcium, was observed in 5.7% of patients with lymphoma. A small percentage (2.7%) of patients with clinical and laboratory TLS tend to experience one or more of the following adverse side effects: an increase in creatinine, cardiac arrhythmia or sudden death, and/or seizure. 18,19 In clinical trials, treatment with VTX has caused grade 3/4 adverse events that include neutropenia (39.6%), thrombocytopenia (29.2%), infection (25.0%), neutropenic fever (7.9%), and diarrhea (6.9%). 19 To overcome such serious adverse events, VTX is administered to patients with AML using a cumbersome daily ramp-up schedule over 4 days from a starting dose (100 mg/body) to the recommended dose (400 or 600 mg/body). 16 In addition, guidance for VTX treatment has been provided to respond to a decrease in neutrophil count via the use of granulocyte colony stimulating factor (G-CSF), dose interruption, or dose reduction. 20 Due to such poor pharmacokinetics and subsequent severe side effects, other approaches are still required in order to improve the usability and efficacy of VTX in clinical settings.
Polyethylene glycol (PEG) is a nonionic synthetic polymer and an excellent tool that can impart favorable pharmacokinetic and pharmacodynamic characteristics to drugs with a low molecular weight. 21,22 Several PEG-anticancer agent conjugates such as PEGylated SN38 (EZN-2208) [23][24][25] and PEGylated irinotecan (NKTR-102) [26][27][28][29] have been in clinical trials. Conjugation of anticancer agents with PEG improves the water solubility and dispersibility of the agents as well as increasing their molecular weights. Following intravenous injection, the resultant PEG-anticancer agent conjugates show prolonged blood circulation via the avoidance of glomerular filtration due to an increase in molecular weight 22 and by preventing interactions with serum proteins by increasing the water solubility. The long circulation properties gained by PEGylation allow the PEG-conjugates extensive accumulation in solid tumors via enhancements in permeability and retention (EPR). 30,31 These effects led us to assume that the conjugation of VTX with PEG could improve the usability and efficacy of VTX, although the PEG-drug conjugate of VTX must be injected intravenously.
In this study, therefore, we designed and synthesized a novel PEG-drug conjugate of VTX (PEG-VTX) using a 4-armed PEG derivative. In our synthetic design, VTX was covalently attached to each of the terminal ends of a 4-armed PEG derivative with an amide bond linker. The cytotoxicity of PEG-VTX was studied in vitro using a human AML cell line. Tumor growth suppression and body weight changes in AML tumor-xenograft mouse models were monitored during treatments with intravenous PEG-VTX and oral free VTX.

| Chemical synthesis and analysis of PEG-VTX
Under a N 2 atmosphere, PEG derivative (4.530 g, 1 eq) was dissolved in dried N,N-dimethylformamide (DMF) (55 ml) at 50 C and mixed with N,N-diisopropylethylamine (DIPEA) (0.296 g, 20 eq), 1-[bis(dimethylamino) methylene]-1H-benzotriazolium 3-oxide hexa-fluorophosphate (HBTU) (0.258 g, 6 eq), and VTX (0.474 g, 4.8 eq) in order. The mixed solution was stirred at 60 C for 6 h and then cooled to 40 C. The reaction solution was dropped into the methyl tert-butyl ether (40 ml) at 30 C and let stand for 20 min. The suspension was slowly cooled to 0 C with stirring for 1.5 h. The precipitation was collected by filtration and washed with 20 ml of methyl tert-butyl ether. The collected cake was dissolved in 20 ml of anhydrous ethanol in a flask at 40 C and mixed with 70 ml of methyl tert-butyl ether. After incubation for 20 min, the suspension was cooled to 0 C with stirring for 1.5 h. The precipitation was again collected by filtration and washed with 20 ml of methyl tert-butyl ether. These recrystallization processes were repeatedly carried out to obtain the final product, which was given the specification of a ≥97.0% PEG-VTX compound with individual impurities ≤1.0%, as verified via HPLC analysis. The resultant cake was dried at 40 C under vacuum for more than 5 h, and the resultant yellowish powder product, PEG-VTX (Figure 1      OCI-AML-2 tumor-bearing mice were either injected with PEG-VTX (100, 200, or 300 mg/kg/day, i.v.) once a week for 2 weeks or orally administered VTX (100 mg/kg/day, p.o.) every day for 2 weeks (n = 8). MV4-11 tumor-bearing mice were either injected with PEG-VTX (300 mg/kg/day, i.v.) once a week for 3 weeks or orally administered VTX (50 mg/kg/day, p.o.) every day for 3 weeks (n = 8). The daily oral administration of VTX was performed using a feeding needle. Tumor volumes and body weights of the treated mice were recorded twice weekly. Tumor growth inhibition (TGI [%]) was calculated using the following formula (TV: tumor volume). 32

| In vitro cytotoxicity on cancer cells of either free VTX or PEG-VTX
The levels of cytotoxicity of both free VTX and PEG-VTX were individually evaluated using MV4-11 human AML cells, Panc-1 human pancreas cancer cells, and A549 human lung cancer cells (Figure 2). To exclude the effect of protein binding, an Opti-MEM medium without the addition of FBS was used because free VTX easily binds to serum proteins (>99.9%). 16 The concentration that was required in order to produce 50% inhibitory effects (IC 50 ) appears in Table 1

| Changes in Bax expression in cytoplasm and mitochondria by treatment with either free VTX or PEG-VTX in vitro
Bax is a member of the Bcl-2 family that accelerates apoptosis and is predominantly present in the cytoplasm of cells. 2 A recent study reported that Bax redistributes from the cytosol to membranes via Bcl-2-mediated apoptosis signaling, and this redistribution of Bax is inhibited by treatment with cycloheximide, which is an apoptosis inhibitor. 33 Thus, as an indication of Bcl-2-mediated apoptosis, the expressions of Bax in the cytoplasm and mitochondria were studied following treatment of MV4-11 cells with either free VTX or PEG-VTX ( Figure 4A upper panel). The unedited raw data of expressions of Bax and β-Actin in cytoplasm is shown in Figure S1, and that of Bax and VDAC1/2 in mitochondria is shown in Figure S2. The signal intensity of each bands was quantified and represented as a relative signal intensity corrected by the expression of a loading control, β-Actin for cytoplasm or VDAC1/2 for mitochondria ( Figure 4A lower panels).

| Activation of caspase by treatment with either free VTX or PEG-VTX in vitro
The activity of caspase, a critical apoptotic factor, was evaluated after treatment with either free VTX or PEG-VTX ( Figure 5). Treatment with free VTX increased caspase activity with an increase in the VTX

MV4-11
Panc-1 A549 F I G U R E 2 Cytotoxic effects of either VTX or PEG-VTX on three different human cancer cell lines in vitro. MV4-11, Panc-1, or A549 cells were seeded onto one of the wells of a 24-well plate (1 Â 10 5 cells/well) and were exposed to either free VTX or PEG-VTX (1 pM $ 100 μM as a VTX concentration). After 48 h of incubation, cell viability was determined via MTT assay. The plot data are reported as the means ± SD (n = 3), and the lines are a fitted curve calculated using the plot data 3.6 | In vivo anti-tumor effects of either free VTX or PEG-VTX on two different AML tumor-bearing mouse models The in vivo anti-tumor effects of PEG-VTX were studied in both OCI-AML-2 and MV4-11 xenograft mouse models. In the OCI-AML-2 xenograft model, the clinical dosage was mimicked by orally administering free VTX daily for 2 weeks at a dose of 100 mg/kg/day: the total VTX dosage was 1400 mg/kg ( Figure 6A upper panel). PEG-VTX was intravenously administered at 100, 200 or 300 mg/kg once a week for 2 weeks: the total VTX dosages were 16, 32, 48 mg/kg since theoretically 100 mg of PEG-VTX contains 8 mg of VTX as an active pharmaceutical ingredient (API). Intravenous PEG-VTX showed a somewhat weaker growth-inhibitory effect even at the highest dose (300 mg/kg), compared with 100 mg/kg of oral free VTX ( Figure 6A middle panel and Table 2). The doses of converted API and VTX, however, were much smaller in PEG-VTX (48 mg/kg) than in free VTX    Table 3). In addition, the intravenous PEG-VTX caused no body weight loss, while the oral free VTX caused >10% body weight loss ( Figure 6B lower panel). These results indicate that PEG-VTX has the potency to treat AML tumors without inducing systemic side effects.

| DISCUSSION
In the current study, we successfully synthesized a PEG-drug conjugate of the marketed anticancer drug VTX ( Figure 1) and confirmed that PEG-VTX promotes the cytotoxicity of MV4-11 human AML cells. This was not the case, however, for other cancer cells such as Panc-1 human pancreas cancer cells and A549 human lung cancer cells in vitro ( Figure 2 and Table 1). The original application of VTX was as a BH3 mimetic highly selective for Bcl-2. 15 39 which speculates that our PEG-VTX could sustain in blood circulation higher than VTX free form. This drastic improvement in the pharmacokinetics of VTX by PEGylation in this study should allow decreases in the injection dosages of API, VTX, as well as a decrease in the dosing frequency, although further investigations into the pharmacokinetics of PEG-VTX in animal models will be necessary.

| CONCLUSIONS
In the present study, a novel PEG-drug conjugate, PEG-VTX, was synthesized and showed highly cytotoxic effects on AML cells both in vitro and in vivo. Most importantly, intravenous PEG-VTX induced sufficient tumor growth suppression without severe adverse side effects with a dosage regimen that was 20-fold less than the total doses of oral free VTX, which induces a somewhat higher level of tumor growth inhibition at the cost of a greater amount of body weight loss. PEG-VTX could be an alternative therapeutic option that is safe and effective for the treatment of patients with AML.

ACKNOWLEDGMENTS
This study was supported in part by a Grant-in-Aid for Young Scientists (19K16415) from the Japan Society for the Promotion of Science.

CONFLICT OF INTEREST
Kiyoshi Eshima is President of Delta-Fly Pharma, Inc. The other authors have no potential conflicts of interest in this study.

DATA AVAILABILITY STATEMENT
The data for this report are available from the corresponding author on reasonable request.