Abstract
JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50 μL plasma was followed by isocratic reverse phase chromatography for a 6 min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1–1000 ng/mL with appropriate accuracy (97.1–107.6%) and precision (3.7–12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2–136.1% with moderate ionization enhancement (10.9–39.5%). Plasma freeze-thaw stability (98.8–104.2%), stability for 13.5 months at −80 °C (93.1–105.6%), and stability for 4 h at room temperature (94.2–97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20 mg/kg JP4-039 and will be extended to other studies of this agent.
Keywords: JP4-039, nitroxide, oxoammonium cation, hydroxylamine species, tandem mass spectrometry, pharmacokinetics, assay, validation, mice, radiation mitigation, radiation protection
Graphical Abstract
INTRODUCTION
Ionizing irradiation results in the generation of oxidative stress in both its acute and chronic late effects. JP4-039 is the lead compound of a promising class of mitochondrial targeting nitroxides (structure provided in Fig. 1). These compounds were designed to accumulate in mitochondria and neutralize free radicals and electrons escaping from the electron transfer chain by cycling from nitroxide to hydroxylamine and, depending on the redox environment, to an oxoammonium species, thereby reducing free radicals and counteracting oxidative stress generated after ionizing radiation exposure. The oxoammonium cation is highly oxidizing and therefore unlikely to exist for long in biologicals samples if generated [1]. There is strong evidence that JP4-039 can prevent the acute effects of ionizing irradiation and mitigate chronic effects of ionizing irradiation when administered after irradiation [2–8]. Thus, targeted nitroxides such as JP4-039 could be used to protect normal tissue during clinical radiotherapy as well as to serve as a countermeasure to mitigate damage after radiation exposure. In this paper, we report a method to quantitate JP4-039, validated to FDA guidance in murine plasma and applied to murine plasma and tissue homogenates, and present the pharmacokinetics and tissue distribution of JP4-039 after administration of an effective dose of 20 mg/kg JP4-039 to male C57BL/6 mice.
Fig. 1.
Structures of JP4-039 in nitroxide, hydroxylamine and oxoammonium cation forms, and [D17]-JP4-039 internal standard in nitroxide form. Fragmentation sites leading to the m/z values monitored are indicated.
EXPERIMENTAL
Chemicals and reagents
JP4-039 and [D17]-JP4-039 were synthesized in the laboratory of Dr. Peter Wipf (mainly in the nitroxide form). DMSO, methanol, and water (all HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Sodium Acetate was purchased from Mallinckrodt Pharmaceuticals (St Louis, MO). Control mouse plasma was purchased from Lampire Biological Labratories (Piperville, PA, USA). Nitrogen for mass spectrometrical applications was purified with a Parker Balston Nitrogen Generator (Parker Balston, Haverhill, MA, USA). Cremophor EL and EDTA were purchased from Sigma–Aldrich (St Louis, MO). Sterile saline (0.154 M NaCl), sterile water, and phosphate-buffered saline (PBS) were purchased from Baxter Healthcare Corporation (Deerfield, IL). Ethanol (200 Proof) was purchased from Pharmaco Products (Brookfield, CT).
Chromatography
The LC system consisted of an Agilent (Palo Alto, CA, USA) 1200 SL autosampler and binary pump, a Phenomenex (Torrance, CA USA) Synergi Hydro-RP (4 μm, 100 × 2 mm) column, and a gradient mobile phase. Mobile phase solvent A was methanol, and mobile phase solvent B was 0.1 μM sodium acetate in water. The initial mobile phase composition is 75% solvent A pumped at 0.3 mL/min and held for a total run time of 6 min.
Mass spectrometry
Mass spectrometric detection was carried out using an ABI SCIEX (Concord, ON, Canada) 4000Q hybrid linear ion trap tandem mass spectrometer with electrospray ionization in positive multiple reaction monitoring (MRM) mode. The settings of the mass spectrometer were as follows: curtain gas 50, CAD 10 Ion transfer voltage 5000 V, probe temperature 550 °C, GS1 40, GS2 40, declustering potential 50 V, entrance potential 10 V collision energy 30 V, and collision cell exit potential 10 V. The MRM m/z transitions monitored were: 447.0>332.0 for JP4-039-Na+; 464.5>408.5 for [D17]-JP4-039-Na+. The LC system and mass spectrometer were controlled by Analyst software (version 1.4.2), and data were collected with the same software. The analyte-to-internal standard ratio was calculated for each standard by dividing the area of each analyte peak by the area of the respective internal standard peak for that sample. Standard curves of the analytes were constructed by plotting the analyte-to-internal standard ratio versus the known concentration of analyte in each sample. Standard curves were fit by linear regression with weighting by 1/y2, followed by back calculation of concentrations.
Preparation of calibration standards and quality control samples
Stock solutions were prepared independently at 1 mg/mL in DMSO and stored at −80 °C. JP4-039 was diluted 10-fold in methanol to obtain a mixture working stock of 0.1 mg/mL and also stored at −80 °C. On the day of assay, these solutions were serially diluted (in steps of 10-fold) in methanol to obtain the lower calibration working solutions. These calibration working solutions were diluted in mouse plasma to produce the concentrations: 1, 3, 10, 30, 100, 300, 1000 ng/mL. For each calibration series, zero and blank samples were also prepared from 50 μL of control plasma. Quality control (QC) stock solutions were prepared independently and stored at −80 °C. These solutions were diluted in murine plasma to produce the following QC samples of either: Lower Limit of Quantitation (LLOQ) 1 ng/mL, QC Low (QCL) 2 ng/mL; QC Mid (QCM) 50 ng/mL, and QC High (QCH) 800 ng/mL.
Sample preparation
To each microcentrifuge tube, 50 μL of each sample (standard, QC, sample plasma, or homogenate of 3 parts PBS to each part tissue (v/g)), 10 μL of 1 μg/mL [D17]-JP4-039 and 300 μL methanol were added and vortexed for 1 min on a Vortex Genie-2 set at 8 (Model G-560 Scientific Industries, Bohemia, NY, USA). Samples were centrifuged at 12,000 × g at room temperature for 5 min. An aliquot of 100 μL of the resulting supernatant was transferred to an autosampler vial that contained 10 μL of 1 mg/mL potassium ferricyanide in water, followed by brief vortexing and injection of 10 μL into the LC-MS/MS system. Study mouse plasma or tissue homogenate samples that exceeded our upper limit of quantitation were diluted in control mouse plasma to within the calibration range. To quantitate JP4-039 in bone marrow cell pellet, we added 200 μL of PBS and sonicated for 15 min followed by a brief vortex. A 50 μL aliquot was taken and processed the same as plasma and tissue.
Validation procedures
Calibration curve and LLOQ, accuracy and precision, selectivity and specificity, extraction recovery and matrix effect, stability, and dilutional integrity were studied as described before [9].
Application of the assay
To cross-validate our heparin plasma based assay to EDTA plasma from study mice and tissue homogenates, plasma calibration curves were used to quantitate JP4-039 spiked to such sample matrices in duplicate at the 3 QC concentrations in male and female tissues separately.
To document the applicability of the assay, we evaluated the pharmacokinetics of JP4-039 in plasma and tissues after administration of a 20 mg/kg IV dose of JP4-039 to male C57B/6 mice. C57BL/6 mice (male, 4–6 weeks of age, SPF) were purchased from Taconic Biosciences (Germantown, NY) and allowed to acclimate to the Hillman Cancer Center Animal Facility for 1 week prior to use as previously described [10]. Mice were housed in microisolator cages, allowed access to food and water ad libitum, and handled on a protocol approved by the University of Pittsburgh IACUC, Rooms were controlled on a 12 h light dark cycle, temperature was maintained between 72 ±6 °F and at least 12 air changes/h.
Mice (N=3/time point) were dosed with 20 mg/kg JP4-039 in the vehicle (1:1:8 cremophor:ethanol:saline (v/v/v)) or with vehicle only and euthanized by CO2 inhalation at the following time points after a 30 s IV bolus administration: 5, 10, 15, 30 min, 1, 2, 3, 4, 6, 16, 24, and 48 h, and at 5 min, and 24 and 48 h after vehicle administration (0.01 mL/g exact body weight). Blood was collected by cardiac puncture with EDTA flushed syringes and plasma was isolated by centrifugation at 12,000 × g for 4 min. The following tissues were collected, weighed, snap frozen in liquid N2 and stored at −80 °C until analyzed: liver, kidneys, spleen, heart, lungs, brain, skeletal muscle, abdominal fat, and small intestines (contents were removed by flushing with 3 ml of PBS). Bone marrow was obtained by flushing both femurs from each mouse with PBS. The bone marrow from each mouse was isolated by centrifugation at 12,000 × g and the supernatant was removed except for approximately 50 μL. Prior to analysis of the bone marrow, the bone marrow was resuspended, sonicated and a 20 μl aliquot was taken for determination of protein using the Bio-Rad Protein assay following the manufacturer’s instructions with bovine serum albumin as the standard. Pharmacokinetic parameters were derived from the average concentration values per time point (N=3 per time point) by non-compartmental modeling using PK Solutions 2.0 (Summit Research Services, Montrose, CO; www.summitPK.com).
RESULTS AND DISCUSSION
Validation of the assay
Chromatography
The approximate retention times of JP4-039 and [D17]-JP4-039 were both at 3.9, corresponding to a retention factor of 2.9, with a void time of 1 min. Representative chromatograms of JP4-039 (at the LLOQ), and internal standard in plasma are displayed in Fig. 2.
Fig. 2.
Representative chromatograms of: A) JP4-039 (m/z 447.0>332.0; 4.1 min) added to control plasma at the LLOQ concentration of 1 ng/mL (top trace with an offset of 200 counts) and control plasma (bottom trace); B) [D17]-JP4-039 internal standard (m/z 464.5>408.5; 4.0 min) added to control plasma at a concentration of 200 ng/mL (top trace with an offset of 15000 counts) and control plasma (bottom trace with an offset of 5000 counts). All masses are sodium adducts.
Calibration curve and LLOQ
The selected assay range of 1–1000 ng/mL fulfilled the FDA criteria for the LLOQ concentration and the calibration curve [11]. Accuracies and precisions at the different concentrations determined from triplicate calibration curves on 3 separate days are reported in Error! Reference source not found.. At some concentrations, the mean square of the within runs was greater than the mean square of the between runs, indicating that there was no significant additional variability due to the performance of the assay in different runs [12]. A representative calibration curve and corresponding correlation and regression coefficients are shown in Fig. 3.
Fig. 3.
Representative calibration curve (N=3 for each concentration) used to quantitate JP4-039 murine plasma samples (response JP4-039 = 0.00635•conc + 0.000163; R2=0.9972). Calibration curves are depicted as response ratio versus nominal concentration (A), and as % residuals of the back-calculated relative to the nominal concentrations versus the log transformed concentration (B), the log-transformation for visual purposes.
Accuracy and precision
FDA guidance specify that the accuracies for all tested concentrations should be within ±15%, and the precisions should not be > 15% CV except for the LLOQ, in which case these parameters should not exceed 20% [11].
The accuracies and intra- and inter-assay precisions for the tested concentrations (LLOQ, QCL, QCM, QCH) were all within the defined acceptance criteria (Error! Reference source not found.).
Selectivity and specificity
According to FDA guidance, the signal at the LLOQ must be at least 5 times the signal of any co-eluting peaks [11]. Chromatograms of six individual mouse control plasma samples contained no co-eluting peaks >20% of the analyte areas at the LLOQ concentration. Mean interference relative to the LLOQ for JP4-039 was 5.1%.
Cross-talk calculations were performed and revealed that cross talk of JP4-039 into the [D17]-JP4-039 channel was <0.75%, and cross talk of [D17]-JP4-039 into the JP4-039 channel was <0.04% (at an applied concentration of 200 ng/mL, this corresponds to <8% of the LLOQ level), indicating cross talk does not appear to be a major issue.
Extraction recovery and matrix effect
A recovery of ≥70% with a variation of 15% is generally accepted [11, 12], and a large and/or variable ion-suppression may result in lack of assay robustness.
The recoveries of JP4-039 ranged from 77.2% to 136.1%, with a CV <23%. Matrix effect ranged from 10.9 to 39.5% with a CV <21% (Error! Reference source not found.), suggesting signal enhancement in the presence of matrix.
Stability
Stability in biological samples is acceptable when ≥85% of the analyte is recovered. The stability of JP4-039 stock solutions at room temperature for 4 h was 104.0% (Table 1). Stability in stock solution for 6 months at −80 °C was 96.0%. The stability of the JP4-039 after 3 freeze thaw cycles (−80°C to RT) were between 98.8 and 104.2%. Long-term stabilities of the analytes in plasma at −80 °C for 13.5 months were adequate with recoveries between 93.1 and 105.6%. The absolute responses of plasma extracts of JP4-039 at the quality control concentrations, when reconstituted and kept in the autosampler for 72 h, were 94.3 to 108.7% of the initial responses (CV 4.3–13.0%).
Table 1.
Stability of JP4-039 stock and mouse plasma under varying conditions.
| Storage Condition | Concentration (ng/mL) | Stability (%) | CV (%) | Replicates |
|---|---|---|---|---|
|
| ||||
| Stock Solution | ||||
| 4 h ambient temp | 100 | 104.0 | 11.2 | 3 |
| 6 months −80°C | 200 | 96.0 | 7.8 | 6 |
| Plasma | ||||
| 4 h ambient temp | 2 (QCL) | 94.2 | 6.1 | 4 |
| 50 (QCM) | 96.2 | 4.9 | 4 | |
| 800 (QCH) | 97.6 | 2.2 | 4 | |
| 3 freeze-thaw cycles −80°C | 2 (QCL) | 98.8 | 3.1 | 6 |
| 50 (QCM) | 104.2 | 5.0 | 6 | |
| 800 (QCH) | 99.1 | 3.4 | 6 | |
| 13.5 months at −80°C | 2 (QCL) | 105.6 | 7.7 | 5 |
| 50 (QCM) | 96.5 | 6.7 | 5 | |
| 800 (QCH) | 93.1 | 4.2 | 5 | |
Dilutional Integrity
The samples diluted from 5,000 ng/mL to 1,000 ng/mL displayed an accuracy of 102.7%, with a CV of 1.5%.
Development
Because JP4-039 and most nitroxides can exist as three redox species (hydroxylamine, nitroxide, and oxoammonium species) [1], we initially attempted to develop an assay that would quantitate each separately. We could readily observe the hydroxylamine and nitroxide species, not the oxoammonium cation. Ultimately, we chose to quantitate JP4-039 as one species after driving the redox equilibrium to the nitroxide form, and monitor its sodium adduct. Below we detail the stepwise development process of our assay, initially attempting separate quantitation of hydroxylamine and nitroxide species.
Chromatography of separate redox species
JP4-039 (mainly in the nitroxide form, NO) was dissolved in methanol/water, 50:50 (v/v). First the detector was tuned to the NO-JP4-039 and hydroxylamine HA-JP4-039 m/z ratios. In positive ionization, high intensity protonated ions (+1), and ammonium adducts (+18) were observed. Chromatographic retention was assessed on three columns: Synergi Hydro RP 4 μm, 100×2 mm; Luna 3 μm phenyl-hexyl 50×2 mm; and Synergi 4 μm polar RP, 50×2 mm. The phenyl-hexyl column resulted in unacceptably asymmetric peak shapes. The Synergi Hydro RP column could retain the analytes best, and resulted in separation of HA-JP4-039 (eluting early), and NO-JP4-039 (eluting late (see Fig. 4)). Before the validation phase, the runtime was shortened to 6 min under isocratic conditions.
Fig. 4.
Chromatograms of two redox species of JP4-039, the nitroxide (NO-JP4) and the hydroxylamine (HA-JP4).
Stability of the nitroxide and hydroxylamine equilibrium in the autosampler
Initially, the stability of the equilibrium of JP4-039 nitroxide and hydroxylamine species was assessed in the autosampler at 4 °C. Over the course of 20 h, the signal for NO-JP4 decreased 13% (from a baseline of 140•106 counts), while the signal for the reduced HA-JP4 increased more than 10-fold (from a baseline of 5.7•106 counts). Next, we attempted to manipulate this condition-dependent equilibrium by the addition of either ascorbic acid, ferricyanide, or pentetic acid (a potent chelator of metal ions which can catalyze or drive redox reactions). Ferricyanide and ascorbic acid could drive the equilibrium to NO-JP4, and HA-JP4, respectively, and pentetic acid did not adequately lock the equilibrium.
Sample preparation for two redox species
[D17]-JP4-039 stable isotope internal standard (IS) was synthesized for this analysis, and used to probe the linear range in plasma. To prevent possible oxidation of any present HA-JP4 during extraction and evaporation of supernatant, we chose to employ protein precipitation with methanol (after addition of IS), followed by injection of the clear supernatant. No stabilizing additives were used. The composition of the supernatant (75% methanol, 25% aqueous) corresponded to the composition of the starting mobile phase employed. Reinjection of these extracts revealed that over the course of 24 h, JP4-039 to IS ratios remained relatively constant, but in terms of absolute peak area, NO-JP4 somewhat decreased, whereas HA-JP4 increased (approximate doubling at 1000 ng/mL). These results suggested that the IS was adequately correcting for the drift in redox equilibrium, i.e. the deuterated internal standard underwent the same drift in redox state. Next, we chose to evaluate the ratio of NO-JP4 to HA-JP4 in different plasma lots, which showed wide variation (mean 5.2; range 0.7–9.6, 72 %CV, N=6). The corresponding ratio of redox species for the IS also varied by plasma lot, but this did not correlate with the ratio of the redox species of the analyte. The relative variability in redox species ratio per plasma lot, the inability to retain this ratio for quantitation after normalizing to IS response, and the inability to “lock” the redox equilibrium by the metal complexing agent pentetic acid for a reasonable period of time were considered potential barriers to a successful assay that could independently quantitate both redox species. In addition, a given biological equilibrium of NO-JP4 to HA-JP4 (possibly dependent on dietary intake of the animal) might be expected to shift upon the addition of IS (>95% in the nitroxide form) during sample preparation. We therefore chose to continue the development of this assay with a step involving addition of ferricyanide to drive all JP4-039 to NO-JP4. We chose the NO-JP4 species over the HA-JP4 species because of its much longer retention time under the standard analytical conditions, ensuring better chromatography, and a lower likelihood of interference by early eluting matrix components.
Sample preparation for a single redox species
Final plasma concentrations exceeding 100 μg/mL potassium ferricyanide resulted in maximum reductions in HA-JP4 peak areas. However, even a final concentration of 1,000 μg/mL potassium ferricyanide could not prevent a 25% loss in NO-JP4 over 15 min. Addition of ferricyanide to the clear supernatant after transfer to the autosampler vial did result in maximal and stable NO-JP4 response. In all likelihood, ferricyanide is reduced away by numerous plasma components that are not present in the supernatant. With a midpoint potential of 740 mV or higher for the nitroxide–oxoamminumcation redox couple [1], it is unlikely that the nitroxide was being converted to the oxoammonium cation by the action of the ferricyanide used in our assay, as the midpoint potential of the ferrocyanide-ferricyanide redox couple is approximately 416 mV [13].
Internal standard cross-talk
Initially, the MRM m/z transitions monitored were: 425>308 for JP4-039; 442>325 for [D17]-JP4-039. In analyzing triplicate calibration curves, there was between-run variability in the performance of the upper calibration samples of 300 to 1000 ng/mL, which were occasionally underestimated (the calibration curve seemed to be “tipping over” with accuracies as low as 60%). Close inspection of the data revealed that in these cases, the calibration curve performed better when not using IS correction. Because the m/z value of the protonated [D17]-JP4-039 IS is identical to the ammonium adduct of the non-deuterated compound, and [JP4-039 + H]+ also happens to have a product ion at m/z 325, athigher analyteconcentrations, the ammonium adduct of JP4 -039 (Q1 mass) fragmenting to the alternate product ion (Q3 mass) started to significantly add to the response of protonated [D17]-JP4-039, resulting in a decrease in the JP4-039/IS ratio, and “tipping over” of the calibration curve. Initially, this effect was minimized by increasing the amount of IS added to samples. Subsequently, we also changed the [D17]-JP4-039 IS Q3 m/z from 325 to 342, to ensure a more robust mass spectrometric detection method.
Use of the sodium adduct for quantitation
After successful validation to FDA guidance utilizing the proton adduct ions, and applying this assay to samples, we noticed a decrease in signal intensity, resulting in loss of the lower limit of quantitation. Varying the concentration of ferricyanide, replacing HPLC columns, tubing, and transferring to another AB SCIEX 4000 LC-MS/MS system occasionally improved sensitivity, but without consistency. Retuning efforts revealed a significant increase in sodium adduct intensity in the Q1 scan relative to the original Q1 scans that formed the basis for initial validation based on the proton adduct ions. After careful review of our procedures and ingredients, we were unable to identify and eliminate any ingredient responsible for introduction of sodium ions, and decided to purposely introduce this ion by addition of a trace concentration of sodium acetate in the mobile phase (final concentration 0.1 μM sodium acetate in mobile phase B). We then retuned on the sodium adduct ions and utilized these to partially revalidate the assay. Sensitivity and robustness over numerous runs was confirmed.
Carry-over
Carry-over of the final method was assessed by injecting 10,000 ng/mL of JP4-039 and [D17]-JP4-039, followed by injection of 4 control plasma samples. Carry-over amounted to less than 0.03%.
Application of the assay
Recoveries of JP4-039 from study plasma and various tissue homogenates are depicted in Error! Reference source not found.. We were surprised to find that female muscle (as opposed to male muscle and other tissues) showed recoveries of less than 80%. Several repeat experiments were performed, consistently showing this sex-specific effect in female muscle homogenate only. These results were consistent between the assay when employing proton adduct ions and sodium adduct ions for quantitation. In future studies, female muscle homogenate samples will be analyzed for JP4-039 concentrations against a calibration curve prepared in control female muscle homogenate.
We applied the assay to samples of plasma and tissues obtained from C57BL/6 male mice administered 20 mg/kg JP4-039 or vehicle administered IV over 30 sec. As seen in Fig. 5, the assay was capable of quantitating JP4-039 in the plasma and tissues. The tissue concentrations of JP4-039 were higher than the plasma concentrations for rapidly equilibrating tissues such as lungs, liver, kidneys and heart. Non-compartmental analysis yielded the following plasma pharmacokinetic parameters for JP4-039: Cmax 6.86 μg/mL, tmax 5 min, AUC0-t 277 μg/mL•min, AUC0-inf 278 μg/mL•min, t½ 16 h; and CL 72.1 mL/h/kg.
Fig. 5.
Plasma (ng/mL) and tissue (ng/μg bone marrow protein; ng/g other tissues) concentrations of JP4-039 in male mice following a 30 sec IV bolus administration of 20 mg/kg JP4-039. (A) Plasma (○), fat (□), bone marrow (◇), spleen (△), lung (x), and brain (+); (B) Plasma (○), small intestines (□), skeletal muscle (◇), kidney (△), heart (+), and liver (x).
Incurred sample reanalysis
Upon completion of the validation, incurred sample reanalysis was performed on plasma samples. Reanalysis of 20 samples yielded an average difference of 9.8%, an average absolute difference of 19.2%, a range of −15.7% to 79.9%, with four samples (20% of samples) exceeding the 20% difference limit [14].
CONCLUSION
The objective of this study was to develop and validate an analytical method for the quantitation of JP4-039 in murine plasma and to apply this assay to plasma and tissue homogenate samples to evaluate the pharmacokinetics of JP4-039 after administration to mice. This is part of a long-term effort to characterize the pharmacology of JP4-039, including dose-linearity, PK-PD relationships and bioavailability. We accomplished our goal using reversed phase chromatography with triple quadrupole mass spectrometric detection. Our development successfully addressed a number of peculiar hurdles, including how to deal with a redox active compound, having an internal standard that experienced cross-talk from the ammonium adduct of the analyte, and doping the mobile phase with a sodium salt to achieve robust quantitation of sodium adduct ions. Previous reports on JP4-039 have included quantification of JP4-039 by electron paramagnetic resonance (EPR) spectra of nitroxide radicals after mixing with acetonitrile (1:1 v/v) after 5 min incubation with 2 mM K3Fe(CN)6 [3]. As we have shown, ferricyanide is not necessarily able to maintain JP4-039 in the nitroxide redox state, and the method lacked sufficient detail on these and other validation aspects. Furthermore, the EPR assay would not be able to distinguish JP4-039 parent drug from metabolites that still retain the nitroxide moiety, and as such our LC-MS/MS assay is more specific. Our preliminary pharmacokinetic evaluation suggests a relatively short plasma half-life with concentrations above our LLOQ out to 6 h, and good tissue distribution, including the brain. Quantitative data generated with the aforementioned EPR method suggested JP4-039 was cleared from plasma by 10 min, and was detected in lung (and intestine) for over 30 min [3]. Our data for plasma, lung and small intestines go out to 6 h, and allow a better and more quantitative evaluation of the tissue pharmacokinetics of JP4-039.
To our knowledge, this is the first assay for the specific and sensitive quantification of JP4-039 published to date, that is validated according to FDA guidance [11]. The analytical method presented herein will be a valuable tool in quantitating JP4-039 as this compound moves forward as a radiation protective agent and as a radiation mitigator through additional preclinical studies and advances toward the clinic.
Supplementary Material
Highlights.
JP4-039 traps reactive oxygen species generated by ionizing radiation
An LC-MS/MS assay from 1–3000 ng/mL in 0.05 mL plasma was validated
The assay was successfully cross-validated to several tissue homogenates.
We present the first JP4-039 mouse disposition data
Acknowledgments
FUNDING
Support: Grant U19 AI068021-11 (NIAID CMCR). This project used the UPCI Cancer Pharmacokinetics and Pharmacodynamics Facility (CPPF) and the HCC Animal Facility and was supported in part by award P30-CA47904and R50-CA211241.
Footnotes
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