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Pharmacokinetics and biodistribution of a novel anticancer thyrointegrin αvβ3 antagonist: triazole modified tetraiodothyroacetic acid conjugated to polyethylene glycol (P-bi-TAT)

Pharmacokinetics and biodistribution of a novel anticancer thyrointegrin αvβ3 antagonist:... We previously developed a triazole modified tetraiodothyroacetic acid (TAT) conjugated to a polyethylene glycol (PEG)-based thyrointegrin αvβ3 antagonist targeted compound, called P-bi-TAT. It exhibited potent anti-angiogenic and anticancer activities in vivo. The objective of the current study is to develop a quantitative bioanalytical method for P-bi-TAT using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to elucidate pharmacokinetics (PK) and biodistribution of P-bi-TAT in animals. We used in-source collision-induced dissociation (CID) for ionization of P-bi-TAT in the positive mode, followed by multiple reaction monitoring (MRM) for quantification. P-bi-TAT was quantified using P-mono-TAT as an internal standard because of its similarity in structure and physicochemical properties to P-bi-TAT. The LOQ for P-bi-TAT was 30 ng/μL and the recovery efficiency was 76% with the developed method. Cmax and AUC results at different doses (1, 3, 10 mg/kg) in rats suggest that P-bi-TAT is dose-dependent within the range administered. Results for Cmax and AUC in monkeys at a low dose (25 mg/kg) were comparable to those in rats. Biodistribution of subcutaneously administered P-bi-TAT in the brain of rats ranged from 7.90 to 88.7 ng/g brain weight, and levels of P-bi-TAT in the brain were dose- dependent. The results suggest that P-bi-TAT is a potential candidate as a molecular-targeted anticancer therapeutic with blood-brain barrier permeability and acceptable PK parameters. Its accumulation in organs, toxicokinetic, and pharmacodynamics needs to be further investigated. Keywords: PEGylated, Triazole tetrac, Integrin αvβ3 antagonist, Pharmacokinetic, Toxicokinetic, LC-MS/MS, Anti- angiogenic, Blood-brain barrier Introduction difficult to cure because of its poor prognosis, and the Cancer was the second leading cause of death in the blood-brain barrier and blood tumor barrier that restrict USA in 2018, after heart disease (Xu et al. 2020). It is the delivery of therapeutics (Fox et al. 2019). There are not a single disease but a variety of different cancer types FDA-approved anticancer agents against brain cancers including myeloma, thyroid, leukemia, esophagus, non- such as temozolomide; however, their efficacies are not Hodgkin lymphoma, stomach, and prostate cancer enough to stop brain tumor progression beside their (Henley et al. 2020). Among cancers, brain cancer is significant adverse effects (Anthony et al. 2019). Thus, more efficient, and safer anticancer therapeutics and improved drug delivery systems are under investigation * Correspondence: shaker.mousa@acphs.edu Pharmaceutical Research Institute, Albany College of Pharmacy and Health in the pharmaceutical industry and in academia. Sciences, 1 Discovery Drive, Rensselaer, NY 12144, USA © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Fujioka et al. AAPS Open (2021) 7:2 Page 2 of 9 ALL Targeted anticancer drugs have been developed and column derivatization and MS technique and ap- applied to multiple cancer types, such as glioblastoma plied to PK study of PEGylated gemcitabine in rats and neuroblastoma (Laquintana et al. 2009; Le Rhun (Yin et al. 2020). In this study, we used in-source et al. 2019; Maeda et al. 2016). Among them, collision-induced dissociation (CID) for ionization of nanoparticle-conjugated compounds show selective effi- P-bi-TAT, followed by multiple reaction monitoring cacy and potency to tumors via the enhanced permeabil- (MRM) (Warrack et al. 2013). P-mono-TAT (2, Fig. ity retention (EPR) effect (Maeda et al. 2016). We 1), which is another conjugate of PEG4000 with one previously synthesized a conjugate of tetraiodothyroace- TAT moiety, was used as an internal standard (IS) tic acid (tetrac) and poly (lactic-co-glycolic acid) (PLGA) because its structure and physicochemical properties polymer, PLGA-diamino-propane-tetraiodothyroacetic are similar to P-bi-TAT. We also optimized the acid (DAT), and nanoparticles of DAT (NDAT) showed extraction procedure from biological matrices using high potency and efficacy on multiple cancers, such as solid-phase extraction (SPE) with modification of the glioblastoma and neuroblastoma (Lin et al. 2016; Lin developed method for CTAT. et al. 2018; Sudha et al. 2017a). We then developed a conjugate of β-cyclodextrin with triazole-modified- Experimental tetraiodothyroacetic acid (TAT), named β-C-TAT Materials (CTAT). It showed anti-angiogenic in a chicken embryo Chemicals and biologicals chorioallantoic membrane (CAM) assay and anticancer P-bi-TAT and P-mono-TAT were synthesized and puri- activities in human primary GBM cells and in glioblast- fied in our laboratory. Acetonitrile, methanol, water, and oma xenografted mice (unpublished data). Encouraged formic acid were LC-MS grade and purchased from by the results, we then synthesized a polyethylene glycol Sigma-Aldrich (St. Louis, MO). o-Phosphoric acid (85%) (PEG)-based targeted drug, P-bi-TAT (1, Fig. 1), which was HPLC grade and purchased from Fisher Scientific is a conjugate of PEG4000 with two TAT moieties (Pittsburgh, PA). Rat and monkey plasma were pur- (Sudha et al. 2017b; Rajabi et al. 2019). It showed high chased from Bioreclamation IVT (New York, NY). Rats affinity with a receptor on integrin αvβ3 (IC 0.14 nM), were purchased from Charles River (Kingston, NY). anti-angiogenic activity in a CAM assay, and anticancer activities in human primary GBM cells and in glioblast- oma xenografted mice (10 mg/kg) (Rajabi et al. 2019). In the current work, we developed a bioanalytical method Methods for P-bi-TAT to determine its pharmacokinetics (PK) P-bi-TAT-treatment of rats and monkeys and biodistribution in experimental animals, i.e., rats P-bi-TAT-treatment of rats was carried out in the ani- and monkeys. mal facility of the Veterans Affairs (VA) Medical Center Liquid chromatography-tandem mass spectrometry (Albany, NY), and the experimental protocol was ap- (LC-MS/MS) is a gold standard for quantification of proved by the Institutional Animal Care and Use Com- drug molecules. However, typical quadrupole-type mittee of the VA. Rats were housed under controlled mass spectrometers are not applicable for P-bi-TAT conditions (temperature, 20–24 °C; humidity, 60–70%). because its molecular weight is more than 3000, and Rats were allowed to acclimatize for at least 1 week prior the quantification of high molecular weight PEGylated to the start of treatments. For the PK study, P-bi-TAT drug conjugates is a challenge by mass spectrometry. (10 mg/kg body weight) was administered subcutane- Yin et al. reported a quantitative method for PEGy- ously to 4 rats, and plasma was collected at 0.25, 0.5, 1.5, lated gemcitabine (Mw 70-76 kDa) using LC-time of 3, 6, 12, 24, and 48 h. Plasma samples were stored at flight mass spectrometer (TOFMS) coupled with pre- −80 °C for further analysis. For the biodistribution study, P-bi-TAT (1, 3, 10 mg/kg) was administered subcutane- ously to 4 rats once a day for 3 weeks, and brain samples were collected 24 h after the last dose. Brain samples were stored at −80 °C for further analysis. The monkey study was conducted by a contract re- search organization. Briefly, P-bi-TAT (low dose, 25 mg/ kg body weight; high dose, 82.5 mg/kg) was adminis- tered subcutaneously to male and female cynomolgus monkeys (n = 2 each) daily for 7 days, and plasma was collected at 0.5, 2, 6, 10, 16, and 24 h on day 1 and day Fig. 1 Chemical structures of P-bi-TAT (1) and P-mono-TAT (2); and 7. Plasma samples were stored at −80 °C for further n means the average number of repeating oxyethylene units (90) analysis. Fujioka et al. AAPS Open (2021) 7:2 Page 3 of 9 Optimization of solid-phase extraction for sample the mass spectrometer were as follows: declustering po- preparation of P-bi-TAT in plasma tentials (DP), 130 V; entrance potentials (EP), 10; colli- Fifty microliters of rat and monkey plasma with or with- sion energies (CE), 51 eV; collision cell exit potential out P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, 150 (CXP), 18 V; curtain gas (CUR), 30 psi; gas 1 (GS1, ng/μL) was vortex-mixed with 100 μL4%H PO and nebulizer gas) 30 psi; gas 2 (GS2, heater gas) 50 psi; ion 3 4 loaded on a C18 or an OASIS HLB SPE column spray voltage (IS), 5000 V; temperature (TEM), 400 °C; (Waters, Milford, MA) in triplicate, followed by washing collision activate dissociation (CAD) gas, 10 psi; dwell with 1 mL of 5% methanol in water. P-bi-TAT and IS time, 150 ms. Nitrogen was used for the gases. were eluted with 1 mL of acetonitrile, acetonitrile/water A standard curve was obtained using standard solu- (80/20 v/v), acetonitrile/water (50/50 v/v), and aceto- tions of P-bi-TAT with concentrations of 10.24, 25.6, 64, nitrile/water (20/80 v/v). After centrifugation, solvent 160, 400, and 1000 ng/mL. was evaporated under a nitrogen stream at 50 °C. Extracts were reconstituted with 50 μL of acetonitrile/ Determination of recovery efficient and matrix effect water (80/20 v/v). Recovery efficient was calculated with the response of extracts from spiked plasma sample (sample) and blank Sample preparation of plasma with solid-phase extraction plasma sample (blank) with spike after extract as follows: Fifty microliters of rat and monkey plasma with or with- Recovery efficientðÞ % ¼ Response in sample=Response in blank  100 out P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, 150 ng/μL) was vortex-mixed with 100 μL4%H PO and 3 4 Similarly, matrix effect was calculated with the re- loaded on an OASIS HLB SPE column (Waters, Milford, sponse of extracts from spiked plasma sample (sample) MA), followed by washing with 1 mL of 5% methanol in and standard solution (standard) as follows: water. P-bi-TAT and IS were eluted with 1 mL of aceto- nitrile/water (80/20 v/v). After centrifugation, solvent Matrix effectðÞ % ¼ Response in sample=Response in standard  100 was evaporated under a nitrogen stream at 50 °C. Ex- tracts were reconstituted with 50 μL of acetonitrile/ Determination of limit of quantification (LOQ) and limit of water (80/20 v/v). quantification (LOD) Standard curve was obtained using standard solutions of Sample preparation of brain homogenate with liquid- β-C-TAT with IS. The limit of quantification (LOQ) and liquid extraction the limit of quantification (LOD) was determined using Brain samples were thawed, weighed, and homogenized data of six injections of a standard solution (1000 ng/ with the same volume of ice-cold water with a hand mL). LOQ and LOD were calculated using the standard homogenizer. Rat brain homogenate (100 μL) with or deviations (SD) of signals for the analyte and the slope without P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, of linear regression curve as follows: 150 ng/μL) was vortex-mixed with 1 mL of acetonitrile/ water (80/20 v/v) for 30 min. After centrifugation, solv- LOQ ¼ 10  SD=slope ent was evaporated under a nitrogen stream at 50 °C. Extracts were reconstituted with 50 μL of acetonitrile/ LOD ¼ 3  SD=slope water (80/20 v/v). LC-MS/MS instrumentation Results An API-4000 mass spectrometer (Sciex, Framingham, Development of bioanalytical method for P-bi-TAT MA) equipped with Shimadzu UPLC system (Kyoto, The ionization of P-bi-TAT was studied in positive and Japan) was used for LC-MS/MS analyses. A Kinetex 2.6 negative modes. Deprotonated pseudo-molecular ions 2− μm Biphenyl 100 LS column (50 × 2.1 mm, Phenom- [M-2H] of P-bi-TAT were detected in negative mode enex, Torrance, CA) was used for reversed-phase separ- (Fig. 2). The mean difference in m/z of ions is 22.0, ation. Mobile phases were (A) water containing 0.1% which is corresponding to the half of an ethylene oxide formic acid and 5% acetonitrile and (B) acetonitrile with unit (44 DA). The calculated MW of P-bi-TAT using m/ 0.1% formic acid. The flow rate was 0.5 mL/min and the z of deprotonated pseudo-molecular ions was consistent gradient was linear from 0% B for 1 min to 95% B for 5- with the theoretical values and ranged from 4899.6 (n = 7 min. The column oven temperature was 40 °C and the 74) to 5998.2 (n = 99). The mathematical mean MW of injection volume was 5 μL. Electro-spray ionization (ESI) P-bi-TAT calculated with strong thirteen m/z of depro- was used in positive MRM mode. Mass transitions for tonated pseudo-molecular ions was 5736.0 and is close analytes were as follows: Q1/Q3, 1358.1/582.4 (P-bi- to the theoretical Mw of P-bi-TAT, 5620. The mass TAT); 1402.1/626.4 (IS). The operative parameters of spectrum of P-bi-TAT in negative mode is useful for Fujioka et al. AAPS Open (2021) 7:2 Page 4 of 9 Fig. 2 Mass spectrum of P-bi-TAT in negative ESI scan mode identification of the molecule; however, the intensities of mathematical mean MW of P-bi-TAT calculated with 2− deprotonated molecular ions [M-2H] were very weak, strong thirteen m/z of deprotonated pseudo-molecular so they were not applicable to MRM. ions was 5605.3 and is close to the theoretical Mw of P- When positive mode was applied, multiply protonated bi-TAT, 5620. The region 1 of mass spectrum of P-bi- pseudo-molecular ions and fragmental protonated mo- TAT in positive mode is useful for identification of the lecular ions [MH] of P-bi-TAT were detected by in- molecule; however, the intensities of multiply protonated n+ source CID (Fig. 3). The calculated MW of P-bi-TAT molecular ions [M+nH] were weak, so they were not using m/z of multiply protonated pseudo-molecular ions applicable to MRM. (z = 5) was consistent with the theoretical values and Next, DP was increased from 45 V to 130 V to ranged from 5077.0 (n = 78) to 6130.0 (n = 102). The enhance in-source CID, fragmental protonated Fig. 3 Mass spectrum of P-bi-TAT in positive ESI scan mode Fujioka et al. AAPS Open (2021) 7:2 Page 5 of 9 molecular ions [MH] of P-bi-TAT were dominantly PK and biodistribution of P-bi-TAT in rats and monkeys detected (Fig. 4). The mean difference in m/z of ions The bioanalytical method was applied to PK studies in is 44.0, which is equal to the ethylene oxide unit (44 rats and monkeys. The time course of P-bi-TAT levels da). The fragmental protonated ions ranged from in rat plasma (over 48 h) is shown in Fig. 7 and in mon- 1182.1 (n = 8) to 2415.9 (n = 36). Two of the strong key plasma (on day 1 and day 7) in Fig. 8. Cmax and fragmental protonated ions (m/z 1358, 1402) were area under the curve (AUC) in rats and monkeys (day selected as precursor ions of MRM. MRM 1), PK parameters in rats and monkeys, and the dose optimization was performed for each precursor ion, proportionality ratios of Cmax and AUC are shown in and MS parameters were optimized with flow injec- Table 1. tion analysis (FIA) optimization and an LC-MS/MS Biodistribution of P-bi-TAT in the brain of rats varied method was developed with the biphenyl column. from 7 (low) to 90 (high) ng/g brain weight (Fig. 9). The The MRM was also applied to the IS because of the levels of P-bi-TAT in the brain were dose-dependent. similarity in chemical structures of P-bi-TAT and IS. One of each MRM was used for quantification for P- Discussion bi-TAT and IS, and the other was used as a qualifier Development of bioanalytical method for P-bi-TAT for each. Under these conditions, the retention times P-bi-TAT has both hydrophilic (PEG) and lipophilic for P-bi-TAT and IS were 5.7 and 5.2 min, respect- (triazole tetrac) structures with high affinity for αvβ3 in- ively (Fig. 5). tegrin (Rajabi et al. 2019), resulting in favorable pharma- The LC-MS/MS method for P-bi-TAT with IS was ceutical properties as a targeted anticancer therapeutic. linear from a concentration of 25 to 1000 ng/mL However, it was difficult to develop a quantitative (Fig. 6). The method detection limit (MDL) for P-bi- method in MRM in both positive and negative modes TAT was estimated to be 30 ng/mL under these con- because there are multiple molecular weights of P-bi- ditions. Recovery efficiency was 76% and the matrix TAT and multiple charged ions (n = 1-5) simultaneously effect was 19%. formed by ESI. In the positive mode, it shows multiple- Fig. 4 Full scan mass spectrum (Q1MS) of P-bi-TAT by in-source CID in positive mode Fujioka et al. AAPS Open (2021) 7:2 Page 6 of 9 Fig. 5 Typical LC-MS/MS for P-bi-TAT (5.7 min) and IS (5.2 min) in positive mode charged ions and fragmented ions. Multiple-charged (Sarver et al. 1997). The long half-life in monkeys ions are dominant under the low declustering potential and possibly in humans suggests the prolonged effi- (DP, ~45 V); however, those ions were too weak as a cacy of P-bi-TAT as a targeted anticancer treatment precursor ion to develop an MRM method. The inten- (Miyazaki et al. 2021). sities of fragmented ions showed a maximum under high The dose ratio between low and high was 3.3 in the DP (~130 V), so we were able to develop the MRM monkey study. The results in dose proportionality ratios method under those conditions. The LOQ and recovery for Cmax and AUC in the monkey study suggest that of the bioanalytical method was satisfactory. the bioavailability of P-bi-TAT is dose-dependent within In the negative mode, deprotonated pseudo-molecular the dose range administered. Accumulation of P-bi-TAT 2− ions [M-2H] of P-bi-TAT were not strong enough for in target organs and in off-target organs, such as liver MRM method development. However, they were useful and kidney, needs to be investigated further. for calculating the exact mass of individual P-bi-TAT by The dose-dependent biodistribution of P-bi-TAT in multiplying m/z of the deprotonated ion by z plus z the brain suggests that it is applicable to the therapeutics (Mw = m/z × z = z). of brain cancers. Fernandes and coworkers reported that PEGylated caffeic acid and ferulic acid (MW ~1000) PK and biodistribution of P-bi-TAT in rats and monkeys were permeable across hCMEC/D3 monolayer cells, a The bioanalytical method was successfully applied to the model of the blood-brain barrier, by transcellular passive PK studies in rats and monkeys. Tmax values were the diffusion due to the hydrophobicity (Fernandes et al. same value of 6 h in rats and monkeys. The Cmax and 2018). Yang et al. showed that a PEGylated amifostine AUC values in rats were similar to those in monkeys at had cytoprotective effects in vitro (Yang et al. 2016). low dose. These results of Cmax and AUC suggest that Fleming et al. reported that the chemotherapy drug monkeys may be a better model animal than rats for P- Camptothecin conjugated with PEG3400 was delivered bi-TAT, and higher equivalent doses of P-bi-TAT may into the brain of rats (Fleming et al. 2004). The biodistri- be required for monkeys (and possibly humans) than for bution of P-bi-TAT in the brains in the current study rats to achieve the targeted concentrations in plasma may be attributed to the involvement of transthyretin and tissues. Half-lives (T ) were longer in monkeys (TTR) and thyroid hormone transporters, such as MCTs 1/2 than in rats. Human T was estimated to be 49 h using and OATPs (Bernal et al. 2015; Horn et al. 2013; Kinne 1/2 asimpleallometric formula, Y =a(Xb),where Y is et al. 2010), in addition to passive diffusion because of human T ; X is rat T ;and a and b are constants the high molecular weight of P-bi-TAT (MW > 5000). 1/2 1/2 Fujioka et al. AAPS Open (2021) 7:2 Page 7 of 9 Fig. 6 Standard curve for P-bi-TAT with IS by positive LC-MS/MS Table 1 Parameters in rats (n = 4) and monkeys (n = 2 female and 2 male) dosed with P-bi-TAT Rats Monkeys, Monkeys, Dose † ǂ low dose high dose proportionality ratio Tmax (h) 6 6 7 - Cmax (μg/mL) 225 222 727 3.3 M/F ratio, Cmax - 1.1 1.0 - T (h) 8.1 19.2 14.5 - 1/2 AUC (μg/mL*h) 3564 3649 11733 3.2 M/F ratio, AUC - 1.2 1.3 - Low dose, 25 mg/kg body weight Fig. 7 Time course of plasma levels of P-bi-TAT (10 mg/kg) in rats n High dose, 82.5 mg/kg = 4. Error bars represent standard errors of the mean M/F ratio (AUC) = male/female Fujioka et al. AAPS Open (2021) 7:2 Page 8 of 9 Acknowledgements Special thanks to both NanoPharmaceuticals LLC and from the Pharmaceutical Research Institute for their funding. Special thanks to PRI chemists and biologists for their teamwork. Authors’ contributions Kazutoshi Fujioka contributed to the analytical and bioanalytical method development, validation, and drafted the manuscript; Kavitha Godugu contributed to the animal studies for PK and biodistribution; and Shaker A. Mousa contributed the study design, concepts, data review, supervision, and edits of the manuscript. The authors read and approved the final manuscript. Funding Funding was received from both NanoPharmaceuticals LLC and from the Pharmaceutical Research Institute at Albany College of Pharmacy and Health Sciences. Fig. 8 Time courses of plasma levels of P-bi-TAT in monkeys on Availability of data and materials day 1 and day 7, n = 2 for female and n = 2 for males. Low dose All data are available and stored at Pharmaceutical Research Institute 25 mg/kg body weight, high dose 82.5 mg/kg body weight. Error (PRI)intranet. All materials related to the project are available at PRI laboratories. bars represent standard errors of the mean. Day 1, low dose: closed circles; day 7, low dose: open circles; day 1, high dose: closed squares; Declarations day 7, high dose: open squares Competing interests S.A. Mousa has a patent related to P-bi-TAT and he is a founder of NanoPhar- maceuticals LLC, which is developing anticancer drugs. All other authors de- clare no conflicts of interest. Conclusion The PK parameters and biodistribution of P-bi-TAT in Received: 5 May 2021 Accepted: 7 July 2021 rats and monkeys showed its preferable characteristics as a targeted anticancer pharmaceutical. A quantitative References bioanalytical method for P-bi-TAT was developed using Anthony C, Mladkova-Suchy N, Adamson DC (2019) The evolving role of LC-MS/MS and SPE. 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Biochem Pharmacol 108:11–21. https://doi.org/10.1016/j.bcp.2016.02.014 Yin L, Ren T, Zhao S, Shi M, Gu J (2020) Comparative pharmacokinetic study of PEGylated gemcitabine and gemcitabine in rats by LC-MS/MS coupled with pre-column derivatization and MSALL technique. Talanta 206:120184. https://doi.org/10.1016/j.talanta.2019.120184 Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AAPS Open Springer Journals

Pharmacokinetics and biodistribution of a novel anticancer thyrointegrin αvβ3 antagonist: triazole modified tetraiodothyroacetic acid conjugated to polyethylene glycol (P-bi-TAT)

AAPS Open , Volume 7 (1) – Aug 16, 2021

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Abstract

We previously developed a triazole modified tetraiodothyroacetic acid (TAT) conjugated to a polyethylene glycol (PEG)-based thyrointegrin αvβ3 antagonist targeted compound, called P-bi-TAT. It exhibited potent anti-angiogenic and anticancer activities in vivo. The objective of the current study is to develop a quantitative bioanalytical method for P-bi-TAT using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to elucidate pharmacokinetics (PK) and biodistribution of P-bi-TAT in animals. We used in-source collision-induced dissociation (CID) for ionization of P-bi-TAT in the positive mode, followed by multiple reaction monitoring (MRM) for quantification. P-bi-TAT was quantified using P-mono-TAT as an internal standard because of its similarity in structure and physicochemical properties to P-bi-TAT. The LOQ for P-bi-TAT was 30 ng/μL and the recovery efficiency was 76% with the developed method. Cmax and AUC results at different doses (1, 3, 10 mg/kg) in rats suggest that P-bi-TAT is dose-dependent within the range administered. Results for Cmax and AUC in monkeys at a low dose (25 mg/kg) were comparable to those in rats. Biodistribution of subcutaneously administered P-bi-TAT in the brain of rats ranged from 7.90 to 88.7 ng/g brain weight, and levels of P-bi-TAT in the brain were dose- dependent. The results suggest that P-bi-TAT is a potential candidate as a molecular-targeted anticancer therapeutic with blood-brain barrier permeability and acceptable PK parameters. Its accumulation in organs, toxicokinetic, and pharmacodynamics needs to be further investigated. Keywords: PEGylated, Triazole tetrac, Integrin αvβ3 antagonist, Pharmacokinetic, Toxicokinetic, LC-MS/MS, Anti- angiogenic, Blood-brain barrier Introduction difficult to cure because of its poor prognosis, and the Cancer was the second leading cause of death in the blood-brain barrier and blood tumor barrier that restrict USA in 2018, after heart disease (Xu et al. 2020). It is the delivery of therapeutics (Fox et al. 2019). There are not a single disease but a variety of different cancer types FDA-approved anticancer agents against brain cancers including myeloma, thyroid, leukemia, esophagus, non- such as temozolomide; however, their efficacies are not Hodgkin lymphoma, stomach, and prostate cancer enough to stop brain tumor progression beside their (Henley et al. 2020). Among cancers, brain cancer is significant adverse effects (Anthony et al. 2019). Thus, more efficient, and safer anticancer therapeutics and improved drug delivery systems are under investigation * Correspondence: shaker.mousa@acphs.edu Pharmaceutical Research Institute, Albany College of Pharmacy and Health in the pharmaceutical industry and in academia. Sciences, 1 Discovery Drive, Rensselaer, NY 12144, USA © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Fujioka et al. AAPS Open (2021) 7:2 Page 2 of 9 ALL Targeted anticancer drugs have been developed and column derivatization and MS technique and ap- applied to multiple cancer types, such as glioblastoma plied to PK study of PEGylated gemcitabine in rats and neuroblastoma (Laquintana et al. 2009; Le Rhun (Yin et al. 2020). In this study, we used in-source et al. 2019; Maeda et al. 2016). Among them, collision-induced dissociation (CID) for ionization of nanoparticle-conjugated compounds show selective effi- P-bi-TAT, followed by multiple reaction monitoring cacy and potency to tumors via the enhanced permeabil- (MRM) (Warrack et al. 2013). P-mono-TAT (2, Fig. ity retention (EPR) effect (Maeda et al. 2016). We 1), which is another conjugate of PEG4000 with one previously synthesized a conjugate of tetraiodothyroace- TAT moiety, was used as an internal standard (IS) tic acid (tetrac) and poly (lactic-co-glycolic acid) (PLGA) because its structure and physicochemical properties polymer, PLGA-diamino-propane-tetraiodothyroacetic are similar to P-bi-TAT. We also optimized the acid (DAT), and nanoparticles of DAT (NDAT) showed extraction procedure from biological matrices using high potency and efficacy on multiple cancers, such as solid-phase extraction (SPE) with modification of the glioblastoma and neuroblastoma (Lin et al. 2016; Lin developed method for CTAT. et al. 2018; Sudha et al. 2017a). We then developed a conjugate of β-cyclodextrin with triazole-modified- Experimental tetraiodothyroacetic acid (TAT), named β-C-TAT Materials (CTAT). It showed anti-angiogenic in a chicken embryo Chemicals and biologicals chorioallantoic membrane (CAM) assay and anticancer P-bi-TAT and P-mono-TAT were synthesized and puri- activities in human primary GBM cells and in glioblast- fied in our laboratory. Acetonitrile, methanol, water, and oma xenografted mice (unpublished data). Encouraged formic acid were LC-MS grade and purchased from by the results, we then synthesized a polyethylene glycol Sigma-Aldrich (St. Louis, MO). o-Phosphoric acid (85%) (PEG)-based targeted drug, P-bi-TAT (1, Fig. 1), which was HPLC grade and purchased from Fisher Scientific is a conjugate of PEG4000 with two TAT moieties (Pittsburgh, PA). Rat and monkey plasma were pur- (Sudha et al. 2017b; Rajabi et al. 2019). It showed high chased from Bioreclamation IVT (New York, NY). Rats affinity with a receptor on integrin αvβ3 (IC 0.14 nM), were purchased from Charles River (Kingston, NY). anti-angiogenic activity in a CAM assay, and anticancer activities in human primary GBM cells and in glioblast- oma xenografted mice (10 mg/kg) (Rajabi et al. 2019). In the current work, we developed a bioanalytical method Methods for P-bi-TAT to determine its pharmacokinetics (PK) P-bi-TAT-treatment of rats and monkeys and biodistribution in experimental animals, i.e., rats P-bi-TAT-treatment of rats was carried out in the ani- and monkeys. mal facility of the Veterans Affairs (VA) Medical Center Liquid chromatography-tandem mass spectrometry (Albany, NY), and the experimental protocol was ap- (LC-MS/MS) is a gold standard for quantification of proved by the Institutional Animal Care and Use Com- drug molecules. However, typical quadrupole-type mittee of the VA. Rats were housed under controlled mass spectrometers are not applicable for P-bi-TAT conditions (temperature, 20–24 °C; humidity, 60–70%). because its molecular weight is more than 3000, and Rats were allowed to acclimatize for at least 1 week prior the quantification of high molecular weight PEGylated to the start of treatments. For the PK study, P-bi-TAT drug conjugates is a challenge by mass spectrometry. (10 mg/kg body weight) was administered subcutane- Yin et al. reported a quantitative method for PEGy- ously to 4 rats, and plasma was collected at 0.25, 0.5, 1.5, lated gemcitabine (Mw 70-76 kDa) using LC-time of 3, 6, 12, 24, and 48 h. Plasma samples were stored at flight mass spectrometer (TOFMS) coupled with pre- −80 °C for further analysis. For the biodistribution study, P-bi-TAT (1, 3, 10 mg/kg) was administered subcutane- ously to 4 rats once a day for 3 weeks, and brain samples were collected 24 h after the last dose. Brain samples were stored at −80 °C for further analysis. The monkey study was conducted by a contract re- search organization. Briefly, P-bi-TAT (low dose, 25 mg/ kg body weight; high dose, 82.5 mg/kg) was adminis- tered subcutaneously to male and female cynomolgus monkeys (n = 2 each) daily for 7 days, and plasma was collected at 0.5, 2, 6, 10, 16, and 24 h on day 1 and day Fig. 1 Chemical structures of P-bi-TAT (1) and P-mono-TAT (2); and 7. Plasma samples were stored at −80 °C for further n means the average number of repeating oxyethylene units (90) analysis. Fujioka et al. AAPS Open (2021) 7:2 Page 3 of 9 Optimization of solid-phase extraction for sample the mass spectrometer were as follows: declustering po- preparation of P-bi-TAT in plasma tentials (DP), 130 V; entrance potentials (EP), 10; colli- Fifty microliters of rat and monkey plasma with or with- sion energies (CE), 51 eV; collision cell exit potential out P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, 150 (CXP), 18 V; curtain gas (CUR), 30 psi; gas 1 (GS1, ng/μL) was vortex-mixed with 100 μL4%H PO and nebulizer gas) 30 psi; gas 2 (GS2, heater gas) 50 psi; ion 3 4 loaded on a C18 or an OASIS HLB SPE column spray voltage (IS), 5000 V; temperature (TEM), 400 °C; (Waters, Milford, MA) in triplicate, followed by washing collision activate dissociation (CAD) gas, 10 psi; dwell with 1 mL of 5% methanol in water. P-bi-TAT and IS time, 150 ms. Nitrogen was used for the gases. were eluted with 1 mL of acetonitrile, acetonitrile/water A standard curve was obtained using standard solu- (80/20 v/v), acetonitrile/water (50/50 v/v), and aceto- tions of P-bi-TAT with concentrations of 10.24, 25.6, 64, nitrile/water (20/80 v/v). After centrifugation, solvent 160, 400, and 1000 ng/mL. was evaporated under a nitrogen stream at 50 °C. Extracts were reconstituted with 50 μL of acetonitrile/ Determination of recovery efficient and matrix effect water (80/20 v/v). Recovery efficient was calculated with the response of extracts from spiked plasma sample (sample) and blank Sample preparation of plasma with solid-phase extraction plasma sample (blank) with spike after extract as follows: Fifty microliters of rat and monkey plasma with or with- Recovery efficientðÞ % ¼ Response in sample=Response in blank  100 out P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, 150 ng/μL) was vortex-mixed with 100 μL4%H PO and 3 4 Similarly, matrix effect was calculated with the re- loaded on an OASIS HLB SPE column (Waters, Milford, sponse of extracts from spiked plasma sample (sample) MA), followed by washing with 1 mL of 5% methanol in and standard solution (standard) as follows: water. P-bi-TAT and IS were eluted with 1 mL of aceto- nitrile/water (80/20 v/v). After centrifugation, solvent Matrix effectðÞ % ¼ Response in sample=Response in standard  100 was evaporated under a nitrogen stream at 50 °C. Ex- tracts were reconstituted with 50 μL of acetonitrile/ Determination of limit of quantification (LOQ) and limit of water (80/20 v/v). quantification (LOD) Standard curve was obtained using standard solutions of Sample preparation of brain homogenate with liquid- β-C-TAT with IS. The limit of quantification (LOQ) and liquid extraction the limit of quantification (LOD) was determined using Brain samples were thawed, weighed, and homogenized data of six injections of a standard solution (1000 ng/ with the same volume of ice-cold water with a hand mL). LOQ and LOD were calculated using the standard homogenizer. Rat brain homogenate (100 μL) with or deviations (SD) of signals for the analyte and the slope without P-bi-TAT (150 ng/μL) and IS (P-mono-TAT, of linear regression curve as follows: 150 ng/μL) was vortex-mixed with 1 mL of acetonitrile/ water (80/20 v/v) for 30 min. After centrifugation, solv- LOQ ¼ 10  SD=slope ent was evaporated under a nitrogen stream at 50 °C. Extracts were reconstituted with 50 μL of acetonitrile/ LOD ¼ 3  SD=slope water (80/20 v/v). LC-MS/MS instrumentation Results An API-4000 mass spectrometer (Sciex, Framingham, Development of bioanalytical method for P-bi-TAT MA) equipped with Shimadzu UPLC system (Kyoto, The ionization of P-bi-TAT was studied in positive and Japan) was used for LC-MS/MS analyses. A Kinetex 2.6 negative modes. Deprotonated pseudo-molecular ions 2− μm Biphenyl 100 LS column (50 × 2.1 mm, Phenom- [M-2H] of P-bi-TAT were detected in negative mode enex, Torrance, CA) was used for reversed-phase separ- (Fig. 2). The mean difference in m/z of ions is 22.0, ation. Mobile phases were (A) water containing 0.1% which is corresponding to the half of an ethylene oxide formic acid and 5% acetonitrile and (B) acetonitrile with unit (44 DA). The calculated MW of P-bi-TAT using m/ 0.1% formic acid. The flow rate was 0.5 mL/min and the z of deprotonated pseudo-molecular ions was consistent gradient was linear from 0% B for 1 min to 95% B for 5- with the theoretical values and ranged from 4899.6 (n = 7 min. The column oven temperature was 40 °C and the 74) to 5998.2 (n = 99). The mathematical mean MW of injection volume was 5 μL. Electro-spray ionization (ESI) P-bi-TAT calculated with strong thirteen m/z of depro- was used in positive MRM mode. Mass transitions for tonated pseudo-molecular ions was 5736.0 and is close analytes were as follows: Q1/Q3, 1358.1/582.4 (P-bi- to the theoretical Mw of P-bi-TAT, 5620. The mass TAT); 1402.1/626.4 (IS). The operative parameters of spectrum of P-bi-TAT in negative mode is useful for Fujioka et al. AAPS Open (2021) 7:2 Page 4 of 9 Fig. 2 Mass spectrum of P-bi-TAT in negative ESI scan mode identification of the molecule; however, the intensities of mathematical mean MW of P-bi-TAT calculated with 2− deprotonated molecular ions [M-2H] were very weak, strong thirteen m/z of deprotonated pseudo-molecular so they were not applicable to MRM. ions was 5605.3 and is close to the theoretical Mw of P- When positive mode was applied, multiply protonated bi-TAT, 5620. The region 1 of mass spectrum of P-bi- pseudo-molecular ions and fragmental protonated mo- TAT in positive mode is useful for identification of the lecular ions [MH] of P-bi-TAT were detected by in- molecule; however, the intensities of multiply protonated n+ source CID (Fig. 3). The calculated MW of P-bi-TAT molecular ions [M+nH] were weak, so they were not using m/z of multiply protonated pseudo-molecular ions applicable to MRM. (z = 5) was consistent with the theoretical values and Next, DP was increased from 45 V to 130 V to ranged from 5077.0 (n = 78) to 6130.0 (n = 102). The enhance in-source CID, fragmental protonated Fig. 3 Mass spectrum of P-bi-TAT in positive ESI scan mode Fujioka et al. AAPS Open (2021) 7:2 Page 5 of 9 molecular ions [MH] of P-bi-TAT were dominantly PK and biodistribution of P-bi-TAT in rats and monkeys detected (Fig. 4). The mean difference in m/z of ions The bioanalytical method was applied to PK studies in is 44.0, which is equal to the ethylene oxide unit (44 rats and monkeys. The time course of P-bi-TAT levels da). The fragmental protonated ions ranged from in rat plasma (over 48 h) is shown in Fig. 7 and in mon- 1182.1 (n = 8) to 2415.9 (n = 36). Two of the strong key plasma (on day 1 and day 7) in Fig. 8. Cmax and fragmental protonated ions (m/z 1358, 1402) were area under the curve (AUC) in rats and monkeys (day selected as precursor ions of MRM. MRM 1), PK parameters in rats and monkeys, and the dose optimization was performed for each precursor ion, proportionality ratios of Cmax and AUC are shown in and MS parameters were optimized with flow injec- Table 1. tion analysis (FIA) optimization and an LC-MS/MS Biodistribution of P-bi-TAT in the brain of rats varied method was developed with the biphenyl column. from 7 (low) to 90 (high) ng/g brain weight (Fig. 9). The The MRM was also applied to the IS because of the levels of P-bi-TAT in the brain were dose-dependent. similarity in chemical structures of P-bi-TAT and IS. One of each MRM was used for quantification for P- Discussion bi-TAT and IS, and the other was used as a qualifier Development of bioanalytical method for P-bi-TAT for each. Under these conditions, the retention times P-bi-TAT has both hydrophilic (PEG) and lipophilic for P-bi-TAT and IS were 5.7 and 5.2 min, respect- (triazole tetrac) structures with high affinity for αvβ3 in- ively (Fig. 5). tegrin (Rajabi et al. 2019), resulting in favorable pharma- The LC-MS/MS method for P-bi-TAT with IS was ceutical properties as a targeted anticancer therapeutic. linear from a concentration of 25 to 1000 ng/mL However, it was difficult to develop a quantitative (Fig. 6). The method detection limit (MDL) for P-bi- method in MRM in both positive and negative modes TAT was estimated to be 30 ng/mL under these con- because there are multiple molecular weights of P-bi- ditions. Recovery efficiency was 76% and the matrix TAT and multiple charged ions (n = 1-5) simultaneously effect was 19%. formed by ESI. In the positive mode, it shows multiple- Fig. 4 Full scan mass spectrum (Q1MS) of P-bi-TAT by in-source CID in positive mode Fujioka et al. AAPS Open (2021) 7:2 Page 6 of 9 Fig. 5 Typical LC-MS/MS for P-bi-TAT (5.7 min) and IS (5.2 min) in positive mode charged ions and fragmented ions. Multiple-charged (Sarver et al. 1997). The long half-life in monkeys ions are dominant under the low declustering potential and possibly in humans suggests the prolonged effi- (DP, ~45 V); however, those ions were too weak as a cacy of P-bi-TAT as a targeted anticancer treatment precursor ion to develop an MRM method. The inten- (Miyazaki et al. 2021). sities of fragmented ions showed a maximum under high The dose ratio between low and high was 3.3 in the DP (~130 V), so we were able to develop the MRM monkey study. The results in dose proportionality ratios method under those conditions. The LOQ and recovery for Cmax and AUC in the monkey study suggest that of the bioanalytical method was satisfactory. the bioavailability of P-bi-TAT is dose-dependent within In the negative mode, deprotonated pseudo-molecular the dose range administered. Accumulation of P-bi-TAT 2− ions [M-2H] of P-bi-TAT were not strong enough for in target organs and in off-target organs, such as liver MRM method development. However, they were useful and kidney, needs to be investigated further. for calculating the exact mass of individual P-bi-TAT by The dose-dependent biodistribution of P-bi-TAT in multiplying m/z of the deprotonated ion by z plus z the brain suggests that it is applicable to the therapeutics (Mw = m/z × z = z). of brain cancers. Fernandes and coworkers reported that PEGylated caffeic acid and ferulic acid (MW ~1000) PK and biodistribution of P-bi-TAT in rats and monkeys were permeable across hCMEC/D3 monolayer cells, a The bioanalytical method was successfully applied to the model of the blood-brain barrier, by transcellular passive PK studies in rats and monkeys. Tmax values were the diffusion due to the hydrophobicity (Fernandes et al. same value of 6 h in rats and monkeys. The Cmax and 2018). Yang et al. showed that a PEGylated amifostine AUC values in rats were similar to those in monkeys at had cytoprotective effects in vitro (Yang et al. 2016). low dose. These results of Cmax and AUC suggest that Fleming et al. reported that the chemotherapy drug monkeys may be a better model animal than rats for P- Camptothecin conjugated with PEG3400 was delivered bi-TAT, and higher equivalent doses of P-bi-TAT may into the brain of rats (Fleming et al. 2004). The biodistri- be required for monkeys (and possibly humans) than for bution of P-bi-TAT in the brains in the current study rats to achieve the targeted concentrations in plasma may be attributed to the involvement of transthyretin and tissues. Half-lives (T ) were longer in monkeys (TTR) and thyroid hormone transporters, such as MCTs 1/2 than in rats. Human T was estimated to be 49 h using and OATPs (Bernal et al. 2015; Horn et al. 2013; Kinne 1/2 asimpleallometric formula, Y =a(Xb),where Y is et al. 2010), in addition to passive diffusion because of human T ; X is rat T ;and a and b are constants the high molecular weight of P-bi-TAT (MW > 5000). 1/2 1/2 Fujioka et al. AAPS Open (2021) 7:2 Page 7 of 9 Fig. 6 Standard curve for P-bi-TAT with IS by positive LC-MS/MS Table 1 Parameters in rats (n = 4) and monkeys (n = 2 female and 2 male) dosed with P-bi-TAT Rats Monkeys, Monkeys, Dose † ǂ low dose high dose proportionality ratio Tmax (h) 6 6 7 - Cmax (μg/mL) 225 222 727 3.3 M/F ratio, Cmax - 1.1 1.0 - T (h) 8.1 19.2 14.5 - 1/2 AUC (μg/mL*h) 3564 3649 11733 3.2 M/F ratio, AUC - 1.2 1.3 - Low dose, 25 mg/kg body weight Fig. 7 Time course of plasma levels of P-bi-TAT (10 mg/kg) in rats n High dose, 82.5 mg/kg = 4. Error bars represent standard errors of the mean M/F ratio (AUC) = male/female Fujioka et al. AAPS Open (2021) 7:2 Page 8 of 9 Acknowledgements Special thanks to both NanoPharmaceuticals LLC and from the Pharmaceutical Research Institute for their funding. Special thanks to PRI chemists and biologists for their teamwork. Authors’ contributions Kazutoshi Fujioka contributed to the analytical and bioanalytical method development, validation, and drafted the manuscript; Kavitha Godugu contributed to the animal studies for PK and biodistribution; and Shaker A. Mousa contributed the study design, concepts, data review, supervision, and edits of the manuscript. The authors read and approved the final manuscript. Funding Funding was received from both NanoPharmaceuticals LLC and from the Pharmaceutical Research Institute at Albany College of Pharmacy and Health Sciences. Fig. 8 Time courses of plasma levels of P-bi-TAT in monkeys on Availability of data and materials day 1 and day 7, n = 2 for female and n = 2 for males. Low dose All data are available and stored at Pharmaceutical Research Institute 25 mg/kg body weight, high dose 82.5 mg/kg body weight. Error (PRI)intranet. All materials related to the project are available at PRI laboratories. bars represent standard errors of the mean. Day 1, low dose: closed circles; day 7, low dose: open circles; day 1, high dose: closed squares; Declarations day 7, high dose: open squares Competing interests S.A. Mousa has a patent related to P-bi-TAT and he is a founder of NanoPhar- maceuticals LLC, which is developing anticancer drugs. All other authors de- clare no conflicts of interest. Conclusion The PK parameters and biodistribution of P-bi-TAT in Received: 5 May 2021 Accepted: 7 July 2021 rats and monkeys showed its preferable characteristics as a targeted anticancer pharmaceutical. A quantitative References bioanalytical method for P-bi-TAT was developed using Anthony C, Mladkova-Suchy N, Adamson DC (2019) The evolving role of LC-MS/MS and SPE. 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Journal

AAPS OpenSpringer Journals

Published: Aug 16, 2021

Keywords: PEGylated; Triazole tetrac; Integrin αvβ3 antagonist; Pharmacokinetic; Toxicokinetic; LC-MS/MS; Anti-angiogenic; Blood-brain barrier

References