The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984's antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (-40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Identification of a Novel SSTR3 Full Agonist for the Treatment of Nonfunctioning Pituitary Adenomas.

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted ß-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Health alliance for prudent prescribing and yield of antibiotics in a patient-centred perspective (HAPPY PATIENT): a before-and-after intervention and implementation study protocol.

BACKGROUND: Excessive and inappropriate use of antibiotics is the most important driver of antimicrobial resistance. The aim of the HAPPY PATIENT project is to evaluate the adaptation of European Union (EU) recommendations on the prudent use of antimicrobials in human health by evaluating the impact of a multifaceted intervention targeting different categories of healthcare professionals (HCPs) on common community-acquired infectious diseases, especially respiratory and urinary tract infections. METHODS/DESIGN: HAPPY PATIENT was initiated in January 2021 and is planned to end in December 2023. The partners of this project include 15 organizations from 9 countries. Diverse HCPs (doctors, nurses, pharmacists, and pharmacy technicians) will be audited by the Audit Project Odense (APO) method before and after an intervention in four different settings: general practice, out of hours services, nursing homes and community pharmacies in four high antibiotic prescribing countries (France, Poland, Greece, and Spain) and one low prescribing country (Lithuania). About 25 individuals from each professional group will be recruited in each country, who will register at least 25 patients with community-acquired infections during each audit period. Shortly before the second registration participants will undertake a multifaceted intervention and will receive the results from the first registration to allow the identification of possible quality problems. At these meetings participants will receive training courses on enhancement of communication skills, dissemination of clinical guidelines with recommendations for diagnosis and treatment, posters for the waiting rooms, and leaflets for patients. The results of the second registration will be compared with those obtained in the first audit. DISCUSSION: HAPPY PATIENT is an EU-funded project aimed at contributing to the battle against antibiotic resistance through improvement of the quality of management of common community-acquired infections based on interventions by different types of HCPs. It is hypothesized that the use of multifaceted strategies combining active intervention will be effective in reducing inappropriate prescribing and dispensing of antibiotics. STUDY REGISTRATION: EU Health programmes project database https://webgate.ec.europa.eu/chafea_pdb/health/projects/900024/summary ; date of registration: 1 January 2021.

Novel Benzohydroxamate-Based Potent and Selective Histone Deacetylase 6 (HDAC6) Inhibitors Bearing a Pentaheterocyclic Scaffold: Design, Synthesis, and Biological Evaluation.

Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as autoimmune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Specific inhibition of histone deacetylase 8 reduces gene expression and production of proinflammatory cytokines in vitro and in vivo.

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1ß and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1ß, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1ß was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1ß and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1ß independent of inhibition of caspase-1 activity; however, synthesis of the IL-1ß precursor was reduced by 40% without significant decrease in IL-1ß mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1ß by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.

Modulation of gamma globin genes expression by histone deacetylase inhibitors: an in vitro study.

Induction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of ß-thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ-globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34⁺ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non-toxic concentrations.

Pharmacokinetics, safety and inducible cytokine responses during a phase 1 trial of the oral histone deacetylase inhibitor ITF2357 (givinostat).

ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50 mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1ß, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.

ITF1697, a stable Lys-Pro-containing peptide, inhibits weibel-palade body exocytosis induced by ischemia/reperfusion and pressure elevation.

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo.

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.

Mycobacterium tuberculosis chaperonin 10 is secreted in the macrophage phagosome: is secretion due to dissociation and adoption of a partially helical structure at the membrane?

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a beta-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately beta-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.

Preventive administration of Mycobacterium tuberculosis 10-kDa heat shock protein (hsp10) suppresses adjuvant arthritis in Lewis rats.

Adjuvant arthritis (AA) can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt) in oil. We have investigated the modulation of AA by mycobacterial 10-kDa heat shock protein (hsp10), administered according to several protocols known to induce immune tolerance and immune deviation. Subcutaneous immunization with hsp10 in aqueous solution did not induce a cellular immune response, evaluated as delayed-type hypersensitivity (DTH) reaction, although anti-hsp10 antibodies, mainly of the IgG2a isotype, were detected in serum of treated animals. When rats were pretreated with hsp10 in aqueous solution before AA induction, no effects were seen on arthritis-induced joint swelling, although osteolysis and lymphocyte infiltration were slightly decreased. When other routes of administration were attempted, the strongest suppression was seen in the group of animals which received four intranasal (i.n.) administrations of protein and a subsequent challenge of hsp10 in incomplete Freund's adjuvant (IFA). We also found that the extent of disease suppression among the different groups of animals correlated with serum anti-hsp10 antibody levels. These antibodies mostly belonged to the IgG2a subtype, suggesting that immune deviation may play a role in the mechanism of disease suppression by hsp10.