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Supplementary Information
The online version contains supplementary material available at 10.1186/s12913-022-08994-0.
PMC9807970
Keywords
PMC9807970
Background
SECONDARY
Out-of-hours primary care service is a crucial part of a well-functioning health care system, which provides health care when general practitioners’ (GPs) offices are closed. The member countries of the Organisation for Economic Co-operation and Development have selected different models to provide this kind of service to their populations [The non-urgent use of out-of-hours service, both in primary care and emergency department setting, is of concern, because it may crowd out patients with the highest need and lead to inefficient use of resources [Some countries, such as the UK, have a national telephone triage and advice system. Norway has one national telephone number for out-of-hours service; however, nurses in local out-of-hours GP cooperatives handle the calls from the local population. A review from 2017 suggested that more than 50% of calls to the telephone triage and advice system could be dealt with by providing telephone advice alone [High variation has been reported in measured appropriateness of advice provided by the telephone triage and advice services [Overall, telephone triage in out-of-hours care appears to be safe because high-risk patients are mostly identified and handled correctly [Norwegian citizens are entitled to have a list-holding GP who offer planned and emergent service for their listed patients in-hour [High primary care physician continuity leads to lower costs in health care, fewer hospitalizations, and lower mortality [Specific training may strengthen telephone triage nurses’ gatekeeping capacity. However, insufficient evidence is available to conclude the effectiveness of interventions aimed at reducing emergency department attendance [The primary aim of this study was to assess the effect of an educational intervention about RTIs and communication skills for telephone triage nurses on out-of-hours attendance for RTIs. The secondary aims were to describe the distribution of RTIs between list-holding GPs and out-of-hours GP cooperatives and assess the intervention’s effect on list-holding GPs’ attendance.
PMC9807970
Methods
PMC9807970
Study design
This is a pragmatic randomized controlled educational intervention among Norwegian telephone triage nurses working in out-of-hours GP cooperatives. All Norwegian out-of-hours GP cooperatives that met our inclusion criteria were included in the study (Table Inclusion criteria for out-of-hours GP cooperativesThe out-of-hours services in the two largest cities of Norway, Bergen, and Oslo, were not included owing to their policy of direct attendance. Out-of-hours cooperatives serving less than 10,000 inhabitants were excluded as they normally will have less than 6 consultations per 24 hours and therefore are less busy [A total of 64 out-of-hours GP cooperatives serving 3.4 million inhabitants (median population size per out-of-hours GP cooperative 36.048, range 11.490–236.202) met the inclusion criteria and were randomized to intervention or control. An independent researcher performed the randomization using the RAND command in Microsoft Excel (2016) in November 2018, with stratification by the population size (small 10,000–40,000; large > 40,001).We contacted the administration of the 32 out-of-hours cooperatives in the intervention group through e-mail or telephone and invited them to participate in a meeting during the annual leadership conference for the primary care out-of-hours service in Norway (Lederkonferansen 2019) in March 2019. In this meeting, we presented the educational intervention and explained how they could implement it locally without any external support. Nineteen persons from 16 out-of-hours GP cooperatives participated. BHL contacted the other 16 out-of-hours GP cooperatives, three by personal contact during the conference and the remaining via telephone calls after the conference. One of the 32 cooperatives refused to participate due to time constraint, while the other 31 accepted the invitation and were included in the study. Data from one out-of-hours cooperative in the intervention group, in addition to one out-of-hours cooperative and one municipality in the control group, were excluded from the study because of the merging of municipalities across the two groups during the study period. Data from two out-of-hours cooperatives (serving 180,989 inhabitants) in the control group were excluded because of data error. As a result, 59 out-of-hours GP cooperatives serving 59% of the Norwegian population, were included in the intention-to-treat analysis (Fig. Flow chart of out-of-hours cooperatives included and reasons for exclusion after randomization
PMC9807970
The intervention
RTIs
MAY, APPENDIX
The intervention consisted of two parts: A 90 minute e-learning course about acute RTIs and general communication techniques for telephone triage nurses (see Appendix A 90 minute group discussion for the telephone triage nurses. Dedicated local nurses led the group sessions based on a written guide (Appendix The intervention was piloted by ten telephone triage nurses at one out-of-hours GP cooperative in May 2019 and adjusted based on the feedback from these nurses.The unique activity reports (Appendix
PMC9807970
Variables
infection, pneumonia
PNEUMONIA, ACUTE BRONCHITIS, ACUTE TONSILLITIS, URINARY TRACT INFECTIONS, ACUTE SINUSITIS, EAR INFECTIONS, INFECTION, SECONDARY, ACUTE LARYNGITIS, INFLUENZA
GP offices and out-of-hours GP cooperatives send electronic compensation claims to the Norwegian Health Economics Administration. The claims contain data on date and type of contact (phone, consultation, or home visit) with GP offices and out-of-hours GP cooperatives as well as age, gender, and diagnosis of the caller/patient. We retrieved these data from the National Directory of Health [We did not have access to the number of nurses working in each out-of-hours cooperative during the intervention. The number of nurses who had just started or completed the e-learning course per 1000 inhabitants in each region was therefore used as a proxy for the proportion of trained nurses in each out-of-hours GP cooperative.The primary outcome was the change in the number of out-of-hours doctor’s consultations for RTIs per 1000 inhabitants in the intervention group versus the control group during the winter months before (December 2018–February 2019) and after (December 2019–February 2020) the intervention. The secondary outcomes per 1000 inhabitants during the same two periods were:the change in the number of list-holding GPs’ consultations for all diagnosesthe change in the number of list-holding GPs’ consultations for RTISthe change in the number of telephone contacts for all diagnoses for list-holding GPsthe change in the number of nurses’ telephone consultations for all diagnoses in the out-of-hours GP cooperativesWe defined RTIs according to the International Classification of Primary Care, 2nd edition, as the following groups: ‘respiratory symptoms’ (R01–05, R08–09, R21 and R23–29), acute tonsillitis (R72 and 76), acute RTIs (R74), acute sinusitis (R75), acute laryngitis (R77), acute bronchitis (R78), influenza infection (R80), pneumonia (R81–82), ‘other RTIs’ (R71, R83 and R99) and ear infections (H01, H29, H70–72 and H74). We also defined the groups urinary tract infections (U01–02, U07, U13 and U70–72), ‘other conditions’ (A03, A76–78), ‘unspecified’ (A99) and ‘all other diagnoses’ (all International Classification of Primary Care codes not included in any of the above-mentioned groups).
PMC9807970
Statistical analysis
REGRESSION
We used frequencies and percentages to describe the distribution of the sample population and count data relating to contacts and consultations made in the control and intervention arms, and StataSE 17 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC) for the statistical analyses.The Poisson regression is the basic model used for modelling count data. It assumes that the mean and the variance of the count variable are approximately equal. We checked this model assumption using the dispersion statistic, which should be equal to one. However, our Poisson model output gave the dispersion statistic equal to 24.2. In addition, the appropriateness of the negative binomial model over the Poisson regression model was checked using the Bayesian information criterion (BIC), which states that the model with the smaller BIC estimate is preferred over the model with a larger BIC. The negative binomial model had the smaller BIC estimate. Thirdly, we obtained a Z score test of 16.1, with a t-probability < 0.01. This test evaluates whether the data are Poisson or negative binomial. Based on this result, we rejected the null hypothesis of no overdispersion in the data. Further, we used the likelihood-ratio test of alpha = 0, to test if the dispersion parameter, was equal to zero, which would have reduced the model to a Poisson regression. Our results showed that alpha was significantly greater than zero. This implies that the data were over-dispersed, hence using the negative binomial model instead of the Poisson model was appropriate.The negative binomial regression model was used with random effects at the level of out-of-hours GP cooperatives to check for any baseline differences between the two groups (Table We obtained estimates of incidence rate ratios (IRRs) for the negative binomial regression model. An IRR > 1 indicates an increase in counts in one group relative to the reference group or one period relative to the reference period, whereas an IRR < 1 indicates a decrease in these parameters. An IRR = 1 indicates no difference in the number of counts between the groups or periods.
PMC9807970
Results
PMC9807970
Population and epidemiology at baseline
sore throat, Laryngitis
EAR INFECTIONS, RTI, SORE THROAT, LARYNGITIS
The 59 out-of-hours cooperatives served 3.12 million inhabitants (59% of the Norwegian population) in 198 (of 356) municipalities. There were 4.72 million contacts in primary health care in these municipalities in the baseline period, of which 4.37 million were list-holding GPs’ consultations and 0.35 million were GP consultations in the out-of-hours service.Women constituted 52.5 and 57.8% of the patients who received service in out-of-hours cooperatives and GP offices, respectively. Patients were relatively younger in the out-of-hours cooperatives than in the GP offices (Fig. Proportion of age groups in GP offices and out-of-hours GP cooperatives, all diagnoses. For RTIs, 34.4 and 16.5% of children under 5 years of age received a diagnosis in out-of-hours cooperatives and GP offices, respectively (Fig. Proportion of age groups in GP offices and out-of-hours GP cooperatives, RTIs. Respiratory symptoms and acute upper RTI were the two most frequently diagnosed RTIs (Fig. Proportion of different RTIs in GP offices and out-of-hours GP cooperatives. All the differences between GP offices and out-of-hours GP cooperatives, except for ear infections, were statistically significant (The proportion of RTIs diagnosed was higher in out-of-hours cooperatives than in the GP offices (22.9% versus 14.0%). Laryngitis, other conditions (viral RTIs frequently diagnosed in children) and sore throat were the most common diagnoses in the out-of-hours GP cooperatives (Fig. Proportion of conditions diagnosed in out-of-hours GP cooperatives
PMC9807970
Discussion
The patient population attending the out-of-hours service during the winter season was younger and had a higher proportion of RTIs than the population attending the regular GP offices. The educational intervention about RTIs and communication skills for telephone triage nurses did not decrease the number of consultations for RTIs in the out-of-hours GP cooperatives.
PMC9807970
Population and epidemiology at baseline
sore throat, sinusitis, streptococci
SORE THROAT, SINUSITIS, ACUTE LARYNGITIS, STREPTOCOCCAL TONSILLITIS
The out-of-hours patient population is characterized by a higher proportion of children and teenagers compared with list-holding GPs’ patient population, especially for RTIs. Danish parents reported a perceived need for prompt action, unpleasant symptoms and worry as some of the main reasons for contacting the out-of-hours service [The proportion of sore throat diagnosed in the out-of-hours cooperatives is high. In our data, sore throat is consistent with suspected or detected streptococcal tonsillitis. There is a strong Norwegian tradition of performing rapid strep A test and initiating treatment with penicillin V if streptococci are confirmed. This tradition may contribute to high expectations in the population of a doctors’ assessment for sore throat, leading to high out-of-hours consultation rates [Acute laryngitis is a hyperacute condition and tends to arise during night-time. Therefore, the high proportion of patients being diagnosed with this condition out-of-hours is unsurprising. However, the proportion of sinusitis cases was low. This finding is consistent with the low-urgency nature of sinusitis and the existing guidelines recommending a wait-and-watch attitude for this condition [
PMC9807970
Effect of the intervention
SECONDARY
Dutch GPs believe that a stricter triage and annual feedback to triage nurses will reduce the number of non-urgent visits to the out-of-hours cooperatives [The decision to seek health care is a complex process influenced by several factors, such as personal, social, and cultural, as well as characteristics of the health care system [Hence, there may be several reasons for the lacking effect of our educational intervention on the primary and secondary outcomes. Strategies aimed at reducing out-of-hours attendance by improving the skills of individual nurses imply that they perform an active gatekeeping role, keeping low-urgency patients out of the cooperatives. From this perspective, an educational intervention similar to the one described in the present study could be expected to have a direct effect on the out-of-hours attendance, particularly because nurses report that the decision regarding the need for a doctor’s consultation is generally made in co-operation between the nurse and caller [However, our e-learning course and group discussion of 90 minutes each may be too limited to oust the external factors telephone triage nurses describe as decisive and non-controllable [
PMC9807970
Methodological considerations
coronavirus disease 2019
REGRESSION, DISEASE, CORONAVIRUS DISEASE 2019, SECONDARY
The data from the Norwegian Directory of Health are reliable and a good source of information about the study population as well as activity in primary health care [The large proportion of the population being covered by the included out-of-hours GP cooperatives, in addition to narrow confidence intervals, indicate that the present study has statistical power to answer our research question.The e-learning course encouraged the nurses to not overrule the triage system in use in their own out-of-hours GP cooperative. Because many triage systems are less specific with regard to RTIs, generally with high sensitivity and low specificity for serious disease, this may have been an obstacle for change [The pragmatic intention-to-treat design yielded limited control on the implementation of the intervention. We do not know how many nurses completed the two educational parts, and how many nurses had access to the course without attending it. This makes the assessment of the effect of the intervention more uncertain. To compensate for this weakness, we included the number of nurses who had started on or completed the e-learning course per 1000 inhabitants as a variable in the regression model. This variable did not have a significant effect (IRR 0.98, 95% CI 0.80–1.19). The sensitivity analysis of this variable was not possible to perform, since the nurses in the control group were not exposed and the proportion of nurses in this group is zero.A longer intervention period might have increased the likelihood of a higher participation rate among nurses, but it would also increase the risk of external bias from the coronavirus disease 2019 pandemic. Hence, it is difficult to conclude about a lacking effect of the intervention based only on the primary and secondary outcomes. A pragmatic design is closer to real life and healthcare. We therefore considered it a pertinent method.
PMC9807970
Conclusion
SECONDARY
The described educational intervention for telephone triage nurses did not influence the out-of-hours attendance for RTIs or list-holding GPs’ attendance. Changing a population’s health service attendance is complicated, and the intervention’s pragmatic design with a lack of control on the proportion of participating nurses, and its limited scope, may be reasons for the missing effect on the primary and secondary outcomes. Future studies on educational interventions should include a process evaluation, be planned for a longer period, and involve both health planning councils and leaders at different levels, as well as GPs and nurses in out-of-hours cooperatives. Furthermore, a need exists to explore the perspective of callers with RTIs who are advised to wait-and-watch or seek their list-holding GPs to reduce non-urgent use of the out-of-hours service.
PMC9807970
Acknowledgements
RESPIRATORY TRACT INFECTIONS, SCHMIDT, EMERGENCY, HANSEN
Ibrahimu Mdala (Researcher, Department of General Practice, Institute of Health and Society, University of Oslo) provided invaluable help with the statistical analyses and descriptions. Arngeir Berge (Senior Adviser and Educationalist, National Centre for Emergency Primary Health Care, Norway) converted our texts about communication and respiratory tract infections into a vivid and interesting e-learning course. Elisabeth Holm Hansen (Professor, Faculty of Health and Social Sciences, University of South-Eastern Norway) and Arnstein Finset (Professor Emeritus, Faculty of Medicine, University of Oslo) validated the content of the e-learning course. A group of experienced out-of-hours telephone triage nurses (Siri-Linn Schmidt Fotland, Astrid Onarheim Spjeldnæs and Kjersti Stadshaug) provided valuable feedback, and the course was adjusted accordingly.
PMC9807970
Authors’ contributions
All authors contributed to the study design and the educational intervention. IKR identified the out-of-hours cooperatives that met the inclusion criteria. BHL administrated the intervention, prepared the raw data for analysis, and performed the analysis with support from SH. All the authors contributed to the interpretation of the findings. BHL wrote the initial manuscript draft. All the authors reviewed and refined the manuscript and have approved the final version.
PMC9807970
Funding
This work was supported by The Norwegian Research Fund for General Practice as a PhD grant to the first author.
PMC9807970
Availability of data and materials
The raw data analysed during the current study contains person sensitive information and publication of these data has not been approved by The Norwegian Data Protection Authority. However, a modified dataset is available from the corresponding author on reasonable request.
PMC9807970
Declarations
PMC9807970
Ethics approval and consent to participate
The study protocol was assessed by The Regional Committee for Research Ethics (2018/1080/REK sør-øst C), which concluded that the study, according to the Norwegian Act on medical and health research, did not need ethical approval [
PMC9807970
Consent for publication
Not applicable.
PMC9807970
Competing interests
The authors declare that they have no competing interests.
PMC9807970
References
PMC9807970
Background
migraines
MIGRAINES
The phase 3 randomized PERSIST study demonstrated the efficacy and tolerability of galcanezumab, a humanized anti-calcitonin gene-related peptide (CGRP) monoclonal antibody for prevention of episodic migraines. We present findings from the open-label extension (OLE) of PERSIST, which evaluated the long-term efficacy and safety of galcanezumab in patients from China, India, and Russia.
PMC10401806
Methods
migraine headache
MIGRAINE HEADACHE
Patients completing the 3-month double-blind period of PERSIST were eligible for the 3-month OLE. Patients previously randomized to galcanezumab (GMB/GMB group) continued to receive galcanezumab 120 mg at all three visits during the OLE whereas patients randomized to placebo received a 240 mg loading dose of galcanezumab and then two 120 mg doses (PBO/GMB group). The primary outcome was the mean change (from double-blind baseline) in the number of monthly migraine headache days (MHDs) to month 6. Other endpoints included percent reduction in monthly MHDs from double-blind baseline to month 6, functional outcomes, safety and tolerability.
PMC10401806
Results
ADVERSE EVENT
Overall, 99% of patients completing the double-blind period entered the OLE, and 96% completed through month 6. Patients in the GMB/GMB group achieved continued improvements in efficacy, with the reduction from baseline in the mean number of monthly MHDs, and slightly increasing from 4.01 days at the end of the double-blind period to 4.62 at the end of the OLE. Of patients who were ≥ 50% responders to galcanezumab at month 3, 66% maintained this response through to month 6. Patients in the PBO/GMB group experienced a rapid reduction in the number of monthly MHDs after initiation of galcanezumab, with a mean reduction from baseline of 4.56 days by month 6. The long-term benefits of galcanezumab were also supported by improvements in other efficacy and functional endpoints. All safety findings were consistent with the known long-term safety profile of galcanezumab; no patients experienced a treatment-related serious adverse event.
PMC10401806
Conclusions
migraine
MIGRAINE
Galcanezumab was efficacious and well-tolerated in patients with episodic migraine from China, India and Russia, for up to 6 months.
PMC10401806
Trial registration
MAY
ClinicalTrisABSTRACT_pals.gov NCT03963232, registered May 24, 2019.
PMC10401806
Supplementary Information
The online version contains supplementary material available at 10.1186/s10194-023-01613-1.
PMC10401806
Keywords
PMC10401806
Background
migraine, Migraine, migraines
MIGRAINE, MIGRAINE, MIGRAINES
Migraine was estimated to affect more than one billion individuals worldwide in 2019 [Among individuals affected by migraines, around one-third (34–39%) may be candidates for preventive therapy [Calcitonin gene-related peptide (CGRP) has a key pathophysiological role in migraine and is expressed widely in the central and peripheral nociceptive system [Galcanezumab has been assessed in multiple phase 3 placebo-controlled trials for episodic, chronic, and treatment resistant migraine [
PMC10401806
Methods
PMC10401806
Study design and treatment
The phase 3 PERSIST study (NCT03963232) was conducted at 26 centers in China, 20 in India, and 4 in Russia (40 total). There were five study periods (Suppl. Figure 
PMC10401806
Patients
Eligibility criteria for the PERSIST trial have been described previously [
PMC10401806
Assessments and endpoints
reductions of ≥, deaths, migraine-associated, headaches
ADVERSE EVENTS
The primary efficacy measure was mean change in the number of monthly MHDs from double-blind baseline (the prospective baseline period) to month 6. Secondary endpoints included response rates (based on percent reduction in monthly MHDs from double-blind baseline to month 6), functional outcomes, safety and tolerability.The mean change in monthly MHDs was derived from the ePRO system, in which patients recorded information about headaches (including medication used) and migraine-associated symptoms each day. Response rates were then estimated as the percentage of patients with reductions of ≥ 50%, ≥ 75% and 100% in monthly MHDs from double-blind baseline. A maintained ≥ 50% response was defined as a ≥ 50% reduction in monthly MHDs from baseline to month 3 that was maintained throughout the OLE. Functional outcomes were assessed as described previously [Safety was assessed by monitoring TEAEs, SAEs, deaths, adverse events leading to discontinuation, laboratory tests, electrocardiograms, vital signs, and body weight. Levels of antidrug antibodies (ADA) and neutralizing ADAs were measured to assess immunogenicity. Treatment-emergent ADAs (TE-ADAs) were defined as a negative baseline ADA result followed by a positive post-baseline ADA result with a titer ≥ 1:20 (treatment-induced) or positive baseline and post-baseline ADA results with a ≥ fourfold increase in titer (treatment-boosted ADA).
PMC10401806
Statistical analysis
Efficacy and safety analyses were performed in all randomized patients who received at least one dose of the study drug in groups defined by treatment assignment during the double-blind period, i.e., patients previously randomized to galcanezumab (GMB/GMB group) or to placebo (PBO/GMB group). The efficacy analysis included data from both the double-blind and OLE periods. Patients who have a baseline (from the double-blind phase) and at least one post-baseline observation were included in the analysis. Continuous efficacy endpoints were analyzed using a restricted maximum likelihood-based mixed model for repeated measures (MMRM). Binary efficacy endpoints with repeated measurements were analyzed with a categorical, pseudo-likelihood-based repeated measures analysis implemented using a generalized linear mixed model procedure (GLIMMIX). Except for the efficacy analyses on MHDs or categorical analysis of response rate (such as 50% response rate) derived from MHDs, in which the continuous value of baseline MHDs was used as a covariate, all other efficacy analyses included baseline number of MHDs category (< 8 vs ≥ 8) as a covariate in the MMRM and GLIMMIX model.Comparisons were claimed to be statistically significant if two-sided
PMC10401806
Results
PMC10401806
Immunogenicity
Among 482 evaluable patients treated with galcanezumab during the double-blind and/or OLE period, 71 (14.7%) had ADAs present at baseline, with 37 patients (7.7%) having neutralizing ADAs. TE-ADAs were detected during galcanezumab treatment in 63 (13.1%) patients, including 59 patients (12.2%) who developed neutralizing ADAs. During the entire study, including the 4-month post-treatment period, 188 galcanezumab-treated patients developed TE-ADAs, including 186 patients (37.6%) who developed neutralizing ADAs. No meaningful relationship was observed between ADAs and efficacy or tolerability of galcanezumab.
PMC10401806
Discussion
migraine
MIGRAINE
In this 3-month OLE of the PERSIST study, galcanezumab 120 mg continued to be efficacious in patients from China, India and Russia with episodic migraine for up to 6 months, with a generally good safety profile.The findings in predominantly Asian patients are consistent with published results from the OLE of the phase 3 CONQUER episodic migraine populations [In the present study, the PBO/GMB group showed a rapid improvement in all efficacy endpoints following their transition to open-label galcanezumab. This result is in-line with the findings from the double-blind periods of the PERSIST and other phase 3 trials [Galcanezumab also had a maintained response during this OLE study, with two-thirds of patients (66.2%) who achieved a clinically meaningful ≥ 50% response during the double-blind phase of the trial maintaining this level of response during the OLE. This suggests patients achieving a good initial response to galcanezumab are likely to continue to show a good response for up to 6 months of treatment.Galcanezumab previously showed acceptable tolerability during the double-blind phase of the PERSIST trial and this continued throughout longer-term treatment in the OLE. No patients experienced a treatment-related SAE and only three patients (0.6%) discontinued treatment due to a TEAE during the OLE. Furthermore, treatment compliance was high (> 97%) during the OLE. Based on the high treatment adherence and acceptable tolerability observed in the PERSIST study it is likely that patient adherence would be high in a clinical setting, this has important clinical implications, as patients with migraine typically exhibit poor adherence to standard preventive treatments [Limitations of this study included that, as for all OLE studies, there was no comparator arm or blinding, thus limiting the interpretability of results from open-label period. Furthermore, the study was only six months in duration, and the longer-term safety and efficacy of galcanezumab in Asian patients remain unknown.
PMC10401806
Conclusions
migraine
MIGRAINE
Once-monthly galcanezumab 120 mg was efficacious and well-tolerated for up to 6 months in patients from China, India, and Russia with episodic migraine. Our results support the long-term findings from OLEs of prior pivotal phase 3 studies of galcanezumab in patients with migraine [
PMC10401806
Acknowledgements
The authors wish to acknowledge Stephanie Carter PhD, CMPP and Jake Burrell PhD (Rude Health Consulting Ltd.) for medical writing support, which was paid for by Eli Lilly and Company. The authors thank Yu Mao from Eli Lilly and Company for project management and medical writing assistance. The authors thank Yan Cheng and Fei Ji from Eli Lilly and Company for review and critical suggestions.”
PMC10401806
Authors’ contributions
SY was involved in study design, data acquisition and interpretation. JZ, LZ, DC, KS, GL, XY, and MZ were involved in data acquisition and interpretation. LS, HL and CQ were involved in data analysis and interpretation. All the authors revised the manuscript critically and approved the final version of the manuscript.
PMC10401806
Funding
The PERSIST study was funded by Eli Lilly and Company.
PMC10401806
Availability of data and materials
Lilly provides access to all individual participant data collected during the trial, after anonymization, except for pharmacokinetic or genetic data. Data are available upon reasonable request. Access is provided after a proposal has been approved by an independent review committee identified for this purpose and after receipt of a signed data-sharing agreement. Data and documents, including the study protocol, statistical analysis plan, clinical study report, and blank or annotated case report forms, will be provided in a secure data-sharing environment. For details on submitting a request, see the instructions provided at
PMC10401806
Declarations
PMC10401806
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki, Council for International Organizations of Medical Sciences International Ethical Guidelines, International Conference on Harmonisation guidelines for Good Clinical Practice, and applicable local laws and regulations. Patients provided written informed consent before undergoing any study procedures. The appropriate institutional review board for each participating site approved the study protocol, patient consent form and other relevant documents.
PMC10401806
Consent for publication
Not applicable.
PMC10401806
Competing interests
Headache, Pain
LS, HL and CQ are full-time employees of Eli Lily and Company. SY serves as associated editor of the Journal of Headache and Pain and as a member of the International Headache Society. MZ has no competing interests. JZ, LZ, DC, KS, GL, XY and SY declare receiving clinical research fees from Eli Lilly and Company for participating as investigators in the PERSIST study.
PMC10401806
References
PMC10401806
Purpose:
CB-103 selectively inhibits the CSL–NICD (Notch intracellular domain) interaction leading to transcriptional downregulation of oncogenic Notch pathway activation. This dose-escalation/expansion study aimed to determine safety, pharmacokinetics, and preliminary antitumor activity.
PMC10501326
Experimental Design:
toxicities, tumor, DLT, hematologic malignancies
DISEASE PROGRESSION, TUMOR, SOLID TUMORS, HEMATOLOGIC MALIGNANCIES, ADENOID CYSTIC CARCINOMA
Patients ≥18 years of age with selected advanced solid tumors [namely, adenoid cystic carcinoma (ACC)] and hematologic malignancies were eligible. CB-103 was dosed orally in cycles of 28 days at escalating doses until disease progression. Notch-activating mutations were required in a dose confirmatory cohort. Endpoints included dose-limiting toxicities (DLT), safety, tumor response, pharmacokinetics, and pharmacodynamics. Exploratory analyses focused on correlates of Notch and target gene expression.
PMC10501326
Results:
tumor, DLTs, toxicity, anemia, deaths
ADVERSE EVENTS, TUMOR, ANEMIA, DISEASE
Seventy-nine patients (64, 12 dose-escalation cohorts; 15, confirmatory cohort) enrolled with 54% receiving two or more lines of prior therapy. ACC was the dominant tumor type (40, 51%). Two DLTs were observed [elevated gamma-glutamyl transferase (GGT), visual change]; recommended phase II dose was declared as 500 mg twice daily (5 days on, 2 days off weekly). Grade 3–4 treatment-related adverse events occurred in 15 patients (19%), including elevated liver function tests (LFTs), anemia, and visual changes. Five (6%) discontinued drug for toxicity; with no drug-related deaths. There were no objective responses, but 37 (49%) had stable disease; including 23 of 40 (58%) patients with ACC. In the ACC cohort, median progression-free survival was 2.5 months [95% confidence interval (CI), 1.5–3.7] and median overall survival was 18.4 months (95% CI, 6.3–not reached).
PMC10501326
Conclusions:
CB-103 had a manageable safety profile and biological activity but limited clinical antitumor activity as monotherapy in this first-in-human study.
PMC10501326
Significance:
DISEASE
CB-103 is a novel oral pan-Notch inhibitor that selectively blocks the CSL–NICD interaction leading to transcriptional downregulation of oncogenic Notch pathway activation. This first-in-human dose-escalation and -confirmation study aimed to determine the safety, pharmacokinetics, and preliminary antitumor efficacy of CB-103. We observed a favorable safety profile with good tolerability and biological activity but limited clinical single-agent antitumor activity. Some disease stabilization was observed among an aggressive
PMC10501326
Introduction
human cancer, cancer, T-ALL/LBL, hematologic cancer, T-cell acute lymphoblastic leukemia/lymphoma
PROLIFERATION, CANCER, METASTASIS, HEMATOLOGIC CANCER
Notch signaling plays a critical role in many cellular processes during development to promote cell-cell communication, whereas dysregulation leads to sustained cell proliferation and potential for invasion or metastasis—the hallmarks of cancer. Signaling of the pathway occurs when ligands bind one of four Notch receptors (The oncogenic role of Notch signaling was first observed in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) (Given the importance of Notch signaling in human cancer, several therapeutic approaches have been investigated to inhibit pathway activation, including mAbs against Notch receptors and small-molecule γ-secretase inhibitors (GSI; ref. Here we present the results of the first-in-human, dose-escalation and -confirmation trial of CB-103 across multiple solid and hematologic cancer types to primarily assess safety and tolerability, pharmacokinetics, and evaluate preliminary antitumor efficacy.
PMC10501326
Materials and Methods
PMC10501326
Study Population
malignant glomus tumor, T-ALL/LBL, cardiac disease, tumor
GASTROINTESTINAL TUMORS, TUMOR, THROMBOEMBOLIC EVENT, BREAST CANCER, DISEASE, SOLID TUMORS, OSTEOSARCOMA, HEPATOCELLULAR CARCINOMA, CARDIAC DISEASE, ONCOLOGY
The study enrolled adults with histologically confirmed, locally advanced and/or metastatic solid tumors who had progressed on at least one line of prior systemic therapy (except for ACC) and relapsed/refractory T-ALL/LBL for whom no standard therapy was available. In dose escalation, participants with solid tumors with known or frequent Notch pathway–activating mutations were eligible (breast cancer, gastrointestinal tumors, hepatocellular carcinoma, osteosarcoma, malignant glomus tumor, and ACC), while the confirmatory cohort planned to enroll participants with selected tumor types (including T-ALL/LBL) and confirmed Notch pathway activation. Key eligibility criteria included patients ≥18 years of age with evaluable disease, Eastern Cooperative Oncology Group (ECOG) performance status 0–1, able to swallow capsules, and adequate organ function. Participants were excluded if they had clinically significant cardiac disease or thromboembolic events within the preceding 6 months, and drugs prolonging the QTc were avoided. A complete list of eligibility criteria is outlined in the Supplementary Materials and Methods.
PMC10501326
Study Design
cancer, intercurrent illness, ≤28
ADVERSE EVENT, DISEASE PROGRESSION, CANCER, ADVERSE EVENT, STAGGERED, REGRESSION
The study was an open-label, nonrandomized, phase I/II dose-escalation study with planned expansion cohorts. In Part A, participants received CB-103 orally on a once daily schedule (28-day cycle length) which could be adapted during escalation to twice daily or intermittent dosing based on pharmacokinetic and safety signals, to determine the MTD or recommended phase II dose (RP2D). Part B was a potential expansion phase at the MTD/RP2D to determine preliminary evidence of antitumor activity and to confirm safety among patients stratified by preselected cancer indications.Part A was based on a two-parameter Bayesian logistic regression model (BLRM) to investigate safety and tolerability of sequentially enrolled dose cohorts of 3–6 patients. The first 2 participants of each cohort were enrolled in a staggered approach with at least 1 day apart between first dosing. Each dose cohort had to complete a DLT assessment period (one treatment cycle, or 28-days for at least 3 patients) and be reviewed by a Cohort Review Committee before opening a subsequent dose cohort. DLT was defined as any grade 3–4 adverse event (AE) or abnormal lab value [according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v4.03] assessed as unrelated to disease progression, intercurrent illness, or concomitant medications that occurred ≤28 days following the first dose of CB-103.Following a 28-day screening period, a starting dose of 13 mg once daily CB-103 was based on nonclinical toxicology and biochemical studies (Supplementary Data) and expected to achieve an exposure area under the curve (AUCSafety was assessed via monitoring of DLTs during the first treatment cycle, and of AEs, physical exam, and clinical lab results (hematology, coagulation, blood chemistry, urinalysis, and cardiac markers) throughout the study (
PMC10501326
Study Endpoints
SD
DISEASE
The primary endpoint for dose escalation was the number of patients experiencing DLT during the first 28-day cycle of CB-103 and for the confirmatory phase, the incidence rate, severity, and relationship of AEs to CB-103. Secondary endpoints included assessment of clinical benefit rate [CBR; defined as achieving complete and/or partial response and/or stable disease (SD) at prespecified benchmarks], duration of response, progression-free survival (PFS) and overall survival (OS), and plasma pharmacokinetics.
PMC10501326
Pharmacokinetics
Plasma pharmacokinetics were assessed for CB-103 via blood samples taken on days 1 (predose, 0.5, 1, 2, 4, 6, 8 hours postdose ± 5 minutes, and 12 hours postdose ± 15 minutes), 2 (24-hour post-day 1, predose), 3 (predose), 8 (predose, 0.5, 1, 2, 4, 6, and 8 hour postdose ± 5 minutes), 9 (24-hour post-day 8, predose), 15, and 22 (predose, and 1 hour postdose) in cycle 1. In cycle 2, on day 1 (predose, 0.5, 1, 2, 6, and, 8 hours postdose ± 5 minutes) and 15 (predose); in cycles 3–6 once at each visit (predose or postdose). For a twice daily dose schedule, pharmacokinetic timepoints were adjusted to evaluate concentration-versus-time curves after morning and evening doses on days 1 and 8.CB-103 concentrations were determined by validated high-performance LC/MS-MS method and analyzed descriptively using Phoenix WinNonlin v6.3 (Pharsight Corporation). Data at timepoints collected were used to generate a population pharmacokinetic model to estimate AUC and C
PMC10501326
Pharmacodynamics and Biomarker Assessments
tumor
DISEASE PROGRESSION, TUMOR
Archival tumor tissue (not older than 6 months prior to screening) or fresh tumor biopsy was required to characterize Notch alteration status by targeted genomic sequencing and/or NICD1 expression (via IHC) among the confirmatory cohort at baseline. Repeat biopsy on cycle 2 day 15 and at disease progression was optional. Serial whole blood samples (and/or bone marrow samples and saliva among patients with T-ALL/T-LBL) were obtained on day 1 of cycles 1–6 to evaluate the dynamics of Notch gene expression. Peripheral blood was collected with PaxGene tubes and frozen at −20°C until processing. RNA was extracted (Qiagen RNeasy Micro kit) from peripheral whole blood and analyzed by Nanostring with percent change in expression levels reported with respect to baseline normalized values.
PMC10501326
Statistical Analysis
The safety set (SS) consisted of all patients who received at least one dose of CB-103 and who had at least one postbaseline safety assessment. The SS was the primary population for all safety and efficacy analyses, except for determination of dose-DLT relationship. The dose-determining set was used to determinate the MTD and included all patients in the SS who had experienced a DLT at any time during cycle 1 and/or met the minimum requirements in cycle 1 (CB-103 dosed for ≥21 days, observed for ≥28-days after day 1, and completed cycle 1 safety evaluations).The objective of the BLRM design (
PMC10501326
Study Oversight and Data Availability
The study was performed in accordance with the Declaration of Helsinki statement on ethical biomedical research and with the International Conference on Harmonization Guidelines for Good Clinical Practice. The study was approved by the local Institutional Review Boards for each study site. All patients provided written informed consent. The trial is registered at ClinicalTrials.gov (NCT03422679) and the full protocol is provided in the Supplementary Materials and Methods. The data generated in the study are available within the article and from the corresponding author upon request.
PMC10501326
Results
PMC10501326
Patient Characteristics
ALL
ACUTE LYMPHOBLASTIC LEUKEMIA, LYMPHOBLASTIC LYMPHOMA, ONCOLOGY, ADENOID CYSTIC CARCINOMA
From December 2017 to January 2022, a total of 79 patients enrolled to the study, including 64 subjects to 12 escalating dose cohorts (ranging from 13 mg once daily to 500 mg twice daily 5 days on and 2 days off each week), and 15 subjects to a single confirmatory cohort dosed at 500 mg once daily (Patient demographicsAbbreviations: ACC = adenoid cystic carcinoma, ALL = acute lymphoblastic leukemia, LBL = lymphoblastic lymphoma, ECOG = Eastern Cooperative Oncology Group, y = years.
PMC10501326
Safety and Tolerability
DLTs, TRAEs
Two DLTs were observed across 12 dose-escalation cohorts (Among the SS population (TRAEs reported per-patient with incidence ≥10%NOTE:
PMC10501326
Efficacy and Survival
ADENOID CYSTIC CARCINOMA, SOLID TUMOR
Among all 76 solid tumor patients (including the confirmatory cohort and all patients with ACC), there were 37 (49%) demonstrating SD but no objective responses (Efficacy and survival outcomes for patients with solid tumorAbbreviations: ACC = adenoid cystic carcinoma, CI = confidence interval, NR = not reached, PFS = progression-free survival, OS = overall survival. Waterfall (At a median follow-up of 5.4 months (range: 0.1–20.5+), median PFS for the entire solid tumor cohort was 1.9 months (95% CI, 1.4–3.2) with a 6-month PFS estimate of 15.5% (95% CI, 7.5–26.0), which was similar across dose-escalation and confirmatory dose cohorts. Among the ACC cohort, median PFS was 2.5 months (95% CI, 1.5–3.7) with a 6-month PFS estimate of 18.6% (95% CI, 7.9–32.8; PFS (
PMC10501326
Pharmacokinetics
Robust pharmacokinetic sampling was performed across 7 days in cycle 1, 2 days in cycle 2, and before and after CB-103 dosing in cycles 3–6. Mean pharmacokinetic plasma concentrations (ng/mL) increased across dose-escalation cohorts 1 through 8 (522 mg once daily) demonstrating a peak (t
PMC10501326
Exploratory Biomarkers
Among the study cohort (Percent change (%) in peripheral blood cell Notch target gene expression was monitored among a subset of patients with ACC receiving twice daily dosing (
PMC10501326
Discussion
ACC-I, fatigue, diarrhea, toxicity, toxicities, anemia, aggressive disease, deaths
DISEASE, ANEMIA, SOLID TUMOR
This phase I/II study of the novel, oral pan-Notch inhibitor CB-103 determined the RP2D as 500 mg twice daily utilizing a 5 day on and 2 day off weekly schedule. Overall, CB-103 was well tolerated with 15 of 79 patients (19%) experiencing grade 3 or 4 AEs which were all reversible, with no deaths related to study drug, and only 5 patients (6%) discontinuing study drug for toxicity. In addition, CB-103 demonstrated favorable pharmacokinetic properties with mean plasma concentrations increasing across dose escalation.Among all 76 evaluable solid tumor patients (most with ACC) there were no objective responses observed, but 58% of ACC patients exhibited SD with a 3- and 6-month CBR of 58% and 28%, respectively. Notably, 68% (27/40) of patients with ACC in our trial had confirmed GSIs can result in diarrhea and fatigue in >60% of patients but grade 3+ AE rates were reportedly low. Gastrointestinal toxicities associated with GSIs were not observed with CB-103. CB-103 has an independent mechanism from GSIs, resulting in a distinct AE profile (anemia, visual changes) which also appears manageable. This is important to highlight as the broader advanced ACC population (regardless of Notch status) is often offered treatment with oral VEGFR tyrosine kinase inhibitors (TKI) such as lenvatinib and axitinib which have reported discontinuation rates of 20% or greater for toxicity (Ferrarotto and colleagues have characterized the poor prognosis and aggressive disease phenotype that distinguishes Another potential target in ACC-I is the apoptotic protein In conclusion, the novel oral pan-NOTCH inhibitor CB-103 demonstrated a manageable safety profile with good tolerability and biological activity despite limited antitumor activity as monotherapy in this first-in-human study. We observed some evidence of disease stabilization in an aggressive ACC-I type population where prognosis is poor, and therapies are urgently needed. Further enrichment for
PMC10501326
Supplementary Material
toxicity
Study design, dose escalation, and confirmatory dosing cohorts (Part A) for CB-103Click here for additional data file.Pharmacokinetic parametersClick here for additional data file.Biomarkers of Clinical Benefit for ACC patientsClick here for additional data file.Safety and toxicity monitoring throughout the CB-103 clinical tria1Click here for additional data file.Pharmacokinetic parameters for CB-103Click here for additional data file.Pharmacodynamic and biomarker assessments: percent change in peripheral Notch target gene expression in circulation across individual patients (each row) on twice daily dosing of CB-103Click here for additional data file.Representativeness of Study ParticipantsClick here for additional data file.
PMC10501326
Acknowledgments
tumor
TUMOR
The authors thank the patients and their families, all coinvestigators, and research coordinators for participating in the study. We thank Professor Jon Aster for his support and scientific contribution to evaluating the tumor
PMC10501326
Authors’ Disclosures
Cancer, Pierre, Leukemia, fromAstraZeneca, P., Schönborn-Kellenberger, Oncology/Cancer
ONCOLOGY, LEUKEMIA, CANCER
G. J. Hanna reports relevant consulting/advisory for Kura, Prelude, and Remix; research support to institution from AACRF, Actuate, Elevar, Gateway for Cancer Research, Genentech, Immunitybio, and V Foundation. A. Stathis reports other, institutional, travel grant from AstraZeneca, Incyte; expert testimony, institutional from Eli Lilly and Bayer; advisory board, institutional from Janssen and Roche; local PI, institutional, financial interest from Abbvie, ADC Therapeutics, Amgen, Bayer, Cellestia, Loxo Oncology, Merck MSD, Novartis, Pfizer, Philogen, Roche; trial chair, institutional, financial interest from AstraZeneca, Incyte. E. Lopez-Miranda reports consultant/advisory board role in AstraZeneca, Daiichi Sankyo, Novartis, Roche and has received travel, inscription, and accommodation funding from Roche and Seagen. F. Racca reports speakers bureau from Roche and Bristol-Myers Squibb; travel, accommodation and expenses Roche, Bristol-Myers Squibb, Merck Sharp & Dohme; consulting and advisory role: Bristol-Myers Squibb; expert testimony: Pharmore research, Psyma. S. Leyvraz reports support for congress and travel from Bayer. D. Hess reports to be shareholder of the following pharmaceutical companies: Roche, Novartis, and Bayer. B. Keam reports honoraria from Merck, Yuhan, Lilly; consulting/advisory from Handok, CBS Bioscience, Trial Informatics, NeoImmuneTech, ImmuneOncia; research funding: Ono Pharmaceutical, MSD Oncology, AstraZeneca. J. Rodon reports non-financial support and reasonable reimbursement for travel from European Society for Medical Oncology; receiving consulting and travel fees from Peptomyc, Kelun Pharmaceuticals/Klus Pharma, Ellipses Pharma, Molecular Partners, IONCTURA (including serving on the scientific advisory board); consulting fees from Vall d'Hebron Institute of Oncology/Ministero De Empleo Y Seguridad Social, Chinese University of Hong Kong, Boxer Capital, LLC, Tang Advisors, LLC receiving research funding from Blueprint Medicines, Black Diamond Therapeutics, Merck Sharp & Dohme, Hummingbird, Yingli and Vall d'Hebron Institute of Oncology/Cancer Core Europe; and serving as investigator in clinical trials with Novartis, Spectrum Pharmaceuticals, Symphogen, BioAlta, Pfizer, GenMab, CytomX, Kelun-Biotech, Takeda-Millenium, GalxoSmithKline, Taiho, Roche Pharmaceuticals, Hummingbird, Yingli, Bicycle Therapeutics, Merus, Curis, Bayer, AadiBioscience, Nuvation, ForeBio, BioMed Valley Discoveries, Loxo Oncology, Hutchinson MediPharma, Cellestia, Deciphera, Ideaya, Amgen, Tango Therapeutics, Mirati Linnaeus Therapeutics, and Cancer Core Europe. M.J. Ahn reports honoraria fromAstraZeneca, BMS, MSD, Lilly, Merck, Ono, Roche, Takeda, Yuhan, Amgen and consultant or Advisor for AstraZeneca, BMS, ONO, Takeda, Lilly, Merck, MSD, Amgen, Novartis, Roche, Yuhan, Arcus, Pfizer, Daichi-Sankyo, Alpha Pharmaceutical, Boronoi. A. Schneeweiss reports grants from Celgene, Roche, AbbVie; personal fees from Celgene, Roche, Pfizer, AstraZeneca, Novartis, MSD, Tesaro, Lilly, Seagen, Gilead, GSK, Bayer, Amgen, Pierre Fabre. J.-M. Ribera reports speaker and advisory boards honoraria, clinical trials from Ariad and Pfizer; speaker and advisory boards honoraria, research support, clinical trials from Amgen; speaker and advisory boards honoraria from Shire. D. Deangelo serves as a consultant for Autolos, Blueprint, Gilead, Incyte, Jazz, Kite, Novartis, Pfizer, Servier, and Takeda; receives research funding from Abbvie, Glycomimetics, Novartis, and Blueprint Pharmaceuticals; serves on the DSMB for Daiichi-Sankyo, Fibrogen, and Mt Sinai MPN Consortium; and is the co-chair of the NCI (CTEP) Leukemia Steering Committee. J.M. Perez Garcia reports advisory role from Lilly, Roche, Eisai, Daichii Sankyo, Seattle Genetics, Gilead; travel expenses from Roche. J. Cortes reports consulting/advisor to Roche, Celgene, Cellestia, AstraZeneca, Seattle Genetics, Daiichi Sankyo, Erytech, Athenex, Polyphor, Lilly, Merck Sharp&Dohme, GSK, Leuko, Bioasis, Clovis Oncology, Boehringer Ingelheim, Ellipses, Hibercell, BioInvent, Gemoab, Gilead, Menarini, Zymeworks, Reveal Genomics, Expres2ion Biotechnologies; honoraria from Roche, Novartis, Celgene, Eisai, Pfizer, Samsung Bioepis, Lilly, Merck Sharp&Dohme, Daiichi Sankyo, AstraZeneca; research funding to the Institution from Roche, Ariad pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer Healthcare, Eisai, F.Hoffman-La Roche, Guardanth health, Merck Sharp&Dohme, Pfizer, Piqur Therapeutics, Puma C, Queen Mary University of London. Stock: MedSIR, Nektar Pharmaceuticals, Leuko (relative); travel, accommodation, expenses from Roche, Novartis, Eisai, pfizer, Daiichi Sankyo, AstraZeneca, Gilead, Merck Sharp&Dhome. O. Schönborn-Kellenberger is a Cellestia Biotech AG consultant. D. Weber is a Cellestia Biotech AG shareholder. P. Pisa is a Cellestia Biotech AG shareholder. M. Bauer is a Cellestia Biotech AG shareholder. L. Beni is a Cellestia Biotech AG shareholder. M. Bobadilla is a Cellestia Biotech AG employee. R. Lehal is a Cellestia Biotech AG employee. M. Vigolo is a Cellestia Biotech AG employee. F.D. Vogl is a Cellestia Biotech AG employee. E. Garralda reports research support from Novartis, Roche, Thermo Fisher Scientific, AstraZeneca, Taiho, BeiGene, Janssen; consultant/advisor for Roche, Ellipses Pharma, Boehringer Ingelheim, Janssen Global Services, Seattle Genetics, Alkermes, Thermo Fisher Scientific, MabDiscovery, Anaveon, F-Star Therapeutics, Hengrui, Sanofi, Incyte; speakers bureau: Merck Sharp & Dohme, Roche, Thermo Fisher Scientific, Lilly, Novartis, SeaGen; employment: NEXT Oncology. No disclosures were reported by other authors.
PMC10501326
Authors’ Contributions
PMC10501326
References
PMC10501326
Introduction
CORONAVIRUS, CORONAVIRUS DISEASE 2019, SEVERE ACUTE RESPIRATORY SYNDROME
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health crisis that is driving efforts to identify alternative medicinal plants to be used in COVID-19 treatment. There is evidence that the Acanthaceae family member Liquid chromatography-tandem mass spectrometry (LC–MS/MS) is a powerful technique widely used in biological analysis, including in the screening of lead candidates, determination of metabolites, and pharmacokinetic investigationTaken together, the previous method was applicable for determining the four major diterpenoids (AP1, AP3, AP4, and AP6) in only plasma samplesThe present study is the first investigation of oral dosing of a high dose administration of
PMC9924185
Materials and methods
PMC9924185
Materials
Standards of andrographolide, AP1 (purity = 100.00%); 14-deoxy-11, 12-didehydroandrographolide, AP3 (purity = 99.80%); neoandrographolide, AP4 (purity = 99.67%); and 14-deoxyandrographolide, AP6 (purity = 100.00%) were supplied by Phytolab GmbH & Co.KG (Vestenbergsgreuth, Germany). The internal standard (IS) of digoxin (purity = 96.6%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The tested product, For enzymatic digestion,
PMC9924185
Methods
PMC9924185
Screening and identification of the analyte and metabolite profiling using LC-QTOF/MS analysis
SEPARATION
For qualitative determination of untargeted compounds, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was performed on an Agilent 6540 QTOF/MS (Agilent Technologies, USA) equipped with an Agilent 1260 infinity liquid chromatography system. The stationary phase of the chromatographic separation was performed on a 150 mm × 4.6 mm, 5.0 µm Phenomenex Luna C18 column (Phenomenex, USA) using a mobile phase consisting of 0.1% formic acid in water pH 2.5–2.7 (A) and 100% acetonitrile (B) at a constant flow rate of 0.5 mL/min with a controlled oven temperature at 35 °C, (and 10 µL) of injection volume. The elution gradient was started with 30%B, and was increased to 90%B within 20 min, then maintained at 95%B during 25–30 min followed by a post-run for 5 min.Mass spectrometric analysis was performed using an ESI negative and positive modes with varying collision energies (10, 20, and 40 V) over a mass range of m/z 100–1200 Da. The MS conditions were as follows: capillary voltage = 3500 V; flow rate of drying gas (N
PMC9924185
Development of analytical and instrument conditions for quantitative determination of four major diterpenoids using LC-QqQ-MS/MS analysis
PMC9924185
LC and MS condition optimization
SEPARATION
Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed using a Nexera X2 LCMS-8060NX triple quadrupole mass spectrometer (Shimadzu, Japan) equipped with a SIL-40C XR autosampler, CTO-40C column oven, CBM-40lite system controller, FCV-20AH2 switching valve, DGU-403 degasser, and LC-40D XR solvent delivery (pump unit). The LC separation was conducted on a VertiSep AQS C18 column (100 mm × 3.0 mm, 3.0 µm) using a gradient mobile phase. The different solvents (including acetonitrile, methanol, and water, with or without formic acid) were tested to optimize sample elution systems.Mass spectrometry (MS) and ionization was optimized and operated in Multiple Reaction Monitoring (MRM) mode. MS detection was fully scanned by directly injecting each standard (10.00–100.00 ng/mL) into the MS instrument without separation on a column. MS optimization was performed under the positive and negative conditions of electrospray ionization (ESI) mode. The MRM transitions and the fragmentations of parent and daughter ions for each analyte, with the corresponding collision energy, are shown in Table MRM transitions and fragmentations of parent ions and daughter ions of andrographolide (AP1); 14-deoxy-11, 12-didehydroandrographolide (AP3); neoandrographolide (AP4); 14-deoxyandrographolide (AP6); and digoxin (IS).*Quantitative ion.
PMC9924185
Preparation of stock standards and working solutions
The primary stock solution (1.00 mg/mL) of the four standard diterpenoids (AP1, AP3, AP4, and AP6) and an internal standard (digoxin) were prepared and then stored at − 20 °C. Briefly, each standard was accurately weighed and dissolved in the appropriate volume of methanol (HPLC grade). Working solutions containing the four diterpenoids were then prepared from the stock solutions by serial dilution with methanol to obtain the appropriate concentrations of these four analytes. For the internal standard (IS), a further dilution of the stock solution was prepared at a concentration of 50.00 ng/mL.
PMC9924185
Preparation of calibration standards and quality control (QC) samples
A calibration curve was generated from different standard concentrations that were prepared by spiking a small volume of the working solution (not more than 5% of the total volume) into blank human plasma or urine to obtain twelve final concentrations ranging between 0.98 and 1000.00 ng/mL for each analyte. Quality Control (QC) samples were also prepared by using the same method as the calibration standards; with concentrations of 2.50, 500.00, and 900.00 ng/mL for low (LQC), medium (MQC), and high (HQC) concentrations, respectively. These spiked plasma samples (calibration standards and QC samples) were then similarly extracted following the sample preparation protocol with the addition of 50.00 ng/mL of IS.
PMC9924185
Plasma and urine samples extraction
An aliquot of 50 µL of plasma or urine was placed in a 1.5 mL of polypropylene centrifuge micro-tube. Protein was removed from the sample by adding 200 µL of methanol (containing 50.00 ng/mL of IS). The mixture was vortexed for 10 min and was then centrifuged at 12,000 rpm, 4 °C for 10 min. The supernatant was filtered through a 0.2-µM PVDF membrane (Chrom Tech) and transferred into a glass insert in a vial before LC‑MS/MS analysis.
PMC9924185
Method validation
The bioanalytical method of LC-QqQ-MS/MS analysis was fully validated for (1) selectivity and specificity, (2) accuracy and precision, (3) linearity, range and lower limit of quantification, (4) recovery, (5) matrix effect, (6) stability, and (7) hemolyzed plasma effect, according to the US FDA Bioanalytical Method Validation Guideline
PMC9924185
Key performance characteristics of method validation
PMC9924185
Selectivity and specificity
The selectivity and specificity were evaluated by comparing between the retention time of the extracted blank plasma or blank urine and the retention time of each spiked standard or IS in the extracted samples. If any interferences were observed, the signal of their peak area should be less than 20% of the peak area of LLOQ, and less than 5% of the average peak area of IS in blank samples.
PMC9924185
Accuracy and precision
For within-day accuracy and precision, four concentration levels at LLOQ, LQC, MQC, and HQC in five replicates, were determined in the same day. For determination of inter-day accuracy and precision, the same concentrations were extracted and analyzed on separate days (three different days) and used for three batch runs in five replicates. The accuracy was shown as a percentage, and the acceptance criteria was that each concentration should be within ± 15% for QC samples, and within ± 20% for the LLOQ. The precision of testing was expressed as percentage of the coefficient of variation (%CV), and the acceptance criteria was that each concentration should be less than 15% for QC samples and less than 20% for LLOQ.
PMC9924185
Linearity, range and lower limit of quantification (LLOQ)
REGRESSION
A twelve-point calibration curve was constructed by plotting the peak-area ratio (analyte:IS) against the concentration of the calibration standards. Determination of linearity was done using 3 calibration curves from each day (three separate days). The analysis requirement of linear regression was determined by the coefficient of determination (RTo determine the lower limit of quantification (LLOQ), 5 samples at LLOQ were analyzed for 3 batch runs on separate days. The analytical signal of LLOQ should be at least five times higher than the signal of the blank plasma or blank urine. The acceptable limits of accuracy should be within 80–120% and %CV ≤ 20.
PMC9924185
Recovery
The recovery of the four diterpenoids in plasma and urine was measured at three different QC concentrations: LQC, MQC and HQC (n = 5 for each concentration) for both pre-spiked standard of QC samples and post-spiked standard of blank plasma or urine. Extraction recoveries were calculated by comparing the peak area of pre-spiked standard in a blank matrix with the area of post-spiked standard in an extracted matrix at corresponding concentrations. To meet acceptance criteria, the recovery should be precise, accurate, and repeatable with %CV of recovery less than 15%.
PMC9924185
Matrix effect
Matrix effects at three levels of QC concentrations (LQC, MQC and HQC) were determined by comparing the mean peak area of the samples prepared by spiking post-extracted samples with the analytes and IS in solution (without extraction) at corresponding concentrations. The %CV of the matrix effect should be within the acceptance criteria, which is less than 15%. The matrix factor (MF) was estimated based on the equation,The IS normalized MF was then calculated asThe acceptance value of IS normalized MF should be within 0.8–1.2.
PMC9924185
Stability
The stability of plasma and urine samples were evaluated at three different QC concentration levels (LQC, MQC and HQC, n = 3 for each level); the conditions were freeze–thaw stability, long-term stability, short-term stability, and post preparative stability in an autosampler. The evaluation of stability under freeze–thaw condition was carried out over five cycles of − 80 °C for at least 12 h of each cycle. Long-term stability was analyzed after storing the samples at − 80 °C. Short-term stability of each analyte, for up to 8 h during processing on the bench at room temperature, was also evaluated. For evaluation of post-preparative stability, the extracted samples were stored at ambient temperature (4 °C) in an autosampler for 24 h. The analyte was considered as a stable sample when the average percentage of accuracy of the QC sample was in the range of 85–115% and the %CV was not over 15%.
PMC9924185
Hemolyzed plasma effect
hemolysis
HEMOLYSIS
To evaluate the effect of hemolysis of red blood cells on the quantification of analytes, hemolyzed plasma was prepared by adding hemolyzed whole blood (3% V/V) into blank plasma
PMC9924185
Quantitative determination of conjugated metabolites using enzymatic digestion assay
PMC9924185
Optimized enzymatic digestion
To optimize the hydrolysis process for determination of conjugated metabolites, enzymatic digestion with glucuronidase and sulfatase was performed under different incubation conditions. The hydrolysis reaction was initiated by adding 50 µL of either enzyme into a 50 µL of plasma or urine sample using different enzyme concentrations (25, 50, 100, 200, and 400 units/mL for sulfatase and 200, 500, 1000, 2000 and 4000 units/mL for The standard calibration curves of each analyte for determining conjugated glucuronide and sulfate metabolites were carried out using the same optimized method as plasma and urine incubation. Briefly, working standard solutions were added into blank plasma and urine samples and then the process of incubation was performed before extraction with protein precipitation.
PMC9924185
Method application to pharmacokinetic study
The validated method was applied in pharmacokinetic investigation of four major diterpenoids in healthy subjects after oral administration of
PMC9924185
Institutional review board and informed consent statement
The study protocol was approved by the Institutional Review Board of the Chulabhorn Research Institute (approval date: 28/08/2020, IRB number: 062/2563) and also registered with the Thai Clinical Trials Registry (approval date: 01/02/2021, TCTR20210201005). All clinical procedures were performed in compliance with the International Conference on Harmonization-Good Clinical Practice (GCP) under the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study.
PMC9924185
Study design and eligibility criteria
The clinical pharmacokinetic study was designed as an open-labeled, with a single oral dosing, and conducted under a fasting condition. Four healthy subjects were recruited according to the following inclusion criteria: 18—55 years old with a body mass index (BMI) of 18–30 kg/m
PMC9924185