Prolonging the stability of cetuximab (Erbitux®) and panitumumab (Vectibix®): An orthogonal assessment of physicochemical, biological and microbiological properties of original opened glass vials and diluted saline preparations.

The two anti-epidermal growth factor receptor monoclonal antibodies (mAbs) cetuximab and panitumumab are the pillars for the treatment of EGFR-positive, KRAS wild-type metastatic colorectal cancers. However, stability data of these mAbs are generally missing or incomplete. Here, we report for the first time an orthogonal analysis of the stability of cetuximab (Erbitux®) and panitumumab (Vectibix®), either undiluted vial leftovers or saline dilutions in polyolefin/polyamide infusion bags. All samples were stored at 2-8 °C protected from light, according to their summary of product characteristics (SmPCs). Alternatively, opened vials and preparations were maintained at 25 °C for 15 h, and then stored again at 2-8 °C protected from light to mimic a temporary interruption of the cold chain. Vial leftovers proved stable up to 180 days when stored according to their SmPCs, while compounded preparations in infusion bags maintained their physiochemical, biological and microbiological stability up to 30 days. Additionally, no changes were detected up to 30 days for the same samples undergoing a thermal excursion. Our results provide additional rationale to the SmPCs, crucial especially in the case of reassignment and pre-preparation of bags. This information will allow hospitals to achieve significant cost savings, and better organization of the entire therapeutic process.

A Method for Risk Assessment Evaluating the Safety, Stability and Efficacy in Clinical Practice of Anticancer Drug Preparations in the Centralized Compounding Unit of the Veneto Institute of Oncology-IRCCS.

BACKGROUND: Preparation of injectable anticancer drugs in hospital pharmacies is a high-risk activity that requires a proper risk assessment (RA) and quality assurance system (QAS) to ensure both a decrease in risk associated with chemotherapy compounding and high quality of the final product, especially in terms of its microbiological stability. METHODS: At the centralized compounding unit (UFA) of the Italian Hospital IOV-IRCCS, a quick and deductive method was applied to evaluate the "added value" provided by each prescribed preparation, and its RA was calculated applying a formula that integrates different pharmacological, technological and organizational aspects. According to specific RA range values, the preparations were divided into different risk levels, in order to determine the QAS to be adopted, according to the Italian Ministry of Health guidelines, whose adherence was meticulously evaluated through a specific self-assessment procedure. A review of the scientific literature was carried out to integrate the risk-based predictive extended stability (RBPES) of drugs with data concerning their physiochemical and biological stability. RESULTS: Based on the self-assessment comprising all microbiological validations of the working area, personnel and products, the microbiological risk level within the IOV-IRCCS' UFA was defined through the creation of a transcoding matrix, conferring a microbiological stability to preparations and vial leftovers of a maximum of 7 days. The calculated RBPES were successfully integrated with stability data from the literature, leading to the drafting of a stability table of drugs and preparations in use in our UFA. CONCLUSIONS: Our methods allowed us to perform an in-depth analysis of the highly specific and technical process of anticancer drug compounding in our UFA, ensuring a certain grade of quality and safety to preparations, especially in terms of microbiological stability. The resulting RBPES table represents an invaluable tool with positive repercussions at organizational and economic levels.

Analysis of the relation between adverse events and overall survival in patients treated with pembrolizumab as a first-line treatment for metastatic NSCLC.

BACKGROUND: Many trials supported pembrolizumab as a first-line monotherapy to significantly improve overall survival (OS) in selected patients with previously untreated metastatic Non-Small Cell Lung Cancer (mNSCLC) and a PD-L1 TPS of ≥50% without EGFR/ALK mutations. The aim of this study was to reveal the correlation between OS and adverse events in real-world settings after 42 months. METHODS: This retrospective observational study involved 98 patients with mNSCLC, TPS ≥ 50%, and no EGFR/ALK aberrations. Patients were treated with pembrolizumab (200 mg q3w) as a first-line treatment. Clinical data, including PD-L1 expression, Performance Status (ECOG-PS), treatment duration, toxicity, and outcomes were retrieved from local electronic medical records and from the Italian Regulatory Agency Registry. RESULTS: The cohort's main characteristics were as follows: median age 73 [44-89] years, 64.3% were male and 35.7% were female, an ECOG-PS score of 0 (n = 73) and 1 or 2 (n = 25), and a PD-L1 > 90% in 29.6% of patients. The entire cohort had stage IV NSCLC at diagnosis. The median number of cycles was 8.5 at a median follow-up of 13 months. The median OS of 13.6 months (95% CI: 11.7-NA) was not influenced by sex and PD-L1, but was significantly associated with ECOG-PS (p = 0.02). Immune-Related Adverse Events (irAEs) occurred in 77.5% of patients (30.1% cutaneous, 27.5% gastrointestinal, and 20.4% endocrinological), but no grade 4 or 5 irAEs were identified. Patients experiencing any type of toxicity had a significantly longer median OS (20.39 months, 95% CI: 13.08-NA) than those with no toxicities (6.46 months, 95% CI: 1.41-NA, p = 0.006). CONCLUSION: The percentage of irAEs detected was comparable to that reported in KEYNOTE-024 and KEYNOTE-042. These real-world findings demonstrated the significant correlation between OS and cutaneous toxicities.

Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer.

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel 99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH2 (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(-) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and 99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of 99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of 99mTc-HscFv1 and 99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of 99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of 99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

A Review on the Current State and Future Perspectives of [99mTc]Tc-Housed PSMA-i in Prostate Cancer.

Recently, prostate-specific membrane antigen (PSMA) has gained momentum in tumor nuclear molecular imaging as an excellent target for both the diagnosis and therapy of prostate cancer. Since 2008, after years of preclinical research efforts, a plentitude of radiolabeled compounds mainly based on low molecular weight PSMA inhibitors (PSMA-i) have been described for imaging and theranostic applications, and some of them have been transferred to the clinic. Most of these compounds include radiometals (e.g., 68Ga, 64Cu, 177Lu) for positron emission tomography (PET) imaging or endoradiotherapy. Nowadays, although the development of new PET tracers has caused a significant drop in single-photon emission tomography (SPECT) research programs and the development of new technetium-99m (99mTc) tracers is rare, this radionuclide remains the best atom for SPECT imaging owing to its ideal physical decay properties, convenient availability, and rich and versatile coordination chemistry. Indeed, 99mTc still plays a relevant role in diagnostic nuclear medicine, as the number of clinical examinations based on 99mTc outscores that of PET agents and 99mTc-PSMA SPECT/CT may be a cost-effective alternative for 68Ga-PSMA PET/CT. This review aims to give an overview of the specific features of the developed [99mTc]Tc-tagged PSMA agents with particular attention to [99mTc]Tc-PSMA-i. The chemical and pharmacological properties of the latter will be compared and discussed, highlighting the pros and cons with respect to [68Ga]Ga-PSMA11.

Impact of Different [Tc(N)PNP]-Scaffolds on the Biological Properties of the Small cRGDfK Peptide: Synthesis, In Vitro and In Vivo Evaluations.

Background: The [99mTc][Tc(N)(PNP)] system, where PNP is a bisphosphinoamine, is an interesting platform for the development of tumor 'receptor-specific' agents. Here, we compared the reactivity and impact of three [Tc(N)(PNP)] frameworks on the stability, receptor targeting properties, biodistribution, and metabolism of the corresponding [99mTc][Tc(N)(PNP)]-tagged cRGDfK peptide to determine the best performing agent and to select the framework useful for the preparation of [99mTc][Tc(N)(PNP)]-housing molecular targeting agents. Methods: cRGDfK pentapeptide was conjugated to Cys and labeled with each [Tc(N)(PNP)] framework. Radioconjugates were assessed for their lipophilicity, stability, in vitro and in vivo targeting properties, and performance. Results: All compounds were equally synthetically accessible and easy to purify (RCY ≥ 95%). The main influences of the synthon on the targeting peptide were observed in in vitro cell binding and in vivo. Conclusions: The variation in the substituents on the phosphorus atoms of the PNP enables a fine tuning of the biological features of the radioconjugates. ws[99mTc][Tc(N)(PNP3OH)]- and [99mTc][Tc(N)(PNP3)]- are better performing synthons in terms of labeling efficiency and in vivo performance than the [99mTc][Tc(N)(PNP43)] framework and are therefore more suitable for further radiopharmaceutical purposes. Furthermore, the good labeling properties of the ws[99mTc][Tc(N)(PNP3OH)]- framework can be exploited to extend this technology to the labeling of temperature-sensitive biomolecules suitable for SPECT imaging.

PSMA-Specific CAR-Engineered T Cells for Prostate Cancer: CD28 Outperforms Combined CD28-4-1BB "Super-Stimulation".

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific second-generation (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3ζ. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such "superstimulation" resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case.

Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines.

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.

Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells.

Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells. They represent an attractive approach for cell-based immunotherapy, as they do not require antigen-specific priming for tumor cell recognition, and can be rapidly expanded in vitro. Their relevant expression of FcγRIIIa (CD16a) can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires therapeutic options. Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or injecting MDA-MB-231 cells orthotopically or intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either in patient-derived tumor xenografts or MDA-MB-231 cell line models. Moreover, this approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and led to an enrichment of the CD16a+ subset.Data highlight the potentiality of this novel immunotherapy strategy where a nonspecific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells. List of abbreviations: ACT, Adoptive Cell Transfer; ADCC, Antibody-Dependent Cell-mediated Cytotoxicity; ADP, Adenosine diphosphate; BLI, Bioluminescence Imaging; CAR, Chimeric Antigen Receptor; CIK, Cytokine Induced Killer cells; CTX, Cetuximab; DMEM, Dulbecco's Modified Eagle Medium; EGFR, Human Epidermal Growth Factor 1; ER, Estrogen; FBS, Fetal Bovine Serum; FFPE, Formalin-Fixed Paraffin-Embedded; GMP, Good Manufacturing Practices; GVHD, Graft Versus Host Disease; HER2, Human Epidermal Growth Factor 2; HRP, Horseradish Peroxidase; IFN-γ, Interferon-γ; IHC, Immunohistochemistry; IL-2, Interleukin-2; ISO, Irrelevant antibody; i.t., intratumoral; i.v., intravenous, mAbs, Monoclonal Antibodies; mIHC, Multiplex Fluorescence Immunohistochemistry; MHC, Major Histocompatibility Complex; NK, Natural Killer; NKG2D, Natural-Killer group 2 member D; NSG, NOD/SCID common γ chain knockout; PARP, Poly ADP-ribose polymerase; PBMCs, Peripheral Blood Mononuclear Cells; PBS, Phosphate-buffered saline; PDX, Patient-derived xenograft; PR, Progesterone; rhIFN-γ, Recombinant Human Interferon-γ; RPMI, Roswell Park Memorial Institute; STR, Short tandem Repeat; TCR, T Cell Receptor; TNBC, Triple Negative Breast Cancer; TSA, Tyramide Signal Amplification.

Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen.

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177Lu-scFvD2B was obtained in high yield and stability. In vitro, 177Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer.

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

[99mTc][Tc(N)PNP43]-Labeled RGD Peptides As New Probes for a Selective Detection of αvß3 Integrin: Synthesis, Structure-Activity and Pharmacokinetic Studies.

New integrin-selective molecules suitable for therapeutic or imaging purposes are currently of interest in development of effective personalized medical platforms. RGDechi is a bifunctional peptide selective for integrin αvß3. Herein, RGDechi and three truncated derivatives functionalized with a cysteine (1-4) were synthesized and labeled with the [99mTc][Tc(N)PNP43]-synthon ([PNP43 = (CH3)2P(CH2)2N(C2H4OCH3)(CH2)2P(CH3)2]) (99mTc1-4) as a basis for selective integrin recognition. The pharmacological parameters of all radiolabeled peptides were assessed along with the pharmacokinetic profiles of the most promising 99mTc1 and 99mTc2 compounds both on healthy and melanoma-bearing mice. Their metabolism and metabolite identification are also reported. 99mTc1-2 are able to discriminate between endogenously expressed integrins αvß3 and αvß5 and possess favorable pharmacokinetics characterized by low liver uptake and rapid elimination from nontarget tissues resulting in positive target-to-nontarget ratios. Results are encouraging; the presented construct can be considered the starting point for the development of agents for the selective detection of αvß3 expression by SPECT.

The nitrobenzoxadiazole derivative MC3181 blocks melanoma invasion and metastasis.

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts. Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively. The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice. Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line. To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e. AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2). Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.

Melanoma targeting with [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analogs: Effects of cyclization on the radiopharmaceutical properties.

The purpose of this study was to evaluate the effect of cyclization on the biological profile of a [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analog. A lactam bridge-cyclized H-Cys-Ahx-ßAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11-Pro-Val-NH2 (NAP-NS2) and the corresponding linear H-Cys-Ahx-ßAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16/F10 melanoma cells. The consistent [99mTc(N)(PNP3)]-labeled compounds were readily obtained in high specific activity and their stability and biological properties were assessed. As an example, the chemical identity of [99mTc(N)(NAP-NS1)(PNP3)]+ was confirmed by carrier added experiments supported by radio/UV HPLC analysis combined with ESI(+)-MS. Compared with the linear peptide, cyclization negatively affected the biological properties of NAP-NS2 peptide by reducing its binding affinity for MC1R and by decreasing the overall excretion rate of the corresponding [99mTc(N)(PNP3)]-labeled peptide from the body as well as its in vivo stability. [99mTc(N)(NAP-NS1)(PNP3)]+ was evaluated for its potential as melanoma imaging probe in murine melanoma model. Data from in vitro and in vivo studies on B16/F10 melanoma model of [99mTc(N)(NAP-NS1)(PNP3)]+ clearly evidenced that the radiolabeled linear peptide keeps its biological properties up on the conjugation to the [99mTc(N)(PNP3)]-building block. The progressive increase of the tumor-to-nontarget ratios over the time indicates a quite stable interaction between the radio-complex and the MC1R.

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer.

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer.

Folic acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells.

We report the synthesis of three optical probes (Eu(3+)⊂1, Eu(3+)⊂2, and Eu(3+)⊂3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu(3+)⊂1 and Eu(3+)⊂2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu(3+)⊂2 > Eu(3+)⊂1 > Eu(3+)⊂3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu(3+)⊂2 and FA or Eu(3+)⊂2 versus (3)H-FA, indicate that internalization is receptor-mediated and that Eu(3+)⊂2 shows high selectivity and specificity for FR.

A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts.

We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo.