GREENER Pharmaceuticals for More Sustainable Healthcare.

Medicines are essential to human health but can also impact the aquatic and terrestrial environment after use by patients and release via excreta into wastewater. We highlight the need for a GREENER approach to identify and meet important environmental criteria, which will help reduce the impact of medicinal residues on the environment. These criteria include effect reduction by avoiding nontarget effects or undesirable moieties, exposure reduction via lower emissions or environmental (bio)degradability, no PBT (persistent, bioaccumulative, and toxic) substances, and risk mitigation. With all of these criteria, however, patient health is of primary importance as medicines are required to be safe and efficacious for treating diseases. We discuss the feasibility of including these criteria for green by design active pharmaceutical ingredients in the process of drug discovery and development and which tools or assays are needed to accomplish this. The integrated GREENER approach can be used to accelerate discussions about future innovations in drug discovery and development.

Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test.

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.

Nine prohibited stimulants found in sports and weight loss supplements: deterenol, phenpromethamine (Vonedrine), oxilofrine, octodrine, beta-methylphenylethylamine (BMPEA), 1,3-dimethylamylamine (1,3-DMAA), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylbutylamine (1,3-DMBA) and higenamine.

BACKGROUND: Weight loss and sports supplements containing deterenol have been associated with serious adverse events including cardiac arrest. OBJECTIVE: To determine the presence and quantity of experimental stimulants in dietary supplements labeled as containing deterenol sold in the United States. METHODS: Dietary supplements available for sale in the US and labeled as containing deterenol or one of its synonyms (e.g., isopropylnorsynephrine and isopropyloctopamine) were purchased online. For each brand, one container or subsample was analyzed by NSF International (Ann Arbor, MI) and one container or subsample by the Netherland's National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands). When differences existed between the two containers or subsamples of the same brand, both products were reanalyzed by Sciensano (Brussels, Belgium). NSF International carried out qualitative and quantitative analyses using ultra-high-performance liquid chromatography (UHPLC) quadrupole-Orbitrap mass spectrometry. RIVM performed qualitative and quantitative analysis using UHPLC quadrupole time-of-flight mass spectrometry. Sciensano carried out qualitative analysis using UHPLC quadrupole-Orbitrap mass spectrometry. RESULTS: Seventeen brands of supplements were analyzed. Many brands included more than one prohibited stimulant in the same product: 4 brands (24%, 4/17) included 2 stimulants, 2 (12%, 2/17) combined 3 stimulants, and 2 (12%, 2/17) combined 4 stimulants. The range of quantities per recommended serving size of the 9 stimulants detected were 2.7 mg to 17 mg of deterenol; 1.3 mg to 20 mg of phenpromethamine (Vonedrine); 5.7 mg to 92 mg of beta-methylphenylethylamine (BMPEA); 18 mg to 73 mg of octodrine; 18 mg to 55 mg of oxilofrine; 48 mg of higenamine; 17 mg of 1,3-dimethylamylamine (1,3-DMAA); 1.8 mg to 6.6 mg of 1,3-dimethylbutylamine (1,3-DMBA); and 5.3 mg of 1,4-dimethylamylamine (1,4-DMAA). CONCLUSION: Weight loss and sports supplements listing deterenol as an ingredient contained 9 prohibited stimulants and 8 different mixtures of stimulants, with as many as 4 experimental stimulants per product. These cocktails of stimulants have never been tested in humans and their safety is unknown.

Current challenges in the detection and analysis of falsified medicines.

Falsified medicines affect public health all around the globe. Complex distribution routes, illegal online webshops and reuse of packaging materials make them hard to detect. In order to tackle this problem, detection methods for the recognition of suspicious medicines and subsequent confirmation of falsification by analytical techniques is required. In this review, we focus on the developments and challenges that existed in the last five years (2015-2020) in the detection and analysis of falsified medicines. These challenges might have not been solved yet or arisen with new types of falsifications, new analytical techniques or detection strategies. Detection of suspicious medicines starts with visual inspection of packaging materials. However, re-use of packaging materials and high-quality imitations complicate visual inspection. Recent developments in the analysis of packaging by microscopic and spectroscopic techniques such as optical microscopy, X-ray fluorescence, infrared spectroscopy and Raman spectroscopy or microscopy, in combination with multivariate analysis show promising results in the detection of falsified medicines. An ongoing big challenge in the analysis of falsified medicines is the affordability of analytical devices. Yet, recent reports showed that lower cost devices, such as Counterfeit Drug Indicator or Counterfeit Detection device version 3 show promising use in the detection of falsified medicines. Furthermore, combining the outcomes of different low-cost analytical techniques, such as Minilab, colorimetry and Counterfeit Drug Indicator significantly increased selectivity and sensitivity in the detection of falsified medicines. Also, recent developments make it possible to link a low-cost technique, such as TLC, to mobile phones. Proper training of personnel has shown room for improvement and remains a challenge, even for relatively simple techniques. With an increased use of analytical fingerprints, an upcoming challenge is the accessibility of the growing pool of data. There is also the need of validated reference libraries on both national and international levels. Developments of the last few years bring us a step closer in the fight against falsified medicines, however challenges remain in the worldwide accessibility of affordable, easily operable and sensitive techniques.

Baseline characteristics of the HAARLEM study: 100 male amateur athletes using anabolic androgenic steroids.

BACKGROUND: The use of anabolic androgenic steroids (AAS) is common among visitors of fitness centers. Knowledge about health risks of AAS use is limited due to lack of clinical studies. METHODS: One hundred men, at least 18 years old, intending to start a cycle of AAS were recruited. Baseline demographical data and reasons for AAS use were recorded. Subjects provided samples of AAS for analysis with UPLC-QTOF-MS/MS. RESULTS: One hundred and eleven men were seen for a baseline visit. Nineteen percent had competed in bodybuilding competitions. Recent illicit drug use was reported by 56%. Seventy-seven percent of participants had used AAS in the past, and 97% of them had experienced side effects. After exclusion, 100 men comprised the cohort for follow-up. The AAS cycle performed had a median duration of 13 weeks (range 2-52), and the average dose of AAS equivalents was 901 mg per week (range 250-3.382). Subjects used other performance and image-enhancing drugs (PIEDs) such as growth hormone (21%). In total, 272 AAS samples were analyzed and 47% contained the AAS indicated on the label. The principal reason for AAS use was gain of muscle mass (44%). Forty-eight percent self-reported to being addicted to AAS. CONCLUSION: The HAARLEM study cohort shows that strength athletes use AAS in a wide variety of cycles and often also use illicit drugs and other potentially harmful PIEDs. The quality of the AAS used is strikingly low. Follow-up of the cohort will provide novel data regarding health risks of AAS use.

The stimulant higenamine in weight loss and sports supplements.

BACKGROUND: Higenamine is a stimulant with cardiovascular properties recently prohibited in sport by the World Anti-Doping Agency (WADA). Higenamine is also a natural constituent of several traditional botanical remedies and is listed as an ingredient in weight loss and sports supplements sold over-the-counter in the United States. OBJECTIVES: We analyzed dietary supplements available for sale in the United States prior to WADA's prohibition of higenamine in sport for the presence and quantity of higenamine. METHODS: All supplements labeled as containing higenamine or a synonym (i.e., norcoclaurine or demethylcoclaurine) available for sale in the United States were identified. For each brand, one sample was analyzed by NSF International (Ann Arbor, MI) and one sample by the Netherland's National Institute for Public Health and the Environment (RIVM). NSF International carried out qualitative and quantitative analyses using ultra high performance liquid chromatography (UHPLC) with tandem mass spectrometry. RIVM carried out qualitative analysis using UHPLC quadrupole time of flight mass spectrometry for an independent confirmation of identity. RESULTS: Twenty-four products were analyzed. The majority of supplements were marketed as either weight loss (11/24; 46%) or sports/energy supplements (11/24; 46%); two brands did not list a labeled indication. The quantity of higenamine (±95% CI) ranged from trace amounts to 62 ± 6.0 mg per serving. Consumers could be exposed to up to 110 ± 11 mg of higenamine per day when following recommended serving sizes provided on the label. Five products (5/24; 21%) listed an amount of higenamine, but none were accurately labeled; the quantity in these supplements ranged from <0.01% to 200% of the quantity listed on the label. CONCLUSION: Dosages of up to 62 ± 6.0 mg per serving of the stimulant higenamine were found in dietary supplements sold in the United States.

A high crosslinking grade of hyaluronic acid found in a dermal filler causing adverse effects.

Facial treatments with dermal fillers for medical or esthetic purposes occasionally give rise to adverse effects, ranging from temporary effects such as reddening of the skin, to long term effects such as hardening of tissue. There appears to be a relationship between the lifetime of the filler product and the risk for adverse effects. The lifetime of hyaluronic acid-based fillers is dependent on the presence and amount of crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE). It would therefore make sense to establish methodology to analyze the crosslinking grade of HA-based filler products on a routine basis. To this end, an analytical method was developed and validated to identify HA-BDDE-based fillers and to quantify their modification and crosslinking grade. The method was subsequently applied to products from the legal supply chain and the illegal market. It was found that the product Hyacorp H 1000, previously taken from the market, indeed contains a high modification grade and crosslinking grade, as was the assumed reason for the increased risk for adverse effects of this product. However, it was also shown that the Hyacorp products are highly unreliable in relation to their product composition in general. In this study, authentic products could not be distinguished from the illegal market products based on their modification and crosslinking grade.

Oncology drugs in the crosshairs of pharmaceutical crime.

Oncology drugs clearly have become a target for pharmaceutical crime. In 2016, falsified oncology drugs ranked fifth in the most commonly falsified drug category among the reports received by the Pharmaceutical Security Institute. Although the prevalence of illicit oncology drugs in the legal supply chains appears to be small, these drugs are difficult to detect, particularly in clinical practice. Forthcoming countermeasures to detect illicit drugs in high-income countries include compulsory antitampering devices and product verification technology for a risk-based selection of medicines. Health-care professionals must implement these new procedures into their workflow and remain vigilant about those medicines that are not selected. Although countermeasures should firmly tighten supply chain security, there are concerns about how quickly pharmaceutical crime will adapt to these protections. Because patients and health-care professionals have shown a lenient attitude towards purchasing medicines from unreliable sources, measures against the highly accessible illegal medicine supply chain remain necessary. To improve detectability in clinical practice, reporting of ineffectiveness and unusual drug effects as adverse events or adverse drug reactions is essential.

Fatalities, Cerebral Hemorrhage, and Severe Cardiovascular Toxicity After Exposure to the New Psychoactive Substance 4-Fluoroamphetamine: A Prospective Cohort Study.

STUDY OBJECTIVE: We study adverse health effects after use of the new psychoactive substance 4-fluoroamphetamine. METHODS: All patients who reported 4-fluoroamphetamine exposure and for whom the Dutch Poisons Information Center was consulted by their physician in 2016 were included in a prospective cohort study. The clinical course was investigated through telephone interviews with the physician and/or patient, using standardized questionnaires. 4-Fluoroamphetamine was analyzed in remaining drug material and biological samples with liquid and gas chromatography-mass spectrometry techniques. RESULTS: We included 45 patients, and follow-up with the physician and/or patient was performed in 33 cases. All patients experienced adverse effects after 4-fluoroamphetamine use. Severe toxicity was reported in 8 patients. In 5 of these patients, 4-fluoroamphetamine exposure was confirmed in biological specimens. Severe toxicity that was reported included 2 fatalities, 4 patients with cerebral hemorrhage (1 fatal), 2 patients with inverted Takotsubo's cardiomyopathy, 1 patient with myocardial infarction, 1 patient with acute heart failure, and an overall high prevalence of pronounced hypertension and tachycardia. CONCLUSION: Since the introduction of 4-fluoroamphetamine to the Dutch drug market in 2007, its use continues to increase, possibly because users perceive it as "ecstasy light" and thus relatively safe. However, the proportion of patients with severe toxicity after 4-fluoroamphetamine use is relatively large in our study population. Therefore, users should be warned about the risks of 4-fluoroamphetamine.

Four experimental stimulants found in sports and weight loss supplements: 2-amino-6-methylheptane (octodrine), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylamylamine (1,3-DMAA) and 1,3-dimethylbutylamine (1,3-DMBA).

BACKGROUND: The United States Food and Drug Administration banned the stimulant 1,3-dimethylamylamine (1,3-DMAA) from dietary supplements and warned consumers that the stimulant can pose cardiovascular risks ranging from high blood pressure to heart attacks. OBJECTIVES: We designed our study to determine if a new stimulant similar in structure to 1,3-DMAA has been introduced as an ingredient in supplements sold in the United States (US). METHODS: We analyzed six brands of supplements that listed an ingredient on the label (e.g., Aconitum kusnezoffii, DMHA or 2-amino-isoheptane) that might refer to an analog of 1,3-DMAA. Supplements were analyzed by two separate laboratories using ultra-high-performance liquid chromatography mass spectrometry and reference standards. RESULTS: Two previously unidentified 1,3-DMAA analogs (2-amino-6-methylheptane [octodrine] and 1,4-dimethylamylamine [1,4-DMAA]) and two banned stimulants (1,3-DMAA and 1,3-dimethylbutylamine [1,3-DMBA]) were identified. Octodrine was found at a dose (±95% CI) of 72 ± 7.5 mg per serving. In Europe, octodrine was previously sold as a pharmaceutical in multi-ingredient medications at dosages from 8 to 33 mg. The quantity of octodrine found in our study was more than twice the largest pharmaceutical dose. The other new stimulant, 1,4-DMAA, has not previously been approved for human consumption, and its safety in humans is unknown. 1,4-DMAA was found at dosages between 21 ± 11 mg to 94 ± 48 mg per serving. In addition, two banned stimulants - 1,3-DMAA and 1,3-DMBA - were also identified: 24 ± 7.6 mg to 35 ± 11 mg of 1,3-DMAA and 51 ± 16 mg of 1,3-DMBA. In one product, 24 ± 7.6 mg of 1,3-DMAA was combined with 21 ± 11 mg of 1,4-DMAA. 1,3-DMAA has been investigated as potentially contributing to hemorrhagic strokes and sudden death, whereas the safety of 1,3-DMBA in humans is unknown. CONCLUSION: Two banned stimulants (1,3-DMAA and 1,3-DMBA) and two previously unidentified stimulants (1,4-DMAA and octodrine) were identified in supplements sold in the United States.

Adverse effects of levamisole in cocaine users: a review and risk assessment.

The immunomodulatory adjuvant and antihelminth levamisole is increasingly used as an adulterant in cocaine worldwide. An accumulating body of clinical and toxicological literature has appeared since 2010 describing neutropenia, agranulocytosis, leukoencephalopathy and vasculitis in cases associated with levamisole-adulterated cocaine. Mostly, neutropenia and agranulocytosis were reported, characterized by a decimation of neutrophils. A large proportion of cases also involved vasculopathy, characterized by pronounced black and purple skin purpura with cutaneous necrosis. Females are more susceptible for both agranulocytosis and vasculitis. Another complication reported with levamisole-adulterated cocaine is leukoencephalopathy, a disabling and potentially fatal neurological disorder caused by cerebral demyelination. In this review, all adverse effects associated with therapeutic levamisole and levamisole-adulterated cocaine are described. In addition, this review provides an update of the pharmacology of levamisole, its metabolism, including toxic metabolites and metabolites that are relevant for levamisole's addition to cocaine. Special emphasis is put on the immunopathology and the dose-effect relationship of chronic levamisole exposure. Finally, a risk assessment is provided based on the current level of levamisole adulteration in street cocaine, the dose range calculated per gram and the pattern of chronic exposure in heavy or dependent users.

Pharmaceutical doses of the banned stimulant oxilofrine found in dietary supplements sold in the USA.

Oxilofrine (4-[1-hydroxy-2-(methylamino)propyl]phenol) is a pharmaceutical stimulant prescribed in dosages of 16 to 40 mg to stimulate the heart and increase blood pressure. It has never been approved for use in the USA as a prescription drug or as a dietary supplement. Several athletes, however, have been banned from sport for testing positive for oxilofrine and have claimed that they inadvertently consumed oxilofrine in sports supplements. Consumption of supplements containing oxilofrine may also pose serious health risks. For example, one brand of supplements containing oxilofrine has been linked to serious adverse events including vomiting, agitation, and cardiac arrest. We designed our study to determine the presence and quantity of oxilofrine in dietary supplements sold in the USA. A validated ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry method was developed for the identification and quantification of oxilofrine. The separation was achieved using a reversed phase column, mass spectrometry detection, and a water/acetonitrile gradient as the mobile phase. The presence of oxilofrine was confirmed using a reference standard. We analyzed 27 brands of supplements labelled as containing a synonym of oxilofrine ('methylsynephrine') and found that oxilofrine was present in 14 different brands (52%) at dosages ranging from 0.0003 to 75 mg per individual serving. Of the supplements containing oxilofrine, 43% (6/14) contained pharmaceutical or greater dosages of oxilofrine. Following instructions on the label, consumers could ingest as much as 250 mg of oxilofrine per day. The drug oxilofrine was found in pharmacological and greater dosages in supplements labelled as containing methylsynephrine. Copyright © 2016 John Wiley & Sons, Ltd.

The quality of sildenafil active substance of illegal source.

There must be a large market for active pharmaceutical ingredients of illegal source to support the huge and lucrative business of trade in illegal medicines. The active substances found in illegal pharmaceuticals may differ from their legal counterparts concerning purity and associated risks for the health of the user. In this study we show two examples in which the active substance sildenafil, used in erectile dysfunction products, was not of European Pharmacopeia quality. In one case milligram-scale amounts of a 2-mercaptobenzothiazole contamination were found, in another case the mesylate salt rather than the monograph based citrate was used. For the user of products containing these active substances, the risks of side effects increase through the inherent properties of the impurity and the chance of overdosing. The fact that the users are most likely not aware of the poor quality of the products adds up to the health risk of using prescription medication without consulting medical professionals.

Operation resistance: A snapshot of falsified antibiotics and biopharmaceutical injectables in Europe.

Operation Pangea is an annual international week of action combating pharmaceutical crime. In this study, called Operation Resistance, we asked the national agencies in Europe to search for falsified antibiotics and biopharmaceutical injectables (peptides and proteins) amongst the medicines seized in Pangea 7 (2014). Reports were received from Belgium, Cyprus, Czech Republic, Denmark, France, the Netherlands, Portugal, Sweden, Spain, the United Kingdom, Norway, and Switzerland. The countries reported seizing about 21,000 dose units (e.g. tablets, capsules) of falsified antibiotics in total. Most of the antibiotics were unlicensed medicines with common antibiotic drugs. In this study week, very few falsified biopharmaceutical injectables were reported. Laboratories reported human growth hormone, sermorelin, melanotan II, and no active ingredients. The average shipment size seemed too large for personal use indicating that a substantial part was intended for resale. This study provides a snapshot of the falsified antibiotics and biopharmaceuticals that enter European countries. How much is actually reaching users during Pangea week - in on other weeks - remains unknown. The shipment sizes indicate falsified antibiotics and biopharmaceuticals are imported for both personal use and resale. The use of antibiotics from unreliable sources is a health risk, contributes to antimicrobial resistance, and may obscure a source of infection from health agencies. The falsified biopharmaceuticals are a health risk because they lack all labelling and may contain unlicensed drugs for injection. It seems important to raise awareness among health-care professionals that falsified medicines in Europe are not restricted to erectile dysfunction drugs. Copyright © 2015 John Wiley & Sons, Ltd.

A synthetic stimulant never tested in humans, 1,3-dimethylbutylamine (DMBA), is identified in multiple dietary supplements.

A synthetic stimulant never before studied in humans, 1,3-dimethylbutylamine (DMBA), was suspected of being present in dietary supplements. DMBA is an analogue of the pharmaceutical stimulant, 1,3-dimethylamylamine (DMAA), which was recently banned by the US Food and Drug Administration. We obtained all dietary supplements sold by US distributors that listed an ingredient on the label, such as AMP Citrate, that might be a marketing name for DMBA. Supplements were analyzed for the presence and quantity of DMBA. Fourteen supplements met our inclusion criteria and were analyzed by two separate laboratories using ultra high performance liquid chromatography (UHPLC) - mass spectrometry and a reference standard. The identity of DMBA was confirmed in 12 supplements in the range of 13 to 120 mg DMBA per serving. Following recommendations on the supplement label for maximum daily intake, customers would consume from 26 to 320 mg of DMBA per day. Supplements containing DMBA were marketed to improve athletic performance, increase weight loss and enhance brain function. DMBA has never before been detected in supplements. The stimulant has never been studied in humans; its efficacy and safety are entirely unknown. Regulatory agencies should act expeditiously to warn consumers and remove DMBA from all dietary supplements.

Chemical fingerprinting of silicone-based breast implants.

With millions of women worldwide carrying them, silicone-based breast implants represent a large market. Even though silicone breast implants already have a history of use of more than 50 years, the discussion on their safety has not yet come to an end. To improve safety assessment, regulatory authorities should have the availability of a set of tests to be able to determine parameters of implant identity and quality. Therefore, the gels and envelopes of various brands and types of silicone-based breast implants have been subjected to infrared, Raman and NMR spectroscopy. We show that by using a combination of complementary spectroscopic techniques breast implants of various origins can be distinguished on typical chemical hallmarks. It was found that typical silicone-based implants display a surplus of vinyl signals in the gel, cyclosiloxane impurities are tolerable at low levels only and a barrier layer is present in the implant envelope. The techniques presented here and the results obtained offer a good starting point for market surveillance studies.