Anti-tumor necrosis factor α: originators versus biosimilars, comparison in clinical response assessment in a multicenter cohort of patients with inflammatory arthropathies.

OBJECTIVE: To compare etanercept and adalimumab biosimilars (SB4 and ABP501) and respective bioriginators in terms of safety and efficacy in a real-life contest. METHODS: We consequently enrolled patients affected by rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, treated with SB4, and ABP501, or with corresponding originators, belonging to the main biological prescribing centers in the Lazio region (Italy), from 2017 to 2020. Data were collected at recruitment and after 4, 8, 12, and 24 months of therapy. RESULTS: The multicenter cohort was composed by 455 patients treated with biosimilars [SB4/ABP501 276/179; female/male 307/146; biologic disease-modifying anti-rheumatic drug (b-DMARD) naïve 56%, median age/ interquartile range 55/46-65 years] and 436 treated with originators (etanercept/adalimumab 186/259, female/ male 279/157, b-DMARD naïve 67,2%, median age/interquartile range 53/43-62 years). No differences were found about safety, but the biosimilar group presented more discontinuations due to inefficacy (p<0.001). Female gender, being a smoker, and being b-DMARD naïve were predictive factors of reduced drug survival (p=0.05, p=0.046, p=0.001 respectively). The retention rate at 24 months was 81.1% for bioriginators and 76.5% for biosimilars (median retention time of 20.7 and 18.9 months, respectively) (p=0.002). Patients with remission/low disease activity achievement at 4 months showed a cumulative survival of 90% to biosimilar therapy until 24 months (p=0.001); early adverse reactions instead represented a cause of subsequent drug discontinuation (p=0.001). CONCLUSIONS: Real-life data demonstrated a similar safety profile between biosimilars and originators, but a reduced biosimilar retention rate at 24 months. Biosimilars could be considered a valid, safe, and less expensive alternative to originators, allowing access to treatments for a wider patient population.

Analysis of the Necrosis-Inducing Components of the Venom of Naja atra and Assessment of the Neutralization Ability of Freeze-Dried Antivenom.

Patients bitten by Naja atra who are treated with bivalent freeze-dried neurotoxic antivenom in Taiwan have an improved survival rate but develop necrotic wound changes. The World Health Organization (WHO) has suggested using the minimum necrotizing dose (MND) of venom as a method of evaluating the neutralization effect of antivenom. The aim of this study was to evaluate the effectiveness of antivenom for the prevention of necrosis based on the MND and clarify which component of the venom of N. atra induces necrosis. The neurotoxins (NTXs) were removed from the crude venom (deNTXs), and different concentrations of deNTXs were injected intradermally into the dorsal skin of mice. After three days, the necrotic lesion diameter was found to be approximately 5 mm, and the MND was calculated. A reduction in the necrotic diameter of 50% was used to identify the MND50. Furthermore, both phospholipase A2 (PLA2) and cytotoxins (CTXs) were separately removed from the deNTXs to identify the major necrosis-inducing factor, and the necrotic lesions were scored. All mice injected with deNTXs survived for three days and developed necrotic wounds. The MND of the deNTXs for mice was 0.494 ± 0.029 µg/g, that of the deNTXs-dePLA2 (major component retained: CTXs) was 0.294 ± 0.05 µg/g, and that of the deNTX-deCTX (major component retained: PLA2) venom was greater than 1.25 µg/g. These values show that CTX is the major factor inducing necrosis. These results suggest that the use of the deNTXs is necessary to enable the mice to survive long enough to develop venom-induced cytolytic effects. CTXs play a major role in N. atra-related necrosis. However, the MND50 could not be identified in this study, which meant that the antivenom did not neutralize venom-induced necrosis.

Assessment of metals induced histopathological and gene expression changes in different organs of non-diabetic and diabetic rats.

Diabetes is a complex metabolic disorder and different environmental toxicants including heavy metals have been involved in diabetes induction. Therefore, assessment of the environmental risk factors and heavy metals induced toxicity have become critical for reducing the consequences of metals pollutants. Previously, we reported heavy metals induced nephrotoxicity in non-diabetic and diabetic rats. Here, we extended our analysis by examining the heavy metals induced organs (heart, kidney, liver, pancreas, and spleen) damage in diabetic and non-diabetic Wistar rats using histopathology and quantitative real-time PCR (qRT-PCR). Following the generation of the diabetic rat model, the animals were exposed to heavy metals including lead (Pb), arsenic (As), manganese (Mn) and cadmium (Cd). Both non-diabetic and diabetic rats were exposed to heavy metals for 30 days and subsequently, the heart, kidney, liver, pancreas and spleen tissues were examined. Heavy metal treatment resulted in irregularly arranged myofibrils and vacuolization in the heart tissue of metal treated groups as evident from hematoxylin and eosin (H & E) staining. The kidney tissue of rats treated with heavy metals showed tubular degeneration, fibrosis, hemorrhage, and vacuolation. The liver of the heavy metals treated rats exhibited cellular degeneration and necrosis. The pancreatic tissue of streptozotocin injected untreated and metal treated rats revealed severe degeneration, necrosis, degranulation, shrinkage, and depression in the islets of Langerhans. Increased red pulp area and congestion were observed in the spleen of the metal mixture treated non-diabetic and diabetic rats. In line with the histological data, the qRT-PCR analysis showed downregulated expression of Bcl2 and upregulation of Caspase-3 in non-diabetic and diabetic metal treated rats as compared to the non-diabetic untreated rats. In conclusion, the present study revealed, diabetic rats are more prone to metal alone as well as metal mixture induced organ damage as compared to non-diabetic rats.

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models.

Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan.

In vitro assessment of cytotoxic, genotoxic and mutagenic effects of antimalarial drugs artemisinin and artemether in human lymphocytes.

Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50 µg/mL) and artemether (7.46; 14.92 and 29.84 µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p < 0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p < 0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

Responsiveness assessment of a 3D tetra-culture alveolar model exposed to diesel exhaust particulate matter.

The aim of the current study was to evaluate the responses of a 3D tetra-culture alveolar model cultivated at the air-liquid-interface (ALI) after apical exposure to diesel exhaust particulate matter (DEPM) based on the three-tiered oxidative stress concept. The alveolar model exposed to increasing doses of DEPM (1.75-5 µg/cm2) responded with increasing activity of the anti-oxidant defense mechanisms (Nrf2 translocation, increased gene expression for anti-oxidant proteins and increased HMOX-1 synthesis) (tier 1). Higher exposure generated a proinflammatory response (NF-kB translocation, increased gene expression of pro-inflammatory cytokines and adhesion molecules, and increased IL-6 and IL-8 synthesis) (tier 2) and, finally, the highest doses applied resulted in a decrease of cell viability due to necrosis (extra-cellular release of LDH) or apoptosis (increased expression of the pro-apoptotic genes CASP7 and FAS) (tier 3). Overall, the results of our study demonstrate that the 3D tetra-culture model when directly exposed to DEPM potently generates a realistic response according to the three-tiered oxidative stress concept. Further evaluation and benchmarking against currently used in vivo rodent models is needed to show its suitability, and to serve in the future as an alternative for in vivo studies in the hazard evaluation of inhalable irritants.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

BACKGROUND/AIMS: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. METHODS: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. RESULTS: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. CONCLUSION: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

Best practices for application of attachment cells to in vitro micronucleus assessment by flow cytometry.

This work seeks to provide users with guidance on cell culture, treatment, processing and analytical conditions for achieving optimal performance of the in vitro micronucleus assay using the In Vitro MicroFlow(®) method. Experimental data are provided to support the advice described. The information provided covers specific topics or issues that are identified as critical to the methodology and thus is meant to work with instruction manuals, published papers and other references, and not as a replacement for these documents. The content is divided into several sections. Cell culture and treatment describes conditions for routine maintenance of cells as well as treatment with test articles. Preparation and processing of samples details steps found to be critical in execution of the procedure. Instrument parameters and analysis covers set-up of the flow cytometer and evaluation of the samples. General assay considerations and interpretation of results describes examination of data in terms of assay validity, viability and genotoxicity assessment. The goal is to educate users and enable them to design, conduct and interpret flow cytometric in vitro micronucleus (MN) studies. Readers should obtain an understanding of specific cell culture practices, options for assay formatting and execution and the information required to successfully integrate and validate the in vitro MN assay into their existing safety program.

Toxicity assessment due to sub-chronic exposure to individual and mixtures of four toxic heavy metals.

Humans are exposed to a cocktail of heavy metal toxicants in the environment. Though heavy metals are deleterious, there is a paucity of information on toxicity of low dose mixtures. In this study, lead (Pb) (0.01mg/L), mercury (Hg) (0.001mg/L), cadmium (Cd) (0.005mg/L) and arsenic (As) (0.01mg/L) were administered individually and as mixtures to 10 groups of 40 three-week old mice (20 males and 20 females), for 120 days. The study established that low dose exposures induced toxicity to the brain, liver, and kidney of mice. Metal mixtures showed higher toxicities compared to individual metals, as exposure to low dose Pb+Hg+Cd reduced brain weight and induced structural lesions, such as neuronal degeneration in 30-days. Pb+Hg+Cd and Pb+Hg+As+Cd exposure induced hepatocellular injury to mice evidenced by decreased antioxidant activities with marginal increases in MDA. These were accentuated by increases in ALT, AST and ALP. Interactions in metal mixtures were basically synergistic in nature and exposure to Pb+Hg+As+Cd induced renal tubular necrosis in kidneys of mice. This study underlines the importance of elucidating the toxicity of low dose metal mixtures so as to protect public health.

Assessment of in vitro cellular responses of monocytes and keratinocytes to tannic acid modified silver nanoparticles.

Hydrolyzable tannins are known to exhibit diverse biological effects, which can be used in combination with silver nanoparticles (AgNPs). In this study, we tested toxic and inflammatory properties of tannic-acid modified 13, 33, 46 nm and unmodified 10-65 nm AgNPs using murine 291.03C keratinocyte and RAW 264.7 monocyte cell lines. Both cell lines exposed for 24h to 1-10 µg/ml of 13 nm, 33 nm, 46 nm and unmodified AgNPs showed dose-dependent toxicity and decreased cell proliferation. Only small-sized AgNPs induced production of ROS by monocytes, but not keratinocytes. Monocytes internalized large aggregates of 33, 46 nm and 10-65 nm AgNPs in cytoplasmic vacuoles, whereas keratinocytes accumulated less particles. AgNPs of 13 nm were localized ubiquitously within both cell types. The tested AgNPs strongly down-regulated production of tumor necrosis factor-α (TNF-α) by monocytes, whereas keratinocytes exposed to AgNPs showed an opposite effect. Unmodified but not tannic acid-modified AgNPs increased production of the pro-inflammatory MCP-1 by monocytes and keratinocytes. In summary, low inflammatory potential and lack of ROS production by tannic-acid modified AgNPs sized above 30 nm suggests that tannic acid modification of large silver nanoparticles may help to increase AgNPs biosafety.

In vivo activity assessment of a "honey-bee pollen mix" formulation.

Honey-bee pollen mix (HBM) formulation is claimed to be effective for the treatment of asthma, bronchitis, cancers, peptic ulcers, colitis, various types of infections including hepatitis B, and rheumatism by the herb dealers in northeast Turkey. In the present study, in vivo antinociceptive, anti-inflammatory, gastroprotective and antioxidant effects of pure honey and HBM formulation were evaluated comparatively. HBM did not show any significant gastroprotective activity in a single administration at 250 mg/kg dose, whereas a weak activity was observed after three days of successive administration at 500 mg/kg dose. On the other hand, HBM displayed significant antinociceptive (p <0.01) and anti-inflammatory (p <0.01) activities at 500 mg/kg dose orally without inducing any apparent acute toxicity or gastric damage. HBM was also shown to possess potent antilipidperoxidant activity (p <0.01) at 500 mg/kg dose against acetaminophen-induced liver necrosis model in mice. On the other hand, pure honey did not exert any remarkable antinociceptive, anti-inflammatory and gastroprotective activity, but a potent antilipidperoxidant activity (p <0.01) was determined. Results have clearly proved that mixing pure honey with bee pollen significantly increased the healing potential of honey and provided additional support for its traditional use. Total phenolic and flavonoid contents of HBM were found to be 145 and 59.3 mg/100 g of honey, which were estimated as gallic acid and quercetin equivalents, respectively.

Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds.

Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.

Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice.

The role of the cytokines TNF-alpha, IL-1beta and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice. Intramuscular injection of this venom induced increments in IL-1beta and IL-6 in muscle, but no elevations of TNF-alpha were detected. Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-alpha, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects. As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-alpha increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1beta and IL-6 in such endotoxic model. PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B. asper venom. Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage. It is concluded that TNF-alpha, IL-1beta and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B. asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.

Physiologically based pharmacokinetic/pharmacodynamic modeling of chemical mixtures and possible applications in risk assessment.

Human exposure to chemicals, be it environmental or occupational, is rarely, if ever, limited to a single chemical. Therefore, it is essential that we consider multiple chemical effects and interactions in our risk assessment process. However, with the almost infinitely large number of chemical mixtures in the environment, systematic studies of the toxicology of these chemical mixtures with conventional methodologies and approaches are impossible because of the immense resources and unrealistically long durations required. Thus, the development of predictive and alternative toxicology method is imperative. In order to have a reasonable chance to deal with the complex issue of toxicology of chemical mixtures, we believe that the following concepts must be considered: (1) the exploitation of recent advances in computational technology; (2) the utilization of mathematical/statistical modeling; (3) coupling computer modeling with very focused, mechanistically based, and short-term toxicology studies. Our approach is, therefore, the utilization of physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) modeling, coupled with very focused, model-directed toxicology experiments as well as other statistical/mathematical modeling such as isobolographic analysis and response surface methodology. Tissue dosimetry at the pharmacokinetic and pharmacodynamic levels is achievable with simple and complex, but chemically defined, mixtures. Our long-term goal is to formulate innovative risk assessment methodologies for chemical mixtures. In this presentation, one of our specific research projects is described: PB-PK/PD modeling of toxicologic interactions between Kepone and carbon tetrachloride (CCl4) and the coupling of Monte Carlo simulation for the prediction of acute toxicity.

Pathogenesis of diquat-induced liver necrosis in selenium-deficient rats: assessment of the roles of lipid peroxidation and selenoprotein P.

A dose of diquat below the amount injurious to selenium-replete animals causes lipid peroxidation and massive liver necrosis in selenium-deficient rats. The current study was undertaken to characterize the lipid peroxidation with respect to the liver injury and to correlate the presence of several selenoproteins with the protective effect of selenium. Lipid peroxidation was assessed by measurement of F2 isoprostanes. Diquat caused an increase in liver and plasma F2 isoprotanes. A gradient of these compounds was detected across the liver in some animals, indicating that this organ was a source of some of the plasma F2 isoprostanes. A time-course experiment showed that liver F2 isoprostane concentration increased before plasma alanine transaminase (ALT) levels rose. Selenium-deficient rats were injected with selenium doses from 2 to 50 micrograms/kg and studied 12 hours later. A dose of 10 micrograms/kg or more prevented diquat-induced lipid peroxidation and liver injury. This dose increased plasma selenoprotein P substantially, and a dose-response was present. Liver cellular and plasma glutathione peroxidase activities remained below 2% of their values in control rats for all selenium doses. In selenium-deficient rats given diquat, hepatic lipid peroxidation precedes hepatic necrosis and could therefore be an important mechanism of the necrosis. Selenoprotein P levels were increased by selenium injections, which protected against diquat injury, but glutathione peroxidase activity was not increased. This is consistent with selenoprotein P being the mediator of the selenium effect.

Classical syndromes in occupational medicine: Phosphorus necrosis--a classical occupational disease.

A disease nearly extinct in occupational health history is phosphorus necrosis, previously seen in near-epidemic proportions among workers making phosphorus-containing matches. Similar destructive lesions were encountered early in the 20th century among personnel fabricating fireworks. Through the diligent efforts of an economist and a supportive congressman, legislation was passed in 1912 placing a tax on phosphorus matches, and because of the fiscal burden resulting, a nontoxic substitute for elemental phosphorus was adopted by all manufacturers. Today phosphorus necrosis is extremely rare, but the former presence of the disease points up both apathy and courage in the identification and eradication of a remarkably disfiguring work-caused disease.

Why do people use paracetamol for suicide?

A questionnaire to assess motives for choosing paracetamol as a suicidal agent was completed by 107 patients admitted after an overdose of the drug. None of the 48 patients interviewed would have chosen paracetamol had they known that there would be an interval of two to three days before the onset of serious symptoms. Only five of the patients had obtained the drug on prescription, but the remainder had obtained it easily from a retail pharmacy. There was no apparent reason for the preference for paracetamol. It would be difficult to restrict the availability of paracetamol, and educating the public about the effects of an overdose would be more appropriate.