Amiloride Is Effective in the Management of Abiraterone-Induced Mineralocorticoid Excess Syndrome without Interfering with Its Antineoplastic Activity.

BACKGROUND: The administration of abiraterone acetate (abiraterone) leads to an adrenocorticotropic hormone (ACTH)-driven increase in mineralocorticoid hormones, requiring glucocorticoid supplementation that may stimulate the growth of prostate cancer (PCa). Amiloride is a drug that selectively reduces the aldosterone-sensitive Na+/K+ exchange and could be effective in the management of mineralocorticoid excess syndrome (MCES). METHODS: The efficacy of amiloride + hydrochlorothiazide (HCT) in the clinical management of abiraterone-induced MCES was assessed in 5 consecutive patients with castration-resistant PCa (CRPC). Then, using the in vitro experimental model of PCa cell lines, the possible effects of drugs usually used in the clinical management of CRPC patients on PCa cell viability were investigated. RESULTS: Amiloride/HCT led to a complete disappearance of all clinical and biochemical signs of abiraterone-induced MCES in the 5 treated patients. The in vitro study showed that abiraterone treatment significantly decreased cell viability of both androgen receptor (AR)-expressing VCaP (vertebral-cancer of the prostate) and LNCaP (lymph node carcinoma of the prostate) cells, with no effect on AR-negative PC-3 cells. Prednisolone, spironolactone, and eplerenone increased LNCaP cell viability, while amiloride reduced it. The non-steroid aldosterone antagonist PF-03882845 did not modify PCa cell viability. CONCLUSIONS: The combination of amiloride/HCT was effective in the management of abiraterone-induced MCES. Amiloride did not negatively interfere with the abiraterone inhibition of PCa cell viability in vitro.

REDUCTION OF VITREOUS PROSTAGLANDIN E2 LEVELS AFTER TOPICAL ADMINISTRATION OF INDOMETHACIN 0.5%, BROMFENAC 0.09%, AND NEPAFENAC 0.1.

PURPOSE: To assess vitreous concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) and prostaglandin E2 (PGE2) in patients treated with NSAIDs before vitrectomy for macular pucker. METHODS: A prospective, investigator-masked, randomized study was performed in 64 patients scheduled to undergo vitrectomy. The patients were randomized 1:1:1:1 to receive indomethacin 0.5%, bromfenac 0.09%, nepafenac 0.1%, or placebo three times a day. NSAIDs and PGE2 levels were evaluated in vitreous samples collected at the beginning of surgery. RESULTS: Mean (SD) vitreous concentrations of the study drugs were 503.13 (241.1) pg/mL for indomethacin, 302.5 (91.03) pg/mL for bromfenac, and 284.38 (128.2) pg/mL for nepafenac. Mean (SD) vitreous PGE2 levels were 247.9 (140.9) pg/mL for indomethacin, 322.12 (228.1) pg/mL for bromfenac, 448.8 (261.1) pg/mL for nepafenac, and 1,133 (323.9) pg/mL for placebo. All three NSAIDs reduced vitreous PGE2 levels to a statistically significant extent, without a significant difference among them. CONCLUSION: All assessed NSAIDs penetrated the vitreous and lowered basal PGE2 levels. A greater penetration was associated with pseudophakic eyes. The important inhibition of prostaglandins in the retina may have a clinical effect on the management of inflammatory retina diseases.

Are the NPS commonly used? An extensive investigation in Northern Italy based on hair analysis.

New psychoactive substances (NPS) are present on the Italian illicit markets, but data from the analysis of biological samples to evaluate their real consumption are rare. For this reason, an epidemiological study was carried out by means of a ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of 115 NPS on the keratin matrix. A total of 847 hair samples were collected in 2020 and 2021 and analyzed. The sample donors were in the age range of 18-40 years, from both genders, and were tested either for driving relicensing or for drug withdrawal monitoring. The UPLC-MS-MS system consisted of a Waters ACQUITY UPLC® I-Class, coupled with a Waters XEVO TQ-XS triple quadrupole mass spectrometer. The method was developed and fully validated according to international guidelines. Limits of detection were set as the minimum criterion to identify positive samples. Overall, 56 samples resulted positive for ketamine, 35 for norketamine, 6 for fentanyl, 3 for norfentanyl, 3 for 4-ANPP, 3 for MDMB-4en-PINACA, 2 for N,N-DMT, 2 for 5-chloro AB-PINACA, 1 for α-PHP and 1 for methcathinone. NPS were detected in a small part of samples (8.4%), which seems in contrast with their apparent wide diffusion in Italy, yet it is congruent with similar investigations based on hair analysis. Future studies will be performed to expand the investigated population, especially in terms of age and origin.

A fatal case of self-poisoning through the ingestion of oleander leaves.

BACKGROUND: Benefits and even dangers of plants are known since time began. The ancients used plants and herbs because of their effects on the human body. Poisoning is a logical consequence of their use: history is full of episodes of plants and herbs poisoning, whether intentional or accidental. AIM: Oleander poisoning is generally accidental; an intentional assumption of its leaves to commit suicide is uncommon because the population is not aware of the harmfulness of its cardiotoxic glycosides, therefore we report a fatal case of self-poisoning through the voluntary ingestion of oleander leaves. METHODS: A diagnosis of oleander self-poisoning was highly suspected on the basis of the circumstantial evidence and the autopsy findings. Toxicological investigations were performed on the samples collected during the autopsy and aimed at confirm the presence of oleandrin at a toxic level. RESULTS: The autopsy revealed a piece of oleander leaf on the posterior third of the tongue's body and several plant residues, similar to the one recovered on the tongue, into the gastric content; petechiae on the deep surface of the scalp, multi-organ congestion, and pulmonary edema were also observed. The histological study corroborated the pulmonary edema macroscopically observed but did not provide any other information. The detection of oleandrin in biological cadaveric samples revealed high, fatal, concentrations. CONCLUSIONS: Cases of voluntary ingestion of oleander with a suicidal intent prove to be uncommon: in the case reported the victim was aware about the possibility to commit suicide through the ingestion of oleander leaves.

Comparative assessment of intraocular inflammation following standard or heavy silicone oil tamponade: a prospective study.

PURPOSE: To evaluate the inflammation associated with the use of standard silicone oil (polydimethylsiloxane; PDMS) and heavy silicone oil (HSO) Densiron-68™ in patients undergoing vitrectomy for retinal detachment. MATERIALS AND METHODS: A prospective study was performed involving 35 patients scheduled to undergo vitrectomy for retinal detachment. Patients received PDMS or Densiron-68™ HSO according to superior or inferior retinal localization of the tears, respectively. For assessing the inflammation, prostaglandin E2 (PGE2 ) and interleukin-1α (IL-1α) levels were evaluated in the aqueous. RESULTS: Thirty-five eyes of 35 patients completed the study: 20 eyes received HSO, and 15 eyes received PDMS. The mean aqueous PGE2 level was significantly higher in HSO patients than in PDMS patients (869.16 ± 242.83 pg/ml versus 369.38 ± 209.7 pg/ml, respectively; p < 0.0001). The mean aqueous IL-1α level was also significantly higher in HSO patients than in PDMS patients (81.40 ± 36.9 pg/ml versus 40.8 ± 32.5 pg/ml, respectively; p = 0.002). In HSO, a moderate positive correlation between the endotamponade duration and both PGE2 (r = 0.44; p = 0.05) and IL-1α (r = 0.48; p = 0.033) levels was observed. In PDMS, a strong positive correlation between the endotamponade duration and both PGE2 (r = 0.89; p < 0.0001) and IL-1α (r = 0.68; p = 0.006) levels was observed. CONCLUSION: Although both HSO and PDMS yielded favourable success rates in the surgical treatment of complicated retinal detachments, HSO triggered a more severe inflammatory reaction, in a time-dependent manner.

Abiraterone acetate exerts a cytotoxic effect in human prostate cancer cell lines.

To study the capability of the CYP17A1 inhibitor abiraterone acetate (AER) to antagonize the androgen receptor (AR) activation in human prostate cancer (PCa) cell lines. T877A-AR-LNCaP, WT-AR-VCaP, AR-negative DU145, and PC3 PCa cell lines were used by MTT and cell count to study the ability of AER and enzalutamide (ENZ) to modify cell viability. The role of ARs in LNCaP was demonstrated through a gene-silencing experiment. The mechanism of AER cytotoxicity in LNCaP cells was studied, as well as the ability of AER to modulate AR gene expression. The in silico docking approach was applied to study the interaction of AER and ENZ with T877A-AR. Through high-performance liquid chromatography, the production of the AER main metabolite Δ4A was studied. AER bound AR in an almost identical manner to that of dihydrotestosterone (DHT). The higher binding energy for AER in T877A-AR could explain the major cytotoxic effect observed in LNCaP cells. The capability of LNCaP cells to synthesize Δ4A could mediate, at least in part, this effect. AER cytotoxicity in LNCaP cells was mainly due to the activation of apoptosis. Further, AER induced modification of AR target gene expression, suggesting a direct effect on AR activity. AER-induced cytotoxicity on PCa cell lines seemed to be mediated by binding with AR. The higher affinity of AER for T877A-AR may suggest a potential role of AER in the management of CRPC carrying this mutation; however, T877A-AR expressing CRPC patients developed AER resistance, probably due to the increase of progesterone.

In vitro antitumor activity of progesterone in human adrenocortical carcinoma.

PURPOSE: The management of patients with adrenocortical carcinoma (ACC) is challenging. As mitotane and chemotherapy show limited efficacy, there is an urgent need to develop therapeutic approaches. The aim of this study was to investigate the antitumor activity of progesterone and explore the molecular mechanisms underlying its cytotoxic effects in the NCI-H295R cell line and primary cell cultures derived from ACC patients. METHODS: Cell viability, cell cycle, and apoptosis were analyzed in untreated and progesterone-treated ACC cells. The ability of progesterone to affect the Wnt/ß-catenin pathway in NCI-H295R cells was investigated by immunofluorescence. Progesterone and mitotane combination experiments were also performed to evaluate their interaction on NCI-H295R cell viability. RESULTS: We demonstrated that progesterone exerted a concentration-dependent inhibition of ACC cell viability. Apoptosis was the main mechanism, as demonstrated by a significant increase of apoptosis and cleaved-Caspase-3 levels. Reduction of ß-catenin nuclear translocation may contribute to the progesterone cytotoxic effect. The progesterone antineoplastic activity was synergically increased when mitotane was added to the cell culture medium. CONCLUSIONS: Our results show that progesterone has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of progesterone with mitotane provides the rationale for testing this combination in a clinical study.

In vitro cytotoxicity of cabazitaxel in adrenocortical carcinoma cell lines and human adrenocortical carcinoma primary cell cultures☆.

Adrenocortical cancer (ACC) is a rare and aggressive malignancy with a poor prognosis. The overall 5-year survival rate of patients with ENS@T stage IV ACC is less than 15%. Systemic antineoplastic therapies have a limited efficacy and new drugs are urgently needed. Human ACC primary cultures and cell lines were used to assess the cytotoxic effect of cabazitaxel, and the role of P-glycoprotein in mediating this effect. Cabazitaxel reduced ACC cell viability, both in ACC cell lines and in ACC primary cell cultures. Molecular and pharmacological targeting of ABCB1/P-gp did not modify its cytotoxic effect in NCI-H295R cells, while it increased the paclitaxel-induced toxicity. Cabazitaxel modified the expression of proteins involved in cellular physiology, such as apoptosis and cell cycle regulation. The drug combination cabazitaxel/mitotane exerted an additive/moderate synergism in different ACC cell experimental models. These results provide a rationale for testing cabazitaxel in a clinical study.

Death after 25C-NBOMe and 25H-NBOMe consumption.

A teenager male was found dead in a waterway after he was spotted jumping off into the water stream. The boy looked agitated and confused after a party with friends. At the gathering place, investigators seized packages of blotter papers. A complete autopsy and a histological evaluation of the main tissues were performed; although the death occurred by drowning, the prosecutor requested toxicological exams, in order to evaluate the potential role of drugs of abuse in the episode. Blood (both peripheral and central) and urine samples as well as seized blotter papers were collected and analyzed as follows. The blotter paper, analyzed through a GC-MS method, revealed the presence of 25-NBOMes. A liquid chromatography tandem mass spectrometric (LC-MS/MS) system was used to identify and quantify 5 different 25-NBOMes (namely 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe) in blood and urine. 25E-NBOMe was used as internal standard (IS). 1mL of urine and 1mL of blood (both peripheral and cardiac) were diluted in 2mL phosphate buffer at pH 6.0, containing IS and purified on a solid phase extraction (SPE) cartridge. LOD and LOQ for the five 25-NBOMes were calculated at 0.05 and 0.1ng/mL respectively. Linearity, accuracy, precision, ion suppression, carry over and recovery were tested and all parameters fulfilled the acceptance criteria. Blood and urine provided positive results for 25C-NBOMe and 25H-NBOMe. Eventually, the seized blotter papers were analyzed by means of LC-MS/MS and the presence of the two NBOMes was confirmed: 25C-NBOMe and 25H-NBOMe were measured at the concentration of 2.80 and 0.29ng/mL in peripheral blood, of 1.43 and 0.13ng/mL in central blood and of 0.94 and 0.14ng/mL in urine, respectively. THC and THCCOOH were also detected in biological fluids, at the concentration of 15.5 and 56.0ng/mL in peripheral blood, 9.9 and 8.5ng/mL in central blood, respectively. NBOMes can produce severe hallucination even at very low doses, and the 25C-NBOMe levels measured in the subject's blood are considered potentially toxic.

Down-regulation of coasy, the gene associated with NBIA-VI, reduces Bmp signaling, perturbs dorso-ventral patterning and alters neuronal development in zebrafish.

Mutations in Pantothenate kinase 2 and Coenzyme A (CoA) synthase (COASY), genes involved in CoA biosynthesis, are associated with rare neurodegenerative disorders with brain iron accumulation. We showed that zebrafish pank2 gene plays an essential role in brain and vasculature development. Now we extended our study to coasy. The gene has high level of sequence identity with the human ortholog and is ubiquitously expressed from the earliest stages of development. The abrogation of its expression led to strong reduction of CoA content, high lethality and a phenotype resembling to that of dorsalized mutants. Lower doses of morpholino resulted in a milder phenotype, with evident perturbation in neurogenesis and formation of vascular arborization; the dorso-ventral patterning was severely affected, the expression of bone morphogenetic protein (Bmp) receptors and activity were decreased, while cell death increased. These features specifically correlated with the block in CoA biosynthesis and were rescued by the addition of CoA to fish water and the overexpression of the human wild-type, but not mutant gene. These results confirm the absolute requirement for adequate levels of CoA for proper neural and vascular development in zebrafish and point to the Bmp pathway as a possible molecular connection underlining the observed phenotype.

Antisecretive and Antitumor Activity of Abiraterone Acetate in Human Adrenocortical Cancer: A Preclinical Study.

CONTEXT: Patients with adrenocortical carcinoma (ACC) frequently suffer from cortisol excess, which portends a negative prognosis. Rapid control of cortisol hypersecretion and tumor growth are the main goals of ACC therapy. Abiraterone acetate (AA) is a potent inhibitor of 17alpha-hydroxylase/17,20-lyase, a key enzyme of adrenal steroidogenesis. OBJECTIVE: This study sought to investigate the therapeutic use of AA in preclinical models of ACC. DESIGN: AA antisecretive and antiproliferative effects were investigated in vitro using NCI-H295R and SW13 ACC cell lines and human primary ACC cell cultures, as well as in vivo using immunodeficient mice. METHODS: Steroid secretion, cell viability, and proliferation were analyzed in untreated and AA-treated ACC cells. The ability of AA to affect the Wnt/beta-catenin pathway in NCI-H295R cells was also analyzed. Progesterone receptor (PgR) gene was silenced by the RNA interference approach. The antitumor efficacy of AA was confirmed in vivo in NCI-H295R cells xenografted in immunodeficient mice. RESULTS: AA reduced the secretion of both cortisol and androgens, increased production of progesterone, and induced a concentration-dependent decrease of cell viability in the NCI-H295R cells and primary secreting ACC cultures. AA also reduced beta-catenin nuclear accumulation in NCI-H295R cells. AA administration to NCI-H295R-bearing mice enhanced progesterone levels and inhibited tumor growth. The cytotoxic effect of AA was prevented by either blocking PgR or by gene silencing. CONCLUSION: AA is able to inhibit hormone secretion and growth of ACC both in vitro and in vivo. It also reduces beta-catenin nuclear accumulation. The cytotoxic effect of AA seems to require PgR.

Ethyl glucuronide in vitreous humor and blood postmortem specimens: analysis by liquid chromatography-electrospray tandem mass spectrometry and interpreting results of neo-formation of ethanol.

INTRODUCTION: The determination of ethyl glucuronide (EtG), a stable and sensitive marker that is specific to alcohol intake, finds many applications both in the forensic toxicology and clinical fields. AIM: The aim of the study is to examine the possibility of using a cadaveric biological matrix, vitreous humor (VH), to determine EtG as a marker of recent ethanol use. METHODS: The blood, taken from the femoral vein, and the VH were obtained from 63 autopsy cases. Analysis of the EtG was performed using an LC/MS/MS system. Analyses of the ethanol and putrefaction biomarkers, such as acetaldehyde and n-propanol, were performed using the HS-GC/FID technique in both the matrices. RESULTS: In 17 cases, both ethanol and EtG were absent in both matrices.Nineteen cases presented ethanol in blood from 0.05 to 0.30 g/L, EtG-Blood concentration from 0.02 to 3.27 mg/L, and EtG-VH concentration from 0.01 mg/L to 2.88 mg/L. Thirteen cases presented ethanol in blood > 0.05 g/L but EtG concentration in blood and VH lower than 0.01 mg/L, are part of these 8 samples presented acetic aldehyde and n- propanol in blood or VH, means identification of putrefaction indicators. Fourteen cases presented ethanol in blood > 0.46 and EtG concentration in blood and VH higher than 0.01 mg/L. CONCLUSIONS: The determination of EtG in biological material is important in those cases where the intake of ethanol appears doubtful, as it allows us to exclude the possibility of any post-mortem formation of ethanol.

Efficacy and vitreous levels of topical NSAIDs.

INTRODUCTION: Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed medications and are routinely used for their analgesic, antipyretic, and anti-inflammatory properties. Because of their potent cyclooxygenase-inhibitory activity, they can inhibit pro-inflammatory prostaglandin synthesis, leading to complex inflammatory cascades. NSAIDs have been broadly used systemically for many decades and have recently become commercially available in the form of topical ophthalmic formulations. NSAIDs are weak acids with pKa values mostly between 3.5 and 4.5 and are poorly water-soluble. New, aqueous ophthalmic solutions of NSAIDs that afford better tissue penetration have recently been developed. In ophthalmological practice, topical NSAIDs are mostly used to stabilize pupillary dilation during intraocular surgery, manage postoperative pain and inflammation, and treat pseudophakic cystoid macular edema. AREAS COVERED: This review focuses on the vitreous penetration of topical NSAIDs and their potential clinical applications in the treatment of retinal diseases. EXPERT OPINION: A growing body of evidence suggests that NSAIDs may be beneficial in the treatment of age-related macular degeneration, diabetic retinopathy, and ocular tumors. Recent studies from our group and other authors have shown that the vitreous levels of NSAID exceed the median inhibitory concentration, which can significantly decrease vitreous PGE2 levels.

An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.

BACKGROUND: Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses. OBJECTIVE: In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed. METHODS: Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos. RESULTS: Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view. CONCLUSION: These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed.