An improved automated one-pot synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) based on a purification by cartridges.

INTRODUCTION: O-(2-[18F]Fluoroethyl)-L-tyrosine ([18F]FET) is an established radiotracer used for oncology investigations by Positron Emission Tomography (PET). Main limitations to its widespread use are the synthesis itself (time; cost; radiochemical yield; complexity) and a troublesome and time-consuming HPLC purification. Aim of this work was to improve the preparation overall efficiency and, most important, to achieve an efficient and reliable purification by means of disposable cartridges. METHODS: [18F]FET was synthesized by direct nucleophilic radiofluorination of O-(2-tosyloxy-ethyl)-N-trityl-L-tyrosine t-butylester (TET) followed by acid hydrolysis with HCl. Several conditions and materials were tested for the synthesis and purification step. For the latter, a number of different commercial cartridges, varying in amount, particulate size and adsorbent, were examined. Best results were obtained by a combination of STRATA-X, tC18 and QMA cartridges. RESULTS: Starting from only 5 mg of TET, up to 11 GBq of injectable solutions of [18F]FET were produced within 36 min with 54-65% radiochemical yields and radiochemical purities >99%. No D-form was observed by chiral HPLC. Chemical purity was 1-2 order of magnitude below the limits imposed by the European Pharmacopoeia's monograph on [18F]FET. A radiochemical purity decrease by radiolysis, observed only on relatively large batches of [18F]FET, was efficiently suppressed by preloading in the receiving final vial a small amount of ethanol (<2% v/v). CONCLUSIONS: By combining improvements to a known synthetic route with a novel cartridge-based purification, [18F]FET was obtained in a very efficient and reproducible way. The whole process was easily implemented on a commercial automated module presently used for [18F]FDG production. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: A few drawbacks regarding the HPLC conditions recommended in the European Pharmacopoeia were highlighted. An alternative method able to cope with them is herein proposed The simplified preparation herein described is expected to encourage a more widespread clinical use of [18F]FET.

Infants vertically infected with hepatitis C: a long term longitudinal follow-up.

HCV vertically acquired infection is asymptomatic and characterized by a high chronic infection rate; only 9% of HCV infected children shows spontaneous remission. As far as a mild course of the disease has been observed during childhood, we hypothesize that any eventual treatment intervention could be postpone until adolescent age.

[Immunological development in asymptomatic HIV infected children]. / Follow-up immunologico di bambini con infezione asintomatica da HIV.

In HIV infected children, CD45+CD4+ T lymph. reconstitution has been related to efficient thymopoiesis. Because human thymus undergoes spontaneous involution at a relatively young age, institution of antiretroviral therapy early in the course of infection has been recommended. 12 HIV vertically infected children aged 4-8 years were investigated T-cell subsets for four years. 7 children were naive for therapy (group NT); 5 experienced nucleoside analogues only (group T). CD45RA+ and CD45RO+ CD4+ values were compared to predicted values of healthy children. The two groups showed similar clinical and immunological baseline characteristics (CDC class N-A). Mean VL at t0 was 4.26 log10 (SD 0.71) in gr. NT and 4.01 log10 (SD 0.57) in gr. T; median CD4 T lymph values were 27% in gr. NT and 23.5% in gr. T. Median CD45RA+ values were 62.8% in gr. NT and 71.3% in gr. T. No differences in VL, CD4+ T lymph., CD45RA+, CD45RO+ were found in between groups or within each group at each time evaluation. Median CD45RA+ values were not different from predicted values of healthy children. None of the children changed CDC class during the study period. Although the number of subjects was small, our study evidenced the possibility of a normal immunological development in HIV-1 vertically infected asymptomatic children naive for HAART during the first decade, even in the presence of significant viremia.

Proposal of a step-wise follow-up for hepatitis C seropositive mothers and their infants.

Mother-to-child transmission of hepatitis C virus can take place in utero, during labour or after birth. Rate of vertical transmission varies widely between surveys but is around 5-6%. Maternal risk factors which may condition perinatal transmission risk are HIV/HCV coinfection, drug use, viral load, viral genotype, type of delivery and breastfeeding. On the basis of recent data, we propose a step-wise follow-up for HCV seropositive mothers and their infants. This proposal might represent an important occasion to unify behaviors in different Obstetrics-Gynecology and Neonatology Units.

[Sedation with magnetic resonance imaging in pediatric patients]. / Tracheostomia in età pediatrica.

219 children (ASA I-II, age 30 days-12 yrs), underwent deep sedation with intravenous thiopental for magnetic resonance imaging in a hospital setting. Sedation strategies and monitoring are described. The procedure showed to be safe for the patients and cause low artefacts by movements.

A 1-year trial of lamivudine for chronic hepatitis B in children.

We carried out a 1-year trial to evaluate the efficacy and tolerability of lamivudine, an oral nucleoside analogue, in a small group of children with vertically acquired chronic hepatitis B. Patients were assessed for serum alanine aminotransferase (ALT) and serum hepatitis B virus (HBV) DNA at baseline and every 4 weeks thereafter, and for hepatitis B s antigen, hepatitis B e antigen and their antibodies every 12 weeks. Analysis of HBV mutation was undertaken at entry and on the occasion of the last positive control of HBV DNA. Lamivudine suppressed serum HBV DNA to undetectable levels in all treated patients within 24 weeks. Serum ALT levels returned to normal values within 36 weeks. Therapy was well tolerated, and although nausea and vomiting were reported in one child, it was not necessary to stop treatment. A new observation was that, contrary to previous data, seroconversion appeared to occur earlier in children with lower ALT levels at baseline.

[Bispectral index: clinical effectiveness and role in reducing anesthetic drug consumption]. / Bispectral index: efficacia clinica e ruolo nella riduzione del consumo di farmaci anestetici.

A numeric value derived from bispectral analysis of EEG, the bispectral index (BIS), has been recently introduced as a monitor of the hypnotic component of anaesthesia. Application of BIS monitoring in anaesthesia appears extremely interesting to drugs titration and drugs economy, and in the evaluation of time for discharge in a one day surgery regimen. In this prospective study ASA I-II patients for Day-Surgery were enrolled in three groups: subaracnoid anaesthesia (SA), general anaesthesia (GA) with BIS and GA without BIS monitoring. At present 35 patients for DS operative hysteroscopy were enrolled. No significant differences were measured between the GA groups. A longer time of recovery was necessary in the SA group. A greater amount of anaesthetic in BIS group could be attributed to a better monitoring of sedation which prevents intraoperative awakening.

Methotrexate and gamma-tert-butyl methotrexate transport in CEM and CEM/MTX human leukemic lymphoblasts.

In a continuing investigation of determinants of their 200-fold methotrexate resistance and their collateral sensitivity to gamma-tert-butyl methotrexate, the ability of CEM/MTX cells to transport the two drugs was analyzed and compared with that of CEM cells. The Km and Vmax values for the influx of methotrexate into CEM cells did not differ significantly from those of CEM/MTX cells, and this was the case for gamma-tert-butyl methotrexate as well. Surface binding and influx rates were proportional to cell surface area, but differences in efflux rates and methotrexate uptake were too large to be explained on this basis. Neither methotrexate nor trimetrexate competed with gamma-tert-butyl methotrexate influx in CEM cells. However, both drugs perturbed the gamma-tert-butyl methotrexate steady state in CEM cells, resulting in slightly less uptake than with gamma-tert-butyl methotrexate alone. However, the major difference between the two cell types was in the methotrexate uptake plateau, which was much greater in the case of the parental cell line. A related observation was the more rapid efflux of methotrexate from CEM/MTX cells than from CEM cells. The poor uptake, the associated meager capacity to polyglutamylate methotrexate and the enhanced methotrexate efflux appear to be responsible for its decreased activity against CEM/MTX cells. Half-lives for gamma-tert-butyl methotrexate efflux were the same in both cell lines, allowing the drug to accumulate to cytotoxic levels despite its inability to form polyglutamates.

Utilization of phytohaemagglutinin for in vivo karyological studies in teleost fish.

A simple technique is described for increasing the number of mitoses in circulating blood and in the kidney of teleost fish. This method employed phytohaemagglutinin-P solution, a T-like cell mitogen, which was injected (1 mg/100 g body wt) intraperitoneally, and 53 h later many blasts suitable for karyological studies, blocked in metaphase, were present both in the circulating blood and in the kidney.

[Utilization of CoCl2 for karyological studies in teleosts]. / Utilizzazione del CoCl2 per studi cariologici nei Teleostei.

This paper presents a simple method of increasing the number of mitoses in the kidney of fish. This method is based on the erythropoietic activity of CoCl2. The specimens of Ictalurus sp. were injected intraperitoneally with 2 mg CoCl2 per 100 g body weight. After 24 h the slides of kidney show a proliferation of erythroblasts. A large number of these cells are arrested in metaphase for karyotype preparations.

[Effect of DDT on hepatocytes in Ictalurus sp. and Carassius carassius]. / Azione del DDT sugli epatociti di Ictalurus sp. e Carassius carassius.

During fifteen days low dosages of DDT on two different LC50-teleost fishes (Ictalarus sp. and Carassius carassius) were studied. Treatment with DDT, after three days, induces in Carassius hepatocytes a loss of glycogen granules, an increase of ergastoplasm, a mitochondrial swelling and wide cytoplasmic vacuoles. In Ictalurus there is no glycogen loss, but an increase of ergastoplasm and a great increase of lipidic inclusions are observed.

Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity.

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination of N,N',N"-triethylenethiophosphoramide and cyclophosphamide in vitro and in vivo.

In vitro and in vivo studies with the drug combination thioTEPA and cyclophosphamide (CPA) were carried out using the MCF-7 human breast carcinoma cell line and the EMT6 mouse mammary carcinoma cell line. In vitro, survival curves were essentially linear. The EMT6 cell line was less sensitive to thioTEPA than the MCF-7 cell line, with concentrations which reduce cell survival to 10% of 440 and 140 microM, respectively. The response of both cell lines to 4-hydroperoxycyclophosphamide was similar. Simultaneous and immediate sequential treatments with these drugs produced supraadditive cell killing of both cell lines, although the magnitude of the supraadditivity was greater in the MCF-7 cell line than in the EMT6 cell line. Both of these drugs appeared to be as effective as thiol-depleting agents as is diethyl maleate. By DNA alkaline elution, there was a pattern of increasing DNA cross-linking similar to the increasing levels of cytotoxicity of this drug combination with increasing thioTEPA concentrations. In the EMT6 tumor in vivo, the maximally tolerated combination therapy (5 mg/kg x 6 thioTEPA and 100 mg/kg x 3 CPA) produced about 25 days of tumor growth delay which was not significantly different than expected for additivity of the individual drugs. The survival of EMT6 tumor cells after treatment of the animals with various single doses of thioTEPA and CPA was assayed. Tumor cell killing by thioTEPA produced a very steep, linear survival curve through 5 logs. The tumor cell survival curve for CPA out to 500 mg/kg gave linear tumor cell kill through almost 4 logs. In all cases, the combination treatment tumor cell survivals fell well within the envelope of additivity. Both of these drugs are somewhat less toxic toward bone marrow cells by the granulocyte-macrophage colony-forming unit in vitro assay method than to tumor cells. The combination treatments were subadditive or additive in bone marrow granulocyte-macrophage colony-forming unit killing. When bone marrow is the dose-limiting tissue, there is a therapeutic advantage to the use of this drug combination.

Collateral methotrexate resistance in cultured human head and neck carcinoma cells selected for resistance to cis-diamminedichloroplatinum(II).

A human head and neck squamous cell carcinoma line (SCC25) derived from a patient with no prior history of radiotherapy or chemotherapy was made resistant to cis-diamminedichloroplatinum(II) (CDDP) by continuous escalation of weekly 30-min pulses of the CDDP from 0 to 0.2 mM over 20 months and then cloned and pulsed weekly with 0.2 mM CDDP for another 20 months. This afforded a resistant subline, SCC25/CP[1], with an IC50 for CDDP 12-fold higher than that of the parental cells. The SCC25/CP[1] cells unexpectedly proved to be cross-resistant to methotrexate (MTX) (24-fold for 30-min treatment and 8-fold for continuous treatment). Resistance was associated with a modest (about 2-fold) increase in the dihydrofolate reductase (DHFR) content according to radioligand-binding assay, and in the rate of cell division. In addition there was a 4-fold decrease in the fraction of long-chain MTX polyglutamates MTX(G4-6) in the cell after 24 h exposure to either 0.2 or 2.0 microM MTX. When the SCC25/CP[1] cells were kept out of CDDP for 8-9 months and 12 months to give the sublines SCC25/CP[2] and SCC25/CP[3], respectively, MTX sensitivity to continuous exposure returned to normal. The SCC25/CP[3] cells still exhibited a slightly elevated DHFR level, but their generation time became shorter than that of the parental SCC25 line. In addition the SCC25/CP[3] cells had an initial uptake velocity (V0) for MTX that was 9-fold greater than the V0 of the SCC25 or SCC25/CP[1] cells, while its ability to form MTX(G4-6) was comparable to that of the SCC25 cells. When SCC25/CP[2] cells were rechallenged with weekly 0.2 mM CDDP pulses for 4-6 months, a MTX-resistant line, SCC25/CP[4], was produced. The SCC25/CP[4] cells retained a slightly elevated DHFR content and a high proliferation rate, but the V0 for MTX influx was intermediate between SCC25 and SCC25/CP[3] cells. The ability to form the longer-chain polyglutamates MTX(G4-6) was again impaired. Thus, MTX cross-resistance can develop in cultured head and neck carcinoma cells when CDDP is used as the selecting agent for primary resistance. MTX resistance is multifactorial, as it is when MTX itself is used as the selecting agent, and appears to involve various combinations of altered growth rate, DHFR content, MTX uptake, and ability to form noneffluxing long-chain MTX polyglutamate species. These results are potentially of clinical relevance, since CDDP and MTX are often used in combination with other drugs or with radiation to treat patients with squamous cell carcinoma of the head and neck.

Metabolism of methotrexate and gamma-tert-butyl methotrexate by human leukemic cells in culture and by hepatic aldehyde oxidase in vitro.

The cellular uptake and metabolism of methotrexate (MTX) and gamma-tert-butyl methotrexate (TBM) were compared in CEM human leukemic lymphoblasts and a highly MTX-resistant subline (CEM/MTX) in which MTX uptake is defective. The CEM/MTX cells were found previously to be as sensitive as the parent line to TBM. While MTX was polyglutamylated extensively in the CEM cells, giving abundant levels of non-effluxing conjugates, polyglutamylation in CEM/MTX cells was reduced severely, even after exposure to a high MTX concentration (100 microM) in the medium. This treatment provided free intracellular MTX in greater than 100-fold excess over the dihydrofolate reductase level. In contrast to MTX, the ester TBM was unmetabolized in either cell line. Uptake levels after incubation of CEM and CEM/MTX cells with 2 microM TBM for 24 hr were 17 and 15 pmol/mg protein respectively. Thus, TBM accumulated equally in both cells and was well retained despite the lack of polyglutamylation. These results, together with the previously observed affinity of the drug for dihydrofolate reductase, provide a plausible rationale for the comparable sensitivity of CEM and CEM/MTX cells to TBM. Experiments were also performed to determine the susceptibility of TBM to metabolic detoxification by hepatic aldehyde oxidase. Km values were 8-fold lower for TBM than for MTX in assays using an enzyme preparation from rabbit liver, and Vmax values were 8-fold higher. Neither MTX nor TBM was oxidized to its 7-hydroxy derivative in intact CEM or CEM/MTX cells. Because TBM is capable of overcoming at least one of the modalities of MTX resistance, defective polyglutamylation, and may be more efficiently detoxified than MTX by the action of hepatic aldehyde oxidase, it has the potential to be a useful agent for the treatment of MTX-resistant tumors.