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.

First preclinical imaging of primary cartilage neoplasm and its local recurrence using 99mTc-NTP 15-5 radiotracer.

UNLABELLED: This study on a rat model of grade II chondrosarcoma aimed to determine whether the radiotracer N-(triethylammonium)-3-propyl-[15]ane-N5 radiolabeled with (99m)Tc ((99m)Tc-NTP 15-5), which binds to cartilage proteoglycans, has pathophysiologic validity for in vivo imaging of cartilage tumoral tissue. METHODS: We used 2 experimental approaches with the Swarm chondrosarcoma rat model: that is, a primary paratibial location and local recurrence after intralesional curettage. (99m)Tc-NTP 15-5 scintigraphy and (99m)Tc-hydroxymethylenediphosphonate ((99m)Tc-HMDP) scanning were performed at regular intervals during 50 d after tumor implantation in a paratibial location (primary model; n = 12 animals) and after intralesional curettage in a femoral condyle location (recurrence model; n = 9 animals). For each animal, positive scans were analyzed at each time point using the target-to-background ratio (TBR), with the target region of interest delineated over the tumor and the background region of interest over muscle. In each model, the TBR time course was followed against primary tumoral growth or recurrence. Tumor volume was monitored for 2 mo by measuring the 2 perpendicular diameters. At study end, animals were sacrificed for histopathologic analysis. RESULTS: For both models, (99m)Tc-NTP 15-5 scans showed tracer accumulation at the site of implantation or curettage. For the primary tumor model, the mean TBR was 1.6 +/- 0.14 by day 4 after implantation and increased over time as the disease progressed, with a mean TBR of 4.25 +/- 0.25 on day 45. For the recurrence model, mean TBR was 3.27 +/- 0.24 by day 4 after curettage and increased with recurrence, with a mean value of 5.25 +/- 0.49 on day 50. (99m)Tc-HMDP bone scans were negative for both models throughout the study; at a later stage of the study, an area of (99m)Tc-HMDP accumulation was seen in the diaphysis of the bone adjacent to the tumor and was attributed to remodeling. CONCLUSION: These experimental results in 2 preclinical models of grade II chondrosarcoma bring forward data in favor of (99m)Tc-NTP 15-5 radiotracer for imaging primary growth of chondrosarcoma and its local recurrence after surgery.

A new O6-alkylguanine-DNA alkyltransferase inhibitor associated with a nitrosourea (cystemustine) validates a strategy of melanoma-targeted therapy in murine B16 and human-resistant M4Beu melanoma xenograft models.

Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O(6)-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O(6)-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.

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.

A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

BACKGROUND: Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). RESULTS: MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.

Design, synthesis and preliminary biological evaluations of novel amphiphilic drug carriers.

The synthesis of a new fluorocarbon amphiphilic drug carrier is described. A polyfunctional amino acid endowed with a fluorocarbon chain and a sugar moiety providing the amphiphilic character constitutes the central element of this structure. A (14)C-radiolabelled acetyl group was grafted onto the third function and the bioavailability of this molecule was specified in mice after IV administration. This amphiphilic drug carrier exhibits a rapid and homogeneous distribution to the whole tissues and slow elimination half-lives (higher than one day) through a biliary excretion without any toxicity (no measured DL 50 for concentrations up to 500 mg/kg).

Synthesis and pharmacokinetic profile of a quaternary ammonium derivative of chlorambucil, a potential anticancer drug for the chemotherapy of chondrosarcoma.

As a part of our targeting program based on the affinity of the quaternary ammonium moiety for cartilage, our objective was to develop more selective anticancer drugs towards chondrosarcoma that would concentrate in this malignant cartilaginous tissue and so improve the therapeutic index through a reduction of side effects. For this purpose we have synthesized and labeled with 14C a quaternary ammonium (QA) derivative of chlorambucil. Biological studies performed in rats showed that [14C]-CQA and [14C]-chlorambucil exhibited different pharmacokinetic profiles. The blood elimination of [14C]-CQA was faster than that of parent compound. [14C]-CQA was principally excreted by the fecal way. However, until 15 min after administration, levels of radioactivity were measured in cartilaginous tissues of rats given [14C]-CQA which was not the case for the rats which had received [14C]-chlorambucil. Although rates of radioactivity were quantified only during 15 min, these results prove that the functionalization of chlorambucil by a quaternary ammonium group allows the molecule to be carried selectively to cartilaginous tissues.

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.

Simulation of neutron interactions at the single-cell level.

We recently reported that the exposure of cancer cells to 14 MeV neutrons at a very low dose rate (0.8 mGy min(-1)) produced a marked increase in cell killing at 5 cGy, followed by a plateau in survival and chromosomal damage. Simulation of the energy deposition events in irradiated cells may help to explain these unusual cell responses. We describe here a Monte Carlo simulation code, Energy Deposition in Cells Irradiated by Neutrons (EDCIN). The procedure considered the experimental setup and a hemispheric cell model. The simulation data fitted the dosimetric measurements performed using tissue-equivalent ionization chambers, Geiger-Müller counters, fission chambers, and silicon diodes. The simulation showed that 80% of the energy deposited in a single cell came from the interactions of neutrons outside the cell and only 20% came from neutron interactions inside the cell. Thus the "external" interactions that result in the production of recoil protons and secondary electrons may induce most of the biological damage, which may be repaired efficiently at low dose rate. The repair process may be triggered from a threshold level of damage, which would explain an initial increase cell death due to unrepaired sublethal damage, and then may compensate for induced damage, resulting in the plateaus.

Synthesis and in vitro evaluation of quaternary ammonium derivatives of chlorambucil and melphalan, anticancer drugs designed for the chemotherapy of chondrosarcoma.

To enhance affinity for malignant cartilaginous tumors (chondrosarcomas), quaternary ammonium (QA) conjugates of chlorambucil and melphalan were prepared by linking the QA moiety to nitrogen mustards via an amide bond. They exhibited closely similar and sometimes more favorable values than their parent compounds. In the cell lines tested, the two QA conjugates displayed appreciable cytotoxicity, the QA conjugate of chlorambucil even showing an enhanced efficiency against chondrosarcoma compared with chlorambucil.

Comparisons of carboplatin and cisplatin as potentiators of 5-fluorouracil and radiotherapy in the mouse L1210 leukaemia model.

BACKGROUND: Carboplatin (CBDCA), an analogue of cisplatin (CDDP), has synergistic antitumour activity in combination with 5-fluorouracil (5-FU) and radiotherapy and is better tolerated than CDDP by patients. In an in vivo model, we evaluated the doses of CBDCA and CDDP required to obtain, in these combinations, equal curative effects. MATERIALS AND METHODS: Groups of 6-week-old B6D2F1 mice were injected with L1210 leukaemia. The chemotherapy was administered 24 hours after L1210 inoculation, while the radiotherapy was performed 1 or 24 hours later. Treatment results were evaluated in terms of prolonged survival. RESULTS: The same degree of potentiation was obtained at the dose ratio CBDCA/CDDP of 4 in association with 5-FU and 3 in combination 5-FU/radiotherapy, with a longer delay between CBDCA administration and radiotherapy. CONCLUSION: CBDCA could be used as a potentiator of 5-FU and radiosensitizer at a daily dose of 20 mg/m2 in clinical therapy.

Synthesis and preliminary biological assessments of RGD bearing biocompatible telomers.

Work reported herein deals with the synthesis and preliminary biological assessments of a new class of biocompatible telomeric carriers bearing peptidic RGDSK sequences as tumor cell targeting and tyrosine moieties labelled with 125I as in vivo probe. The radioactivity levels obtained in several tissues, after the intravenous injections of these telomers in mice bearing grafted B16 syngenic melanoma showed that the addition of a RGD residue to a telomeric structure confers it an increased affinity for the highly vascularized zone surrounding the tumor.