Pharmacokinetic evaluation of gemcitabine and 2',2'-difluorodeoxycytidine-5'-triphosphate after prolonged infusion in patients affected by different solid tumors.

BACKGROUND: The study determined pharmacokinetic parameters, toxicity profile and preliminary clinical activity of gemcitabine administered i.v. at different infusion rates in patients with a range of solid tumors. PATIENTS AND METHODS: Twenty patients were enrolled for both pharmacokinetic and clinical studies. Gemcitabine 300 mg/m(2) was administered during 1 h, 2 h or 3 h, and as a conventional dose of 1000 mg/m(2) during 30 min infusion. Administration was on days 1, 8 and 15 every 4 weeks. RESULTS: Patients were randomly assigned to one of the four arms. After 30 min infusion of 1000 mg/m(2) gemcitabine the plasma concentration remained above the saturation level of 10-20 microM, whereas after 1, 2 or 3 h infusion 300 mg/m(2) gemcitabine it remained below the saturation level for most of the time (being in the range 2.5-10 microM). Gemcitabine triphosphate was determined in the four arms in white blood cells; for infusion times from 0.5 to 3 h there was a progressive enhancement of gemcitabine triphosphate levels. In all evaluable patients the toxicity was mild, myelosuppression being the main toxicity. No grade 3 or 4 toxicities occurred. Clinical response was similar in patients receiving 300 mg/m(2) gemcitabine in 2 and 3 h and in the 1000 mg/m(2) arm. CONCLUSIONS: 300 mg/m(2) gemcitabine during 3 h infusion produced the highest accumulation of gemcitabine triphosphate. Thus, to achieve the highest possible gemcitabine triphosphate level, prolonged infusion time would appear to be more important than a high dose administered as a short infusion. However, there was no substantial difference in toxicity or antitumoral activity in the all different patient groups.

Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: physical characterization.

Ultrasonic imaging is a widely available, noninvasive, and cost-effective diagnostic modality, but vessels smaller than 200 mum in diameter are impossible to visualize. Commercial ultrasound contrast agents (UCAs), consisting of encapsulated gas microbubbles injected intravenously, enable only a qualitative visualization of the microvascularization for a short period of time since they are rather unstable. In a strategy to develop more stable UCAs, we designed a process to obtain nano/microcapsules with a single core of liquid perfluorocarbons within a biodegradable polymeric shell of homogeneous thickness. The polymer shell should improve the stability of the capsules as compared to UCAs stabilized by a monomolecular layer, while the acoustic impedance of the perfluorocarbons should ensure their echogenicity. These capsules have been optimized to encapsulate several liquid perfluorocarbons: perfluorohexane, perfluorodecalin, and perfluorooctyl bromide. The system is rather versatile: the mean size of the capsules can be adjusted between 70 nm and 25 microm and the thickness-to-radius ratio (T/R) can be easily modulated by simply modifying the polymer-to-perfluorocarbon ratio. T/R does not depend on the size of the capsules and is between 0.2 and 0.6. The dependence of the echogenic properties of the capsules with their size and their T/R has yet to be studied experimentally before this system can be evaluated in vivo.

Clinical and pharmacokinetic phase II study of pegylated liposomal doxorubicin and vinorelbine in heavily pretreated recurrent ovarian carcinoma.

BACKGROUND: This multicenter phase II study evaluated feasibility, clinical efficacy, toxicity and pharmacokinetics of the combination of pegylated liposomal doxorubicin (PLD) and vinorelbine (VNR) in patients with platinum-paclitaxel pretreated recurrent ovarian cancer. PATIENTS AND METHODS: All patients received prior treatment with platinum and paclitaxel. Thirty-two heavily pretreated (median number of chemotherapy regimens two, range one to six) ovarian cancer patients received treatment with PLD 30 mg/m(2) and VNR 30 mg/m(2) every three weeks for six cycles. Ten patients entered the pharmacokinetic study, five receiving the PLD-VNR and five the VNR-PLD sequence. RESULTS: In 30 patients evaluated for response and toxicity, the overall response rate was 37% and 10% of patients achieved stable disease. Median time to progression and overall survival were 5.5 months (range 1-10) and 9 months (range 2-16), respectively. Toxicity was generally mild and reversible. VNR AUC(tot) and plasma levels were considerably higher in the PLD-VNR sequence. CONCLUSIONS: The PLD-VNR regimen exhibits significant activity in heavily pretreated patients, is well tolerated and is associated with encouraging survival. Preliminary pharmacokinetic results suggest the PLD-VNR sequence for further clinical applications. This regimen should be considered as a treatment option in patients with chemotherapy-resistant ovarian cancer.

From conventional to stealth liposomes: a new Frontier in cancer chemotherapy.

In this review, the different types of liposome used in medicine, in particular in the field of antitumor therapy, are focalised, emphasizing their structures, pharmacological action, pharmacokinetics and biodistribution, toxicity profiles and in the main clinical applications. The first-generation liposomes (conventional liposomes) comprised a liposome-containing amphotericin B, Ambisome, and Myocet, doxorubicin-containing liposome used in clinical trials to treat metastatic breast cancer. The last generation liposomes were pegylated liposomal doxorubicin (Caelix), called "stealth liposomes" because of their ability to evade interception by the immune system, characterized by very long-circulation half-life, favourable pharmacokinetic behaviour and specific accumulation in tumor tissues.

Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast.

Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.

A sequence-dependent combination of docetaxel and vinorelbine: pharmacokinetic interactions.

We studied possible pharmacokinetic interactions between docetaxel (DTX) and vinorelbine (VNR) in patients affected by different types of cancer. Patients with metastatic breast cancer or recurrent head and neck cancer received the following schedules: Protocol A: 11 patients were i.v. infused for 1 h with DTX (80 mg/m2) at once, followed by VNR (25 mg/m2) as slow i.v. bolus; Protocol B: VNR (25 mg/m2) as a slow 10 min i.v. bolus was administered to 12 patients, immediately followed by 1 h i.v. infusion of DTX (80 mg/m2). In both schedules, VNR and DTX plasma concentrations versus time were analysed by HPLC obtaining the corresponding non-compartmental pharmacokinetic parameters. VNR appeared pharmacokinetically affected by the sequential administration of DTX, since with protocol B, Cmax and AUC were significantly higher and clearance lower than in protocol A. Moreover, a significant increase in the VNR plasma level was observed in correspondence with the peak plasma level of DTX. By contrast, Cmax, AUC and clearance of DTX did not vary in the two protocols. Also the number of neutrophils at nadir on day 8 of treatment varied significantly in the two schedules. In conclusion we observed altered pharmacokinetic parameters between protocol A (DTX, VNR) and protocol B (VNR/DTX). In particular, patients following protocol B seemed to be exposed to higher VNR plasma concentration and to higher haematological toxicity.

A preliminary pharmacokinetic study of docetaxel, carboplatin and concurrent radiotherapy for regionally advanced squamous cell carcinoma of the head and neck.

This work investigates the pharmacokinetics and toxicity resulting from the concomitant use of low dose carboplatin (CBCA)/docetaxel (DTX) plus concurrent radiotherapy in patients with head and neck cancer. The study comprised 11 patients with stage III-IV head and neck cancer. All patients received 2 Gy radiotherapy daily, 5 fractions per week, up to a planned total of 70 Gy over 7 weeks. CBCA (AUC 0.4 mg/ml, min/day) was also administrated as 20 min i.v. infusion, starting 1 day before the first radiotherapy fraction. CBCA was administered for 5 consecutive days every 2 weeks (weeks 1, 3, 5 and 7). DTX 30 mg/m2 (1 h i.v. infusion) was given as a single dose on days 10, 24 and 38. CBCA on day 1 and DTX on day 10 were analysed to determine the concentration-time curves during the first 24 h. CBCA Cmax and Cmin in 2-5 days and on day 15 and 29, as well as total plasma platinum on days 2, 3, 4, 5, 29 and 43 were also assayed. By calculating the non-compartmental pharmacokinetic parameters of the two drugs from the available plasma concentrations we found in the first week values similar to those reported in the literature as single agents. In contrast, during subsequent weeks (weeks 3 and 5), a significant and progressive increase of platinum levels was observed. So, it could be assumed that after 2 weeks of CBCA and DTX treatment a bias in dose calculation occurred because the linear relationship between creatinine clearance (used to calculate the expected AUC through the Calvert formula) and CBCA clearance was no longer observed.

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.

Melphalan monitoring during hyperthermic perfusion of isolated limb for melanoma: pharmacokinetic study and 99mTc-albumin microcolloid technique.

BACKGROUND: The kinetics of melphalan leakage from extracorporeal fluid to the peripheral blood was studied in ten patients undergoing hyperthermic isolation perfusion of the lower limbs as an adjuvant treatment in high-risk melanoma. MATERIALS AND METHODS: Systemic leakage was monitored by a new technique using 99mTc-albumin microcolloid. Serial samples were drawn from a peripheral vein and from the perfusion circuit during surgical treatment and analysed by HPLC. RESULTS: The leakage measured with 99mTc-albumin microcolloid ranged from 1.5 to 18%/h (mean 8%/h). The average concentrations in the perfusate were 200-300-fold those found in the systemic circulation. A good correlation (R=0.945) was obtained between systemic AUC (0 to 1 hour) and leakage measured through the 99mTc procedure. Negligible toxicity was found and the survival rate yielded 92% of objective response. CONCLUSION: By studying the pharmacokinetic data of melphalan in the circuit and in the systemic circulation, we were able to validate the 99mTc procedure used during clinical perfusion. Moreover, considering the efficiency of the system as well as the minimum toxicity and the high survival rate, a reduction of perfusion time may be considered.

Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases.

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

Clinical and pharmacokinetic data of a docetaxel-epirubicin combination in metastatic breast cancer.

In order to explore activity and pharmacokinetic data of a docetaxel-epirubicin combination we analyzed a population of 60 metastatic breast cancer patients. All the patients had an ECOG performance status < 3; 41 patients (68%) had visceral metastases as dominant site of disease, including 33% with liver metastases. Three or more involved organs were present in 43% of patients; 35% had received prior hormonotherapy; 10% for metastatic disease. Twenty-five patients (42%) had received prior adjuvant chemotherapy; 15% a CAF regimen. Twenty per cent of patients had less than 12 months disease-free interval. Docetaxel and epirubicin were both given at a dose of 75 mg/m2 i.v. d. 1 every 3 weeks. After a median of six cycles we had 5 CR (8.3%), 40 PR (66.6%), 7 NC (11.6%), and 8 PD (13.3%). Response rates in patients with visceral and liver metastases were 78% and 55% respectively. Premenopausal status, < 1 year disease free survival and > 3 metastatic sites were associated with a lower response rate. After a median follow-up of 19 months (12-36), median disease-free survival is 11 months and median overall survival has not been reached. Grade 4 neutropenia was observed in 75% of courses but with febrile neutropenia in 6.2% of courses only. Non-hematologic toxicity wasn't clinically important. A NYHA class III reversible cardiac failure was observed in one patient (1.6%). The pharmacokinetic evaluation in 16 patients has shown that docetaxel transiently interfered with epirubicin plasma level when docetaxel was administered 1 h after epirubicin.

Design of folic acid-conjugated nanoparticles for drug targeting.

The new concept developed in this study is the design of poly(ethylene glycol) (PEG)-coated biodegradable nanoparticles coupled to folic acid to target the folate-binding protein; this molecule is the soluble form of the folate receptor that is overexpressed on the surface of many tumoral cells. For this purpose, a novel copolymer, the poly[aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H(2)NPEGCA-co-HDCA)] was synthesized and characterized. Then nanoparticles were prepared by nanoprecipitation of the obtained copolymer, and their size, zeta potential, and surface hydrophobicity were investigated. Nanoparticles were then conjugated to the activated folic acid via PEG terminal amino groups and purified from unreacted products. Finally, the specific interaction between the conjugate folate-nanoparticles and the folate-binding protein was evaluated by surface plasmon resonance. This analysis confirmed a specific binding of the folate-nanoparticles to the folate-binding protein. This interaction did not occur with nonconjugated nanoparticles used as control. Thus, folate-linked nanoparticles represent a potential new drug carrier for tumor cell-selective targeting.

Stereospecific syntheses of trans-vinyldioxidosqualene and 3-hydroxysulfide derivatives, as potent and time-dependent 2,3-oxidosqualene cyclase inhibitors.

trans-Vinyldioxidosqualene and beta-hydroxysulfide derivatives were synthesized stereospecifically and evaluated as inhibitors of animal and yeast oxidosqualene cyclases. Only trans-vinyldioxidosqualene and 2,3-epoxy-vinyl-beta-hydroxysulfides, having the reactive function at crucial positions 14,15 and 18,19, were active as inhibitors of animal and yeast cyclases. (14-trans)-28-Methylidene-2,3: 14,15-dioxidoundecanorsqualene 27 was the most potent inhibitor of the series of pig liver cyclase, with an IC50 of 0.4 microM, and it behaved also as the most active time-dependent inhibitor of the animal enzyme.

Pharmacokinetic monitoring of mycophenolate mofetil in kidney transplanted patients.

Mycophenolate mofetil (MMF) is a new immunosuppressant drug used in association with cyclosporin and oral corticosteroids to prevent acute rejection following renal allograft transplantation. MMF is an ester pro-drug of mycophenolic acid (MFA), the true active species, into which it is completely transformed after oral administration. The recommended initial dose to prevent kidney transplant rejection is 2 g/day irrespective of body weight, 1 g twice daily. The goal of this study was to correlate dosage (fixed or by body weight) and toxic effects to some non-compartmental values such as peak level (Cmax), time to peak level (Tmax) and trough level (Cmin). In a small number of patients who had already reached the plasma steady state, we found a large inter-patient variability, while the same qualitative pharmacokinetic profile (as Tmax) was conserved. At plasma trough level > 4 microg/ml some serious toxic effects were observed, whereas at Cmin < 2 microg/ml, there were some cases of interstitial rejection. There was also a negative correlation between dosage and body weight, suggesting that dosages related to body weight might be better than fixed ones. Finally, monitoring plasma level of drug from transplantation to at least 12 months after surgery, at fixed MFA dosage, a small but significant decline of MFA plasma levels was found.

Rationally designed inhibitors as tools for comparing the mechanism of squalene-hopene cyclase with oxidosqualene cyclase.

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drug's antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 2'-succinyl, 2'-methylpyridinium acetate and 2'-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 2'-mPEG (5000)-paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mgml(-1)). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t(1/2)beta, plasma lifetime, which was longer in liposomes containing 2'-mPEG (5000)-paclitaxel.

19-Azasqualene-2,3-epoxide and its N-oxide: metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae.

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7+/-2 and 25+/-5 microM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [14C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 microM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and >100 microM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in "ultrasonically stimulated" cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.

Systematic analysis of yeast strains with possible defects in lipid metabolism.

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild-type strains, among them FY1679, CEN.PK2-1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism.

Synthesis of novel 4,5-diphenylthiazole derivatives as potential acyl-CoA:cholesterol O-acyltransferase inhibitors.

Several novel N-(4,5-diphenylthiazol-2-yl)-N'-aryl or alkyl (thio)ureas and N-(4,5-diphenylthiazol-2-yl)alkanamides were prepared as potential acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors. Synthesis was accomplished by reaction of 2-amino-4,5-diphenylthiazole with the suitable isocyanate, isothiocyanate or acyl chloride. Some analogues without the 5-phenyl substituent or both the phenyl groups in 4 and 5 position of the thiazole ring were also prepared. Moreover, some bioisosters of the title compounds in which the thiazole ring was replaced by an imidazole were synthesized starting from the 2-amino-4,5-diphenyl-1H-imidazole. The ability of synthesized compounds to inhibit ACAT was evaluated in vitro by measuring the formation of cholesteryl[14C]oleate from cholesterol and [1-14C]oleoyl-CoA in rat liver microsomes. Among the tested compounds, only some thiazole ureas were able to inhibit ACAT in a reasonable degree. N-(4,5-diphenylthiazol-2-yl)- N'-[2,6-bis(2-methylethyl)phenyl] urea (11) and N-(4,5-diphenylthiazol-2-yl)-N'-n-butyl urea (16) were the most active compounds in the series showing IC50 values in the low micromolar range.