Farnesyltransferase inhibitors (FTIs) represent a new class of signal transduction inhibitors that block the processing of cellular polypeptides that have cysteine terminal residues and, by so doing, interdict multiple pathways involved in proliferation and survival of diverse malignant cell types. Tipifarnib is an orally bioavailable, nonpeptidomimetic methylquinolone FTI that has exhibited clinical activity in patients with myeloid malignancies including elderly adults with acute myelogenous leukemia (AML) who are not candidates for traditional cytotoxic chemotherapy, patients with high-risk myelodysplasia, myeloproliferative disorders, and imatinib-resistant chronic myelogenous leukemia. Because of its relatively low toxicity profile, tipifarnib provides an important alternative to traditional cytotoxic approaches for elderly patients who are not likely to tolerate or even benefit from aggressive chemotherapy. In this review, we will focus on the clinical development of tipifarnib for treatment of newly diagnosed AML, both as induction therapy for elderly adults with poor-risk AML and as maintenance therapy following achievement of first complete remission following induction and consolidation therapies for poor-risk AML. As with all other malignancies, the optimal approach is likely to lie in rational combinations of tipifarnib with cytotoxic, biologic and/or immunomodulatory agents with non-cross-resistant mechanisms of action. Gene expression profiling has identified networks of differentially expressed genes and gene combinations capable of predicting response to single agent tipifarnib. The clinical and correlative laboratory trials in progress and under development will provide the critical foundations for defining the optimal roles of tipifarnib and in patients with AMl and other hematologic malignancies.

OBJECTIVE

The optimal radiofrequency (RF) power and lesion duration using contact force (CF) sensing catheters for atrial fibrillation (AF) ablation are unknown. We evaluate 50 W RF power for very short durations using CF sensing catheters during AF ablation.

METHODS

We evaluated 51 patients with paroxysmal (n = 20) or persistent (n = 31) AF undergoing initial RF ablation.

RESULTS

A total of 3961 50 W RF lesions were given (average 77.6 ± 19.1/patient) for an average duration of only 11.2 ± 3.7 s. As CF increased from < 10 to>> 40 g, the RF application duration decreased from 13.7 ± 4.4 to 8.6 ± 2.5 s (p < 0.0005). Impedance drops occurred in all ablations, and for patients in sinus rhythm, there was loss of pacing capture during RF delivery suggesting lesion creation. Only 3% of the ablation lesions were at < 5 g and 1% at>> 40 g of force. As CF increased, the force time integral (FTI) increased from 47 ± 24 to 376 ± 102 gs (p < 0.0005) and the lesion index (LSI) increased from 4.10 ± 0.51 to 7.63 ± 0.50 (p < 0.0005). Both procedure time (101 ± 19.7 min) and total RF energy time (895 ± 258 s) were very short. For paroxysmal AF, the single procedure freedom from AF was 86% at 1 and 2 years. For persistent AF, it was 83% at 1 year and 72% at 2 years. There were no complications.

CONCLUSIONS

Short duration 50 W ablations using CF sensing catheters are safe and result in excellent long-term freedom from AF for both paroxysmal and persistent AF with short procedure times and small amounts of total RF energy delivery.

The purpose of this study is to clarify the effects of intestinal drug supersaturation on solubility-limited nonlinear absorption. Oral absorption of a novel farnesyltransferase inhibitor (FTI-2600) from its crystalline free base and its HCl salt was determined in dogs. To clarify the contribution of supersaturation on improving drug absorption, in vivo intraluminal concentration of FTI-2600 after oral administration was estimated from the pharmacokinetics data using a physiologically based model. Dissolution and precipitation characteristics of FTI-2600 in a biorelevant media were investigated in vitro using a miniscale dissolution test and powder X-ray diffraction analysis. In the in vitro study, the HCl salt immediately dissolved but precipitated rapidly. The metastable amorphous free base precipitant, which did not convert into the stable crystalline free base in the simulated intestinal fluids for several hours, generated a 5-fold increase in dissolved concentration compared to the equilibrium solubility of the crystalline free base. By computer simulation, the intraluminal drug concentration after administration of the free base was estimated to reach the saturated solubility, indicating solubility-limited absorption. On the other hand, administration of the HCl salt resulted in an increased intraluminal concentration and the plasma concentration was 400% greater than that after administration of the free base. This in vivo/in vitro correlation of the increased drug concentrations in the small intestine provide clear evidence that not only the increase in the dissolution rate, but also the supersaturation phenomenon, improved the solubility-limited absorption of FTI-2600. These results indicate that formulation technologies that can induce supersaturation may be of great assistance to the successful development of poorly water-soluble drugs.

The present work evaluated the key enzymes involved in xylitol production (xylose reductase [XR] and xylitol dehydrogenase [XDH]) and their correlation with xylose, arabinose, and acetic acid assimilation during cultivation of Candida guilliermondii FTI 20037 cells in sugarcane bagasse hemicellulosic hydrolysate. For this purpose, inocula previously grown either in sugarcane bagasse hemicellulosic hydrolysate (SBHH) or in semidefined medium (xylose as a substrate) were used. The highest xylose/acetic acid consumption ratio (1.78) and the lowest arabinose consumption (13%) were attained in the fermentation using inoculum previously grown in semidefined medium (without acetic acid and arabinose). In this case, the highest values of XR (1.37 U mg prot(-1)) and XDH (0.91 U mg prot(-1)) activities were observed. The highest xylitol yield (approximately 0.55 g g(-1)) and byproducts (ethanol and glycerol) formation were not influenced by inoculum procedure. However, the cell previously grown in the hydrolysate was effective in enhancing xylitol production by keeping the XR enzyme activity at high levels (around 0.99 U.mg(prot) (-1)), reducing the XDH activity (34.0%) and increasing xylitol volumetric productivity (26.5%) with respect to the inoculum cultivated in semidefined medium. Therefore, inoculum adaptation to SBHH was shown to be an important strategy to improve xylitol productivity.

Increasing evidence shows that exosomes are key regulators in cancer cell-to-cell communication. Several reports on Epstein-Barr virus (EBV)-related malignancies demonstrate that latent membrane protein 1 (LMP1) secreted by exosomes derived from EBV- or LMP1-positive cells can promote cancer progression and metastasis. However, the mechanism by which LMP1 is loaded into exosomes is still poorly understood. Here, we examined whether the process of LMP1 loading into exosomes is linked to the multifunctional molecule of the ubiquitin system-ubiquitin C-terminal hydrolase-L1 (UCH-L1). For the first time, we demonstrate that LMP1 is physically associated with UCH-L1 and that directing of LMP1 to exosomes is mediated by C-terminal farnesylation of UCH-L1. Additionally, we found that the FTI-277 farnesyltransferase inhibitor reduces motility- and anchorage-independent growth of EBV-positive cells in functional assays. On the basis of our results, we conclude that C-terminal farnesylation of UCH-L1 is one of the key mechanisms by which LMP1 is sorted to exosomes. We hypothesize that inhibition of farnesylation with specific small-molecule inhibitors blocks exosome-mediated transfer of prometastatic molecules such as LMP1 during cancer cell-to-cell communications and thereby impedes the process of cancer invasion. IMPORTANCE Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion.

Imatinib mesylate (Gleevec, Novartis Pharmaceuticals Corp, East Hanover, NJ; Glivec, Novartis Pharma AG, Basel, Switzerland), a signal transduction inhibitor with preferential effects against the tyrosine kinase activity of the protein product of the ABL proto-oncogene, induced hematologic responses in>>or=90% of patients with chronic-phase chronic myeloid leukemia (CML). In Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), the BCR-ABL translocation is the main transforming event, making it another hematologic malignancy targeted by this ABL-tyrosine kinase inhibitor. In an international multicenter phase II trial, imatinib-induced hematologic responses (typically brief) were achieved in 60% of patients with relapsed or refractory Ph(+) ALL. Subsequently, the German Multicenter Study Group for Adult ALL (GMALL) analyzed 59 patients treated in two successive nonrandomized phase II trials of imatinib in patients with relapsed or refractory Ph(+) ALL. Peripheral blood blasts cell clearance occurred within 8 to 14 days in most patients. However, in a significant proportion, blast counts subsequently increased 16 to 50 days after treatment onset. Imatinib mesylate was particularly effective in patients with relapse after stem cell transplantation (SCT); 75% of patients achieved complete leukemic response. Rapid development of resistance during treatment with imatinib mesylate remains a major problem. Further research efforts should explore the mechanisms of resistance to imatinib mesylate; effectiveness of other targeted therapies (eg, farnesyl transferase inhibitors [FTIs]); combination therapies; and inclusion of strategies for immune response modification (eg, donor lymphocyte infusions, interferon-alpha) for Ph/BCR-ABL-positive leukemias.

The mechanism of action of farnesyltransferase inhibitors (FTIs) has not been fully clarified. We investigated the cytotoxic effects of various FTIs in chronic myeloid leukemia (CML), using LAMA cells and marrow cells from 40 CML patients in chronic phase. FTI-mediated cytotoxic effect was observed in LAMA cells and in 65% of primary CML cells, whereas marrow cells from controls were only weakly affected. Cytotoxic effects were partially related to enhanced apoptosis; however, Fas-receptor (FasR) and Fas-ligand (FasL) expression were not modified by FTIs. Susceptibility to FTI-mediated inhibition did not correlate with FasR/FasL expression in CD34+ CML cells. Moreover, intra-cellular activation of caspase-1 and -8 were not altered by FTIs, and their blockade did not reverse FTI toxicity. However, we observed FTI-induced activation of caspase-3, and its inhibition partially reverted FTI-induced apoptosis. FTIs did not modulate bcl2, bclxL, and bclxS expression, whereas they increased inducible nitric oxide (iNOS) mRNA and protein levels, resulting in higher NO production. Furthermore, C3 exoenzyme, a Rho inhibitor, significantly increased iNOS expression in CML cells, suggesting that FTIs may up-regulate NO formation at least partially through FTI-mediated inhibition of Rho. We conclude that FTIs induce selective apoptosis in CML cells via activation of iNOS and caspase-3.

Although the prognosis of pediatric leukemias has improved considerably, many patients still have relapses. Tipifarnib, a farnesyl transferase inhibitor (FTI), was developed to target malignancies with activated RAS, including leukemia. We tested 52 pediatric acute myeloid leukemia (AML) and 36 pediatric acute lymphoblastic leukemia (ALL) samples for in vitro sensitivity to tipifarnib using a total cell-kill assay and compared these results to those obtained with normal bone marrow (N BM) samples (n = 25). AML samples were significantly more sensitive to tipifarnib compared to B-cell precursor ALL (BCP ALL) or N BM samples. Within AML, French-American-British (FAB) M5 samples were most sensitive to tipifarnib. T-cell ALL samples were significantly more sensitive than BCP ALL and N BM samples. In AML there was a marked correlation between tipifarnib resistance and daunorubicin or etoposide resistance, but not to cytarabine or 6-thioguanine. RAS mutations were present in 32% of AML and 18% of ALL samples, but there was no correlation between RAS mutational status and sensitivity to tipifarnib. Future studies are needed to identify biomarkers predictive of tipifarnib sensitivity. In addition, clinical studies, especially in T-cell ALL, seem warranted.

HGPS (Hutchinson-Gilford progeria syndrome) is a rare genetic disease affecting children causing them to age and die prematurely. The disease is typically due to a point mutation in the coding sequence for the nuclear intermediate-type filament protein lamin A and gives rise to a dominant-negative splice variant named progerin. Accumulation of progerin within nuclei causes disruption to nuclear structure, causes and premature replicative senescence and increases apoptosis. Now it appears that accumulation of progerin may have more widespread effects than previously thought since the demonstration that the presence and distribution of some nucleolar proteins are also adversely affected in progeria cells. One of the major breakthroughs both in the lamin field and for this syndrome is that many of the cellular defects observed in HGPS patient cells and model systems can be restored after treatment with a class of compounds known as FTIs (farnesyltransferase inhibitors). Indeed, it is demonstrated that FTI-277 is able to completely restore nucleolar antigen localization in treated progeria cells. This is encouraging news for the HGPS patients who are currently undergoing clinical trials with FTI treatment.

Advances in our understanding of the molecular pathways and genetic mutations that control tumor cell proliferation and metastasis present an opportunity to develop novel, mechanism-based therapeutic strategies. Ras mutations are the most frequently activated oncogenes in human tumors, with over 30% expressing ras mutations. Molecular dissection of the signaling pathway and the mechanisms of ras anchorage, post-translational modification, and downstream effector signaling of ras now under intensive investigation will help us to design additional methods for ras-directed therapy in an effort to reach an optimal treatment for human tumors that will most likely comprise a combination of modalities targeted at the different underlying genetic defects. The successes and limitations of ras-targeted therapies must be viewed in light of the increasing understanding of the complexity of the ras-signaling pathway. Only now are we beginning to discover the many functions of this integrated pathway, such as the differences between the actions of various ras isoforms that may affect our choice of therapeutic approach. Many of these Ras therapeutic targets have shown success in preclinical studies, and some have shown efficacy in clinical trials with minimal toxicities. Compounds that block ras-transforming activity without affecting normal ras function seem more attractive for the future development of ras-targeted therapy. FTIs may partially fulfill such requirements. Based on their specific, novel, and mechanism-based action; minimal toxicity; and encouraging responses in clinical trials, the development of Ras therapeutic targets as single agents or in combination with conventional chemotherapy and radiotherapy should be pursued.

The efficacy of 131I therapy in achieving euthyroidism has been studied in a group of 264 patients followed for up to 10 yr. One hundred and eighty-six were given a dose adjusted for thyroid size and radioactive iodine uptake (Protocol 1), and a second group received the same dosage followed by antithyroid drug therapy plus potassium iodide for 15 days (Protocol 2). At 10-yr follow-up, 50-60% of patients were euthyroid. 25-29% of patients required 2 doses of 131I, and 4-5% required 3 doses. Fewer patients became hypothyroid when their pretreatment FTI was above the average value. More patients became hypothyroid, if their pretreatment test for antimicrosomal antibodies was positive. Patients who required a second dose of radioactive iodide had a significantly greater chance of having worsening of their ophthalmopathy than those who became hypothyroid after the first dose. Treatment with radioactive iodide under either protocol appears to achieve euthyroidism at 10 yr with an incidence higher than that achieved by antithyroid drugs and comparable to that reported for subtotal thyroidectomy.

Complete endocrinologic evaluation of 9 women (ages 24-41) with idiopathic melasma (melasma not associated with pregnancy nor ingestion of oral contraceptives) was performed and compared to age- and sex-matched normal controls. Serum cortisol, adrenocorticotropin, plasma immunoreactive alpha and beta melanocyte-stimulating hormones, luteinizing hormone, follicular-stimulating hormone, estradiol and progesterone levels were performed in the basal state. Additionally, total T4, T3RU, FTI, prolactin, 2-h postprandial blood sugar, and 24-h urine for 17-hydroxysteroids and 17-ketosteroids were done and found to be normal. The melasma patients presented statistically significant increased levels of LH (p less than 0.001) and lower levels of serum estradiol (p less than 0.025) than normal controls. It is proposed that these hormonal alterations may represent subclinical evidence of a mild ovarian dysfunction which may underlie the pathogenesis of some cases of idiopathic melasma.

Like Ras, farnesylation of the IP (prostacyclin receptor) is required for its efficient intracellular signalling, and hence the IP represents a potential target for inhibition by FTIs [FTase (farnesyl protein transferase) inhibitors]. Herein, the effect of SCH66336 on the isoprenylation and function of the human and mouse IPs overexpressed in human embryonic kidney 293 cells, and by the IP endogenously expressed in human erythroleukaemia cells, was investigated. SCH66336 yielded concentration-dependent decreases in IP-mediated cAMP generation (IC50 0.27-0.62 nM), [Ca2+]i mobilization (IC50 26.6-48.3 nM) and IP internalization, but had no effect on signalling by the non-isoprenylated beta2 adrenergic receptor or b isoform of the TP (prostanoid thromboxane A2 receptor). Additionally, SCH66336 impaired IP-mediated crossdesensitization of TPa signalling (IC50 56.1 nM) and reduced farnesylation of the molecular chaperone protein HDJ-2 (IC50 3.1 nM). To establish whether farnesylation of the IP is inhibited and/or whether its 'CaaX motif' might undergo alternative geranylgeranylation in the presence of SCH66336, a series of chimaeric Ha (Harvey)-Ras fusions were generated by replacing its CaaX motif (-CVLS) with that of the IP (-CSLC) or, as controls, of Ki (Kirsten)-Ras 4B (-CVIM) or Rac 1 (-CVLL). Whereas SCH66336 had no effect on Ha-RasCVLL isoprenylation in vitro or in whole cells, it supported alternative geranylgeranylation of Ha-RasCVIM, but completely impaired isoprenylation of both Ha-RasCVLS and Ha-RasCSLC. These data confirm that the -CSLC motif of the IP is a direct target for inhibition by the FTI SCH66336, and in the presence of strong FTase inhibition, the IP does not undergo compensatory geranylgeranylation

The shortage of geranylgeranyl-pyrophosphate (GGPP) was associated to an increased IL-1β release in the autoinflammatory syndrome mevalonate kinase deficiency (MKD), a rare inherited disease that has no specific therapy. Farnesyltransferase inhibitors (FTIs) act at the end of mevalonate pathway. Two FTIs, tipifarnib (Tip) and lonafarnib (Lon), were therefore evaluated as possible therapeutical choices for the treatment of MKD. FTIs could lead to a redirection of the limited available number of mevalonate intermediates preferentially to GGPP synthesis, eventually preventing the uncontrolled inflammatory response. The effect of Tip and Lon on intracellular cholesterol level (ICL) and on proinflammatory cytokines secretion was evaluated in a cellular model of MKD, chemically obtained treating RAW 264.7 cells with lovastatin (Lova) and alendronate (Ald). The combination of FTIs with the isoprenoid geraniol (GOH) was also tested both in this model and in monocytes isolated from MKD patients. Tip and Lon proved to revert the ICL lowering and to significantly reduce the lipopolysaccharide-induced cytokines secretion in Ald-Lova -RAW 264.7 cells. This anti-inflammatory effect was amplified combining the use of GOH with FTIs. The effect of GOH and Tip was successfully replicated in MKD patients' monocytes. Tip and Lon showed a dramatic anti-inflammatory effect in monocytes where mevalonate pathway was chemically or genetically impaired.

Our recent findings on Rheb and eIF4E address key questions of translational control in cancer and have implications for tumor therapy.(1) Briefly, we find that Rheb a proximal activator of mTORC1 and protein translation can cooperate with c-Myc in tumorigenesis in vivo in a manner resembling Akt or the oncogenic eIF4E translation initiation factor. Rheb is highly expressed in some human lymphomas as well as other cancers and likely contributes to malignancies in different tissues.(2) The cancer-relevant activities emanating from increased Rheb depend on activation of mTORC1 and are sensitive to rapamycin. Moreover, farnesyltransferase inhibitors (FTIs) can directly block Rheb activity and this is responsible for the therapeutic effect of these drugs in certain tumors. We will discuss here how translational control mechanisms contribute to oncogenesis and speculate on the potential and limitations of targeting these co-operating oncogenic events for therapy.

OBJECTIVE

Protein isoprenylation is an important step in the intracellular signalling pathway conducting cell growth and differentiation. In bone, protein isoprenylation is required for osteoclast differentiation and activation. However, its role in osteoblast differentiation and function remains unknown. In this study, we assessed the role of protein isoprenylation in osteoblastogenesis in a model of mesenchymal stem cells (MSC) differentiation.

METHODS

We tested the effect of an inhibitor of farnesylation [farnesyl transferase inhibitor-277 (FTI-277)] and one of geranylgeranylation [geranylgeranyltransferase inhibitor-298 (GGTI-298)] on osteoblast differentiating MSC. In addition, we tested the effect of alendronate on protein isoprenylation in this model either alone or in combination with other inhibitors of isoprenylation.

RESULTS

Initially, we found that levels of unfarnesylated proteins (prelamin A and HDJ-2) increased after treatment with FTI-277 concomitantly affecting osteoblastogenesis and increasing nuclear morphological changes without affecting cell survival. Furthermore, inhibition of geranylgeranylation by GGTI-298 alone increased osteoblastogenesis. This effect was enhanced by the combination of GGTI-298 and alendronate in the osteogenic media.

CONCLUSIONS

Our data indicate that both farnesylation and geranylgeranylation play a role in osteoblastogenesis. In addition, a new mechanism of action for alendronate on protein isoprenylation in osteogenic differentiating MSC in vitro was found. In conclusion, protein isoprenylation is an important component of the osteoblast differentiation process that could constitute a new therapeutic target for osteoporosis in the future.

Oxidized low-density lipoprotein (Ox-LDL) might be involved in the progression of renal disease. Ox-LDL stimulation of plasminogen activator inhibitor-1 (PAI-1) expression via transforming growth factor-beta (TGF-beta)/Smad signaling in mesangial cells required activation of extracellular signal-regulated kinase (ERK). Mevalonate depletion by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, decreases the levels of farnesyl pyrophosphate (FPP) for isoprenylation of Ras. We postulate that statins may ameliorate the Ox-LDL-induced mesangial matrix accumulation by inhibiting Ras/ERK activation with subsequent downregulation of TGF-beta target genes. Quiescent mesangial cells were incubated for 18 h with and without the presence of lovastatin before 50 microg/mL of Ox-LDL treatment for 1 h. Lovastatin inhibited markedly the stimulatory effects of Ox-LDL on ERK1/2 activation, nuclear Smad3 expression, TGF-beta1 and PAI-1 mRNA and protein expression, and PAI-1 luciferase activity. These inhibitory effects of lovastatin were reversed almost completely by mevalonate or FPP. Similar to lovastatin, FTI-277, which is an inhibitor of Ras farnesylation, decreased the Ox-LDL-induced activation of ERK/Smad3 and induction of TGF-beta1/PAI-1. These results indicate that lovastatin prevents the Ox-LDL-induced Ras/ERK activation that results in inhibition of Smad3 activation in mesangial cells with subsequent downregulation of TGF-beta target genes. Thus, statins seem to have antifibrotic effects through their anti-TGF-beta response that are relevant in the treatment of chronic renal disease with dyslipidemia.

Ras is a member of a super-family of guanine-binding or G-proteins. Ras functions as a molecular switch in the transduction of signals generated by the activation of a variety of cell surface receptors and relays the signals to downstream effectors. Little is known about signal transduction in schistosomes. In order for Schistosoma mansoni to survive different immune responses triggered by the host as well as to migrate from the site of penetration at the skin to the final destination in portal circulation, they must receive signals from the host environment and respond to them in a way that allows their survival. We have isolated the schistosome Ras cDNA by using sequence information of the schistosome Ras homologue submitted to the Genbank database. Analysis of the encoded peptide revealed 81% identity and 92% similarity with K-Ras from various species. Ras is a single copy gene as determined by quantitative hybridization experiments. The cDNA was cloned into pGEX-4T and the expressed peptide was used to generate specific antibody reagents. Affinity purified antibodies identified a 23 kDa native protein that localizes to the subtegument. Ras is not associated with the tegument. Ras is expressed in all the developmental stages of the parasite. However, Ras is over-expressed in female worms compared to males. Schistosome Ras was also shown to be post-translationally modified by addition of farnesyl isoprenoid moiety to the cysteine residue in the C-terminal box. Using a schistosome extract in vitro SmRas farnesylation was inhibited by the farnesyl transferase inhibitor, FTI-277, at concentrations comparable to those required to inhibit K-Ras processing. These initial studies on signal transduction in schistosomes should provide a solid basis for improving our understanding of schistosome-host interactions.

Hexachlorobenzene (HCB) residue levels in dosed rats (50.0 mg kg-1 body wt.day-1, n = 9) were significantly (P < 0.05) greater in the periovarian fat compared to the thyroid gland. Hexachlorobenzene residue levels were significantly (P < 0.05) greater in the thyroid versus the adrenal and ovary. Ovarian HCB residue levels were greater than those found in the thymus, liver and lung. Serum thyroxin (T4) and the free T4 index (FTI) were significantly (P < 0.05) suppressed in HCB-treated rats compared to the control group (n = 8). In contrast, no significant differences in serum concentrations of oestradiol (E2), progesterone (P4) or percentage triiodothyronine uptake (%T3) were observed, thus suggesting an HCB-induced hypothyroid-like state. In a second experiment, adult female Sprague Dawley rats (n = 16) were dosed as above and superovulated with pregnant mare serum gonadotrophin (PMSG, 10 IU s.c.) and human chorionic gonadotrophin (hCG, 20 IU s.c.). Circulating levels of P4 were significantly (P < 0.05) elevated compared to the control group (n = 8). The %T3 uptake and serum T4 levels were significantly (P = 0.05) suppressed compared to controls. Hexachlorobenzene treatment had no effect on circulating levels of E2 or on the FTI. These results suggest that HCB-induced changes found in the spontaneously cycling rat are augmented by ovulation induction strategies. We also conclude that HCB concentrates in the endocrine tissues in addition to the fat.

Nineteen patients with essential hypertension (EH) were studied as outpatients. After administration of chlorthalidone, 50 mg/day for 4 weeks, prazosin 1-4 mg/day (1.82 +/- 0.33 mg/day) was added for a period of 12 weeks. Prazosin lowered supine blood pressure from 149.7 +/- 2.85/102.0 +/- 2.75 mm Hg to 128.2 +/- 3.0/86.1 +/- 1.04 mm Hg (p less than 0.001). Prazosin did not alter heart rate significantly. Prazosin increased the thyroid stimulating hormone (TSH) from 3.63 +/- 0.33 microunits/ml to 4.83 +/- 0.45 microunits/ml (p less than 0.025), thyroxine (T4) from 10.03 +/- 0.29 micrograms/ml to 10.85 +/- 0.42 micrograms/ml (p less than 0.005), and decreased triiodothyronine (T3) from 36.65 +/- 0.62% to 35.42 +/- 0.56%, which was not significant. The free thyroxine index (FTI) increased slightly from 3.67 +/- 0.12 to 3.83 +/- 0.14 (p less than 0.025). However, all values remained within the normal range for the laboratory. Serum cholesterol increased insignificantly. Triglycerides decreased significantly from 223.4 +/- 50.6 mg/dl to 161.7 +/- 29.0 mg/dl (p less than 0.05). High density lipoproteins (HDL) increased significantly from 30.1 +/- 2.1% to 36.0 +/- 3.06 (p less than 0.025). Low density lipoproteins (LDL) decreased insignificantly and very low density lipoproteins (VLDL) decreased from 20.9 +/- 3.44 to 16.3 +/- 2.85 (p less than 0.005). The cholesterol ratio increased from 45.51 +/- 4.3 to 64.71 +/- 10.7 (+42.1%). These results indicate that, in patients with essential hypertension, prazosin is an effective antihypertensive agent and that it significantly increases HDL, decreases VLDL, and improves the cholesterol ratio.

Epothilones are a new class of natural products that bind to tubulin and prevent the depolymerization of microtubules, although they have no structural similarity to paclitaxel. Taxanes are only marginally effective in the treatment of disseminated prostate cancer, although they may have useful activity when administered in combination with estramustine. Unlike paclitaxel, epothilones are not substrates for P-glycoprotein and are active in multidrug resistant cells. Epothilones A and B (EA, EB) have recently been synthesized in toto. In this report, we examine the effects of synthetic epothilones and their desoxy derivatives, as well as paclitaxel, on prostate cancer cell lines. EB was the most active of these compounds in tissue culture (IC(50): 50-75 pM), four to ten-fold more potent than paclitaxel. EA and the desoxyderivatives of EA and EB (dEA, dEB) were also active, but less potent than EB. Each of these compounds causes mitotic block followed by apoptotic cell death. The relative potencies for cell cycle arrest and cytotoxicity directly correlate with the ability of the drugs to bind microtubules, stabilize mitotic spindles and induce the formation of interphase microtubule bundles. Therefore, synthetic epothilones are potent inhibitors of prostate cancer cell lines and work in a fashion similar to paclitaxel. Recently, we showed that farnesyl transferase inhibitors sensitize tumor cells to paclitaxel-induced mitotic arrest. We now have extended these observations to show that paclitaxel and the epothilones synergize with FTI to arrest the growth of prostate cancer cells. Moreover, this occurs in DU145, a cell line that is not particularly sensitive to the FTI. The combination of FTI and epothilone represent a new potential clinical strategy for the treatment of advanced prostatic cancer.

As deregulation of RAS signaling is important in the pathogenesis of myeloid leukemias, molecular targeting of RAS signaling may be a promising therapeutic strategy. Farnesyl transferase inhibitors (FTIs) are the most promising class of these new cancer therapeutics. Several FTIs have entered phase II clinical trials in acute myeloid leukemia (AML). Since geranylgeranylation of K-RAS and N-RAS in the presence of FTIs may represent an important mechanism of FTI resistance, 6 geranylgeranyl transferase-I inhibitors (GGTIs) were screened alone and in combination with FTI for growth inhibition of myeloid leukemia cells. Significant growth inhibition (>70%) in myeloid cell lines was observed for GGTI-286 (9/19), GGTI-298 (14/19), GGTI-2147 (16/19) and FTI L-744,832 (17/17). GGTI treatment of NB-4 cells resulted in an accumulation of cells in G(0)/G(1), whereas FTI L-744,832 primarily caused an increase in G(2)/M. FTI and GGTIs both induced apoptosis. In all cases, FTI/GGTI cotreatment led to synergistic cytotoxic effects in both myeloid cell lines (5/5) and primary AML cells (6/6). This synergy coincided with increased apoptosis. FTI/GGTI cotreatment caused an accumulation of unprocessed N-RAS and inactive N-RAS-RAF complexes. Our results suggest that alternative geranylgeranylation of N-RAS may represent an important mechanism of resistance to FTI monotherapy in myeloid leukemia cells.

The effect of acetic acid concentration on xylose-fermentation to xylitol by Candida guilliermondii FTI 20037 was evaluated in semisynthetic medium containing different concentrations of the acid. Increasing acetic acid concentration up to 1.0 g/l favored xylitol yield and productivity, with maximum values of 0.82 g/g and 0.57 g/l.h, respectively. The presence of acetic acid reduced cell production at all concentration. Furthermore, acetic acid was assimilated by the yeast together with the sugars and was depleted from the medium at concentrations of less than 3.0 g/l. The ability of this yeast to assimilate acetic acid suggests that these cells act as agents of medium detoxification. This behavior may lead to a viable microbiological process of xylitol production by C. guilliermondii FTI 20037 using xylose-rich lignocellulosic hydrolysates in which acetic acid is commonly present, causing inhibition of fermentative activity.

Farnesyltransferase inhibitors (FTIs) target multiple pathways including the Ras pathway implicated in the pathogenesis of some hematologic malignancies. R115777 and BMS-214662, selective FTIs in clinical development, exhibit preclinical activity against cell lines and tumor xenografts with or without ras mutations. Phase I dose-escalating trials at M.D. Anderson Cancer Center have explored the potential of these agents as monotherapy for leukemias and myelodysplastic syndrome (MDS). In 20 patients with MDS, two cycles of oral R115777 for 3 consecutive weeks followed by a 1-week rest produced an overall response rate of 30%, consistent with 29% reported in poor-prognosis acute leukemia or blast-phase chronic myelogenous leukemia (CML). Administration of BMS-214662 as a weekly intravenous infusion produced a decrease in bone marrow blasts of greater than 50% in 23% of patients with acute leukemia or MDS; 18% achieved normalization of blast counts to less than 5%. In both studies, most responding patients did not have ras mutations. The most common side effects at maximum tolerated doses of R115777 (400 mg twice daily) and BMS-214662 (118 mg/m(2) weekly) were myelosuppression and nausea, respectively. Further evaluation of FTIs for hematologic malignancies clearly is warranted. Future research should address whether molecular techniques can identify patients most likely to respond to an FTI, optimal administration schedules for these agents, and the value of incorporating an FTI into combination regimens for difficult-to-treat hematologic malignancies.