Reported hepatotoxicity and hepatotoxicity guidance in the product information of protein kinase inhibitors in oncology registered at the European Medicines Agency.

Protein kinase inhibitors (PKIs) used in oncology can induce severe and even fatal hepatotoxicity. Several PKIs are registered within a certain class to target a specific kinase. No systematic comparison of the reported hepatotoxicity and clinical guidance for monitoring and management of hepatotoxic events between the various PKI summaries of product characteristics (SmPC) is yet available. A systematic analysis of data on 21 hepatotoxicity parameters obtained from the SmPCs and European public assessment reports (EPARs) of European Medicines Agency-approved antineoplastic PKIs (n = 55) has been conducted. The median reported incidence (range) of all grades of aspartate aminotransferase (AST) elevations was 16.9% (2.0%-86.4%) for PKI monotherapy, with 2.1% (0.0%-10.3%) being grade 3/4 and for all grades alanine aminotransferase (ALT) elevations 17.6% (2.0%-85.5%), with 3.0% (0.0%-25.0%) being grade 3/4. Fatalities due to hepatotoxicity were reported for 22 out of 47 PKIs (monotherapy) and for 5 out of 8 PKIs (combination therapy). A maximum grade of grade 4 and grade 3 hepatotoxicity was reported for 45% (n = 25) and 6% (n = 3), respectively. Liver parameter monitoring recommendations were present in 47 of the 55 SmPCs. Dose reductions were recommended for 18 PKIs. Discontinuation was recommended for patients meeting Hy's law criteria (16 out of 55 SmPCs). Severe hepatotoxic events are reported in approximately 50% of the analyzed SmPCs and EPARs. Differences in the degree of hepatotoxicity are apparent. Although liver parameter monitoring recommendations are present in the vast majority of the analyzed PKI SmPCs, the clinical guidance for hepatotoxicity was not standardized.

Interchangeability of generic drugs for subpopulations: Bioequivalence simulation from a nonparametric PK model of gabapentin generic drugs.

Patients are often switched between generic formulations of the same drug, but in some cases generic interchangeability is questioned. For generic drugs to be approved, bioequivalence with the innovator drug should be demonstrated, but evidence of bioequivalence is not required in the intended patient population or relative to other approved generics. AIM: We aim to identify pathophysiological pharmacokinetic subpopulations for whom there is a difference in comparative bioavailability compared to a healthy population. METHODS: We used simulated exposures from a nonparametric model of multiple generics and the originator gabapentin. Exposure was simulated for virtual populations with pharmacokinetic characteristics beyond those of healthy subjects with regard to rate of absorption, volume of distribution and reduced renal function. Virtual parallel design bioequivalence studies were performed using a random sample of 24 simulated subjects, with standard acceptance criteria. RESULTS: Results indicated increased pharmacokinetic variability for patient populations with a lower rate of absorption or a reduced renal function, but no change in the average comparable bioavailability ratio. This increased variability results in a reduced likelihood of demonstrating bioequivalence. Observations were similar for comparisons between all different formulations, as well as between subjects who received the identical formulation in a repeated fashion. No relevant effect was observed for simulations with increased volume of distribution. CONCLUSION: Our simulations indicate that the reduced likelihood of demonstrating bioequivalence for subjects with altered pharmacokinetics is not influenced by a formulation switch, nor does the average comparable bioavailability ratio change, therefore these results support generic interchangeability and current approval requirements for generics.

Impact of the new European Union In Vitro Diagnostics Regulation on the practice of hospital diagnostic laboratories.

OBJECTIVES: The In Vitro Diagnostics Regulation 2017/746 (IVDR) coming into force from May 2022, creates the first European regulatory recognition for biomarker tests linked to medicinal products, so-called companion diagnostics (CDx). Since the introduction of the IVDR is associated with uncertainties about its impact on hospital practice, it is urgent and valuable to investigate how and why CDx are currently used in hospital practice, which factors influence the choice for applying in-house or commercial CDx, and what the expectations are about how the IVDR may affect current practice. METHODS: We investigated these questions using an interview-based approach and focused on 15 hospital laboratories in the Netherlands, including 7 academic and 8 general hospitals. All types of CDx were considered relevant for this research, including both genetic and protein-based biomarkers. RESULTS: Factors found included: costs and convenience, complexity of application, and compatibility with existing workflows. Next to in-house and commercial CDx, hospital laboratories addressed compatibility by tweaking existing CDx. CONCLUSION: Although increased quality of CDx is welcomed, worries toward increased costs and administrative work, and decreased quality were expressed. Further, the IVDR might also hinder using optimized in-house and tweaked CDx. Additionally, increased administrative burden could decrease innovativeness toward CDx.

Evaluation of Companion Diagnostics in Scientific Advice and Drug Marketing Authorization Applications by the European Medicines Agency.

With the implementation of the new EU regulation on in vitro diagnostics (IVDR) in May 2022, notified bodies will be required to assess Companion Diagnostics (CDx). The EMA and national medicines agencies will be consulted on the performance and safety of CDx. In this paper, we report on our systematic review on how the EMA has dealt with CDx in dossiers for marketing authorization procedures, in 2017-2019, and in scientific advice procedures in 2016-2020, prior to the implementation of the new IVDR. Out of 167 medicines approved or refused by the EMA, CDx played a role for 20 medicines during assessment. Both European public assessment reports (EPARs) and the internal day 80 and day 120 assessment reports (ARs) of the EMA centralized marketing authorization procedures for these 20 medicines were analyzed in detail to determine how CDx were assessed. Likewise, in 46 of 159 cases in which scientific advice was provided, CDx were mentioned in the question-and-answer section of the scientific advice, and these were analyzed in an analogous manner. Our analysis indicates that clinical performance and analytical performance of the CDx were the most-discussed topics, being discussed 11 and seven times in the 20 EPARs and 59 and 29 times in the ARs, respectively. For scientific advice, clinical and analytical performance was discussed 65 and 22 times in the 46 retrieved mentions of scientific advice. Other aspects in relation to CDx were discussed as well, although at a lower frequency, in assessment reports and scientific advice. Overall, our analysis demonstrates that, despite the absence of an obligation from a legal point of view, EMA has gained experience on the assessment of CDx, most notably regarding its analytical and clinical performance. This experience may be useful in situations in which the EMA and national agencies of EU member states will formally be consulted by notified bodies regarding the performance and safety of CDx. In addition, the issues raised in the EPARs, ARs and scientific advice reports provide insight for applicants on aspects of CDx that need careful consideration.

Drug switching in the Netherlands: a cohort study of 20 active substances.

BACKGROUND: For a patient, drug switches are not desirable (either between a brand-name drug and a generic drug, or between two generic drugs of the same active substance). Research into the causes of drug switches, and related adverse drug reactions, is hampered by the absence of quantitative data on drug switches. METHODS: We describe the frequency of drug switches in the Netherlands for a selection of active substances. A retrospective cohort study was conducted using the Drug Information System of the National Health Care Institute in the Netherlands. We studied the Dutch patient population from mid-2009 to 2016. The selection of active substances (n = 20) was made based on a report by Lareb, the Netherlands Pharmacovigilance Centre, on adverse drug reactions related to drug switching, and we used qualitative and quantitative descriptive analyses. A drug switch is defined as the replacement of a patient's prescribed drug with a similar drug from a different manufacturer. RESULTS: We identified 23.8 million drug switches on a total of 206 million (11.6%) similar drug dispenses. The frequency of drug switches demonstrated a yearly peak in the period from January to March. In some months, for atorvastatin, losartan, pantoprazole, and irbesartan, more than 60% of similar drug dispenses were drug switches. Most drug switches (80.3%) were between two generic drugs, and 0.12% of these involved a drug from a European parallel import. The proportion of drug switches between two brand-name drugs decreased from 14.5 to 5.53% during our study period, and of these, 86.5% involved a drug from a European parallel import. CONCLUSIONS: Drug switching is common in the Netherlands, and most of the drug switches we studied are between generic drugs. The observed annual peak of drug switches is most likely explained by a specific Dutch reimbursement policy. Not only are the data valuable as is, but they also serve as a first step towards elucidating the reasons for the occurrence of these drug switches. In addition, these data can be used to put into perspective the adverse drug reactions associated with drug switching.

Pharmacokinetics and Generic Drug Switching: A Regulator's View.

There appears to be a mismatch between the assumed therapeutic equivalence of generic drugs, their interchangeability, and reported clinical discomfort following generic drug use and drug switches. In this article, we describe why we are of the opinion that the current regulatory approach to the evaluation of generic drugs based on average bioequivalence is sufficient to expect therapeutic equivalence in the clinical setting. This has often been debated, specifically as adverse drug reactions related to generic drug switches are regularly reported. We agree that clinical discomfort during a bioequivalent drug switch may indeed be caused by different exposures to the active substance. However, this difference in exposure is not a result of the characteristics or quality of generic drugs; it is caused by the pharmacokinetic within-subject variability of the active substance, i.e., the variability on the bioavailability of the active substance, when comparing two occasions of administration of the same drug product, to the same patient. Therefore, reported clinical discomfort following generic drug use and drug switches does not warrant a change in the regulatory approach to the evaluation of the bioequivalence of generic drugs. Switching from a brand-name drug to currently approved generic drugs, or between different generic drugs, will in principle result in comparable exposure, within boundaries determined by the within-subject variability of the pharmacokinetics of the active substance involved.

Pharmacogenetic-Pharmacokinetic Interactions in Drug Marketing Authorization Applications via the European Medicines Agency Between 2014 and 2017.

This study aimed to determine to which extent data on potential pharmacogenetic-pharmacokinetic (PG-PK) interactions are provided to, and assessed by, the European Medicines Agency (EMA) in novel drug marketing authorization applications (MAAs), and whether regulatory assessment of PG-PK interactions is adequate or could be optimized. For this purpose, we retrospectively analyzed MAAs of small molecule drugs assessed by the EMA between January 2014 and December 2017. As per two key requirements in the EMA's guideline, we analyzed cases where (i) a single functionally polymorphic drug metabolizing enzyme (DME) metabolizes > 25% of the drug, or (ii) the drug's PK shows high interindividual variability not explained by other factors than PG. Results showed that, of 113 drugs analyzed, 53 (47%) had ≥ 1 functionally polymorphic DME accounting for > 25% of the drug's metabolism, yielding 55 gene-drug pairs. For 36 of 53 (68%) of the products, CYP3A4 was the major DME. Compliance with European Union (EU) guidance on PG-PK issues in drug development was notably different for CYP3A4 substrates vs. non-CYP3A4 substrates. Adequate PG-PK data were provided during registration in 89% (16/18) of cases concerning non-CYP3A4 substrates, compared with 32% (12/37) of cases concerning CYP3A4 substrates. Concluding, PG-PK interactions related to non-CYP3A4 substrate drugs were, in general, addressed adequately in EU MAAs. PG-PK information on CYP3A4 substrates was available less frequently, despite some available evidence on the functional relevance of CYP3A4 polymorphisms. A more harmonized approach toward assessment of PG-PK issues in EU MAAs seems warranted, and a discussion on the relevance of CYP3A4 polymorphisms, such as CYP3A4*22, is recommended.

Quantification of Adverse Drug Reactions Related to Drug Switches in The Netherlands.

We performed a retrospective cohort study in the Dutch patient population to identify active substances with a relatively high number of adverse drug reactions (ADRs) potentially related to drug switching. For this, we analyzed drug switches and reported ADRs related to switching between June 1, 2009, and December 31, 2016, for a selection of 20 active substances. We also compared pharmacovigilance analyses based on the absolute, switch-corrected, and user-corrected numbers of ADRs. In total, 1,348 reported ADRs and over 23.8 million drug switches were obtained from the National Health Care Institute in The Netherlands and from Lareb, which is The Netherlands Pharmacovigilance Centre. There was no correlation between the number of ADRs and the number of switches, but, on average, we found 5.7 reported ADRs per 100,000 switches. The number was relatively high for rivastigmine, levothyroxine, methylphenidate, and salbutamol, with 74.9, 50.9, 47.6, and 26.1 ADRs per 100,000 switches, respectively. When comparing analyses using the absolute number and the switch-corrected number of ADRs, we demonstrate that different active substances would be identified as having a relatively high number of ADRs, and different time periods of increased numbers of ADRs would be observed. We also demonstrate similar results when using the user-corrected number of ADRs instead of the switch-corrected number of ADRs, allowing for a more feasible approach in pharmacovigilance practice. This study demonstrates that pharmacovigilance analyses of switch-related ADRs leads to different results when the number of reported ADRs is corrected for the actual number of drug switches.

The mechanisms of pharmacokinetic food-drug interactions - A perspective from the UNGAP group.

The simultaneous intake of food and drugs can have a strong impact on drug release, absorption, distribution, metabolism and/or elimination and consequently, on the efficacy and safety of pharmacotherapy. As such, food-drug interactions are one of the main challenges in oral drug administration. Whereas pharmacokinetic (PK) food-drug interactions can have a variety of causes, pharmacodynamic (PD) food-drug interactions occur due to specific pharmacological interactions between a drug and particular drinks or food. In recent years, extensive efforts were made to elucidate the mechanisms that drive pharmacokinetic food-drug interactions. Their occurrence depends mainly on the properties of the drug substance, the formulation and a multitude of physiological factors. Every intake of food or drink changes the physiological conditions in the human gastrointestinal tract. Therefore, a precise understanding of how different foods and drinks affect the processes of drug absorption, distribution, metabolism and/or elimination as well as formulation performance is important in order to be able to predict and avoid such interactions. Furthermore, it must be considered that beverages such as milk, grapefruit juice and alcohol can also lead to specific food-drug interactions. In this regard, the growing use of food supplements and functional food requires urgent attention in oral pharmacotherapy. Recently, a new consortium in Understanding Gastrointestinal Absorption-related Processes (UNGAP) was established through COST, a funding organisation of the European Union supporting translational research across Europe. In this review of the UNGAP Working group "Food-Drug Interface", the different mechanisms that can lead to pharmacokinetic food-drug interactions are discussed and summarised from different expert perspectives.

Ethnicity-Specific Drug Safety Data in European Medicines Agency Registration Dossiers, European Public Assessment Reports, and European and Singapore Drug Labels: Lost in Translation?

BACKGROUND: Information on drug safety in different ethnic populations reported in public documents such as the European Public Assessment Reports (EPARs), European Summary of Product Characteristics (SmPCs) or Singapore Package Inserts (SGPIs) generally appears limited. OBJECTIVE: This study aimed to clarify the extent of drug safety data in ethnic populations that is available in drug registration dossiers used for registration in the European Union (EU) and Singapore, and how much of this information is then included in the EPARs and SmPCs or SGPIs. METHODS: For this purpose, drug registration dossiers and these public documents for a selection of 25 drugs authorized both in the EU and Singapore were compared (note, the number of available full registration dossiers was only 24 due to a technical issue). RESULTS: Detailed safety data in ethnic groups were present in 23/24 (96%) of the drug registration dossiers, but was only present in 12/25 (48%) of the EPARs, 8/25 (32%) of the SmPCs, and 9/25 (36%) of the SGPIs. Furthermore, in many cases where ethnicity-specific safety information was provided in the SmPC or SGPIs, details on the ethnic subpopulations was not provided. CONCLUSIONS: Despite the fact that safety data analyzed with respect to ethnic populations are available in almost all screened registration dossiers, this information is often unknown to patients or prescribers as it was often not included in the EPARs, EU SmPCs or SGPIs. In order to increase the availability of such potentially important safety information, it is recommended to at least provide ethnic populations and group size in these public documents. In this way, trust in the registered drugs in different ethnic populations may be increased, and more robust treatment decisions may be obtained in clinical practice.

Interchangeability of Generic Drugs: A Nonparametric Pharmacokinetic Model of Gabapentin Generic Drugs.

Substitution by generic drugs is allowed when bioequivalence to the originator drug has been established. However, it is known that similarity in exposure may not be achieved at every occasion for all individual patients when switching between formulations. The ultimate aim of our research is to investigate if pharmacokinetic subpopulations exist when subjects are exposed to bioequivalent formulations. For that purpose, we developed a pharmacokinetic model for gabapentin, based on data from a previously conducted bioavailability study comparing gabapentin exposure following administration of the gabapentin originator and three generic gabapentin formulations in healthy subjects. Both internal and external validation confirmed that the optimal model for description of the gabapentin pharmacokinetics in this comparative bioavailability study was a two-compartment model with absorption constant, an absorption lag time, and clearance adjusted for renal function, in which each model parameter was separately estimated per administered formulation.

A comparison of the intrasubject variation in drug exposure between generic and brand-name drugs: a retrospective analysis of replicate design trials.

AIMS: The aim of the present study was to investigate whether differences in total and peak drug exposure upon generic substitution are due to differences between formulations or to intrasubject pharmacokinetic variability of the active substance. METHODS: The study was designed as a retrospective reanalysis of existing studies. Nine replicate design bioequivalence studies representing six drug classes - i.e. for alendronate, atorvastatin, cyclosporin, ebastine, exemestane, mycophenolate mofetil, and ropinirole - were retrieved from the Dutch Medicines Regulatory Authority. RESULTS: In most studies, the intrasubject variability in total and peak drug exposure was comparable for the brand-name [in the range 0.01-0.24 for area under the concentration-time curve (AUCt ) and 0.02-0.29 for peak plasma concentration (Cmax ) on a log scale] and generic (0.01-0.23 for AUCt and 0.08-0.33 for Cmax ) drugs, and was comparable with the intrasubject variability upon switching between those drugs (0.01-0.23 for AUCt and 0.06-0.33 for Cmax ). The variance related to subject-by-formulation interaction could be considered negligible (-0.069 to 0.047 for AUCt and -0.091 to 0.02 for Cmax ). CONCLUSION: In the investigated studies, the variation in total and peak exposure seen when a patient is switched from a brand-name to a generic drug is comparable with that seen following repeated administration of the brand-name drug in the patient. Only the intrasubject variability seems to play a crucial and decisive role in the variation in drug exposure seen; no additional formulation-dependent variation in exposure is observed upon switching. Thus, our data support that, for the medicines that were included in the present investigation, from a clinical pharmacological perspective, the benefit-risk balance of a generic drug is comparable with that of the brand-name drug.

Investigation into the interchangeability of generic formulations using immunosuppressants and a broad selection of medicines.

PURPOSE: To date, the interchangeability of generic drugs has only been investigated for a limited number of medicines. The objective of this study was to investigate generic-generic drug interchangeability in a large subset of generic formulations in order to cover a broad spectrum of drugs. METHODS: Orally administered drugs for investigation in this study were selected using strict, predefined criteria, with the purpose to avoid bias. This selection procedure yielded atorvastatin, bicalutamide, naratriptan, olanzapine, perindopril, and venlafaxine. Further, ciclosporin, tacrolimus, and mycophenolate mofetil were investigated as test immunosuppressants. Adjusted indirect comparisons were conducted between generic drugs containing the same active substance, and the 90% confidence interval (CI) for AUC and Cmax was calculated. RESULTS: In total, 120 bioequivalence studies were identified in the Dutch medicine regulatory agency's database, allowing 292 indirect comparisons between generic drugs. The indirect comparison results indicated that in the vast majority of cases, i.e., 80.5%, the 90% CIs for both AUCt and Cmax fell within the bioequivalence criteria (in 90.1 and 87.0% for AUCt and Cmax, respectively). In 1% of the 292 indirect comparison for AUCt and 3% for Cmax, a wider range of 75-133% (or 80-125%) was exceeded. CONCLUSIONS: Overall, our study suggests that exposure-related risks associated with the exchange of different generic drugs in clinical practice are not increased to a relevant extent compared to the situation in which a generic is exchanged with the innovator.

Pharmacogenetics in the evaluation of new drugs: a multiregional regulatory perspective.

Pharmacogenetics, one of the cornerstones of personalized medicine, has the potential to change the way in which health care is offered by stratifying patients into various pretreatment categories, such as likely responders, likely non-responders or likely to experience adverse drug reactions. In order to advance drug development and regulatory science, regulatory agencies globally have promulgated guidelines on pharmacogenetics for nearly a decade. The aim of this article is to provide an overview of new guidelines for the implementation of pharmacogenetics in drug development from a multiregional regulatory perspective - encompassing Europe, the United States and Japan - with an emphasis on clinical pharmacokinetics.

Interchangeability of generic anti-epileptic drugs: a quantitative analysis of topiramate and gabapentin.

PURPOSE: The objective of this study was to determine whether the so-called "shift" or "drift" problem might occur when generic anti-epileptic drugs are interchanged, and thus to assess if generic anti-epileptic drugs are interchangeable and can be used in an efficacious and safe way on the basis of their bioequivalence to one and the same reference product. METHODS: The bioequivalence of topiramate and gabapentin generics was evaluated. For proper interstudy comparison, individual exposure data (AUC and C(max)) for each bioequivalence study present in the registration dossier was normalized based on the absolute exposure data of one of two innovators. The exposure-normalized plasma concentration curves of the generic product arms between studies were compared, providing indirect evidence of bioequivalence of the different generics. Additionally, comparisons were made for generic-generic as well as innovator-innovator exchange based on absolute exposure data from individual bioequivalence studies. RESULTS: In almost all cases, estimated 90% confidence intervals of the AUC and C(max) ratios for generic-generic interchange were within the routine 80-125% criterion. When absolute, non-corrected exposure data were used for this interstudy comparison, in a number of cases 90% confidence intervals outside the 80-125% criterion were found upon interchanging generics from two studies. However, a similar pattern of 90% confidence intervals outside the 80-125% criterion was observed for the comparison of innovator arms, despite the fact that the innovator was identical in all studies. CONCLUSION: Our results strongly indicate that the so-called drifting problem upon generic-generic substitution does not result in important differences in exposure upon exchanging topiramate generics or gabapentin generics.

Randomized phase I clinical and pharmacologic study of weekly versus twice-weekly dose-intensive cisplatin and gemcitabine in patients with advanced non-small cell lung cancer.

PURPOSE: To establish the maximum dose intensity of cisplatin plus gemcitabine on a weekly or two-weekly schedule in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Patients with NSCLC stage IIIB or IV were randomized to receive weekly or two-weekly courses of gemcitabine on day 1 and cisplatin on day 2. An interpatient dose escalation scheme was used, and pharmacokinetics were determined for both agents in plasma and WBCs. RESULTS: Seventy-three patients were included, 32 on the weekly schedule and 41 on the two-weekly schedule. Fifty patients received all planned courses. Dose-limiting toxicities were leukocytopenia, neutropenia, and trombocytopenia on the weekly schedule and ototoxicity on the two-weekly schedule. Most common nonhematological toxicities consisted of nausea, vomiting, and fatigue. The highest dose intensity of cisplatin could be achieved on the two-weekly schedule, and therefore, further development of the weekly schedule was abandoned. The maximum tolerated dose was established at 1500 mg/m(2) gemcitabine in combination with cisplatin 90 mg/m(2). More than half (53%) of patients achieved an objective response on the two-weekly schedule, versus 23% in the weekly treatment arm. The pharmacokinetic studies revealed a significant interaction: gemcitabine reduced both GG and AG platinum-DNA intrastrand adducts in WBCs. CONCLUSION: The combination of gemcitabine (1500 mg/m(2)) with cisplatin at a dose intensity of 50 mg/m(2)/week is feasible on a two-weekly administration scheme in NSCLC patients.

Frequent expression of the multi-drug resistance-associated protein BCRP/MXR/ABCP/ABCG2 in human tumours detected by the BXP-21 monoclonal antibody in paraffin-embedded material.

Breast cancer resistance protein (BCRP/MXR/ABCP/ABCG2; hereafter ABCG2) is a member of the ATP-binding-cassette family of transporters that causes multi-drug resistance to various anticancer drugs. The expression of ABCG2 in human tumours and its potential involvement in clinical drug resistance are unknown. Recently, two monoclonal antibodies against human ABCG2 were produced, BXP-34 and BXP-21. This study describes an immunohistochemical method using BXP-21 to study ABCG2 expression in formalin-fixed, paraffin-embedded tissues. No staining was seen using BXP-34 with the same protocols. The expression of ABCG2 was then investigated in a panel of 150 untreated human solid tumours comprising 21 tumour types. Overall, ABCG2 expression was frequent. Specificity of immunohistochemistry was confirmed by the detection of a 72 kD band in western blotting. ABCG2 expression was seen in all tumour types, but it seemed more frequent in adenocarcinomas of the digestive tract, endometrium, and lung, and melanoma. Positive tumours showed membranous and cytoplasmic staining. In certain adenocarcinomas, prominent membranous staining was seen. Endothelial cells frequently displayed moderate to strong staining. ABCG2 is widely present in untreated human solid tumours and may represent a clinically relevant mechanism of drug resistance. Future studies in specific tumour types are needed to ascertain its clinical relevance. BXP-21 and the immunohistochemical protocol described here will be of value in these investigations.