Compare to non-obese children, the rates in obese children were higher for CO (odds ratio (OR) 3.54, 95% confidence interval (CI) 1.15 ~ 10.92); PM10  and PM2.5 . In obese children, correlations between higher concentrations of CO, PM10, PM2.5 and higher nasal discomfort (higher PRQLQ); and correlations between higher concentrations of CO, PM10, PM2.5, NMHC (non-methane hydrocarbon) and higher nasal mucosa inflammation (lower NPEF) were noted. Obesity negatively affected AR severity when AR children experienced higher concentrations of CO, PM10, and PM2.5. Increased nasal inflammation induced by air pollutants might be the underlying mechanism.The association between air pollution, allergic rhinitis (AR), and obesity has not been studied. From 2007 to 2011, 52 obese and 152 non-obese children (7–17 years old) with AR were recruited. Pediatric-Rhinoconjunctivitis-Quality-of-Life Questionnaire (PRQLQ) and nasal peak expiratory flow (NPEF) were tested. Association between the scores and rates of the two tests and mean air pollutant concentrations within 7 days before the tests were compared. When exposed to higher concentrations of CO, PM Therefore, determining the association between air pollution, obesity, and AR is crucial. However, this association has not been previously studied.Recently, more attention has focused on the impact of air pollution on individual health. Obesity2 and biological3 studies had find their association. Regarding the relationship between obesity and AR, epidemiological5 and biological6 studies also find their association. Air pollution can induce the recruitment of inflammatory cells and inflammatory cytokines7. AR and obesity are considered chronic inflammatory illnesses9. Studies also found that the aforementioned inflammatory cells and cytokines induced by air pollutants are also increased in people with obesity or AR10. It indicated that air pollution, AR, and obesity share the same immune and inflammatory effects on the human body.Regarding the relationship between air pollution and AR, epidemiological2; or AR and obesity5; or air pollution and obesity11 were studied. The combined associations between air pollution and obesity and AR were not studied. Therefore, a retrospective cross-sectional, case–control study was conducted to find the association. We aimed to investigate the influence of air pollutants on the severity of rhinitis  and degree of nasal obstruction and nasal mucosa inflammation among AR children when they are obese or non-obese.Therefore, it is reasonable to speculate that air pollutants might have a stronger adverse effect on the severity of AR when they are obese. However, only the separate relationship between air pollution and AR2), carbon monoxide (CO), particles ≤ 10 μm in diameter (PM10), particles ≤ 2.5 μm in diameter (PM2.5), nitric oxide (NO), nitric dioxide (NO2), and non-methane hydrocarbon (NMHC).The Environmental Protection Administration established five air pollution monitoring stations in Taichung, Taiwan. Concentrations of air pollutants, ambient temperature and relative humidity were measured continuously around the clock and reported hourly. Data on the mean daily levels of air pollutants, ambient temperature and relative humidity were supported and the data between 2007 and 2011 were gathered for the study. The following seven air pollutants were assessed: nitrogen dioxide  were measured and recorded in the chart, and the body mass index  was calculated. Based on the growth charts for Taiwanese children and adolescents13, these participants were classified into (1) obese AR children (BMI > 85th percentile) group and (2) non-obese AR children (BMI < 85th percentile) group.From chart review, children aged 7–17 years with AR residing in Taichung, Taiwan, were recruited from a teaching hospital in the same city, from January 1, 2007, to December 31, 2011. AR was diagnosed by two pediatric allergy specialists. Based on the AR and its Impact on Asthma guidelines14 and nasal peak expiratory flow 15 were tested on the same day and were done in the same setting for each patient. They were tested once and randomly on different days over the 5 years. NPEF was measured three times in the seated position using a Mini-Wright peak flow meter  connected to an anesthetic face mask. Subsequently, the highest value was eventually selected. The questionnaire was writ by children and under the assistance of their parents.In these AR children, the Pediatric Rhinoconjunctivitis Quality of Life Questionnaire The mean concentrations of each air pollutant within 7 days before the tests were calculated for the study. A higher PRQLQ indicates that patients suffer more discomfort from nasal symptoms . Correlations between PRQLQ scores and mean pollutant concentrations were compared to determine the influence of obesity on the quality of life of AR children when they attained higher air pollutant concentrations. A lower NPEF indicates that the patients suffered a higher degree of nasal obstruction and nasal mucosa inflammation. Correlations between NPEF rates and mean pollutant concentrations were also compared to determine the influence of obesity on nasal obstruction and inflammation when they exposed to higher air pollutant concentrations. Potential confounders were adjusted, including age, sex, second-hand smoke exposure, combined with asthma or not and parental occupation. Parental occupation included white-collar , blue-collar (company employee and others), and others . Mean ambient temperature and relative humidity within 7 days before the test were also adjusted.10 was divided to 34.0–67.2 μg/m3(low level) and 67.3–100.5 μg/m3 (high level); and PM2.5 was divided to 20.0–40.7 μg/m3(low level) and 40.8–61.5 μg/m3 (high level). PRQLQ contain 23 items and each item on a scale of 0 (not troubled) to 6 (extremely troubled)14. Scales euqal or higher then 4 (quite troubled) were equal to more severe AR symptoms in our clinical experience. Therefore, children whose PRQLQ scores were equal or higher than 92 (sceles 4 multiply by 23 items) were considered as those with obvious AR discomfort on the study day. When children are exposed to high pollutant level as mentioned above, the rates of subjects with obvious AR discomfort (higher PRQLQ scores) were compared between obese and non-obese groups. We aimed to determine the association between obesity and AR discomfort when children with AR are exposed to higher air pollutant concentrations.To find the association, mean air pollutant concentrations within 7 days before the tests were divided into low and high levels. For example, the concentration range of CO was divided to 0.35–0.60 ppm (low level) and 0.61–0.86 ppm (high level); PMt-test was used for continuous variables. Spearman’s rank correlations were used for the correlation test between PRQLQ scores, NPEF rates, and pollutant concentrations because the seven pollutant concentrations were not normally distributed. Multivariate regression and multivariate logistic regression analysis was used to adjust for the confounding factors. Statistical significance was set at p < 0.05 for a two-tailed test. We interpreted the correlation coefficient values with the method reported by Portney and Watkins16, as follows: (1) 0.00–0.25 and − 0.25–0.00, no relationship; (2) 0.25–0.5 and − 0.25 to − 0.5, fair relationship (3) 0.5–0.75 and − 0.5 to − 0.75, moderate-to-good relationship; and (4) < 0.75 and < − 0.75, good-to-excellent relationship. Statistical significance was set at p < 0.05 for a two-tailed test, and a correlation coefficient of > 0.25 or < − 0.25. This study was approved by the Institutional Review Board of Chung Shan Medical University Hospital, Taichung, Taiwan . All experiments were performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s).All analyses were performed using Statistical Analysis Software (SAS)  and PASW Statistics 18 . Chi-square tests were used for categorical variables, and the The flow diagram is presented in Fig. 10, and PM2.5 concentrations within 7 days before the test. The results were: CO Spearman’s ρ = 0.396, p = 0.026; PM10 Spearman’s ρ = 0.502, p = 0.025; PM2.5 Spearman’s ρ = 0.506, p = 0.022. In contrast, no correlation between PRQLQ scores and the seven pollutants was noted in non-obese AR children. Additionally, regardless of statistical significance, the correlations of the seven pollutants were stronger in obese AR children (with a higher correlation coefficient).The Spearman’s rank correlation test was used to calculate the Spearman’s rank correlation coefficient (Spearman’s ρ). Table 10, and PM2.5 concentrations were noted in non-obese AR children.When ambient temperature and relative humidity were not added for adjustment , a positive correlation between PRQLQ scores and CO, PM10, PM2.5, and NMHC concentrations within 7 days before the test. The results were: CO Spearman’s ρ = − 0.379, p = 0.048; PM10 Spearman’s ρ = − 0.429, p = 0.013; PM2.5 Spearman’s ρ = − 0.489, p = 0.005; NMHC Spearman’s ρ = − 0.415, p = 0.043. No correlation was observed between the NPEF rates and the seven air pollutants in non-obese AR children.Table 2, PM10, and PM2.5 concentrations. The results were: SO2 Spearman’s ρ = 0.290, p = 0.016; PM10 Spearman’s ρ = 0.540, p = 0.030; PM2.5 Spearman’s ρ = 0.517, p = 0.024. In boys with obesity, no correlations were found. In girls with obesity, the NPEF rates were statistically negatively correlated with PM10 and PM2.5 concentrations. The results were: PM10 Spearman’s ρ = − 0.532, p = 0.013; PM2.5 Spearman’s ρ = − 0.561, p = 0.016. In boys with obesity, no correlations were found. In non-obese AR children, and in both boys and girls, no correlation was observed between PRQLQ scores, NPEF rates, and air pollutant concentrations.Table p-value = 0.028; Odds Ratio (OR) 3.54, 95% confidence interval (CI) 1.15 ~ 10.92). The rates of obvious AR discomfort were also higher in obese children when they were exposed to higher PM10 and PM2.5 levels, compare to the rates in non-obese children.The results are presented in the Table 18. In obese AR children in our study, we find a relationship between higher CO, PM10, PM2.5, NMHC concentrations and higher degree of nasal obstruction/inflammation (lower NPEF rates). Our study results were compatible with other studies. These studies suggest that air pollution could induce nasal obstruction3 and nasal mucosa inflammation19. In obese AR children in our study, we also find a relationship between higher CO, PM10, PM2.5 concentrations and higher AR discomfort or symptoms (higher PRQLQ scores). Therefore, we speculate that, through the induction of nasal obstruction and mucosa inflammation, CO, PM10, and PM2.5 had a negative impact on nasal discomfort and symptoms in obese AR children. We also found that, in non-obese AR children, there were no correlations between air pollution and AR discomfort/symptoms and nasal obstruction/mucosa inflammation.A lower NPEF rates indicates a higher degree of nasal obstruction and mucosa inflammation3. In our study, air pollution data were gathered within 7 days before the tests. Because of the short duration, the impact of air pollution on AR might have been caused by the neurogenic effects in our study. The trigeminal nerve innervates the nasal cavity. A study indicated that, through the activating of trigeminal nerve endings, air pollution exposure can trigger rhinitis20. Another study indicated that that obesity can increase the nociceptive activation of the trigeminal system under external stimulation21. The abovementioned findings might explain our result, which showed that air pollution had a more detrimental effect on AR when they were obese, compared to those without obesity.When exposed to pollutants, rhinitis symptoms can be exacerbated by immunological (long-term effect) and neurogenic (short-term effect) mechanisms22. Another study indicated that particles less than 100 nm were observed in human trigeminal ganglia capillaries23. This implies that PM could influences the trigeminal nerve and might result in the exacerbating of AR. In children with obesity in our study, we found a stronger correlation between PM10, PM2.5, PRQLQ (more severe AR symptoms), and NPEF . The two aforementioned studies by might explain the stronger correlation between PM10, PM2.5, and AR, compared to those of other air pollutants.A rats and mouse study found that airborne particulate matter (PM) can be transported via the trigeminal nerve. PM may also induce neurotoxicity, oxidative stress, and neuro-inflammation of nerves24. It was suggested that men have higher sympathetic tone and women have higher parasympathetic autonomic activity25. A study indicated that women appear to be more sensitive to intranasal trigeminal stimulation than men26. Another study reported a sex-differentiated electrophysiological response to intranasal trigeminal stimuli27. These findings could explain the stronger correlation observed between PRQLQ, NPEF, and air pollution  in female children with obesity and AR in our study, compared to male children.A reduction in the sympathetic tone can cause venous sinusoid dilatation, which can contribute to nasal obstruction symptomsTwo molecular mechanisms might explain our study results. First, air pollution could induce more nasal inflammation because obese children are in the chronic inflammatory states. Second, air pollution could induce more neuro-inflammation in obese children. Our study results  could explain the first speculation. However, the role of neuro-inflammation was not investigated in our study. Future study is necessary.28. Meteorological conditions including temperature and humidity were also report to be correlated with concentration of air pollutants29. Therefore, temperature and humidity could influence both the severity of AR and concentration of air pollutants, and should be adjusted when investigating the association between air pollution and AR. However, most studies investigating the relationship between air pollution and AR in children did not adjust for the two co-factors30. The two co-factors were adjusted in our study, so we can find the association between obesity, AR and air pollution more accurate.Ambient temperature and relative humidity was reported to influence the severity of ARFurthermore, our study had some limitations. First, the study was conducted at a single center. Therefore, it cannot reflect the general profile of the entire population owing to selection bias. Second, potential confounders  were not analyzed. Third, only children with moderate to severe AR were assessed in this study. Therefore, future studies including more children with AR from different clinics and areas, patients with mild AR, and gathering more confounding factors such as dietary habits, physical activity, and emotional factors are required.10, and PM2.5 negatively impacted discomfort and symptoms of rhinitis in obese AR children, rather than non-obese AR children. When obese AR children attain higher pollutant concentrations, increased nasal obstruction and inflammation might be the mechanism underlining the present study findings. This suggests that children with obesity and AR should avoid exposure to air pollutants, specifically CO, PM10, and PM2.5. Although we observed a significant correlation between air pollutants, obesity, and AR in Taiwan, regional differences in air pollution concentration, socioeconomic status, and race could have also altered the outcomes. Therefore, future studies should incorporate more diverse groups of participants with more significant variations in geographical location, socioeconomic status, race, and sex. Biological study to explain the association between AR, obesity and air pollution are required.The association between air pollution, AR, and obesity remain unclear to date. This study first demonstrated that air pollutant CO, PM


Andrographis paniculata, a medicinal plant in Thailand national list of essential medicines, has been proposed for treatment of patients with mild to moderate coronavirus disease 2019. This study aims to develop a highly selective and sensitive liquid chromatography triple quadrupole tandem mass spectrometry method for quantitative determination of major diterpenoids in plasma and urine with application in pharmacokinetics. Chromatographic separation was performed on C18 column using a gradient mobile phase of water and acetonitrile. Mass spectrometry was analyzed using multiple reaction monitoring with negative ionization mode. This validated analytical method was very sensitive, less time consuming in analysis, and allowed the reliability and reproducibility on its application. The clinical pharmacokinetics was evaluated after single oral administration of A. paniculata extract . The disposition kinetics demonstrated that major diterpenoids could enter into systemic circulation, but they are mostly biotransformed (phase II) into conjugated glucuronide and sulfate metabolites. These metabolites are predominantly found in plasma and then extremely eliminated, in part through urinary excretion. The successful application of this analytical method supports its suitable uses in further clinical benefits after oral administration of A. paniculata. Andrographis paniculata (Burm.f.) Nees, can alleviate the severity of symptoms and shorten the duration of treatment in patients with mild to moderate COVID-192. The recommended dose of A. paniculata extract for relief of symptoms in uncomplicated upper respiratory tract infections (URTIs) is 20 mg orally administered three times a day 5. However, a higher dose of the extract, 60 mg/dose of andrographolide three times a day (180 mg/day of andrographolide), has been used in a pilot study of patients who tested positive for COVID-19 in Thailand2. While the clinical benefits of the extract (60 mg of andrographolide/dose) have been evaluated, the data on the pharmacokinetics of this dose remain limited. Therefore, one of the objectives of this study was to investigate the clinical pharmacokinetics of andrographolide (AP1), a major phytoconstituent of this plant, and of other diterpenoid derivatives8 including: 14-deoxy-11, 12-didehydroandrographolide (AP3), neoandrographolide (AP4), and 14-deoxyandrographolide (AP6) after a single oral administration of A. paniculata extract .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 9. Additionally, the advanced liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) technique was established for compound screening and the metabolic pathways of all related metabolites were proposed in this study. Multiple reaction monitoring (MRM) is a targeted highly specific and sensitive MS technique10. The MRM selection coupled with triple quadrupole MS/MS (LC-QqQ-MS/MS) enables the selective quantification of specific compounds in complex mixtures. Literature reviews considering the existing quantitative determination of active compounds of A. paniculata extract in biological samples illustrated that most of the analytical inquiry has focused on andrographolide13, with only one study providing the determination of four major diterpenoids in plasma6. The previous method, however, limits the quantifiable detection of andrographolide with LLOQ at 2.50 ng/mL6. Due to this limitation, there were several restricted undetectable plasma levels of diterpenoid derivatives during the elimination phase7. As indicated in the previous reports14, the concentrations of major diterpenoids in human blood circulation were observed to be very low due to their poor oral bioavailability. Therefore, andrographolide and its derivatives were less than a quantifiable limit when blood sampling was carried out over 8 h until 24 h6. In case of this limitation, the desired pharmacokinetic parameters cannot be obtained during this elimination period. To support the complete pharmacokinetic investigation, the present study has further developed and fully validated a rapid, highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with the lower limit of quantification < 1.00 ng/mL, that simultaneously determined the four major diterpenoids in plasma samples. Their conjugated metabolites were also determined by using enzymatic digestion.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 investigation6. For this reason, the application of present newly developed analytical method was then expanded to urine samples. The method was validated for plasma and urine samples in terms of selectivity, specificity, accuracy, precision, linearity, range, limit of quantification, recovery, matrix effects, stability tests, and hemolysis assessment, according to the acceptance criteria of the US FDA Bioanalytical Method Validation Guideline15 and the International Conference on Harmonization ICH Guideline M10 on Bioanalytical Method Validation16.Taken together, the previous method was applicable for determining the four major diterpenoids  in only plasma samplesA. paniculata extract (60 mg of andrographolide/dose) in which the pharmacokinetics of the parent compounds and their conjugated glucuronide and sulfate metabolites with the presumptive phase II metabolic pathways pathways have been simultaneously determined in both plasma and urine samples.The present study is the first investigation of oral dosing of a high dose administration of A. paniculata aqueous extract capsules  was manufactured by Panaosod Co., Ltd.  in accordance with the quality standards of Good Manufacturing Practice (GMP). The assay contents of capsule composition were determined using a validated HPLC–DAD method17, and calculated as 20.26, 6.62, 5.28, 3.02 mg/capsule for AP1, AP3, AP4, and AP6, respectively.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 . The internal standard (IS) of digoxin (purity = 96.6%) was purchased from Sigma-Aldrich . The tested product, β-glucuronidase type IX-A (lyophilized powder) from Escherichia coli, glucuronidase activity of 1,000,000–5,000,000 units/g protein , and sulfatase type H-1 from Helix pomatia, sulfatase activity ≥ 10,000 units/g solid  were obtained from Sigma-Aldrich . For preparation of phosphate and acetate buffers, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), glacial acetic acid (CH3COOH), and sodium acetate (CH3COONa), were obtained from Sigma-Aldrich . Acetonitrile and methanol (HPLC grade) were purchased from Merck . Milli-Q water  was used in the LC–MS/MS system throughout the analysis procedures.For enzymatic digestion, 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  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  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.2) = 10.0 L/min, 350 °C; nebulizer pressure = 30 psi, fragmentation voltage = 250 V for negative mode and 100 V for positive mode. All acquisition data were analyzed using MassHunter Software B.07.01 and MassHunter Qualitative Analysis Software B.08.00 . To support the metabolite identification, the MS chromatograms were determined by comparing the fragmentation patterns of metabolites and library searches in Mass Hunter, and Human Metabolome Database (HMDB). For sample preparation, pooled urine samples at 0–4 and 4–8 h were prepared using the same extraction process described in the pharmacokinetic study.Mass spectrometric analysis was performed using an ESI negative and positive modes with varying collision energies  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 (NLiquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed using a Nexera X2 LCMS-8060NX triple quadrupole mass spectrometer  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  using a gradient mobile phase. The different solvents  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 The primary stock solution (1.00 mg/mL) of the four standard diterpenoids  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.A calibration curve was generated from different standard concentrations that were prepared by spiking a small volume of the working solution  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  were then similarly extracted following the sample preparation protocol with the addition of 50.00 ng/mL of IS.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.15 and the International Conference on Harmonization ICH Guideline M10 on Bioanalytical Method Validation16.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 GuidelineThe 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.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.2 ≥ 0.99). The acceptance criteria for accuracy and precision of the calibration curve should be within 85–115% and ± 15%CV of the nominal value, except for LLOQ, which should be within 80–120% and ± 20%CV.A twelve-point calibration curve was constructed by plotting the peak-area ratio  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.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%.Matrix effects at three levels of QC concentrations  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.The stability of plasma and urine samples were evaluated at three different QC concentration levels ; 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%.18, which was then vigorously vortexed. Thereafter, the working standard solution was added to the hemolyzed plasma at LQC and HQC samples (n = 6 replicates). The determinations for the analytes were compared between concentrations of each analyte in the blank plasma without hemolysis and the measured concentration in hemolyzed plasma. The measurement of all analytes was considered not to be interfered with hemolysis effect when the average of %accuracy was within a range of 85–115% and %CV was ≤ 15%.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 plasmaβ-glucuronidase). Then the mixture was incubated at different time points . The reaction was terminated by adding 150 µL of cold methanol containing internal standard (50.00 ng/mL). After vortex mixing for 10 min, the extracted samples were centrifuged at 12,000 rpm, 4 °C, for 10 min. Lastly, 150 µL of supernatant was collected by filtering the mixture through a 0.2-µM PVDF membrane (Chrom Tech), and then transferred to an insert vial for LC–MS/MS analysis.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  and also registered with the Thai Clinical Trials Registry . 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.2. Study participants were identified as healthy based on laboratory testing and physical examinations. The participants received 3 capsules containing A. paniculata extract  before breakfast with 240 mL of drinking water. Blood samples, in EDTA tubes, were taken after oral administration at 0, 10, 20, 30, 45 min, and 1, 1.5, 2, 4, 6, 8, 10, 12, 24 h post dose. All blood samples were then centrifuged at 4000 ± 100 rpm, 4 °C for 5 min. Plasma was pipetted and stored in cryotubes at − 80 °C until further analysis. Urine samples were collected during 0–4, 4–8, 8–12, 12–24, 24–32, 32–40 and 40–48 h after dosing and were then kept at − 80 °C before sample extraction.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/mmax) and the time to reach maximum concentration (Tmax) were taken directly from the individual plasma concentration versus time profiles. The area under the curve from time zero to the last sampling time (AUC0-t) was estimated using the linear trapezoidal rule. The apparent volume of distribution (Vd/F) and the elimination half-life (t1/2) were determined using non-compartmental analysis.Pharmacokinetic parameters were determined by non-compartmental analysis using PK solutions software, version 2.0 (Summit Research Services). The maximum concentration of four targeted compounds .Statistical analysis was performed using STATA statistical software . Continuous data were illustrated as mean ± SD, while half-life and TThe datasets generated and/or analyzed during the present study are not publicly available due to ethical concerns and confidentiality agreements but are available from the corresponding author on reasonable request that needs a consensus from colleagues.A. paniculata extract was analyzed in urine samples. A total of four compounds were tentatively identified using the chromatographic spectra from LC-QTOF/MS. The proposed metabolic pathways were associated with phase II conjugation. Three compounds (M1-3) involved in the glucuronidation pathway, and one compound (M4) related to the sulfation pathway  in negative ESI mode. This m/z was corresponded to the molecular formula of C26H38O11. The MS/MS fragmentation showed a successive loss of glucuronic acid and H2O (C26H38O11–176 Da–18 Da) at 349.2196 and 331.1827, and the product ions of M1 were consistent with the fragment pattern of andrographolide . The eliminated glucuronide (-176 Da) of M2 exhibited a fragmented molecule at 331.1843. The specific fragmentation (507 → 331 → 239 → 108) (Table The extracted-ion chromatogram (EIC) at 507.2188  in negative ESI of a deprotonated molecule [M-H]− was observed at m/z 509.2264 , which corresponded to a base formula of C26H38O10. The MS fragmentation indicated a deduction of glucuronide in M3 (C26H38O10-176 Da) at m/z 333.2002. The MS product ions were observed at 333, 305, and 285, which were determined to be a characteristic fragmentation of 14-deoxyandrographolide (AP6) . This m/z was 80 mass units (SO3) higher than that of andrographolide (AP1). A subsequent fragmentation product was detected at m/z 349.1469, which was due to the neutral loss of SO3 molecule. The product ions were 349/287/96 (Table Compound 4 (M4) at retention time 10.15 min, showed a deprotonated molecule [M–H]A. paniculata including andrographolide, deoxyandrographolide, 11,12-didehydro-14-deoxyandrographolide, neoandrographolide, andrographiside, deoxyandrographiside, and andropanoside8. However, the aqueous extract of A. paniculata used in this clinical study composed of andrographolide (AP1), 14-deoxy-11, 12-didehydroandrographolide (AP3), neoandrographolide (AP4), and 14-deoxyandrographolide (AP6). Based on the finding of compound screening, the conjugated glucuronide and sulfate were detected in the metabolite profiling after oral administration of A. paniculata extract. Considering this evidence and integrating all supporting data, the four major diterpenoids  and their respective conjugated glucuronide and sulfate metabolites have been selected as candidate analytes for determining their concentrations in plasma and urine samples, subsequently applied in this pharmacokinetic interpretation.The WHO monograph on selected medicinal plants indicated the major compounds of 11. Therefore, this study achieved LC separation by using water and acetonitrile, without adding formic acid in mobile phase.Chromatographic conditions were optimized to obtain a maximum intensity, and a high sensitivity with well-quantified chromatograms of each candidate analyte . Addition of formic acid resulted in peak distortion and a decrease in negative signal intensity of the AP1 parent compound, as described by Xu et al.Finally, optimal chromatographic separation was achieved by using a mobile phase of Milli-Q water (A) and acetonitrile (B) following elution gradients: during 0–1.5 min maintained at 40%B, then increased to 100%B during 1.5–3.0 min, and maintained at 100%B for 3.0–4.5 min. The gradient was returned to 40%B at 4.6–6.0 min. Column temperature was maintained at 40 °C during the LC separation with a constant flow rate of 0.5 mL/min, and 10 μL of injection volume.The MS system, coupled with a (-)ESI ion source, was used in this analytical method due to the significant intensity observed in the negative ionization. The MS quantification was obtained by monitoring precursor/product ion transitions (m/z) at 349.1/287.2, 331.1/239.2, 479.2/161.0, 333.1/285.2 and 779.3/649.2 for AP1, AP3, AP4, AP6, and IS, respectively  and only requiring a short analysis time (6 min) with the lower limit of quantification < 1.00 ng/mL.There was no interference effect from the endogenous matrix at the retention time of all analytes and IS. The representative chromatograms of blank plasma and urine; and the chromatograms with the retention times of the spiked four diterpenoid standards or IS in methanol, blank plasma and blank urine, are shown in Figs. S2, S3 and S4, respectively.The %CV for intra-day and inter-day precision of each analyte in plasma sample Table a were wiThree calibration curves were calculated based on data from three consecutive days using twelve concentrations for each standard in plasma and urine. The coefficients of variation of AP1, AP3, AP4, and AP6, were 0.24–9.76, 0.58–11.48, 0.20–10.64, 0.53–10.26 for plasma, and 2.71–13.43, 2.57–10.46, 2.55–10.13, 1.69–10.88 for urine, respectively. Percent accuracies were 85.50–109.02%, 94.26–106.46%, 90.93–101.04%, 92.75–110.08% for plasma, and 91.14–103.01%, 92.41–106.39%, 90.39–105.42%, 91.22–110.17% for urine, respectively. The results show good linearity (> 0.99) for both plasma and urine over the range of 0.98–1000.00 ng/mL. This method established LLOQ for the four diterpenoids at 0.98 ng/mL in both plasma and urine. .Mean extraction recovery for each analyte and IS in plasma and urine is summarized in Table The matrix effects at three levels of QC concentrations are presented in Table The stability of the four major diterpenoids in biological matrices was evaluated under different conditions. The results showed that AP1, AP3, AP4 and AP6 were stable in plasma and urine during five cycles of freeze–thaw storage. The analytes were also found to be stable after 3 months of “long-term” storage at − 80 °C. The extracted samples kept in the autosampler (at 4 °C) were stable for at least 24 h. No significant levels of degradation products were detected during the sample preparation process. The samples were stable for up to 8 h at room temperature as determined by quantitative analysis of all four analytes. The stability results in plasma and urine are summarized in Table There was no interference observed in the hemolyzed plasma blank at the retention time of each analyte and of IS. As indicated in Table The method for deconjugation was developed and optimized to obtain a complete hydrolysis reaction in plasma and urine samples. The results indicate that incubation time and enzyme concentration influence the hydrolysis reaction as observed in the measured concentration of each compound Fig. a–h.Figurβ-glucuronidase in phosphate buffer medium was obtained in 30 min  from Escherichia coli and 100 units/mL of sulfatase type H-1 from Helix pomatia (containing sulfatase 100 units/mL and glucuronidase 2193 units/mL).The highest completed hydrolysis of min Fig. a, c, whemin Fig. b, d. Themin Fig. e–h. The β-glucuronidase in phosphate buffer pH 6.8 (composed of 0.1 M sodium phosphate monobasic and 0.1 M sodium phosphate dibasic) at 37 °C. For the hydrolysis of sulfatase, 50 µL of 100 units/mL of sulfatase in an acetate buffer at pH 5.0 (composed of 0.1 M acetic acid and 0.1 M sodium acetate) was added into 50 µL of sample; the mixture was then incubated at 37 °C for 2 h. The reaction was stopped in all hydrolysis samples with 150 µL of cold methanol, and then protein precipitation was performed prior to LC–MS/MS analysis. However, due to unavailable standard of the conjugated forms (AP-glucuronide and sulfate-glucuronide). The quantitative analysis of conjugated metabolites was determined after the hydrolysis reaction by enzymatic digestion, and the free form was analyzed without hydrolysis. The conjugated standards of AP-glucuronide or AP-sulfate should be synthesized for determining the exact amount of the glucuronide or sulfate.Finally, the hydrolysis of glucuronide metabolites in the pharmacokinetic study was performed according to the following process: 50 µL of plasma or urine sample was incubated for 30 min with 50 µL of 1000 U/mL In this study, the concentrations of the total conjugated metabolites were used to generate plasma and urine concentration–time profiles. Standard curves were calculated for both plasma and urine samples and were within a range of 0.98–1000.00 ng/mL. The coefficients of variation of AP1, AP3, AP4, and AP6, were within 1.60–7.18%, 1.04–9.84%, 3.84–10.49%, 2.80–12.03%, for plasma; and 2.76–8.84%, 4.05–11.76%, 2.65–9.67%, 3.07–10.78%, for urine, respectively. Percentage of accuracies were within 92.98–107.22%, 92.25–107.47%, 94.11–103.41%, 97.73–107.46% for plasma, and 92.74–105.31%, 92.10–105.98%, 91.24–103.81%, 94.72–107.69% for urine, respectively.A. paniculata extract  are shown in Fig. max), while Tmax parameters for the conjugated metabolites were observed within 1.25–1.50 h after dosing  and their respective conjugated metabolites after oral administration of a single dose of A. paniculata extract, all four major compounds  of the extract were primarily excreted renally. A portion of these parent compounds underwent biotransformation in the liver to become the conjugated metabolites that were also eliminated via the urine. The exception to this was neoandrographolide (AP4). Similar to the results in plasma, conjugated metabolites of AP4 were negligible in the urine. The accumulation of parent diterpenoids in urine occurred within 32 h after administration 6. Due to these limitations, a bioanalytical method for quantitative determination of all four major diterpenoids, in both plasma and urine, was developed and validated for application in pharmacokinetic study. The method was completely validated at the quantifiable limit of LLOQ = 0.98 ng/mL. The plasma concentration of andrographolide was detectable until a completion of the elimination phase. This method minimized the sample volume necessary for extraction. The additional process was expanded to cover not only quantification of parent compounds but also of their conjugated metabolites in plasma and urine by hydrolysis reaction. In addition, this method also shortens the run time required for chromatographic separation to 6 min on the High-Performance Liquid Chromatography (HPLC). It is, therefore, beneficial to use this analytical method for the quantification of major parent compounds and conjugated metabolites of A. paniculata extract in biological samples, even when using a limited volume of plasma or urine obtained in animal or human studies. Moreover, this newly developed method is useful in clinical pharmacokinetics, when a large number of samples must be analyzed in a short time period.In a review of the literature, most studies determined only andrographolideA. paniculata extract, the parent compounds were extensively biotransformed by phase II hepatic metabolic pathways, where the compounds were conjugated with glucuronic acid and sulfate to increase their polarity. These conjugated metabolites then entered into blood circulation and finally were eliminated partly through renal excretion. These results are consistent with the findings of earlier preclinical pharmacokinetics studies in rats, in which sulfate metabolites were detected in both the livers and urine20. In addition, another pharmacokinetics study in humans demonstrated that glucuronide and sulfate conjugated metabolites were present in urine after oral administration of A. paniculata extract22.The validated analytical method in this study was applied to determine four active diterpenoids in preliminary testing of four subject samples. The results of the pilot pharmacokinetic study of 4 subjects show that the four major parent compounds are partially absorbed into blood circulation, resulting in low detectable concentrations of the free form of each analyte in plasma samples. The analysis of plasma levels of the parent compounds increased after deconjugation by glucuronidase and sulfatase enzymes, suggesting the presence of glucuronide and sulfate metabolites. This finding implies that after oral administration of A. paniculata extract. The one exception was the conjugation of neoandrographolide (AP4). The metabolites in terms of AP4 glucuronide and sulfate were negligible in plasma and urine samples. This finding suggests that glucuronidation and sulfation may not be the major metabolites of neoandrographolide. One reason for this may be that the chemical structure of neoandrographolide (diterpene glucoside) contains a sugar moiety at C-19 . Quantitative analysis of conjugated metabolites was determined using an enzyme hydrolysis reaction. The findings concerning the identification of novel metabolites of A. paniculata and the related compounds fill a gap in the knowledge of human metabolic pathways involved in their phase II biotransformation including glucuronidation and sulfation. Further, the findings of this pilot clinical pharmacokinetic study support future research efforts examining the use of A. paniculata extract in human  for the treatment of COVID-19 symptoms and other therapeutic uses.This newly developed LC–MS/MS method provides a thorough analytical method for the intended purposes of quantitative determination of the four major parent compounds  in both plasma and urine samples. In this way, this method offers a solution to the limitations inherent in other methods and enables a more complete pharmacokinetic analysis. This highly sensitive method (LLOQ < 1.00 ng/mL), with requiring a short analysis time (6.0 min), was successfully applied in a pharmacokinetic study of orally administered Supplementary Information.


The variables of interest were sex, office work, and sedentary work, with age, clothing thermal insulation, and alcohol consumption as adjustment factors. The mean CT was 14.6 °C. Office workers had a 6.8 °C higher mean CT than other workers, but the difference ranged from 3.8 °C to 10.0 °C from P17 to P83. Sedentary workers had a 2.0 °C higher mean CT than others, but the difference increased from 0.5 °C to 3.0 °C through P17–P83. The mean CT did not differ between sexes, but men had a 1.6–5.0 °C higher CT at P17–P50 (>20 °C) and a 5.0 °C lower CT at P83 (<10 °C). The CT was relatively high at warm (≥10 °C), dry (relative humidity <41%), and drafty (air velocity > 0.35 m/s) worksites. We conclude that office, sedentary, and female workers and those working at warm, dry, and draughty sites are sensitive to the coldest temperatures, whereas male workers are sensitive even to moderate temperatures.The self-assessed threshold temperature for cold in the workplace is not well known. We asked 392 chicken industry workers in Thailand what they regard as the cold threshold (CT) and compared subgroups of workers using linear and quantile regressions by CT sextiles (percentiles P Poultry industry workers, who often work at temperatures down to −20 °C, frequently suffer from cold-related harm. In Thailand, for example, most of them report poor work performance, or musculoskeletal or respiratory symptoms ,2,3,4,5.Thailand is a major producer of chicken meat and its products, worldwide. In 2020–2021, the country was ranked as the fourth largest exporter after Brazil, the USA, and the EU ,16. TherIn our previous study, we asked poultry industry workers in Thailand what temperature they perceived as cold at the workplace . The mosWe used a database of poultry industry workers described previously in detail . To detecl) was calculated as Icl = 0.161 + 0.835 ∑ Iclu, where Iclu denotes the clo value for each clothing item.Information on each participant’s personal details, work-related factors, living habits, and cold-related symptoms was obtained through a structured interview conducted by trained interviewers . The sel® 9545 . The technical details of the instruments used are in Air temperature (Ta) and relative humidity (RH) were measured using a 303 C thermo-hygrometer , and air velocity (AV) was measured using a VelociCalc17, P33, P50, P67, and P83, from warmest to coldest), and quantile regressions were conducted using these percentiles as the response variates. Sextiles were used because they described the response pattern in more detail than lower quantiles (tertiles to quintiles) and were less erratic than higher quantiles (septiles to deciles). The explanatory factors of main interest were sex, job category, and physical work strain, whereas age, thermal insulation of clothing, and alcohol consumption were included as adjustment factors. Additional analyses allowed for workplace Ta, RH and AV, but no attempt was made to quantify their independent effects. Education, smoking, leisure-time exercise, and moving between cold and warm sites were also considered as adjustment factors, but they had only marginal effects on the parameters of interest and were omitted from the final analyses. The models were fitted using a variant of the Barrodale and Roberts algorithm [https://CRAN.R-project.org/, accessed on 21 January 2023). The results were also presented as marginal means for each variable of interest, involving the calculation of averages of all model-estimated CTs for the outcome [The self-assessed CTs (°C) were first described in terms of means, medians, SDs, and ranges; their group-wise differences were tested using one-way analysis of variance and linear regression coefficients. The subgroup differences were further elaborated graphically by presenting the cumulative distribution of CT in each subgroup, with values accumulating from warm to cold. These suggested that group-wise differences in CT may be different at various locations of its distribution. Therefore, quantile regression  was usedlgorithm . The res outcome ,25. Each2 , and 147 participants (38%) were classified as obese (BMI ≥ 25.0 kg/m2). Ninety-six participants (24%) were smokers, 46 (12%) consumed alcohol on a weekly basis, and 182 (46.4%) exercised at least once a week during leisure time. Twenty-six participants (6.7%) reported diagnosed cardiovascular disease, 20 (5.1%) reported a respiratory disease, and 25 (6.4%) reported a back, joint, skin, or mental condition or diabetes mellitus.The body mass index (BMI) averaged 25.2 kg/mThe mean daily working time was 8.7 h per day , with 54% working > 8 h/day. Office workers did slightly fewer hours (8.3 h/day) than other workers (8.9 h/day) and fewer of them worked > 8 h/day . Sedentary and non-sedentary workers showed no major difference in average working time  nor percentage working > 8 h/day .The mean thermal insulation of clothing was 1.15 clo , and it was lower in women (1.08 clo) than in men (1.20 clo), lower among office workers (0.85 clo) than others (1.27 clo), and lower among sedentary (1.03 clo) than non-sedentary workers (1.19 clo). The mean clo was similarly low among office workers independent of whether the work was sedentary or non-sedentary , and it was similarly high among sedentary and non-sedentary non-office workers .Altogether, 308 subjects (78.8%) moved daily between cold and warmer sites at least four times a day, with 78.5% and 79.0% for men and women, and 70.3% and 82.9% for office workers and other workers, respectively. The participants spent an average of 1.59 h per day (range 0–10) at temperatures <0 °C, with men and women spending 2.92 h/day and 0.40 h/day, respectively, office workers and other workers spending 0.19 h/day and 2.87 h/day, respectively, and sedentary and non-sedentary workers spending 0.81 h/day and 1.99 h /day, respectively. All participants reported that they had air conditioning in the workplace.Measurements of Ta, RH, and AV for a subsample of 284 workers are described in Women worked at warmer temperatures than men , and their worksites were more humid  while AV was similar for women and men . Office workers experienced higher temperatures than other workers , and their worksites were more humid  and drafty . Sedentary and non-sedentary workers showed little difference regarding worksite Ta  and RH , but sedentary workers experienced more drafty sites than others . Highly educated workers had higher worksite temperatures than low-educated workers , and they worked at more humid  and drafty sites .The mean CT was 14.6 °C , but the distribution was skewed to the warmer side, with a median of 18.0 °C, and 90% of the values located within the range −4.0–27.0 °C . Relativ80–P90), while no sex differences were seen at CT values ≥ 10 °C. Office workers reported CTs of at least 10 °C higher than other workers, mainly at colder values of CT, although differences existed throughout the distribution. The CT was as much as 10–15 °C higher among the highly educated than the low-educated workers, but mainly at percentiles P90 and higher.Subgroup differences larger than those between the mean CT values are shown in 40 and above (Sedentary workers reported 5–15 °C higher CTs than non-sedentary workers at percentiles Pnd above . Partici80 and higher), while little differences were seen at lower percentiles. Participants at dry working sites (RH < 41%) reported as cold 5–8 °C higher temperatures than those working at more humid sites but mainly at central values of the CT distribution. Workers at drafty working sites (air velocity > 0.38 m/s) assessed as cold 5–20 °C higher temperatures than those at less drafty sites but only at P40 or higher.83. However, the adjustment for personal and work-related factors reduced this deficit at P83 to 5.0 °C and turned the non-existent crude differences at P17–P50 to male excesses of 1.6–5.0 °C, with no marked changes after further adjustments for workplace Ta, RH, and AV. 17–P50, the contrary difference at P83, and the resulting crossover of male and female CTs.The crude sex differences in 17 to P33 among the office workers (While the crude mean CT in  workers .17) remaining after all adjustments. The latter trends were based on a slightly steeper accumulation of CTs across percentiles among sedentary workers than among non-sedentary workers  among sedentary than non-sedentary workers, a difference that reduced to 2.0 °C (95% CI 0.1–3.8) after adjusting for personal and workplace factors, and almost vanished when further adjusted for Ta, RH, and V . However workers .67 and also elevated at P83; however, almost no significant coefficients remained after adjustments for Ta, RH, and AV.Low thermal insulation of clothing (clo < 1.15) was associated with a 3.1 C (CI 1.2–5.1) higher mean CT compared with higher insulation (clo ≥ 1.15), with inconsistent variations of coefficients at various percentiles; however, this was reduced to insignificance after all adjustments . The mea83 (Working at warmer sites (Ta ≥ 10 °C) was associated with a 1.8 °C higher mean CT (CI −0.2–3.9) compared with working at colder sites, but a 7.0 °C higher CT (CI 2.0–12.0) was seen at P83 . A dry wThere was wide variation in the temperatures that the participants regarded as cold, from −28 °C to 30 °C, with 90% of values located within the range of −4.0 °C to 27 °C and one half at 18.0 °C or higher. Temperatures warmer than 10 °C, the ISO-defined temperature for workplace cold , were acAlthough the mean and median CTs observed, i.e., 14.5 °C and 18.0 °C, respectively, were not directly based on actual cold exposure, but rather on the worker’s image of his/her past thermal sensations of cold, they are close to or slightly below various estimates defined as cool or cold, for example: 18 °C in Taiwan , 18 °C iOur main finding was the 6.8 °C higher CT among office workers compared with that among other workers. Our previous study found that a higher proportion of office workers regarded temperatures > 20 °C as cold, and more office workers suffered from cold-related cardiac symptoms and musculoskeletal pain, as well as poorer concentration and motivation ,2. In adThe thermal insulation of clothing as low as 0.85 clo among office workers provides an obvious explanation for their relatively high CT. Clo values lower than the median 1.15 were associated with a 3.1 °C higher CT in the crude analysis, although this factor did not appear as an independent factor, likely due to its effect mixing with other factors. Thermal insulation was allowed in the analysis; however, office workers frequently suffer from cold symptoms in the cheeks, ears, and elbows, which often remain unprotected . Other eMuch of the reason why office workers have a high CT relates to the question of why they do not wrap up adequately. One could assume that they have mistaken beliefs of office temperatures of around 20 °C not being harmful because these are much higher than the standard definition for workplace cold of 10 °C . In addiWhile 61% of the office staff were sedentary, sedentariness was also associated with CT independent of office work. We have previously seen that sedentariness among chicken industry workers is associated with cold-related pain in the knees, lower back, and thighs . A higheAccording to previous reports, women are more sensitive to cold than men ,44,45,46This implies that, among all workers who regard temperatures warmer than approximately 15 °C as cold, CT is up to 5.0 °C higher in men, while among those assessing temperatures <15 °C as cold, the threshold is 5.0 °C lower in men. This may be interpreted in terms of men possibly using adequate protection only at temperatures they consider very cold, but fail to do so at more moderate temperatures, which women, in turn, recognise as cold enough to require protection. Men are known to accept very cold temperatures more likely than women, while fewer sex differences exist regarding moderate cold . Our resIt has been reported that older people prefer slightly higher temperatures than younger people because of decreasing metabolic rates  and impaAs obese people are less sensitive to cold than others owing to their thicker subcutaneous fat layer and greater lean body mass, which increases heat production , we expeChicken industry workers who consume alcohol weekly or more often have an increased occurrence of cold-related performance problems . We obseSmokers had a 1.8 °C lower mean CT than non-smokers, although this was not a significant factor in the multivariable analysis. This finding was unexpected because smoking reduces cutaneous circulation  and micrSince repeated daily exposure to cold leads to blunting of physiological responses and adjustments to thermal stimuli ,57, coldThe main strength of our study is that we focused on variations in the self-assessed cold between subgroups of workers. The need for subgroup analyses to identify vulnerable groups has been recognised ,33, but The validity of the question regarding the measurement of CT can only be assessed in terms of face validity because the answers cannot be compared with any gold standard. The question was clear and easily understandable, although we did not specify the meaning of cold: a general feeling of cold, feeling cold while breathing, or touching cold surfaces. The question relates to a true experience of cold at the participant’s regular worksite or other sites in his/her workplace and is likely to detect group-wise differences in cold sensitivity. However, establishing the content validity and predictive value of subjectively assessed CT requires further study, preferably in terms of prospective studies.Convenience sampling was used, which was dictated by the availability of workers during their regular working hours and the working schedules of the study team. This may have affected the results in an unknown way, but any major bias in the group-wise differences is unlikely because we adjusted for relevant confounders. One limitation is that the independent effects of all explanatory factors may not have been identified with certainty because of collinearity. Several medical conditions and medications may further affect cold sensitivity , but we Asking food industry workers about their subjective cold threshold is useful not only because it reflects the actual experience of cold but also because people are likely to take protective action at temperatures which they regard as cold. Our study provides an obvious case for preventive action, especially since we identified subgroups of workers who perceive markedly higher temperatures as cold compared to that of others. Occupational health care should advise office staff and sedentary workers to wear thicker clothing and more clothing layers, even if they do not regard them as appropriate or necessary. Male workers should be informed that even moderate cold requires protection, and female workers who regard only very low temperatures as cold need more clothing. The recommended values for workplace temperatures should preferably be customised based on their entire distribution, and not only on any single value or range. Additional measures to be targeted to office and sedentary workers and to men and women include drinking hot beverages, keeping hydrated, avoiding unnecessary stays in the cold, and setting appropriate air conditioning. The regulation of indoor temperatures, allowing for different perceptions of cold among various categories of workers, should be considered. Research-based recommendations and standards for cold work that do not currently exist in Thailand should be established, considering variations between subgroups of workers. Further research is needed to explain the reasons for variations in cold perception. This information is especially relevant in view of ongoing climate change, which will affect this region the most  and will


HES is recommended for arthritis, gout, and joint pain. The current endeavor is an attempt to put it to the test and verify its efficacy scientifically. It was tested for DPPH, hydroxyl, and nitric oxide scavenging activities. It was shown that HES had the greatest TAC and FRAC values when compared to catechin and ascorbic acid. HES exhibited DPPH and hydroxyl radical scavenging activity that was dose-dependent. Incubation of sodium nitroprusside solutions in PBS at 25°C for 150 min resulted in the production of nitric oxide. Nitric oxide production was effectively decreased by HES. Anti-inflammatory medications boosted the migration of PMN cells toward the chemoattractant FMLP in an agarose experiment of PMN chemotaxis. In carrageenan-induced rat paw edema, in the HES-treated group, paw thickness was 3.021 ± 0.084 at t = 0, but it showed an increase in paw inflammation after one hour, i.e., 3.195 ± 0.082 cm which again showed a decrease in paw thickness up to 4th hour, i.e., 3.018 ± 0.078, 2.98 ± 0.032, and 2.684 ± 0.061 at t = 2, 3, and 4, respectively. It showed again getting back to the normal thickness of paw at t = 24 hrs, i.e., 3.029 ± 0.118 cm. It is concluded that the formulation is potent enough and can be used effectively for the treatment of inflammation and associated health issues. Moreover, there is much scope to evaluate its effectiveness using different in vitro and in vivo models.Habb-e-Suranjan (HES), an Unani formulation, has been studied for its anti-inflammatory properties in both  Unani System of Medicine is Graeco-Arabic medicine founded by Hippocrates and Galen and refined by Arabian and Persian doctors such as Rhazes  and Avicenna (Ibn-e-Sina), Al-Zahrawi, and Ibn Nafis throughout the Middle Ages. Buqrat 460-377 BC), also known as Hippocrates, was a descendant of Aesculapius and is regarded as the “Father of Unani Medicine” , 2. It b77 BC, alMany researchers are working on herbal medicine to prove its effectiveness in a variety of diseases including COVID-19 –14. It iFor the promotion and maintenance of health, the Unani system of medicine places a strong focus on lifestyle management. Diet, lifestyle, emotions, interactions with the environment, and even spiritual considerations are all taken into account . It takeAloe barbadensis), Halela , and Suranjan (Colchicum luteum) were combined in equal amounts in the test formulation, Habb-e-Suranjan (HES), which was made under the supervision of a GMP-certified pharmacist at CRIUM, Hyderabad, and sent to the research site. Three times a day, patients were instructed to apply 5 ml of lukewarm oil to the afflicted joints in the morning and evening twice a day for two weeks after taking HES (500 mg each) for two weeks  × 100.n et al. . The capA0 is the absorbance of the control, whereas the drug's/absorbance standard is A1. The IC50 was then determined by plotting the percentage of inhibition versus the concentration. At each concentration, the experiment was carried out three times.Here, (2) Hydroxyl Radical Scavenging Activity. By using the technique of Rahman et al., the hydroxyl radical scavenging activity of the medicines was determined  × 100/A0.termined . This syA0 is the control's absorbance without a sample present. Sample and 2-deoxy-D-ribose were added to A1 and the absorbance was measured. When 2-deoxy-D-ribose is omitted from the sample, its absorbance is equal to A2. The IC50 was then determined by plotting the percentage of inhibition versus the concentration. At each concentration, the experiment was carried out three times.In the equation, (3) In Vitro Activity of HES on Human PMN Cell. Samples of fresh, anticoagulated blood taken from healthy human volunteers were used to isolate PMN cells. In order to extract PMN cells, 5 ml of blood was combined with 1.75 percent Dextran T-500 solution in saline and placed for 45 minutes at room temperature for sedimentation. The remaining RBCs were washed three times with PBS before being suspended in a 10% fetal bovine serum-containing RPMI-1640 medium, which was then centrifuged. The PMN cell density was set using RPMI-1640 media containing 10% fetal calf serum. As part of the in vitro testing, dilutions of the active ingredients in each of the assays were made in RPMI-1640 medium by adding 10 μl of the dilution to 990 μl medium. Effect on the chemotaxis of PMN cells/human PMN cell chemotaxis produced by formyl-N-methyl-leucyl-phenylalanine (FMLP) was studied in vitro using the chemotaxis test technique under agarose. The agarose solution (1.2 w/v) was prepared in Eagle's minimal essential medium (MEM) and heated to 56°C. The microscopic slides (50 × 75 mm) were produced by putting 6.0 cc of the aforementioned solution over each slide and allowing this to settle at room temperature. The slides were kept at 2-8°C for six hours. After that, 5 mm diameter wells were drilled into the settled gel, each one 4 mm in diameter. The wells were added with 20 μl FMLP solution in RMPI-1640 medium whose concentration was 1 × 10−8 for determining the chemotaxis in response to FMLP. The surrounding wells contained 10 μl PMN  and 10 μl of each drug solution. The slides were then incubated at 37°C in the presence of 5% CO2 for 2 h. The neutrophils were fixed for 30 min using methanol, after the process of incubation. The gels were gently removed after fixation, and the neutrophils were stained with Giemsa stain.The slides were then examined at a 100x magnification using a digital microscope. Motic-3.2 software was used to measure the distance traveled by the leading front of cells toward the FMLP-containing wells .(4) Nitric Oxide Scavenging Assay. Borra and Gurumurthy explained the procedure in detail, in which a solution of 10 mM sodium nitroprusside in phosphate-buffered saline (PBS) pH 7.4 was combined with 0.5 ml of drug solutions at different concentrations ranging from 10 to 50 μg/ml and ascorbic acid at concentrations of 25 to 200 μg/ml. This combination was kept at a temperature of 25°C. 0.5 ml of the incubation solution was collected after 150 minutes and mixed with 0.5% of the Griess reagent, 0.5% of the sulfanilic acid reagent , and 0.1 percent w/v of the naphthyl ethylene diamine dihydrochloride (1.0 ml). After 30 minutes of incubation at room temperature, the mixture's absorbance at 540 nm was recorded [recorded .The HES pills were light olive brown and oval (solid) with a distinctive agreeable taste and astringent smell. The pills were kept in a cold, dark area in firmly covered containers, shielded from moisture, light, and temperature. Physicochemical examination found a slightly basic pH of 6.5 as stated in Tables The presence of numerous secondary metabolites like alkaloids, tannins, flavonoids, proteins, and the mucilage may explain the therapeutic benefits of HES tablets. As a result, preliminary screening assays can aid in the detection of bioactive components, which can lead to medication development and discovery. Furthermore, these tests make it easier to estimate the quantity of pharmacologically active chemical substances and to separate them qualitatively. In the HES extract, preliminary phytochemical screening with several qualitative chemical assays revealed the presence of reducing sugars, protein, amino acid, alkaloids, and tannins, whereas carbohydrates, glycosides, flavonoids, saponins, fats and oils, and steroids were lacking.t = 0, paw thickness was observed at 3.026 ± 0.89 cm which remained the same after 24 hrs. In the carrageenan control group, paw thickness showed an exponential increase and displayed significance at p < 0.001. It was 3.031 ± 0.076 cm at t = 0, which was increased after 24 hrs to 4.261 ± 0.123 cm. In the HES-treated group, paw thickness was 3.021 ± 0.084 at t = 0, but it showed an increase in paw inflammation after one hour, i.e., 3.195 ± 0.082 cm which again showed a decrease in paw thickness up to 4th hour, i.e., 3.018 ± 0.078, 2.98 ± 0.032, and 2.684 ± 0.061 at t = 2, 3, and 4, respectively. It showed again getting back to the normal thickness of paw at t = 24 hrs, i.e., 3.029 ± 0.118 cm. In diclofenac sodium-treated groups, it displayed the same pattern as demonstrated by the HES-treated group. All the results obtained were significant with the control group. The values are tabulated in In a carrageenan-induced paw edema test, rats received subplantar injections of carrageenan that displayed increased and decreased paw thickness with time. In the normal control group at p < 0.001) showed a significant decrease in pain from 0 to 60 min, and similar treatment with standard oxycodone also showed a significant decrease (p < 0.001) in pain from 0 to 60 min. Thus, HES and the standard drug showed complete blockade of carrageenan-induced hypersensitivity.In p < 0.001) and metformin (p < 0.001) have shown significant inhibition of dye leakage . From All three samples have shown significant inhibition concerning control . Paw edema was significantly reduced in the HES and MTX-treated groups compared to the untreated controls (p < 0.001)  .Arthritis-related hematological alterations are shown in μg/ml, the absorbance of HES was in the range of 0.584 ± 0.053 to 1.89 ± 0.076, while at 150 μg/ml, the range was 0.874 ± 0.043 to 2.394 ± 0.064. An increase in therapeutic dosage resulted in an increase in total antioxidant activity.The TAC and FRAC of HES are shown in μg/ml, the absorbance was in the range of 0.975 ± 0.056 to 3.409 ± 0.065, while at 150 μg/ml, the range was 1.345 ± 0.086 to 3.225 ± 0.067. Results show that HES has the greatest TAC and FRAC values, which is similar to the catechin and ascorbic acid standards.A moderate to high FRAC was seen in HES when drug concentrations were boosted in the animal. At 100 μg/ml, the scavenging activity of HES and BHT was 78 ± 0.64 and 95 ± 0.94 μg/ml. By suppressing the DPPH radical with a high inhibition value, the extract demonstrated concentration-dependent antioxidant activity. The approach relies on the creation of the nonradical form DPPH-H by the reaction, which reduces the DPPH solution in the presence of a hydrogen donor antioxidant. The extract was able to convert the DPPH radical into yellow diphenyl picrylhydrazine, which was stable. Because of its propensity to contribute hydrogen, ascorbic acid has been discovered to decrease and discolor 1,1-diphenyl-2-picrylylhydrazyl. HES extract appears to be able to give hydrogen while also acting as an antioxidant. As the concentration of the extract rises, so does the effect of elimination.μg/ml was 47 ± 0.75, while that of catechin was 63 ± 0.84 μg/ml.HES has exhibited a dose-dependent hydroxyl radical scavenging activity as illustrated in p < 0.001).In an agarose experiment of PMN cell chemotaxis, the drugs stimulated the migration of PMN cells toward the chemoattractant FMLP. According to 50 for HES was 41.59 ± 2.45 μg/ml, while the IC50 for ascorbic acid was 186.34 ± 3.28 μg/ml.In tests, the formulation had just a little ability to reduce levels of nitric oxide (NO). Drug concentration raises the percentage of inhibition. After 150 minutes of incubation at 25°C with sodium nitroprusside solutions in PBS, NO production was detected. As shown in in vitro and in vivo models. A battery of tests evaluating a range of activities such as carrageenan-induced rat paw edema, Von Frey test, adjuvant-induced arthritis (AIA) in rats, hydroxyl radical scavenging activity test, nitric oxide scavenging activity test, free radical scavenger DPPH, and in vivo and in vitro anti-inflammatory activities have been demonstrated. The inclusion of diverse polyherbal components, which is a recognized powerful antioxidant and anti-inflammatory, could explain Habb-e-Suranjan's effectiveness. Overall, the current study shows the anti-inflammatory and antioxidant ability of formulation, which not only rationalizes some ethnomedicinal claims but also identifies a promising candidate for further research, particularly in chronic inflammatory disorders like rheumatoid arthritis. More study is needed to be done to find the biomolecules that are responsible for the anti-inflammatory and antioxidant effects. In the future, we are aiming to investigate its effectiveness using more quality in vivo models and by estimating more precise biochemical parameters followed by histological examination.One of the most frequently prescribed Unani formulations for the treatment of inflammation and arthritis is Habb-e-Suranjan. In the present investigation, we tried a scientific attempt to evaluate its anti-inflammatory potential using different


In the subsequent single ascending dose parts in HVs  and patients with PD , participants were randomized to placebo or AZ‐009 up to 4 mg. In patients, after medication withdrawal, Movement Disorder Society‐Unified Parkinson's Disease Rating Scale part III and on within 45 minutes postdose increased dose dependently: 0% (placebo), 17% (2 mg), 50% (3 mg), and 83% (4 mg).AZ‐009 was rapidly absorbed with peak plasma concentrations at 2 minutes, as compared to 30 minutes for subcutaneous apomorphine. Adverse events for AZ‐009 were comparable to subcutaneous apomorphine, except for mild and transient throat irritation. Adverse events limited AZ‐009 dose escalation in HVs to 3 mg. Patients tolerated up to 4 mg. In patients with PD, 2, 3, and 4 mg AZ‐009 reduced mean Movement Disorder Society‐Unified Parkinson's Disease Rating Scale part III score (standard deviation) by 10.7 (13.6), 12.8 (7.9), and 10.3 (3.7) points, respectively, compared to 4.8 (4.9) after placebo at 10 minutes postdose. The percentage of patients achieving full off periods. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyAZ‐009 appears to be a rapid‐acting and reasonably well‐tolerated formulation for treating  Initial therapy is selected based on a number of criteria, including patient age, comorbid conditions, disease severity, and degree of functional disability.l‐dopa therapy, and a large proportion of patients experience motor complications within a few years of starting its use.off episodes (“wearing off”), prolonged latency to on, inability to turn on, sudden on/off fluctuations, and/or dyskinesia. These fluctuations in therapeutic effects can be predictable or unpredictable and do not only involve fluctuations in motor symptoms but also in nonmotor symptoms, such as anxiety/panic attacks, mood changes, slow thinking, and pain.Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects movement, cognition, emotion, and autonomic activity. Patients with PD are usually treated with dopaminergic drugs, such as levodopa  of AZ‐009 and compare it with a registered subcutaneous apomorphine injection (part A) and to study the safety and PK of single ascending doses of AZ‐009 in healthy volunteers (HVs) (part B) and patients with PD (part C). The last study part also evaluated the efficacy of AZ‐009 during an induced morning ClinicalTrials.gov (NCT03822364). The protocol was approved by the Independent Ethics Committee of Foundation Beoordeling Ethiek Biomedisch Onderzoek. Before any study‐related activity, all participants provided written informed consent. The study was conducted at the Centre for Human Drug Research between October 2018 and May 2019.The study was conducted in accordance with European Medicines Agency guidelines for Good Clinical Practice and registered in off state as assessed by a physician.This study was divided into three parts: parts A, B, and C. Refer to Supporting Information Fig. The study consisted of a screening visit; at‐home pretreatment with an antiemetic (domperidone) three times daily; a single stay of 7, 3, or 2 days  at the clinical research unit; and a follow‐up telephone call. In part A, participants received 10 mg domperidone three times daily from 3 days before dosing until after last dose. In part B, domperidone dose was increased to 20 mg on the evening and morning before dosing. At other time points, domperidone intake remained 10 mg as in part A. In part C, participants received 20 mg domperidone three times daily from 2 days before dosing until after dosing.2 were eligible to participate. In study part C, nonsmoking patients with PD with recognizable off periods aged 30–85 years with Hoehn & Yahr stage I–IV were eligible for participation. Patients were excluded if their systolic blood pressure (BP) was below 100 mm Hg at screening or baseline, they had symptomatic clinically relevant and medically uncontrolled orthostatic hypotension, or a history of long QT syndrome and/or a QTcF of >470  or >480 ms .In study parts A and B, healthy nonsmoking men and women aged 18–60 years with a body mass index of 18–32 kg/mAZ‐009 was available in two dose strengths (1 and 2 mg apomorphine hydrochloride). A dose of 3 mg was delivered by three consecutive oral inhalations of 1 mg and a dose of 4 mg by two consecutive inhalations of 2 mg. Matching Staccato placebo  was identical to AZ‐009 but without a coated apomorphine film. AZ‐009 and matching placebo were manufactured by Alexza Pharmaceuticals Inc. Participants were instructed to inhale through the mouthpiece with a steady deep breath and to hold their breath for as long as possible, up to 10 seconds.Inhalation through the product initiates the controlled rapid heating of a thin film of excipient‐free apomorphine to form a thermally generated drug vapor. The vapor condenses into aerosol particles with a particle size distribution appropriate for efficient delivery to the deep lung, ie, with a mass median aerodynamic diameter in the range of 0.5 to 3.5 μm.In study part A, apomorphine was also administered subcutaneously with the APO‐go PEN. APO‐go was provided as the commercially available product with the appropriate country‐specific labeling by the Leiden University Medical Centre pharmacy. A volume of 0.2 mL (2 mg) was injected in the thigh.For all study parts, a medical screening was performed to assess eligibility based on medical history, concomitant medications, electrocardiogram, vital signs, routine hematology, chemistry and urinalysis, and physical examination. Electrolytes and QTcF were assessed at screening (before domperidone initiation) and again at baseline (after domperidone initiation and before apomorphine administration). During the study, safety was evaluated by monitoring of AEs , vital signs, electrocardiograms, physical examination, and clinical laboratory tests. Orthostatic hypotension was defined as a systolic BP decline of ≥20 mm Hg or a diastolic BP decline of ≥10 mm Hg on standing. Postural dizziness was defined as dizziness on standing that was not accompanied by a decline in BP (at the scheduled measurement time) as defined for orthostatic hypotension.Blood samples for PK analysis were obtained predose; at 1, 2, 5, 10, 20, 30, and 45 minutes postdose; and 1, 2, 4, 8, and 24 hours postdose in parts A and B. In part C, samples were obtained predose; at 2, 5, 15, 30, and 45 minutes postdose; and at 1, 1.5, 4, and 5 hours postdose. A lower sampling frequency and shorter sampling duration were chosen in part C to allow time for efficacy measurements and to reduce patient burden. Plasma samples were analyzed for apomorphine using a validated liquid chromatography–tandem mass spectrometry method.Cmax), time to Cmax (Tmax), apparent terminal elimination half‐life (t1/2), and area under the plasma concentration time curve from zero to infinity (AUC0‐inf).Plasma concentrations of apomorphine were analyzed using noncompartmental analysis in Phoenix WinNonlin version 8.1. PK parameters that were calculated include maximum plasma concentration  was performed using an analysis of variance model and the two one‐sided t tests procedure. The analysis of variance model included factors for sequence, subject within sequence, treatment, and period. Point estimates and 90% confidence intervals for the geometric mean ratios (AZ‐009/subcutaneous apomorphine) were calculated for PK parameters by back transformation to the original scale.For part A, the comparison of the dose‐normalized log‐transformed PK parameters Cmax and AUC0‐inf for apomorphine were compared across dose levels (1–4 mg) to assess dose proportionality. Statistical analyses were conducted using a power model with mixed effects.For parts A to C combined, Motor function was assessed using part III of the licensed Movement Disorder Society‐Unified Parkinson's Disease Rating Scale (MDS‐UPDRS). Physicians administering the scale were trained and certified in its use. To the degree feasible, the same physician evaluated a patient at day −1 (day before dosing), day 1 predose, and 10, 30, and 60 minutes postdose. Mean change from baseline MDS‐UPDRS part III total score was calculated and presented graphically.on with disabling dyskinesia, on with nondisabling dyskinesia, on with no dyskinesia and normal motor function, partial on and off. The first three categories were combined, classified as full on, and presented graphically as percentage of patients turning full on.The disease state of a patient was assessed by a physician predose and at 2, 10, 20, and 45 minutes postdose. Possible categories were See Supporting Information Figs. Cmax 1 to 2 minutes after dosing and showed a biexponential elimination phase. In contrast, apomorphine concentrations after subcutaneous apomorphine injection increased over time with a median Tmax of 30 minutes. When normalized for dose, the Cmax and AUC0‐inf geometric mean ratios  of AZ‐009/subcutaneous apomorphine were 2.9 (1.6–5.4) and 0.8 (0.5–1.2), respectively. Mean apomorphine t1/2 ± standard deviation (SD) of AZ‐009 was shorter (39 ± 7 minutes) than that of subcutaneous apomorphine (55 ± 22 minutes). Intersubject variability (CV%) in apomorphine Cmax and AUC0‐inf was higher for AZ‐009 (53.7% and 47.2%) than for subcutaneous apomorphine (36.4% and 22.7%).Apomorphine was rapidly absorbed into the systemic circulation after administration of AZ‐009 and subcutaneous apomorphine in HVs Fig. . DescripTmax in HVs was similar as in part A, ie, 1 minute. The first PK sample in patient with PD was taken at 2 minutes postdose. Median Tmax in patients with PD was 2 or 3 minutes depending on the dose group  AUC0‐inf was 5.1 (1.5), 12.6 (4.5), and 11.3 (5.1) h/ng/mL for 2, 3, and 4 mg AZ‐009, respectively.AZ‐009 was rapidly systemically absorbed in HVs Fig. , as wellCmax and 0.77 (0.41–1.13) for the AUC0‐inf.Dose proportionality was assessed on the combined data of parts A to C. The estimated exponent  was 0.57 (0.15–1.00) for the The incidence of moderate AEs was 62.5% after AZ‐009 and 100% after subcutaneous apomorphine treatment  to 50% (3 mg) to 83% (4 mg). No patients presented with disabling dyskinesias.All patients were assessed by a physician as being in an ing Fig. . None ofoff periods. Even though subcutaneous apomorphine is efficacious, it can be painful and/or difficult to self‐administer and often results in injection‐site reactions.off periods. This three‐part study was designed to evaluate the PK of AZ‐009 and compare it with the subcutaneous injection and to examine the safety and PK of ascending doses of AZ‐009 in HVs and patients with PD. The last study part also aimed to evaluate the efficacy of AZ‐009 in patients with PD during an induced morning off state.Subcutaneous apomorphine injections have long been used by patients with PD for the treatment of sudden or early‐morning Tmax of 2 minutes based on the combined data of HVs and patients with PD. In contrast, the subcutaneous apomorphine injection resulted in a Tmax of 30 minutes. The PK profile of AZ‐009 makes it especially suitable for fast onset of action, which is preferential in the treatment of sudden off periods. Dosing with 1 mg AZ‐009 resulted in a mean (SD) Cmax of 14.3 (7.7) ng/mL and 2 mg subcutaneous apomorphine in 8.6 (3.1) ng/mL. A difference in total exposure (AUC0‐inf) between inhalable and subcutaneous apomorphine could not be confirmed because of the relatively high variability and small sample size. Similarly, no definitive conclusions could be drawn on dose proportionality. Future larger trials will need to be conducted to gain more information on this.AZ‐009 led to rapid systemic exposure with a median Despite comparable PK, AZ‐009 resulted in a less favorable safety profile in HVs than in patients with PD. This was not unexpected because patients with PD are likely to have developed tolerance because of daily dopaminergic medication use. Also, patients with PD were administered a higher domperidone dose compared with HVs. The most frequently reported AEs in patients with PD were throat irritation, orthostatic hypotension, and yawning. Throat irritation occurred immediately after dosing and usually resolved within minutes. Orthostatic hypotension was mostly asymptomatic and was observed in the placebo group as well. This can likely be partly explained by autonomic dysregulation in PD.One patient with PD receiving 3 mg AZ‐009 presented with severe hypotension shortly after dosing that was treated with ephedrine. Hypotension is a known side effect of apomorphine,During this trial, a prototype of the inhalation device was used. Of 25 patients with PD, 23 (92.0%) indicated they liked how the drug was delivered. Whether they also found the device easy to use could not be adequately evaluated because of the prototype being used. Future trials should therefore focus on ease of use of the commercial device in patients with PD.off periods in patients with PD after overnight medication withdrawal. At 10 minutes postdose, all three AZ‐009 dose groups showed a clear reduction (10.3–12.8 points) from baseline in mean MDS‐UPDRS part III score that was greater than for placebo (4.8 points). These reductions were larger than 3.25 points, which has been described as the minimal but clinically relevant improvement.off periods or from delayed on. Findings on the MDS‐UPDRS part III were supported by the physician's on/off state assessment. Whereas none of the placebo patients achieved a full on response, the AZ‐009–treated patients dose‐dependently converted from off to full on. For future studies, assessing on/off states after 45 minutes is advised to determine duration of clinical effect. Because patients were randomized to their AZ‐009 dose, it is likely that they did not reach their maximum possible improvement. In clinical practice, the dose of apomorphine is titrated to reach a dose with optimal efficacy and minimal side effects. Whereas this study demonstrates a beneficial effect of AZ‐009 over placebo, future studies should further investigate the efficacy of AZ‐009 at the patient's optimal dose.Treatment with 2, 3, and 4 mg AZ‐009 showed promise in controlling morning off periods.Taken together, AZ‐009 is reasonably well tolerated by patients with PD pretreated with domperidone. AZ‐009 is rapidly absorbed into the systemic circulation and can provide rapid relief from early‐morning 1. Research project: A. Conception, B. Organization, C. Execution;2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique;3. Manuscript: A. Writing of the first draft, B. Review and Critique.E.T.: 1A, 1B, 1C, 2A, 2C, 3AJ.d.H.: 1A, 1B, 1C, 2A, 2C, 3BD.P.: 1A, 1B, 2A, 2C, 3BL.M.: 1C, 3BM.L.: 3BD.H.: 3BK.K.: 3BK.M.: 3BM.E.S.: 3BT.A.: 3BW.Z.: 3BE.v.B.: 1A, 1B, 2A, 2C, 3BT.N.: 1A, 1B, 2A, 2C, 3BG.J.G.: 1A, 1B, 1C, 2A, 2C, 3BEva Thijssen: Employee, Centre for Human Drug Research (CHDR) and Leiden University Medical Centre (LUMC), Leiden, the Netherlands. Jonas den Heijer: Employee, CHDR and LUMC, Leiden, the Netherlands. David Puibert: Employee, Ferrer, Barcelona, Spain. Laurence Moss: Employee, CHDR and LUMC, Leiden, the Netherlands. Mingzu Lei: Employee, Alexza Pharmaceuticals, Mountain View, CA, USA. David Hasegawa: Employee, Alexza Pharmaceuticals, Mountain View, CA, USA. Kyo Keum: Alexza Pharmaceuticals, Mountain View, CA, USA. Ken Mochel: Employee, Alexza Pharmaceuticals, Mountain View, CA, USA. Mohammed Ezzeldin Sharaf: Employee, Ferrer, Barcelona, Spain. Tom Alfredson: Employee, Alexza Pharmaceuticals, Mountain View, CA, USA. Wenxiang Zeng: Employee, Alexza Pharmaceuticals, Mountain View, CA, USA. Emilie van Brummelen: Employee, CHDR, Leiden, the Netherlands. Tatjana Naranda: President and COO, Alexza Pharmaceuticals, Mountain View, CA, USA. Geert Jan Groeneveld: CSO/CMO at CHDR and professorship at LUMC, Leiden, the Netherlands.Figure S1 Overview of study designs.Click here for additional data file.Figure S2 CONSORT flow diagram for study part A.Click here for additional data file.Figure S3 CONSORT flow diagram for study part B.Click here for additional data file.Figure S4 CONSORT flow diagram for study part C.Click here for additional data file.Table S1 PK parameters of apomorphine after single‐dose administrations of 1 mg AZ‐009 inhalation and 2 mg subcutaneous (sc) injection to healthy volunteers.Click here for additional data file.Table S2 PK parameters of apomorphine after single‐dose administrations of 2 and 3 mg AZ‐009 to healthy volunteers (part B) and 2, 3, and 4 mg AZ‐009 to Parkinson's disease patients (part C).Click here for additional data file.