A proposed management algorithm for late-onset efavirenz neurotoxicity.

A high proportion of HIV-positive patients in South Africa receive concomitant efavirenz (EFV) and isoniazid (INH) therapy. EFV is metabolised in the liver via CYP2B6, and genetic polymorphism of CYP2B6 is known to result in slowed metabolism of the drug. INH is also metabolised in the liver, causing inhibition of a pathway that plays an important role in slow EFV metabolisers. Concomitant INH use therefore affects plasma levels of EFV. EFV is well known to cause neuropsychiatric side-effects on initiation, and a recent adult case series described late-onset neurotoxicity in the form of subacute ataxia and encephalopathy in patients treated with EFV for a median of 2 years, in association with toxic plasma levels of the drug. We have seen an increase in cases of EFV toxicity presenting to our neurology referral unit. All cases have been in the context of recent initiation of concomitant INH. We therefore conducted a retrospective case record audit to describe these seven cases with the additional advantage of tertiary-level assessment. We outline the clinical features and investigation results, as well as outcomes after EFV was stopped. Our main objectives are to highlight the probable role of concomitant INH use in the development of this syndrome, and to suggest that only limited work-up may be warranted in suspected cases.

Effect of a fluorinated pyrimidine on cachexia and tumour growth in murine cachexia models: relationship with a proteolysis inducing factor.

The fluorinated pyrimidine nucleoside, 5'-deoxy-5-fluorouridine (5'-dFUrd) has been shown to effectively attenuate the progress of cachexia in the murine adenocarcinomas MAC16 and colon 26 as well as in the human uterine cervical carcinoma xenograft, Yumoto. Although concomitant inhibition of tumour growth was observed in all three models this was not sufficient to account for the preservation of body weight. An attempt has been made to correlate the anti-cachectic activity of 5'-dFUrd with the presence of a tumour produced proteolysis-inducing factor (PIF), thought to be responsible for the development of cachexia in the MAC16 model. Two variants of colon 26 adenocarcinoma were employed, clone 20 which produces profound cachexia, and clone 5 which produces no change in body weight in recipient animals. Mice bearing the colon 26, clone 20 variant showed evidence for the presence of PIF in tumour, serum and urine, while there was no evidence for the presence of PIF in tumour or body fluids of mice bearing the clone 5 tumours. Treatment of animals bearing the clone 20 variant with 5'-dF Urd led to the disappearance of PIF from the tumour, serum and urine concomitant with the attenuation of the development of cachexia. The human cervical carcinoma, Yumoto, which also induced cachexia in recipiant animals, showed expression of PIF in tumour, serum and urine in control and vehicle-treated mice, but was absent in mice treated with 5'-dFUrd. Thus in these experimental models cachexia appears to be correlated with the presence of PIF.

Effect of the specific cyclooxygenase-2 inhibitor meloxicam on tumour growth and cachexia in a murine model.

The effects of the cyclooxygenase-2 (COX-2) inhibitor, meloxicam, on tumour growth and cachexia have been determined in 2 established murine adenocarcinomas (MAC). At a dose level of 2.5 and 5.0 mgkg(-1), meloxicam produced pronounced inhibition of the growth of the MAC13 tumour, increasing the tumour volume doubling time from 2 to 5 days. Meloxicam also suppressed growth of the MAC16 tumour, which is generally refractory to standard cytotoxic agents, increasing the tumour volume doubling time from 1.5 to 2.5 days at dose levels of 0.5 and 1.0 mgkg(-1). Cachexia was also effectively attenuated at these dose levels. To investigate whether meloxicam exerted a direct effect on the cachectic process, studies on protein degradation were carried out using C(2)C(12) mouse myoblasts in response to a proteolysis-inducing factor (PIF). PIF produced maximum protein degradation at a concentration of 4.2 nM, and this was effectively attenuated by meloxicam at concentrations greater than 1 microM. This suggests that meloxicam may be capable of directly antagonizing the process of muscle catabolism in cachexia.

Effect of a cachectic factor on carbohydrate metabolism and attenuation by eicosapentaenoic acid.

The effect of a proteolysis-inducing factor (PIF), produced by cachexia-inducing tumours on glucose utilization by different tissues and the effect of pretreatment with the polyunsaturated fatty acid eicosapentaenoic acid (EPA), has been determined using the 2-deoxyglucose tracer technique. Mice receiving PIF showed a profound depression of body weight (2.3 g) over a 24-h period, which was completely abolished by pretreatment with a monoclonal antibody to PIF or by 3 days pretreatment with EPA at 500 mg kg(-1). Animals receiving PIF exhibited a marked hypoglycaemia, which was effectively reversed by both antibody and EPA pretreatment. There was an increase in glucose utilization by brain, heart and brown fat, but a decrease by kidney, white fat, diaphragm and gastrocnemius muscle after administration of PIF. Changes in organ glucose consumption were attenuated by either monoclonal antibody, EPA, or both. There was a decrease in 2-deoxyglucose uptake by C2C12 myoblasts in vitro, which was attenuated by EPA. This suggests a direct effect of PIF on glucose uptake by skeletal muscle. These results suggest that in addition to a direct catabolic effect on skeletal muscle PIF has a profound effect on glucose utilization during cachexia.

Biological evaluation of a lipid-mobilizing factor isolated from the urine of cancer patients.

We have previously shown human lipid-mobilizing factor (LMF) to be homologous with the plasma protein Zn-alpha2-glycoprotein in amino acid sequence, electrophoretic mobility, and immunoreactivity. In this study, both LMF and Zn-alpha2-glycoprotein have been shown to stimulate glycerol release from isolated murine epididymal adipocytes with a comparable dose-response profile. Both LMF and Zn-alpha2-glycoprotein caused a stimulation of adenylate cyclase in murine adipocyte plasma membranes in a GTP-dependent process, with maximum stimulation at 0.1 microM GTP and with saturation at protein concentrations of >5 microg/assay. Administration of LMF to exbreeder male mice over a 89-h period produced a decrease in body weight without a change in food and water intake. Body composition analysis showed a 42% reduction in carcass lipid when compared with controls. Treatment of ob/ob mice with human LMF over a 160-h period also produced a decrease in body weight, with a 19% reduction in carcass fat, without a change in body water or nonfat mass. Serum levels of glycerol and 3-hydroxybutyrate were significantly increased, as was oxygen uptake by interscapular brown adipose tissue, providing evidence of increased lipid mobilization and utilization. Human white adipocytes responded to both LMF and isoprenaline to the same extent, although the maximal response was lower than that for murine white adipocytes. These results suggest that LMF not only has the capacity to induce lipid mobilization and catabolism in mice, but it also has the potential to exert similar effects in cachectic cancer patients.

Mechanism of the anti-tumour effect of 2,3,5-trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504).

2,3,5-Trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase, effectively suppressed growth of the MAC16 tumour in vivo and prevented the accompanying cachexia, when administered daily at a dose of 10 mg kg(-1). There was a reduction in the tumour concentration of linoleic (LA), arachidonic (AA), oleic, stearic and palmitic acid. In order to elucidate the mechanism of the anti-tumour action, the effect of CV-6504 on the metabolism of AA through the 5-, 12- and 15-lipoxygenase pathways has been determined in cell lines sensitive (MAC16, MAC13, MAC26 and Caco-2) and resistant (A549 and DU-145) to CV-6504. Incubation of all cell lines with [3H]AA led to the appearance of [3H]5-, 12- and 15-HETE. Preincubation of MAC16, MAC13, MAC26 and Caco-2 with 10 microM CV-6504 inhibited the conversion of AA to 5-, 12- and 15-HETE, while in A549 and DU-145 cells there was no effect on metabolism through any lipoxygenase pathway. Two other cell lines, MDA-MB-231 and PC-3, sensitive to growth inhibition by CV-6504, are known to require LA for growth, while DU-145, which was insensitive to growth inhibition by CV-6504, showed no growth response to LA. These results suggest that some tumours are dependent on lipoxygenase metabolites of LA and AA for their continual growth, and interference with this pathway produces a specific growth inhibition.

Metabolism and pharmacokinetics of the anti-tumour agent 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504).

2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504) is an effective inhibitor of the growth of established murine adenocarcinomas (MACs) and is shortly to enter clinical investigation. When administered to mice bearing the MAC16 tumour, CV-6504 rapidly disappeared from the plasma and tissues and there was an accumulation of the sulphate and glucuronide metabolites. After 24 h, the concentration of free CV-6504 in the tumour (3.3 microM) was higher than that in the liver (0.24 microM) and equal to the IC50 value for the inhibition of the growth of MAC16 cells in vitro (3 microM). The concentration of glucuronide and sulphate metabolites in both tumour and liver decreased with time. Both the MAC16 tumour and the liver possessed similar beta-glucuronidase activity, which could account for the accumulation of free CV-6504. Although the sulphate and glucuronide conjugates of CV-6504 were ineffective inhibitors of the growth of MAC13 cells in vitro at concentrations up to 100 microM, in vivo at a concentration of 50 mg kg-1 day-1 the conjugates produced a similar anti-tumour effect to CV-6504 at a concentration of 5 mg kg-1 day-1. The MAC13 tumour possessed both beta-glucuronidase and sulphatase activity capable of converting the sulphate and glucuronide conjugates to free CV-6504. Using MAC13 cells ex vivo, CV-6504 inhibited conversion of arachidonic acid to 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE). The percentage reduction in formation of 12- and 15-HETE exceeded that of 5-HETE. Inhibition of HETE formation may be responsible for the anti-tumour activity of CV-6504.

Inhibition of tumour growth by lipoxygenase inhibitors.

The potential involvement of lipoxygenase metabolites in the tumour growth stimulatory activity of arachidonic and linoleic acid has been studied using the 5-lipoxygenase inhibitors, BWA4C, BWB70C and Zileuton. In vitro the former two agents were relatively potent inhibitors of growth of murine adenocarcinomas (MACs) with IC50 values < 10 microM, whereas Zileuton was less effective. In vivo studies showed BWA4C to be an effective inhibitor of the growth of both the MAC26 and MAC16 tumours at dose levels between 5 and 25 mg kg-1 (b.d.). The growth rate of the MAC26 tumour was also decreased by BWB70C at 25 mg kg-1, whereas lower doses were either ineffective or stimulated tumour growth. This differential effect of the 5-lipoxygenases inhibitors on tumour growth may arise from effects on the 12- and 15-lipoxygenase pathways. To quantify the effect cells were labelled with [3H]arachidonic acid and the biosynthesis of 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE) was analysed by high-performance liquid chromatography. All three agents caused a decrease in 5-HETE production, although the effect was less pronounced with Zileuton. In MAC26 cells both BWA4C and BWB70C caused a decrease in 12-HETE formation whereas Zileuton had no effect on the other lipoxygenase pathways. The inhibitory effect of these agents on cell growth may result from an imbalance of metabolism of arachidonic acid between the 5-, 12- and 15-lipoxygenase pathways.

Novel anti-tumour activity of 2,3,5-trimethyl-6-(3-pyridylmethyl)-1,4- benzoquinone (CV-6504) against established murine adenocarcinomas (MAC).

2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase and thromboxane A2 synthase and a scavenger of active oxygen species, has been shown to exhibit profound anti-tumour activity against three established murine adenocarcinomas (MACs) that are generally refractory to standard cytotoxic agents. For the cachexia-inducing MAC16 tumour, optimal anti-tumour activity was seen at dose levels of 10 and 25 mg kg-1 day-1, together with a reversal of cachexia and a doubling of the time to sacrifice of the animals through cachexia from 8 days to 17 days. The remaining tumour fragments showed extensive necrosis in regions distal from the blood supply. Growth of the MAC13 tumour was also effectively suppressed at dose levels between 5 and 50 mg kg-1 day-1, resulting in a specific growth delay between 1.0 and 1.2. Growth of the MAC26 tumour was also inhibited a concentration-related manner, with doses of 25-50 mg kg-1 day-1 being optimal. Anti-tumour activity towards all three tumours at low dose levels of CV-6504 was effectively suppressed by concurrent administration of linoleic acid (1 g kg-1 day-1), suggesting that inhibition of linoleate metabolism was responsible for the anti-tumour effect. Tumour sensitivity may be correlated with increased DT-diaphorase that are required to metabolise CV-6504 to the active hydroquinone, which inhibits 5-lipoxygenase activity.

Effect of polyunsaturated fatty acids on the growth of murine colon adenocarcinomas in vitro and in vivo.

The effect of the polyunsaturated fatty acids (PUFAs) linoleic acid (LA) and arachidonic acid (AA) on the growth of two murine colon adenocarcinoma cell lines (MAC26 and MAC13) has been determined both in vitro and in vivo. When the serum concentrations in the medium became growth limiting, low concentrations (18-33 microM) of both PUFAs were growth stimulatory to both cell lines, while higher concentrations were growth inhibitory. Growth stimulation by AA in both cell lines, and by LA in MAC13, was effectively inhibited by both the cyclo-oxygenase and lipoxygenase inhibitor indomethacin, and the lipoxygenase inhibitor BWA4C in a dose-dependent manner. The most effective inhibition was exerted by BWA4C, suggesting metabolism of both PUFAs through the lipoxygenase pathway for growth stimulation. In vivo studies using the MAC26 tumour showed a significant stimulation of tumour growth when LA was administered orally at concentrations higher than 0.4 g kg-1 day-1. Higher concentrations did not produce a further increase in tumour growth rate. This suggests that there is a threshold dose for growth stimulation by LA which, together with that in the diet, amounted to 3.8% of the total caloric intake. The increase in tumour volume induced by LA arose from a reduction in the potential doubling time from 41 to 28 h and was effectively reversed by indomethacin (5 mg kg-1). These results suggest that PUFAs may play an important role in tumour growth and may offer a potential target for the development of chemotherapeutic agents.

Control of synthesis of wall teichoic acid during balanced growth of Bacillus subtilis W23.

Enzymes involved in the synthesis of teichoic acid and its linkage to the wall in Bacillus subtilis W23 were measured in chemostat cultures growing at equilibrium at a dilution rate of 0.2 h-1 in different concentrations of inorganic phosphate. All the enzymes, except teichoic acid glucosyl transferase, which was insensitive to changes in phosphate concentration, were almost undetectable at 0.5 mM-phosphate. At higher phosphate concentrations the changes in activity of the enzymes of linkage unit synthesis were sufficient to account for the changes in the rate of incorporation of teichoic acid into the wall in vivo. Between 3.5 and 4.5 mM-phosphate the amount of teichoic acid synthesized in vivo increased, but no increase in the ability of toluenized bacteria to synthesize teichoic acid could be detected. Allosteric regulation might therefore be important at high phosphate concentrations. Bacteria maintained a constant ATP content and a constant adenylate energy charge during chemostat growth at all phosphate concentrations.

Control of synthesis of wall teichoic acid in phosphate-starved cultures of Bacillus subtilis W23.

CDP-glycerol pyrophosphorylase, CDP-ribitol pyrophosphorylase and poly(ribitol phosphate) synthetase activities have been measured in cultures of Bacillus subtilis W23 as they became phosphate-starved either in batch culture or during changeover from potassium limitation to phosphate limitation in a chemostat. The results indicated that repression of synthesis of all three enzymes occurred at the onset of phosphate starvation and that this was accompanied by inhibition of inactivation of CDP-glycerol pyrophosphorylase and poly(ribitol phosphate) synthetase. These results show that the initial response to phosphate starvation involves more than inhibition of one enzyme as proposed by Glaser and Loewy [Glaser L. and Loewy, A. (1979) J. Biol. Chem. 254, 2184-2186]. Synthesis of both linkage unit and poly(ribitol phosphate) are inhibited independently.