Probenecid increases renal retention and antitumor activity of DFMO in neuroblastoma
Chad R. Schultz1 · Matthew A. Swanson2 · Thomas C. Dowling3 · André S. Bachmann1
Received: 2 March 2021 / Accepted: 30 May 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Background Neuroblastoma (NB) is the most common extracranial solid tumor in children. Interference with the polyamine biosynthesis pathway by inhibition of MYCN-activated ornithine decarboxylase (ODC) is a validated approach. The ODC inhibitor α-difluoromethylornithine (DFMO, or Eflornithine) has been FDA-approved for the treatment of trypanosomiasis and hirsutism and has advanced to clinical cancer trials including NB as well as cancer-unrelated human diseases. One key challenge of DFMO is its rapid renal clearance and the need for high and frequent drug dosing during treatment.
Methods We performed in vivo pharmacokinetic (PK), antitumorigenic, and molecular studies with DFMO/probenecid using NB patient-derived xenografts (PDX) in mice. We used LC–MS/MS, HPLC, and immunoblotting to analyze blood, brain tissue, and PDX tumor tissue samples collected from mice.
Results The organic anion transport 1/3 (OAT 1/3) inhibitor probenecid reduces the renal clearance of DFMO and signifi- cantly increases the antitumor activity of DFMO in PDX of NB (P < 0.02). Excised tumors revealed that DFMO/probenecid treatment decreases polyamines putrescine and spermidine, reduces MYCN protein levels and dephosphorylates retinoblas- toma (Rb) protein (p-RbSer795), suggesting DFMO/probenecid-induced cell cycle arrest. Conclusion Addition of probenecid as an adjuvant to DFMO therapy may be suitable to decrease overall dose and improve drug efficacy in vivo. Keywords Probenecid · DFMO/Eflornithine · Repurposing drugs · Pediatric cancer · Renal drug clearance · Pharmacokinetics Background Neuroblastoma (NB) is the most common extracranial solid tumor of the sympathetic nervous system and establishes almost exclusively in children. The average age at diagnosis is 1–2 years and nearly 90% of cases are diagnosed by age 5. There are about 700 children per year in the United States diagnosed with NB, which accounts for ~ 6% of all pediat- ric cancers. Almost all patients with MYCN-amplification fall into the category of high-risk NB patients, with lower chances of therapeutic success and higher risk of relapse. The 5-year survival rate of high-risk NB patients is 40–50%. Aggressive multimodal chemotherapy along with surgery, stem cell transplant, immunotherapy and retinoid therapy remains the standard care for treatment of patients with high- risk NB [1–8]. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines (putrescine, spermidine, spermine), which are positively charged metabolites that contribute to normal cell cycle regulation and when elevated can stimulate increased cell proliferation [9–14]. ODC is a validated drug target in NB  and other MYC-deregu- lated cancers [15–17]. The ODC gene is directly activated by MYC transcription factors through interactions with the E-box elements of ODC1 gene [18–21]. Given the high rel- evance of MYCN gene amplification in NB, we postulated in 2004 that ODC is an important drug target for this cancer type and that inhibition of polyamine biosynthesis via ODC inhibition might be a novel therapeutic strategy for NB [22, 23]. We found that α-difluoromethylornithine (DFMO), a well-established ODC inhibitor, is effective against NB by inducing p27/Rb-mediated G1/S cell cycle arrest [22–30]. Our initial observations were confirmed independently by two other groups assessing DFMO in the TH-MYCN NB transgenic mouse model [31, 32]. Importantly, DFMO has been FDA-approved for the treatment of West African sleep- ing sickness (trypanosomiasis) [33–36] as well as hirsutism [37, 38]. We repositioned DFMO and performed the first phase I study in NB , followed by a phase II study to evaluate the potential use of DFMO as a chemopreventive agent with the goal to prevent/delay the relapse of high- risk NB patients . Currently, a Children’s Oncology Group (COG) phase II randomized study (ANBL 1821)  is underway to assess chemo-immunotherapy (irinotecan, temozolomide, dinutuximab) with or without DFMO (Eflo- rnithine) in children with relapsed, refractory or progressive NB. While DFMO has a high safety profile [25, 26, 39, 40], this useful oral drug has pharmacokinetic (PK) limita- tions with regard to dose efficacy and renal drug retention. To be efficacious, DFMO is given at high concentrations (1–5 mM in cell cultures, 1–2% (w/v) in animal studies, and 500–1500 + mg/m2 BID in patients), which in part is due to its rapid renal clearance. It is estimated that over 80% of non-metabolized DFMO is secreted into the urine, thus call- ing for the frequent administration of high doses of DFMO [41, 42]. To improve efficacy and reduce drug dosing, we hypothesized that the use of an adjuvant such as probenecid could improve DFMO retention. Probenecid is known to competitively inhibit renal excretion of certain organic ani- onic drugs (e.g., penicillin), thereby increasing their plasma concentration and prolonging their effect . In this study, we performed PK and antitumor efficacy studies in mice to determine the impact of probenecid on the retention of DFMO in plasma and brain and to measure the antitumor effect of DFMO/probenecid in NB tumor patient- derived xenografts (PDX) in mice. Materials and methods Chemicals, reagents, and antibodies The irreversible ODC inhibitor DFMO was provided by Dr. Patrick Woster (Medical University of South Caro- lina, Charleston, SC). Probenecid was purchased from Millipore-Sigma. Mouse monoclonal antibody for retino- blastoma protein (Rb) (4H1), rabbit polyclonal antibody for phosphorylated Rb (p-RbSer795) and rabbit monoclonal antibody for p-RbSer807/811 (D20B12) were purchased from Cell Signaling Technology. Mouse monoclonal antibodies for MYCN (B8.4B) and GAPDH (C4) were purchased from Santa Cruz Biotechnology. Goat anti-rabbit and Goat anti- mouse secondary antibodies conjugated to IRDye®680 RD or IRDye®800CW were obtained from Licor. Protein assay dye reagent was obtained from Bio-Rad Laboratories. Sol- vents utilized in DFMO quantification were LC/MS grade water, HPLC grade acetonitrile, and LC/MS grade formate obtained from Fisher Chemical®. Western blot Tumor lysates were prepared in radioimmuno-precipita- tion assay (RIPA) buffer [20 mM Tris–HCl (pH 7.5), 0.1% sodium lauryl sulfate, 0.5% sodium deoxycholate, 135 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA], supplemented with complete protease inhibitor cocktail (Roche Molecular Biochemicals), and phosphatase inhibi- tors, 20 mM sodium fluoride, and 0.27 mM sodium vana- date. Total protein concentration was determined using the Bradford dye reagent protein assay (Bio-Rad Laborato- ries). Tumor lysates in SDS sample buffer were boiled for 10 min and equal amounts of protein were resolved by 10% SDS-PAGE. Protein was electro-transferred onto 0.45 µM polyvinylidene difluoride Immobilon-P membrane (Milli- pore). Primary antibodies were incubated overnight at 4 °C in 5% BSA in Tris-buffered saline containing 0.1% Tween 20. Secondary antibodies were incubated for 1 h at room temperature in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20. Blots were imaged using an Odyssey Clx (Licor) Western blot scanner. Relative protein signal quantification was performed using Image Studio Lite (Licor) software. In vivo pharmacokinetic studies Animal treatment Six- to eight-week-old female athymic nu/nu mice were obtained from the Van Andel Research Institute (Grand Rap- ids, MI). Mouse PK studies were assessed under Michigan State University approved IACUC protocol 07-18-090-00. Mice were administered vehicle control (0.1 mM Tris/0.7% saline) or 400 mg/kg probenecid via intraperitoneal (i.p.) injection 30 min prior to dosing with 500 mg/kg DFMO in water via oral gavage. Blood was harvested from three ani- mals per group by intracardial puncture at 10 and 30 min, 1, 2, 4, and 6 h post administration of DFMO. The brains of the mice were also removed (up to the 4-h time point) and snap- frozen with liquid nitrogen. The blood samples were spun at 1000 × g for 15 min. The supernatant plasma and brain tissue samples were frozen at – 80 °C for LC–MS/MS determina- tion of DFMO concentration ± probenecid treatment. Bioanalysis DFMO concentrations in mouse plasma and brain were determined by LC–MS/MS analysis. Briefly, samples (calibration standards, quality control and study samples) were processed by protein precipitation (acetonitrile). Following centrifugation, supernatant was transferred to a clean vial and placed in a refrigerated injector com- partment. The isocratic mobile phase consisted of ace- tonitrile in formic acid. Analyte separation was achieved using an Atlantis HILIC Silica column preceded by an Atlantis HILIC 3 µm VanGuard Cartridge. A Shimadzu LC–MS/MS 8040 equipped with electrospray ioniza- tion (ESI) was used to quantify DFMO using Multiple Reaction Monitoring (MRM). The DFMO precursor ion (183 m/z) was used to quantify daughter ions at transitions of: 183 > 120.10, 183 > 166.10, and 183 > 80.05.Standard curve concentrations were linear over the range of 250 ng/ mL to 50,000 ng/mL in mouse plasma. Additionally, three levels of quality control were applied at 500, 7500, and 40,000 ng/mL in mouse plasma. Standard curves (n = 10) were linear with r2 values between 0.9960 and 0.9999. All plasma standards (n = 20) exhibited accuracy and preci- sion with maximum deviation and coefficient of variation values of ± 11% and ± 5%, respectively. Quality control samples (n = 70) were accurate within ± 12%, with ± 4% precision. In brain samples, DFMO was extracted from tissue using cold acetonitrile and verapamil as the internal standard control.
Pharmacokinetic data analysis
Since all time-points were terminal, mean DFMO concen- trations per time point were used to calculate the compos- ite PK parameters by non-compartmental analysis using Phoenix WinNonlin version 7.6 (Certara, Princeton, NJ). The maximum plasma concentration (Cmax) and the time to reach maximum plasma concentration (Tmax) were obtained by visual inspection of the data from the concentration–time curve. The area under the plasma concentration versus time curve (AUC) was calculated using the linear trapezoidal method (linear interpolation). The terminal elimination phase of the PK profile was estimated based on the best fit (r) using at least the last three observed concentrations. PK parameters describing the systemic exposure of DFMO were estimated from observed concentrations, the dosing regimen, the AUC, and the terminal elimination phase rate constant (kel) for each group. The portion of the AUC from the last measurable concentration to infinity (AUC∞) was estimated from the equation Ct/kel, where Ct represents the last measurable concentration. The extrapolated portion of the AUC was used for the determination of AUC∞.
Polyamines (putrescine, spermidine, spermine) from termi- nal tumor tissues were isolated, dansylated, and analyzed by HPLC as previously described [44–46]. Briefly, poly- amines were extracted and protonated in perchloric acid/ sodium chloride buffer. To 100 μL of sample, 4.5 nmol of 1,7 diaminoheptane internal standard and 200 μL of 1 M sodium carbonate was added prior to dansylation with 400 μL of 5 mg/mL dansyl chloride (Sigma Aldrich, St. Louis, MO). Samples were analyzed using a Thermo Sci- entific/Dionex Ultimate 3000 HPLC equipped with a Syn- cronis C18 column (250 × 4.6 mm, 5 μM pore size). The dansylated polyamine derivatives were visualized by excita- tion at 340 nM and emission at 515 nM. Using the relative molar response derived from N-dansylated polyamine and 1,7 diaminoheptane standards, the amount of N-dansylated polyamine derivatives was calculated and normalized to total sample protein.
In vivo tumor studies
Five-week-old female athymic nude mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Tumor volume studies were assessed under Michigan State Uni- versity’s IACUC approved protocol 201900240. PDX COG-N-623 (Children’s Oncology Group Childhood Cancer Repository) was derived from a MYCN-amplified NB tumor established at time of disease progression after chemotherapy . Mice were flank injected with 15 × 106 PDX NB cells in 200 μL of 1:1 mixture of serum free RPMI and matrigel. Tumor volume was measured using calipers and calculated using the formula (length ×width2)/2. Once tumors were 100 mm3 in size, mice were randomized into 4 treatment groups: (1) Control (n = 9): normal drink- ing water; probenecid vehicle control (0.1 mM Tris/0.7% saline) injected i.p. twice daily 6 h apart. (2) Probenecid alone (n = 9): normal drinking water; 200 mg/kg probenecid (Millipore Sigma) in 1 mM Tris/0.7% saline injected i.p. twice daily 6 h apart. (3) DFMO alone (n = 10): 2% DFMO in drinking water; probenecid vehicle control injected i.p. twice daily 6 h apart. (4) DFMO + Probenecid (n = 10): 2% DFMO in drinking water; 200 mg/kg probenecid injected i.p. twice daily 6 h apart. Mouse weights were recorded weekly. Tumor volume measurements were taken 3 times per week. Fresh normal drinking water or 2% DFMO drinking water was exchanged 3 times per week. The volume of water consumed per mouse was recorded during water exchanges. Tumors were treated for 3 weeks, at which point control tumors had reached approximately 2500 mm3 in size. Mice were euthanized with CO2 using a Euthanex Prodigy system. Tumors were removed and weighed. One half of the tumors were snap-frozen with liquid nitrogen, while the other half was fixed with 10% neutral-buffered formalin.
The statistical significance for the PK study, tumor treat- ment study, and polyamine analysis was determined using an unpaired Student’s t test assuming the null hypothesis. Statistical significance in protein levels as measured by Western blot was determined by ANOVA with multiple comparison analysis. For all tests, a value of P ≤ 0.05 was considered statistically significant. All data points are the means of N replicates and error bars show standard devia- tion (± S.D.) or standard error (± S.E.) as noted in each legend.
Probenecid slows DFMO elimination and increases plasma exposure
To investigate if DFMO clearance is reduced in the pres- ence of probenecid, we performed an in vivo single-dose PK study. A summary of the PK parameters obtained fol- lowing oral dosing in mice is shown in Table 1. The plots of the mean DFMO plasma and brain concentrations over time are shown in Fig. 1A, B, respectively. Following oral administration, absorption was rapid (Tmax observed within 60 min) in both groups, with the Cmax being lower in the probenecid group. The overall DFMO exposure, as measured by AUClast, AUC∞, tended to be higher in the probenecid group, reflected by a slightly lower (10%) clearance. The elimination half-life of DFMO was slightly higher in the probenecid group, likely explained by a ~ 15% reduction in volume of distribution (Vz/F). In brain tissue, the maximum concentration achieved was higher in the probenecid group (14.6 mcg/g) compared to the DFMO control (11.0 mcg/g). Overall exposure in brain was slightly higher in the probenecid group compared to the control group, with AUC values of 2.15 mcg*min/g vs. 2.22 mcg*min/g, respectively.
Fig. 1 DFMO concentrations in mouse plasma and brain tissues from in vivo PK study. A Analysis of mouse plasma. DFMO adminis- tered as a single oral dose (500 mg/kg) via gavage; Values represent mean and standard deviation (S.D.); n = 3 per time point. B Analy- sis of relative quantity of DFMO in brain tissue (ng DFMO per gram of tissue). Values represent mean and S.D; n = 3 per time point. *— denotes statistically significant difference as compared to control (P < 0.05). **—Denotes statistically trending difference as compared to control (P = 0.058) Probenecid improves antitumor potency of DFMO in PDX of NB In the first part of this study, our results suggest that addi- tion of probenecid can prolong the presence of DFMO in the blood plasma and the brain. To investigate if DFMO retention results in improved antitumorigenic potency, we performed in vivo studies in mice using PDX of human NB. Our data suggest that addition of probene- cid indeed increased DFMO potency against NB PDX in mice if compared with DFMO alone (Fig. 2). Probenecid alone had no effect. All PDX tumors were treated for 20–21 days, at which point the control and probene- cid alone tumors reached greater than 2500 mm3, thus requiring euthanasia of these animals per internal IACUC guidelines. At this time point, DFMO-treated tumors displayed a slight decrease in tumor size and weight, while those tumors that were treated with DFMO plus probenecid were significantly smaller in size compared to control (P < 0.005) or DFMO-treated tumors (P < 0.02) (Fig. 2A–C). Mice consumed similar amounts of water in all treatment groups (Supplementary Figure 1). Fig. 2 Antitumor growth effect of DFMO/Probenecid in NB PDX in mice. A Tumors were grown to 100 mm3 in size and then ran- domly assigned to four treatment groups: (1) Control, (2) DFMO, (3) Probenecid, and (4) DFMO + Probenecid. During treatment, tumor volume (mm3) was measured three times a week at various time points (days) over a maximum of 21 days. B Mean tumor volume (mm3) and C tumor weight (g) at the end of study, after tumor excision. Data represent the mean ± standard error (S.E.) Control (n = 9), DFMO (n = 10), Probenecid (n = 9), DFMO + Probenecid (n = 10). DFMO/probenecid-treated tumors show significantly reduced tumor volume and tumor weight. *—denotes statistically significant dif- ference between control and DFMO/probenecid treatment groups (P < 0.005). **—denotes statistically significant difference between DFMO and DFMO/probenecid treatment groups (P < 0.02). DFMO/probenecid decrease polyamine levels in PDX tumor tissues To better understand the pharmacodynamic (PD) response to drug treatments, we measured the polyamine content (putrescine, spermidine, spermine) of excised NB PDX tumors from all treatment groups, as a surrogate marker of drug activity. Putrescine and spermidine levels were lowered not probenecid- or vehicle-treated PDX tumors (Fig. 3). This confirms that DFMO has reached the tumor target site in which it specifically inhibits ODC, the enzyme that directly converts ornithine to putrescine. DFMO/probenecid reduce MYCN protein levels and dephosphorylates Rb in PDX tumor tissues To study signaling proteins affected by DFMO ± probene- cid treatments, NB PDX tumors were excised and studied by Western blot. MYCN protein levels were significantly reduced (P < 0.05) in DFMO/probenecid-treated mice (Fig. 4). Furthermore, Rb phosphorylation at Ser795 (p-RbSer795) was significantly decreased (P = 0.05) with DFMO/probenecid combination treatments (Fig. 5). Rb phosphorylation at Ser807/811 (p-RbSer807/811) did not change (not shown). The dephosphorylation of p-RbSer795 suggests G1/S cell cycle arrest, thus confirming our previous results with DFMO in cultured NB cells . Fig. 4 DFMO/Probenecid decreases MYCN protein in NB PDX tumors. A Analysis of excised PDX tumors for MYCN protein lev- els by Western blot. Numbers represent individual mice. GAPDH is used as a loading control. B Quantification of MYCN protein in four groups (control, DFMO, Probenecid, and DFMO + Probenecid) from two separate experiments. MYCN/GAPDH signal ratios were deter- mined using Image Studio Lite (Ver 5.2) and normalized to control. DFMO + Probenecid-treated tumors had significantly lower levels of MYCN as compared to control. *—denotes statistically significant differences as compared to control (P < 0.05). Fig. 3 DFMO- and DFMO/Probenecid-treated tumors have decreased levels of putrescine. A portion of terminal tumor tissue from all four treatment groups was harvested and analyzed for polyam- ine levels. NB tumors treated with DFMO or DFMO + Probene- cid had significantly reduced levels of putrescine as compared to control-treated tumors. Spermidine levels were reduced in DFMO or DFMO + Probenecid treatment groups but were not statisti- cally significant. Spermine levels were unchanged. Data represent the mean ± standard error (S.E.) of polyamine levels of Control (n = 9), DFMO (n = 10), Probenecid (n = 9) and DFMO + Probenecid (n = 10)-treated tumors. *—denotes statistically significant differ- ences as compared to control (P < 0.05). Discussion DFMO is an enzyme-activated, irreversible inhibitor of ODC that acts by covalently binding to the enzyme . The serum half-life of DFMO is 2–4 h and over 80% is elimi- nated unchanged in the urine [41, 42]. To address this dosing limitation, approaches to mitigate the rapid elimination of DFMO are being explored. Probenecid is an FDA-approved the tubular cell membrane, probenecid interferes with organic anion transporter 1/3 (OAT 1/3) and urate trans- porters (URAT). For example, OAT1/3 preferentially binds probenecid with higher affinity than uric acid which conse- quently prevents reabsorption of uric acid in the plasma and increases uric acid in the urine. Although it is primarily used to treat gout and hyperuricemia, recent interest in repurpos- ing this drug has emerged based on its anti-viral and anti- cancer properties . For example, probenecid has been shown to augment the anti-cancer activity of folate analogs, such as methotrexate and 10-deazaaminopterin . The ability of probenecid to inhibit the renal uptake of drugs also plays a role in protecting the kidneys from nephrotoxic agents such as cidofovir . The major adverse effect of cidofovir, nephrotoxicity, is attributed to its uptake into renal proximal tubular cells by organic anion transporters such as OAT1/3 along the basolateral membrane (Fig. 6). This accumulation in the renal proximal tubular cell is the likely cause of tubular necrosis. Probenecid acts as a competitor inhibitor of cidofovir uptake, reducing intracellular levels of cidofovir and increasing cidofovir plasma levels (Fig. 6). Similarly, it has been used to increase systemic exposure for other drugs including cephalosporins, the anti-viral agent oseltamivir and methotrexate . Since the rapid renal clearance in patients is one of DFMO’s main deficiencies, we reasoned that addition of probenecid may prolong the presence of DFMO in the blood plasma and hence improve its antitumorigenic potency. Our data suggest that addition of probenecid extended the half- life of DFMO in the plasma and increased its potency against NB PDX in mice (Figs. 1A, 2). Since complete inhibition of renal secretion was not observed, it is likely that either additional organic anionic transporters are involved in the renal handling of DFMO, the dosing strategy of probenecid was not optimized, or that a transition to non-renal clearance mechanisms occurred. Nonetheless, these results suggest that the renal handling of DFMO can be modified by renal drug transport-inhibiting agents. Fig. 5 DFMO/Probenecid dephosphorylates Rb protein in NB PDX tumors. Analysis of excised PDX tumors for retinoblastoma (Rb) pro- tein by Western blot. Numbers represent individual mice. A Rb and phosphorylated Rb (p-RbSer795) are shown. GAPDH was used as a loading control. B Quantification of p-RbSer795 in four groups (con- trol, DFMO, Probenecid, and DFMO + Probenecid). Rb/GAPDH and p-RbSer795/GAPDH signal ratios were first determined using Image Studio Lite (Ver 5.2), and p-RbSer795/Rb values were then plotted rela- tive to control. DFMO + Probenecid-treated tumors had significantly reduced levels of p-RbSerS795. *—denotes statistically significant dif- ferences compared to control ( P = 0.05). The translatability of mouse to human dosing regimens needs to be addressed in future studies. This study was not designed to provide a thorough characterization of probene- cid administration (i.e., dose, plasma exposure, and time course) and impact on DFMO disposition. We selected the probenecid regimen based on prior studies in mice demon- strating OAT transport inhibition for several drugs including topotecan and methotrexate at doses of 200–1200 mg/kg [50, 53–56]. In humans, the Cmax for probenecid is approximately 100–200 mg/L after a single 1000 mg oral dose. The concen- tration required to inhibit the OAT3 transporter (IC50) is esti- mated at 4.4 µM (1.3 mg/L) . Additional PK modeling studies assessing the influence of probenecid administration, as well as additional covariates (age, gender, renal function, etc.) on the PK of DFMO, are required to determine the or more MYCN bands were detected in these more heterog- enous PDX tumor tissues (Fig. 4A), possibly representing MYCN isoforms. Similar results were obtained when using another commercial MYCN antibody (not shown). MYCN gene amplification is a key prognostic marker associated with aggressive NB and unfavorable outcomes [5, 27] and the MYCN protein is considered an undruggable drug tar- get. Therefore, the observation that DFMO, besides directly inhibiting ODC, indirectly also reduces MYCN protein lev- els by yet unidentified mechanisms is of great importance. In summary, this study shows for the first time how a renal drug transport inhibitor could potentially be used as an adjuvant for NB in cancer treatment and chemopreven- tion studies in combination with DFMO. Various clinical trials with DFMO are underway for NB, intracranial brain. Probenecid. Probenecid inhibits Organic Anion Transporters (OAT1/3) in the basolateral membrane of cells in the proximal tubule. This can lead to reduced tubular uptake, reduced elimina- tion and increased plasma levels of drugs normally secreted by this mechanism (e.g., penicillin, cidofovir). In addition, probenecid also inhibits Urate Transporters (URAT) in the apical membrane of the proximal tubule, which decreases uric acid reabsorption, resulting in an increased urinary excretion of uric acid, which is beneficial in patients with uric acid handling disorders such as gout optimal means of co-administering these two drugs when treating NB, and whether there is a role for dosing DFMO in combination with probenecid, such as that described for oseltamivir for prophylaxis of influenza virus . Although not an intracranial brain tumor, NB can metas- tasize to the brain and our data showed also higher levels of DFMO in mouse brains in the presence of probenecid com- pared to DFMO alone (Fig. 1B). Of note, the probenecid- dependent increase of DFMO in the brain might also be of interest for future clinical trials with DFMO, for example, in recurrent anaplastic astrocytoma (NCT02796261), medul- loblastoma (NCT03581240), and Alzheimer’s disease  to improve its overall efficacy. While it was previously shown that DFMO inhibits the growth of tumors in the transgenic TH-MYCN NB mouse model [32, 59] and NB cell line xenografts , this is the first study to examine the effect of DFMO in human PDX of NB. We found that DFMO reduces the tumor size when compared to controls with statistically significant greater effects when combined with probenecid (Fig. 2). Analysis of excised PDX tumors suggested that DFMO had reached the tumor target tissue, because the levels of the ODC reaction product putrescine were significantly reduced (Fig. 3). Fur- thermore, molecular analyses revealed that DFMO/probene- cid reduces MYCN total protein levels and dephosphorylates p-RbSer795 in vivo (Figs. 4, 5), which is consistent with our previous in vitro data, suggesting DFMO-mediated G1/S cell cycle arrest [23, 28]. Unlike NB cell lines, in which we typi- cally detect a single MYCN protein band at 60–63 kDa, two tumors, prostate cancer, and colorectal cancer as well as cancer-unrelated diseases, such as Alzheimer’s disease, diabetes and Bachmann–Bupp syndrome (OMIM:619075), a neurodevelopmental, ODC1-linked genetic disorder we recently discovered [45, 61]. Alternative OATs and inhibi- tors including ciprofloxacin, itraconazole, omeprazole and others may be studied in a more systematically manner to find the most effective adjuvant to boost DFMO effects. Potential advantages of effective DFMO adjuvant therapy would be increased drug retention of DFMO in the blood and target tissues, lowering of currently high oral doses (500–1500 mg/m2 BID), cost savings and reduced adverse effects. Similar retention improvements might be achieved with other anticancer therapeutics (e.g., methotrexate, etopo- side, cisplatin, topotecan, dacarbazine, melphalan) that have significant (> 30%) renal elimination.
Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00280-021-04309-y.
Acknowledgements This study was supported by the St. Baldrick’s Foundation (Grant No. 591704) and by a generous gift from the Alex Mandarino Foundation (St. Joseph, Michigan) to ASB. The PDX model of NB was kindly provided by the Children’s Oncology Group Childhood Cancer Repository (Dr. Patrick Reynolds), powered by the Alex’s Lemonade Stand Foundation. We thank Dr. Patrick Woster (Medical University of South Carolina, Charleston, SC) for providing DFMO. We are grateful to April M. Stafford (Dr. Daniel Vogt labora- tory, Michigan State University) for excellent technical support with in vivo gavage experiments. We thank Dr. Sky Pike in the Ferris State University Shimadzu Core Lab for assistance with analytical methods development.
Authors’ contributions ASB and TCD supervised and designed the study and developed the technical protocols. CRS performed all experi- ments except LC–MS/MS, which was developed and performed by MLS. ASB, TCD, MLS, and CRS interpreted data and wrote the manu- script. All authors reviewed all drafts of the manuscript including the final draft.
Data availability All data generated or analyzed during this study are included in this published article.
Conflict of interest ASB is the sole inventor of U.S. patent (US 9,072,778) issued on July 7, 2015 entitled “Treatment Regimen for N- Myc, C-Myc, and L-Myc amplified and overexpressed tumors”. ASB was an oncology consultant for Lodo Therapeutics (New York, NY). No potential conflicts of interest were disclosed by the other authors.
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