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Laura Brambilla,1 Valeria Frangione,2 Marisa Meloni1 1VitroScreen, in vitro Research Laboratory, Milan, Italy; 2IBSA Institut Biochimique SA, Pambio-Noranco, SwitzerlandCorrespondence: Marisa Meloni, VitroScreen, Via Mosè Bianchi, 103, Milan, 20149, Italy, Tel +39 02 89077608, Email marisa.meloni@vitroscreen.comPurpose: Medical Device Regulation (EU) 2017/745 requires the principal mode of action (MoA) to be demonstrated by experimental data. The MoA of Ialuril® Prefill (combined as HA+CS+CaCl2: sodium hyaluronate 1.6%, sodium chondroitin sulphate 2% w/v and calcium chloride 0.87%) Class III medical device, indicated for intravesical instillation to reduce urinary tract infections, has been evaluated on a 3D reconstructed human bladder epithelium (HBE).Methods: Three experimental designs; i) E. coli strain selection (DSM 103538, DSM 1103) to investigate the HA+CS+CaCl2 properties in modifying bacterial growth in liquid broth (CFU 4h and 24h) at 80%, 50% and 25% concentrations; ii) evaluation of film forming properties on HBE after 15 min exposure by quantifying caffeine permeation across the epithelium; iii) capacity to counteract E. coli adhesion and biofilm formation on colonized HBE by viable counts and ultrastructural analysis by scanning electron microscopy (SEM) using ciprofloxacin as the reference antimicrobial molecule.Results: No significant differences were observed in bacterial viability for both the E. coli strains. HA+CS+CaCl2 reduced caffeine permeation of 51.7% and 38.1% at 1h and 2h, respectively and determined a significant decrease in caffeine permeation rate at both timepoints supporting HA+CS+CaCl2 capacity to firmly adhere to the bladder epithelium creating a physical barrier on the surface. The viable counts in HBE treated tissues then infected with E. coli resulted not different from the negative control suggesting that the device did not inhibit E. coli growth. SEM images showed homogenous product distribution over the HBE surface and confirmed the capacity of HA+CS+CaCl2 to adhere to the bladder epithelium, counteracting biofilm formation.Conclusion: The results support the capacity of HA+CS+CaCl2 to counteract bacterial invasion by using a physico-mechanical mode of action: this medical device represents a valid alternative to antibiotics in the treatment of recurrent UTIs.Keywords: recurrent cystitis, urinary tract infections, E. coli UPEC, E. coli biofilm, hyaluronic acid, chondroitin sulphate, film forming properties, anti-bacterial adhesion, reconstructed 3D human bladder epithelium

Milko Radicioni,1 Chiara Castiglioni,1 Andrea Giori,2 Irma Cupone,3 Valeria Frangione,4 Stefano Rovati4 1CROSS Research S.A., Phase I Unit, Arzo, Switzerland; 2IBSA Farmaceutici Italia, Lodi, Italy; 3Bouty S.p.A., Strada Padana Superiore, Cassina De’ Pecchi, Italy; 4IBSA Institut Biochimique S.A., Pambio-Noranco, Switzerland Abstract: A new orodispersible film formulation of the phosphodiesterase type 5 inhibitor, sildenafil, has been developed to examine the advantages of an orally disintegrating film formulation and provide an alternative to the current marketed products for the treatment of erectile dysfunction. The pharmacokinetics of the sildenafil 100 mg orodispersible film (IBSA) was compared to that of the conventional marketed 100 mg film-coated tablet (Viagra®) after single-dose administration to 53 healthy male volunteers (aged 18–51 years) in a randomized, open, two-way crossover bioequivalence study. Each subject received a single oral dose of 100 mg of sildenafil as test or reference formulation administered under fasting conditions at each of the two study periods according to a randomized crossover design. There was a washout interval of ≥7 days between the two administrations of the investigational medicinal products. Blood samples for pharmacokinetic analysis were collected up to 24 h post-dosing. The primary objective was to compare the rate (peak plasma concentration; Cmax) and extent (area under the curve [AUC] from administration to last observed concentration time; AUC0–t) of sildenafil absorption after single-dose administration of test and reference. Secondary endpoints were observed to describe the plasma pharmacokinetic profiles of sildenafil and its metabolite N-desmethyl-sildenafil relative bioavailability and safety profile after single-dose administration. The mean sildenafil and N-desmethyl-sildenafil plasma concentration–time profiles up to 24 h after single-dose administration of sildenafil 100 mg orodispersible film and film-coated tablet were nearly superimposable. The bioequivalence test was fully satisfied for sildenafil and N-desmethyl-sildenafil in terms of rate and extent of bioavailability. Adverse events occurred at similar rates for the two formulations and were of mild-to-moderate severity. The results suggest that the new orodispersible film formulation can be used interchangeably with the conventional film-coated formulation. Keywords: sildenafil, pharmacokinetics, bioequivalence, orodispersible film, PDE5 inhibitor, N-desmethyl-sildenafil

The serendipitous discovery that inhibiting type 5 phosphodiesterase (PDE5) using sildenafil, a potent PDE5 inhibitor (PDE5i) initially developed for cardioprotection, introduced the possibility of orally managing erectile dysfunction (ED) led to an increase in research data, which are currently considered groundbreaking for the new discipline of sexual medicine. Findings from a number of laboratories and clinics around the world unanimously demonstrated the following: (i) the major cause of ED is directly or indirectly related to cardiovascular disease (CVD); (ii) ED and CVDs share the same risk factors, which are related mainly to lifestyle choices; (iii) the first therapeutic approach to both ED and CVDs is to transform harmful lifestyles into virtuous lifestyles; and (iv) PDE5is in general, particularly sildenafil, are very safe, if not protective, for use in CVD patients. However, the use of PDE5is has faced several challenges. Many patients and some healthcare providers (HCPs) often share the misconception that using these drugs can increase the risk of CVD. Some patients might desire to fulfill the unmet need for privacy linked to the stigma of being treated for ED or might be enticed by the idea of buying drugs online, either because of shame or cheaper prices, without knowing the risks associated with counterfeit drugs. The aim of this narrative revision of the current literature is to demonstrate that (i) the orodispersible film of sildenafil is safe from a CV perspective; (ii) it is a discreet formulation that respects the need for privacy; and (iii) it is virtually the unique PDE5i formulation too expensive to produce outside the correct channels, making it impossible to be counterfeit., Competing Interests: Declarations. Competing interests: AA and GB are employees of IBSA Farmaceutici Itala, and VF is an employee of IBSA SA Switzerland. EAJ is or has been a consultant and/or paid speaker for Bayer, Ibsa, Menarini, Otsuka, Recordati, Pfizer, Viatris. IBSA Farmaceutici Italia sponsored the medical writing and the publication fees., (© 2025. The Author(s).)

Purpose: Medical Device Regulation (EU) 2017/745 requires the principal mode of action (MoA) to be demonstrated by experimental data. The MoA of Ialuril ® Prefill (combined as HA+CS+CaCl 2 : sodium hyaluronate 1.6%, sodium chondroitin sulphate 2% w/v and calcium chloride 0.87%) Class III medical device, indicated for intravesical instillation to reduce urinary tract infections, has been evaluated on a 3D reconstructed human bladder epithelium (HBE)., Methods: Three experimental designs; i) E. coli strain selection (DSM 103538, DSM 1103) to investigate the HA+CS+CaCl 2 properties in modifying bacterial growth in liquid broth (CFU 4h and 24h) at 80%, 50% and 25% concentrations; ii) evaluation of film forming properties on HBE after 15 min exposure by quantifying caffeine permeation across the epithelium; iii) capacity to counteract E. coli adhesion and biofilm formation on colonized HBE by viable counts and ultrastructural analysis by scanning electron microscopy (SEM) using ciprofloxacin as the reference antimicrobial molecule., Results: No significant differences were observed in bacterial viability for both the E. coli strains. HA+CS+CaCl 2 reduced caffeine permeation of 51.7% and 38.1% at 1h and 2h, respectively and determined a significant decrease in caffeine permeation rate at both timepoints supporting HA+CS+CaCl 2 capacity to firmly adhere to the bladder epithelium creating a physical barrier on the surface. The viable counts in HBE treated tissues then infected with E. coli resulted not different from the negative control suggesting that the device did not inhibit E. coli growth. SEM images showed homogenous product distribution over the HBE surface and confirmed the capacity of HA+CS+CaCl 2 to adhere to the bladder epithelium, counteracting biofilm formation., Conclusion: The results support the capacity of HA+CS+CaCl 2 to counteract bacterial invasion by using a physico-mechanical mode of action: this medical device represents a valid alternative to antibiotics in the treatment of recurrent UTIs., Competing Interests: VF is an employee of IBSA. The authors report no other conflicts of interest in this work., (© 2024 Brambilla et al.)

Background: The newly devised orodispersible film (ODF) of sildenafil is the first phosphodiesterase type 5 inhibitor (PDE5i) available in a 75-mg dose. This intermediate dose and the particular properties of the ODF formulation can improve the clinical management of erectile dysfunction (ED) patients., Aim: We investigated the effects of the sildenafil ODF 75-mg dose on both sexual quality of life and erectile function based on the results from an observational study in daily practice in Italy., Methods: This study was a post hoc analysis of results from an observational, real-life study carried out in ED patients at 6 treatment centers in Italy. All subjects were asked to take the prescribed dose of sildenafil ODF at inclusion (visit 1) and to return for a control visit (visit 2) to confirm or adapt the prescribed dose after a minimum of 4 weeks. An end of study control visit (visit 3) was performed after additional 4 weeks., Outcomes: Erectile function, assessed by the International Index of Erectile Function-Erectile Function (IIEF-EF) domain; sexual quality of life, measured using the sexual quality of life instrument for men (SQoL-M)., Results: Among the 36 subjects initially recruited for the 75-mg dose, 5 patients dropped out of the study (2 at visit 2 and 3 at visit 3), none of whom due to treatment inefficacy or serious adverse events. At visit 2, the mean (SD) IIEF-EF scores significantly increased (∆ = 7.97 [4.71], P  < 0.0001) as SQoL-M scores also did (∆ = 10.76 [10.46], P  < 0.0001). At visit 3, IIEF-EF and SQoL-M scores were still significantly improved compared to baseline (∆ = 10.64 [7.01], P  < 0.0001, and ∆ = 18.15 [12.32], P  < 0.0001, respectively). By ANCOVA, we found no significant effects for age, BMI, previous use of PDE5i, presence of metabolic comorbidities, or smoking habits on study outcomes at both visits 2 and 3., Clinical Implication: The new 75-mg ODF sildenafil formulation is a safe and effective treatment for ED, significantly improving both erectile function and sexual quality of life in patients undergoing treatment., Strengths and Limitations: This is the first study assessing the efficacy of the sildenafil ODF 75-mg dose in a real-life setting. However, the small sample size, possible underlying cultural factors, and limited availability of clinically relevant data may have affected the reliability of our results., Conclusion: The use of the 75 mg ODF formulation for sildenafil represents an effective and safe novel treatment option for ED patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society of Sexual Medicine.)

Background and Objective: A topical formulation of diclofenac (FLECTOR diclofenac epolamine topical system (FDETS)) is approved in adults for the treatment of acute pain due to minor strains, sprains, and contusions; however, its safety and efficacy have not been investigated in a pediatric population. This study assessed the safety and efficacy of the FLECTOR (diclofenac epolamine) topical system in children., Methods: This was an open-label, single-arm, phase IV study at ten USA-based family medicine or pediatric practices in children aged 6-16 years with a clinically significant minor soft tissue injury sustained within the preceding 96 h and at least moderate spontaneous pain on the Wong-Baker FACES ® Pain Rating Scale. The FLECTOR topical system was applied twice daily until pain resolution or Day 14. The primary endpoint was local tolerability and systemic safety. Key secondary endpoints were diclofenac plasma concentrations and analgesic efficacy., Results: 104 patients were enrolled; 52 were 6-11 years old, and 52 were 12-16 years old (mean age 11.6 years). The maximum tolerability score experienced by any patient was 1 (faint redness). Fourteen adverse events (none serious) in nine patients (8.7%) were considered possibly treatment-related. Reduction in pain during the study was somewhat greater for patients aged 6-11 versus 12-16 years (p < 0.011). The diclofenac plasma concentration tended to be higher in the younger age group compared with older patients: 1.83 versus 1.46 ng/mL at the first assessment and 2.49 versus 1.11 ng/mL at the last assessment (p = 0.002)., Conclusion: The FLECTOR topical system safely and effectively provided pain relief for minor soft tissue injuries in the pediatric population, with minimal systemic nonsteroidal anti-inflammatory drug exposure and low potential risk of local or systemic adverse events., Clinical Trial Registration: ClinicalTrials.gov identifier NCT02132247., (© 2021. The Author(s).)

Background: A 1-mL aqueous solution for parenteral injection containing diclofenac sodium and hydroxypropyl-β-cyclodextrin, presently on the market for intramuscular and subcutaneous administration (Akis ® /Dicloin ® ), was further developed for intravenous (i.v.) bolus administration., Objectives: The study objective was to compare the tolerability and diclofenac pharmacokinetics after a single i.v. bolus of the investigational solution to those of other parenteral diclofenac products., Methods: The study comprised three parts: (i) Part 1: an exploratory dose-escalation study to evaluate the tolerability of 25 mg/1 mL, 50 mg/1 mL and 75 mg/1 mL diclofenac sodium formulations administered as a single 5-s i.v. bolus; (ii) Part 2: an exploratory, randomised, crossover study to evaluate the pharmacokinetics of diclofenac following 5-, 15-, and 30-s i.v. bolus injections of diclofenac sodium 75 mg/1 mL; (iii) Part 3: a randomised crossover study to compare the pharmacokinetics of diclofenac following a 5-s i.v. bolus of the 75 mg/1 mL solution to the pharmacokinetics of diclofenac following a 30-min i.v. infusion or intramuscular administration of a 75 mg/3 mL reference formulation., Results: The extent of exposure to diclofenac sodium afforded by the 5-s i.v. bolus of 75 mg/1 mL was equivalent to that provided by the 30-min i.v. infusion of 75 mg/3 mL, since the 90% confidence interval of the geometric mean ratio (GMR) of the area under the curve (AUC) from time 0 to the last plasma concentration time t (AUC 0-t ) was within the limits 80.00-125.00%, as was the 90% confidence interval of the GMR of the AUC from time 0 extrapolated to infinity (AUC 0-∞ ). The maximum observed plasma concentration (C max ) was approximately 2.7-fold higher and was achieved earlier (0.05 vs. 0.50 h) with the 1 mL than with the 3 mL formulation, and was similar to data published for a 75 mg/2 mL formulation given as a 15-s i.v. bolus., Conclusions: Diclofenac sodium 75 mg/1 mL solution administered as a 5-s i.v. bolus was well tolerated. The pharmacokinetic profile, which showed a faster onset and a higher concentration peak than seen for other products and administration routes, suggests a superior analgesic effect.

Background: Sildenafil was the first selective drug available on the market as oral therapy for erectile dysfunction (ED). A novel sildenafil orodispersible film (ODF) for ED treatment, containing sildenafil citrate, has recently been marketed., Objectives: Study objective was to investigate sildenafil bioavailability of the novel ODF formulation after sublingual and supralingual administration., Methods: In this randomised, two-way cross-over study, 12 healthy male volunteers received a single 50 mg sildenafil dose by the sublingual and supralingual administration routes. Plasma sildenafil was determined up to 12 h post-dose. Peak concentration (C max ) and area under concentration-time curve (AUC 0-t ) were calculated and compared between the two administration routes by analysis of variance (ANOVA)., Results: Sublingual and supralingual administration can be claimed equivalent regarding the extent of sildenafil exposure since AUC 0-t 90 % CIs corresponded to 94.90-110.58% and were within the pre-specified acceptance range. C max 90% CIs (79.92-125.57%) were only slightly outside the 80.00-125.00% limits, due to the small sample size, while the time to achieve C max did not differ between treatments (p = 0.9277). Rate of exposure of the two administration routes was therefore similar. Reported treatment-related adverse events were mild to moderate headache (33.3% of subjects) and vomiting (8.3%)., Conclusions: In healthy men, sublingual and supralingual administration of sildenafil ODF resulted in a remarkably similar pharmacokinetic profile and confirmed the safety of both study treatments. The recently marketed sildenafil ODF, administered by both investigated routes, can provide a valuable alternative to the marketed solid oral forms (tablets) in ED treatment.

Aims: Intravesical instillation of hyaluronic acid (HA) plus chondroitin sulfate (CS) in women with bladder pain syndrome/interstitial cystitis (BPS/IC) has shown promising results. This study compared the efficacy, safety, and costs of intravesical HA/CS (Ialuril ® , IBSA) to dimethyl sulfoxide (DMSO)., Methods: Randomized, open-label, multicenter study involving 110 women with BPS/IC. The allocation ratio (HA/CS:DMSO) was 2:1. Thirteen weekly instillations of HA (1.6%)/CS (2.0%) or 50% DMSO were given. Patients were evaluated at 3 (end-of-treatment) and 6 months. Primary endpoint was reduction in pain intensity at 6 months by visual analogue scale (VAS) versus baseline. Secondary efficacy measurements were quality of life and economic analyses., Results: A significant reduction in pain intensity was observed at 6 months in both treatment groups versus baseline (P < 0.0001) in the intention-to-treat population. Treatment with HA/CS resulted in a greater reduction in pain intensity at 6 months compared with DMSO for the per-protocol population (mean VAS reduction 44.77 ± 25.07 vs. 28.89 ± 31.14, respectively; P = 0.0186). There were no significant differences between treatment groups in secondary outcomes. At least one adverse event was reported in 14.86% and 30.56% of patients in the HA/CS and DMSO groups, respectively. There were significantly fewer treatment-related adverse events for HA/CS versus DMSO (1.35% vs. 22.22%; P = 0.001). Considering direct healthcare costs, the incremental cost-effectiveness ratio of HA/CS versus DMSO fell between 3735€/quality-adjusted life years (QALY) and 8003€/QALY., Conclusions: Treatment with HA/CS appears to be as effective as DMSO with a potentially more favorable safety profile. Both treatments increased health-related quality of life, while HA/CS showed a more acceptable cost-effectiveness profile., (© 2016 Wiley Periodicals, Inc.)

Objective: to investigate the efficacy and safety of a medicated plaster containing betamethasone valerate (BMV) 2.25 mg in patients with chronic elbow tendinopathy., Methods: randomized, double-blind, placebo-controlled study with assignment 2:2:1:1 to BMV medicated plaster applied daily for 12 hours, daily for 24 hours or matched placebo. 62 patients aged ≥18 years with chronic lateral elbow tendinopathy were randomized. The primary efficacy variable was pain reduction (VAS) at day 28. Secondary objectives included summed pain intensity differences (SPID), overall treatment efficacy and tolerability., Results: mean reduction in VAS pain score at day 28 was greater in both BMV medicated plaster groups, -39.35±27.69 mm for BMV12-h and -36.91±32.50 mm for BMV24-h, than with placebo, -20.20±27.32 mm. Considering the adjusted mean decreases, there was a statistically significant difference between BMV12-h and placebo (p=0.0110). Global pain relief (SPID) and overall treatment efficacy were significantly better with BMV. BMV and placebo plasters had similar local tolerability and there were few treatment-related adverse events., Conclusions: BMV plaster was significantly more effective than placebo at reducing pain in patients with chronic elbow tendinopathies. The BMV plaster was safe and well tolerated.

Objective: To evaluate whether a diclofenac epolamine + heparin topical (plaster) is more effective than diclofenac plaster alone in reducing deep somatic hyperalgesia in subjects without spontaneous pain and whether the effect is linked to or independent of the anti-edematous action of heparin., Design: Prospective, double-blind, randomized and controlled, four-arm parallel design trial., Subjects: One hundred and four patients (84 women, 20 men, mean age 42.2 ± 13.3 years), with deep somatic hyperalgesia in one thigh, randomly assigned to one of 4 groups of 26 each., Intervention: Each group underwent one of the following plaster treatments on one thigh: diclofenac+heparin; diclofenac; heparin; placebo, for 7 days, renewing the plaster every 24 hours., Outcome Measures: Before treatment (day 1), at day 4 and day 8, assessment of (a) pressure and electrical pain thresholds of vastus lateralis and overlying subcutis and skin; and (b) structure/thickness of subcutis and muscle with ultrasounds at the same level., Results: During treatment, in placebo and heparin, no significant threshold changes, except subcutis thresholds which increased slightly (P < 0.02); in diclofenac and diclofenac+heparin, significant increase in all thresholds (0.0001 < P < 0.04). Electrical muscle pain thresholds increased significantly more in diclofenac+heparin than in diclofenac, heparin, and placebo (0.0001 < P < 0.04). In all groups: no edema and thickness changes at ultrasounds in muscle and subcutis., Conclusions: Topical diclofenac+heparin is significantly more effective than diclofenac alone in reducing muscle hyperalgesia in subjects without spontaneous pain, independently of the anti-edematous action of heparin. The results provide a rationale for the use of diclofenac+heparin also in algogenic conditions without evident signs of injury/edema/hematoma., (© 2014 World Institute of Pain.)

Aim: Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac., Methods: Microdialysis was used to determine the local interstitial concentration of PGE2 and 8-iso-PGF2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days., Results: At baseline PGE2 concentrations were 1169 ± 780 pg ml(-1) at rest and 1287 ± 459 pg ml(-1) during dynamic exercise and increased to 2005 ± 1126 pg ml(-1) during recovery. After treatment average PGE2 concentrations were 997 ± 588 pg ml(-1) at rest and 1339 ± 892 pg ml(-1) during exercise. In contrast with the baseline phase no increase in PGE2 concentrations was recorded during the recovery period after treatment (PGE2 1134 ± 874 pg ml(-1)). 8-iso-PGF2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies., Conclusions: We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE2 induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases., (© 2013 The Authors. British Journal of Clinical Pharmacology © 2013 The British Pharmacological Society.)

Objective: To investigate the efficacy and safety of a topical plaster containing diclofenac epolamine (DHEP) 1.3% in the treatment of patients with acute minor soft tissue injuries in China., Research Design and Methods: This prospective, randomized, double blind, placebo-controlled study had balanced random assignment to DHEP medicated plaster and placebo plaster. A total of 384 patients, aged 18-74 years, with minor soft tissue injury occurring within 72 hours of study entry were enrolled and randomized. Plasters were applied twice daily for seven consecutive days. Outcomes were assessed in three visits over 7 days, in addition to patients' daily self-assessment and an adverse events follow-up visit on day 21., Main Outcome Measures: The primary efficacy endpoint was the mean change from baseline in pain on movement on a 100 mm Visual Analogue Scale (VAS) after 7 days of treatment. Secondary efficacy endpoints included pain on movement day-by-day evaluation, summed pain intensity difference, overall treatment efficacy, rescue medication consumption, and treatment tolerability., Results: Reduction in pain on movement after 7 days of treatment, the primary efficacy endpoint, was statistically significantly greater in the DHEP plaster group than with placebo (reduction in VAS pain scores -53.78 ± 16.96 vs -37.02 ± 18.30 for DHEP vs placebo, p < 0.0001). The greater analgesic effect of DHEP plaster was evident by day 1 and increased progressively throughout the treatment period. Global pain relief and overall treatment efficacy were significantly better with DHEP. Both DHEP and placebo plaster were well tolerated with few adverse events, mostly application site reactions., Conclusions: A medicated plaster containing DHEP applied to the affected site in Chinese patients with minor soft tissue injury, such as sprains, strains and contusions, was significantly more effective than placebo at reducing pain scores. Onset of action was rapid and the DHEP plaster was safe and well tolerated. The main limitation was the use of a subjective, though validated, self-reported VAS to assess the primary endpoint.

In our study, we have investigated whether tumors of distinct histological origin can be rejected if expressing CIITA-driven MHC class II molecules. Moreover, we assessed whether antitumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Stable CIITA-transfectants of C51colon adenocarcinoma, RENCA renal adenocarcinoma, WEHI-164 sarcoma as well as TS/A mammary adenocarcinoma were generated. Tumor cells transfectants were injected in vivo, and their growth kinetics and recipient's immune response were analyzed. Tumor rejection and/or retardation of growth was found for the first 3 CIITA-transfected tumor cell lines and confirmed for TS/A-CIITA. Animals rejecting CIITA-transfected tumors acquired specific immunological memory as demonstrated by resistance to challenge with parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with the efficient priming of CD4(+) T helper cells and the consequent activation of CD8(+) T lymphocytes. T cells from TS/A-vaccinated mice were used in an adoptive immunotherapy model of established tumors. The results showed the cure at early stages and significantly prolonged survival at later stages of tumor progression. Importantly, CD4(+) T cells were clearly superior to CD8(+) T cells in antitumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. These results establish the general application of our tumor vaccine model and disclose the additional application of this strategy for producing better lymphocyte effectors for adoptive antitumor immunotherapy.

A new strategy of vaccination against mammary tumors, extendible to tumors of distinct histological origin, based on the administration of tumor cells genetically modified to express major histocompatibility complex (MHC) class II gene products, will be described. Expression of MHC class II molecules in solid tumors, generally lacking these molecules, is achieved by transfecting tumor cells with the MHC class II transactivator (CIITA), the major regulator of the entire family of MHC class II genes. CIITA is encoded by the AIR-1 locus, discovered in our laboratory. The rationale underlying this approach consists in making the tumor cells a sort of surrogate antigen presenting cells for MHC-II-restricted CD4 + T helper (TH) cells. Indeed, it is known that an efficient adaptive immune response against cancer cells can only be achieved if tumor-specific TH cells, the key lymphocyte subpopulation required to trigger both humoral and cellular effector mechanisms, are optimally stimulated. Results from our group show that: (a) CIITA-modified tumor cells can be rejected in vivo by syngeneic immunocompetent mice; (b) this rejection is mediated primarily by CD4 + TH lymphocytes that activate cytolytic CD8 + T cell effectors ; (c) tumor-rejecting mice are resistant to challenge with parental unmodified tumor cells and display long term immune memory; (d) anti-tumor vaccination can be reproduced by using inactivated, nonreplicating CIITA-transfected tumor cells; (e) immune effectors and particularly primed CD4 + TH cells can be used successfully in approaches of immunotherapy of established tumors. These results open the way to envisage a possible use of CIITA-modified mammary tumor cells as a vaccine for increasing both the inducing and the effector phase of the anti-tumor immune response in human settings., (© 2010 Wiley Periodicals, Inc.)

The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the Na(+)/Cl(-)-dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the NH(2) or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeuT(Aa) atomic structure and confirm that the COOH termini of the adjacent subunits are closer than NH(2) termini.