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Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors gamma-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

Background and Objectives: In neuraxial anesthesia, increase of skin temperature is an early sign of successful block. Yet, during peripheral nerve block of the lower extremity, increase in skin temperature is a highly sensitive, but late sign of a successful block. We hypothesized that after interscalene brachial plexus block, a rise in skin temperature follows impairment of sensation during successful nerve block and occurs only distally, as observed in the lower extremity., Methods: In the present study, we prospectively evaluated the changes in skin temperature after interscalene brachial plexus blockade in 45 patients scheduled for elective shoulder surgery. We assessed pinprick and cold sensation as well as skin temperature at sites of the skin innervated by the median, ulnar, radial, axillary and musculocutaneous nerve., Results: At the skin areas innervated by the axillary and musculocutaneous nerve, skin temperature did not increase after successful block. At the distal sites, innervated by the median, ulnar, and radial nerve, skin temperature increased significantly (1.9-2.1 degrees C within 30 min) after successful block while it did not after failed nerve block or on the contralateral side. In these areas attenuation of skin sensation preceded a measurable rise in skin temperature (> or =1 degrees C) in 56.3% of nerve blocks, occurred at the same time in 35.2%, and in 8.5% the temperature rise occurred first., Conclusions: Assessment of skin temperature cannot predict the success of an interscalene brachial plexus block of the axillary and musculocutaneous nerve. Distally, the increase of skin temperature has a high sensitivity and specificity but occurs later than the loss of sensory and motor functions. Therefore, the measurement of skin temperature during interscalene blockade is of limited clinical value.

Objective: Stimulating catheters have been introduced into clinical practice to confirm perineural localization of the catheters. The muscular twitch induced over the catheter may be used to evaluate nerve function intraoperatively. Therefore, the function of the sciatic nerve was evaluated during major cancer surgery of the femur., Case Report: A 7-year-old boy (29 kg) was scheduled for hip rotationplasty for resection of an osteosarcoma of the left femur under general anesthesia and postoperative pain therapy with an epidural stimulating catheter. In hip rotationplasty the femur is resected, the lower limb and foot are rotated 180 degrees and the tibia plateau is attached to the pelvic acetabulum to form a new hip joint. During preparation of the left thigh and the sciatic nerve, motor responses to stimulation of the catheter were preserved, but the stimulation threshold increased. After vascular anastomosis the foot remained cold, therefore ropivacaine was applied epidurally and subsequently a warming of the foot was observed. At the end of the operation, the patient was free of pain, a good capillary pulse of the leg was observed, and the patient was able to move the foot and toes of the rotated leg., Conclusions: The use of epidural stimulating catheters as a tool to monitor nerve function is a novel and simple procedure to monitor nerve function intraoperatively and to enable good postoperative pain control.

Background: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis., Methods: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells., Results: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells., Conclusions: Apoptosis is triggered by concentrations of lidocaine occurring intrathecally after spinal anesthesia, whereas higher concentrations induce necrosis. The data indicate that death receptors are not involved in lidocaine-induced apoptosis. In contrast, the observation that B-cell lymphoma-2 protein overexpression or the lack of caspase 9 abolished apoptosis clearly implicates the intrinsic mitochondrial death pathway in lidocaine-induced apoptosis.

The pentacyclic acridinium methosulfate salt RHPS4 induces the 3'single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase to the telomere and thus G-quadruplex ligands can effectively inhibit both the catalytic and capping functions of telomerase. In this study, we examined mechanisms underlying telomere uncapping by RHPS4 in uterus carcinoma cells (UXF1138L) with short telomeres and compared the susceptibility of bulk and clonogenic cancer cells to the G-quadruplex ligand. We show that treatment of UXF1138L cells with RHPS4 leads to the displacement of the telomerase catalytic subunit (hTERT) from the nucleus, induction of telomere-initiated DNA-damage signalling and chromosome fusions. We further report that RHPS4 is more potent against cancer cells that grow as colonies in soft agar than cells growing as monolayers. Human cord blood and HEK293T embryonic kidney cell colony forming units, however, were more resistant to RHPS4. RHPS4-treated UXF1138L xenografts had a decreased clonogenicity, showed loss of nuclear hTERT expression and an induction of mitotic abnormalities compared with controls. Although single-agent RHPS4 had limited in vivo efficacy, a combination of RHPS4 with the mitotic spindle poison Taxol caused tumour remissions and further enhancement of telomere dysfunction.

Interaction of 2-iodoaniline or 5-fluoro-2-iodoaniline with a range of arylsulfonyl chlorides affords sulfonamides that undergo Sonogashira couplings under thermal or microwave conditions with the alkyne 4-ethynyl-4-hydroxycyclohexa-2,5-dien-1-one followed by cyclization to 4-[1-(arylsulfonyl-1H-indol-2-yl)]-4-hydroxycyclo-hexa-2,5-dien-1-ones. This method allows for incorporation of a range of substituents into the arylsulfonyl moiety, and compounds showed selective in vitro inhibition of cancer cell lines of colon and renal origin, a feature of compounds bearing the quinol pharmacophore.

Background: Intraoperative monitoring of the spinal cord via cortical somatosensory-evoked potentials (SSEP) is a routine during spinal surgery. However, especially in neuromuscular scoliosis, the reliability of cortical SSEP has been questioned. Therefore, we compared the feasibility of cortical SSEP in idiopathic and neuromuscular scoliosis using anaesthetics known to have only minimal effect on SSEP recordings., Methods: Total intravenous anaesthesia with propofol and remifentanil as continuous infusion was standardized for all the patients. Median and tibial nerve cortical SSEP were monitored in 54 patients who underwent surgery for spinal deformity. Twenty-seven had idiopathic scoliosis and 27 had neuromuscular scoliosis. The portion of reproducible results and intraoperative changes were compared between the groups., Results: In both groups, cortical SSEP could be monitored with sufficient reliability. Only in two patients with idiopathic and four patients with neuromuscular scoliosis no reproducible traces could be obtained. The amplitudes in patients with neuromuscular scoliosis were lower than in those with idiopathic scoliosis, but not statistically significant. There were no postoperative neurological deficits. The number of false positive and true positive did not differ between the groups., Conclusions: Assessment of cortical SSEP during spine surgery was equally effective and reliable in patients with neuromuscular scoliosis and in patients with idiopathic scoliosis, possibly as a result of propofol-remifentanil anaesthesia.

Regeneration of peripheral nerves involves an essential contribution by surrounding tissues. This study focuses on the role of the target tissue on the regeneration of afferent peripheral nerves. We hypothesized that nerves implanted into the appropriate target tissue regain their function, whereas they degenerate when implanted into a different tissue. Therefore, aortic nerves of rabbits were transected and implanted into arteries or veins, and their function and structure was reevaluated after 1.5, 3, and 10 months. In a subset of animals, the nerves were again severed and implanted into the other vessel. Twelve of 18 nerves implanted into arteries regained typical neurophysiological activity, but none of those implanted into veins. Two times even baroreflexes were elicited through the newly built nerve endings. The structure of the nerve endings implanted into arteries resembled baroreceptors, whereas no fiber growth was detected in veins. Morphometrically, the fiber number and diameter increased over the observed time period after implantation into arteries. Nerves implanted into veins, transected after 3 months, and then implanted into arteries also regained neurophysiological activity. Again, they rebuilt baroreceptors and significantly increased their fiber number and diameter. In conclusion, when severed baroafferents are implanted into arteries, they regenerate new baroreceptors and restore the normal myelination and fiber size of the nerve over time, whereas veins seem to inhibit nerve fiber sprouting and regeneration of severed fibers.

The plant Andrographis paniculata found throughout Southeast Asia contains Andrographolide 1, a diterpenoid lactone, which has antitumour activities against in vitro and in vivo breast cancer models. In the present study, we report on the synthesis of andrographolide derivatives, 3,19-isopropylideneandrographolide (2), 14-acetyl-3,19-isopropylideneandrographolide (3) and 14-acetylandrographolide (4), and their in vitro antitumour activities against a 2-cell line panel consisting of MCF-7 (breast cancer cell line) and HCT-116 (colon cancer cell line). Compounds 2 and 4 were also screened at the US National Cancer Institute (NCI) for their activities against a panel of 60 human cancer cell lines derived from nine cancer types. Compound 2 was found to be selective towards leukaemia and colon cancer cells, and compound 4 was selective towards leukaemia, ovarian and renal cancer cells at all the dose-response parameters. Compounds 2 and 4 showed non-specific phase of the cell cycle arrest in MCF-7 cells treated at different intervals with different concentrations. NCI's COMPARE and SOM mechanistic analyses indicated that the anticancer activities of these new class of compounds were not similar to that of standard anticancer agents, suggesting novel mechanism(s) of action.

Background: In this prospective, randomized, double-blind trial we investigated the use of stimulating catheters in patients during and after shoulder surgery; functional improvement being the primary outcome measurement., Methods: After eliciting an adequate muscular twitch at < or =0.5 mA nerve stimulation output, the perineural catheter was advanced either blindly (conventional catheter = CC group, n = 20) or guided by stimulation via the catheter (stimulating catheter = SC group, n = 20). A bolus of 40 mL prilocaine 1% and 10 mL ropivacaine 0.75% was injected, followed by a patient-controlled infusion of ropivacaine 0.2% (8 mL/h infusion rate, bolus 2 mL, lockout time 20 min)., Results: Onset of motor block was faster in the SC group, whereas sensory block did not differ between groups. Median pain scores on two postoperative days were equal. Improvement of the objective shoulder function score (Constant Murley Score) 6 wk postoperatively was enhanced to a clinically relevant extent in the SC group compared to the CC group (P < 0.01)., Conclusions: We conclude that the use of a stimulating catheter results in a faster onset of motor block, unaltered postoperative pain, and a significantly improved functional outcome 6 wk after shoulder surgery.

Three routes have been explored to synthesise the telomere-targeted agent 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate . Application of a 6-(2-azidophenyl)phenanthridine precursor gave an entry to the indazolo[2,3-f]phenanthridine ring system not the required quino[4,3,2-kl]acridine. A six step synthesis starting from 2,6-dibromo-4-methylbenzonitrile via a 1-arylacridin-9(10H)-one intermediate, or , gave the required in low overall yield (<10%). The most efficient route entailed the one-pot (five step) conversion of 1,2-dimethyl-6-fluoroquinolinium methosulfate to in 33% yield employing triethylamine as base and nitrobenzene as solvent.

Background and Objectives: Skin-temperature increase is a reliable but late indicator of success during regional-anesthesia techniques. The goal of this study is to determine the distribution of skin-temperature changes during different regional techniques. Does skin temperature increase in the whole area innervated by the blocked neural structures or only in certain regions within this area with the capability to react preferentially to sympathetic block (i.e., vessel-rich skin)? Although onset time may vary between different regional-anesthetic techniques, we hypothesized that the distribution of skin warming is equal., Methods: Skin temperature was assessed continuously by infrared thermography in 24 patients who received either combined femoral-nerve and sciatic-nerve block, epidural anesthesia, or spinal anesthesia., Results: Apart from differences in time of onset, no differential spatial distribution of skin-temperature changes could be detected. The earliest and greatest rise of skin temperature occurred at the great toe (10.6 degrees C +/- 0.4 degrees C), became smaller proximally, and was negligible above the ankles, irrespective of the type and extent of block. Video-thermography revealed that cold blood flows through subcutaneous veins immediately after onset of sympathetic block and initially decreases skin temperature (0.6 degrees C +/- 0.3 degrees C) during onset of spinal anesthesia., Conclusion: Irrespective of the applied regional-anesthetic technique, skin-temperature changes are more pronounced distally. Thermography prevents false measurements of skin temperature above subcutaneous veins and displays flow of cold blood as the mechanism of initial skin-temperature drop after regional anesthesia. Measurements of skin-temperature increase cannot be used to evaluate the extent of analgesia or sympathetic block.

The synthesis and in vitro antitumour evaluation of a new series of fluorinated benzothiazole-substituted 4-hydroxycyclohexa-2,5-dienones ('quinols') is described. The new compounds were found to be of comparable activity compared to the non-fluorinated precursor PMX 464, in terms of antiproliferative activity in sensitive human cancer cell lines (nanomolar GI(50) values) and inhibitory activity against the thioredoxin signalling system.

Purpose: Hypoxia-inducible factor-alpha (HIF-alpha) is a transcription factor that regulates the response to hypoxia. HIF-alpha protein is found at high levels in many cancers, and the redox protein thioredoxin-1 (Trx-1) increases both aerobic and hypoxia-induced HIF-alpha. Therefore, Trx-1 and HIF-alpha are attractive molecular targets for novel cancer therapeutics., Experimental Design: We investigated whether two novel anticancer drugs AJM290 and AW464 (quinols), which inhibit Trx-1 function, can inhibit the HIF pathway., Results: Treatment of several cancer cell lines with AJM290 or AW464 prevented the hypoxia-induced increase of vascular endothelial growth factor (VEGF) at subtoxic concentrations. AJM290 and AW464 also decreased VEGF in pVHL mutant renal cell carcinoma cells that constitutively overexpress HIF-alpha protein. They surprisingly up-regulated HIF-alpha expression in breast cancer cell lines in normoxia and hypoxia as well as in pVHL mutant cells. In the MDA-MB-468 breast cancer cell line, the compounds inhibited RNA and protein expression of the HIF-alpha target genes, carbonic anhydrase IX, VEGF, and BNIP3, concordantly with HIF-alpha up-regulation. Both compounds specifically inhibited HIF-alpha-dependent induction of hypoxia regulatory element-luciferase and HIF-1alpha hypoxia regulatory element-DNA binding. To analyze the HIF-1alpha domain inhibited by AJM290, we transfected cells with plasmids expressing a fusion protein of Gal linked to HIF-1alpha or HIF-1alpha COOH-terminal transactivation domain (CAD) with a Gal4-responsive luciferase reporter gene. AJM290 inhibited both the full-length HIF-1alpha and HIF-1alpha CAD transcriptional activity., Conclusions: AJM290 and AW464 are inhibitors of HIF-1alpha CAD transcription activity and DNA binding, but they also inhibit degradation of HIF, in contrast to other Trx inhibitors.

Background: Neuraxial anaesthesia in adults decreases the dose of i.v. or inhalational anaesthetic needed to reach a desired level of sedation. Furthermore, spinal anaesthesia alone has a sedative effect. The mechanism behind this phenomenon is presumed to be decreased afferent stimulation of the reticular activating system after sympatholysis. We hypothesized that this mechanism is equally active in infants undergoing spinal anaesthesia., Methods: In total, 20 unpremedicated former preterm infants underwent surgery under spinal anaesthesia with hyperbaric bupivacaine 0.5% 1 mg kg(-1) with epinephrine 10 microg kg(-1). No additional sedatives or anaesthetics were administered. Sedation was evaluated using the bispectral index (BIS) score and the 95% spectral edge frequency (SEF(95))., Results: After spinal anaesthesia, mean (SD) BIS began to decrease significantly from baseline 97.0 (1.1) to 83.9 (14.4) after 15 min (P=0.006). BIS decreased further, reaching the lowest values after 30 min [62.2 (14.0); P<0.00001]. Mean (SD) SEF(95) declined from baseline 26.1 (1.8) Hz to 24.3 (3.1) after 5 min (P=0.02) and further to 9.9 (3.8) after 30 min (P<0.00001). Mean arterial pressure also decreased significantly from 66.5 (4.7) mm Hg within 10 min to 56.1 (5.6) after spinal anaesthesia (P=0.0002), while heart rate remained stable., Conclusions: These results suggest that sedation after spinal anaesthesia in infants is at least as pronounced as in adults. The sedative effect of spinal anaesthesia should be kept in mind when additional sedatives are administered, especially in former preterm infants.

The inhibition of the telomere-binding protein TRF2, by expressing the dominant negative form TRF2(DeltaBDeltaC), has been used as a model of anti-telomere strategy to induce a reversion of the malignant phenotype of M14 and JR5 human melanoma lines. Over-expression of TRF2(DeltaBDeltaC) induced apoptosis and reduced tumourigenicity exclusively in JR5 cells. p53 and Rb status and apoptotic response to DNA damage did not seem to account for the different response of the two lines to TRF2 inhibition. Interestingly, JR5 cells possess shorter and more dysfunctional telomeres compared to M14 line. Moreover, the treatment with the G-quadruplex-interacting agent (G4-ligand) RHPS4 sensitises M14 cells to TRF2 inhibition. These results demonstrate that TRF2 can impair tumuorigenicity of human cancer cells. They further suggest that a basal level of telomere instability favours an efficient response to TRF2 inhibition and that a combined anti-TRF2 and G4-ligand therapy would have synergistic inhibitory effects on tumour cell growth.

Novel heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) are promising novel anticancer agents. They exhibit in vitro antiproliferative activity against colon, renal, and breast carcinoma cell lines as well as in vivo antitumor activity in colon, renal, and breast tumor xenografts. Elucidation of the mechanism of antitumor action of these compounds is of great importance. We show in this study that the compounds induced apoptosis as demonstrated by caspase 3 and PARP cleavage at doses causing G(2)/M cell cycle arrest. Glutathione was found to play an important role in modulating quinol-mediated cytotoxicity. In HCT 116 cells, treatment with 1 and 2 caused a 2- to 3-fold increase in the total glutathione content, suggestive of a glutathione-mediated antioxidant response. Indeed, buthionine sulfoximine (BSO)-induced glutathione depleted cells were 6-10 times more sensitive to 1 and 2, while glutathione monoethyl ester supplementation decreased the antitumor potencies by 2-3 times. In further studies we determined other cellular proteins which bind to an immobilized quinol analog, and identified several proteins including beta-tubulin, heat shock protein 60, and peroxiredoxin 1 as potential molecular targets of quinols that may contribute to their proapoptotic and antiproliferative effects.

A new series of fluoro-, methoxyl-, and amino-substituted isoflavones have been synthesized as potential antitumor agents based on structural similarities to known flavones and isoflavones (quercetin and genistein respectively) and antitumor 2-phenylbenzothiazoles. Target compounds were synthesized using palladium-catalyzed coupling methodologies to construct the central aryl carbon-carbon single bond. The new isoflavone derivatives were tested for in vitro activity in human breast (MDA-MB-468 and MCF-7) and colon (HT29 and HCT-116) cancer cell lines. Low micromolar GI50 values were obtained in a number of cases, with the MDA-MB-468 cell line being the most sensitive overall. Notably, significant potentiation of growth inhibitory activity (GI50<1 microM for 12d, 12f, 12h, 12k, 12l, 12o but not the methylene-bridged derivative 12i) was observed when MDA-MB-468 cells were co-incubated with TBDD, a powerful inducer of cytochrome P450 (CYP)-1A1 activity, suggesting that isoflavone derivatives can act as substrates for CYP1A1 bioactivation.

Patients with Duchenne muscular dystrophy (DMD) are likely to suffer from cardiac insufficiency. Subclinical cardiac insufficiency may decompensate intraoperatively. During spinal surgery, recording of somatosensory evoked potentials (SSEP) is the standard method of spinal cord monitoring. Assessment of SSEP has proven to be a highly prognostic measure of neurological outcome after cardiopulmonary resuscitation (CPR). In the case presented, scalp SSEP as response to stimulation of both median and tibial nerves were recorded during spinal surgery in a 15-year-old boy with DMD. The patient developed severe hypotension and circulatory collapse intraoperatively. SSEP were measured before, during and up to 3h after circulatory collapse. He was successfully resuscitated and fully recovered. Latencies of SSEP remained stable from all extremities whereas amplitudes significantly decreased during CPR, but recovered completely within 3h. The amplitudes of SSEP serve as a more sensitive marker for brain ischaemia than latencies. Stability of latencies and full recovery of amplitudes within 3h indicated sufficient CPR and predicted a good neurological recovery.

The Schwartz-Jampel syndrome, also known as myotonia chondrodystrophica, is a rare autosomal recessive disorder characterized by bone dysplasia, growth retardation and generalized myotonia. Laryngoscopy and intubation may be difficult because of micrognathia and limited mouth opening due to myotonia of the masseter muscles. As regional anaesthesia reduces myotonic contractions and avoids administration of opioids causing respiratory depression, it appears to be the ideal method for postoperative analgesia. We report on a 5-year-old girl who underwent osteotomy of both hips and received intraoperative and postoperative analgesia via a caudal catheter. Excellent analgesia without myoclonic episodes could be achieved by continuous infusion of ropivacaine.

Increase in skin temperature (Ts) occurs early during neuraxial blocks. However, the reliability of Ts to predict successful peripheral block is unknown. Therefore, we investigated whether an increase in Ts more than 1 degrees C precedes or follows an impairment of sensation after combined femoral and sciatic nerve block as well as after epidural anesthesia. In this prospective, nonrandomized study we determined Ts changes in 33 patients undergoing knee or foot surgery under femoral and sciatic nerve block and 10 patients undergoing epidural anesthesia. Perception and motor function were assessed every 5 min. An increase in Ts (> or =1 degrees C) at the foot occurred later after sciatic nerve block than after epidural anesthesia (10.3 +/- 2.8 versus 5.0 min; P < 0.01). Alterations of Ts at skin innervated by the femoral nerve were <1 degrees C. Ts increase preceded sensory block after sciatic nerve block in 6.6% of patients but indicated a successful block (sensitivity, specificity, and accuracy = 100%). We conclude that an increase of Ts is a reliable, but late, sign of successful sciatic nerve block. Therefore it is of limited clinical value. Ts changes after femoral nerve block are negligible and late.

Andrographolide 1, a diterpenoid lactone of the plant Andrographis paniculata, known to possess antitumour activity in in vitro and in vivo breast cancer models was subjected to semisynthesis leading to the preparation of a number of novel compounds. These compounds exhibited in vitro antitumour activity with moderate to excellent growth inhibition against MCF-7 (breast) and HCT-116 (colon) cancer cells. Compounds 3,19-(2-chlorobenzylidene)andrographolide(5), 3,19-(3-chlorobenzylidene)andrographolide(6), 3,19-(3-fluorobenzylidene) andrographolide(7), 3,19-(4-fluorobenzylidene)andrographolide(8), 3,19-(2-fluorobenzylidene)andrographolide(10), 3,19-(2-chloro-5-nitrobenzylidene)andrographolide (21), 3,19-(4-chlorobenzylidene)andrographolide(30) and 3,19-(2-chloro-4-fluorobenzylidene) andrographolide(31) were also screened against 60 NCI (National Cancer Institute, USA) human tumour cell lines derived from nine cancer cell types.

Objective: To evaluate the safety, tolerability and efficacy of mirtazapine in patients with the primary diagnosis of chronic pain and concomitant depression in an open post-marketing surveillance study., Research Design and Methods: 594 patients with a primary diagnosis of at least one chronic pain syndrome (minimum duration of 3 months) and the diagnosis of concomitant depression, appropriately made by a neurologist or psychiatrist, were recruited at psychiatric and/or neurological outpatient facilities throughout Germany. The primary efficacy parameter was pain at baseline and endpoint using a patient self-assessment scale. Secondary analyses were performed at baseline, week 1 (day 7 +/- 2), week 4 (day 28 +/- 4) and at endpoint (day 42 +/- 4 or early termination) and included safety and tolerability assessments. Investigators rated the severity of different potential co-morbidities (including depression) with a four-step rating scale (not present, mild, moderate, severe)., Results: 594 patients were enrolled and treated with mirtazapine (mean daily dose of 34.5 +/- 10.4 mg at study endpoint). A statistically significant (p < 0.0001; one sample sign test) reduction of pain from baseline to endpoint was found for the overall population. The percentage of patients free of pain or with only moderate pain increased significantly, irrespective of patients' age or pain syndromes. Furthermore, we found a substantial improvement from baseline to endpoint regarding co-morbidities such as sleep disturbance, irritability and exhaustion. The number of adverse events was low (<7%; n = 37), with fatigue (n = 13) and weight gain (n = 11) occurring most frequently. No previously-unknown side effects occurred. One hundred and six patients (18%) discontinued mirtazapine during the study. The main reason was lack of efficacy (6%, n = 33), which may be a reflection of sub-optimal response to the anti-depressant or analgesic effect of the drug, but no appropriate rating scale was used to clarify this question. Only a small number of patients stopped the drug due to adverse events (3%; n = 15). At study endpoint, the majority of physicians and patients rated the overall efficacy and tolerability of mirtazapine as good or very good. Most patients (80%) continued the therapy after 6 weeks., Conclusions: Despite the limitations of an open observational study, our findings suggest that mirtazapine is a safe and well-tolerated drug for use in daily clinical practice. It still remains unclear whether the reduction of pain, the enhancement of the depressed mood or the combination of both effects led to these results. Nevertheless, our data point to a potential beneficial effect of mirtazapine in the treatment of patients with pain and concomitant depression. However, more systematic research, including placebo-controlled studies, and further empirical testing are necessary.

A series of new 2-phenylbenzothiazoles has been synthesized on the basis of the discovery of the potent and selective in vitro antitumor properties of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (8n; GW 610, NSC 721648). Synthesis of analogues substituted in the benzothiazole ring was achieved via the reaction of o-aminothiophenol disulfides with substituted benzaldehydes under reducing conditions. Compounds were evaluated in vitro in four human cancer cell lines, and compound 8n was found to possess exquisitely potent antiproliferative activity (GI(50) < 0.1 nM for MCF-7 and MDA 468). Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. Structure-activity relationships established that the compound 8n stands on a pinnacle of potent activity, with most structural variations having a deactivating in vitro effect. Mechanistically, this new series of agents contrasts with the previously reported 2-(4-aminophenyl)benzothiazoles; compound 8n is not reliant on induction of CYP1A1 expression for antitumor activity.

Telomeric integrity is required to maintain the replicative ability of cancer cells and is a target for the G-quadruplex-stabilizing drug 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4). We report a senescent-like growth arrest in MCF-7 breast cancer cells, within 14 to 17 days, and a reduction in telomere length (from 5.2 kilobases (kb) to 4.7 and 4.3 kb after 17 days of treatment at 0.5 and 1 microM, respectively). These effects occurred at noncytotoxic drug concentrations (doses < 1 microM over a 14-day exposure) compatible with long-term drug dosing. The telomere length of cancer cells influences their sensitivity to growth inhibition by RHPS4: mutant (mt) human telomerase reverse transcriptase (hTERT)-expressing MCF-7 cells [short telomere restriction fragment (TRF) length, 1.9 kb; IC50, 0.2 microM] were 10 times more sensitive to RHPS4 compared with wild-type (wt) hTERT-expressing, vector-transfected control cells (longer TRF-length 5.2 kb; IC50 2 microM) in the 5 day SRB assay. This relationship was corroborated in a panel of 36 human tumor xenografts grown in vitro showing a positive correlation between telomere length and growth inhibitory potency of RHPS4 (15-day clonogenic assay, r = 0.75). These observations are consistent with loss of the protective capping status of telomeres mediated by RHPS4 G-quadruplex-stabilization, thus leading to greater susceptibility of cells with shorter telomeres. In combination studies, paclitaxel (Taxol), doxorubicin (Adriamycin), and the experimental therapeutic agent 17-(allylamino)-17-demethoxygeldanamycin, which inhibits the 90-kDa heat shock protein, conferred enhanced sensitivity in RHPS4 treated MCF-7 cells, whereas the DNA-interactive temozolomide and cisplatin antagonized the action of RHPS4. Our results support the combined use of certain classes of cytotoxic anticancer agents with RHPS4 to enhance potential clinical benefit.

Palladium(0)-mediated Suzuki-Miyaura and Heck transformations have been exploited to provide examples of 8-methylquino[4,3,2-kl]acridines and 8,13-dimethylquino[4,3,2-kl]acridinium iodides bearing bulky saturated (3-acetoxy)propyl or (E)-3-(morpholin-4-yl)-3-oxopropenyl substituents variously in the 3-, 6-, or 10-positions of the pentacyclic nucleus. The pharmacological/pharmaceutical properties of four compounds (4, RHPS4), (5, IH383), (6, RHPS16), and (17, RHPS19) were measured to assess their clinical potential as DNA G-quadruplex-stabilizing/telomerase inhibitory agents. The following properties were measured: stability in tissue culture media in the presence of A549 lung and MCF-7 breast tumor cells, metabolic stability when incubated with rat liver microsomes, and rate of uptake and subcellular location in A549 and MCF-7 cells. Compound 17 was unstable in tissue culture media, failed to achieve nuclear access, and was excluded from further consideration. Of the other agents, 4 exhibited the most favorable pharmaceutical profile: the agent has appropriate stability in the presence of tumor cells and rat liver microsomes and achieves rapid ingress into cell nuclei where the putative molecular target is located.

Novel reaction pathways for the hypervalent iodine-mediated oxidation of bioactive phenols containing extended conjugated pi-systems are described. Oxidation of 4-hydroxystilbenes in methanol using a hypervalent iodine-based oxidant led to the formal 1,2-addition of methoxy groups across the central stilbene double bond. Treatment of the structurally related 4-hydroxyisoflavone with di(trifluoroacetoxy)iodobenzene leads to the surprising formation of 2,4'-dihydroxybenzil. Potential mechanisms for these new reaction pathways are discussed, and the X-ray crystal structure of 2,4'-dihydroxybenzil is presented. In contrast, oxidation of the corresponding 3-hydroxystilbenes and 3-hydroxyisoflavone led to conventional dienone oxidation products. The antitumour implications of these oxidation processes are briefly highlighted; the novel 4-substituted phenolic oxidation products were found to be inactive in terms of in vitro antitumour cellular activity, whereas the 3-substituted phenol products gave novel agents with potent and enhanced antitumour activity in the HCT 116 cancer cell line.

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.

The parallel solution-phase synthesis of a series of 30 monohydroxylated (E)-stilbene analogues is described. In vitro screening revealed low micromolar activity (GI(50)) against the MDA MB 468 breast cancer cell line. Activity in MDA MB 468 cells correlated with the ability to induce apoptosis following drug treatment by the most potent agents in the series, e.g., 5dy and 5jy, an observation further reinforced by AnnexinV-FITC analysis and fluorescence microscopy.

AW464 (NSC 706704) is a novel benzothiazole substituted quinol compound active against colon, renal and certain breast cancer cell lines. NCI COMPARE analysis indicates possible interaction with thioredoxin/thioredoxin reductase, which is upregulated under hypoxia. Through activity on HIF1alpha, VEGF levels are regulated and angiogenesis controlled. A thioredoxin inhibitor could therefore exhibit enhanced hypoxic toxicity and indirect antiangiogenic effects. In vitro experiments were performed on colorectal and breast cancer cell lines under both normoxic and hypoxic conditions and results compared against those obtained with normal cell lines, fibroblasts and keratinocytes. Antiangiogenic effects were studied using both large and microvessel cells. Indirect antiangiogenic effects (production of angiogenic growth factors) were studied via ELISA. We show that AW464 exerts antiproliferative effects on tumour cell lines as well as endothelial cells with an IC(50) of approximately 0.5 microM. Fibroblasts are however resistant. Proliferating, rather than quiescent, endothelial cells are sensitive to the drug indicating potential antiangiogenic rather than antivascular action. Endothelial differentiation is also inhibited in vitro. Hypoxia (1% O(2) for 48 h) sensitises colorectal cells to lower drug concentrations, and in HT29s greater inhibition of VEGF is observed under such conditions. In contrast, bFGF levels are unaffected, suggesting possible involvement of HIF1alpha. Thus, AW464 is a promising chemotherapeutic drug that may have enhanced potency under hypoxic conditions and also additional antiangiogenic activity.

A series of substituted 4-(1-arylsulfonylindol-2-yl)-4-hydroxycyclohexa-2,5-dien-1-ones (indolylquinols) has been synthesized on the basis of the discovery of lead compound 1a and screened for antitumor activity. Synthesis of this novel series was accomplished via the "one-pot" addition of lithiated (arylsulfonyl)indoles to 4,4-dimethoxycyclohexa-2,5-dienone followed by deprotection under acidic conditions. Similar methodology gave rise to the related naphtho-, 1H-indole-, and benzimidazole-substituted quinols. A number of compounds in this new series were found to possess in vitro human tumor cell line activity substantially more potent than the recently reported antitumor 4-substituted 4-hydroxycyclohexa-2,5-dien-1-ones(1) with similar patterns of selectivity against colon, renal, and breast cell lines. The most potent compound in the series in vitro, 4-(1-benzenesulfonyl-6-fluoro-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1h), exhibits a mean GI(50) value of 16 nM and a mean LC(50) value of 2.24 muM in the NCI 60-cell-line screen, with LC(50) activity in the HCT 116 human colon cancer cell line below 10 nM. The crystal structure of the unsubstituted indolylquinol 1a exhibits two independent molecules, both participating in intermolecular hydrogen bonds from quinol OH to carbonyl O, but one OH group also interacts intramolecularly with a sulfonyl O atom. This interaction, which strengthens upon ab initio optimization, may influence the chemical environment of the bioactive quinol moiety. In vivo, significant antitumor activity was recorded (day 28) in mice bearing subcutaneously implanted MDA-MB-435 xenografts, following intraperitoneal treatment of mice with compound 1a at 50 mg/kg.

Triple-stranded DNA structures have been implicated in a number of major biological processes, including the transcription and translation of a number of genes, as well as in the interaction of DNA with a number of proteins. Furthermore, antigene therapies under development are based on the recognition and binding of a single oligonucleotide strand to a double-stranded sequence, thus forming a triple helix. Triplex DNA formation is a relatively weak and temporary phenomenon; therefore, molecules that selectively bind to and stabilize triple helices may show a variety of novel biological effects. The biophysical and biological characterization of a series of antitumor polycyclic acridines that bind to triplex DNA is reported. These compounds, whose synthesis has been previously reported, have been tested for their interaction with both purine and pyrimidine type triple helices and compared with the relevant double-stranded DNA. As a pyrimidine triplex model we have used the T*AT sequence, which we have compared with the AT duplex, whereas the purine triplex oligonucleotide d[G3A4G3]*d[G3A4G3].d[C3T4C3] has been compared with the duplex d[G3A4G3].d[C3T4C3]. The compounds demonstrate various degrees of preferential binding to triplex DNA over normal duplex DNA, as measured by UV, fluorescence, circular dichroism, and thermal denaturation. Tri-substituted acridine derivatives demonstrated the highest affinity and ability to stabilize triplex DNA structures. Furthermore, structure/affinity analysis gives insights into the structural features that optimize affinity and selectivity for triplex DNA, and may play a role in their profile of antitumor activity.

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any "resistant" cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.

Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L-10 micromol/L; 24-72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.

This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G(2)/M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and beta-galactosidase activity. The short-term biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.

Phortress (the dihydrochloride salt of the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203)) is an experimental antitumor agent with potent and selective activity against human-derived carcinomas of breast, ovarian and renal origin. UK clinical trials of Phortress are scheduled to begin in 2004. The mechanism of action of Phortress is distinct from all classes of chemotherapeutic agents currently in the clinic, and involves metabolic activation by cytochrome P450 (CYP) 1A1 to electrophilic species, which generate DNA adducts in sensitive tumors only. In the present study, the antitumor efficacy of Phortress has been compared with that of doxorubicin (Dox) in nine human-derived mammary carcinoma xenograft models, cultivated subcutaneously in the flanks of nude mice. In addition, cyp1a1 mRNA expression was measured in tumors of control and treated animals. Phortress compared favorably with Dox: significant activity, independent of estrogen receptor (ER) status, was established in 7/9 xenografts; in one xenograft model, Phortress elicited superior antitumor activity; no model demonstrated complete resistance to Phortress. In accordance with this observation, all xenografts available for examination (8) displayed clear induction of cyp1a1 expression upon treatment of mice with Phortress whereas Dox failed to induce cyp1a1 expression in all models. Prolonged viability of tumor fragments, recovered for treatment ex vivo could not be sustained; thus correlations between tumor cells' response to Phortress and cyp1a1 or cyp1b1 inducibility following 5F 203 treatment could not be determined with confidence.

Benzimidazole-N-oxide modifications of potent lipophilic dihydrofolate reductase (DHFR) inhibitors (e.g., methylbenzoprim 1 and dichlorobenzoprim 2) have been prepared by base-promoted cyclization of the nitrophenylbenzylamino groups to explore the possibility that abrogation of DHFR-inhibitory activity might reveal clues to an alternative anti-ras mechanism. Examples of the new series had only low growth inhibitory activities (GI(50) generally >50 microM) against colon HCT116 and lung HT29 cell lines but, unlike methylbenzoprim, this activity was unaffected by hypoxanthine/thymidine rescue.

The c-MYC oncogene, containing the DNA 5'-GGAGG repeat sequence, is emerging as an attractive target for anti-cancer therapeutics with evidence to suggest that DNA quadruplex formation is involved in regulating gene transcription. We describe NMR studies and molecular dynamics (MD) simulations on intermolecular quadruplex structures containing the purine-rich GGAGG motif which we show readily assembles to form stable parallel-stranded quadruplexes containing A- and G-tetrads with evidence for weak sequence-specific K(+) ion binding at the 5'-GA step.

Background: In isolated nerves, tetrodotoxin (TTX) blocks nerve conduction longer than bupivacaine. In vivo, however, both substances block nerve conduction to an equal duration, presumably because the hydrophilic TTX binds only weakly to the perineural tissue. High molecular weight hyaluronic acid (HA) prolongs the action of local anaesthetics several-fold. We tested whether admixture of HA enhances the binding of TTX to the perineural tissue and thus induces an ultra-long conduction block after a single application., Methods: In 12 anaesthetized rabbits, the minimal blocking concentrations of TTX, TTX and HA (TTX/HA) and bupivacaine with HA (bupivacaine/HA) were determined by blocking the natural spike activity of the aortic nerve. In 18 other animals, equipotent concentrations of either TTX, TTX/HA or TTX/bupivacaine/HA were applied topically to the aortic nerve. After disappearance of the spike activity, the wound was closed to simulate the clinical situation of a single shot nerve block. The time until recovery of spike activity was determined. The nerves were examined for signs of neurotoxicity 24 h after the application of the drugs. Data are presented as means +/- SD and compared by ANOVA and Student's t-test for unpaired data., Results: The conduction block by TTX/bupivacaine/HA (10.1 +/- 1.9 h) or TTX/HA (9.3 +/- 1.0 h) was significantly longer than that of plain TTX (7.9 +/- 1.0 h). Neurotoxicity was not observed., Conclusions: Both HA and HA/bupivacaine prolong the TTX-induced conduction blockade of the aortic nerve of rabbits in vivo. No signs of neurotoxicity were observed.

Cyclisation of 9-(benzotriazol-1-yl)acridine to the pentacycle 8H-quino[4,3,2-kl]acridine in a range of low-boiling solvents is mechanistically distinct from previously published photochemical (carbene) and thermolytic (radical) cyclisations. Fragmentation of the triazole ring of to a diazonium intermediate, and its subsequent heterolysis (-N(2)) and cyclisation is facilitated by solvation of intermediate zwitterionic species. Derivatives of 2- and 3-aminoquinoacridines methylated in the 8-position can be converted to 8,13-dimethylquino[4,3,2-kl]acridinium iodide salts with methyl iodide and were required for biological examination as potential telomerase inhibitors. The chloro group in 3-chloro-8-methyl-8H-quino[4,3,2-kl]acridine can be replaced efficiently by benzylamino, 4-morpholinyl and cyano substituents in palladium(0) mediated reactions.

The NMR structure of the parallel-stranded DNA quadruplex d(TTAGGGT)(4), containing the human telomeric repeat, has been determined in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl]acridinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor of telomerase at submicromolar levels (IC(50) value of 0.33(+/-0.13)microM), exhibiting a wide differential between telomerase inhibition and acute cellular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic potency through interaction with, and stabilisation of, four-stranded G-quadruplex structures. RHPS4 forms a dynamic interaction with d(TTAGGGT)(4), as evident from 1H and 19F linewidths, with fast exchange between binding sites induced at 318 K. Perturbations to DNA chemical shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify the 5'-ApG and 5'-GpT steps as the principle intercalation sites; a structural model has been refined using NOE-restrained molecular dynamics. The central G-tetrad core remains intact, with drug molecules stacking at the ends of the G-quadruplex. The partial positive charge on position 13-N of the acridine ring appears to act as a "pseudo" potassium ion and is positioned above the centre of the G-tetrad in the region of high negative charge density. In both ApG and GpT intercalation sites, the drug is seen to converge to the same orientation in which the pi-system of the drug overlaps primarily with two bases of each G-tetrad. The drug is held in place by stacking interactions with the G-tetrads; however, there is some evidence for a more dynamic, weakly stabilised A-tetrad that stacks partially on top of the drug at the 5'-end of the sequence. Together, the interactions of RHPS4 increase the t(m) of the quadruplex by approximately 20 degrees C. There is no evidence for drug intercalation within the G-quadruplex; however, the structural model strongly supports end-stacking interactions with the terminal G-tetrads.

The dithiol-reducing thioredoxin/thioredoxin reductase system normally maintains the reduced state of key enzymes responsible for the cell's anti-oxidant defences. We therefore addressed the question of whether AW 464--a novel thioredoxin inhibitor--as well as broad spectrum dithiol ligands diamide and phenylarsine oxide are able to induce and execute a regular apoptotic sequence of events without overwhelming the cell's ability to detoxify reactive oxygen species. All three agents were found to target the thioredoxin system in a cell-free assay. In HL-60 leukaemia cells, they were also found to induce Bak activation, cytochrome c release from mitochondria, decreasing Delta Psi m, chromatin condensation, phosphatidyl serine exposure and Tdt-sensitive DNA nicks. At the onset of apoptosis there was no evidence of increases in oxygen free radicals or peroxide in cells treated with AW 464 or diamide. Phenylarsine oxide induced both free radicals and hydrogen peroxide, but this did not appear to interfere with apoptosis. We conclude that pharmacological targeting of thioredoxin can induce a well-orchestrated apoptotic programme.

Pd(0) mediated couplings between substituted 2-(pivaloylamino)benzeneboronic acids and 3,6-disubstituted-10-methylacridones 13 bearing a bromo or trifluoromethylsulfonyloxy substituent in the 1-position yield intermediate 1-arylacridones 16 which can be can be cyclised to new 8-methylquino[4,3,2-kl]acridines 17 with phosphorus oxychloride or 6 M HCI in EtOH. Heck reactions between triflate-substituted substrates 17 and acrylic acid derivatives afforded quinoacridines with unsaturated side-chains in the 6-position. Alkylboranes, prepared by interaction of 9-borabicyclo[3,3,1]nonane (9-BBN) and allyl acetate or N-allyltrifluoroacetamide, participated in Suzuki-Miyaura reactions with chloro-substituted 8-methylquinoacridines to form derivatives bearing functionalised propyl groups in the 6- and 10-positions. Representative 8-methylquinoacridines were methylated with methyl iodide to yield telomerase-inhibitory 8,13-dimethylquinoacridinium iodides 24.

The purpose of this research was to quantitate and confirm the mechanism of in vivo metabolic activation of temozolomide. The secondary aims were to evaluate the tumor, normal tissue, and plasma pharmacokinetics of temozolomide in vivo, and to determine whether such pharmacokinetics resulted in tumor targeting. [(11)C]temozolomide kinetics were studied in men using positron emission tomography (PET). It has been postulated that temozolomide undergoes decarboxylation and ring opening in the 3-4 position to produce the highly reactive methyldiazonium ion that alkylates DNA. To investigate this, a dual radiolabeling strategy, with [(11)C]temozolomide separately radiolabelled in the 3-N-methyl and 4-carbonyl positions, was used. We hypothesized that (11)C in the C-4 position of [4-(11)C-carbonyl]temozolomide would be converted to [(11)C]CO(2) if the postulated mechanism of metabolic conversion was true resulting in lower [(11)C]temozolomide tumor exposure. Paired studies were performed with both forms of [(11)C]temozolomide in 6 patients with gliomas. Another PET scan with (11)C-radiolabelled bicarbonate was performed and used to account for the metabolites of temozolomide using a data-led analytical approach. Plasma was analyzed for [(11)C]temozolomide and [(11)C]metabolites throughout the scan duration. Exhaled air was also sampled throughout the scan for [(11)C]CO(2). The percentage ring opening of temozolomide over 90 min was also calculated to evaluate whether there was a differential in metabolic breakdown among plasma, normal tissue, and tumor. There was rapid systemic clearance of both radiolabelled forms of [(11)C]temozolomide over 90 min (0.2 liter/min/m(2)), with [(11)C]CO(2) being the primary elimination product. Plasma [(11)C]CO(2) was present in all of the studies with [4-(11)C-carbonyl]temozolomide and in half the studies with [3-N-(11)C-methyl]temozolomide. The mean contributions to total plasma activity by [(11)C]CO(2) at 10 and 90 min were 12% and 28% with [4-(11)C-carbonyl]temozolomide, and 1% and 4% with [3-N-(11)C-methyl]temozolomide, respectively. There was a 5-fold increase in exhaled [(11)C]CO(2) sampled with [4-(11)C-carbonyl]temozolomide compared with [3-N-(11)C-methyl]temozolomide (P < 0.05). A decrease in tissue exposure [area under the curve between 0 and 90 min (AUC(0-90 min))] to [(11)C]temozolomide was also observed with [4-(11)C-carbonyl] temozolomide compared with [3-N-(11)C-methyl]temozolomide. Of potential therapeutic advantage was the higher [(11)C]radiotracer and [(11)C]temozolomide exposure (AUC(0-90 min)) in tumors compared with normal tissue. [(11)C]temozolomide ring opening over 90 min was less in plasma (20.9%; P < 0.05) compared with tumor (26.8%), gray matter (29.7%), and white matter (30.1%), with no differences (P > 0.05) between tumor and normal tissues. The significantly higher amounts of [(11)C]CO(2) sampled in plasma and exhaled air, in addition to the lower normal tissue and tumor [(11)C]temozolomide AUC(0-90 min) observed with [4-(11)C-carbonyl]temozolomide, confirmed the postulated mechanism of metabolic activation of temozolomide. A higher tumor [(11)C]temozolomide AUC(0-90 min) in tumors compared with normal tissue and the tissue-directed metabolic activation of temozolomide may confer potential therapeutic advantage in the activity of this agent. This is the first report of a clinical PET study used to quantify and confirm the in vivo mechanism of metabolic activation of a drug.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

The synthesis and antitumor evaluation of a series of new heteroaromatic- and aromatic-substituted hydroxycyclohexadienones ("quinols"), and their imine counterparts, are described. The quinols were synthesized via the addition of a lithiated aromatic moiety to a quinone ketal followed by deprotection. When the aromatic portion of the molecule is a fused heterobicyclic structure (e.g., benzothiazole derivative 7a), potent in vitro antitumor activity was observed in HCT 116 (GI50 = 40 nM) and HT 29 (GI50 = 380 nM) human colon as well in as MCF-7 and MDA 468 human breast cancer cell lines. When examined on the NCI Developmental Therapeutics Screening Program in vitro screen (60 human cancer cell lines), active compounds in this series consistently displayed a highly unusual pattern of selectivity; cytotoxicity (LC50) was concentrated in certain colon and renal cell lines only. Analogue 7a also showed in vivo antitumor activity against human RXF 944XL renal xenografts in nude NMRI mice and is the focus of further study.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 microM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 microM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 microM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) > or = 100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 microM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 microM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (< or = 10 microM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg(-1)). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.