Stability of nafamostat in intravenous infusion solutions, human whole blood and extracted plasma: implications for clinical effectiveness studies
Abstract
Aim: To describe the stability of nafamostat in infusion solutions, during blood sample collection and in extracted plasma samples in the autosampler. Methods: Nafamostat infusion solutions were stored at room temperature in the light for 24 h. For sample collection stability, fresh blood spiked with nafamostat was subjected to combinations of anticoagulants, added esterase inhibitor and temperature. Nafamostat was monitored in the extracted plasma samples in the autosampler. Results: Nafamostat was stable in infusion solutions. Nafamostat in whole blood was stable for 3 h before centrifugation when collected in sodium fluoride/potassium oxalate tubes (4°C). Nafamostat in extracted plasma samples degraded at 4.7 ± 0.7% per h. Conclusion: Viable samples can be obtained using blood collection tubes with sodium fluoride, chilling and processing promptly.
Graphical abstract
Nafamostat is a synthetic protease inhibitor drug, with recent studies demonstrating that nafamostat has antiviral properties [1–3]. Nafamostat has been identified as one of the most potent drugs against SARS-CoV-2 [2]. This has led to the development of clinical studies investigating the potential use of nafamostat as a therapeutic treatment for COVID-19 in hospitalized patients [4–8].
Nafamostat is a highly polar drug that is rapidly hydrolyzed by esterases to form two inactive metabolites, 6-amidino-2-naphthol and p-guanidinobenzoic acid [9–11]. The instability of nafamostat presents a challenge for sample collection, storage and during process to support reliable quantification. The enzymes that hydrolyze nafamostat are found predominantly in erythrocytes but are also found in plasma [9]. This means that although centrifugation is an important step, it will not completely arrest hydrolysis.
There is a paucity of information about the stability of nafamostat in biological samples with only a few analytical methods to quantify nafamostat in plasma reported in the literature [10,11]. In the study published by Cao et al. [10], nafamostat was stable in human plasma at 4°C for 30 min, and hydrolysis was measured at 8% in human blood at 4°C for the time required for sample processing (not specified). Oh et al. [11] examined the stability of nafamostat in rat plasma, and their results indicated that an acidic pH was essential to inhibit enzymatic hydrolysis of nafamostat.
A phase 1b/2a study on the safety, pharmacokinetics and pharmacodynamics of nafamostat in patients with COVID-19 pneumonitis reported unmeasurable concentrations of nafamostat in plasma for almost all subjects [5]. However, nafamostat stability and sample collection and storage conditions were not reported.
To confidently advance research in this field, an understanding of the stability of nafamostat during dose administration, blood sample collection, and sample processing is required so that measured concentrations of nafamostat are reliable.
Therefore, the aim of our study was to establish appropriate conditions to support the administration and reliable measurement of nafamostat in whole blood and plasma. To achieve this, we sought to describe the stability of nafamostat in infusion solutions, the impact of the use of different blood collection tubes, the presence of an esterase inhibitor and different storage temperatures during sample collection and in extracted plasma samples in the autosampler. The presence of degradation product 6-amidino-2-naphthol was used to confirm degradation.
Materials & methods
Chemicals & reagents
Nafamostat mesylate (pharmaceutical formulation) was supplied by Chong Kun Dang Pharmaceutical (Seoul, Korea), and 6-amidino-2-naphthol hydrochloride (purity 95%) was purchased from Toronto Research Chemicals (Toronto, Canada). Gabapentin (purity 99.8%) and sodium fluoride (≥99%) were purchased from Sigma-Aldrich (MO, USA). Formic acid (≥99%) was of LCMS grade (Thermo Fisher Scientific, NJ, USA), and methanol (≥99.9%) of HPLC grade (Merck, Darmstadt, Germany). Ultrapure water from a Milli-Q system was used. Drug-free human plasma containing lithium heparin as anticoagulant was obtained from Innovative Research (MI, USA). Drug-free human whole blood for stability testing was obtained from a single donor (ethics approval no. HREC/15/QRBW/249).
Chromatographic conditions & detection
A Nexera ultra-high performance liquid chromatography (UHPLC) system consisting of dual pumps, degassers and autosampler (sample compartment set at 5°C) was used (Shimadzu, Kyoto, Japan). The stationary phase was an XBridge BEH C18 (2.1 × 30 mm, 2.5 μm) analytical column (Waters, CT, USA) preceded by a C18 SecurityGuard ULTRA HPLC cartridge (Phenomenex, CA, USA).
Mobile phase A was prepared in water containing 0.1% formic acid (v/v) and mobile phase B was methanol containing 0.1% formic acid (v/v). A gradient elution with a constant flow rate of 0.25 ml/min was used. The gradient started at 10% of mobile phase B for the first 0.75 min and then increased linearly to 45% of mobile phase B from 0.75 to 1 min. This condition was held from 1 to 1.5 min. The gradient was then decreased linearly returning to 10% of mobile phase B from 1.5 to 2 min. This final condition was held for 2 min. The total run time was 4 min and produced a backpressure of approximately 3000 psi.
The analyte detection was performed using a Shimadzu 8030+ triple quadrupole mass spectrometer equipped with an electrospray ionization turbo ion source operated in positive mode using LabSolutions version 5.99 (Shimadzu Corporation, Kyoto, Japan). A 100 ms dwell time was used for the detection of all analytes using a sum of multiple reaction monitoring mode with m/z transitions of 174.55→114.10, 174.55→118.05 and 174.55→140.95 for nafamostat, 187.00→170.05, 187.00→115.05 and 187.00→143.15 for 6-amidino-2-naphthol and 172.05→67.05 for gabapentin (internal standard). Nitrogen was used as nebulizing gas, with an interface setting consisting of the nebulizing gas flow of 3 l/min, a desolvation line temperature of 250°C, heat block temperature of 400°C, and a drying gas flow of 15 l/min. The collision gas was argon.
Preparation of calibration standards & quality controls
Nafamostat calibration standards and quality control samples (QCs) were prepared using drug-free human plasma containing lithium heparin as an anticoagulant. Calibration standards were prepared at nafamostat concentrations of 5, 10, 20, 50, 100 and 200 ng/ml, and QCs were prepared at nafamostat concentrations of 5, 15, 80 and 160 ng/ml. A separate set of calibration standards were prepared containing 6-amidino-2-naphthol at concentrations of 1, 2, 5, 10, 20, 50 and 100 ng/ml. No QCs were prepared containing 6-amidino-2-naphthol.
Plasma samples processing method
All plasma sample processing was performed in an ice-water bath. A volume of 50 μl of plasma (calibration standard, QC or test sample) was mixed with 10 μl of sodium fluoride (50 mg/ml). A 150 μl aliquot of internal standard solution (75 ng/ml of gabapentin in methanol) was added to the mixture, which was then vortex-mixed (5 s) and promptly centrifuged at 13,800 × g for 5 min at 4°C. The supernatant was transferred to an autosampler vial and a 2 μl aliquot sampled by the instrument for analysis.
Inter-assay precision of nafamostat
QCs of nafamostat in plasma at nominal concentrations of 15, 80 and 160 ng/ml were assayed in duplicate in three batches on three separate days. Acceptance criteria were precision and accuracy within ±15% at each concentration.
Stability of nafamostat
Intravenous infusion solutions
Nafamostat was prepared at low (385 mg/l) and high (909 mg/l) concentrations with two commonly used intravenous infusion solutions, 0.9% w/v sodium chloride and 5% w/v glucose. The infusion bags were stored in the light at room temperature (24 ± 2°C) and sampled in 0.5 ml aliquots at 5 min, 1, 2, 3, 4, 6 and 24 h. Aliquots of infusion solution samples were stored at -80°C until analysis. The aliquots were diluted 1:20 in water, and 30 μl of the diluted solution were mixed with 400 μl of internal standard (250 ng/ml of gabapentin in water); 2 μl were directly injected onto the LCMS with the same instrumental conditions as plasma to obtain nafamostat, 6-amidino-2-naphthol and internal standard peak areas. Nafamostat was considered stable in infusion solutions if the content was within 95–105%.
Sample collection stability
Drug-free whole blood was obtained from a single donor on the day of the experiment via venipuncture into a syringe and immediately split into six conditions (A–F):
| A. | 2.5 mg/ml sodium fluoride/potassium oxalate anticoagulant tube stored at 4°C | ||||
| B. | 2.5 mg/ml sodium fluoride/potassium oxalate anticoagulant tube +2.5 mg/ml sodium fluoride stored at 4°C | ||||
| C. | Lithium heparin anticoagulant tube stored at 4°C | ||||
| D. | 2.5 mg/ml sodium fluoride/potassium oxalate anticoagulant tube stored at room temperature (24 ± 2°C) | ||||
| E. | Lithium heparin anticoagulant tube stored at room temperature (24 ± 2°C) | ||||
| F. | Lithium heparin anticoagulant tube +2.5 mg/ml sodium fluoride stored at 4°C | ||||
Each blood collection tube (A–F) was prepared with a 3 ml volume, comprising 2.82 ml of the donated blood; 0.15 ml of 50 mg/ml sodium fluoride for tubes B and F or 0.15 ml water for tubes A, C, D and E; and 0.03 ml of 10 μg/ml nafamostat solution (to obtain a nafamostat blood concentration of 100 ng/ml). Tubes were mixed for 5 min on a rotatory mixer. After mixing was complete, each tube (A–F) was used to prepare five aliquots of 0.5 ml blood in labelled polypropylene vials. One vial from each set was immediately centrifuged at 3700 × g for 5 min at 4°C (0 h). The remaining blood vials were placed in the corresponding test conditions (A, B, C and F at 4°C, and D and E at room temperature). Vials were removed from the test condition and centrifuged, as previously described, at 1, 2, 3 and 4 h. The resulting plasma from all samples were stored at -80°C until analysis within a single batch. Plasma samples were processed using the procedure described in the plasma samples processing method section. Samples centrifuged immediately after preparation (0 h) were considered to have 100% nafamostat remaining. The proportion remaining in the subsequent injections was then calculated (accounting for autosampler degradation), with stability criteria of ≥90% remaining since initial processing deemed acceptable.
Extracted plasma samples stored in the autosampler
A set of six calibration standards, four QCs in duplicate, two internal standard-only samples (containing internal standard but no nafamostat) and one drug-free sample (containing neither internal standard nor nafamostat) in plasma were prepared and immediately extracted using the methodology described earlier. The 17 extracted samples were placed in the autosampler at 5°C and injected into the chromatographic system, taking 1.28 h (4.5 min per injection, with 32 s for the injection cycle time and 4 min of run time) to inject the batch. The batch injection was repeated another six times without pause, so that each sample was injected every 1.28 h.
The peak area ratio of nafamostat/internal standard from the initial injection batch were considered as 100% nafamostat remaining for each of the 14 non-zero samples. The proportion of nafamostat remaining was calculated for subsequent injections of each of the non-zero samples based on the peak area ratio of nafamostat/internal standard. The proportion remaining was plotted against time since initial injection for each sample, and the rate of degradation taken from the mean slope value. The acceptance criterion for autosampler stability was ±15% accuracy.
Results
Calibration
The calibration curve was linear in the range of 5–200 ng/ml; quadratic regression analysis with 1/concentration2 weighting was applied (mean r2 = 0.9978, all calibrators were within ± 11.7%). The limit of detection (based as 3 times signal/noise) and lower limit of quantification were 2.6 and 5 ng/ml, respectively.
Inter-assay precision
The inter-batch precision was calculated for quality control samples (n = 6) at 15, 80 and 160 ng/ml with a resulting precision of 10.1, 11.5 and 3.3% and accuracy of 96.9, 102.7 and 98.8%, respectively (Table 1).
| Nominal concentration (ng/ml) | 15 | 80 | 160 |
| Mean concentration (ng/ml) | 14.5 | 82.1 | 158 |
| Standard deviation | 1.5 | 9.4 | 5 |
| Precision | ±10.1% | ±11.5% | ±3.3% |
| Accuracy | 96.9% | 102.7% | 98.8% |
Stability in infusion solutions
The four infusion conditions provided similar results over 24-h storage at room temperature when exposed to light. Figure 1 presents the percent drug remaining over time (mean ± standard deviation [SD]) of the low and high concentration solutions for 0.9% w/v sodium chloride (A) and 5% w/v glucose (B). These results show the achievement of the pre-established acceptance criteria. 6-amidino-2-naphthol was detectable in these solutions, but the area of the peak represented <0.1% of the nafamostat peak area.
Sample collection stability
The proportion of nafamostat remaining in the blood samples collected under different conditions in the sample collection stability test is shown in Figure 2A. The measured concentrations of 6-amidino-2-naphthol in the sample collection stability test is shown in Figure 2B. Table 2 shows the percent remaining for each condition, highlighting the duration that nafamostat remains stable.
| Time (h) | 0 | 1 | 2 | 3 | 4 | |
|---|---|---|---|---|---|---|
| Nafamostat remaining (%) | A. 2.5 mg/ml sodium fluoride/potassium oxalate at 4°C | 100† | 103† | 99† | 91† | 89 |
| B. 2.5 mg/ml sodium fluoride/potassium oxalate +2.5 mg/ml sodium fluoride at 4°C | 100† | 102† | 97† | 103† | 98† | |
| C. Lithium heparin at 4°C | 100† | 94† | 94† | 81 | 73 | |
| D. 2.5 mg/ml sodium fluoride/potassium oxalate at room temperature (24 ± 2°C) | 100† | 89 | 76 | 64 | 48 | |
| E. Lithium heparin at room temperature (24 ± 2°C) | 100† | 90† | 76 | 81 | 54 | |
| F. Lithium heparin +2.5 mg/ml sodium fluoride at 4°C | 100† | 104† | 100† | 95† | 92† |
Autosampler stability of nafamostat in extracted plasma
Figure 3 presents the changes to the nafamostat and 6-amidino-2-naphthol peak area ratio (calculated against the internal standard area) over time for the nafamostat quality control sample at 160 ng/ml as an illustration. The percent of nafamostat remaining over time based on peak area ratio for sample extracts are presented in Figure 4. The rate of nafamostat degradation is 4.7 ± 0.7% per hour (mean ± SD). Autosampler stability is acceptable (±15%) for approximately 3 h.
Discussion
In this work, we studied the stability of nafamostat in infusion solutions, the impact of the use of different blood collection tubes, the presence of an esterase inhibitor and different storage temperatures during sample collection and in extracted plasma samples in the autosampler. The results obtained suggest that nafamostat is stable in 0.9% w/v sodium chloride and 5% w/v glucose infusion solutions stored in the light at room temperature for up to 24 h; nafamostat was most stable when blood was collected into sodium fluoride/potassium oxalate anticoagulant when stored at 4°C until centrifugation within 3 h, and stability was further improved in presence of an esterase inhibitor (+2.5 mg/ml sodium fluoride added); and nafamostat in extracted plasma samples stored in the autosampler at 5°C for up to 8 h degrades at a rate of approximately 4.7 ± 0.7% per h.
The infusion solutions are free of esterases or other conditions to hydrolyze nafamostat, which is why nafamostat showed excellent stability in infusion solutions. However, this study only evaluated the stability of nafamostat in infusion solutions exposed to light at room temperature for 24 h; the stability of nafamostat in these solutions has yet to be comprehensively demonstrated under different temperature conditions and for longer periods of time.
In the sample collection stability test, nafamostat stability was improved when blood was collected into sodium fluoride/potassium oxalate versus heparin anticoagulant tubes (with 91 vs 81% remaining after 3 h, respectively).
The presence of the esterase inhibitor sodium fluoride in the blood collection tube was expected to reduce the hydrolysis of nafamostat. In fact, additional sodium fluoride further increased the stability of nafamostat (92% remaining after 4 h when blood was collected into lithium heparin tubes and chilled, and 98% remaining after 4 h when blood was collected into sodium fluoride/potassium oxalate tubes and chilled).
Temperature of storage was a significant factor for nafamostat stability; room temperature storage led to poor stability after only 1 h (with 89% remaining when blood was collected into sodium fluoride/potassium oxalate tubes and 90% remaining when blood was collected into lithium heparin tubes). Storing the vials at 4°C before centrifugation led to improved nafamostat stability (with 94% remaining after 2 h when blood was collected into lithium heparin tubes, and 91% remaining after 3 h when blood was collected into sodium fluoride/potassium oxalate tubes).
Although the concentrations of 6-amidino-2-naphthol observed in Figure 2B demonstrate the difficulty in preventing degradation of nafamostat in biological specimens, our results indicate that further degradation can be limited in the presence of an esterase inhibitor on collection and chilling.
Sodium fluoride has the significant advantage of being a commercially available component of blood collection tubes, either as sodium fluoride/potassium oxalate or sodium fluoride-K2EDTA. Whereas the addition of extra sodium fluoride (or alternate enzyme inhibitors) to the blood collection tube before or immediately after blood collection can improve the stability of nafamostat, an off-the-shelf blood collection tube that is chilled until centrifugation is a simple and practical solution for sample collection.
In the extracted plasma samples, the peak area of nafamostat reduced in subsequent injections over the almost 8 h of storage in the autosampler at 5°C and was accompanied by an increase in the 6-amidino-2-naphthol area. Although nafamostat degradation meets the criteria (±15%) for 3 h, we have chosen to correct for this degradation to control this predictable error (4.7% per hour) when measuring nafamostat in biological samples. In all cases, the presence of 6-amidino-2-naphthol was measurable, although the concentration of 6-amidino-2-naphthol does not fully account for the concentration of nafamostat lost. In this study, we have monitored 6-amidino-2-naphthol as did Cao et al., whereas Quinn et al. measured p-guanidinobenzoic acid (also known as 4-guanidinobenzoic acid) [5].
The recent investigation by Quinn et al. into the pharmacokinetics of nafamostat in COVID-19 patients reported unmeasurable levels of nafamostat (lower limit of quantitation not stated) in 19 of 21 subjects (sample collection not reported) but levels of the hydrolysis product p-guanidinobenzoic acid were detectible [5]. Furthermore, blood spiked with nafamostat (50 ng/ml) showed little degradation and negligible hydrolysis product over 80 min, although the anticoagulation and temperature conditions were not specified [5]. The degradation of nafamostat in whole blood at 37°C is rapid, and Quinn and colleagues postulated that in patients hospitalized with COVID-19 pneumonitis, the breakdown is such that there are very low levels of circulating nafamostat [5].
Oh et al. tackled the instability of nafamostat in animal studies by promptly centrifuging rat blood and spiking the resultant plasma with hydrochloric acid [11]. Stability testing results were excellent for plasma at pH 2.2 or below. However, this intervention has not been tested in human blood and does not tackle instability before centrifugation.
Limitations of the study include that the analytical technique to quantify nafamostat in plasma by UHPLC–MS/MS is only partially validated, matrix effect was not evaluated, the stability in infusion solutions and sample collection stability experiments were not done in any replicates and no robust statistical analyses were performed. Furthermore, we did not assess whole blood partitioning and nonspecific binding of nafamostat to plastic tubes, which might be useful in strengthening the assumption of stability of nafamostat in various tubes and autosampler conditions. It is possible that alternate sample preparation techniques, such as that by Cao et al., that extract nafamostat from biological moieties such as esterases, or the addition of hydrochloric acid as used by Oh et al. with rat plasma, may enhance the stability of extracted samples. Additionally, the blood used in the experiments was sourced from a single healthy volunteer and the plasma from a limited number of volunteers, so we were unable to test blood from special sources such as COVID-19 patients and donors with extremely high or low esterase levels.
Conclusion
The stability of nafamostat in infusion solutions, during sample collection under different conditions (different blood collection tubes, the presence of an esterase inhibitor and different storage temperatures) and in extracted plasma samples in the autosampler was investigated. Stable specimens of nafamostat in plasma can be obtained by sampling into sodium fluoride/potassium oxalate blood collection tubes and storing them at 4°C for up to 3 h before centrifugation. Further stability can be obtained through the use of higher concentrations of sodium fluoride.
Other interventions such as acidification with hydrochloric acid (as used by Oh et al.) as a condition for human blood sample stability before centrifugation should be investigated.
Preventing degradation of nafamostat in biological specimens is difficult.
The instability of nafamostat presents a challenge to support reliable quantification.
Nafamostat is stable in infusion solutions exposed to light at 24 ± 2°C for 24 h.
Nafamostat in blood collected in sodium fluoride tubes showed improved stability.
Further stability was attained when adding an esterase inhibitor.
Chilling the vials to 4°C before centrifugation improved nafamostat stability.
Room temperature storage led to poor nafamostat stability after only 1 h.
The rate of nafamostat degradation in extracted plasma is 4.7 ± 0.7% per hour.
Author contributions
All authors made substantial contributions to the conception or design of the work, drafted and revised the work, approved the final version to be published and will ensure that questions related to the accuracy or integrity of the work are appropriately resolved.
Financial & competing interests disclosure
Nafamostat mesylate was kindly donated by CKD Pharma. JA Roberts acknowledges funding from the Australian National Health and Medical Research Council for a Centre of Research Excellence (APP2007007), and an Investigator Grant (APP2009736) and an Advancing Queensland Clinical Fellowship. Unrelated to this article, JA Roberts declares the following items: Consultancies/Advisory Boards – Qpex (2022); Gilead (2022); Pfizer (2020); Sandoz (2020); Wolters Kluwer (2021); MSD (2019); Summit Pharma (2021). Speaking Fees – MSD (2022); Gilead (2022); Pfizer (2021); Cipla (2021). Industry Grants – QPEX (2021), British Society of Antimicrobial Chemotherapy (2021); Biomerieux (2019); Pfizer (2019). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Open access
This work is licensed under the Creative Commons Attribution 4.0 License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Papers of special note have been highlighted as: • of interest; •• of considerable interest
References
- 1. Nafamostat mesylate blocks activation of SARS-CoV-2: new treatment option for COVID-19. Antimicrob. Agents Chemother. 64(6), 1–3 (2020).
- 2. . Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells: nafamostat is the most potent antiviral drug candidate. J. Med. Virol. 93(3), 1403–1408 (2020).
- 3. The anticoagulant nafamostat potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. Viruses 12(629), 1–9 (2020).
- 4. Nafamostat in hospitalized patients with moderate to severe COVID-19 pneumonia: a randomised phase II clinical trial. EClinicalMedicine 41, 101169 (2021).
- 5. Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: phase 1b/2a experimental study to investigate safety, pharmacokinetics and pharmacodynamics. eBioMedicine 76, 103856 (2022).
- 6. Multicenter, single-blind, randomized controlled study of the efficacy and safety of favipiravir and nafamostat mesilate in patients with COVID-19 pneumonia. Int. J. Infect. Dis. 128, 355–363 (2022).
- 7. ASCOT ADAPT study of COVID-19 therapeutics in hospitalised patients: an international multicentre adaptive platform trial. Trials 23(1), 1–11 (2022).
- 8. Nafamostat mesylate for treatment of COVID-19 in hospitalised patients: a structured, narrative review. Clin. Pharmacokinet. 61(10), 1331–1343 (2022).
- 9. Involvement of human blood arylesterases and liver microsomal carboxylesterases in nafamostat hydrolysis. Drug Metab. Pharmacokinet. 21(2), 147–155 (2006). • Study that identified the enzymes that metabolize nafamostat in human blood.
- 10. A method for quantifying the unstable and highly polar drug nafamostat mesilate in human plasma with optimized solid-phase extraction and ESI-MS detection: More accurate evaluation for pharmacokinetic study. Anal. Bioanal. Chem. 391(3), 1063–1071 (2008). •• Bioanalysis of nafamostat by HPLC–MS to quantify nafamostat in human plasma.
- 11. Pharmacokinetics of nafamostat, a potent serine protease Inhibitor, by a novel LC–MS/MS analysis. Molecules 27, 1–14 (2022). • LC–MS/MS method to quantify nafamostat in rat plasma.
