Comparable efficacy of oral bendamustine versus intravenous administration in treating hematologic malignancies

Abstract

Purpose

The purpose of this study was to analyze potential differences in antitumor efficacy and pharmacokinetics between intravenous (IV) bendamustine and a novel orally administered (PO) bendamustine agent that is utilizing the beneficial properties of superstaturated solid dispersions formulated in nanoparticles.

Methods

Pharmacokinetics of IV versus PO bendamustine were determined by analysis of plasma samples collected from NSG mice treated with either IV or PO bendamustine. Plasma samples were analyzed using liquid chromatography–mass spectrometry following a liquid–liquid extraction to determine peak bendamustine concentration, area under the concentration–time curve, and the half-life in-vivo. In-vitro cytotoxicity of bendamustine against human non-Hodgkin Burkitt’s Lymphoma (Raji), multiple myeloma (MM.1s), and B-cell acute lymphoblastic leukemia (RS4;11) cell lines was determined over time using MTS assays. Luciferase-tagged versions of the aforementioned cell lines were used to determine in-vivo bendamustine cytotoxicity of IV versus PO bendamustine at two different doses.

Results

Bendamustine at a high dose in-vitro causes cell death. There was no significant difference in antitumor activity between IV and novel PO bendamustine at a physiologically relevant concentration in all three xenograft models. In-vivo pharmacokinetics showed the oral bioavailability of bendamustine in mice to be 51.4%.

Conclusions

The novel oral bendamustine agent tested exhibits good oral bioavailability and systemic exposure for in-vivo antitumor efficacy comparable to IV bendamustine. An oral bendamustine formulation offers exciting clinical potential as an additional method of administration for bendamustine and warrants further evaluation in clinical studies.

Introduction

Bendamustine is an intravenously (IV) administered chemotherapeutic agent used primarily to treat hematologic malignancies. Early testing examined the use of bendamustine in the context of multiple myeloma as well as breast and small cell lung cancers [1–5]. Later, bendamustine was found to have activity against chronic lymphocytic leukemia, Hodgkin’s and non-Hodgkin lymphomas [2, 6–8]. Market availability of the drug began in Germany in the 1970s under the name Cytostasan^®, and later was re-marketed as Ribomustin^® [2, 3]. Today, bendamustine is sold in the United States under the brand names Belrapzo^®, Bendeka^®, and Treanda^®, and is approved for the treatment of chronic lymphocytic leukemia and non-Hodgkin lymphomas [9].

Nitrogen mustard analogues are synthesized from mustard gas with the replacement of sulfur with nitrogen. Bendamustine (4-(5-[bis(2-chloroethyl)amino]-1-methyl-2-benzimidiazolyl) butyric acid hydrochloride) contains a benzimidazole ring which is thought to confer anti-metabolite properties in addition to the mechlorethamine group and butyric acid side chain [2, 7, 10, 11]. The butyric acid side chain confers hydrophilic properties to bendamustine, allowing for solubility in water [11]. As an alkylating agent, bendamustine can create inter- and intra- strand lesions in DNA, leading to cell death in proliferative cells such as cancerous and hematopoietic cells [2, 7]. Compared to other alkylating agents such as cyclophosphamide, bendamustine induces more DNA lesions at equitoxic concentrations and has been shown in early studies to activate a base excision DNA repair pathway, a unique method of action for an alkylating agent [2, 3, 8]. While specific mechanisms are still to be determined, bendamustine has been shown to induce apoptosis through a p53-dependant pathway, and exposure to bendamustine in-vitro can lead to mitotic catastrophe whereby cells are inhibited at several mitotic checkpoints with severe DNA damage and subsequent cell death [2, 3, 8].

Bendamustine is considered safe and well tolerated, with adverse effects including neutropenia, lymphopenia, nausea, and vomiting [6]. We have studied the immunomodulatory properties of bendamustine as a chemotherapeutic in the contexts of pre- allogeneic hematopoietic cell transplant conditioning and as a post-transplant graft-versus-host-disease chemotherapeutic, targeting alloreactive T-cells [12–19]. Recently, bendamustine has been used in place of fludarabine and cyclophosphamide combination regimens for lymphodepletion in the context of chimeric antigen receptor therapies (CAR-T) [20–22].

In humans, bendamustine is typically given on two consecutive days, intravenously over 30 to 60 min. Pharmacokinetic data in humans have shown the effective half-life to be about forty minutes [11]. The current parenteral administration of bendamustine requires treatment to take place in the clinic or hospital. The presence of an effective and pharmacologically comparable oral formulation of bendamustine would have advantages for the patient and the clinic. An oral (PO) form of bendamustine would allow for patients to receive treatment at home reducing travel, time commitment and financial burden on patients and caretakers. Previously, Shimizu et al. demonstrated that bendamustine given through liquid-filled hard capsules was a safe method of delivery with mild gastrointestinal distress in patients with advanced solid tumors [23]. Herein we investigated the pharmacokinetics and anti-tumor effects of a novel, PO form of bendamustine that is a supersaturated solid dispersion formulated in nanoparticles and is administered by oral gavage to immunodeficient NSG mice bearing human hematologic cancers and compared its efficacy and toxicity to commercially available IV bendamustine.

Materials and methods

Mice

NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, RRID: BCBC_4142) mice were purchased from The Jackson Laboratory and bred at the University of Arizona Experimental Mouse Shared Resource. Female mice ages 6–14 weeks were randomized by weight and used for experiments. All mice had ad libitum access to food and water and were maintained on a twelve-hour light–dark cycle in specific-pathogen free conditions. The University of Arizona Institutional Animal Care and Use Committee (IACUC) approved all protocols and plans for the experiments.

In-vivo pharmacokinetics study

Sixteen NSG mice were allocated to two groups (8 mice per group) and administered a single dose of bendamustine either IV or by oral gavage. Bendamustine for IV administration (SelleckChem no. S1212) was solubilized in dimethyl sulfoxide (DMSO), diluted with Phosphate-Buffered Saline (PBS, Cytiva, SH30256.01) containing 0.2% carboxymethylcellulose and 0.25% polysorbate 80 and dosed at 15 mg/kg. PO bendamustine was obtained from Exinda Therapeutics and sealed with desiccant in amber glass containers to avoid exposure to air or moisture. Oral bendamustine was obtained by the method described on example 4 of patent US11701344. It is a supersaturated amorphous dispersion obtained through spray drying and is a dry powder that consists of bendamustine HCL monohydrate and Polyvinylpyrrolidone PVP-K17 at a ratio of 1:3 by weight (instead of 1:5 as indicted in the patent example). To prepare PO bendamustine (17.65 mg/kg), the appropriate amount of drug was weighed in a sterile hood and solubilized with filtered water. DMSO was added to solubilized PO bendamustine aliquots at a 5% concentration to control for the difference in the IV versus PO preparations. No precipitation was observed post solubilization. PO bendamustine was administered to mice by oral gavage within one hour of preparation. IV bendamustine was administered to mice via lateral caudal tail vein injection. At 0.25, 0.5, 1, 2, 4, and 6 h after bendamustine administration, mice (4 mice per sampling time point) were bled via tail tip bleed into EDTA tubes. Peripheral blood was centrifuged, and plasma collected and stored at −80 degrees. Plasma samples were transferred to the University of Arizona Cancer Center Analytical Chemistry Shared Resource for analysis using previously validated methods via liquid/liquid extraction and quantification with tandem mass spectrometry [24, 25]. The calibration range for bendamustine is 1.9–3800 ng/mL, with a limit of quantification of 1.9 ng/mL. The average plasma concentration versus time data were analyzed using PKSolver 2.0 using a noncompartmental approach.

Cell culture

Raji (ATCC, CCL-86, 70,053,718, RRID:CVCL_0511), MM.1s (ATCC, CRL- 2 9 7 4, 70,042,525, RRID:CVCL_8792), and RS4;11 (ATCC, CRL-1873, 70,036,117, RRID:CVCL_0093) were thawed from liquid nitrogen and cultured in RPMI-1640 (Cytiva, SH30027.01) with 10% fetal bovine serum (FBS), HEPES (Gibco, 15,630-080), sodium pyruvate (Cytiva, SH30239.01), and penicillin/streptomycin (P/S, Gibco, 15,140-122). Cells were kept at 37 °C and 5% CO₂. Cells were maintained by the addition of media every one to three days.

MTS assays

For the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, Raji (50,000 cells/well), MM.1s (40,000 cells/well), or RS4;11(40,000 cells/well) were plated in a 96-well plate in 100uL total volume. Bendamustine (75 mg/mL stock) was diluted in media and added in a serial dilution in triplicate. Control wells containing media only and media with bendamustine were plated for control. Plates were kept in an incubator at 37 °C and 5% CO₂ for 24 to 72 h. No additional media replacement occurred during the incubation period. The plates were read at 490 nm (PowerWave XS, BioTek) four hours after 20 uL of reagent was added to each well using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (VWR, Promega Corporation, Madison, WI, USA). Data was read using BioTek Gen 5 Microplate Software. Background absorbance from the media-only or media and drug wells was subtracted from each corresponding concentration data point. The percent viability was normalized to the cell-only control. Data was averaged from the three triplicate values per each plate. Each experimental plate was repeated three times in total. Data was inputted and graphed using GraphPad Prism 9 (La Jolla, CA, RRID:SCR_002798).

Tumor models

Raji-Luciferase (Raji-Luc, ATCC, CCL-86-LUC2, 70,059,299, RRID:CVCL_C9CV), MM.1s-Luc (Fenics Bio, CL-1617, 080721), and RS4;11-Luc (Fenics Bio, CL-1221) were thawed from liquid nitrogen and maintained in RPMI-1640 with 10% FBS, HEPES, Sodium Pyruvate, and P/S. Following three washes in 1 × PBS, cells were administered to mice via tail vein injection in 100 uL total volume 24 h following a 150 cGy total body irradiation with a RadSource X-ray irradiator. For the Raji-Luc or MM.1s-Luc model, 0.2 × 10⁶ cells were given one day after thaw. For RS4;11-Luc, 1 × 10⁶ cells were administered after a six day expansion culture. Cells were spun down and washed in 1xPBS three times before being resuspended in the aforementioned concentrations. NSG mice were given either IV bendamustine (15 mg/kg or 30 mg/kg) or PO bendamustine (30 mg/kg or 60 mg/kg) on days 1 and 2 for the MM.1s-Luc and RS4;11-Luc models and on days 3 and 4 for the Raji-Luc model. The dose difference between IV and PO is due to bioavailability adjustment to maintain equivalence. Mice were monitored daily for morbidity and weighed two to three times weekly. Animals losing more than thirty percent of starting weight for two consecutive weight scores or those experiencing total hind-limb paralysis were sacrificed.

Bioluminescence imaging

Two to three times weekly, mice received a 200uL intraperitoneal injection of D-luciferin (15 mg/mL, Gold Bio), were anesthetized with 2% isoflurane, and imaged using the Spectral Instruments LagoX (Tucson, AZ, USA) system five to ten minutes later. Images were taken over a 5-min exposure time. For visual representative images, scales were adjusted to a radiance scale minimum of 1.7 × 10⁴ and maximum of 5.5 × 10⁷ and saved as JPEG files. Bioluminescence was quantified using the Aura imaging software (Spectral Instruments Imaging, Tucson, AZ, USA) and are presented as photons/second in a region of interest that included the entire animal.

Statistics

GraphPad Prism 9 (La Jolla, CA, RRID:SCR_002798) was used for statistical analysis. IC50 values were determined for each cell line at each time point using a nonlinear fit [Inhibitor] vs. response—variable slope (four parameter). Survival Kaplan–Meir curves were analyzed using a Log-rank Mantel-Cox test[26, 27]. P-values of < 0.05 were considered significant, and the asterisks indicate increasing levels of significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Results

Comparison of PO versus IV administration of bendamustine

The plasma concentration versus time profiles with IV and PO bendamustine are shown (Fig. 1). The highest plasma bendamustine concentrations were observed at the first sample time of 0.25 h with both routes of administration. Plasma concentrations declined slowly as a function of time with an effective terminal half-life of 0.53 and 2.22 h, following IV and PO doses, respectively. A PO bendamustine dose of 17.65 mg/kg produced an average maximum plasma concentration (Cmax) of 4872.1 ng/mL compared to an average Cmax with IV bendamustine dose of 15 mg/kg of 10,518.4 ng/mL. The area under the plasma concentration versus time curve from time 0 extrapolated to infinity (AUC_0–∞) was 9973.3 ng h/mL with IV bendamustine compared to 6031.9 ng h/mL with PO bendamustine, indicating an oral bioavailability of 0.514.

Fig. 1.

Pharmacokinetics of Intravenous and Oral Administrations of Bendamustine in NSG mice. Plasma concentrations of bendamustine delivered either intravenously (IV) by tail vein injection or orally (PO) by gavage. Plasma concentrations of bendamustine were determined by liquid chromatography-mass spectrometry following liquid–liquid extraction

Bendamustine has a concentration dependent cytotoxicity against hematologic cancer cell lines in-vitro

After 24, 48, and 72 h of exposure to bendamustine in-vitro, MTS data showed sensitivity of RS4;11, MM.1s, and Raji cell lines to bendamustine induced cell death. RS4;11, a B-cell acute lymphoblastic leukemia, showed decreased cell viability as compared to Raji, a Non-Hodgkin Burkitt’s Lymphoma, and MM.1s, a multiple myeloma, for all time points (Fig. 2). At 24 h, RS4;11 had the lowest IC50 (80 uM) while MM.1s and Raji had IC50s of 210 uM and 270 uM, respectively. At 48 h, the IC50 concentrations were 32 uM (RS4;11), 87 (MM.1s), and 143 (Raji). Finally, at 72-h, all cell lines exposed to bendamustine resulted in the lowest IC50 values across the three time points. RS4;11, MM.1s, and Raji had 72-h IC50 values of 25 uM, 54 uM, and 133 uM respectively. All cell lines maintained some resistance to bendamustine-induced killing at low concentrations below 12.34 uM.

Fig. 2.

In vitro cytotoxicity of bendamustine against human hematologic malignancy cell lines. Dose response curves for RS4;11, MM.1s, and Raji. Tumor lines were plated in triplicate with various concentrations of bendamustine (BEN) as indicated for 24, 48, or 72 h. Plates were read using the CellTiter 96 AQueous One Solution Cell Proliferation (MTS) Assay and read at 490 nm on a plate reader 4 h after administration of reagent to each well. Normalized percent viability for three averaged experiments is shown at each BEN concentration

RS4;11 B-cell acute lymphoblastic leukemia (B-ALL) xenograft model

In-vivo murine experiments were performed as depicted in Fig. 3. In the mouse xenograft model of a B-cell acute lymphoblastic leukemia, RS4:11, there was no difference between IV and PO bendamustine at both the lower doses (Fig. 4a, IV 15 mg/kg or PO 30 mg/kg) or higher doses (Fig. 4b, IV 30 mg/kg or PO 60 mg/kg). Surprisingly, given its increased sensitivity to bendamustine in-vitro only the higher doses of IV and PO bendamustine impacted survival. Using IV 30 mg/kg and PO 60 mg/kg, both treatments resulted in a modest yet significantly improved survival over the control group. All treatment groups showed a modest decrease in body weight following bendamustine dosing, with a recovery to near starting weight, and there was no difference in appearance of the mice between the groups. (Data not shown). Additionally, bioluminescence imaging quantified to photons/second shows no difference between the low doses and higher doses (Fig. 4c, IV 15 mg/kg or PO 30 mg/kg) and lower doses of bendamustine (Fig. 4d, IV 15 mg/kg or PO 30 mg/kg).

Fig. 3.

Schematic diagram of experimental layout. NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, RRID: BCBC_4142) mice received 150 cGy total body irradiation (TBI) on day-2 and were injected on day-1 with tumor cells by tail vein injection. Bendamustine (BEN) was administered on Days 3 and 4 either intravenously by tail vein injection (IV, 15 mg/kg or 30 mg/kg) or by oral gavage (PO, 30 mg/kg or 60 mg/kg) for the MM.1s-Luc and RS4;11-Luc models. For the Raji-Luc model, BEN was administered on days 1 and 2. Image created with BioRender.com

Fig. 4.

In vivo application of IV versus PO bendamustine (BEN) treatment in a B-Cell Acute Lymphoblastic Leukemia xenograft model. NSG mice received 150 cGy TBI on Day-2 and were injected on Day-1 with 1 × 10⁶ luciferase-tagged RS4;11 cells by tail vein injection. BEN was administered on Days 3 and 4 either intravenously by tail vein injection (IV, 15 mg/kg or 30 mg/kg) or by oral gavage (PO, 30 mg/kg or 60 mg/kg). a,b Kaplan–Meier curves shows survival of NSG mice with comparable doses of IV or PO BEN. Mice were sacrificed if they reached two consecutive weight loss scores greater than thirty percent of starting weight or if moribund. c,d,e Bioluminescence imaging was conducted two to three times weekly and c,d photons/second in a region of interest encompassing the entire animal are shown as average over time, n = 10 per group. e Representative images from one experiment are shown with the same rainbow coloring set to the same min and max radiance. Experiments were repeated twice. A log-rank (Mantel-Cox) test was performed for survival analysis. P-values of < 0.05 were considered significant, and the asterisks indicate increasing levels of significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

MM.1s multiple myeloma xenograft model

The xenograft multiple myeloma model using MM.1s-Luciferase cells demonstrated no survival difference between control (No Rx) and any treatment group (Fig. 5a,b). Despite no significance in survival, the high dose (30 mg/kg) of IV bendamustine showed a modest trend toward decreased bioluminescence imaging signal (Fig. 5d). There was no visual difference between the mice in any group, and weight was comparable between treatment groups with weight loss seen in conjunction with development of tumor for all groups (Data not shown).

Fig. 5.

In vivo application of IV versus PO bendamustine (BEN) treatment in a Multiple Myeloma xenograft model. NSG mice received 150 cGy TBI on Day-2 and were injected on Day-1 with 2 × 10.⁵ luciferase-tagged MM.1s cells by tail vein injection. BEN was administered on Days 1 and 2 either intravenously by tail vein injection (IV, 15 mg/kg or 30 mg/kg) or by oral gavage (PO, 30 mg/kg or 60 mg/kg). a,b Kaplan–Meier curves shows survival of NSG mice with equivalent doses of IV or PO BEN. Mice were sacrificed if they reached two consecutive weight loss scores greater than thirty percent of starting weight or if hind limb paralysis was reached. c,d,e Bioluminescence imaging was conducted two to three times weekly and c,d photons/second are shown as average over time, n = 9–10 per group. e Representative images are shown according to the same rainbow coloring set to the same min and max radiance. Experiments were repeated twice. A log-rank (Mantel-Cox) test was performed for survival analysis. P-values of < 0.05 were considered significant, and the asterisks indicate increasing levels of significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Raji Burkitt’s lymphoma xenograft model

Using Raji, a Burkitt’s lymphoma, NSG mouse model we found no significant difference between pharmacokinetically comparable lower doses of IV vs PO bendamustine (Fig. 6a, 15 mg/kg or 30 mg/kg, respectively) however, both treatment groups had significantly extended survival as compared to the control (IV 15 mg/kg vs. No Rx; PO 30 mg/kg vs. No Rx). In this Burkitt’s lymphoma, which was the most resistant in-vitro (Fig. 2), doubling the dose of both IV and PO bendamustine conferred prolonged survival, with greater improvement seen with IV over PO, leading to a significant difference between them (Fig. 6b). Both at the lower doses and higher doses, modest weight loss was seen in the treatment groups, however there was no difference in appearance of the mice between groups (Data not shown). For bioluminescent imaging, IV bendamustine at 30 mg/kg showed a decreased tumor burden at week 3 compared to other treatment groups (Fig. 6d,e). However, mice only survived approximately one more week when compared to PO bendamustine.

Fig. 6.

In vivo application of IV versus PO bendamustine (BEN) treatment in a non-Hodgkin’s Burkitt’s Lymphoma xenograft model. NSG mice received 150 cGy TBI on Day-2 and were injected on Day-1 with 2 × 10.⁵ luciferase tagged Raji cells by tail vein injection. BEN was administered on Days 1 and 2 either intravenously by tail vein injection (IV, 15 mg/kg or 30 mg/kg) or by oral gavage (PO, 30 mg/kg or 60 mg/kg). a,b Kaplan–Meier curves shows survival of NSG mice with equivalent doses of IV or PO BEN. Mice were sacrificed if they reached two consecutive weight loss scores greater than thirty percent of starting weight or if hind limb paralysis was reached. c,d,e Bioluminescence imaging was conducted two to three times weekly and c,d photons/second are shown as average over time, n = 10–15 per group. e Representative images are shown according to the same rainbow coloring set to the same min and max radiance. Experiments were repeated twice. A log-rank (Mantel-Cox) test was performed for survival analysis. P-values of < 0.05 were considered significant, and the asterisks indicate increasing levels of significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Discussion

We have successfully shown that a newly developed orally bioavailable formulation of bendamustine, when appropriately dosed and administered, yields comparable cytotoxic effects to bendamustine administered IV against hematologic malignancies in our mouse models for lymphoma, multiple myeloma, and leukemia. As bendamustine is currently administered exclusively through IV routes in clinical practice, patients are constrained to in-hospital treatments. Therefore, the availability of an effective oral form of bendamustine is essential to enable the expansion of out-of-hospital treatment options for patients. It would further reduce the overall cost of treatment for patients and healthcare systems and enhance treatment compliance especially on certain classes of older and frail patients. Oral bendamustine could also facilitate easier exploration of alternative dosing regimens that might enhance anticancer activity and/or improve treatment tolerability. For example, a phase II trial of a 5-day cycle of daily bendamustine 60 mg/m² intravenously, every 4 to 6 weeks, demonstrated a 76.5% partial and complete remission rate with disease stabilization in 19.6% of 102 patients with pre-treated low-grade non-Hodgkin lymphomas [28]. Bendamustine’s unique pharmacologic activities as both an alkylating agent and a purine nucleoside analog makes it a candidate for exploring extended daily dosing regimens, which are not practical for intravenous dosing. Overall, the possibility of an oral bendamustine formulation could beneficially impact quality of life and convenience for many patients.

While our pharmacokinetic data in mice is promising, analysis of the novel oral bendamustine pharmacokinetics must be examined in humans to determine safety and efficacy in adult and pediatric patients. Comparing IV to PO doses of bendamustine (15 mg/kg IV and 17.65 mg/kg PO, respectively) the Cmax values of 10,518.4 ng/mL and 4872.1 ng/mL correspond to concentrations of 29 uM and 14 uM respectively. Previous pharmacokinetic data of bendamustine in humans reported peak plasma concentrations of 5746 ng/mL or 5320 ng/mL for doses of 120 mg/m², and an effective half-life of around 40 min [11, 29]. With our IV bendamustine pharmacokinetic data showing a half-life of 0.53 h, the IV duration of the drug is similar. However, our PO bendamustine showed a half-life of 2.22 h, with a maximum plasma concentration that is consistent with a bioavailability of 51.4%. Although the longer half-life observed with PO bendamustine was longer compared to that with IV bendamustine, it likely represents continued absorption from the gastrointestinal tract, characteristic of “flip-flop” pharmacokinetics. With flip-flop pharmacokinetics, the rate of drug absorption is slower than the rate of drug elimination [30, 31]. For most immediate-release drugs, the rate of absorption from the gastrointestinal tract is faster than the rate of drug elimination. Flip-flop pharmacokinetics occurs frequently with drugs absorbed by a slow first-order process, such as extended- or sustained-release dose formulations, where the terminal linear slope of the plasma concentration-versus time curve suggests a longer half-life as compared to the half-life of the drug administered intravenously. However, drugs that exhibit low permeability rates across the intestinal membrane, such as drugs with low oil-to-water partition coefficients or substrates for uptake and/or efflux transporters in the gastrointestinal tract, also exhibit flip-flop pharmacokinetics [30, 31]. Bendamustine has previously been shown to be a substrate for P-glycoprotein and breast cancer resistance protein [9]. Although extensive information is unavailable, based on measured and in silico data, bendamustine injection is a proposed Biopharmaceutics Drug Disposition Classification System (BDDCS) Class 1 drug, which is highly soluble but extensively metabolized [32]. Transporter effects in the gut and liver are predicted to have minimal or clinically insignificant effects on Class 1 drug disposition, but the role of uptake or efflux transport systems in mediating bendamustine activity or transport has not been elucidated. Whereas the BDDCS system may be useful for prediciting characteristics of individual drugs and their disposition potential brain penetration and food effects on oral absorption, studies to evaluate these factors should be conducted.

We also considered the possibility that the plasma concentration time data might reflect enterohepatic recycling of bendamustine with oral dosing. Bendamustine undergoes biliary excretion in mice, rats, and dogs, without about 90% of intravenously administered drug recovered in the feces [33]. If a difference in the terminal slopes between oral and intravenous dosing might reflect enterohepatic recycling, no prominent secondary peaks were evident in the individual plasma concentration time profiles for individual animals, perhaps in part due to the sampling times obtained. Previous work on the pharmacokinetics of bendamustine have shown that phase 1 oxidation by cytochrome P450 1A2 (CYP1A2) yields byproducts that do not contribute significantly to toxicity, and CYP1A2 plays a minor role in eliminating bendamustine. Limited involvement of hepatic metabolism through CYP1A2 has also led to conclusions that drug interactions with CYP1A2 inhibitors are unlikely [11, 23]. In humans, bendamustine undergoes N-dealkylation and hydroxylation catalyzed by CYP1A2, to two active metabolites, M3 and M4 [34]. Mice, rats, dogs, monkeys, and humans exhibit appreciable interspecies differences in cytochrome P450 (CYP450)-mediated drug metabolism [35]. CYP1A2 comprises about 13% of total hepatic CYP450 content in humans but is more variable in rodents. Bendamustine also undergoes nonenzymatic hydrolysis to 2 products, HP1 and HP2, which possess low antitumor activity [11]. These mechanisms may in part account for incomplete bendamustine oral bioavailability, which cannot be fully explored through preclinical studies.

Published data with oral bendamustine is limited. In a phase 1 study of an orally-formulated bendamustine, the half-life with oral bendamustine was 0.71 ± 0.12 h compared to 0.47 ± 0.5 h following a 60-min intravenous infusion, indicating a slightly longer half-life with oral administration [23]. These investigators utilized a liquid-filled hard capsule for bendamustine in humans that cannot be compared to the oral bendamustine formulation that we studied, a proprietary solid dispersion solution designed to enhance oral bioavailability, in mice. Additional studies are needed to better describe the systemic exposure and terminal half-life with PO bendamustine with extended sampling times and multiple dose levels.

Our in-vitro data of bendamustine’s cytotoxic effects against three hematological malignancy cell lines demonstrated surprising results. Bendamustine is a chemotherapeutic agent typically given for chronic lymphocytic leukemia, indolent B-cell non-Hodgkin lymphoma, and multiple myeloma. We demonstrated here that across varying concentrations of bendamustine, RS4;11, a B-cell acute lymphoblastic leukemia, was most sensitive to bendamustine-induced killing in-vitro. However, our in-vivo data demonstrated that only some experimental groups had a minimal but statistically significantly increased survival compared to untreated controls. MM.1s, a multiple myeloma cell line, displayed intermediate sensitivity in our in-vitro testing. This outcome is in line with expectations, given the established efficacy of bendamustine against multiple myeloma, both as a standalone treatment and as part of combination therapy, particularly in cases of relapsed or refractory multiple myeloma [36, 37]. In line with our data, previous studies have reported MM.1s sensitivity to bendamustine in-vitro at concentrations higher than 100 umol/L at 72 h [38]. In our mouse xenograft model, however, there was no survival advantage for mice receiving bendamustine as compared to control. A slight decrease in bioluminescence signal at IV bendamustine 30 mg/kg demonstrates that high doses of bendamustine may decrease early tumor burden in NSG mice. However, the MM.1s-Luciferase model resulted in saturating signal (photons/second) for every group at our given dose of tumor cells. Lastly, the most resistant cell line in-vitro was Raji, a non-Hodgkin Burkitt’s lymphoma. Despite resistance in our MTS assays, bendamustine in-vivo prolonged survival of mice in all treatment groups as compared to the controls. No cell line in the xenograft mouse models demonstrated complete clearance of tumor, offering a window for possibilities of studying the immunomodulatory effects of bendamustine with immunocompetent mice, or NSG mice engrafted with human immune cells.

Across our models, there were limited differences between IV and PO formulations of bendamustine, especially at the lower doses of 15 mg/kg and 30 mg/kg respectively. After examining the pharmacokinetic data and comparing the area under the curve (AUC inf, ng/mL*h), the oral bioavailability was determined to be 0.514 of the IV dose. Using this value, equivalent PO doses were given as double that of IV, with the assumption that the oral bioavailability data is similar at higher doses of PO bendamustine (30 mg/kg and 60 mg/kg). Differences were seen between IV and PO bendamustine in the Raji-Luc model when doses of bendamustine were doubled to 30 mg/kg and 60 mg/kg for IV and PO respectively. While we have yet to elucidate why these differences exist, one can speculate that differences in peak plasma concentration or differences in absorption time profiles may play a role in IV extending survival against the tumor.

The availability of oral bendamustine has the potential to significantly enhance accessibility and reshape the options for dosing schedules for patients. Administering the medication orally enables the possibility of safe and effective therapy that is more convenient and uses fewer resources, opening new avenues for novel treatment regimens. With the addition of pharmacokinetic studies in rats and dogs completed by Exinda Therapeutics, the utilization of oral bendamustine warrants human trials to assess its safety, validate its pharmacokinetics, and investigate any potential nutritional or drug interactions that may arise due to this alternative delivery method.

Data availability

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