'Under pressure': The role of therapeutic drug monitoring in the treatment of hypertension.

Antihypertensive drugs do not qualify as optimal candidates for therapeutic drug monitoring (TDM), given their obvious physiological effect, the absence of a clear relationship between drug concentrations and pharmacodynamic outcomes and their wide therapeutic range. However, since non-adherence is a major challenge in hypertension management, using drug concentrations can be of value to identify non-adherence as a first step towards better blood pressure control. In this article we discuss the key challenges associated with measuring and interpreting antihypertensive drug concentrations that are important when TDM is used to improve non-adherence. Additionally, we elaborate on the role of TDM in optimizing antihypertensive drug treatment besides addressing non-adherence by highlighting its value in specific patient groups with altered pharmacokinetic parameters such as female vs. male or elderly patients.

Developing practical recommendations for drug-disease interactions in patients with hypertension.

Background: Hypertension, a significant risk factor for cardiovascular diseases, demands proactive management as cardiovascular diseases remain the leading cause of death worldwide. Reducing systolic and diastolic blood pressure levels below recommended reference values of <140/90 mmHg can lead to a significant reduction of the risk of CVD and all-cause mortality. However, treatment of hypertension can be difficult and the presence of comorbidities could further complicate this treatment. Drugs used to manage these comorbidities may inadvertently have an impact on blood pressure, resulting in a phenomenon known as drug-disease interaction. This study aims to assess the safety of medication that can affect blood pressure in patients with hypertension and provide practical recommendations for healthcare professionals. Methods: For the development of recommendations for the drug-disease interaction (DDSI) hypertension, a six-step plan that combined literature selection and multidisciplinary expert opinion was used. The process involved (1) defining the scope of the DDSI and selecting relevant drugs, (2) collecting evidence, (3) data-extraction, (4) reaching of expert consensus, (5) publication and implementation of the recommendations in healthcare systems and (6) updating the information. Results: An increase of 10 mmHg in systolic blood pressure and 5 mmHg in diastolic blood pressure was defined as clinically relevant. Corticosteroids, danazol, and yohimbine caused a clinically relevant DDSI with hypertension. Several other drugs with warnings for hypertension in the official product information were assessed to have no clinically relevant DDSI due to minor influence or lack of data on blood pressure. Drugs with evidence for a relevant change in blood pressure which are prescribed under close monitoring of blood pressure according to clinical guidelines, were deemed to be not clinically relevant for signalling. Conclusion: This study provides specific recommendations that can be implemented directly in clinical practice, for example, in clinical decision support systems, potentially resulting in safer drug use in patients with hypertension and better healthcare by reducing alert fatigue. Future research should focus on evaluating the effectiveness of implementation strategies and their impact on reducing unsafe use of medication in patients with hypertension.

Quality-of-life and beliefs about medication in relation to a therapy adherence intervention in resistant hypertension: the Resistant HYpertension: MEasure to ReaCh Targets trial.

OBJECTIVE: To assess the impact of personalized feedback on therapy adherence testing results on quality of life and beliefs about medication in patients with resistant hypertension, as well as to identify patient-oriented predictors of therapy adherence. METHODS: This study was a prespecified post hoc analysis of the multicenter randomized controlled trial Resistant HYpertension: MEasure to ReaCh Targets (RHYME-RCT). Patients were randomized to a personalized feedback conversation on measured antihypertensive drug levels additional to standard-of-care, or standard-of-care only. The primary outcomes consisted of EuroQol EQ-5D-5L and Beliefs about Medicine Questionnaire (BMQ) scores at 12 months. RESULTS: A total of 56 patients with median age 61.5 [25th-75th percentile: 55.8-69.3] years (21.4% women) were included. Mean blood pressure ±SD was 149.8/84.1 ±â€Š14.9/13.8 mmHg while being on a median of 5.6 [4.8-7.3] defined daily dosages (DDD) of antihypertensive drugs. At 12 months, no differences were observed in EQ-5D-5L index (0.81 [0.69-0.89] vs. 0.89 [0.73-1.00]; P = 0.18) and visual analogue scale score on general patient-perceived health (70 [60-80] vs. 70 [60-82]; P = 0.53) between the intervention-arm and the standard-of-care only-arm. Likewise, individual EQ-5D-5L domain scores and BMQ scores did not differ between both arms. Irrespective of the intervention, independent positive predictors of the percentage adherence were patient age, EQ-5D-5L index score, BMQ-specific necessity score and concern score, whereas the total number of drugs prescribed was a negative predictor. CONCLUSION: Within this prespecified subanalysis of the randomized RHYME-RCT trial, implementation of a personalized feedback conversation targeting therapy adherence did not improve health-related quality-of-life and beliefs about medication in patients with resistant hypertension.

The BMP7-Derived Peptide p[63-82] Reduces Cartilage Degeneration in the Rat ACLT-pMMx Model for Posttraumatic Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks. DESIGN: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis. RESULTS: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery. CONCLUSIONS: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up.

Antihypertensive drug concentration measurement combined with personalized feedback in resistant hypertension: a randomized controlled trial.

BACKGROUND: Adherence to antihypertensive drugs (AHDs) is crucial for controlling blood pressure (BP). We aimed to determine the effectiveness of measuring AHD concentrations using a dried blood spot (DBS) sampling method to identify nonadherence, combined with personalized feedback, in reducing resistant hypertension. METHODS: We conducted a multicenter, randomized, controlled trial (RHYME-RCT, ICTRP NTR6914) in patients with established resistant hypertension. Patients were randomized to receive either an intervention with standard of care (SoC) or SoC alone. SoC consisted of BP measurement and DBS sampling at baseline, 3 months (t3), 6 months (t6), and 12 months (t12); AHD concentrations were measured but not reported in this arm. In the intervention arm, results on AHD concentrations were discussed during a personalized feedback conversation at baseline and t3. Study endpoints included the proportion of patients with RH and AHD adherence at t12. RESULTS: Forty-nine patients were randomized to receive the intervention+SoC, and 51 were randomized to receive SoC alone. The proportion of adherent patients improved from 70.0 to 92.5% in the intervention+SoC arm ( P  = 0.008, n  = 40) and remained the same in the SoC arm (71.4%, n  = 42). The difference in adherence between the arms was statistically significant ( P  = 0.014). The prevalence of resistant hypertension decreased to 75.0% in the intervention+SoC arm ( P  < 0.001, n  = 40) and 59.5% in the SoC arm ( P  < 0.001, n  = 42) at t12; the difference between the arms was statistically nonsignificant ( P  = 0.14). CONCLUSION: Personalized feedback conversations based on DBS-derived AHD concentrations improved AHD adherence but did not reduce the prevalence of RH.

The impact of a summative national prescribing assessment and curriculum type on the development of the prescribing competence of junior doctors.

PURPOSE: The primary aim of this study was to investigate the effect of including the Dutch National Pharmacotherapy Assessment (DNPA) in the medical curriculum on the level and development of prescribing knowledge and skills of junior doctors. The secondary aim was to evaluate the relationship between the curriculum type and the prescribing competence of junior doctors. METHODS: We re-analysed the data of a longitudinal study conducted in 2016 involving recently graduated junior doctors from 11 medical schools across the Netherlands and Belgium. Participants completed three assessments during the first year after graduation (around graduation (+ / - 4 weeks), and 6 months, and 1 year after graduation), each of which contained 35 multiple choice questions (MCQs) assessing knowledge and three clinical case scenarios assessing skills. Only one medical school used the DNPA in its medical curriculum; the other medical schools used conventional means to assess prescribing knowledge and skills. Five medical schools were classified as providing solely theoretical clinical pharmacology and therapeutics (CPT) education; the others provided both theoretical and practical CPT education (mixed curriculum). RESULTS: Of the 1584 invited junior doctors, 556 (35.1%) participated, 326 (58.6%) completed the MCQs and 325 (58.5%) the clinical case scenarios in all three assessments. Junior doctors whose medical curriculum included the DNPA had higher knowledge scores than other junior doctors (76.7% [SD 12.5] vs. 67.8% [SD 12.6], 81.8% [SD 11.1] vs. 76.1% [SD 11.1], 77.0% [12.1] vs. 70.6% [SD 14.0], p < 0.05 for all three assessments, respectively). There was no difference in skills scores at the moment of graduation (p = 0.110), but after 6 and 12 months junior doctors whose medical curriculum included the DNPA had higher skills scores (both p < 0.001). Junior doctors educated with a mixed curriculum had significantly higher scores for both knowledge and skills than did junior doctors educated with a theoretical curriculum (p < 0.05 in all assessments). CONCLUSION: Our findings suggest that the inclusion of the knowledge focused DNPA in the medical curriculum improves the prescribing knowledge, but not the skills, of junior doctors at the moment of graduation. However, after 6 and 12 months, both the knowledge and skills were higher in the junior doctors whose medical curriculum included the DNPA. A curriculum that provides both theoretical and practical education seems to improve both prescribing knowledge and skills relative to a solely theoretical curriculum.

Monitoring antihypertensive drug concentrations to determine nonadherence in hypertensive patients with or without a kidney transplant.

BACKGROUND: Nonadherence to antihypertensive drugs (AHDs) is a major contributor to pseudo-resistant hypertension. The primary objective of this study was to determine the prevalence of nonadherence to AHDs among patients visiting the nephrology and vascular outpatient clinics. METHODS: Patients were eligible to participate in this prospective observational study if they used at least two AHDs that could be measured with a validated UHPLC-MS/MS method and had an office blood pressure at least 140 and/or at least 90 mmHg. For resistant hypertension, included patients had to use at least three AHDs including a diuretic or four AHDs. Adherence was assessed by measuring drug concentrations in blood. The complete absence of drug in blood was defined as nonadherence. A posthoc analysis was performed to determine the influence of a having a kidney transplant on the adherence rates. RESULTS: One hundred and forty-two patients were included of whom 66 patients fulfilled the definition of resistant hypertension. The overall adherence rate to AHDs was 78.2% ( n  = 111 patients), with the highest adherence rate for irbesartan (100%, n  = 9) and lowest adherence rate for bumetanide ( n  = 69%, n  = 13). In further analysis, only kidney transplantation could be identified as an important factor for adherence (adjusted odds ratio = 3.35; 95% confidence interval 1.23-9.09). A posthoc analysis showed that patients with a kidney transplant were more likely to be adherent to AHDs (non-KT cohort 64.0% vs. KT-cohort 85.7%, χ 2 (2) = 10.34, P  = 0.006). CONCLUSION: The adherence rate to AHDs in hypertensive patients was high (78.2%) and even higher after a kidney transplant (85.7%). Furthermore, patients after kidney transplant had a lower risk of being nonadherent to AHDs.

[When will we treat patients with a polypill shortly after myocardial infarction?] / Wanneer gaan we patiënten een 'polypil' geven?

The 'polypill' stands for fixed-dosed combination pills with generic drugs that act on multiple cardiovascular risk factors. Data from randomized controlled trials show consistent beneficial effects of treatment with a polypill on both cardiovascular risk factors and relevant marjor cardiovascular endpoints. However, polypills are not readily available worldwide and only a limited number of polypills is marketed in Europe. Physicians have to embrace polypills in regular care to let the patient benefit from the advantages of the polypill. Licensing more polypills is an essential step to implement these pills in clinical care. Regulatory agencies need to reduce the dossier content requirements for registrations of new fixed-dosed combination pills so generic pharmaceutical companies can expand the number of marketed polypills.

In Vitro Biotransformation and Anti-Inflammatory Activity of Constituents and Metabolites of Filipendula ulmaria.

(1) Background: Filipendula ulmaria (L.) Maxim. (Rosaceae) (meadowsweet) is widely used in phytotherapy against inflammatory diseases. However, its active constituents are not exactly known. Moreover, it contains many constituents, such as flavonoid glycosides, which are not absorbed, but metabolized in the colon by gut microbiota, producing potentially active metabolites that can be absorbed. The aim of this study was to characterize the active constituents or metabolites. (2) Methods: A F. ulmaria extract was processed in an in vitro gastrointestinal biotransformation model, and the metabolites were characterized using UHPLC-ESI-QTOF-MS analysis. In vitro anti-inflammatory activity was evaluated by testing the inhibition of NF-κB activation, COX-1 and COX-2 enzyme inhibition. (3) Results: The simulation of gastrointestinal biotransformation showed a decrease in the relative abundance of glycosylated flavonoids such as rutin, spiraeoside and isoquercitrin in the colon compartment, and an increase in aglycons such as quercetin, apigenin, naringenin and kaempferol. The genuine as well as the metabolized extract showed a better inhibition of the COX-1 enzyme as compared to COX-2. A mix of aglycons present after biotransformation showed a significant inhibition of COX-1. (4) Conclusions: The anti-inflammatory activity of F. ulmaria may be explained by an additive or synergistic effect of genuine constituents and metabolites.

Do we become better prescribers after graduation: A 1-year international follow-up study among junior doctors.

AIM: The aim of this study was to investigate how the prescribing knowledge and skills of junior doctors in the Netherlands and Belgium develop in the year after graduation. We also analysed differences in knowledge and skills between surgical and nonsurgical junior doctors. METHODS: This international, multicentre (n = 11), longitudinal study analysed the learning curves of junior doctors working in various specialties via three validated assessments at about the time of graduation, and 6 months and 1 year after graduation. Each assessment contained 35 multiple choice questions (MCQs) on medication safety (passing grade ≥85%) and three clinical scenarios. RESULTS: In total, 556 junior doctors participated, 326 (58.6%) of whom completed the MCQs and 325 (58.5%) the clinical case scenarios of all three assessments. Mean prescribing knowledge was stable in the year after graduation, with 69% (SD 13) correctly answering questions at assessment 1 and 71% (SD 14) at assessment 3, whereas prescribing skills decreased: 63% of treatment plans were considered adequate at assessment 1 but only 40% at assessment 3 (P < .001). While nonsurgical doctors had similar learning curves for knowledge and skills as surgical doctors (P = .53 and P = .56 respectively), their overall level was higher at all three assessments (all P < .05). CONCLUSION: These results show that junior doctors' prescribing knowledge and skills did not improve while they were working in clinical practice. Moreover, their level was under the predefined passing grade. As this might adversely affect patient safety, educational interventions should be introduced to improve the prescribing competence of junior doctors.

Best Practices to Implement Dried Blood Spot Sampling for Therapeutic Drug Monitoring in Clinical Practice.

BACKGROUND: Sampling of blood at home to determine the concentration of drugs or other compounds can be effective in limiting hospital-based sampling. This could lower hospital visits and patient burden, improve the quality of life, and reduce health care costs. Dried blood spot (DBS) microsampling is often used for this purpose, wherein capillary blood, obtained by pricking the heel or finger, is used to measure different analytes. Although DBS has several advantages over venous blood sampling, it is not routinely implemented in clinical practice. To facilitate the bench to bedside transition, it is important to be aware of certain challenges that need to be considered and addressed. RESULTS: Here, important considerations regarding the implementation of DBS in clinical practice, the choice of patients, blood sampling, transport, and laboratory analysis are discussed. In addition, we share our experience and provide suggestions on how to deal with these problems in a clinical setting.

Development and Validation of a Dried Blood Spot Assay Using UHPLC-MS/MS to Identify and Quantify 12 Antihypertensive Drugs and 4 Active Metabolites: Clinical Needs and Analytical Limitations.

PURPOSE: As nonadherence to antihypertensive drugs (AHDs) can increase the risk of cardiovascular events, hospitalization, and higher costs, there is a need for a reliable, objective, and easy method to assess nonadherence in patients. The dried blood spot (DBS) sampling method used to measure drug concentrations meets these requirements. For detecting nonadherence, identification is more important than quantification. Owing to their use in clinical practice, it is important to measure multiple AHDs with a single method. Therefore, we developed and validated a single DBS method for 17 commonly used AHDs and 4 active metabolites using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHODS: Analytical validation of the DBS assay was performed in accordance with the guidelines on bioanalytical method validation of the European Medicines Agency and US Food and Drug Administration as well as the International Association of Therapeutic Drug Monitoring and Clinical Toxicology guidelines. RESULTS: We validated 12 of the 17 AHDs according to the European Medicines Agency and Food and Drug Administration requirements for bioanalytical method validation. Eleven AHDs were validated for both identification and quantification of drug concentrations, whereas nifedipine was only validated for identification. However, 5 of the 17 AHDs were excluded due to suboptimal validation results. Lercanidipine was excluded due to nonlinearity, and all 4 AHDs measured in the negative mode of UHPLC-MS/MS were not in accordance with one or more of the acceptance criteria and were therefore excluded. CONCLUSIONS: The described method accurately measured AHDs in DBS and can be used to determine nonadherence in patients. However, method validation revealed a challenging balance between analytical limitations and clinical needs when analyzing multiple drugs using the same method.

DiffErenCes in AntihypertenSive Drug Blood Levels in Patients with HypertensiON (DECISION): Protocol for a Prospective Observational Study Comparing Pharmacokinetics and Pharmacodynamics Between Young and Elderly Patients.

Adequate controlled blood pressure decreases the risk of cardiovascular events. However, the elderly are more vulnerable and thereby more prone to side effects of antihypertensive drugs. A lack of pharmacokinetic and pharmacodynamic (PK/PD) studies in older patients makes specific and tailored advices towards antihypertensive drug therapy difficult. The aim of our study, DiffErenCes In antihypertenSive drug levels In patients with hypertensiON (DECISION), is to fill in this PK/PD knowledge gap and move towards precision dosing. DECISION is a prospective observational PK/PD study set up to determine the difference in exposure to the antihypertensive drugs, losartan and perindopril, measured by drug levels in blood. The area under the curve (AUC; PK) and furthermore the association between the AUC and the effect on blood pressure (PD) will be compared between elderly and younger patients.

Sex Differences in Spironolactone and the Active Metabolite Canrenone Concentrations and Adherence.

We aim to investigate sex differences in blood concentrations of spironolactone and the active metabolite canrenone in resistant hypertension patients. Furthermore, sex differences in adherence for spironolactone and other antihypertensive drugs (AHDs) were studied. The patients in this post hoc study had all participated in a single-blind randomized controlled trial called RHYME-RCT (Dutch Trial Register, NL6736). Concentrations in blood of several AHDs were assessed in RHYME-RCT to investigate adherence to treatment. This allowed for a comparison of drug exposure to spironolactone and canrenone between males and females. In linear regression models, no statistically significant sex differences (N = 35) in spironolactone (B =-10.23, SE = 7.92, p = 0.206) or canrenone (B = 1.24, SE = 10.96, p = 0.911) concentrations after adjustment for dose and time between sampling and intake were found. Furthermore, no statistically significant differences in non-adherence to spironolactone were found between sexes (N = 54, male 15% vs. female 38%, p = 0.100), but non-adherence to spironolactone was associated with non-adherence to other AHDs (p ≤ 0.001). Spironolactone and canrenone concentrations were not different between males and females with resistant hypertension. Although not statistically significant, females were twice as likely to be non-adherent to spironolactone compared to males, and thereby also more likely to be non-adherent to other AHDs.

Comparison of automated office blood pressure measurement with 24-hour ambulatory blood pressure measurement.

PURPOSE: Although 24-hour ambulatory blood pressure measurement (24-h ABPM) is the most important method to establish true hypertension, in clinical practice often repeated automated office blood pressure (AOBP) measurements are used because of convenience and lower costs. We aimed to assess the agreement rate between a 30 and 60 min AOBP and 24-h ABPM. MATERIALS AND METHODS: Patients with known hypertension (cohort 1) and patients visiting the neurology outpatient clinic after minor stroke or transient ischaemic attack (cohort 2) were selected. We performed AOBP for 30-60 min at 5-min intervals followed by 24-h ABPM and calculated average values of both measurements. Agreement between the two methods was studied with McNemar and Bland-Altman plots with a clinically relevant limit of agreement of ≤10 mm Hg difference in systolic BP. RESULTS: Our final cohort consisted of 135 patients from cohort 1 and 72 patients from cohort 2. We found relatively low agreement based on the clinical relevant cut-off value; 64.7% of the measurements were within the limits of agreement for 24-h systolic and 50.2% for 24-h diastolic. This was 61.4% for daytime systolic and 56.6% for daytime diastolic. In 73.5% of the patients, both methods led to the same diagnosis of either being hypertensive or non-hypertensive. This resulted in a significant difference between the methods to determine the diagnosis of hypertension (p < 0.0001). CONCLUSION: We conclude that 30-60 min AOBP measurements cannot replace a 24-h ABPM and propose to perform 24-h ABPM at least on a yearly basis to confirm AOBP measurements.

Antiplasmodial activity of constituents and their metabolites after in vitro gastrointestinal biotransformation of a Nauclea pobeguinii extract.

Nauclea pobeguinii is traditionally used for treatment of malaria. Previous studies on the plant extract and strictosamide, the putative active constituent, showed a profound in vivo activity of the extract but no in vitro activity of strictosamide. This might indicate that one or more compounds present in the extract, most likely alkaloids, act as prodrugs undergoing biotransformation after oral administration resulting in the active compounds. The phytochemical composition of a N. pobeguinii extract was characterized using UHPLC-UV-HRMS (Ultrahigh-Performance Liquid Chromatography-Ultraviolet-High Resolution Mass Spectrometry) data. An in vitro gastrointestinal model was used to simulate biotransformation of the extract allowing monitoring of the relative abundances of individual constituents over time on one hand, while antiplasmodial activity and cytotoxicity of the biotransformed extract could be evaluated on the other hand. A diversity of compounds was (tentatively) identified in the extract, mainly saponins and alkaloids, including 32 compounds that have not been reported before in N. pobeguinii. The automated data analysis workflow used for unbiased screening for metabolites showed that glycosylated compounds decreased in intensity over time. Alkaloids containing no sugar moieties, including angustine-type alkaloids, showed no gastrointestinal biotransformation. In vitro gastrointestinal biotransformation of strictosamide did not result in a major metabolite. Moreover, multivariate data analysis using Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed no in vitro activity of strictosamide or its metabolites suggesting that other compounds or metabolites present in the extract are responsible for the antiplasmodial effect of the N. pobeguinii extract. The OPLS-DA proposes alkaloids with a ß-carboline moiety as active principles, suggesting that antiplasmodial activity of N. pobeguinii derives from an additive or synergistic effect of multiple minor alkaloids and their metabolites present in the bark extract of N. pobeguinii.

Effects of medicagenic acid metabolites, originating from biotransformation of an Herniaria hirsuta extract, on calcium oxalate crystallization in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Herniaria hirsuta is traditionally used in Moroccan folk medicine for treatment of urinary stones and as a diuretic. It is rich in saponins, which are known to be deglycosylated in the colon, whereafter aglycones such as medicagenic acid are absorbed and further metabolized in the liver. AIM OF THE STUDY: A sample of hepatic metabolites of medicagenic acid, with medicagenic acid glucuronide as the most abundant one, was evaluated for in vitro activity against urinary stones. A crystallization assay and a crystal-cell interaction assay were used to evaluate in vitro activity of hepatic metabolites of medicagenic acid on CaC2O4 (calciumoxalate) crystals, present in the majority of urinary stones. MATERIALS AND METHODS: In the crystallization assay the effects on nucleation of Ca2+ and C2O42- and aggregation of the CaC2O4 crystals are studied. In the crystal-cell interaction assay crystal retention is investigated by determining the amount of Ca2+ bound to injured monolayers of MDCK I cells. RESULTS: Results of the crystallization assay showed a tentative effect on crystal aggregation. The crystal-cell interaction assay showed a significant inhibition of crystal binding, which may reduce crystal retention in the urinary tract. CONCLUSIONS: As both formation of crystals by inhibiting aggregation and retention of crystals is affected, the beneficial effect of H. hirsuta against urinary stones may at least in part be attributed to medicagenic acid metabolites, indicating that saponins containing medicagenic acid may act as prodrugs.

Introducing the importance and difficulties of a three-step approach to improve nonadherence to antihypertensive drugs: a case series.

Nonadherence to antihypertensive drugs is an important reason for not reaching blood pressure goals. A possible method to improve nonadherence involves three essential steps: identification of nonadherent patients (step 1), determination of underlying causes (step 2) and a personalized solution (step 3). We present three unique cases to show the importance and difficulties of this three-step approach. Patients participated in a randomized controlled trial to improve nonadherence to antihypertensive drugs (RHYME-RCT, Dutch Trial Register NL6736). Drug level measurements were used to identify nonadherence to antihypertensive drugs and communication on drug levels was supported by a tailored feedback tool in these patients. These cases showed that a three-step approach of identifying nonadherence and determination of the underlying cause, can lead to a personalized solution to improve therapy even when nonadherence was excluded. Open communication with patients remains an essential part when improving nonadherence.

BMP7 reduces the fibrocartilage chondrocyte phenotype.

The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFß-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA)12-reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA)12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule.

Alkaloids from Lepidium meyenii (Maca), structural revision of macaridine and UPLC-MS/MS feature-based molecular networking.

Lepidium meyenii Walp., known as Peruvian ginseng, is widely used in ethnomedicine. To date, L. meyenii is cultivated worldwide at high-altitude and is commonly used as a food supplement. However, its medicinal value is still controversial and its mechanism of action remains unknown, due to limited knowledge about the phytochemical constituents of this plant species. In this study, a multidisciplinary approach comprising conventional NMR- and HRMS-based structure elucidation, quantum mechanical calculation of NMR chemical shifts and UPLC-MS/MS feature-based molecular networking was applied to analyse the phytochemical profile of L. meyenii. In the current work, three previously undescribed imidazole alkaloids were identified using extensive spectroscopic techniques (HRMS, NMR), for which the names lepidiline E, F and G were adopted. In addition, two amidine alkaloids were reported, representing an undescribed class of alkaloids in L. meyenii, and 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid, a well-known ß-carboline alkaloid, was also isolated from L. meyenii for the first time. Molecular networks of imidazole, amidine and ß-carboline alkaloids in L. meyenii were constructed by the Global Natural Products Social Molecular Networking (GNPS) web platform, resulting in the tentative identification of three undescribed analogues. In addition, the structure of a previously reported compound named 'macaridine' was revised as macapyrrolin C based on density functional theory (DFT) calculations and comprehensive comparison of NMR data.