Assessment of 99mTc-NTP 15-5 uptake on cartilage, a new proteoglycan tracer: Study protocol for a phase I trial (CARSPECT).

Background: 99mTc-NTP 15-5 is a SPECT radiotracer targeting proteoglycans (PG), components of the cartilaginous extracellular matrix. Imaging of PGs would be useful for the early detection of cartilage disorders (osteoarthritis, arthritis and chondrosarcoma, Aromatase Inhibitor associated arthralgia (AIA) in breast cancer), and the follow-up of patients under treatment. According to preclinical study results, 99mTc-NTP 15-5, is a good candidate for a specific functional molecular imaging of joints. We intend to initiate a first in-human study to confirm and quantify 99mTc-NTP 15-5 uptake in healthy joints. Methods: As the clinical development of this radiotracer would be oriented toward the functional imaging of joint pathologies, we have chosen to include patients with healthy joints (unilateral osteoarthritis of the knee or breast cancer with indication of AI treatment). This phase I study will be an open-label, multicenter, dose-escalation trial of a radiopharmaceutical orientation to determine the recommended level of activity of 99mTc-NTP 15-5 to obtain the best joint tracer contrasts on images, without dose limiting toxicity (DLT). The secondary objectives will include the study of the pharmacology, biodistribution (using planar whole body and SPECT-CT acquisitions), toxicity, and dosimetry of this radiotracer. The dose escalation with 3 activity levels (5, 10, and 15 MBq/kg), will be conditioned by the absence at the previous level of DLT and of a visualized tracer accumulation on more than 80% of healthy joints as observed on scintigraphy performed at ≤ 2 h post-injection. Discussion: This first in-human phase I trial will be proof-of-concept of the relevance of 99mTc-NTP 15-5 as a cartilage tracer, with the determination of the optimal methodology (dose and acquisition time) to obtain the best contrast to provide a functional image of joints with SPECT-CT. Trial registration number: Clinicaltrials.gov: NCT04481230. Identifier in French National Agency for the Safety of Medicines and Health Products (ANSM): N°EudraCT 2020-000495-37.

Design, synthesis and evaluation of targeted hypoxia-activated prodrugs applied to chondrosarcoma chemotherapy.

The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique hallmarks for developing a chondrosarcoma targeted drug-delivery system. Tumor targeting could be achieved using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged proteoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia-responsive drug release. In a previous work, ICF05016 was identified as efficient proteoglycan-targeting hypoxia-activated prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a first study of the structure-activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second part of the study, namely the modification of the nitro-aromatic trigger and the position of the proteoglycan-targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex, leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-nitrobenzyl one, were showed to undergo efficient cleavage with release of the corresponding mustard. However N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic cells, likely not sufficient for application as hypoxia-activated prodrugs.

Linker structure-activity relationships in fluorodeoxyglucose chlorambucil conjugates for tumor-targeted chemotherapy.

Nitrogen mustards, such as chlorambucil (CLB), can cause adverse side-effects due to ubiquitous distribution in non-target organs. To minimize this toxicity, strategies of tumor-targeting drug delivery have been developed, where a cytotoxic warhead is linked to a tumor-cell-specific small ligand. Malignant cells exhibit marked glucose avidity and an accelerated metabolism by aerobic glycolysis, known as the Warburg effect, and recognized as a hallmark of cancer. A targeting approach exploiting the Warburg effect by conjugation of CLB to 2-fluoro-2-deoxyglucose (FDG) was previously reported and identified two peracetylated glucoconjugates 2 and 3 with promising antitumor activities in vivo. These results prompted us to investigate the importance of the spacer in this tumor-targeting glucose-based conjugates. Here we report the chemical synthesis and an in vitro cytotoxicity evaluation, using a 5-member panel of human tumor cell lines and human fibroblasts, of 16 new CLB glucoconjugates in which the alkylating drug is attached to the C-1 position of FDG via different linkages. We studied the structure-activity relationships in the linker, and evidenced the positive impact of an aromatic linker on in vitro cytotoxicity: compound 51 proved to be the most active FDG-CLB glucoside, characterized by a bis-aromatic spacer tethered to CLB through an amide function.

Development of a freeze-dried kit formulation for the preparation of 99mTc-NTP 15-5, a radiotracer for scintigraphic imaging of proteoglycans.

The cartilage-targeting strategy is based on the strong affinity of quaternary ammonium (QA) functions for cartilage proteoglycans. We use a bifunctional agent containing QA moiety and a polyazamacrocycle structure able to complex technetium-99m. (99m)Tc-NTP 15-5 was selected for its high stability and its high affinity for proteoglycans in vivo. Labeling conditions of NTP 15-5 were optimized, and a lyophilized kit was developed for radiolabeling of (99m)Tc-NTP 15-5 (radiochemical yields 94.6±1.8%). (99m)Tc-NTP 15-5 was stable and resulted in favorable biological evaluations.

Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma.

Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively).

Complementary mass spectrometric approaches and scanning electron microscopy to study the structural stability of polyurethane tunneled dialysis catheters after exposure to ethanol solutions.

RATIONALE: Ethanol lock is an emerging therapeutic option for preventing and/or controlling catheter-associated infection. A previous study of silicone catheters showed they underwent no polymer degradation when kept in 60% ethanol for 15 days at 37 °C. The stability of the more widely used polyurethane catheters was studied here in the same way. METHODS: A qualitative and quantitative study of the stability of Carbothane® catheters was performed following their immersion at 37 °C in different solvents (0.9% sodium chloride as control medium and 40%, 60%, 95% ethanol solutions) for different periods of time (from 5 min to 15 days) using scanning electron microscopy and complementary mass spectrometry techniques. RESULTS: Electron ionization analysis of the 95% ethanol storage solutions revealed the release of about 45 products (8 of which were major) subdivided into two groups according to their fragmentation patterns. Combining all the mass spectrometric data made it possible to propose structures. Group I (major) originated from the polycarbonate diol component (soft segment) and group II (minor) from the dicyclohexylmethane-4,4'-diisocyanate component (rigid segment). Semi-quantitative gas chromatography/mass spectrometry (GC/MS) analysis showed that no significantly higher release was observed after immersion for 30 min at 37 °C in 40% ethanol (mean ratio = 0.677 ± 0.068) than after immersion in reference 0.9% sodium chloride solution for 15 days (0.837 ± 0.127). CONCLUSIONS: A 30 min-40% (v/v) ethanol solution can be considered as safe for preventing the infectious complications of Carbothane® dialysis catheters, and a 30 min-60% (v/v) ethanol treatment can be occasionally used to eradicate established biofilm.

Spacer optimization of new conjugates for a melanoma-selective delivery approach.

In the search for more selective anticancer drugs, we designed and synthesized seven conjugates varying the structure of the linker connecting the 5-iodo-2'-deoxyuridine (IUdR) to the ICF 01012 melanoma-carrier for potential intratumoural specific drug release. Chemical and in vitro metabolic stability evaluations showed that, except for the ester conjugate (1), the ketal (2b), acetal (2a), carbonate (4) and carbamate (3) conjugates were compatible with our approach. The acetal (2a) and its PEGylated derivative (2c) were of particular interest for further in vivo development owing to their respective pH-dependent stability and limited metabolic degradation in order to exploit the acidic subcellular environment of malignant melanocytes to trigger the release of therapeutics upon internalization in cells.

Synthesis, radioiodination and in vivo screening of novel potent iodinated and fluorinated radiotracers as melanoma imaging and therapeutic probes.

In order to develop new iodinated and fluorinated matched-pair radiotracers for Single-Photon Emission Computed Tomography (SPECT)/Positron Emission Tomography (PET) imaging and targeted radionuclide therapy of melanoma, we successfully synthesized and radiolabelled with iodine-125 seven new derivatives, starting from our previously described lead structure 3. The relevance of these radiotracers for gamma scintigraphic imaging of melanoma in rodent was assessed. The tumoural radioactivity uptake was most often high and specific even at early time points (12.1-18.3% ID/g at 3 h p.i. for [(125)I]39-42) and a fast clearance from the non-target organs was observed. Also, calculated effective doses that could be delivered to tumours when using corresponding [(131)I]-labelled analogues were generally higher than 100 cGy/MBq injected (98.9-150.5 cGy/MBq for [(131)I]39-42). These results make compounds 39-42 suitable candidates for (i) PET imaging of melanoma after labelling with fluorine-18 and (ii) targeted radionuclide therapy of disseminated melanoma after labelling with iodine-131.

New aldehyde and vinylsulfone proteasome inhibitors for targeted melanoma therapy.

The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [(123)I]-N-(2-diethylaminoethyl)benzamide ([(123)I]BZA) or [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([(125)I]BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the highest cytotoxicity (IC(50), 0.71 and 0.64 µM respectively), which persisted in the presence of an MTL derived from N-(dialkylaminoalkylenyl)benzamide residue.

Design, synthesis and in vitro drug release investigation of new potential 5-FU prodrugs.

In order to identify new efficient prodrugs of 5-fluorouracil (5-FU) and to develop an original targeting approach using 2-fluoro-2-deoxyglucose (FDG) as a potential drug carrier, eight original 5-FU derivatives were synthesized: 5-FU was attached by the N1 position of the pyrimidinic ring to the C1 position of the FDG structure either by direct coupling (2a) or via various spacers (3, 6a-c, 10b and 19). A new sensitive high-performance liquid chromatography method was developed to simultaneously quantify 5-FU and its derivatives in human plasma and other relevant media at physiological temperatures. Half-lives were determined from the degradation profiles of these conjugates. Slow degradation of compounds 2a, 3, 10b and 19 was observed in vitro at 37 °C, but no 5-FU release was noticed. By contrast, the in vitro drug release profiles of compounds 6a-c followed pseudo-first-order kinetics, and 5-FU was found in all the media. The antiproliferative activity of the eight compounds was assessed in vitro by a fluorometric assay against two human solid cancer cell lines and one healthy cell line. A correlation was found between the activities of the compounds and their ability to release 5-FU efficiently.

Single photon emission computed tomography/positron emission tomography imaging and targeted radionuclide therapy of melanoma: new multimodal fluorinated and iodinated radiotracers.

This study reports a series of 14 new iodinated and fluorinated compounds offering both early imaging ((123)I, (124)I, (18)F) and systemic treatment ((131)I) of melanoma potentialities. The biodistribution of each (125)I-labeled tracer was evaluated in a model of melanoma B16F0-bearing mice, using in vivo serial γ scintigraphic imaging. Among this series, [(125)I]56 emerged as the most promising compound in terms of specific tumoral uptake and in vivo kinetic profile. To validate our multimodality concept, the radiosynthesis of [(18)F]56 was then optimized and this radiotracer has been successfully investigated for in vivo PET imaging of melanoma in B16F0- and B16F10-bearing mouse model. The therapeutic efficacy of [(131)I]56 was then evaluated in mice bearing subcutaneous B16F0 melanoma, and a significant slow down in tumoral growth was demonstrated. These data support further development of 56 for PET imaging ((18)F, (124)I) and targeted radionuclide therapy ((131)I) of melanoma using a single chemical structure.

Development of a high-performance liquid chromatographic method for the determination of a new potent radioiodinated melanoma imaging and therapeutic agent.

N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carbamide (ICF 01012) is a new melanoma imaging agent showing promising properties for application in internal radionuclide therapy. We developed an analytical protocol for detection of ICF 01012 in biological samples using HPLC. The proposed method was first validated using standard of ICF 01012 and four potent metabolites of this compound and then applied to follow the metabolic fate of [(125)I]ICF 01012 after injection in melanoma-bearing mice. The results demonstrate that this method exhibits a good linearity (r(2)=0.9947), specificity and acceptable accuracy. This simple method appears convenient and sufficient for pharmacokinetic studies on [(125)I]ICF 01012.

Evaluation of radiolabeled (hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma.

Targeted radionuclide therapy using radioiodinated compounds with a specific affinity for melanoma tissue is a promising treatment for disseminated melanoma, but the candidate with the ideal kinetic profile remains to be discovered. Targeted radionuclide therapy concentrates the effects on tumor cells, thereby increasing the efficacy and decreasing the morbidity of radiotherapy. In this context, analogues of N-(2-diethylaminoethyl)-4-iodobenzamide (BZA) are of interest. Various (hetero)aromatic analogues 5 of BZA were synthesized and radioiodinated with (125)I, and their biodistribution in melanoma-bearing mice was studied after i.v. administration. Most [ (125)I] 5-labeled compounds appeared to bind specifically and with moderate-to-high affinity to melanoma tumor. Two compounds, 5h and 5k, stood out with high specific and long-lasting uptake in the tumor, with a 7- and 16-fold higher value than BZA at 72 h, respectively, and kinetic profiles that makes them promising agents for internal targeted radionuclide therapy of melanoma.

Synthesis and cytotoxic properties of new fluorodeoxyglucose-coupled chlorambucil derivatives.

Frequently used in the treatment of malignant cells, alkylating agents, like most anticancer substances, produce adverse side effects caused by the toxicity of the agents toward normal tissues and lose efficiency through poor distribution to target sites. Our approach to developing more selective drugs with low systemic toxicity is based on the premise that the body distribution and cell uptake of a drug can be altered by attaching a neoplastic cell-specific uptake enhancer, such as 2-fluoro-2-deoxyglucose (FDG), the radiotracer most frequently used in PET for tumor imaging. Two properties of deoxyglucose, namely preferential accumulation in neoplastic cells and inhibition of glycolysis, underpin this targeting approach. Here, we report the synthesis of 19 new chlorambucil glycoconjugates in which the alkylating drug is attached to the C-1 position of FDG, directly or via different linkages. This set of compounds was evaluated for in vitro cytotoxicity against different human normal and tumor cell lines. There was a significant improvement in the in vitro cytotoxicity of peracetylated glucoconjugates compared with the free substance. Four compounds were finally selected for further in vivo studies owing to their lack of oxidative stress-inducing properties.

Synthesis, radiosynthesis, and biological evaluation of new proteasome inhibitors in a tumor targeting approach.

Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant melanoma cells. Of these, 10 displayed the highest cytotoxicity (0.18 +/- 0.16 microM). A radiosynthesis of the iodine aldehyde was performed. Its drug biodistribution showed that some selectivity of the benzamide group toward malignant melanoma tissue was conserved.

Mass spectrometry and scanning electron microscopy study of silicone tunneled dialysis catheter integrity after an exposure of 15 days to 60% ethanol solution.

Anti-infectious lock is an emerging therapeutic option for preventing and/or controlling catheter-associated infection. Ethanol has widespread bactericidal activity, limited side effects, and low risk of inducing antimicrobial resistance. However, concerns have been raised about ethanol-induced catheter structural degradation. In this study, silicone catheters were immersed at 37 degrees C in three different solvents: 0.9% sodium chloride, 60% ethanol, and 95% ethanol for 4 h, 15 days and 15 days after a first storage of 4 h. Scanning electron microscopy (magnification 1000-20 000 times) of the inner surface of the catheter revealed no damage to the lumen surfaces of catheters immersed in 95% ethanol for 15 days compared with the reference catheter. Gas chromatography/mass spectrometry (GC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the storage solutions revealed a significant release of polydimethylsiloxanes having a number of dimethylsiloxane units lower than 30 in the 95% ethanol solution and a structure highly consistent with a cyclic structure. Most release occurred within the first 4 h of exposure. In contrast, there was no difference in the small amounts of silicone released in 0.9% sodium chloride as reference and 60% ethanol solution, whatever the exposure time. These results should allow the development of clinical trials to assess the efficacy of the 60% ethanol lock technique in preventing or controlling the infectious complications of silicone dialysis catheters.

Preliminary studies of new proteasome inhibitors in the tumor targeting approach: synthesis and in vitro cytotoxicity.

The proteasome is a multicatalytic protease that plays a critical role in the cell. The control of proteasomes could, thus, provide a weapon for the treatment of cancer. Therefore, we have synthesized six new peptide aldehyde inhibitors of the proteasome linked to the N-(2-diethylaminoethyl)benzamide (BZA-CO) structure, in order to target the cytotoxic activity to malignant melanoma cells. Biological studies demonstrated the influence of length and composition of the amino acid chain on the cytotoxicity of our compounds. Among them, compound 19 presents the highest cytotoxicity (IC50 = 0.64 +/- 0.07 micromol): this cytotoxicity was maintained in the presence of BZA-CO but decreased 8-fold compared to the control MG132. Fluorescence activated cell sorter (FACS) and cytotoxic activity analysis demonstrated the selectivity of compound 19 for melanoma cells. Finally, western blottings of ubiquitinated proteins in IPC227F cells as well as proteasome assays confirmed that the cytotoxicity was linked to an inhibition of the proteasome activity.

Identification of degradation products of diclofenac by electrospray ion trap mass spectrometry.

The degradation products of diclofenac in aqueous dosage form in accelerated storage conditions were characterized by electrospray ionization-ion trap mass spectrometry (ESI-MS). Liquid chromatography (LC)-MS analyses revealed the presence of three degradation products. ESI-MS(n) spectra were used to study diclofenac fragmentation in detail and to characterize the structures of degradation products. A previously described degradation product, formed by a cyclization reaction of diclofenac producing the indolinone derivative, was found. As any hydroxylated product was found, no oxidation seems to occur in the dosage form used. On the contrary, two degradates have been detected and identified, leading to a primary alcohol structure or an aldehyde function in place of the acetate group of diclofenac.

Synthesis and in vivo biodisposition of [14C]-quaternary ammonium-melphalan conjugate, a potential cartilage-targeted alkylating drug.

For the purpose of developing more selective anticancer drugs that would concentrate in the malignant cartilaginous tumors (chondrosarcomas), and so improve therapeutic index through a reduction of side effects, a quaternary ammonium (QA) conjugate of melphalan was synthesized and labeled with (14)C by linking the QA moiety to nitrogen mustard via an amide bond. Comparative pharmacokinetic study of [(14)C]-melphalan and its [(14)C]-QA conjugate conducted on rats showed that the two compounds were principally excreted by the urinary way. The blood elimination of the QA conjugate was faster than that of the melphalan. In the other hand a higher rate of radioactivity derived of [(14)C]-MQA was found in feces. In the biodisposition for most organs, no striking differences were found between melphalan and its QA conjugate except for cartilages which exhibited more higher radioactivity level. Amounts of radioactivity derived from [(14)C]-QA conjugates measured in cartilaginous tissues until 1 h after injection demonstrate that the introduction of a QA moiety on melphalan allows the molecule to be carried selectively to cartilaginous tissues. As the [(14)C]-QA conjugate is radiolabeled on the chloroethyl alkylating moiety, levels of radioactivity measured in the cartilaginous tissues results from unchanged compound or metabolite having kept the active group.