Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and ...manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.
In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to ...determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2O2‐impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2O2, and they are analyzed by LC–MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides‐containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC–MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC–MS/MS. Identification and quantification of oxidized species were achieved by high‐resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.
Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system ...response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.
•AIM2 mRNA is highly expressed in tumor tissues of LUAD patients according to the TNM stage.•AIM2 expression in tumor tissues is not related to the smoking status.•AIM2 participates to lung chronic ...inflammation leading to LUAD.•AIM2 positive DCs are in their inactive/resting phenotype in LUAD.•AIM2 expression is correlated to an immunosuppressive environment in LUAD.
The absent in melanoma 2 (AIM2) inflammasome has been demonstrated as involved in tumor growth. In this study we used human samples of lung adenocarcinoma (LUAD) patients, taking advantage of a mouse model of smoking cessation. Human samples were stratified according to the smoking status, high-risk factor for this type of tumor. Both public transcriptomic and human samples obtained by a clinical trial proved that AIM2 was upregulated either in terms of mRNA or protein, respectively, in the tumor mass according to the TNM stage, but it did not relate to the smoking status, age and sex. The upregulation of AIM2 was correlated to an immunosuppressive environment according to resting/non-active dendritic cells (DCs) and T regulatory cells, as demonstrated in both human samples and by means of an experimental model of smoking mice. Computational analysis showed that AIM2 upregulation was correlated to both an inflammasome profile, responsible for the poor prognosis of non-smoker and smoker LUAD patients, and to a non-inflammasome profile for former smoker.
In conclusion, our study demonstrated that AIM2 is involved in lung carcinogenesis either in a canonical and non-canonical manner due to an immunosuppressive microenvironment associated to a dismal prognosis of LUAD patients.
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•Design of a 2-substituted 1,5-benzothiazepine library and its sulphone variants.•Synthesis of enantioenriched forms of benzothiazepine-based compounds.•Evaluation of anticancer ...properties by cell-free and cell-based assays.•Up-regulation of HDAC inhibition-related key targets.•Sulphone-bearing molecules were more effective than their reduced form.
Herein, we report the development of a new series of histone deacetylase inhibitors (HDACi) containing a 2-substituted 1,5-benzothiazepine scaffold. First, a virtual combinatorial library (∼1.6 × 103 items) was built according to a convenient synthetic route, and then it was submitted to molecular docking experiments on seven HDACs isoforms belonging to classes I and II. Integrated computational filters were used to select the most promising ones that were synthesized through an optimized approach, also amenable to generating both racemic and enantioenriched benzothiazepine-based derivatives. The obtained compounds showed potent HDAC inhibitory activity, especially those containing the sulphone moiety, endowed with IC50 in the nanomolar range. In addition, in vitro outcomes of our synthesized compounds demonstrated a cytotoxic effect on U937 and HCT116 cell lines and an arrest in the G2/M phase (13 ≤ IC50 ≤ 18 µM). Finally, Western blot analyses outlined the modulation of the histone acetyl markers such as H3K9/14, acetyl-tubulin, and the apoptotic indicator p21 in both cancer cell lines, disclosing a good HDAC inhibitor activity exerted by the designed items. Given the key role of HDACs in many cellular pathways, which makes these enzymes appealing and “hot” drug targets, our findings highlighted the importance of these 2-substituted 1,5-benzothiazepine-based compounds (both in the reduced and oxidized version) for the development of novel epidrugs.
Introduction. Despite the increasing of number of patients with Sickle Cell Disease (SCD) in Italy, due to multi-ethnic migratory phenomena, a large percentage of Caucasian sickle population is ...already present in Italy mainly with b-thal/HbS genotype. Red cell transfusion is one effective treatment for both acute and chronic complications of SCD, while hydroxycarbamide (HC) is used to reduce the frequency of painful vaso-occlusive crises (VOCs) and decrease the need for blood transfusion.
Through the National Comprehensive Reference Centers for SCD, the Italian Society of Thalassemia and Hemoglobinopathies (SITE), in collaboration with the Society Italian Transfusion Medicine and Immunohematology (SIMTI) and the Italian Association of Hematology and Pediatric Oncology (AIEOP) conducted a national survey to collect information on different therapeutic approaches used for SCD patients.
Aim. To assess therapeutic approaches used a large Italian cohort of patients with SCD, accounting for age, genotype and ethnicity.
Patients and Methods. Observational Longitudinal Systemic Multicentre Study (https://clinicaltrials.gov/ct2/show/NCT03397017). Data were collected from 2015 to 2018 through a standard web-based application (www.SITE-italia.org) encrypted by the Central Server. All the SCD patients, treated or not treated, were included in order to identify the overall number and all gave written informed consent. The study was approved by Ethics Committee of Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico of Milan, Italy.
Results. Thirty-four centers were involved from 14 Italian regions and 1,579 patients were enrolled (802 male and 777 female; median age 23 years - IQR, 25th-75th 10-41 yrs). Genotype, age and ethnicity distribution are shown in Table 1A. As expected, the median age of non-Caucasian patients, mainly HbSS, is significantly lower than Caucasian ones (p<0.001).
Out of 1,579, 365 SCD patients (23%) did not receive any therapy. Acute transfusion regimen (ATR), Chronic transfusion regimen (CTR) and HC were given in monotherapy, respectively in 160, 226 and 197 patients, or in succession/combination in 631 (Table 1B), distributed throughout genotypes. The main reasons for ATR were acute anemia (384 events) and VOCs (352), followed by acute chest syndrome (ACS; 170), surgery (82), pregnancy (64), splenic sequestration (26), stroke (9); multi-organ failure (MOFs 6) and priapism (5). For CRT, it was acute anemia (306 events) and prevention of VOCs (371), ACS (107), primary stroke prevention (78) and secondary prevention stroke (55), pain HC-resistent (39) and leg ulcers (12). For 275 patients out of 631 it was possible to follow the timing of therapy switching (Table 1C). Of 275 patients, 67.6% switched from ATR/CRT to HC, 2.9% from HC to CRT and 6.5% stopped every therapy. Out of 275 patients, 104 were treated with overlapping therapeutic regimen.
Discussion. The significant difference of age in Caucasian and non-Caucasian patients is probably due to the efficacy of the national prevention program of hemoglobinopathies, because the non-Caucasian patients are prevalently born out of Italy.
The transfusional approach is similar in HbSS and b°-thal/HbS and b+-thal/HbS patients regarding both ATR and CTR. HbSC genotype needed less therapies(p <0.001), confirming a less severe clinical pattern. About the combo or sequential therapy, HC was the more frequent chronic therapy used lifelong, mainly in patients with HbSS, because of wide spread of age and transfusional match problems due to different ethnicity.
Summary/Conclusion. The transfusional approach is similar in HbSS, b°-thal/HbS and b+-thal/HbS patients with similar indications, prevalently VOCs and anemia. The significant higher age in Caucasian cohort and the consequent long term follow up could be the cause of variable therapeutic approach observed, however Hydroxycarbamide seemed to be the therapy more frequently used and finally suggested to manage chronic manifestations.
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Origa:Apopharma: Honoraria; Novartis: Honoraria; Bluebird Bio: Consultancy; Cerus Corporation: Research Funding. Forni:Apopharma: Other: DSM Board; Celgene: Research Funding; Novartis: Other: travel expenses, Research Funding; Shire: Research Funding; Roche: Consultancy.
Introduction
Despite the availability of sophisticated devices and suitable recommendations on how to best perform insulin injections, lipohypertrophy (LH) and bruising (BR) frequently occur as a ...consequence of improper injection technique.
Aim
The purpose of this nationwide survey was to check literature-reported LH risk factors or consequences for any association with BR
Method
This was a cross-sectional, observational, multicenter study based on the identification of skin lesions at all patient-reported insulin injection sites in 790 subjects with diabetes. General and injection habit-related elements were investigated as possible BR risk factors.
Results
While confirming the close relationship existing between LH and a full series of factors including missed injection site rotation, needle reuse, long-standing insulin treatment, frequent hypoglycemic events (hypos), and great glycemic variability (GV), the observed data could find no such association with BR, which anyhow came with high HbA1c levels, missed injection site rotation, and long-standing insulin treatment.
Conclusion
BR most likely depends on the patient’s habit of pressing the injection pen hard onto the skin. Despite being worrisome and affecting quality of life, BR seems to represent a preliminary stage of LH but does not affect the rate of hypos and GV.
Trial Registration
207/19.09.2017
Papillary and follicular thyroid cancers, together termed differentiated thyroid cancers (DTC), comprise the majority of thyroid carcinomas and have an optimal prognosis. Most DTCs appear as ...asymptomatic thyroid nodules. Fine-needle aspiration (FNA) cytology is the first diagnostic test for a thyroid nodule in a euthyroid patient. Surgery is the primary treatment for thyroid cancers. Most clinicians recommend near-total or total thyroidectomy, and then 131I ablation therapy, since its consequences are minimal and follow-up is facilitated. A total body scan (TBS) is performed 4 to 7 days after 131I treatment. At a later stage, all patients should be treated with L-tiroxine so as to suppress TSH, and must undergo a periodic evaluation of TSH and thyroglobulin (Tg), the most sensitive and specific marker of DTC. After 6-12 months, TBS with 131I is performed, a technique complementary to serum Tg evaluation. For this technique, it is also necessary to have a high serum TSH concentration, obtained by withdrawing thyroxine therapy for 4 to 6 weeks. This standard method induces hypothyroidism. An alternative method to the withdrawal of thyroid hormones in the follow-up of DTC patients is to administer recombinant human TSH (rh-TSH). After the dose of rhTSH, 131I is administered, and then TBS can be performed 48-72 hours later. Currently, several authors have explored the possibility that rh-TSH-stimulated Tg levels may represent the only necessary test to differentiate patients with persistent disease from disease-free patients, without performing a diagnostic TBS.
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