Corema album Leaves Mediate DNA Damage in Triple-Negative Breast Cancer Cells.

Corema (C.) album is a shrub endemic to the Atlantic coast and has been described as yielding beneficial effects for human health. Nevertheless, studies concerning the bioactivity of C. album leaves are scarce. This study aims at investigating the anticancer potential and mode of action, of an hydroethanolic extract of C. album leaves (ECAL) on triple-negative breast cancer. This is a poor survival breast cancer subtype, owing to its high risk of distant reappearance, metastasis rates and the probability of relapse. The ECAL ability to prevent tumor progression through (i) the inhibition of cell proliferation (cell viability); (ii) the induction of apoptosis (morphological changes, TUNEL assay, caspase-3 cleaved) and (iii) the induction of DNA damage (PARP1 and γH2AX) with (iv) the involvement of NF-κB and of ERK1/2 pathways (AlphaScreen assay) was evaluated. ECAL activated the apoptotic pathway (through caspase-3) along with the inhibition of ERK and NF-κB pathways causing DNA damage and cell death. The large polyphenolic content of ECAL was presumed to be accountable for these effects. The extract of C. album leaves can target multiple pathways and, thus, can block more than one possible means of disease progression, evidencing the anticancer therapeutic potential from a plant source.

Effect of Pd2Spermine on Mice Brain-Liver Axis Metabolism Assessed by NMR Metabolomics.

Cisplatin (cDDP)-based chemotherapy is often limited by severe deleterious effects (nephrotoxicity, hepatotoxicity and neurotoxicity). The polynuclear palladium(II) compound Pd2Spermine (Pd2Spm) has emerged as a potential alternative drug, with favorable pharmacokinetic/pharmacodynamic properties. This paper reports on a Nuclear Magnetic Resonance metabolomics study to (i) characterize the response of mice brain and liver to Pd2Spm, compared to cDDP, and (ii) correlate brain-liver metabolic variations. Multivariate and correlation analysis of the spectra of polar and lipophilic brain and liver extracts from an MDA-MB-231 cell-derived mouse model revealed a stronger impact of Pd2Spm on brain metabolome, compared to cDDP. This was expressed by changes in amino acids, inosine, cholate, pantothenate, fatty acids, phospholipids, among other compounds. Liver was less affected than brain, with cDDP inducing more metabolite changes. Results suggest that neither drug induces neuronal damage or inflammation, and that Pd2Spm seems to lead to enhanced brain anti-inflammatory and antioxidant mechanisms, regulation of brain bioactive metabolite pools and adaptability of cell membrane characteristics. The cDDP appears to induce higher extension of liver damage and an enhanced need for liver regeneration processes. This work demonstrates the usefulness of untargeted metabolomics in evaluating drug impact on multiple organs, while confirming Pd2Spm as a promising replacement of cDDP.

Sulfated Oligomers of Tyrosol: Toward a New Class of Bioinspired Nonsaccharidic Anticoagulants.

Sulfated phenolic polymers have extensively been investigated as anticoagulant agents in view of their higher bioavailability and resistance to degradation compared to heparins, allowing for increased half-lives. In this frame, we report herein the preparation of sulfated derivatives of tyrosol, one of the most representative phenolic constituents of extra virgin olive oil, by different approaches. Mild sulfation of OligoTyr, a mixture of tyrosol oligomers, that has been reported to possess antioxidant properties and osteogenic activity, afforded OligoTyrS I in good yields. Elemental analysis, NMR, and MALDI-MS investigation provided evidence for an almost complete sulfation at the OH on the phenylethyl chain, leaving the phenolic OH free. Peroxidase/H2O2 oxidation of tyrosol sulfated at the alcoholic group (TyrS) also provided sulfated tyrosol oligomers (OligoTyrS II) that showed on structural analysis highly varied structural features arising likely from the addition of oxygen, derived from water or hydrogen peroxide, to the intermediate quinone methides and substantial involvement of the phenolic OH group in the oligomerization. In line with these characteristics, OligoTyrS I proved to be more active than OligoTyrS II as antioxidant in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assays and as anticoagulant in the classical clotting times, mainly in prolonging the activated partial thromboplastin time (APTT). After intraperitoneal administration in mice, OligoTyrS I was also able to significantly decrease the weight of an induced thrombus. Data from chromogenic coagulation assays showed that the anticoagulant effect of OligoTyrS I was not dependent on antithrombin or factor Xa and thrombin direct inhibition. These results clearly highlight how some structural facets of even closely related phenol polymers may be critical in dictating the anticoagulant activity, providing the key for the rationale design of active synthetic nonsaccharidic anticoagulant agents alternative to heparin.

Impact of the Pd2Spm (Spermine) Complex on the Metabolism of Triple-Negative Breast Cancer Tumors of a Xenograft Mouse Model.

The interest in palladium(II) compounds as potential new anticancer drugs has increased in recent years, due to their high toxicity and acquired resistance to platinum(II)-derived agents, namely cisplatin. In fact, palladium complexes with biogenic polyamines (e.g., spermine, Pd2Spm) have been known to display favorable antineoplastic properties against distinct human breast cancer cell lines. This study describes the in vivo response of triple-negative breast cancer (TNBC) tumors to the Pd2Spm complex or to cisplatin (reference drug), compared to tumors in vehicle-treated mice. Both polar and lipophilic extracts of tumors, excised from a MDA-MB-231 cell-derived xenograft mouse model, were characterized through nuclear magnetic resonance (NMR) metabolomics. Interestingly, the results show that polar and lipophilic metabolomes clearly exhibit distinct responses for each drug, with polar metabolites showing a stronger impact of the Pd(II)-complex compared to cisplatin, whereas neither drug was observed to significantly affect tumor lipophilic metabolism. Compared to cisplatin, exposure to Pd2Spm triggered a higher number of, and more marked, variations in some amino acids, nucleotides and derivatives, membrane precursors (choline and phosphoethanolamine), dimethylamine, fumarate and guanidine acetate, a signature that may be relatable to the cytotoxicity and/or mechanism of action of the palladium complex. Putative explanatory biochemical hypotheses are advanced on the role of the new Pd2Spm complex in TNBC metabolism.

P-glycoprotein activation by 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) in rat distal ileum: ex vivo and in vivo studies.

In vitro studies showed that 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) increases P-glycoprotein (P-gp) expression and activity in Caco-2 cells, preventing xenobiotic toxicity. The present study aimed at investigating TX5 effects on P-gp expression/activity using Wistar Han rats: a) in vivo, evaluating intestinal P-gp activity; b) ex vivo, evaluating P-gp expression in ileum brush border membranes (BBM) and P-gp activity in everted intestinal sacs; c) ex vivo, evaluating P-gp activity in everted intestinal sacs of the distal and proximal ileum. TX5 (30 mg/kg, b.w.), gavage, activated P-gp in vivo, given the significant decrease in the AUC of digoxin (0.25 mg/kg, b.w.). The efflux of rhodamine 123 (300 µM), a P-gp fluorescent substrate, significantly increased in TX5-treated everted sacs from the distal portion of the rat ileum, when P-gp activity was evaluated in the presence of TX5 (20 µM), an effect abolished by the P-gp inhibitor verapamil (100 µM). No increases on P-gp expression or activity were found in TX5-treated BBM of the distal ileum and everted distal sacs, respectively, 24 h after TX5 (10 mg/kg, b.w.) administration. In vivo, no differences were found on digoxin portal concentration between control (digoxin 0.025 mg/kg, b.w., intraduodenal) and TX5-treated (digoxin+TX5 20 µM, intraduodenal) rats. The observed discrepancies in digoxin results can be related to differences in TX5 dose administered and used methodologies. Thus, the results show that TX5 activates P-gp at the distal portion of the rat ileum, and, at the higher dose tested (30 mg/kg, b.w.), seems to modulate in vivo the AUC of P-gp substrates.

Vascular impairment of adenosinergic system in hypertension: increased adenosine bioavailability and differential distribution of adenosine receptors and nucleoside transporters.

Adenosinergic system regulates vascular tonicity through the complex system of adenosine, adenosine receptors (ARs) and nucleoside transporters. This work aimed at evaluating the impact of hypertension on adenosine bioavailability and expression/distribution profile of AR subtypes (A1, A2A, A2B, A3) and equilibrative nucleoside transporters (ENT1, ENT2, ENT3, ENT4). Adenosine was measured in vascular tissue extracts by HPLC (fluorescence detection); immunoreactivities (ARs/ENTs) in mesenteric arteries/veins from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were analyzed by histomorphometry. Significantly higher adenosine bioavailability occurred in arteries than in veins. Adenosine bioavailability was even more increased in SHR vessels. Expression/distribution of ARs and ENTs observed in all vascular layers (intima, media, adventitia), with more intensified expression in arteries than in veins. In SHR arteries, a downregulation of all ENT along with downregulated and punctuated distribution of A1 and A2B receptors occurred comparatively to WKY arteries. By contrast, expressions of ARs and ENTs were unaltered, exception for an A2A receptor upregulation, and ENT2 downregulation in SHR veins relatively to WKY veins. Our data evidenced clear alterations of adenosinergic dynamics occurring in hypertension, particularly in arterial vessels. An increased adenosine bioavailability was observed, for the first time, in hypertensive vascular tissues.

Real­world retrospective study of early­stage prostate cancer at a Portuguese Comprehensive Cancer Centre: The PEarlC study.

Despite the high prevalence of localised prostate cancer (LPC) and locally advanced prostate cancer (LAPC), evidence on the characteristics of patients, treatments and clinical outcomes stratified by disease risk is limited. The PEarlC study was conducted to characterise a cohort of patients with early-stage prostate cancer that included real-world clinical outcomes. Retrospective data from a cohort of patients diagnosed with LPC/LAPC between 2015 and 2017 and followed up until December 2020 at a Portuguese comprehensive cancer centre (IPO Porto) was analysed. Patients were classified as LPC (high- or non-high-risk) or LAPC according to European Association of Urology guidelines, were eligible if diagnosed at stage I-III and followed up in Urology, Medical Oncology or Radiation Oncology outpatient clinics of IPO Porto. Data was collected from the medical/administrative records database. Clinical outcomes included prostate-specific antigen (PSA) progression-free survival, metastasis-free survival, disease-free survival, progression-free survival, overall survival (OS), PSA response (palliative) and no evidence of residual tumour (prostatectomy). Time-to-event outcomes were compared between subgroups using the log-rank test. A total of 790 patients were included (54.8% non-high-risk LPC, 30.9% high-risk LPC, 14.3% LAPC) and the median follow-up was 46.7 months. Patients had a median age of 68.0 years. The majority of patients were stage II (52.9%) and Eastern Cooperative Oncology Group 0-1 (99.9%) and received treatment with curative intent (85.4%). The median was only achieved in progression-free survival (29.9 months; 95% CI, 26.5-41.0 months), as evaluated in palliative patients. At year 5, 82.9% were free of PSA progression (curative), 87.5% were metastasis-free, 83.7% were disease-free, all patients in palliative treatment progressed and the 5-year OS rate was 92.9% (CI 95%, 90.2-95.7%). Among patients with LPC, OS was worse in high-risk vs. non-high-risk patients (5-year OS rate, 88.8% vs. 96.8%; hazard ratio=3.34, CI 95%, 1.64-7.05; P=0.001). PSA response rate was 81.4% in the palliative setting. There was no evidence of residual tumour in 61.6% of patients who underwent prostatectomy. Although most patients with early-stage prostate cancer treated at IPO Porto showed positive 5-year real-world outcomes, patients with high-risk LPC showed worse OS compared with patients with non-high-risk LPC and therefore a poorer prognosis. The present large-sample real-world study is an important contribution to reducing the evidence gap on prostate cancer.

Patient-Reported Outcomes and Experiences Assessment in Women with Breast Cancer: Portuguese Case Study.

In 2020, female breast cancer was the most commonly diagnosed cancer worldwide, representing the type of cancer with the highest incidence among women and the second most common cause of cancer death among women in all OECD countries. The conventional measures addressing the burden of breast cancer by measuring mortality, incidence, and survival do not entirely reflect the quality of life and patients experience when receiving breast cancer care. The main objective of this study is to capture patient-reported outcomes and experiences in women with breast cancer in Portugal using methods developed for international benchmarking purposes, such as the OECD Patient-reported Indicators Surveys. The study included 378 women with breast cancer, with the age distribution being 19.8% aged 15 to 49 years and 80.2% aged 50 years and over. The data collection procedure and analysis followed the "OECD Breast Cancer Patient Reported Outcomes Working Group" protocol, allowing subsequent comparability with data from other OECD member countries. Most women were satisfied with the treatment outcome regarding the shape of their lumpectomy breast when wearing a bra (96.1%) and with the equal size of both breasts (78.3%). Findings on the WHO QOL-BREF showed that women manifest a lower score in well-being when compared with the general population or populations living with chronic diseases. This study shows the feasibility of implementing and using patient-reported metrics (PROM and PREM) in breast cancer services in Portugal. Measuring PROMs and PREMs from Portuguese women receiving breast cancer care provides insightful evidence into the quality and value of cancer care.

Metabolic Impact of Anticancer Drugs Pd2Spermine and Cisplatin on the Brain of Healthy Mice.

The new palladium agent Pd2Spermine (Spm) has been reported to exhibit promising cytotoxic properties, while potentially circumventing the known disadvantages associated to cisplatin therapeutics, namely acquired resistance and high toxicity. This work presents a nuclear magnetic resonance (NMR) metabolomics study of brain extracts obtained from healthy mice, to assess the metabolic impacts of the new Pd2Spm complex in comparison to that of cisplatin. The proton NMR spectra of both polar and nonpolar brain extracts were analyzed by multivariate and univariate statistics, unveiling several metabolite variations during the time course of exposition to each drug (1-48 h). The distinct time-course dependence of such changes revealed useful information on the drug-induced dynamics of metabolic disturbances and recovery periods, namely regarding amino acids, nucleotides, fatty acids, and membrane precursors and phospholipids. Putative biochemical explanations were proposed, based on existing pharmacokinetics data and previously reported metabolic responses elicited by the same metal complexes in the liver of the same animals. Generally, results suggest a more effective response of brain metabolism towards the possible detrimental effects of Pd2Spm, with more rapid recovery back to metabolites' control levels and, thus, indicating that the palladium drug may exert a more beneficial role than cDDP in relation to brain toxicity.

Pd2Spermine Complex Shows Cancer Selectivity and Efficacy to Inhibit Growth of Triple-Negative Breast Tumors in Mice.

Pd2Spm is a dinuclear palladium(II)-spermine chelate with promising anticancer properties against triple-negative breast cancer (TNBC), a breast carcinoma subset with poor prognosis and limited treatment options. The present study evaluated the in vitro and in vivo anticancer effects of Pd2Spm compared to the reference metal-based drug cisplatin. Triple-negative breast cancer MDA-MB-231 cells, non-cancerous MCF-12A breast cells and chorioallantoic membrane (CAM) assay were used for antiproliferative, antimigratory and antiangiogenic studies. For an in vivo efficacy study, female CBA nude mice with subcutaneously implanted MDA-MB-231 breast tumors were treated with Pd2Spm (5 mg/kg/day) or cisplatin (2 mg/kg/day) administered intraperitoneally during 5 consecutive days. Promising selective antiproliferative activity of Pd2Spm was observed in MDA-MB-231 cells (IC50 values of 7.3-8.3 µM), with at least 10-fold lower activity in MCF-12A cells (IC50 values of 89.5-228.9 µM). Pd2Spm inhibited the migration of MDA-MB-231 cells, suppressed angiogenesis in CAM and decreased VEGF secretion from MDA-MB-231 cells with similar potency as cisplatin. Pd2Spm-treated mice showed a significant reduction in tumor growth progression, and tumors evidenced a reduction in the Ki-67 proliferation index and number of mitotic figures, as well as increased DNA damage, similar to cisplatin-treated animals. Encouragingly, systemic toxicity (hematotoxicity and weight loss) observed in cisplatin-treated animals was not observed in Pd2Spm-treated mice. The present study reports, for the first time, promising cancer selectivity, in vivo antitumor activity towards TNBC and a low systemic toxicity of Pd2Spm. Thus, this agent may be viewed as a promising Pd(II) drug candidate for the treatment of this type of low-prognosis neoplasia.

Insights into Nuclear G-Protein-Coupled Receptors as Therapeutic Targets in Non-Communicable Diseases.

G-protein-coupled receptors (GPCRs) comprise a large protein superfamily divided into six classes, rhodopsin-like (A), secretin receptor family (B), metabotropic glutamate (C), fungal mating pheromone receptors (D), cyclic AMP receptors (E) and frizzled (F). Until recently, GPCRs signaling was thought to emanate exclusively from the plasma membrane as a response to extracellular stimuli but several studies have challenged this view demonstrating that GPCRs can be present in intracellular localizations, including in the nuclei. A renewed interest in GPCR receptors' superfamily emerged and intensive research occurred over recent decades, particularly regarding class A GPCRs, but some class B and C have also been explored. Nuclear GPCRs proved to be functional and capable of triggering identical and/or distinct signaling pathways associated with their counterparts on the cell surface bringing new insights into the relevance of nuclear GPCRs and highlighting the nucleus as an autonomous signaling organelle (triggered by GPCRs). Nuclear GPCRs are involved in physiological (namely cell proliferation, transcription, angiogenesis and survival) and disease processes (cancer, cardiovascular diseases, etc.). In this review we summarize emerging evidence on nuclear GPCRs expression/function (with some nuclear GPCRs evidencing atypical/disruptive signaling pathways) in non-communicable disease, thus, bringing nuclear GPCRs as targets to the forefront of debate.

2,4,6-trinitrobenzenesulfonic acid-induced colitis in Rattus norgevicus: a categorization proposal.

Reproducibility in animal research is crucial for its reliance and translational relevance. The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced model of inflammatory bowel disease (IBD) is widely used but inconsistently and incompletely characterized throughout the literature. This hinders comparisons between studies and influences the low rate of translation of effective preclinical molecules. The purpose of this study was to categorize TNBS-induced colitis, based on macroscopic and microscopic scoring systems, and to identify basic routine parameters that could anticipate those categories. We retrospectively analysed male Wistar Rattus norvegicus (n=28 for the control group and n=87 for the TNBS group) and categorized TNBS-induced colitis in three phenotypes: Mild, Moderate and Severe colitis, as for human IBD. Also, we showed that the time course of food intake and fecal excretion (but not body weight, fluid intake or welfare scores) could foresee those categories. So, routine evaluation of food intake and fecal excretion may guide researchers in planning their experiments, selecting the animals with the severity of colitis that better matches their aims, or applying early humane endpoints to animals that will not be used in the experiments. In conclusion, categorizing TNBS-induced colitis enhances the reproducibility of data gathered with this experimental model and strengths its translational relevance.

Inflammation as a Possible Trigger for Mitoxantrone-Induced Cardiotoxicity: An In Vivo Study in Adult and Infant Mice.

Mitoxantrone (MTX) is a pharmaceutical drug used in the treatment of several cancers and refractory multiple sclerosis (MS). Despite its therapeutic value, adverse effects may be severe, namely the frequently reported cardiotoxicity, whose mechanisms need further research. This work aimed to assess if inflammation or oxidative stress-related pathways participate in the cardiotoxicity of MTX, using the mouse as an animal model, at two different age periods (infant or adult mice) using two therapeutic relevant cumulative doses. Histopathology findings showed that MTX caused higher cardiac toxicity in adults. In MTX-treated adults, at the highest dose, noradrenaline cardiac levels decreased, whereas at the lowest cumulative dose, protein carbonylation increased and the expression of nuclear factor kappa B (NF-κB) p65 subunit and of M1 macrophage marker increased. Moreover, MTX-treated adult mice had enhanced expression of NF-κB p52 and tumour necrosis factor (TNF-α), while decreasing interleukin-6 (IL-6). Moreover, while catalase expression significantly increased in both adult and infant mice treated with the lowest MTX cumulative dose, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase only significantly increased in infant animals. Nevertheless, the ratio of GAPDH to ATP synthase subunit beta decreased in adult animals. In conclusion, clinically relevant doses of MTX caused dissimilar responses in adult and infant mice, being that inflammation may be an important trigger to MTX-induced cardiotoxicity.

Novel Insights into Mice Multi-Organ Metabolism upon Exposure to a Potential Anticancer Pd(II)-Agent.

Pd(II)-compounds are presently regarded as promising anticancer drugs, as an alternative to Pt(II)-based drugs (e.g., cisplatin), which typically trigger severe side-effects and acquired resistance. Dinuclear Pd(II) complexes with biogenic polyamines such as spermine (Pd2Spm) have exhibited particularly beneficial cytotoxic properties, hence unveiling the importance of understanding their impact on organism metabolism. The present study reports the first nuclear magnetic resonance (NMR)-based metabolomics study to assess the in vivo impact of Pd2Spm on the metabolism of healthy mice, to identify metabolic markers with possible relation to biotoxicity/side-effects and their dynamics. The changes in the metabolic profiles of both aqueous and lipophilic extracts of mice kidney, liver, and breast tissues were evaluated, as a function of drug-exposure time, using cisplatin as a reference drug. A putative interpretation was advanced for the metabolic deviations specifically triggered by Pd2Spm, this compound generally inducing faster metabolic response and recovery to control levels for all organs tested, compared to cisplatin (except for kidney lipid metabolism). These results constitute encouraging preliminary metabolic data suggestive of potential lower negative effects of Pd2Spm administration.

Role of Inflammation and Redox Status on Doxorubicin-Induced Cardiotoxicity in Infant and Adult CD-1 Male Mice.

Doxorubicin (DOX) is a topoisomerase II inhibitor commonly used in the treatment of several types of cancer. Despite its efficacy, DOX can potentially cause fatal adverse effects, like cardiotoxicity. This work aimed to assess the role of inflammation in DOX-treated infant and adult mice and its possible link to underlying cardiotoxicity. Two groups of CD-1 male mice of different ages (infants or adults) were subjected to biweekly DOX administrations, to reach a cumulative dose of 18.0 mg/kg, which corresponds approximately in humans to 100.6 mg/m2 for infants and 108.9 mg/m2 for adults a clinically relevant dose in humans. The classic plasmatic markers of cardiotoxicity increased, and that damage was confirmed by histopathological findings in both groups, although it was higher in adults. Moreover, in DOX-treated adults, an increase of cardiac fibrosis was observed, which was accompanied by an increase in specific inflammatory parameters, namely, macrophage M1 and nuclear factor kappa B (NF-κB) p65 subunit, with a trend toward increased levels of the tumor necrosis factor receptor 2 (TNFR2). On the other hand, the levels of myeloperoxidase (MPO) and interleukin (IL)-6 significantly decreased in DOX-treated adult animals. In infants, a significant increase in cardiac protein carbonylation and in the levels of nuclear factor erythroid-2 related factor 2 (Nrf2) was observed. In both groups, no differences were found in the levels of tumor necrosis factor (TNF-α), IL-1ß, p38 mitogen-activated protein kinase (p38 MAPK) or NF-κB p52 subunit. In conclusion, using a clinically relevant dose of DOX, our study demonstrated that cardiac effects are associated not only with the intensity of the inflammatory response but also with redox response. Adult mice seemed to be more prone to DOX-induced cardiotoxicity by mechanisms related to inflammation, while infant mice seem to be protected from the damage caused by DOX, possibly by activating such antioxidant defenses as Nrf2.

Preclinical Pharmacokinetics and Biodistribution of Anticancer Dinuclear Palladium(II)-Spermine Complex (Pd2Spm) in Mice.

Palladium-based compounds are regarded as potential analogs to platinum anticancer drugs with improved properties. The present study assessed the pharmacokinetics and biodistribution of a dinuclear palladium(II)-spermine chelate (Pd2Spm), which has previously been shown to possess promising in vitro activity against several therapy-resistant cancers. Using inductively coupled plasma-mass spectrometry, the kinetic profiles of palladium/platinum in serum, serum ultrafiltrate and tissues (kidney, liver, brain, heart, lungs, ovaries, adipose tissue and mammary glands) were studied in healthy female Balb/c mice after a single intraperitoneal bolus injection of Pd2Spm (3 mg/kg bw) or cisplatin (3.5 mg/kg bw) between 0.5 and 48 h post-injection. Palladium in serum exhibited biphasic kinetics with a terminal half-life of 20.7 h, while the free palladium in serum ultrafiltrate showed a higher terminal half-life than platinum (35.5 versus 31.5 h). Palladium was distributed throughout most of the tissues except for the brain, with the highest values in the kidney, followed by the liver, lungs, ovaries, adipose tissue and mammary glands. The in vitro cellular accumulation was also evaluated in breast cancer cells, evidencing a passive diffusion as a mechanism of Pd2Spm's cellular entry. This study reports, for the first time, the favorable pharmacokinetics and biodistribution of Pd2Spm, which may become a promising pharmacological agent for cancer treatment.

Fast and reliable ICP-MS quantification of palladium and platinum-based drugs in animal pharmacokinetic and biodistribution studies.

Palladium-(Pd)-based drugs are emerging as alternatives to platinum (Pt) anticancer chemotherapeutics, which increases the need for efficient and suitable procedures of Pd analysis in reduced amounts of pre-clinical animal samples. Herein, an ICP-MS (inductively coupled plasma-mass spectrometry) method was developed and validated for simple and fast analysis of Pd/Pt-based drugs in 11 distinct biological matrices (adipose tissue, muscle, liver, kidney, spleen, testis, heart, lungs, brain, blood and serum). The critical variables affecting sample preparation and Pd/Pt extraction were optimized using two-level (2k) factorial and central composite designs. Biological samples (50 mg) were digested in closed tubes with a screw cap, using a 3 : 1 (v/v) mixture of nitric acid (900 µL) and hydrochloric acid (300 µL) for 60 min in a 90 °C water bath. Full method validation using in-house materials showed a LOD of 0.001 µg L-1, linear dynamic range from 0.025-10 µg L-1 (R2 = 0.9999 for Pd; R2 = 0.9998 for Pt), good repeatability (CV: 0.02-1.9%) and intermediate precision (CV: 0.52-1.53%) for both the studied metals. The accuracy ranged from 83.5-105.1% considering microwave-assisted digestion as the reference method. The developed and validated method allows the processing of hundreds of biological samples simultaneously, with low reagent and sample consumption. Therefore, the method is highly suitable for analysis of novel Pd/Pt-based drugs in pharmaco-toxicokinetic and biodistribution animal studies that involve a large number of multi-organ samples.

Nuclear G-protein-coupled receptors as putative novel pharmacological targets.

Cell surface G-protein-coupled receptors (GPCRs) are targets for ∼ 30% of drugs currently on the market, and are the largest group of gene products targeted by drugs. Until recently, signaling mediated by GPCRs was thought to emanate exclusively from the cell membrane as a response to extracellular stimuli. However, recent research has revealed the existence of nuclear (n)GPCRs with the ability to trigger identical and/or distinct signaling pathways to their respective counterparts on the cell surface. Understanding of the GPCR signaling platform on the nuclear membranes and its involvement in physiology and/or pathophysiology will be important to develop selective pharmacological and pharmaceutical approaches. In this review, we summarize our current understanding of nGPCRs, with emphasis on their potential as novel pharmacological targets.

A Metabolomic Approach for the In Vivo Study of Gold Nanospheres and Nanostars after a Single-Dose Intravenous Administration to Wistar Rats.

Gold nanoparticles (AuNPs) are promising nanoplatforms for drug therapy, diagnostic and imaging. However, biological comparison studies for different types of AuNPs fail in consistency due to the lack of sensitive methods to detect subtle differences in the expression of toxicity. Therefore, innovative and sensitive approaches such as metabolomics are much needed to discriminate toxicity, specially at low doses. The current work aims to compare the in vivo toxicological effects of gold nanospheres versus gold nanostars (of similar ~40 nm diameter and coated with 11-mercaptoundecanoic acid) 24 h after an intravenous administration of a single dose (1.33 × 1011 AuNPs/kg) to Wistar rats. The biodistribution of both types of AuNPs was determined by graphite furnace atomic absorption spectroscopy. The metabolic effects of the AuNPs on their main target organ, the liver, were analyzed using a GC-MS-based metabolomic approach. Conventional toxicological endpoints, including the levels of ATP and reduced and oxidized glutathione, were also investigated. The results show that AuNPs preferentially accumulate in the liver and, to a lesser extent, in the spleen and lungs. In other organs (kidney, heart, brain), Au content was below the limit of quantification. Reduced glutathione levels increased for both nanospheres and nanostars in the liver, but ATP levels were unaltered. Multivariate analysis showed a good discrimination between the two types of AuNPs (sphere- versus star-shaped nanoparticles) and compared to control group. The metabolic pathways involved in the discrimination were associated with the metabolism of fatty acids, pyrimidine and purine, arachidonic acid, biotin, glycine and synthesis of amino acids. In conclusion, the biodistribution, toxicological, and metabolic profiles of gold nanospheres and gold nanostars were described. Metabolomics proved to be a very useful tool for the comparative study of different types of AuNPs and raised awareness about the pathways associated to their distinct biological effects.