Evaluation of non-clinical toxicity studies of COVID-19 vaccines.

In this study we evaluated the outcomes of non-clinical toxicity studies of various SARS-CoV-2 vaccines produced with different manufacturing technologies, with focus on Repeated Dose Toxicity (RDT) and Developmental and Reproductive Toxicity (DART) studies. We found that RDT and DART studies at doses relevant for human treatment showed no adverse effects while remaining observations were expected findings including local reactogenicity, immune response and macroscopic findings at the injection site. We have also reviewed the European Medicines Agency (EMA) nonclinical assessment reports for market authorization. Regardless of utilized vaccine manufacturing technology EMA assessment of the non-clinical studies consisted most frequently of comments related to study design, species selection and missing data. Sponsors have often submitted platform studies (vaccine studies with the same technology/construct but using other antigens) as supplementary data. Animal model-based toxicity testing has shown rather small effects, which have been never serious adverse effects. The translational value to support clinical development is mainly to inflammatory effects, indicative of the primary action of the vaccines. From a 3R perspective supportive platform technology data consisting of previously executed RDT and DART studies from the same platform technology are encouraged to be implemented in the vaccine assessment process.

The pre- and post-authorisation data published by the European medicines agency on the use of biologics during pregnancy and lactation.

AIMS: The effects of biologics on reproduction/lactation are mostly unknown although many patients that receive biologics are women of reproductive age. The first objective of this study was to investigate the publicly available data on pregnancy/lactation before and after marketing authorization in Europe of biologics for the indications of rheumatologic inflammatory autoimmune diseases and inflammatory bowel disease. Secondary objectives included the assessment of the clinical relevance of the provided data and comparison of initial and post-authorization data. METHODS: Initial and post-authorization data were extracted from the European Public Assessment Reports and the latest versions of Summary of Product Characteristics using publicly available documents on the European Medicines Agency's website. Four sections were categorized regarding pregnancy outcomes: pre-clinical/animal studies, human female fertility, pregnancy-related outcomes and congenital malformations in the human fetus. Three sections were categorized regarding lactation outcomes: pre-clinical/animal studies, excretion in human breast milk and absorption in children through breastfeeding. The clinical applicability of each category was scored by specified criteria, based on scientific literature, and further as defined by the authors. RESULTS: For the 16 included biologics, post-authorization data were delivered only for adalimumab, certolizumab pegol, etanercept and infliximab. For the 12 remaining biologics limited data on pregnancy and lactation during the post-marketing period of 2-21 years were available. CONCLUSIONS: In this article several suggestions are provided for improving a multidisciplinary approach to these issues. The initiation of suitable registries by marketing authorization holders and data transparency for clinicians and academics are highly endorsed.

Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

Toward a comparative retrospective analysis of rat and rabbit developmental toxicity studies for pharmaceutical compounds.

Based on a proposal made at the ICH Workshop in Tallinn, Estonia (2010), the value of the rabbit embryo-fetal development (EFD) versus the rodent EFD was examined by the HESI DART group. A cross-industry data survey provided anonymised EFD and toxicokinetic data from EFD studies on over 400 marketed and unmarketed drugs (over 800 studies) that were entered by experts at RIVM into US EPA's ToxRefDB style database. The nature and severity of findings at the lowest observed adverse effect level (LOAEL) are being reviewed to quantitate the frequency with which lesser signs of embryo-fetal effects (e.g., delays in ossification, minor changes in frequency of variants) are driving the LOAELs. Interpretation was based on exposure rather than administered dose. This paper provides an update of this ongoing project as discussed during a workshop of the European Teratology Society in Ispra, Italy (2013). This was the first presentation of the initial data set, allowing debate on future directions, to provide a better understanding of the implications of either delaying a rabbit EFD or waiving the need in particular circumstances.

Classifying the adverse mitogenic mode of action of insulin analogues using a novel mechanism-based genetically engineered human breast cancer cell panel.

Insulin analogues are widely used in clinical practice. Modifications on the insulin molecular structure can affect the affinity and activation towards two closely related receptor tyrosine kinases: the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R). A switch towards higher IGF1R affinity is likely to emphasize mitogenesis rather than glucose metabolism. Relevant well-validated experimental tools to address the insulin analogue activation of either INSR or IGF1R are missing. We have established a panel of human MCF-7 breast cancer cell lines either ectopically expressing the INSR (A or B isoform) in conjunction with a stable knockdown of the IGF1R or ectopically expressing the IGF1R in conjunction with a stable knockdown of the INSR. In these cell lines, we systematically evaluated the INSR and IGF1R receptor activation and downstream mitogenic signalling of all major clinical relevant insulin analogues in comparison with insulin and IGF1R. While most insulin analogues primarily activated the INSR, the mitogenic activation pattern of glargine was highly similar to IGF1 and insulin AspB10, known to bind IGF1R and induce carcinogenesis. Yet, in a long-term proliferation assay, the proliferative effect of glargine was not much different from regular insulin or other insulin analogues. This was caused by the rapid enzymatic conversion into its two metabolic active metabolites M1 and M2, with reduced mitogenic signalling through the IGF1R. In summary, based on our new cell models, we identified a similar mitogenic potency of insulin glargine and AspB10. However, rapid enzymatic conversion of glargine precludes a sustained activation of the IGF1R signalling pathway.

Use of statins is associated with an increased risk of rheumatoid arthritis.

OBJECTIVES: Statins offer significant cardiovascular benefits. Their use, however, influences immune regulation, which may potentially facilitate autoimmunity, eventually resulting in autoimmune diseases such as rheumatoid arthritis (RA).The authors studied whether statin use was associated with an increased risk of developing RA by conducting a case-control study using the Netherlands Information Network of General Practice database. METHODS: The authors identified 508 patients aged 40 years or older with a first-time diagnosis of RA in the period 2001-2006. Each RA case was matched to five controls for age, sex and index date, which was selected 1 year before the first diagnosis of RA. Odds ratios for the first-time diagnosis of RA were verified by a referral to a rheumatologist and/or at least one prescription of disease-modifying anti-rheumatic drugs and/or two prescriptions of corticosteroids after the date of first diagnosis. RESULTS: Cases were more often users of statins (15.9%) compared to controls (8.6%). After adjustment for cardiovascular risk factors and use of comedication, statin use was associated with an increased risk of incident RA (adjusted OR, 1.71 (95% CI 1.16 to 2.53); p=0.007). A consistent trend of increasing risk with increased cumulative duration, cumulative defined daily doses and number of prescriptions was not observed. However, a small trend between the potency of statin treatment and the risk of RA was found. CONCLUSIONS: Statin use seems to be associated with an increased risk of developing RA. Our findings should be replicated by additional studies.

Assessing immunotoxicity: guidelines.

Over the last couple of years the assessment of immunotoxic potential of human pharmaceuticals has drawn considerable attention worldwide. Regulatory agencies entrusted with the registration of pharmaceuticals (or other compounds) found an increased need for guidance on this issue. This has resulted in the release of guidance documents on immunotoxicity in Europe, USA and Japan in close succession. In Europe the CPMP has released their immunotoxicity guidance documents that are now in force. The FDA and the Japanese Authorities are in the process of doing so, and will shortly enforce them. Immune suppression and stimulation, hypersensitivity, photosensitivity, drug-induced autoimmunity and developmental immunotoxicity are the focus of regulatory testing. This review discusses these kinds of immunotoxicity and their clinical implications. The three regional guidelines and screening tools for detection are discussed. Additionally, the scientific background on which these guidelines are based is briefly highlighted.

Xpa and Xpa/p53+/- knockout mice: overview of available data.

DNA repair deficient Xpa-/- and Xpa-/-/p53+/- knock-out mice in a C57BL/6 genetic background, referred to as respectively the XPA and XPA/p53 model, were investigated in the international collaborative research program coordinated by International Life Sciences Institute (ILSI)/Health and Environmental Science Institute. From the selected list of 21 ILSI compounds, 13 were tested in the XPA model, and 10 in the XPA/p53 model. With one exception, all studies had a duration of 9 months (39 weeks). The observed spontaneous tumor incidence for the XPA model after 9 months was comparable to that of wild-type mice (total 6%). For the XPA/p53 model, this was somewhat higher (9%/13% for males/females). The 3 positive control compounds used, B[a]P, p-cresidine, and 2-AAF, gave positive and consistent tumor responses in both the XPA and XPA/p53 model, but no or lower responses in wild-type mice. From the 13 ILSI compounds tested, the single genotoxic carcinogen (phenacetin) was negative in both the XPA and XPA/p53 model. Positive tumor responses were observed for 4 compounds, the immunosuppressant cyclosporin A, the hormone carcinogens DES and estradiol, and the peroxisome proliferator WY-14,643. Negative results were obtained with 5 other nongenotoxic rodent carcinogens, and 2 noncarcinogens tested. As expected, both DNA repair deficient models respond to genotoxic carcinogens. Combined with previous results, 6 out of 7 (86%) of the genotoxic human and/or rodent carcinogens tested are positive in the XPA model. The positive results obtained with the 4 mentioned nongenotoxic ILSI compounds may point to other carcinogenic mechanisms involved, or may raise some doubts about their true nongenotoxic nature. In general. the XPA/p53 model appears to be more sensitive to carcinogens than the XPA model.

Disturbance of cardiovascular circadian rhythms by pertussis vaccine in freely-moving rats.

Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.

Mechanisms of nongenotoxic carcinogenesis and assessment of the human hazard.

Regulatory toxicologists in the pharmaceutical area are faced with many chemical entities to be classified as rodent carcinogens, in most cases on the basis of a nongenotoxic mechanism. The purpose of this paper is to describe some mechanisms for nongenotoxic tumorigenicity and to indicate which type of testing should be done to substantiate why in those cases such a mechanism is not relevant to humans. The increasing attention being given to epigenetic carcinogenesis points at the need for a thorough evaluation during the toxicological program for safety assessment, enabling adequate assessment of the human hazard posed by such compounds. Data to support the nongenotoxic carcinogenesis may be obtained by collecting specific information from current safety assessment programs or from future, separate studies.

Current status and use of short/medium-term models for assessment of carcinogenicity of human pharmaceuticals: regulatory perspectives.

In the summer of 1997 international governmental organizations and industry partners agreed upon a new document on 'Testing for Carcinogenicity of Pharmaceuticals'. The most important element in the new guidance was the acceptability of only one life-time carcinogenicity study in a rodent species (preferably the rat). In addition a choice could be made to test the pharmaceutical in one of the newly developed models, i.e. the newborn mouse assay or one of the various transgenic mouse assays. In the present paper the strengths and weaknesses of various models are discussed from a regulatory point of view. The aim of the new animal models would eventually be replacing animal life-span studies without compromizing human safety. Such studies should supplement the life-span studies and provide additional information not readily available from the long-term assay. At present there is insufficient information to predict or offer guidance on which of the models may be the most suitable. New models are not useful to test the carcinogenic potential of biotechnological products.

Effects of prolonged exposure to morphine and methadone on in vivo parameters of immune function in rats.

In rats, two 6-week repeated dose oral toxicity studies were performed with morphine (250 and 500 mg/kg food) and methadone (200 and 400 mg/kg food), respectively. Alterations in immune function were studied by assessing primary and secondary immune responses to sheep red blood cells. In addition, the ability to resist challenge with infectious agents was measured in host resistance models employing the parasite Trichinella spiralis and the bacterium Listeria monocytogenes. The primary and secondary antibody responses to sheep red blood cells were not affected by treatment with either morphine or methadone. The clearance of L. monocytogenes bacteria in the spleen was not affected either. Prolonged treatment with morphine, however, resulted in a decrease in host resistance to T. spiralis infection, as indicated by a 1.5-fold increase in numbers of muscle larvae counted in the carcass, but did not affect the T. spiralis-specific IgM, IgG and IgE antibody responses. In contrast to morphine, the methadone-treated animals did not show a significant change in host resistance to T. spiralis. Total serum IgG levels, however, were increased in high-dose methadone-treated animals. Apparently, prolonged administration of morphine to rats resulted in immune suppression, mediating a slight, though biologically relevant, exacerbation of the T. spiralis infection, whereas methadone did not.

Repeated administration of whole-cell and acellular pertussis vaccines affects haemodynamics and autonomic responsiveness.

Rats were treated in a repetitive way one to four times with either pertussis toxin, combined Diphtheria-Tetanus-Poliomyelitis-Pertussis vaccine (DTP-IPV vaccine, which includes inactivated polio virus and whole-cell pertussis), DT-IPV vaccine (lacking the whole-cell pertussis component) or acellular pertussis (aP) vaccine or Haemophilus influenzae type b vaccine. Baseline diastolic blood pressure, baseline heart rate and adrenergic and cholinergic responses were evaluated 4 days after last treatment. Pertussis toxin decreased baseline diastolic blood pressure (28-43%) and increased baseline heart rate (28-40%). Adrenergic and cholinergic response were inhibited by 65-75% and 70-78%. Multiple treatments were grossly as effective as single treatment. Similar results were obtained with DTP-IPV, while DT-IPV did not affect any of the four responses measured. Acellular pertussis vaccine did not affect baseline diastolic blood pressure, but significantly increased baseline heart rate (14%) and inhibited the adrenergic (19-23%) and cholinergic response (39-50%). This indicates that the acellular vaccine tested contains pharmacologically active pertussis toxin. As the effects were less pronounced compared to DTP-IPV, it is concluded that acellular pertussis retains less residual toxicological effects than whole-cell pertussis vaccine and may therefore be a safer vaccine. The observed effects on haemodynamics and autonomic control seem to be specific for pertussis toxin and pertussis-related vaccines as Haemophilus influenzae type b vaccine is in this respect virtually inactive.

[Cognitive and psychotic effects after cessation of chronic cannabis use]. / Cognitieve effecten en psychotische effecten na stopzetting van chronisch cannabisgebruik.

Establishment of residual cognitive and psychotic effects (effects present at the time that all active cannabinoids are eliminated from the body) putatively produced by prolonged heavy cannabis use is difficult, because of many confounding variables like slow elimination of active cannabinoids, lack of supervision during abstinence, poor use of well-matched control groups and the presence of withdrawal symptoms. Residual cognitive effects were observed in some but not in all tests after prolonged heavy cannabis use. The effects were mostly mild. The relationship of cannabis use, psychotic effects and schizophrenia was unclear; the cannabis conceivably gave relief, but it also appeared that cannabis caused schizophrenia in young people and (or) enhanced the symptoms, especially in young people poorly able to cope with stress or in whom the antipsychotic therapy was unsuccessful.

The effect of pertussis toxin and whole-cell pertussis vaccine on haemodynamics and autonomic responsiveness in the rat depends on route of administration and age.

Vaccination of children with Diphtheria, Tetanus, Poliomyelitis and pertussis vaccine (DTPoP-vaccine) containing the whole-cell pertussis component is known to be associated with manifestation of side-effects such as acute encephalopathy, convulsions and hypotensive-hyporesponsive episodes. In young and adult rats the effects of pertussis toxin and DTPoP-vaccine on haemodynamics and autonomic responsiveness are evaluated following treatment with high dose via different routes of administration (s.c., i.p. and i.v.). The effect of pertussis toxin is dose-dependent (between 1 and 20 micrograms kg-1) and largest responses are observed after i.v. administration. At 20 micrograms kg-1, i.v. pertussis toxin decreases baseline diastolic blood pressure and increases baseline heart rate by 31% and inhibits autonomic responsiveness (salbutamol-induced increase in diastolic blood pressure and arecoline-induced decrease in heart rate). In adult rats DTPoP-vaccine induces generally more prominent effects than in young rats. In adult rats DTPoP-vaccine reduces baseline diastolic blood pressure by 25% while no response is observed in young rats. In adult rats DTPoP inhibits the adrenergic response though less compared to treatment of pertussis toxin. After treatment with DTPoP-vaccine (single or twice) only minor differences are observed between young and adult rats. Present results show that adult rats are more sensitive to pertussis toxin and pertussis vaccine than young rats and that the responses depend on the route of administration.

New perspectives for alternative approaches to carcinogenicity testing: a regulator viewpoint.

Genotoxic compounds are thought more likely to be transspecies carcinogens inducing carcinomas in more than one species, implying a hazard to humans. Non-genotoxic compounds will have a certain threshold concentration below which they are definitely not carcinogenic. What are we looking for in the case of pharmaceuticals? New animal models would be helpful to enhance the quality of assessment of carcinogenic potential, based on 'weight of evidence', in order to reach the goal of replacing animal life span studies without compromising human safety. Such studies should supplement life span studies and provide additional information not readily available from the long-term assay. Short- or medium-term assays may be helpful in confirming the transspecies character of carcinogens.

The utility of two rodent species in carcinogenic risk assessment of pharmaceuticals in Europe.

For the past 20-30 years, lifespan carcinogenicity studies for pharmaceuticals have been required to be carried out in two rodent species. Due to scientific progress, the necessity/justification of lifespan studies in two species for the assessment of carcinogenic risk of pharmaceuticals is currently under discussion. A study in one species (either rat or mouse) might suffice. To appraise the need for a study in a second species, a database was compiled of all pharmaceuticals tested for carcinogenicity for which a marketing authorization was applied for in Germany and The Netherlands since 1980. The incidence of treatment-related tumor findings was determined in either rat or mouse or in both. Tumor findings occurred for nearly 50% of all compounds, with the rat being more sensitive than the mouse. Specific attention was given to the question whether tumor findings in mice ever caused the regulatory authorities to refuse registration, to restrict the proposed therapeutic indication of a pharmaceutical, or to apply a cautionary label. It was found that no tumor findings in mice alone ever led to such a regulatory action. In addition, whether mouse studies had been important in interpreting the results of rat studies was determined. A negative mouse study (no tumors found) was rarely used to declare the rat findings irrelevant to humans. A mechanistic explanation was used as a much more important argument in the assessment of tumor findings in rats. In case of transspecies findings, the target organs were the usual ones, such as lung and liver, or the tumors occurred as a result of an exaggerated pharmacodynamic action expected from the pharmacology of the compound. The results of the database thus question the need of maintaining the requirement of rodent carcinogenicity studies in two species.

Comparison of the in vivo effects of morphine and methadone on natural killer cell activity in spleen, peritoneal cavity, and lungs in rats.

Opiates and opioid agents are known to affect the immune system. In humans this includes alterations in natural killer (NK) cell activity. Morphine is reported to reduce in vivo spleen NK activity in rats, whereas for methadone only in vitro data have been described. In the present paper we describe a systematic study on the chronic effects of well-known opiates, comparing for the first time the effects of morphine and methadone on NK cell activity in various organs: in addition to spleen, also in the peritoneal cavity, and lungs. In all organs the NK activity was determined using three effector: target cell ratios. Morphine and methadone given by food during 6 weeks decreased the NK cell activity in rat spleen, supporting published data on morphine. The role of the opiate receptor is discussed. However, the overall action of morphine could not be described as suppressive because stimulation of NK cell activity in the peritoneal cavity and lungs by morphine was found. In contrast, methadone induced a decrease in the NK cell activity in these organs. Apart from these differential expressions of morphine- and methadone associated effects on NK cell activity, the findings demonstrate the potential adverse effects of these opiates on an important antiviral defence mechanism.