Using Macrophage Polarization in Human Platelet Lysate to Test the Immunomodulatory Potential of Cells for Clinical Use.

Macrophage-based co-cultures are used to test the immunomodulatory function of candidate cells for clinical use. This study aimed to characterize a macrophage polarization model using human platelet lysate (hPL) as a GMP-compliant alternative to Fetal Bovine Serum (FBS). Primary human monocytes were differentiated into unpolarized (M0) or polarized (M1, M2a, and M2c) macrophages in an hPL- or FBS-based medium. The protein secretion profiles and expression of phenotypic markers (CD80 for M1, CD206 for M2a, and CD163 for M2c) were analyzed. Subsequently, chondrocytes were tested in an hPL-based co-culture model to assess their immunomodulatory function in view of their possible use in patients with osteoarthritis. The results showed similar marker regulation between hPL and FBS cultures, but lower basal levels of CD206 and CD163 in hPL-cultured macrophages. Functional co-culture experiments with chondrocytes revealed increased CD206 expression both in hPL and in FBS, indicating an interaction between macrophages and chondrocytes. While markers in FBS-cultured macrophages were confirmed in hPL-cultured cells, the interpretation of marker modulation in immunomodulatory assays with hPL-based cultures should be carried out cautiously due to the observed differences in the basal marker levels for CD206 and CD163. This research underscores the utility of hPL as a GMP-compliant alternative to FBS for macrophage-based co-cultures and highlights the importance of understanding marker expressions in different culture conditions.

NCX 470 Exerts Retinal Cell Protection and Enhances Ophthalmic Artery Blood Flow After Ischemia/Reperfusion Injury of Optic Nerve Head and Retina.

Purpose: The purpose of this study was to assess the retinal protective activity and ocular hemodynamics after NCX 470 (0.1%) compared to bimatoprost administered as the US Food and Drug Administration (FDA)-approved drug (Lumigan - 0.01% ophthalmic solution, LUM) and at an equimolar dose (0.072%, BIM) to that released by NCX 470. Methods: Endothelin-1 (ET-1) induced ischemia/reperfusion injury model in rabbits was used. ET-1 was injected nearby the optic nerve head (ONH) twice/week for 6 weeks. Starting on week 3, the animals received vehicle (VEH), NCX 470, LUM, or BIM (30 µL/eye, twice daily, 6 days/week) until the end of ET-1 treatment. Intraocular pressure (IOP), ophthalmic artery resistive index (OA-RI), and electroretinogram (ERG) data were collected prior to dosing and at different time points postdosing. Reduced glutathione, 8-Hydroxy 2-deoxyguanosine, and Caspase-3 were determined in the retina of treated eyes. DNA fragmentation was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. Results: ET-1 increased IOP (VEHIOP_Baseline = 20.5 ± 0.8 and VEHIOP_Week6 = 24.8 ± 0.3 mmHg) and OA-RI (VEHOA-RI_Baseline = 0.36 ± 0.02 and VEHOA-RI_Week6 = 0.55 ± 0.01) and reduced rod/cone responses over time. Oxidative stress, inflammation, and apoptotic markers increased in ET-1-treated eyes. NCX 470 prevented IOP (NCX 470IOP_Week6 = 18.1 ± 0.6 mmHg) and OA-RI changes (NCX 470OA-RI_Week6 = 0.33 ± 0.01) and restored ERG amplitude leaving unaltered the respective latency; these effects were only partially demonstrated by LUM or BIM. Additionally, NCX 470 reduced oxidative stress, inflammation, and apoptosis in the retinas of treated eyes. BIM and LUM were numerically less effective on these parameters. Conclusions: NCX 470 repeated ocular dosing ameliorates ocular hemodynamics and retinal cell dysfunction caused by ischemia/reperfusion via nitric oxide- and bimatoprost-mediated mechanisms. Translational Relevance: If confirmed in clinical setting our data may open new therapeutic opportunities to reduce visual field loss in glaucoma.

Human platelet lysate stimulates neurotrophic properties of human adipose-derived stem cells better than Schwann cell-like cells.

BACKGROUND: Trauma-associated peripheral nerve injury is a widespread clinical problem causing sensory and motor disabilities. Schwann cells (SCs) contribute to nerve regeneration, mainly by secreting nerve growth factor (NGF) and brain-derived neurotrophic factor. In the last years, adipose-derived stem cells (ASCs) differentiated into SCs (SC-ASCs) were considered as promising cell therapy. However, the cell trans-differentiation process has not been effectively showed and presents several drawbacks, thus an alternative approach for increasing ASCs neurotrophic properties is highly demanded. In the context of human cell-based therapies, Good Manufacturing Practice directions indicate that FBS should be substituted with a xenogeneic-free supplement, such as Human Platelet Lysate (HPL). Previously, we demonstrated that neurotrophic properties of HPL-cultured ASCs were superior compared to undifferentiated FBS-cultured ASCs. Therefore, as following step, here we compared the neurotrophic properties of differentiated SC-like ASCs and HPL-cultured ASCs. METHODS: Both cell groups were investigated for gene expression level of neurotrophic factors, their receptors and neuronal markers. Moreover, the expression of nestin was quantitatively evaluated by flow cytometry. The commitment toward the SC phenotype was assessed with immunofluorescence pictures. Proteomics analysis was performed on both cells and their conditioned media to compare the differential protein profile. Finally, neurotrophic abilities of both groups were evaluated with a functional co-culture assay, assessing dorsal root ganglia survival and neurite outgrowth. RESULTS: HPL-cultured ASCs demonstrated higher gene expression of NGF and lower expression of S100B. Moreover, nestin was present in almost all HPL-cultured ASCs and only in one quarter of SC-ASCs. Immunofluorescence confirmed that S100B was not present in HPL-cultured ASCs. Proteomics analysis validated the higher expression of nestin and the increase in cytoskeletal and ECM proteins involved in neural regeneration processes. The co-culture assay highlighted that neurite outgrowth was higher in the presence of HPL-ASCs or their conditioned medium compared to SC-ASCs. CONCLUSIONS: All together, our results show that HPL-ASCs were more neurotrophic than SC-ASCs. We highlighted that the HPL triggers an immature neuro-induction state of ASCs, while keeping their stem properties, paving the way for innovative therapies for nerve regeneration.

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

NCX 470 Restores Ocular Hemodynamics and Retinal Cell Physiology After ET-1-Induced Ischemia/Reperfusion Injury of Optic Nerve and Retina in Rabbits.

Purpose: Determine whether NCX 470, a nitric oxide (NO)-donating bimatoprost with clinically demonstrated intraocular pressure (IOP)-lowering effects, improves ocular hemodynamics and retinal physiology. Methods: Endothelin-1 (ET-1)-induced ischemia/reperfusion model in New Zealand white rabbits was used. ET-1 was injected next to the optic nerve twice/week (Monday and Thursday) for 6 weeks. Starting on week 3, animals received NCX 470 (0.1% bid, 6 days/week Monday-Saturday) or vehicle until the end of ET-1 treatment. IOP, ophthalmic artery resistive index (OA-RI) and retina physiology (electroretinogram, ERG) were determined before dosing and at different times post-dosing. All measurements were taken on Mondays before the AM daily dosing (36 h treatment-free). Finally, oxidative stress markers were determined in dissected retina and iris/ciliary body of treated eyes. Results: Injection of ET-1 progressively increased IOP (20.7 ± 0.6, 24.9 ± 1.2, and 27.0 ± 0.6 mmHg at baseline, week 2 and 6, respectively) and OA-RI (0.30 ± 0.02, 0.39 ± 0.02, and 0.42 ± 0.03 at baseline, week 2 and 6, respectively) and reduced rods and/or cones response as indicated by changes in ERG amplitudes under different stimulating conditions. NCX 470 re-established baseline IOP (21.8 ± 1.0 mmHg), OA-RI (0.33 ± 0.02), and ERG amplitude by week 6 (mostly rod response, 0.01Dark_AVeh_6week = 32.2 ± 3.0 µV and 0.01Dark_ANCX470_6week 44.3 ± 4.5 µV; mostly cone response, 3.0Dark_AVeh_6week = 87.6 ± 10.1 µV and 3.0Dark_ANCX470_6week = 122.8 ± 11.4 µV; combined rod/cone response, 3.0Light_AVeh_6week = 49.8 ± 6.5 µV and 3.0Light_ANCX470_6week = 64.2 ± 6.8 µV). NCX 470 also reversed ET-1-induced changes in glutathione and manganese superoxide dismutase (oxidative stress markers) in retina and iris/ciliary body. Conclusions: Repeated ocular topical dosing with NCX 470 reverses ET-1-induced changes in IOP, OA-RI, and ERG suggesting improved ocular hemodynamics and retinal physiology likely independently from its demonstrated IOP-lowering effect.

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival.

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.

NCX 667, a Novel Nitric Oxide Donor, Lowers Intraocular Pressure in Rabbits, Dogs, and Non-Human Primates and Enhances TGFß2-Induced Outflow in HTM/HSC Constructs.

Purpose: NCX 667, a novel nitric oxide (NO) donor with an isomannide core, was characterized for its IOP-lowering ability in animal models of ocular hypertension and glaucoma. Bioengineered human trabecular meshwork/Schlemm's canal (HTM/HSC) constructs were used to explore the mode of action. Methods: Ocular normotensive New Zealand white (NZW) rabbits (ONT-rabbits), spontaneously ocular hypertensive pigmented Dutch-belted rabbits (sOHT-rabbits), hypertonic saline (5%)-induced transient ocular hypertensive NZW rabbits (tOHT-rabbits), ocular normotensive Beagle dogs (ONT-dogs), and laser-induced ocular hypertensive cynomolgus monkeys (OHT-monkeys) were used. NCX 667 or vehicle (30 µL) was instilled in a crossover, masked fashion and intraocular pressure (IOP) measured before dosing (baseline) and for several hours thereafter. The ONT-rabbits were used for cyclic guanosine monophosphate (cGMP) determination in ocular tissues after ocular dosing with NCX 667. Transforming growth factor-beta2 (TGFß2) (2.5 ng/mL, six days)-treated HTM/HSC constructs were used to address changes in outflow facility. Results: NCX 667 resulted in robust and dose-dependent IOP decrease in all models used. Maximal IOP-lowering efficacy at 1% was -4.1 ± 0.6, -12.2 ± 2.7, -10.5 ± 2.0, -5.3 ± 0.8, and -6.6 ± 1.9 mmHg, respectively, in ONT-dogs, sOHT-rabbits, tOHT-rabbits, ONT-rabbits, and OHT-monkeys. In ONT-rabbits NCX 667 (1%) increased cGMP in aqueous humor (AH) but not in retina and iris/ciliary body. NCX 667 concentration-dependently increased outflow facility in TGFß2-treated HTM/HSC constructs (outflow facility, 0.10 ± 0.06 and 0.30 ± 0.10 µL/min/mmHg/mm2, respectively, in vehicle- and NCX 667-treated constructs). Conclusions: NCX 667 leads to robust IOP lowering in several animal models. Evidence in HTM/HSC constructs indicate that the IOP reduction likely results from NO-mediated increase of the conventional outflow pathway. Other mechanisms including changes in AH production and episcleral vein pressure may not be excluded at this time.

NCX 1741, a Novel Nitric Oxide-Donating Phosphodiesterase-5 Inhibitor, Exerts Rapid and Long-Lasting Intraocular Pressure-Lowering in Cynomolgus Monkeys.

Purpose: We studied the IOP-lowering effects of NCX 1741, a novel nitric oxide (NO)-donating derivative of the phosphodiesterase type-5 inhibitor, avanafil, in Cynomolgus monkey with laser-induced ocular hypertension (OHT-monkeys). NCX 1193 (NO-donating moiety), NCX 1744 (NCX 1741 without ester nitrate moiety), and travoprost (PGF2α analogue) were used for comparison. Ocular exposure after NCX 1741 dosing also was addressed. Methods: Vehicle (phosphate buffer pH 6.0, Kolliphor® 5%, DMSO 0.3%, benzalkonium chloride 0.02%), NCX 1741, NCX 1193, NCX 1744, or travoprost were instilled (30 µL; single dose) masked and conscious IOPs were measured by pneumatonometry. LC-MS/MS-based methods were employed to monitor ocular exposure of NCX 1741 and main metabolites after ocular dosing in New Zealand White rabbits. Results: NCX 1741 (2.2%, 0.8 µmol/eye) lowered IOP with an Emax (ΔΔIOP, IOP change vs. pre-dose and vehicle) between 5 and 8 h post-dosing (ΔΔIOP5h, -5.3 ± 2.0 mmHg and ΔΔIOP8h, -6.0 ± 2.1 mmHg). Conversely, equimolar (0.47%, 0.8 µmol/eye) NCX 1193 IOP-lowering effects were maximal 3 h post-dosing (ΔΔIOP3h, -4.7 ± 1.6 mmHg) and declined thereafter (ΔΔIOP5h, -1.6 ± 1.1 mmHg). In a follow-up study, NCX 1741 (1.5%, 0.5 µmol/eye) was more effective than NCX 1744 despite a similar duration. Further, NCX 1741 was as effective as travoprost (0.1%, 0.06 µmol/eye) at 5 and 8 h post-dosing (travoprost, ΔΔIOP5h, -3.4 ± 2.2 mmHg and ΔΔIOP8h, -4.9 ± 1.3 mmHg) but had shorter duration (NCX 1741, ΔΔIOP24h, -1.5 ± 1.1 mmHg; travoprost, ΔΔIOP24h, -7.1 ± 2.8 mmHg). NCX 1741 resulted in significant aqueous humor exposure, as determined by the levels of the main metabolite, avanafil. Conclusions: NCX 1741 rapidly and effectively lowers IOP in OHT-monkeys for several hours post-dosing. How these effects translate in humans is still to be defined.

Prostaglandin analogues and nitric oxide contribution in the treatment of ocular hypertension and glaucoma.

In patients with ocular hypertension or glaucoma, all treatments aim to lower intraocular pressure (IOP) by modulating aqueous humour (AH) production and/or uveoscleral and trabecular meshwork/Schlemm's canal AH drainage. PG analogues are considered to be the 'gold standard' treatment and are the most frequently used IOP-lowering agents. Recent data support an important role for NO in regulating IOP. Thus, novel PG analogues carrying a NO-donating moiety were recently advanced. Latanoprostene bunod (LBN) and NCX 470, NO-donating derivatives of latanoprost and bimatoprost, respectively, are examples of such compounds. LBN ophthalmic solution, 0.024% (Vyzulta™), showed greater IOP-lowering efficacy compared with that of Xalatan® (latanoprost ophthalmic solution, 0.005%) or 0.5% timolol maleate in clinical settings. NCX 470 was found to be more effective than bimatoprost in animal models of ocular hypertension and glaucoma. Selective EP2 receptor agonists (i.e. taprenepag isopropyl, omidenepag isopropyl and aganepag isopropyl) and non-selective prostanoid receptor agonists (i.e. ONO-9054, sepetaprost isopropyl) that concomitantly stimulate FP and EP3 receptors have also been shown to hold promise as effective IOP-lowering agents. LINKED ARTICLES: This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc.

Repeated Dosing with NCX1404, a Nitric Oxide-Donating Pregabalin, Re-establishes Normal Nociceptive Responses in Mice with Streptozotocin-Induced Painful Diabetic Neuropathy.

NCX1404 [(3S)-5-methyl-3-(((1-(4-(nitrooxy)butanoyloxy)ethoxy)carbonylamino) methyl)hexanoic acid] is a novel nitric oxide (NO)-donating pregabalin that is readily absorbed and processed in vivo to pregabalin and NO. We determined the antiallodynic response of NCX1404 after acute or after 7, 14, and 21 days of repeated daily oral dosing in mice with streptozotocin (STZ)-induced painful diabetic neuropathy (PDN). Pregabalin and its combination with the NO donor isosorbide mononitrate (ISMN) were used for comparison. The blood levels of pregabalin and nitrites, used as surrogate marker of NO release, after NCX1404 or pregabalin dosing were monitored in parallel experiments using liquid chromatography with tandem mass spectrometry (LC-MS/MS). NCX1404 and pregabalin resulted in similar pregabalin levels as it was their antiallodynic activity after acute dosing in STZ mice. However, NCX1404 resulted in disease-modifying properties when administered daily for 21 days, as indicated by the time- and dose-dependent reversal of STZ-induced mechanical allodynia (paw withdrawal threshold [PWT]Veh_21d= 1.3 ± 0.15 g for vehicle; PWTNCX1404_21d= 1.4 ± 0.5 g, 2.9 ± 0.2 g* and 4.1 ± 0.2 g*, respectively for 19, 63, and 190µmol/kg, oral gavage [PO] of NCX1404; *P< 0.05 versus vehicle). This effect was not shared by pregabalin at equimolar doses (190µmol/kg, PO, PWTPregab_21d= 1.4 ± 0.1 g*, *P< 0.05 versus equimolar NCX1404). In addition, the NO donor ISMN (52.3µmol/kg, PO) alone or combined with pregabalin (63µmol/kg) was active at 7 days (PWTVeh_7d= 1.7 ± 0.16 g; PWTISMN_7d= 3.9 ± 0.34 g*; PWTPregab_7d= 1.3 ± 0.07 g; PWTISMN+pregab_7d= 3.8 ± 0.29 g*; *P< 0.05) but not at later time points. The long-term effect of NCX1404 was independent of residual drug exposure and lasted for several days after the treatment was stopped. In summary, like pregabalin, NCX1404 is an effective antiallodynic agent. Differently from pregabalin, repeated dosing of NCX1404 re-established normal nociceptive responses in STZ-induced PDN in mice.

"Fit-for-purpose" development of analytical and (semi)preparative enantioselective high performance liquid and supercritical fluid chromatography for the access to a novel σ1 receptor agonist.

A rapid and straightforward screening protocol of chiral stationary phases (CSPs) in HPLC and SFC resulted in three different methods "fit-for-purpose", i.e. analysis and scale-up to semi-preparative enantioselective chromatography. The efficient use of these three methods allowed expedited preparation of an important drug discovery target, (R/S)-1, a potent new sigma 1 (σ1) receptor agonist. The approach taken resulted in significant savings of both time and labor for the isolation of enantiomers compared to the development of a stereo-selective synthesis. The enantiomers of 1 have been isolated allowing studies of their chirooptical properties and an in-deep comparative examination of the pharmacological profile for the individual enantiomers.

Identification of RC-33 as a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells. Part 2: g-scale synthesis, physicochemical characterization and in vitro metabolic stability.

Strong pharmacological evidences indicate that σ1 receptors are implicated in the pathophysiology of all major CNS disorders. In the last years our research group has conducted extensive studies aimed at discovering novel σ1 ligands and we recently selected (R/S)-RC-33 as a novel potent and selective σ1 receptor agonist. As continuation of our work in this field, here we report our efforts in the development of this new σ1 receptor agonist. Initially, we investigated the binding of (R) and (S) enantiomers of RC-33 to the σ1 receptor by in silico experiments. The close values of the predicted affinity of (R)-RC-33 and (S)-RC-33 for the protein evidenced the non-stereoselective binding of RC-33 to the σ1 receptor; this, in turn, supported further development and characterization of RC-33 in its racemic form. Subsequently, we set-up a scaled-up, optimized synthesis of (R/S)-RC-33 along with some compound characterization data (e.g., solubility in different media and solid state characterization by thermal analysis techniques). Finally, metabolic studies of RC-33 in different biological matrices (e.g., plasma, blood, and hepatic S9 fraction) of different species (e.g., rat, mouse, dog, and human) were performed. (R/S)-RC-33 is generally stable in all examined biological matrices, with the only exception of rat and human liver S9 fractions in the presence of NADPH. In such conditions, the compound is subjected to a relevant oxidative metabolism, with a degradation of approximately 65% in rat and 69% in human. Taken together, our results demonstrated that (R/S)-RC-33 is a highly potent, selective, metabolically stable σ1 agonist, a promising novel neuroprotective drug candidate.

Synthesis of novel nitric oxide (NO)-releasing esters of timolol.

A novel class of timolol derivatives with nitric oxide (NO)-donating moieties achieved chemical stability yet under physiologically relevant conditions released timolol and NO. Hindered esters A were designed and synthesized, whose 'triggered' release relied on enzymatic hydrolysis of the nitrate ester in A to B, that in turn cyclized to liberate timolol.