Triton WR-1339, a lysosomotropic compound, is excreted into bile and alters the biliary excretion of lysosomal enzymes and lipids

In these experiments, we tested two hypothesis: first, that Triton WR-1339, a nonionic detergent which is sequestered in hepatocyte lysosomes, undergoes biliary excretion; and second, that Triton WR-1339, which also alters serum lipid levels and modifies hepatic catabolism of lipoproteins, affects the biliary output of proteins and lipids. When 3H-Triton WR-1339 was administered to rats, biochemical and morphologic studies showed that hepatocyte lysosomes sequestered Triton WR-1339: (i) the subcellular distribution of 3H was identical to that of lysosomal enzymes after liver fractionation by differential or isopycnic centrifugation, and (ii) lysosomes appeared engorged with Triton WR-1339 on electron microscopy. 3H was also excreted into bile in parallel to three lysosomal enzymes. Triton WR-1339 administration caused a coordinate increase in the biliary excretion of three lysosomal enzymes and also increased the biliary output of total protein, bile acids, and phospholipid. Triton WR-1339 administration did not affect bile flow or the biliary outputs of cholesterol, plasma membrane, and cytosolic enzymes, but did decrease biliary cholesterol saturation by 50%. These results demonstrate that an exogenous compound which is sequestered in hepatocyte lysosomes may be excreted directly into bile in parallel with endogenous lysosomal constituents. The data also show that such a lysosomotropic agent may also selectively modify the biliary excretion of proteins and lipids. The findings are consistent with the existence of a lysosome-to-bile hepatic excretory pathway and suggest that hepatocyte lysosomes may be important in modulating biliary protein and lipid secretion.     

Schistosomicidal activity of the antimalarial drug, mefloquine, in Schistosoma mansoni-infected mice

Therapeutic effects of racemic mefloquine were assessed in Schistosoma mansoni-infected mice, and evaluated by recording worm burden, the status of egg maturation and viability, and intestinal mast cell recruitment. Age-matched mice were divided into four groups, of which two were infected. At 8 weeks postinfection, one group of infected and one group of uninfected mice were treated with a single dose of mefloquine (150 mg/kg). Ten days after treatment, all animals were killed. Mefloquine at 150 mg/kg had no effect on worm burden, but significantly reduced the number of eggs in the first three developmental egg stages. Analysis of intestinal mast cell numbers showed that mefloquine induced mastocytosis both in infected and control animals. In conclusion, mefloquine significantly reduces egg production in S. mansoni-infected mice, suggesting a therapeutic potency in schistosomiasis therapy. Mefloquine also exerts a significant proinflammatory effect on the intestine. Through its effect on egg production, mefloquine may be a cause of silent schistosomiasis in travelers using mefloquine for malaria chemoprophylaxis. Further study of the anti-schistosomal activity of mefloquine is warranted, as its activity against other helminths.     

Serious adverse events of mefloquine in relation to blood level and gender

Mefloquine is widely used for prophylaxis in areas with chloroquine-resistant falciparum malaria. As the use of mefloquine has increased, so have the reports on its adverse effects. We sought to evaluate the possible association between serum levels of mefloquine and serious side effects caused by this drug by means of a case-control design study. The study population included 17 patients who presented to emergency rooms or travel clinics with symptoms suggesting serious adverse effects of mefloquine and 28 controls (healthy people, still taking mefloquine after travel). The mean age of the patients and the controls was 31.5 +/- 11.6 years and 34 +/- 12.2 years, respectively. The percentage of women among the patients was higher than in the control population (76% versus 40%, respectively; P = 0.03). Most of the complaints were related to the central nervous system (13 of 17); 5 patients interrupted their trip and 2 others were hospitalized. No difference in the level of mefloquine in the blood was found between the patients and the control groups. Also, no significant difference was found between mefloquine levels in the blood of men and women. These results suggest that blood levels of mefloquine do not correlate with its severe adverse events. Women tended to be more susceptible than men, despite having similar blood levels of the drug.     

Artesunate with mefloquine at various intervals for non-severe Plasmodium falciparum malaria

To study the efficacy, tolerance, population pharmacokinetics and pharmacodynamics of artesunate followed by mefloquine at various intervals, 360 patients with Plasmodium falciparum malaria received 4 mg/kg of artesunate and thereafter 15 mg/kg of mefloquine simultaneously (group A), after 8 hours (after group B), and after 24 hours (group C). Three dosages were completed with placebo. Follow-up was 28 days. All patients recovered rapidly except one case of failure within the first 24 hours. Mefloquine pharmacokinetics was similar in the three regimens. Parasites reappeared in 26%, 26%, and 33% of the patients in groups A, B, and C, respectively. Early recrudescence was associated with high initial parasite density, slow parasite clearance, and rapid mefloquine clearance and low plasma concentrations at day 28. Mefloquine plasma concentrations all reached therapeutic ranges, suggesting reduced parasite sensitivity. In conclusion, there is no interaction between artesunate and mefloquine with respect to tolerance, efficacy, and pharmacokinetics. Single-dose combination therapy with artemisinin drugs and 15 mg/kg of mefloquine does not completely prevent parasite recurrence and may not prevent mefloquine resistance.     

Investigation of the in vitro Gender-Specific Partitioning of Mefloquine in Malarial Infected Red Blood Cells and Plasma

The investigation of gender-specific partitioning of the antimalarial drug mefloquine to cellular and fluid blood compartments was performed using blood collected from a female and male healthy subject that were infected with Plasmodium falciparum PCM2 clone and spiked with mefloquine (0.25, 1, and 5 μM). Mefloquine concentrations in red cells of both female and male subjects were significantly higher than plasma, which suggests an intensive uptake by red cells. This was supported by a high ratio of mefloquine concentrations in the parasitized and non-parasitized red cells of about 4-fold. Gender-specific partitioning of mefloquine in parasitized blood was seen only in plasma where significantly higher concentrations were observed in female compared with male plasma. Down-adjusting the therapeutic dose of mefloquine in female patients with malaria is not advisable because mefloquine concentrations in the target cellular compartment are similar in both genders.     

Pharmacokinetics of prophylactic mefloquine

The pharmacokinetics of the prophylactic dose of mefloquine (Lariam: 500 mg every 4 weeks, with a loading dose of 750 mg on the first week) was studied in six healthy Thai male volunteers. Mefloquine was well tolerated during the study period of 16 weeks. The only side-effects found were nausea and diarrhea in 2 volunteers after the first dose of mefloquine. The mean minimum concentration of mefloquine at steady state ranged from 290 to 460 ng/ml. The maximum concentration on week 16 after the last dose was 1558 +/- 48 ng/ml at the mean time of 38 +/- 19 hours. The other pharmacokinetic parameters obtained were: absorption half life = 6.6 +/- 3.0 hours; distribution = 5.1 +/- 3.1 days; terminal half life = 12.9 +/- 2.2 days; apparent volume of distribution = 10.5 +/- 2.3 l/kg; area under the concentration-time curve = 26.9 +/- 2.2 mg/dl. Although this prophylaxis regimen is ideal when considering the compliance, the minimum concentration obtained was much too low for optimum therapeutic concentration. We therefore suggest that weekly prophylaxis schedule should be a better regimen as the difference between minimum and maximum mefloquine concentration would be smaller.     

Cured of fear of flying

A woman is described who developed fear of flying because she took mefloquine as malaria prophylaxis prior to the flight. Mefloquine, because of its potential neurotoxicity, should not be used for persons with fear of flying.     

Ethanol Administration Alters the Proteolytic Activity of Hepatic Lysosomes

Protein accumulation in liver cells contributes to alcohol-induced hepatomegaly and is the result of an ethanol-elicited deceleration of protein catabolism (Alcohol Clin Exp Res 1349, 1989). Because lysosomes are active in the degradation of most hepatic proteins, the present studies were conducted to determine whether ethanol administration altered the proteolytic activities of partially purified hepatic lysosomes. Rats were fed liquid diets containing either ethanol (36% of calories) or isocaloric maltodextrin for periods of 2–34 days. Prior to death, all animals were injected with [³H]leucine to label hepatic proteins. Rats subjected to even brief periods of ethanol feeding (2–8 days) exhibited significant hepatomegaly and hepatic protein accumulation compared with pair-fed control animals. Crude liver homogenates and isolated lysosomal-mitochondrial and cytosolic subfractions were incubated at 37°C, and the acid-soluble radioactivity generated during incubation was measured as an index of proteolysis. At neutral pH, in vitro protein breakdown in incubated liver homogenates and subcellular fractions from control and ethanol-fed rats did not differ significantly. The extent of protein hydrolysis increased when samples were incubated at pH 5.5, which approximates the pH optimum for catalysis by lysosomal acid proteases. Under the latter conditions, partially purified lysosomes from control animals had 2-fold higher levels of proteolysis than corresponding fractions from ethanol-fed rats. The difference in proteolytic capacity appeared to be related to a lower latency and a higher degree of fragility of lysosomes from ethanol-fed rats at the acidic pH. The results suggest that ethanol-induced alterations in lysosomal membranes may be partially responsible for their altered capacities for protein hydrolysis. Such changes may result from ethanol-related alterations in lipid metabolism that may affect lysosome biogenesis or the maturation of lysosomes from autophagic vacuoles.     

Intragastric mefloquine is absorbed rapidly in patients with cerebral malaria

Mefloquine has proved effective in chloroquine- and quinine-resistant falciparum malaria, but it cannot be given parenterally. We have measured the absorption of mefloquine hydrochloride suspension (mean 15.6, range 9.7-28.6 mg/kg) given by nasogastric tube to 19 cerebral malaria patients already receiving intravenous quinine. Absorption was rapid with both dose schedules used; mean absorption half-times were 1.5 and 1.8 hr, and plasma mefloquine concentrations exceeded 200 ng/g within 3 hr of completing administration in all but one exceptionally ill patient who died 40 hr later. Steady state plasma concentrations over 7 days ranged from 300 to 1,050 (mean 561) ng/g. Bioavailability of mefloquine suspension in cerebral malaria therefore appears to be adequate for treatment in all but the most severely ill patients. Although intragastric mefloquine cannot now be recommended as an alternative to intravenous quinine for the treatment of severe chloroquine-resistant falciparum malaria, this situation could change if quinine resistance increases further.     

The effects of mefloquine treatment in pregnancy.

We investigated the relationship between mefloquine antimalarial treatment and the outcome of pregnancy in Karen women living in an area along the western border of Thailand where multidrug-resistant Plasmodium falciparum infections are common. Of 3,587 pregnancies investigated, 208 (5.8%) were exposed to mefloquine, 656 (18.3%) to quinine only, and 909 (25.3%) to other antimalarials, and 2,470 (68.9%) had no documented malaria. There were 61 stillbirths and 313 abortions. Women who received mefloquine treatment during but not before pregnancy had a significantly greater risk of stillbirth than did women treated with quinine alone (odds ratio [OR], 4.72; 95% confidence interval [CI], 1.7-12.7), women exposed to other treatments (OR, 5.10; 95% CI, 2-13.1), and women who had no malaria (OR, 3.50; 95% CI, 1.6-7.6) (P < .01). This association remained after adjustment for all identified confounding factors. Mefloquine was not associated with abortion, low birth weight, neurological retardation, or congenital malformations. Mefloquine treatment during pregnancy was associated with an increased risk of stillbirth.     

The chemotherapy of rodent malaria, XXVII. Studies on mefloquine (WR 142,490).

Mefloquine (WR 142,490) is a potent blood schizontocide active against drug-sensitive and drug-resistant lines of Plasmodium berghei. The ED50 and ED90 against the P. berghei N strain in albino mice are 1.5 and 3.8 mg/kg respectively. The highly chloroquine-resistant RC line is less sensitive and mefloquine is not fully effective at the maximum tolerated dose in the '4-day test'. Mefloquine has a similar mode of action to quinine both in vitro and (as demonstrated by the morphological changes it induces in P. berghei) in vivo, but is some 100 times more potent. Unlike quinine and WR 122,455 it appears not to interact with DNA. It has no causal prophylactic effect. Mixtures of mefloquine with pyrimethamine, sulphaphenazole or primaquine have an additive effect.     

Cost considerations of malaria chemoprophylaxis including use of primaquine for primary or terminal chemoprophylaxis

The costs of mefloquine, chloroquine, doxycycline, primaquine, and atovaquone/proguanil are calculated for various durations of exposure to malaria. The cost is included for detecting glucose 6-phosphate dehydrogenase (G6PD) deficiency before administering primaquine for primary or terminal prophylaxis. For durations of exposure ranging from 3 to 730 days, if no terminal prophylaxis is given, doxycycline (generic) is the least expensive regimen. Compared with doxycycline hyclate, chloroquine costs three to four times more, and primaquine, after screening for G6PD, costs about eight times more. Atovaquone/proguanil is less expensive than mefloquine for a 3-day exposure, but more expensive for 7 or more days. When terminal chemoprophylaxis with primaquine for 14 days is used in addition to doxycycline, mefloquine, chloroquine, or atovaquone/proguanil, primaquine alone is the least expensive regimen for exposures of < 10 days. Thereafter, doxycycline plus 14 days of primaquine is most economical. For subsequent exposures when G6PD status is already known, primaquine alone is the least expensive regimen for up to 9 days of exposure, but doxycycline is less expensive thereafter. In general, generic doxycycline hyclate is the least expensive regimen. Primaquine alone is economically attractive. Mefloquine, doxycyline monohydrate, and atovaquone/proguanil, the most expensive regimens, are similar in cost for a 7-day exposure, but thereafter, atovaquone/proguanil is much more expensive.     

Failure to observe testosterone-induced nucleus-lysosome interaction in rat ventral prostate.

The possibility that sex steroids act to promote the association of lysosomes with nuclei was studied by 2 methods in the secretory epithelium of ventral prostate of castrated rats treated with androgens: (a) electron microscopic (EM) examination of intact tissue and (b) study of fresh nuclear suspensions isolated from prostate homogenates using the fluorescing dye acridine orange (AO) and EM. AO-stained particles in nuclear suspensions were found to correspond to either (i) section granules, (ii) primary lysosomes or (iii) heterogenous dense bodies (HDB), considered to be lysosomes. Intact prostate tissue and crude nuclear suspensions were studied in adult rats 3-21 days after castration and in normal rats. Changes in nuclear architecture are evident 8 days or more after castration; the number of HDB increase while secretion granules and primary lysosomes decrease. Whether from prostate of castrated, normal or hormone-treated animals, a small number of the lysosomal HDB or primary lysosomes are seen closely apposed to epithelial cell nuclei. In response to short-term testosterone administration (15 or 60 min) in 10- and 21-day castrate rats, chromatin distribution appears to become more condensed; yet the number of associations of nuclei with lysosomal elements does not change. These studies of androgen action in rat ventral prostate (a classical target organ for the study of androgen action) provide no evidence to support the idea that lysosomal association with, or invasion of, nuclei in target cells is a general feature of sex steroid hormone action.     

Thyroid hormones control lysosomal enzyme activities in liver and skeletal muscle

Because protein degradation in liver and skeletal muscle is increased by thyroid hormones and decreased by thyroidectomy; we investigated the influence of thyroid hormones on the level of lysosomal enzymes. Hypophysectomized rats received daily injections of L-thyroxine or L-triiodothyronine. After 3 days of this regimen, homogenates of liver and skeletal muscle showed a 2- to 3-fold increase in the activities of cathepsin D, cathepsin B, and other lysosomal enzymes including leucine aminopeptidase, acid phosphatase, beta-galactosidase, N-acetylglucosaminidase, and alpha-mannosidase. In liver, this effect reflected increased enzyme activity in the two subcellular fractions that normally contain lysosomes. Titration of cathepsin D with pepstatin indicated that the increase in this activity resulted from an increase in the number of enzyme molecules. These effects occurred with both pharmacologic (thyrotoxic) and physiologic (growth-promoting) doses of thyroid hormones. Liver and skeletal muscle from thyroidectomized rats had approximately 50% of the normal levels of lysosomal enzyme activities. Under these various conditions, heart and kidney, tissues in which protein degradation does not appear to be influenced by thyroid hormones, showed no significant changes in lysosomal hydrolases. Thus, thyroid hormones regulate proteolytic and other lysosomal enzyme activities in those tissues in which these hormones influence protein degradation. Many characteristic features of hyperthyroidism and hypothyroidism may result from changes in levels of lysosomal enzymes.     

Case of drug-induced pneumonia possibly due to mefloquine (antimalarial drug)]

A 59-year-old man took mefloquine (antimalarial drug), total dose of 1,000 mg, to prevent malaria before and during traveling to South Africa. Three weeks after the first administration, he was admitted complaining of fever and dyspnea. Chest HRCT showed ground-glass opacities and consolidation in both lung fields. Withdrawal of mefloquine and treatment with corticosteroid resulted in improvement of the clinical findings. From the clinical course, we considered this case to be mefloquine-induced pneumonia. Mefloquine may have caused pulmonary toxicity.     

Storage and release of secretory granules in human prolactinomas: modification by bromocriptine

Electron microscopic morphometric evaluation of the effects of bromocriptine on human prolactinomas during and after short-term parenteral (7 days) and long-term peroral (4-6 weeks) treatment showed that a reduction in size of prolactinoma cells occurs within a few days (half-time to maximum shrinkage, 2.2 days). The number of secretory granules discharged into the intercellular space increased after short-term treatment by a factor of three but the total volume of stored secretory granules did not change significantly. The total volume of cellular lysosomes, both primary and secondary, decreased significantly to about one-half of the pretreatment value. The amount of stored lipoids, end-products of lysosomal activity, decreased in specimens treated for 7 days, but returned to pretreatment levels in specimens treated for 4-6 weeks, suggesting that lysosomal material is discharged from the cells together with the secretory material.     

Inhibitory effect of dietary lipids on chaperone-mediated autophagy

Cytosolic proteins can be selectively delivered to lysosomes for degradation through a type of autophagy known as chaperone-mediated autophagy (CMA). CMA contributes to intracellular quality control and to the cellular response to stress. Compromised CMA has been described in aging and in different age-related disorders. CMA substrates cross the lysosomal membrane through a translocation complex; consequently, changes in the properties of the lysosomal membrane should have a marked impact on CMA activity. In this work, we have analyzed the impact that dietary intake of lipids has on CMA activity. We have found that chronic exposure to a high-fat diet or acute exposure to a cholesterol-enriched diet both have an inhibitory effect on CMA. Lysosomes from livers of lipid-challenged mice had a marked decrease in the levels of the CMA receptor, the lysosome-associated membrane protein type 2A, because of loss of its stability at the lysosomal membrane. This accelerated degradation of lysosome-associated membrane protein type 2A, also described as the mechanism that determines the decline in CMA activity with age, results from its increased mobilization to specific lipid regions at the lysosomal membrane. Comparative lipidomic analyses revealed qualitative and quantitative changes in the lipid composition of the lysosomal membrane of the lipid-challenged animals that resemble those observed with age. Our findings identify a previously unknown negative impact of high dietary lipid intake on CMA and underscore the importance of diet composition on CMA malfunction in aging.     

Potent block of Cx36 and Cx50 gap junction channels by mefloquine

Recently, great interest has been shown in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with targeted connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain regions. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major effects on other gap junctions or membrane channels. We show that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC(50) approximately 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC(50) approximately 1.1 microM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fold higher. To further examine the utility and specificity of this compound, we characterized its effects in acute brain slices. Mefloquine, at 25 microM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side effect was an increase in spontaneous synaptic activity. Mefloquine (25 microM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular properties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50.     

Mefloquine chemoprophylaxis of soldiers on the Thai-Cambodian border.

Chemoprophylaxis of malaria on the Thai-Cambodian border is difficult due to the high level of drug resistance. Thirteen separate companies of Royal Thai Marine Militia were placed on 250 mg weekly mefloquine chemoprophylaxis from August 1989 to January 1990. A mean number of 722 soldiers received two or more doses of mefloquine per month for the five month study. The medication was well tolerated and compliance averaged 91%. Substantial numbers of prophylaxis breakthroughs were seen which resulted in 3.2 cases of malaria/100 man-months. Sixty-eight falciparum malaria cases were documented in men who had taken at least two mefloquine doses in the previous four weeks. No serious neuropsychiatric reactions occurred. Mefloquine chemoprophylaxis failures exist on the Thai-Cambodian border and are one sign of the spread of mefloquine resistance.     

The endocytic catalysts,Rab5a and Rab7, are tandem regulators of thyroid hormone production

Rab proteins are small GTPases that control distinct vesicular transport steps. Along the endocytic pathway, Rab5a is a rate-limiting catalyst of internalization, and Rab7 controls trafficking through late endosomes to lysosomes. The dependence of thyroid hormone production by thyrocytes on thyroglobulin endocytosis and intracellular processing in late endosomes/lysosomes suggests that its rate can be regulated by the expression or function of these endocytic catalysts. We compared the expression level and membrane recruitment of Rab5a and Rab7 in autonomous thyroid adenomas (where the cAMP cascade is constitutively activated) and surrounding quiescent tissues. The concentrations of Rab5a and Rab7, but not of Rab8, were coordinately increased up to 6-fold in adenomas, and correlated with a proportionate decrease in soluble thyroglobulin content (reflecting colloid depletion by accelerated endocytic uptake in hyperactive tissue). In adenomas, a higher proportion of Rab5a and Rab7 was membrane associated, and the equilibrium density of particulate Rab7 and iodine shifted toward lysosomal fractions, indicating that progression along the degradation pathway also was promoted. In cultures of polarized human thyrocytes from normal patients, thyroid-stimulating hormone or forskolin increased, to a similar extent, Rab5a and Rab7 but not Rab8 expression, apical endocytosis of thyroglobulin and lucifer yellow, and basolateral secretion of T(3) and T(4). Taken together, these in vivo and in vitro observations demonstrate that thyroid-stimulating hormone, via cAMP, coordinately enhances the expression of Rab5a and Rab7, which promote Tg endocytosis and transfer to lysosomes, respectively, resulting in accelerated thyroid hormone production.