Abstract
Camptothecin (CPT), a potent inhibitor of topoisomerase I and HIF-1α, failed to demonstrate utility as an anti-cancer agent in early clinical trial investigations, primarily due to limited clinical activity and significant toxicity attributable to unfavorable physicochemical properties (e.g. low plasma solubility, pH-labile lactone ring). NLG207 (formerly CRLX101), a nanoparticle-drug conjugate (NDC) of CPT designed to optimize plasma pharmacokinetics and facilitate drug delivery to tumors, is included as part of combination treatment in two Phase II clinical trials ongoing at the National Cancer Institute ( NCT02769962 and NCT03531827). To better understand the potential for drug-drug interactions and to correlate drug exposure to clinical outcomes and pharmacodynamic biomarkers, a robust analytical method was developed to measure CPT in human plasma. Two sample processing methods were developed to quantify both NDC-bound CPT and free CPT, primarily via alteration of pH conditions. A solid-phase extraction recovered >79% of CPT prior to quantitative analysis by ultra HPLC-MS/MS. Dynamic calibration ranges of 10 to 10,000 ng/mL and 1 to 1,000 ng/mL for total and free CPT, respectively were expected to capture clinical ranges. NLG207 NDCs demonstrated significant rates of CPT release in human plasma at room temperature after 2 hours but were shown to be stable at 4°C for 24 hours and through 4 freeze/thaw cycles. This assay was used to quantitate CPT plasma concentrations in clinical samples to confirm clinical utility following NLG207 treatment in a subject with advanced prostate cancer.
Keywords: Bioanalytical, Mass Spectrometry, Pharmacokinetics, Clinical Pharmacology, Oncology, Anti-cancer, Topoisomerase I Inhibitor
1. Introduction
NLG207 (formerly CRLX101) is a nanoparticle-drug conjugate (NDC) of the potent topoisomerase I inhibitor, camptothecin (CPT), developed to overcome issues associated with poor plasma solubility[1–3] and enhance drug delivery to tumor tissue (Figure 1).[4–6] Following deposition into tumors, the rate of CPT release is controlled via pH-dependent hydrolysis of the carboxyl ester linkers while retaining CPT in the active lactone form.[6, 7] The anti-tumor activity of NLG207, both as monotherapy and in combination with other agents, has been characterized in numerous preclinical models,[8–12] with enhanced efficacy demonstrated in comparison to small molecule CPT and irinotecan.[13]
Figure 1.
Subunit of NLG207 nanoparticle-drug conjugates
To date, the treatment of over 304 patients with at least one dose of NLG207 on multiple clinical trials has been reported in the literature.[7, 9, 14–17] Utility of NLG207 has spanned across multiple tumor histologies, including advanced non-small cell lung cancer, advanced ovarian cancer, and metastatic renal cell carcinoma. The most commonly utilized Phase II dose of NLG207 is 15 mg/m2 given via intravenous infusion every two weeks, given either as monotherapy or as combination therapy (e.g. bevacizumab), though lower (12 mg/m2) or more frequent (weekly) dosing strategies have been explored. Characterization of clinical plasma pharmacokinetics via non-compartmental analysis has been conducted using serial samples collected over two weeks from the initial Phase I/II clinical trial (samples quantitated by an unpublished LC/MS method by the company). Pharmacokinetic parameters have been reported for both CPT bound to NLG207 (conjugated) and free CPT (unconjugated), with a relative 11-fold increase in exposure (determined via AUCLAST) of conjugated over unconjugated CPT. Mean clearance and volume of distribution values were found to be dose-independent, with volume of distribution values of conjugated CPT suggesting retention predominantly in the vasculature and highly perfused tissues (2.33 to 4.63 L).[7]
Herein we describe a novel, robust, selective, accurate and precise bioanalytical method for the quantification of CPT, both conjugated and unconjugated to NLG207 NDCs. Previous analytical methods, though accurate and precise, proved to be inefficient. This assay was developed and validated according to FDA guidelines, while also incorporating a solid phase extraction step, including an internal standard (SN-38), and eliminating the use of strong acid in sample preparation to reduce LC column degradation. To further improve assay validation consistency and reliability from run to run, the quantitation ranges were validated between 10 to 10,000 ng/mL and 1 to 1,000 ng/mL for total CPT and free CPT quantitation, respectively, which are reflective of clinically relevant drug concentrations.[7] This assay was demonstrated to be applicable for clinical pharmacokinetic purposes by using the small patient cohorts of NCT02769962 and NCT03531827 (subsequent pharmacokinetic data to be published separately), and used to assess the stability of NDCs in clinically relevant scenarios.
2. Materials and methods
2.1. Materials
S-(+)-Campothecin was provided by Millipore Sigma (Rockville, MD), SN-38 was provided by Selleck Chemicals (Houston, TX), and NLG207 was provided by NewLink Genetics (Ames, IA). The chemical structures of camptothecin and SN-38, analyte and internal standard respectively, are shown in Figure S1. Formic acid and sodium hydroxide were provided by Millipore Sigma (Rockville, MD). Optima-grade methanol was obtained from Fisher Scientific (Fairlawn, NJ), and a Hydro-Reverse Osmosis system (Durham, NC) linked to a Milli-Q UV Plus purifying system (Billerica, MA) was used to generate de-ionized water. Drug-free human heparinized plasma and whole blood was obtained from the NIH Clinical Center Blood Bank (Bethesda, MD).
2.2. Total CPT quantitation
The purpose of this preparative method was to force-release all CPT from NLG207 NDCs using high pH conditions[18] and quantitate the total CPT present in a patient sample.
2.2.1. Preparation of stock solutions
Clinical-grade vials of NLG207 (35 mg CPT equivalents) were re-constituted in Dulbecco’s phosphate buffered saline (DPBS) to a concentration of 1 mg/mL, aliquoted in glass vials, and stored in −20°C. To ensure NDC stability following multiple freeze-thaws, a previously unused (i.e. one freeze-thaw) 1 mg/mL vial was used to generate serial dilutions (working stocks) in DPBS for each individual run. The working stocks were then used for preparation of calibration and quality control (QC) samples. The SN-38 Master Stock at a concentration of 1 mg/mL was prepared individually via dissolution in DMSO, vortex mixing, brief sonication, and storage at 80°C.
Calibration standards were freshly prepared in drug-free human heparinized plasma at concentrations of 10, 50, 100, 500, 1000, 5000, and 10,000 ng/mL in duplicate for each analytical run. The required amount of working stock was added to sufficient drug-free heparinized plasma to generate fresh quality control (QC) samples for each analytical run at concentrations of 10 (lower limit of quantitation; T-LLOQ), 30 (low range; T-LQC), 3000 (medium range; T-MQC), 9000 (high range; T-HQC), and 30,000 ng/mL (10-fold dilution; T-DQC).
2.2.2. Sample preparation
Samples were thawed on wet ice and inverted 3 to 5 times to homogenize. Fifty microliters of 0.1N NaOH were added to 50 μL of plasma calibrator aliquots, QC samples and unknown samples, then vortexed briefly to release CPT from NDCs. Following a 15-minute incubation at room temperature, 200 μL of 1% formic acid (FA) containing 250 ng/mL of SN-38 (internal standard) were added to lower pH and convert CPT to the lactone form. The resulting mixture was vortex-mixed, then centrifuged for 10 minutes at 13,200 rpm (11,700 xg) in a mini-centrifuge. Approximately 250 μL were removed and added to a solid-phase extraction (SPE) plate (Evolute Express ABN, Biotage; Charlotte, NC). Once on the SPE plate, the sample was washed with 1% aqueous methanol, and eluted in 100% methanol. The eluent was dried down and reconstituted in (60/40, v/v) 1% FA (aq)/MeOH).
2.3. Free CPT quantitation
The purpose of this preparative method was to stabilize NLG207 NDCs using low pH conditions[18] and quantitate only the free CPT, unbound to NLG207.
2.3.1. Preparation of stock solutions
CPT Master Stock solutions were prepared via dissolution in DMSO at a concentration of 1 mg/mL. The master stock solution was vortex mixed and briefly sonicated before being stored in glass vials at −80°C. Serial dilutions (working stocks) in DMSO were prepared from the master stock for the generation of calibration and QC samples and stored in glass vials at −80°C. SN-38 Master Stock was prepared as described for the total CPT quantitation.
Calibration standards were freshly prepared in drug-free human heparinized plasma at concentrations of 1, 5, 10, 50, 100, 500, and 1,000 ng/mL in duplicate for each analytical run. The required amount of working stock was added to sufficient drug-free heparinized plasma to generate fresh QC samples for each analytical run at concentrations of 1 (F-LLOQ), 3 (F-LQC), 300 (F-MQC), 900 (F-HQC), and 10,000 ng/mL (10-fold dilution; F-DQC).
2.3.2. Sample preparation
Similarly to the total CPT quantitation, samples were thawed on wet ice and inverted 3 to 5 times to homogenize. To one hundred microliters of plasma calibrator aliquots, QC samples, and unknown samples, 200 μL of 1% formic acid containing 250 ng/mL of SN-38 (internal standard) were added to stabilize NDCs, preventing release of CPT. The resulting mixture was vortexed, then centrifuged for 10 minutes at 13,200 rpm. Mixtures were processed via SPE, dried, and reconstituted as described in the total CPT quantitation procedure.
2.4. Instrument conditions
Instrument conditions for both total and free CPT quantitation calibration methods were consistent. For each sample, five microliters were injected onto a Waters ACQUITY UPLC® system (Waters Corporation, Milford, MA) that included a binary pump, refrigerated autosampler (6°C) and a temperature-controlled column compartment (room temperature). An Acquity UPLC BEH Shield RP18, 2.1×50mm, 1.7 μm column was used for chromatographic separation. The mobile phase comprised of A: 0.1% FA in water, and B: methanol with a 0.4 mL/min flow rate and a 3-minute total run time. A gradient of mobile phase B was implemented via a linear increase from 35% to 75% during the first 1.5 minutes, then a linear decrease back to 35% over 1.0 minute, then maintaining 35% for 0.5 minutes. The column eluent was directed into a SCIEX QTRAP5500® mass spectrometer (SCIEX, Framingham, MA). Selected reaction monitoring (SRM) in the positive ion mode was set to monitor CPT (lactone form) (m/z 349.2 → 305.2), and the internal standard SN-38 (m/z 393.2 → 349.2) (Table S1).[19] Peak integrations generated via SRM, and subsequent data analyses were performed using the Multi-Quant program in Analyst (SCIEX, Framingham, MA).
2.5. Validation
2.5.1. Quadratic regression
Calibration curves for total CPT and free CPT were constructed using least-squares quadratic regression analysis of 7 point curves (10 to 10,000 ng/mL and 1 to 1,000 ng/mL for total CPT and free CPT respectively); the peak area ratio of the analyte to the internal standard were plotted using 1/x2 as a weighting factor (x = ratio of the nominal analyte:IS concentration). Calibrator response functions and choice of regression analysis were investigated via correlation coefficient (r) calculation and percent deviation (% DEV) determination for all calibrators.
2.5.2. Trueness and precision
The trueness (accuracy) and precision of CPT quantification was evaluated for each method on four separate days using separate sets of 5 different concentrations (LLOQ, LQC, MQC, HQC, and DQC 10-fold dilution). Blank plasma, internal standard only, and calibration standard samples were prepared in duplicate for each run; QC and LLOQ samples were prepared in replicates of five for each of the four days (n=20). The percent difference between the mean observed concentration and the nominal concentration was used to determine trueness (%DEV). Assay consistency and reproducibility were evaluated via calculation of within-run precision (WRP) and between-run precision (BRP), per the equations below:
With grand mean represented by GM, within-group mean squared by MSWIT, between-group mean squared by MSBET, and the number of repetitions by n. Bioanalytical evaluation allowed for ±15% variability in trueness and precision, per FDA guidelines.
2.6. Stability
The post-preparative stability at 4 °C of CPT and SN-38 in the 96 well injector plates in the autosampler pending analysis was assessed. For both the total CPT and free CPT quantitation methods, samples were injected and analyzed immediately following preparation, then re-injected and re-analyzed 24 hours after the initial analysis. The original values obtained from those samples were compared to the values obtained 24 hours later.
Stability tests were performed to analyze potential alterations in CPT concentrations in the context of NLG207 conjugation. CPT degradation was analyzed primarily via quantitation of total CPT and NDC stability was assessed via comparisons of free CPT (i.e. drug unbound to the NDC). The stability of NLG207 and CPT in human plasma was assessed following multiple freeze/thaw cycles. Samples were assayed in triplicate at four concentrations of NLG207 (30, 300, 3000, and 9000 ng/mL, per CPT equivalent) following up to 4 freeze/thaw cycles at −80°C (at least 12 hours for each freeze cycle). Samples were processed using both assay validation methods for the measurement of total and free CPT. In the same analytical run, a comparison of analyte concentrations following each storage period to analyte concentrations of freshly prepared samples was generated.
The stability of NLG207 NDCs in plasma at room temperature was evaluated, mimicking conditions of sample processing in the laboratory. NLG207 samples in human plasma at four different concentrations (30, 300, 3000, and 9000 ng/mL, per CPT equivalent) were either extracted immediately (fresh) or incubated at room temperature, each in triplicate. Samples were processed using both assay methods for the measurement of total and free CPT. Following analysis from the same analytical run, the analyte concentrations after 1 or 2 hours at room temperature were compared to the concentrations of immediately extracted samples.
The stability of the NLG207 in whole blood, mimicking post-collection conditions on the inpatient unit, was assessed at room temperature or 4°C. NLG207 samples in whole blood at three different concentrations (50, 500, and 5,000 ng/mL, per CPT equivalent), were either extracted immediately (fresh), kept at room temperature for 3 hours, or kept at 4°C for 24 hours, each in triplicate. Plasma was isolated via inversion of the sample 5 to 8 times and centrifugation for 5 minutes at 2,400 RPM at 4°C. Samples were processed using both assay validation methods for the measurement of total and free CPT. The analyte concentrations (total and free CPT) for each of the incubation conditions were compared to the concentration of freshly prepared samples in the same analytical run.
2.7. Extraction recovery and matrix effects
CPT and SN-38 extraction efficiency, or recovery, following SPE was evaluated by comparison of analyte peak areas before and after placement on the SPE plate at multiple concentrations of CPT (6, 60, 600, and 6,000 ng/mL; each n=5), each with a constant concentration of SN-38 (250 ng/mL). Due to differences in sample volume between the total CPT and free CPT preparative methods, the concentrations listed are consistent with the amount of drug processed via the total CPT method; equivalent concentrations for the free CPT method would be halved (e.g. 6 ng/mL corresponds to 3 ng/mL for the free CPT method). To further characterize selective and matrix effects, CPT and SN-38 were spiked into five lots of plasma at each of the corresponding concentrations and assayed in quintuplet.
Plasma matrix effects on camptothecin and SN-38 mass spectrometric signals were evaluated via comparison of samples spiked in plasma to samples spiked in the reconstitution solution. Camptothecin and SN-38 were spiked into the reconstitution solution (60/40, v/v; 1% FA (aq)/MeOH) at four concentrations of CPT (6, 60, 600, and 6,000 ng/mL; each n=3), each with 250 ng/mL of SN-38. The peak areas of CPT and SN-38 were analyzed and compared to the same concentrations of drug spiked into 5 separate lots of plasma after extraction, removing potential discrepancies attributable to the extraction step.
2.8. Clinical application
The applicability of this method to measure CPT clinically was demonstrated via quantitation of serial plasma samples collected following administration of NLG207 12 mg/m2 dose of via one-hour intravenous infusion. The plasma concentration vs time data was collected in 3 patients with metastatic castration-resistant prostate cancer (mCRPC) during and after the first dose of NLG207 prior to initiating enzalutamide as part of combination therapy (per NCT03531827). Blood was collected in 6 mL sodium heparin tubes (BD, Franklin Lakes, NJ) and immediately placed at 4°C until time of processing (samples were processed at the study site). Plasma was isolated via inversion of the sample 5 to 8 times and centrifugation for 5 minutes at 2,400 RPM at 4°C, then stored at −80°C until time of quantitation. Concentrations of total and free CPT were measured following simultaneous processing of the same samples using both quantitation methods. The pharmacokinetic data was compared to previously published data administering NLG207 as monotherapy to patients with advanced solid tumors.[7] The clinical protocol was approved by the Institutional Review Board of the National Cancer Institute’s Center for Cancer Research (Bethesda, MD).
Incurred sample reanalysis was conducted using plasma samples from the previously mentioned 3 patients with mCRPC. Plasma samples collected during the NLG207 intravenous infusion, at CMAX, and during the elimination phase were included in the analysis (n=27). Nine of the 27 samples were re-analyzed 6 months after original quantitation, whereas the remaining samples were re-analyzed 2 days after original quantitation. The repeated total CPT quantitation was compared to the original quantitation per sample (not pooled) using the following formula:
The repeated free CPT quantitation was also compared to the original quantitation, with free CPT being expressed as the “%free” CPT in relation to the total CPT quantitation of the same plasma sample (i.e. %free CPT = [free CPT]/[total CPT] × 100). Since the free CPT quantitation is impacted by the CPT release from NDCs, differences in the free CPT quantitation were reported as the increase in %free CPT. The percentage of samples within ±20% of the mean (original and repeat) for the total CPT assessment and the percentage of samples with <1% increase for %free CPT assessment were reported.
3. Results and discussion
3.1. Selectivity
Figure S2 depicts several LC-MS/MS chromatograms, including drug-free extract (Fig. S2A), internal standard only (Fig. S2B), LLOQ for both total and free CPT quantitation methods (10 ng/mL and 1 ng/mL, Fig. S2C and S2D, respectively). Additionally, Figure 2 depicts the LC-M/MS chromatograms of a clinical pharmacokinetic sample taken at the end of a 12 mg/m2 intravenous infusion of 60 minutes (total CPT and free CPT, Fig 2A and 2B, respectively); the LC method provided optimal separation of analyte and internal standard for both the total and free CPT procedures (Fig 2C and 2D, respectively). The retention times of CPT lactone and SN-38 were 1.46 and 1.66 min, respectively. For both sample processing methods, the elution following an organic gradient via UPLC yielded sharp, symmetrical peaks without interference from the plasma matrix.
Figure 2.
A clinical pharmacokinetic sample measured at the end of infusion of a 12 mg/m2 dose of NLG207, total (A & C) and free CPT (B & D). Chromatograms correspond to camptothecin lactone form (analyte), and SN-38 (internal standard).
3.2. Validation
The calibration standards, ranging from 10 to 10,000 ng/mL and 1 to 1,000 ng/mL for total and free CPT quantitation respectively, were run in duplicate on each of four days (n=8). The seven calibration standards for each quantitation method were accurate and precise (Table 1), with a mean r2 value of 0.99639 ± 0.00181 and 0.99906 ± 0.00058 for total and free CPT, respectively. It was initially attempted to quantitate the full range of 1 to 10,000 ng/mL for both sample preparation methods, but the final assay ranges were selected for increased between run consistency and more relevant clinical applicability. Reflective of differences in the range of the total and free CPT quantitation methods, four appropriate quality control (QC) samples, at low, medium, high, and tenfold diluted concentrations, and a LLOQ QC were run daily in quintuplet over four days (n=20). The QC concentrations for both quantitation methods were accurate and precise, within the required ≤15% (Table 2). Of note, the quantitation of two samples for the total CPT validation (one calibration sample [50 ng/mL] and one QC sample [T-LQC, 30 ng/mL]), was omitted due to significant deviation from the nominal concentration resulting from inaccuracy in sample preparation.
Table 1.
Calibration quadratic regression
| Total Camptothecin | |||||
|---|---|---|---|---|---|
| Nominal (ng/mL) | GM (ng/mL) | SD (ng/mL) | DEV (%) | CV (%) | n |
| 10.00 | 10.01 | 0.32 | 0.14 | 3.25 | 8 |
| 50.00 | 47.75 | 2.50 | −4.50 | 5.25 | 7 |
| 100.00 | 94.10 | 2.65 | −5.90 | 2.82 | 8 |
| 500.00 | 501.9 | 9.67 | 0.38 | 1.93 | 8 |
| 1000.00 | 1076 | 62.4 | 7.60 | 5.80 | 8 |
| 5000.00 | 5296 | 175 | 5.92 | 3.30 | 8 |
| 10000.00 | 9547 | 233 | −4.53 | 2.44 | 8 |
| Free Camptothecin | |||||
|---|---|---|---|---|---|
| Nominal (ng/mL) | GM (ng/mL) | SD (ng/mL) | DEV (%) | CV (%) | n |
| 1.00 | 1.01 | 0.03 | 0.50 | 3.19 | 8 |
| 5.00 | 4.92 | 0.13 | −1.65 | 2.63 | 8 |
| 10.00 | 9.92 | 0.11 | −0.78 | 1.16 | 8 |
| 50.00 | 49.50 | 0.67 | −1.01 | 1.36 | 8 |
| 100.00 | 101.5 | 1.83 | 1.53 | 1.80 | 8 |
| 500.00 | 515.4 | 15.1 | 3.09 | 2.93 | 8 |
| 1000.00 | 983.6 | 14.1 | −1.64 | 1.43 | 8 |
Abbreviations: GM, grand mean; SD, standard deviation; DEV (%), relative deviation from nominal value; CV (%), coefficient of variation; n, number of replicate observations within each validation run, i.e. for each concentration, two samples were run on four separate occasions (total of 8 samples at each concentration).
Table 2.
Trueness and precision
| Total Camptothecin | |||||||
|---|---|---|---|---|---|---|---|
| Nominal (ng/mL) | GM (ng/mL) | SD (ng/mL) | DEV (%) | CV (%) | WRP (%) | BRP (%) | n |
| 10 (T-LLOQ) | 10.34 | 0.87 | 3.43 | 8.44 | 4.54 | 8.01 | 20 |
| 30 (T-LQC) | 33.03 | 1.71 | 10.09 | 5.18 | 3.39 | 4.29 | 19 |
| 3000 (T-MQC) | 3255 | 101 | 8.49 | 3.10 | 2.66 | 1.79 | 20 |
| 9000 (T-HQC) | 9249 | 386 | 2.77 | 4.17 | 3.73 | 2.10 | 20 |
| 30000 (T-DQC) | 32510 | 585 | 8.36 | 1.80 | 1.88 | -* | 20 |
| Free Camptothecin | |||||||
|---|---|---|---|---|---|---|---|
| Nominal (ng/mL) | GM (ng/mL) | SD (ng/mL) | DEV (%) | CV (%) | WRP (%) | BRP (%) | n |
| 1 (F-LLOQ) | 1.06 | 0.06 | 6.20 | 5.98 | 4.64 | 4.24 | 20 |
| 3 (F-LQC) | 3.16 | 0.09 | 5.18 | 2.72 | 2.55 | 0.99 | 20 |
| 300 (F-MQC) | 319.2 | 12.6 | 6.40 | 3.94 | 2.25 | 3.65 | 20 |
| 900 (F-HQC) | 966.5 | 54.1 | 7.39 | 5.59 | 3.86 | 4.56 | 20 |
| 10000 (F-DQC) | 9703 | 404 | 7.81 | 4.17 | 2.65 | 3.62 | 20 |
Abbreviations: GM, grand mean; SD, standard deviation; DEV (%), relative deviation from nominal value; CV (%), coefficient of variation; WRP (%), within-run precision; BRP (%), between-run precision; n, number of replicate observations within each validation run.
- No additional variation was observed as a result of performing the assay in different runs.
3.3. Stability
The 24-hour post-preparative stability measurements resulted in minimal deviations compared with the initial run for both validation methods. Quality control measurements for both methods were also consistent with the initial run (<6% change), enabling samples extracted from human plasma to be reanalyzed within 24 hours of preparation without significant degradation (Table S2). Additionally, the post-preparative stability permitted the simultaneous processing of samples using both total and free CPT methods, followed by sequential LC-MS/MS quantitation. For all subsequent stability analyses, the samples were processed for both total and free CPT quantitation simultaneously for more accurate and direct data comparisons.
Freeze/thaw stability of CPT and NLG207 NDCs in human plasma was shown following analysis of four freeze/thaw cycles at 4 different concentrations of NLG207 (concentration measured as total CPT equivalents; Table 3A). No significant degradation of small molecule CPT was indicated via comparisons of total CPT concentrations between freeze/thaw cycles (<8% change). Standard processing of NLG207 1 mg/mL (CPT equivalent) stocks stored in DPBS at 80°C followed by immediate transfer to human plasma prior to processing consistently resulted in 3% [2.5 to 3.5%] of CPT to be released from NLG207 NDCs, as indicated by the freeze/thaw dataset for all indicated concentrations. The noted free:total CPT ratio was not meant to be indicative of what is observed clinically, but rather served as an additional internal quality control for stability assessments. The free CPT concentrations and free:total CPT ratios following several freeze/thaws did not deviate significantly from freshly prepared samples, with <13% and <10% change, respectively (Table 3B and 3C).
Table 3.
Freeze/thaw stability of NLG207 NDCs in plasma, including measurements of total camptothecin concentrations (A), free camptothecin concentrations (B), and the calculated ratio of free to total camptothecin concentrations (C).
| A | Total Camptothecin | |||||||
|---|---|---|---|---|---|---|---|---|
| 30 ng/mL | 300 ng/mL | 3000 ng/mL | 9000 ng/mL | |||||
| Free/Thaw Cycle | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) |
| 0 (fresh) | 33.08 | - | 332.0 | - | 3503 | - | 9898 | - |
| 1 | 32.88 | −0.6 | 315.1 | −5.1 | 3231 | −7.8 | 9470 | −4.3 |
| 2 | 35.02 | 5.9 | 317.6 | −4.3 | 3296 | −5.9 | 9746 | −1.5 |
| 3 | 35.34 | 6.8 | 335.1 | 1.0 | 3359 | −4.1 | 9696 | −2.0 |
| 4 | 33.22 | 0.4 | 312.5 | −5.8 | 3272 | −6.6 | 9656 | −2.4 |
| B | Free Camptothecin | |||||||
|---|---|---|---|---|---|---|---|---|
| 30 ng/mL | 300 ng/mL | 3000 ng/mL | 9000 ng/mL | |||||
| Free/Thaw Cycle | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) | GM (ng/mL) | DEV (%) |
| 0 (fresh) | 1.02 | - | 10.83 | - | 110.1 | - | 305.6 | - |
| 1 | 1.08 | 5.6 | 10.68 | −1.4 | 104.7 | −4.9 | 321.9 | 5.3 |
| 2 | 1.08 | 6.2 | 9.98 | −7.8 | 97.13 | −11.8 | 337.6 | 10.5 |
| 3 | 1.09 | 7.2 | 10.12 | −6.6 | 97.05 | −11.9 | 300.5 | −1.7 |
| 4 | 0.99 | −2.6 | 9.91 | −8.5 | 95.93 | −12.9 | 317.0 | 3.7 |
| C | Ratio of Free: Total Camptothecin | |||||||
|---|---|---|---|---|---|---|---|---|
| 30 ng/mL | 300 ng/mL | 3000 ng/mL | 9000 ng/mL | |||||
| Free/Thaw Cycle | % Free CPT | DEV (%) | % Free CPT | DEV (%) | % Free CPT | DEV (%) | % Free CPT | DEV (%) |
| 0 (fresh) | 3.18 | - | 3.37 | - | 3.24 | - | 3.19 | - |
| 1 | 3.27 | 2.9 | 3.39 | 6.5 | 3.24 | 1.9 | 3.40 | 6.8 |
| 2 | 3.09 | −2.8 | 3.14 | −1.2 | 2.95 | −7.4 | 3.46 | 8.9 |
| 3 | 3.09 | −2.8 | 3.02 | −5.1 | 2.89 | −9.2 | 3.10 | −2.6 |
| 4 | 2.99 | −6.0 | 3.17 | −0.3 | 2.93 | −7.9 | 3.28 | 3.1 |
Abbreviations: GM, grand mean; DEV (%), relative deviation from fresh.
Room temperature stability of NLG207 NDCs in human plasma was assessed at timepoints relevant to the timing of sample processing (Table 4). Total CPT concentrations were not affected by room temperature at one or two hours (<10% change), indicating no significant degradation of CPT molecules. Free CPT concentrations were significantly impacted by 2 hours (>33% increase compared to fresh), with >14% change measured for each concentration range after one hour. The data indicates limited NDC stability at room temperature and the importance of immediate sample processing following the thawing of a frozen sample, notably for accurate and precise free CPT quantitation.
Table 4.
Bench-top plasma stability of NLG207 nanoparticles at room temperature
| Nominal | Immediately After Preparation | One Hour Post Preparation | Two Hours Post Preparation | ||
|---|---|---|---|---|---|
| -Total CPT -[Free CPT]* | GM (ng/mL) | GM (ng/mL) | MC (%) | GM (ng/mL) | MC (%) |
| 30 ng/mL [1.0 ng/mL] | 32.07 | 35.03 | 9.2 | 34.05 | 6.2 |
| 1.01 | 1.18 | 17.6 | 1.35 | 33.8 | |
| 300 ng/mL [9 ng/mL] | 342.7 | 330.29 | −3.6 | 337.0 | −1.7 |
| 9.34 | 10.91 | 16.8 | 13.19 | 41.2 | |
| 3000 ng/mL [90 ng/mL] | 3282 | 3449 | 5.1 | 3496 | 6.5 |
| 90.28 | 110.1 | 22 | 130.7 | 44.8 | |
| 9000 ng/mL [270 ng/mL] | 9207 | 9237 | 0.3 | 9395 | 2.0 |
| 300.7 | 345.0 | 14.7 | 422.1 | 40.4 | |
Abbreviations: GM, grand mean; MC, mean change from fresh.
- Based on the totality of data, approximately 3% [2.5 to 3.5%] of total CPT from NLG207 NDCs is present as free CPT under standard conditions. The nominal concentrations of free CPT listed are 3% of the nominal total CPT concentrations. For the 30 ng/mL total CPT concentration range, the nominal free CPT concentration is rounded up to 1.0 ng/mL (3.33% of total CPT) to reflect the lower limit of quantitation.
The stability of NLG207 NDCs in whole blood prior to plasma separation was assessed at room temperature and 4°C (Table 5). Following 3 hours at room temperature, the total CPT concentrations were not impacted (<10% change), whereas the free CPT concentrations increased dramatically (>70% change). When stored at 4°C, the total and free CPT concentrations were not significantly altered after 24 hours (<15% change for both total and free CPT). In summation, though CPT was stable in blood under both conditions, the NDCs were only stable at 4°C after 24 hours and not at room temperature after 3 hours. The data confirms current inpatient hospital procedure, enabling samples to be stored on ice overnight and processed the subsequent morning without significant alterations in CPT concentration measurement.
Table 5.
Pre-processing blood stability of NLG207 nanoparticles at room temperature and at 4°C
| Nominal (CPT Equivalents) | Immediately After Preparation | 3 hours at Room Temperature | 24 Hours at 4°C | ||
|---|---|---|---|---|---|
| -Total CPT -[Free CPT]* | GM (ng/mL) | GM (ng/mL) | MC (%) | GM (ng/mL) | MC (%) |
| 50 ng/mL [1.5 ng/mL] | 49.64 | 51.64 | 4.0 | 53.23 | 7.2 |
| 1.39 | 2.58 | 86.0 | 1.40 | 1.0 | |
| 500 ng/mL [15 ng/mL] | 554.4 | 579.7 | 4.6 | 595.3 | 7.4 |
| 16.83 | 28.74 | 70.8 | 14.40 | −14.4 | |
| 5000 ng/mL [150 ng/mL] | 5240 | 5549 | 4.7 | 5035 | −3.9 |
| 152.5 | 300.0 | 96.7 | 139.4 | −8.6 | |
Abbreviations: GM, grand mean; MC, mean change from fresh.
- Based on the totality of data, approximately 3% [within 2.5 to 3.5%] of total CPT from NLG207 NDCs is present as free CPT under standard conditions. The nominal concentrations of free CPT listed are 3% of the nominal total CPT concentrations.
3.4. Extraction recovery and matrix effects
The assessments of solid phase extraction efficiency, human plasma matrix effects, and overall process efficiency at 6, 60, 600, and 6000 ng/mL CPT with consistent concentrations of SN-38 are summarized in Table S3. The mean overall extraction recoveries of CPT and SN-38, estimated via comparisons of mass spectrometric signal response for the molecules spiked into 5 different lots of human plasma pre- versus post-extraction, ranged from 75.99–85.35% and 86.04–89.19%, respectively. When comparing the effects of human plasma to clean solution (process efficiency), analyte and IS peak areas between post-extraction plasma and mobile phase were reduced by 79.05–80.19% and 82.44–83.57%, respectively, for analyte concentrations ranging from 6 to 600 ng/mL; for the 6000 ng/mL concentrations, the analyte and IS signals were only reduced by 94.49% and 96.67%, respectively. With respect to overall process efficiency, the mean ratio of analyte to IS recoveries was consistent across all concentration levels, however, with respect to extraction efficiency, mean analyte recovery increased with concentration while IS recovery remained relatively constant. The comparison of post-column analyte injection and blank plasma extract injection was consistent with negligible matrix effects.
3.5. Clinical application
Figure 3 depicts the total and free CPT concentration vs. time curves for 3 patients with mCRPC who received a single dose of 12 mg/m2 NLG207 via one-hour intravenous infusion (determined in human plasma). These results are in line with previous data reported from Phase I clinical evaluation of pharmacokinetic evaluation,[7] suggesting the sufficiency of the developed analytical methods to detect CPT concentrations with clinically relevant sensitivity, enabling further evaluation of CPT plasma concentrations as part of ongoing Phase II evaluation of NLG207 treatment combinations. The clinical results presented here demonstrated the applicability of this assay in capturing the range of plasma concentrations achieved with clinical doses given. Further pharmacokinetic analyses will be published separately following the availability of more data.
Figure 3.
Total and free camptothecin (CPT) plasma concentration vs. time curves for three patients with mCRPC administered a one-hour intravenous infusion of NLG207 12 mg/m2.
The incurred sample reanalysis examined a total of 27 plasma samples collected from the previously mentioned patients with mCRPC. Comparisons of total CPT quantitation showed that 100% of samples were within 20% of the mean of original and repeat quantitation. Twenty-three of 27 plasma samples (85.2%) reported less than 1% change increase in %free CPT following repeat quantitation, with the greatest increase in %free CPT reported as 1.64%. Of the samples collected 6 months prior to reanalysis, 3 of 9 plasma samples (33%) reported a >1% change in %free CPT, suggesting some caution with increased retention time of the plasma samples. Additionally, all samples with >1% increase in %free CPT were at later collection time points (e.g. 8, 12, and 24 hours post end of infusion). Results from the incurred sample reanalysis demonstrate reliability of the assay to provide consistent quantitation of NLG207 plasma samples.
4. Conclusions
Outlined here are consistent and reliable analytical methods associated with quantitation of CPT in the context of NLG207, a promising therapeutic agent currently included as part of two combination treatments under investigation at the NCI. This method proved sensitive (T-LLOQ 10 ng/mL, F-LLOQ 1 ng/mL), selective, accurate, and precise for quantitating both NDC-bound and free CPT concentrations in clinically relevant ranges. The solid phase extraction proved to be efficient at recovering CPT (~80%), and approaches used to modify pH were sufficient for the exclusive detection of CPT lactone. This method demonstrated 24-hour post-preparative stability, a factor that enables the simultaneous processing and subsequent analysis of total and free CPT. NLG207 NDCs were found to be stable in plasma through four freeze/thaws and stable in whole blood at 4°C for 24 hours. The assay shows the importance of limiting sample exposure to room temperature, as the amount of CPT release from NLG207 was significant after 2 hours. Assessment of clinical application demonstrated the capability of this assay to produce results in line with previously published pharmacokinetic data. This assay is currently being applied to the pharmacokinetic analyses of two Phase II studies examining the treatment combinations of NLG207 and olaparib, and NLG207 and enzalutamide.
Supplementary Material
Highlights:
NLG207 is a nanoparticle-drug conjugate of camptothecin (topoisomerase I inhibitor)
Analytical methods were developed to measure nanoparticle-bound & free camptothecin
The pharmacokinetic assay to quantitate plasma concentrations was accurate & precise
NLG207 samples were stable up to 24 hours at 4°C and following 4 freeze-thaw cycles
Clinical utility was shown via quantitation of samples from a NLG207-treated patient
Acknowledgements
This study was funded in part by the Intramural Research Program of the NIH, National Cancer Institute, and in part by a CRADA between the National Cancer Institute and NewLink Genetics (#03089). This is US Government work. There are no restrictions on its use. The views expressed within this paper do not necessarily reflect those of the US Government. We thank the nursing staff of the National Cancer Institute and the fellows of the Medical Oncology Branch at the National Cancer Institute for their care of our patients. Most importantly, we appreciate the patients with cancer who enroll in investigational trials to advance the knowledge of this disease.
Footnotes
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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