A novel electrochemiluminescent immunoassay for diclofenac using conductive polymer functionalized graphene oxide as labels and gold nanorods as signal enhancers.

Diclofenac (DCF) is among the pharmaceutical compounds of concern based on its nocuousness in environment. In this work, a novel electrochemiluminescent (ECL) immunosensor for detecting DCF was constructed using poly(etherimide)-poly(3,4-ethylene dioxythiophene): poly(styrene sulfonate) functionalized graphene oxide and CdSe@CdS quantum dots (QDs-PEI-GO/PEDOT) as bioreceptor for conjugating DCF antibody to magnify signal. It is worth noting that this is the first time GO/PEDOT has been applied to ECL sensor field. Compared with GO, GO/PEDOT exhibited a higher conductivity and more stable chemical property, indicating that the proposed immunosensor would possess stronger and more stable luminescence performance. In addition, the electrode was modified with gold nanorods (AuNRs) which increase the load capacity of DCF coating antigen through Au-N bond. Competitive immunoassay method was chosen for structuring immunosensor where polyclonal antibody (pAb) against DCF exhibited high-affinity recognition of DCF, which greatly improved the sensitively and selectivity of sensor. Consequently, the proposed immunosensor gratified in DCF detection with low detection limit (LODs) of 0.33 pg mL-1 (S/N = 3), and displayed high stability and sensitivity, which initiated a new route for DCF determining.

Anaphylaxis to diclofenac: nine cases reported to the Allergy Vigilance Network in France.

Nine cases of diclofenac hypersensitivity recorded by the Allergy Vigilance Network in France from 2002 to 2012 were studied. Data from history, symptoms, skin tests, basophil activation tests, and oral challenge (OC) were recorded. Grade 3 severe anaphylactic reactions occurred in seven cases of nine. IgE-dependent anaphylaxis was confirmed in six cases: positive intradermal tests (n = 4), a syndromic reaction during skin tests (n = 1), and one case with grade 1 reaction and negative skin tests had an anaphylactic shock to the OC. A nonimmune reaction was suspected in one case. An IgE-dependent mechanism may be the predominant cause of adverse reactions to diclofenac. Allergy skin tests must be carried out sequentially at the recommended concentrations. BATs may be helpful because they can support the diagnosis of anaphylaxis. Given the risks of a direct challenge to diclofenac, OC to aspirin should be performed first to exclude a nonimmunologic hypersensitivity to NSAIDs. Tests for specific IgEs to most frequently used NSAIDs such as diclofenac and ibuprofen are urgently needed.

Application of a heterogeneous immunoassay for the quality control testing of release-active forms of diclofenac.

We report on a specially designed diclofenac-ELISA for the determination of diclofenac in the presence of release-active forms of diclofenac in lactose dissolved in water solutions according to a predefined schedule in single-blind experiments. In accordance with the objective of this project, a number of experiments were conducted to determine the optimal ELISA conditions for detecting potential modulatory effects of release-active forms of diclofenac depending on their ability to affect the binding of diclofenac to anti-diclofenac antibodies. As a feature, the diclofenac antibodies were previously incubated with manufactured pharmaceutical samples containing release-active forms of diclofenac or placebo. For comparison of the sample types, measured in ELISA optical densities were chosen. For statistic analysis, Student's two-sample t-test and single-factor ANOVA were applied. The extremely low concentrations of diclofenac of 0.01, 0.05 and 0.1 ng mL(-1) seem most appropriate for routine assay performance. The source of diclofenac used for standard solution preparation is not important but it could be important as the source of diclofenac for release active form of diclofenac preparation. As an outcome, the ELISA appeared to be suitable for the detection of the modifying effects of release-active forms of diclofenac toward the pharmaceutical substance in vitro.

Basophil activation test for investigation of IgE-mediated mechanisms in drug hypersensitivity.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions (1). In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient (2). Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry (3). In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly (4), whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study (1). The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree. As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Suppression of gastric acid increases the risk of developing immunoglobulin E-mediated drug hypersensitivity: human diclofenac sensitization and a murine sensitization model.

BACKGROUND: Hypersensitivity reactions towards non-steroidal anti-inflammatory drugs (NSAID) are common, although true allergies are detectable only in a subgroup of patients. The current study was prompted by a case observation, where a patient experienced generalized urticaria following his second course of diclofenac and proton pump inhibitor medication, and was found to have diclofenac-specific IgE. During recent years, our group has been investigating the importance of gastric digestion in the development of food allergies, demonstrating anti-acid medication as a risk factor for sensitization against food proteins. OBJECTIVE: Here, we aimed to investigate whether the mechanism of food allergy induction described can also be causative in NSAID allergy, using diclofenac as a paradigm. METHODS: We subjected BALB/c mice to several oral immunization regimens modelled after the patient's medication intake. Diclofenac was applied with or without gastric acid suppression, in various doses, alone or covalently coupled to albumin, a protein abundant in gastric juices. Immune responses were assessed on the antibody level, and functionally examined by in vitro and in vivo crosslinking assays. RESULTS: Only mice receiving albumin-coupled diclofenac under gastric acid suppression developed anti-diclofenac IgG1 and IgE, whereas no immune responses were induced by the drug alone or without gastric acid suppression. Antibody induction was dose dependent with the group receiving the higher dose of the drug showing sustained anti-diclofenac titres. The antibodies induced triggered basophil degranulation in vitro and positive skin tests in vivo. CONCLUSION: Gastric acid suppression was found to be a causative mechanism in the induction of IgE-mediated diclofenac allergy.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

NSAIDS-induced anaphylaxis precipitating acute coronary vasospasm.

We report a case of NSAID-induced anaphylaxis precipitating coronary artery vasospasm leading to myocardial infarction in the presence of normal coronaries. Furthermore, we have also discussed the possible mechanisms for this. Attending clinicians should be aware of this potential complication when treating anaphylaxis, especially in patients with known or presumed coronary artery disease.

The effects of non-steroidal anti-inflammatory drug application on incisional wound healing in rats.

OBJECTIVE: There is evidence that non-steroidal anti-inflammatory drugs (NSAIDs) delay both epithelialisation and angiogenesis in the early phases of wound healing because of an antiproliferative effect. We investigated the influence of diclofenac, a non-selective NSAID, on incisional wound healing. METHOD: Ten male Wistar rats were given 5 mg diclofenac per kg bodyweight per day; 10 rats were given placebo pellets. After 10 days, unimpaired healing occurred independently of drug treatment both macroscopically and microscopically. Histomorphometry revealed a significant reduction (p = 0.006) in fibroblasts after diclofenac application (median 3 166 cells per mm2) compared with the placebo group (median 3940 cells per mm2). Epidermal thickness was not statistically different between the two groups. RESULTS: Diclofenac diminished the amount of fibroblasts in connective tissue, reflecting the known antiproliferative effect of NSAIDs on fibroblasts. Clinical healing was not affected. CONCLUSION: We recommend short-term diclofenac application for post-surgical and post-traumatic patients with wounds who would benefit from its antiphlogistic and analgesic effect. However, if wound healing is disturbed, the negative effect of diclofenac on fibroblasts should be considered. This is particularly relevant for patients with chronic wounds or conditions such as diabetes which can delay wound healing.

Effect of prenatal administration of NSAIDs on the immune response in juvenile and adult rats.

In order to investigate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the development of rat immunity, indomethacin (IND; 0.25, 0.5, or 1.0 mg/kg/day), acetyl salicylic acid (ASA; 90, 180, or 360 mg/kg/day), or diclofenac sodium salt (DSS; 0.5, 1.0, or 2.0 mg/kg/day) suspended in 0.5% methylcellulose aqueous solution, was orally administered once daily to five pregnant Sprague-Dawley (IGS) rats per group on days 18-21 of gestation. After parturition, the serum IgM and IgG levels, the spleen weight, and the number of spleen cells were measured in 3- and 8-week-old pups. Afterwards, immunophenotyping analysis of splenocytes or peripheral blood lymphocytes and T-dependent antibody response were performed. The number of spleen cells in 3-week-olds increased when 1.0 mg/kg of IND and 180 mg/kg of ASA were administered. Immunophenotyping analysis using flow cytometry (FCM) indicated that the proportion and number of CD45RA(+) cells increased, and the proportion of CD3(-) NKR-P1A(+) cells decreased in males when dosed with IND at 1.0 mg/kg or ASA at 180 mg/kg. The serum anti-KLH IgG antibody titer decreased in the males of the IND 1.0 mg/kg dosing group, the serum levels of anti-KLH IgM, total IgM, and IgG were not changed at all. These changes disappeared in 8-week-old pups. There were no effects on any of the parameters in the 3- and 8-week-olds of the DSS treatment group. These results suggest that IND or ASA administration to dams during late gestation either causes a change in the lymphocyte subsets, or that they suppress the T-dependent antibody response in juvenile males. Both of these changes eventually recover to intact levels later on during development. These results will contribute to the development of a technique for the assessment of developmental immunotoxicity and generate data on the effect of prenatal administration of NSAIDs on the developmental immune system in pups.

Misdiagnosis in patients with diclofenac-induced hemolysis: new cases and a concise review.

Diclofenac has been implicated in many cases of life-threatening immune hemolytic anemia (IHA). Nevertheless, confusion still occurs at the bedside and in the laboratory. Herein we report nine new patients and summarize all published cases (total, n = 61). Direct and indirect antiglobulin tests were performed according to standard procedures. Tests for drug-dependent antibodies were performed in the presence and absence of the target drugs and their ex vivo antigens (in the urine of patients treated with the drug). Diclofenac- and/or ex-vivo-antigen-dependent red cell antibodies were detected in all new cases. We identified nine new cases with diclofenac-dependent IHA. All cases were initially suspected to have an abdominal illness and/or autoimmune hemolytic anemia of warm type. Acute renal failure was present in three of the 9 new patients and in 20 of 37 published patients. Seven of the 46 patients died (15%), and clinical information is lacking in 15 other published cases. Diclofenac-dependent and/or ex-vivo-antigen-dependent IHA should always be considered when a patient on diclofenac develops acute renal failure and/or IHA.

Drug-induced anaphylaxis : case/non-case study based on an italian pharmacovigilance database.

OBJECTIVE: To identify the number of cases of anaphylaxis reported in association with different classes of drugs and compare it with other reports contained in the same database. METHODS: The data were obtained from a database containing all of the spontaneous reports of adverse drug reactions (ADRs) coming from the Italian regions of Emilia Romagna, Lombardy and the Veneto, which are the main contributors to the Italian spontaneous surveillance system. The ADRs reported between January 1990 and December 2003 with a causality assessment of certainly, probably or possibly drug related (according to the WHO criteria) were analysed using a case/non-case design. The cases were defined as the reactions already coded by the WHO preferred terms of 'anaphylactic shock' or 'anaphylactoid reaction' (this last term also included anaphylactic reaction) and those with a time of event onset that suggested an allergic reaction and involved at least two of the skin, respiratory, gastrointestinal, CNS or cardiovascular systems; the non-cases were all of the other ADR reports. The frequency of the association between anaphylaxis and the suspected drug in comparison with the frequency of anaphylaxis associated to all of the other drugs was calculated using the ADR reporting odds ratio (ROR) as a measure of disproportionality. RESULTS: Our database contained 744 cases (including 307 cases of anaphylactic shock with 10 deaths) and 27 512 non-cases. The percentage of anaphylaxis cases reported in inpatients was higher than that among outpatients (59.1% vs 40.9%). This distribution is significantly different from that of the other ADR reports that mainly refer to outpatients. After intravenous drug administrations, anaphylactic shock cases were more frequent than anaphylactoid reactions or other ADRs, but more than one-third of these reactions were caused by an oral drug. Blood substitutes and radiology contrast agents had the highest RORs. Among the systemic antibacterial agents, anaphylaxis was disproportionally reported more often for penicillins, quinolones, cephalosporins and glycopeptides, but diclofenac was the only NSAID with a significant ROR. As a category, vaccines had a significantly lower ROR, thus indicating that anaphylaxis is reported proportionally less than other ADRs. CONCLUSIONS: Anaphylaxis is a severe ADR that may also occur with commonly used drugs. It represents 2.7% of all of the ADRs reported in an Italian spontaneous reporting database.

Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity.

The advances in the drug development that allowed the replacement of many potentially hepatotoxic agents by safer alternatives have been out-weighed by the vast expansion of the total number of agents now available for use. Now, rare adverse reactions to several commonly prescribed medications contribute to the total burden of drug-induced liver injury. Studies involving well-characterised patients with diclofenac-induced hepatotoxicity indicate that multiple steps are involved in the development of liver injury. Individual susceptibility to idiosyncratic hepatotoxicity is determined by the interaction of metabolic and immunological factors. Immunomodulatory and anti-inflammatory cytokines, such as IL-10, may have a protective role in reducing drug-induced liver injury. Understanding the mechanisms of idiosyncratic hepatotoxicity may increase our ability to identify susceptible individuals and hence, prevent serious adverse reactions.

The flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome.

BACKGROUND: Hypersensitivity reactions to non-steroidal anti-inflammatory drugs (NSAIDs), manifested by cutaneous symptoms and/or airway manifestations represent 20-25% of all hypersensitivity reactions to drugs. Today, it is still claimed that no in vitro diagnostic tests exist for that condition and that the only way to confirm the diagnosis is a provocation challenge. OBJECTIVE: The objective of this study was to assess whether NSAIDs may provoke blood basophil activation in vitro in such patients, as detected by a flowcytometric technique. METHODS: Sixty NSAID hypersensitive patients (38 with cutaneous, 20 with airway and two with cutaneous and airway symptoms) and 30 control patients (15 asthmatics) were selected. Their hypersensitivity was confirmed by documented history indicating at least two clinical episodes to two or more different NSAIDs or by positive oral provocation challenge. Isolated buffy coat leukocytes were stimulated in vitro with aspirin, paracetamol, metamizol, diclofenac, and naproxen. The percentage of activated basophils was evaluated by an anti-CD63. RESULTS: Aspirin showed a sensitivity of 43.3%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 99.4%. For the other NSAIDs, the sensitivity and specificity values were: for paracetamol 11.7% and 100%, for metamizol 15% and 100%, for diclofenac 43.3% and 93.3% and for naproxen 54.8% and 74.1%. When considering the first four NSAIDs, the global sensitivity raised to 63.3% and specificity to 93.3%. If the number of tests is to be limited for practical reasons, the combination of acetylsalicylic acid and diclofenac at two concentrations yields a sensitivity of 58.3% and a specificity of 93.3%. CONCLUSIONS: Flowcytometric determinations of basophil activation following stimulation with NSAIDs show a high sensitivity (60-70%) with specificity above 90%. So this test may help avoiding some cumbersome and dangerous provocation challenges.

Effects of post-operative pain treatment using non-steroidal anti-inflammatory analgesics, opioids or epidural blockade on systemic and local immune responses in children.

BACKGROUND: Many studies have been carried out on the effects of anaesthetic drugs and methods on the immune response, but pain and its relief also affect the immune response. We measured systemic immune responses in the blood circulation and local responses in the surgical wound when non-steroidal anti-inflammatory analgesics (NSAIDs), opioids or epidural blockade was used in the peri-operative treatment of pain. METHODS: Responses were measured in 51 children, aged from 2 to 12 years and undergoing major surgery under balanced anaesthesia. Bolus doses of diclofenac intravenously (i.v.) and rectally (NSAID group), continuous i.v. infusion of oxycodone (opioid group) or continuous epidural infusion of bupivacaine + fentanyl (epidural group) were used peri-operatively for pain relief. RESULTS: The only difference related to the analgesic method was shorter duration of post-operative leucocytosis and lower phytohaemagglutinin (PHA)-induced lymphocyte proliferative responses in peripheral blood in the opioid group than in the NSAID or epidural groups. By contrast, time-related alterations were seen overall in leucocyte and differential counts, lymphocyte and their subset counts, lymphocyte proliferative responses, and in serum cortisol, C-reactive protein, plasma interleukin-6 and group II phospholipase A2 concentrations and in the appearance of different cell types in the wound. CONCLUSIONS: Post-operative pain treatments using diclofenac (NSAID), oxycodone (opioid) and epidural blockade have basically similar effects on systemic and local immune responses with only slight, probably clinically unimportant differences in children undergoing surgery under general anaesthesia.

Massive haemolysis after intramuscular diclofenac in a patient who apparently tolerated oral medication.

BACKGROUND AND OBJECTIVES: Administration of diclofenac may lead to immune haemolytic anaemia (IHA) owing to the presence of drug-dependent antibodies and/or autoantibodies. A relationship with oral or intramuscular drug administration is unknown. Here, we describe a patient who apparently tolerated oral diclofenac but developed severe IHA following intramuscular injection of the drug. PATIENTS AND METHODS: A 66-year-old-female was admitted to hospital because of jaundice and nausea, which were initially presumed to be manifestations of a postcholecystectomy syndrome. The patient soon developed haemolysis and renal failure. Although the symptoms and signs were suggestive of autoimmune haemolytic anaemia (AIHA), the patient had diclofenac-induced IHA. RESULTS: Serological testing, including detection of drug-dependent antibodies, was performed using standard techniques. The patient's serum was found to contain a highly reactive diclofenac-dependent red cell antibody of the immune complex type (titre 256 000). She recovered after 7 weeks of treatment with prednisolone, blood transfusions, haemodialysis and plasma exchange. CONCLUSIONS: Diclofenac-induced IHA should always be considered when a patient on diclofenac develops haemolysis.

Investigating the TNP-OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans.

Using current animal models, it is not possible to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with anaphylaxis. It is generally accepted that the ultimate effector mechanism involves drug-induced IgE antibody. The objective of the present study was to determine if diclofenac, zomepirac and glafenine, which are associated with anaphylaxis in humans, have immunostimulating potential in the murine TNP-OVA (trinitrophenyl-ovalbumin) popliteal lymph node assay (PLNA), and more specifically to determine if the immunostimulation caused by these LMWCs results in IgE antibody production. These LMWCs were chosen because both zomepirac and glafenine were removed from the market due to high association with anaphylaxis, and diclofenac, which remains on the market, is frequently associated with anaphylaxis. In addition to conducting a TNP-OVA PLNA, the immunostimulating potential of these compounds was examined in the direct PLNA. When co-administered with TNP-OVA, all three LMWCs caused dose-dependent (0.25, 0.50, 1.00 and 1.25 mg) increases in popliteal lymph node (PLN) weight and cellularity that were observed beginning with the 0.25-mg dose. In addition, beginning with the 0.25-mg dose, all three compounds caused dose-dependent increases in TNP-OVA specific IgM and IgG(1) antibody-forming cells (AFCs). Diclofenac induced an isotype switch and caused a dose-dependent increase in the number of IgE AFCs with no detectable IgG(2a) AFCs and minimal high-dose-only IgG(2b) AFCs. Zomepirac induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50 mg only, and glafenine induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50-1.00 mg. In the direct PLNA, diclofenac caused dose-dependent increases in PLN weight and cellularity that were observed beginning with dose of 0.50 mg, whereas zomepirac failed to increase any PLN parameter and glafenine only increased the PLN weight. These results suggest that diclofenac, zomepirac and glafenine are immunostimulating LMWCs in the TNP-OVA PLNA with the potential to induce IgE antibody against a co-administered hapten-conjugate. Furthermore, these results suggest that the TNP-OVA PLNA offered significant advantages over the direct PLNA. Although it is not realistic to suggest that a single assay, based on a low number of test compounds, can identify all LMWCs with the potential to cause anaphylaxis in humans, these observations do demonstrate the potential utility of the PLNAs in examining LMWC-induced immunomodulation and support further development and investigation of the assays.

Fatal acute diclofenac-induced immune hemolytic anemia.

We report the case of a 72-year-old woman who developed fatal immune hemolytic anemia with multisystem organ failure and shock caused by diclofenac-dependent red blood cell autoantibodies. The patient described dramatically illustrates the potential severity of this adverse reaction and emphasizes the need for increased awareness of this complication of drug therapy.