Target Attainment and Population Pharmacokinetics of Cefazolin in Patients with Invasive Staphylococcus aureus Infections: A Prospective Cohort Study.

This study aimed to determine cefazolin target attainment in patients with invasive Staphylococcus aureus (S. aureus) infections and to develop a population pharmacokinetic (PK) model. Adult patients with invasive S. aureus infections treated with cefazolin bolus infusions were included. Unbound and total trough and mid-dose cefazolin concentrations were measured, and strain-specific MICs were determined. The primary outcome was the proportion of patients attaining 100% fT>MIC at all time points evaluated. A population PK model was developed, using non-linear mixed-effects modelling. Overall, 51 patients were included, with a total of 226 unbound and total cefazolin concentrations measured (mean: 4.4 per patient). The median daily dosage in patients with an estimated glomerular filtration rate of >60 mL/min/m2 was 8 g. The median age was 74 years (interquartile range (IQR) 57-82) and 26% were female. A history of chronic kidney disease and acute kidney injury were present in 10/51 (19.6%) and 6/51 (11.7%), respectively. Achievement of 100% fT>MIC occurred in 86% of the patients and decreased to 45% when a target of 100% fT>4xMIC was evaluated. The mean unbound cefazolin fraction was 27.0% (standard deviation (SD) 13.4). Measured and estimated mean cefazolin trough concentrations differed significantly [13.1 mg/L (SD 23.5) vs. 7.4 mg/L (SD 7.9), p < 0.001]. In the population PK model, elevated estimated creatinine clearance and bolus instead of continuous application were covariates for target non-attainment. In conclusion, cefazolin target achievement was high, and the measurement of the unbound cefazolin concentration may be favored. The Monte Carlo simulations indicated that target attainment was significantly improved with continuous infusion.

Population pharmacokinetics of flucloxacillin as intermittent bolus infusion in patients with Staphylococcus aureus bloodstream infection.

BACKGROUND: Optimal antibiotic dosing for Staphylococcus aureus bloodstream infections (BSI) is still controversial. One reason is inter-individual variation in pharmacokinetics, which may be influenced by various patient-related factors, particularly in critically ill patients. OBJECTIVES: To describe the population pharmacokinetics (PopPK) of the antibiotic flucloxacillin in patients with S. aureus BSI. Subsequently, we sought to translate the model into a user-friendly app for generating a priori and a posteriori time-concentration curves and dose recommendations to optimize dosing regimens. METHODS: Total and unbound flucloxacillin concentrations were included from 49 patients from a prospective cohort study conducted during clinical routine, including non-critically ill and critically ill individuals who received intermittent bolus applications. These data were analysed using non-linear mixed-effects modelling. RESULTS: Most patients (98%) were treated with 2 g of flucloxacillin every 4 h. We developed a joint model that simultaneously described total and unbound concentrations. The model included an allometric effect of glomerular filtration rate on clearance and albumin on the albumin dissociation constant. The latter was especially important, as in our population the unbound fraction was higher at 11.5% (16.7% for critically ill patients) compared with reported values of approximately 5%. Based on our joint model, we developed a web-based app for optimizing dosing regimens of flucloxacillin. CONCLUSIONS: By utilizing data from clinical routine, we were able to create a predictive PopPK model of flucloxacillin and identify influential covariates. The web-based app is currently being validated in a clinical trial.

Machine Learning-Based Analysis Reveals Triterpene Saponins and Their Aglycones in Cimicifuga racemosa as Critical Mediators of AMPK Activation.

Cimicifuga racemosa (CR) extracts contain diverse constituents such as saponins. These saponins, which act as a defense against herbivores and pathogens also show promise in treating human conditions such as heart failure, pain, hypercholesterolemia, cancer, and inflammation. Some of these effects are mediated by activating AMP-dependent protein kinase (AMPK). Therefore, comprehensive screening for activating constituents in a CR extract is highly desirable. Employing machine learning (ML) techniques such as Deep Neural Networks (DNN), Logistic Regression Classification (LRC), and Random Forest Classification (RFC) with molecular fingerprint MACCS descriptors, 95 CR constituents were classified. Calibration involved 50 randomly chosen positive and negative controls. LRC achieved the highest overall test accuracy (90.2%), but DNN and RFC surpassed it in precision, sensitivity, specificity, and ROC AUC. All CR constituents were predicted as activators, except for three non-triterpene compounds. The validity of these classifications was supported by good calibration, with misclassifications ranging from 3% to 17% across the various models. High sensitivity (84.5-87.2%) and specificity (84.1-91.4%) suggest suitability for screening. The results demonstrate the potential of triterpene saponins and aglycones in activating AMP-dependent protein kinase (AMPK), providing the rationale for further clinical exploration of CR extracts in metabolic pathway-related conditions.

Drug-Disease Severity and Target-Disease Severity Interaction Networks in COVID-19 Patients.

Drug interactions with other drugs are a well-known phenomenon. Similarly, however, pre-existing drug therapy can alter the course of diseases for which it has not been prescribed. We performed network analysis on drugs and their respective targets to investigate whether there are drugs or targets with protective effects in COVID-19, making them candidates for repurposing. These networks of drug-disease interactions (DDSIs) and target-disease interactions (TDSIs) revealed a greater share of patients with diabetes and cardiac co-morbidities in the non-severe cohort treated with dipeptidyl peptidase-4 (DPP4) inhibitors. A possible protective effect of DPP4 inhibitors is also plausible on pathophysiological grounds, and our results support repositioning efforts of DPP4 inhibitors against SARS-CoV-2. At target level, we observed that the target location might have an influence on disease progression. This could potentially be attributed to disruption of functional membrane micro-domains (lipid rafts), which in turn could decrease viral entry and thus disease severity.

Effectiveness of Antiviral Therapy in Highly-Transmissible Variants of SARS-CoV-2: A Modeling and Simulation Study.

As of October 2021, neither established agents (e.g., hydroxychloroquine) nor experimental drugs have lived up to their initial promise as antiviral treatment against SARS-CoV-2 infection. While vaccines are being globally deployed, variants of concern (VOCs) are emerging with the potential for vaccine escape. VOCs are characterized by a higher within-host transmissibility, and this may alter their susceptibility to antiviral treatment. Here we describe a model to understand the effect of changes in within-host reproduction number R0, as proxy for transmissibility, of VOCs on the effectiveness of antiviral therapy with molnupiravir through modeling and simulation. Molnupiravir (EIDD-2801 or MK 4482) is an orally bioavailable antiviral drug inhibiting viral replication through lethal mutagenesis, ultimately leading to viral extinction. We simulated 800 mg molnupiravir treatment every 12 h for 5 days, with treatment initiated at different time points before and after infection. Modeled viral mutations range from 1.25 to 2-fold greater transmissibility than wild type, but also include putative co-adapted variants with lower transmissibility (0.75-fold). Antiviral efficacy was correlated with R0, making highly transmissible VOCs more sensitive to antiviral therapy. Total viral load was reduced by up to 70% in highly transmissible variants compared to 30% in wild type if treatment was started in the first 1-3 days post inoculation. Less transmissible variants appear less susceptible. Our findings suggest there may be a role for pre- or post-exposure prophylactic antiviral treatment in areas with presence of highly transmissible SARS-CoV-2 variants. Furthermore, clinical trials with borderline efficacious results should consider identifying VOCs and examine their impact in post-hoc analysis.

Modeling Structure-Activity Relationship of AMPK Activation.

The adenosine monophosphate activated protein kinase (AMPK) is critical in the regulation of important cellular functions such as lipid, glucose, and protein metabolism; mitochondrial biogenesis and autophagy; and cellular growth. In many diseases-such as metabolic syndrome, obesity, diabetes, and also cancer-activation of AMPK is beneficial. Therefore, there is growing interest in AMPK activators that act either by direct action on the enzyme itself or by indirect activation of upstream regulators. Many natural compounds have been described that activate AMPK indirectly. These compounds are usually contained in mixtures with a variety of structurally different other compounds, which in turn can also alter the activity of AMPK via one or more pathways. For these compounds, experiments are complicated, since the required pure substances are often not yet isolated and/or therefore not sufficiently available. Therefore, our goal was to develop a screening tool that could handle the profound heterogeneity in activation pathways of the AMPK. Since machine learning algorithms can model complex (unknown) relationships and patterns, some of these methods (random forest, support vector machines, stochastic gradient boosting, logistic regression, and deep neural network) were applied and validated using a database, comprising of 904 activating and 799 neutral or inhibiting compounds identified by extensive PubMed literature search and PubChem Bioassay database. All models showed unexpectedly high classification accuracy in training, but more importantly in predicting the unseen test data. These models are therefore suitable tools for rapid in silico screening of established substances or multicomponent mixtures and can be used to identify compounds of interest for further testing.

A Comparison of Nine Machine Learning Mutagenicity Models and Their Application for Predicting Pyrrolizidine Alkaloids.

Random forest, support vector machine, logistic regression, neural networks and k-nearest neighbor (lazar) algorithms, were applied to a new Salmonella mutagenicity dataset with 8,290 unique chemical structures utilizing MolPrint2D and Chemistry Development Kit (CDK) descriptors. Crossvalidation accuracies of all investigated models ranged from 80 to 85% which is comparable with the interlaboratory variability of the Salmonella mutagenicity assay. Pyrrolizidine alkaloid predictions showed a clear distinction between chemical groups, where otonecines had the highest proportion of positive mutagenicity predictions and monoesters the lowest.

Modeling of SARS-CoV-2 Treatment Effects for Informed Drug Repurposing.

Several repurposed drugs are currently under investigation in the fight against coronavirus disease 2019 (COVID-19). Candidates are often selected solely by their effective concentrations in vitro, an approach that has largely not lived up to expectations in COVID-19. Cell lines used in in vitro experiments are not necessarily representative of lung tissue. Yet, even if the proposed mode of action is indeed true, viral dynamics in vivo, host response, and concentration-time profiles must also be considered. Here we address the latter issue and describe a model of human SARS-CoV-2 viral kinetics with acquired immune response to investigate the dynamic impact of timing and dosing regimens of hydroxychloroquine, lopinavir/ritonavir, ivermectin, artemisinin, and nitazoxanide. We observed greatest benefits when treatments were given immediately at the time of diagnosis. Even interventions with minor antiviral effect may reduce host exposure if timed correctly. Ivermectin seems to be at least partially effective: given on positivity, peak viral load dropped by 0.3-0.6 log units and exposure by 8.8-22.3%. The other drugs had little to no appreciable effect. Given how well previous clinical trial results for hydroxychloroquine and lopinavir/ritonavir are explained by the models presented here, similar strategies should be considered in future drug candidate prioritization efforts.

Development and validation of a prognostic COVID-19 severity assessment (COSA) score and machine learning models for patient triage at a tertiary hospital.

BACKGROUND: Clinical risk scores and machine learning models based on routine laboratory values could assist in automated early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients at risk for severe clinical outcomes. They can guide patient triage, inform allocation of health care resources, and contribute to the improvement of clinical outcomes. METHODS: In- and out-patients tested positive for SARS-CoV-2 at the Insel Hospital Group Bern, Switzerland, between February 1st and August 31st ('first wave', n = 198) and September 1st through November 16th 2020 ('second wave', n = 459) were used as training and prospective validation cohort, respectively. A clinical risk stratification score and machine learning (ML) models were developed using demographic data, medical history, and laboratory values taken up to 3 days before, or 1 day after, positive testing to predict severe outcomes of hospitalization (a composite endpoint of admission to intensive care, or death from any cause). Test accuracy was assessed using the area under the receiver operating characteristic curve (AUROC). RESULTS: Sex, C-reactive protein, sodium, hemoglobin, glomerular filtration rate, glucose, and leucocytes around the time of first positive testing (- 3 to + 1 days) were the most predictive parameters. AUROC of the risk stratification score on training data (AUROC = 0.94, positive predictive value (PPV) = 0.97, negative predictive value (NPV) = 0.80) were comparable to the prospective validation cohort (AUROC = 0.85, PPV = 0.91, NPV = 0.81). The most successful ML algorithm with respect to AUROC was support vector machines (median = 0.96, interquartile range = 0.85-0.99, PPV = 0.90, NPV = 0.58). CONCLUSION: With a small set of easily obtainable parameters, both the clinical risk stratification score and the ML models were predictive for severe outcomes at our tertiary hospital center, and performed well in prospective validation.

The effect of early treatment with ivermectin on viral load, symptoms and humoral response in patients with non-severe COVID-19: A pilot, double-blind, placebo-controlled, randomized clinical trial.

BACKGROUND: Ivermectin inhibits the replication of SARS-CoV-2 in vitro at concentrations not readily achievable with currently approved doses. There is limited evidence to support its clinical use in COVID-19 patients. We conducted a Pilot, randomized, double-blind, placebo-controlled trial to evaluate the efficacy of a single dose of ivermectin reduce the transmission of SARS-CoV-2 when administered early after disease onset. METHODS: Consecutive patients with non-severe COVID-19 and no risk factors for complicated disease attending the emergency room of the Clínica Universidad de Navarra between July 31, 2020 and September 11, 2020 were enrolled. All enrollments occurred within 72 h of onset of fever or cough. Patients were randomized 1:1 to receive ivermectin, 400 mcg/kg, single dose (n = 12) or placebo (n = 12). The primary outcome measure was the proportion of patients with detectable SARS-CoV-2 RNA by PCR from nasopharyngeal swab at day 7 post-treatment. The primary outcome was supported by determination of the viral load and infectivity of each sample. The differences between ivermectin and placebo were calculated using Fisher's exact test and presented as a relative risk ratio. This study is registered at ClinicalTrials.gov: NCT04390022. FINDINGS: All patients recruited completed the trial (median age, 26 [IQR 19-36 in the ivermectin and 21-44 in the controls] years; 12 [50%] women; 100% had symptoms at recruitment, 70% reported headache, 62% reported fever, 50% reported general malaise and 25% reported cough). At day 7, there was no difference in the proportion of PCR positive patients (RR 0·92, 95% CI: 0·77-1·09, p = 1·0). The ivermectin group had non-statistically significant lower viral loads at day 4 (p = 0·24 for gene E; p = 0·18 for gene N) and day 7 (p = 0·16 for gene E; p = 0·18 for gene N) post treatment as well as lower IgG titers at day 21 post treatment (p = 0·24). Patients in the ivermectin group recovered earlier from hyposmia/anosmia (76 vs 158 patient-days; p < 0.001). INTERPRETATION: Among patients with non-severe COVID-19 and no risk factors for severe disease receiving a single 400 mcg/kg dose of ivermectin within 72 h of fever or cough onset there was no difference in the proportion of PCR positives. There was however a marked reduction of self-reported anosmia/hyposmia, a reduction of cough and a tendency to lower viral loads and lower IgG titers which warrants assessment in larger trials. FUNDING: ISGlobal, Barcelona Institute for Global Health and Clínica Universidad de Navarra.

In vitro hepatotoxicity of Petasites hybridus extract (Ze 339) depends on the concentration, the cytochrome activity of the cell system, and the species used.

Ze 339, a CO2 extract prepared from the leaves of Petasites hybridus, possesses antispasmodic and anti-inflammatory effects and is proven to be effective in the treatment of allergic rhinitis. To study possible hepatotoxic effects of Ze 339, its main constituents and metabolites, a series of in vitro investigations were performed. Furthermore, different reconstituted fractions of extract (petasins and fatty acid fraction) were examined in three in vitro test systems using hepatocytes: Two human cell lines, with lower and higher activity of cytochrome P450 enzymes (HepG2, HepaRG) as well as a rodent cell line with high cytochrome P450 activity (H-4-II-E), were used. Metabolic activity, assessed by the WST-1 assay, was chosen as indicator of cytotoxicity. To assess potential bioactivation of Ze 339 compounds, metabolic experiments using S9 fractions from rats, dogs, and humans and isolated cytochromes (human/rat) were performed, and the formation of reactive metabolites was assessed by measuring cellular concentrations of glutathione and glutathione disulphide. Our data revealed that the cytotoxicity of Ze 339, its single constituents, and main metabolites depends on the concentration, the cytochrome activity of the cell system, and the species used.

Performance of the UKPDS Outcomes Model 2 for Predicting Death and Cardiovascular Events in Patients with Type 2 Diabetes Mellitus from a German Population-Based Cohort.

BACKGROUND AND OBJECTIVE: Accurate prediction of relevant outcomes is important for targeting therapies and to support health economic evaluations of healthcare interventions in patients with diabetes. The United Kingdom Prospective Diabetes Study (UKPDS) risk equations are some of the most frequently used risk equations. This study aims to analyze the calibration and discrimination of the updated UKPDS risk equations as implemented in the UKPDS Outcomes Model 2 (UKPDS-OM2) for predicting cardiovascular (CV) events and death in patients with type 2 diabetes mellitus (T2DM) from population-based German samples. METHODS: Analyses are based on data of 456 individuals diagnosed with T2DM who participated in two population-based studies in southern Germany (KORA (Cooperative Health Research in the Region of Augsburg)-A: 1997/1998, n = 178; KORA-S4: 1999-2001, n = 278). We compared the participants' 10-year observed incidence of mortality, CV mortality, myocardial infarction (MI), and stroke with the predicted event rate of the UKPDS-OM2. The model's calibration was evaluated by Greenwood-Nam-D'Agostino tests and discrimination was evaluated by C-statistics. RESULTS: Of the 456 participants with T2DM (mean age 65 years, mean diabetes duration 8 years, 56% male), over the 10-year follow-up time 129 died (61 due to CV events), 64 experienced an MI, and 46 a stroke. The UKPDS-OM2 significantly over-predicted mortality and CV mortality by 25% and 28%, respectively (Greenwood-Nam-D'Agostino tests: p < 0.01), but there was no significant difference between predicted and observed MI and stroke risk. The model poorly discriminated for death (C-statistic [95% confidence interval] = 0.64 [0.60-0.69]), CV death (0.64 [0.58-0.71]), and MI (0.58 [0.52-0.66]), and failed to discriminate for stroke (0.57 [0.47-0.66]). CONCLUSIONS: The study results demonstrate acceptable calibration and poor discrimination of the UKPDS-OM2 for predicting death and CV events in this population-based German sample. Those limitations should be considered when using the UKPDS-OM2 for economic evaluations of healthcare strategies or using the risk equations for clinical decision-making.

Prediction of clinically relevant drug-induced liver injury from structure using machine learning.

Drug-induced liver injury (DILI) is the most common cause of acute liver failure and often responsible for drug withdrawals from the market. Clinical manifestations vary, and toxicity may or may not appear dose-dependent. We present several machine-learning models (decision tree induction, k-nearest neighbor, support vector machines, artificial neural networks) for the prediction of clinically relevant DILI based solely on drug structure, with data taken from published DILI cases. Our models achieved corrected classification rates of up to 89%. We also studied the association of a drug's interaction with carriers, enzymes and transporters, and the relationship of defined daily doses with hepatotoxicity. The results presented here are useful as a screening tool both in a clinical setting in the assessment of DILI as well as in the early stages of drug development to rule out potentially hepatotoxic candidates.

The hepatotoxic potential of protein kinase inhibitors predicted with Random Forest and Artificial Neural Networks.

Protein kinases (PKs) play a role in many pivotal aspects of cellular function. Dysregulation and mutations of protein kinases are involved in the development of different diseases, which might be treated by inhibition of the corresponding kinase. Protein kinase inhibitors (PKIs) are generally well tolerated, but unexpected and serious adverse events on the heart, lung, kidney and liver were observed clinically. In this study, the structure-activity relationship of PKIs in relation to hepatotoxicity was investigated. A dataset of 165 PKIs was compiled and the probability of human hepatotoxicity with two different machine learning algorithms (Random Forest and Artificial Neural Networks) was analysed. The estimated probability of hepatotoxicity was generally high for single PKIs. However, depending on the target kinase of the PKI, a difference in hepatotoxic potential could be observed. The similarity of the PKIs to each other is caused by the conserved site of action of the protein kinases. Hepatotoxicity may therefore always be an issue in PKIs.

Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. AIM OF THE STUDY: The aim was to develop an in vitro screening method of their cytotoxicity. MATERIALS AND METHODS: Human and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). RESULTS: Galactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. CONCLUSIONS: If no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Editor's Highlight: Identification of Any Structure-Specific Hepatotoxic Potential of Different Pyrrolizidine Alkaloids Using Random Forests and Artificial Neural Networks.

Pyrrolizidine alkaloids (PAs) are characteristic metabolites of some plant families and form a powerful defense mechanism against herbivores. More than 600 different PAs are known. PAs are ester alkaloids composed of a necine base and a necic acid, which can be used to divide PAs in different structural subcategories. The main target organs for PA metabolism and toxicity are liver and lungs. Additionally, PAs are potentially genotoxic, carcinogenic and exhibit developmental toxicity. Only for very few PAs, in vitro and in vivo investigations have characterized their toxic potential. However, these investigations suggest that structural differences have an influence on the toxicity of single PAs. To investigate this structural relationship for a large number of PAs, a quantitative structural-activity relationship (QSAR) analysis for hepatotoxicity of over 600 different PAs was performed, using Random Forest- and artificial Neural Networks-algorithms. These models were trained with a recently established dataset specific for acute hepatotoxicity in humans. Using this dataset, a set of molecular predictors was identified to predict the hepatotoxic potential of each compound in validated QSAR models. Based on these models, the hepatotoxic potential of the 602 PAs was predicted and the following hepatotoxic rank order in 3 main categories defined (1) for necine base: otonecine > retronecine > platynecine; (2) for necine base modification: dehydropyrrolizidine ≫ tertiary PA = N-oxide; and (3) for necic acid: macrocyclic diester ≥ open-ring diester > monoester. A further analysis with combined structural features revealed that necic acid has a higher influence on the acute hepatotoxicity than the necine base.