Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics

Abstract

Background: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate.

Methods: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis.

Findings: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry.

Interpretation: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events.

Funding: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Keywords: Infectious diseases; Nafamostat mesylate; Respiratory medicine; SARS-CoV-2/COVID-19; Total manuscript word count.

Figure 1

Consort flow diagram.

Figure 2

a: Clinical course for each patient from hospital admission to discharge or death. Study identifier code, age, gender and ISARIC score (score 0–21 points) given on y-axis with clinical course, time on trial as well as key clinical events reported. b: Kaplan-Meier plot reporting duration of hospital stay following randomisation. Numbers at risk in each trial arm are shown at the bottom of the figure (just above the x-axis).

Figure 2

a: Clinical course for each patient from hospital admission to discharge or death. Study identifier code, age, gender and ISARIC score (score 0–21 points) given on y-axis with clinical course, time on trial as well as key clinical events reported. b: Kaplan-Meier plot reporting duration of hospital stay following randomisation. Numbers at risk in each trial arm are shown at the bottom of the figure (just above the x-axis).

Figure 3

Pharmacokinetics of IV nafamostat. a: Ratio of Post-NM to Pre-nafamostat in each participant in the nafamostat arm, measured by mass spectrometry with a detection m/z [M+H]+=348.1455. b: Assessment of nafamostat metabolite 4-GBA in samples by mass spectrometry at detection of m/z [M+H]+= 180.0767. c: 4-GBA concentration in patients samples taken pre-infusion and at early (2 h) and later time points (>6 h) (plotted for 12 patients in whom matched samples available). **** p value <0.0001, * p value = 0.02. Error bars: SD. d: To assess whether the conversion of nafamostat to 4-GBA could have happened ex-vivo prior to spinning, whole blood was ‘spiked’ with an aliquot of nafamostat (50 ng/ml), and samples were incubated for different periods (t0-t80min). Plasma was generated at the time points and the samples were snap-frozen before analysis performed using RP-MS. A standard curve was prepared to quantify 4-GBA levels in each sample. The area under the total ion chromatogram for each compound in each sample was integrated to obtain the intensity. Error bars: SEM.

Figure 4

a and b – copies per mL of E and S gene respectively in nasopharyngeal and oropharyngeal swabs. c and d – copies per mL of E and S gene respectively in saliva. In both groups, the numbers of participants declined with time representing participant discharge or withdrawal (nafamostat D1 n = 10, D3 n = 9, D5 n = 8. SOC D1 n = 11, D3 n = 10, D 5 n = 3).

Figure 5

Thromboelastometry (ClotPro®). Blood tests performed daily on all patients. Presented here as pooled groups depending on treatment given at time of blood draw. Results pooled to three groups- no anticoagulant treatment (No Tx), on IV NM (nafamostat), on other therapeutic anticoagulation e.g. apixaban/ dalteparin (Anti coag). Significance by one-way ANOVA and Student's t-test. a: Panel evaluating anticoagulant activity (clotting time). **** p <0.001, * p = 0.02. 1: CT EX test, 2: CT FIB test, 3: CT IN test, 4: CT RVV test, 5: CT NA test. b: Panel evaluating antifibrinolytic activity (lysis time and maximum lysis percentage). **** p <0.0001. 6: ML EX test, 7: LT TPA test. c: Panel evaluating clot strength (maximum clot firmness). * p = 0.04. 8: MCF FIB test, 9: MCF EX test. Six ClotPro tests were performed on whole blood samples daily: EX-test: assessment of coagulation with extrinsic activation using recombinant tissue factor. FIB-test: assessment of coagulation with extrinsic activation but in the presence of inhibitors of platelet aggregation. Measures the fibrin contribution to clot strength. IN–test: assessment of coagulation with intrinsic activation using ellagic acid. RVV-test: screening test for direct FXa antagonists, also sensitive to thrombin antagonists. NA-test: non-activated test - assessment of coagulation without an activator of coagulation. TPA-test: assessment of coagulation with fibrinolysis activation (using recombinant tissue plasminogen activator). Measures the effect of anti-fibrinolytic drugs.

Figure 6

Biomarkers over time. SOC (blue)- standard of care. NAF (pink)- Nafamostat Mesylate. a-d: Changes of COVID-19 cytokine storm related cytokines IL-6, IL-8, CXCL-10, IL-1RA. e-l: Clinical biomarkers: creatinine, CRP, D-dimer, neutrophil: lymphocyte ratio, AST, GGT, creatinine kinase and potassium. m-p: Biomarkers of coagulation such as protein C, antithrombin, APTT & fibrinogen. Data presented as mean with best-fit line and 95% confidence intervals by linear regression. All NM patients included in analysis including when off infusion.

Figure 7

Peripheral blood immunophenotyping. Flow cytometric analysis of circulating leukocytes was performed on recruitment prior to treatment and on D4 and D7 on patients in hospital. Histograms illustrate the proportion of: a: Activated CD8+ T cells. b: activated CD4+ T cells. c: CD38+CD27+ antibody secreting cells. d: CXCR3+ CD8+ T cells. e: Classical monocytes (CD14+CD16-), f: Transitional monocytes (CD14+CD16+). g: Non-classical monocytes (CD14lowCD16high). h: CX3CR1 monocytes. i: immature neutrophils. Filled histograms show patients on SoC (D1 n = 13, D4 n = 13 D7 n = 7). Open histograms show NM treated patients (D1 n = 12, D4 n = 12, D7 n = 7). Error Bars show standard deviation.