High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs.

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3 µM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5 µM, ≥2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).

Development of a novel large animal model of ischemic heart failure using autologous platelet aggregates.

BACKGROUND: Current animal models of heart failure lack the biomass of thrombus that occurs in patients undergoing myocardial infarction. We propose a novel animal model of ischemic cardiomyopathy developed by sequential direct injections of autologous platelet aggregates into the coronary circulation resulting in development of ischemic cardiac insufficiency. METHODS: Autologous platelets from adult sheep were isolated and aggregated. Aggregated platelets were then injected into the coronary circulation of anesthetized animals under fluoroscopic guidance. Troponin I levels were monitored for first three days after embolization to validate cardiac tissue injury. Progression of heart failure was corroborated by monitoring changes in echo-based assessment of ejection fraction and left ventricular end-systolic and end-diastolic dimensions. Thrombus-based obstruction of coronary artery was confirmed with histopathology review by mepacrine labeling of pre-aggregated platelets. RESULTS: All experimental animals developed heart failure-like cardiac insufficiency confirmed by elevated levels of troponin I and associated with significant drop in the ejection fraction. CONCLUSIONS: Sequential injections of aggregated platelets into coronary circulation lead to progressive development of ischemic cardiac insufficiency. This phenomenon seems to mimic development of ischemic heart failure seen in human patients and opens multiple research opportunities to fill the existing gap between basic research and clinical practice.

Pharmacokinetics of quinacrine in the treatment of prion disease.

BACKGROUND: Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPSc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease. METHODS: Oral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue (brain, liver, spleen) samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended. RESULTS: Quinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPSc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 microg/g) and liver (maximum of 400 microg/g) tissue. CONCLUSIONS: The documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrPC) to PrPSc will be critical toward its potential benefit.

Results of quinacrine administration to patients with Creutzfeldt-Jakob disease.

Several chemicals inhibit the accumulation of abnormal prion proteins in vitro. We administered one, the antimalarial agent quinacrine, to three patients with sporadic Creutzfeldt-Jakob disease (CJD) and to one with iatrogenic CJD. Quinacrine at 300 mg/day was given enterally for 3 months. Within 2 weeks of administration, the arousal level of the patient with akinetic mutism improved. The other 3 patients, insensible before treatment, had integrative responses such as eye contact or voluntary movement in response to verbal and/or visual stimuli restored. Clinical improvement was transient, lasting 1-2 months during treatment. Quinacrine was well tolerated, except for liver dysfunction and yellowish pigmentation. Although its antiprion activity in the human brain has yet to be proved, these modest effects of quinacrine suggest the possibility of using chemical intervention against prion diseases.

Mepacrine accumulation during treatment of chloroquine-resistant falciparum malaria.

Oral mepacrine dihydrochloride, 200 mg (158 mg of the base) six-hourly for five doses followed by 100 mg (79 mg of the base) eight-hourly for six days (half dosage for those less than or equal to 50 kg) was given to 21 patients with high-grade chloroquine-resistant falciparum malaria in eastern Thailand. Fifteen patients (71%) had a clinical response [fever clearance time of 81 +/- 35 hours (mean +/- S.D.)] and 13 (62%) had complete clearance of parasitaemia (clearance time 92 +/- 42 hours). Two patients were cured, but 11 patients returned with recurrent parasitaemia between 11 and 40 days after starting treatment. Five patients had an R2 response and three had an R3 response. Mepacrine retains some activity against chloroquine-resistant falciparum malaria but it cannot be recommended for use in Thailand. The doses used, which are those also recommended for giardiasis, led to progressive and potentially toxic mepacrine accumulation. Further evaluation of regimens which produce safer plasma concentration profiles is needed.

Determination of quinacrine (mepacrine) in plasma by high-performance liquid chromatography with fluorimetric detection.

Quinacrine (mepacrine, Atebrine) is an antiparasitic acridine derivative that is currently used as an intrapleural sclerosing agent. It was determined in plasma by reversed-phase high-performance liquid chromatography of a dichloroethane extract. The mobile phase was methanol-phosphate buffer (65:35) with 0.1 mM decylammonium hydrochloride and 8 mM ethanolammonium hydrochloride. Strong base was added post-column, and the detection of quinacrine was by fluorescence at 270/495 nm. Use of ethacridine lactate as internal standard was feasible but without significant advantage. The coefficient of variation for eight determinations of quinacrine was 4% at 3.4 ng/ml and 5% at 17 ng/ml. The stability of quinacrine in blood, plasma, water and dichloroethane solution was satisfactory for practical work. The usefulness of the analytical procedure for pharmacokinetic studies could be demonstrated.

Exploration of mechanisms of amine storage in the dense bodies of human platelets.

Storage of 5-hydroxytryptamine (5HT) in membrane-bound vesicles (dense bodies) of platelets has been proposed to occur as a result of the formation of macromolecular complexes between nucleotides and 5HT, or because of the existence of an electrochemical proton gradient (delta mu H+) across the vesicle membrane. Tests of the applicability of these hypotheses to 5HT storage in the dense bodies of human platelets have been made by examining the disposition of quinacrine and 5HT in these organelles following varying treatments. Binding seems unlikely, since solid analogues of the dense body core (calcium, adenine nucleotides, and pyrophosphate) do not significantly bind 5HT or quinacrine. Incubation of platelets with substances which disrupt delta mu H+ releases a large percentage of the intra-platelet quinacrine. A much smaller fraction of the total platelet 5HT is released by similar treatment, suggesting that the delta mu H+ may not be required for 5HT storage. Because inhibition of the de novo uptake of 5HT into dense bodies fails to cause the significant loss of the 5HT stored in this compartment, 5HT stores do not appear to be maintained by active 5HT uptake. Several substances which enter the dense bodies equally well at 0 degrees C and 37 degrees C cause release of 5HT at 37 degrees C but not at 0 degrees C. The release observed at 37 degrees C thus cannot be attributable to collapse of delta mu H+ or to the displacement of 5HT from intra-granular binding sites, but may be related to increased membrane permeability to 5HT at 37 degrees C. Based on these observations, it appears as if 5HT taken up into the dense bodies of human platelets is retained because the dense body membrane has a very low passive permeability for 5HT, and that many compounds which cause 5HT release at 37 degrees C may act by increasing this permeability.

Effect of intrauterine administration of tetracyclines on cynomolgus monkeys.

Cynomolgus monkeys were used to screen for chemicals which potentially could be used as tubal occluding agents. Intrauterine administrations of solution or pellets of tetracycline and its analogues (100 mg doses) were tested for their effects on morphologic changes in the reproductive tract of monkeys. These effects were compared to monkeys receiving intrauterine administration of quinacrine pellets (36 mg) since quinacrine has been used successfully in the clinical setting. Blood levels of drugs, blood chemistry and hematology determinations and liver and kidney pathology data were also obtained as indices for toxicity. Morphologic damage to the uterine lining and intramural section of the tube (including necrosis, inflammation or scarring) was elicited by intrauterine tetracycline and doxycycline in the same frequency and severity as quinacrine. In contrast, saline or sham control monkeys showed no morphological damage of the tube or uterus. Although all drugs could be detected in the blood 4 hours after intrauterine administration, levels were near or below the limit of detection by one week. No evidence was found for toxicity of tetracycline or its analogues for the dosage given. Because of these results and the extensive literature on tetracycline toxicity, further studies should be directed toward the use of tetracycline as a sterilizing agent in women.

The uptake of mepacrine by horse polymorphonuclear leucocytes in vitro.

The uptake of mepacrine by isolated horse polymorphonuclear leucocytes (PMN) was measured using spectrophotofluorimetry. Two phases of uptake were observed, the first, rapid fraction, essentially complete by 10 min, and a second, slow fraction, which was still proceeding after 60 min. The appearance of mepacrine within the PMN was also visualized by fluorescence microscopy. Discrete yellow points of fluorescence were observed in the cytoplasm of PMN within 30 s. These discrete points corresponded both in size and number to the PMN granules. After 5 min, the nuclei showed faint fluorescence which increased in intensity with time. It was concluded that the accumulation of mepacrine by horse PMN consists of at least two phases, an initial rapid component which represents uptake of the drug through the plasma membrane into the cytoplasmic granules, and a subsequent slower component, possibly representing nuclear binding.

Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid.

Mepacrine has been used as an inhibitor of the activation of endogenous phospholipases in many systems. These endogenous phospholipases are important in the modification of the lipid environment of membrane proteins and in the release of locally active oxygenated arachidonic acid metabolites. In both human platelets and erythrocytes, mepacrine blocks the release of fatty acid from phospholipid by endogenous phospholipases. However, mepacrine also interacts directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives. This interaction occurs rapidly and is maximal at concentrations of mepacrine greater than 0.2 mM. Such drug-phospholipid interaction may perturb membrane architecture and function and be responsible for the inhibitory effects of mepacrine on cellular responses observed in many systems. Since the alteration in membrane phospholipid composition occurs under the same conditions as phospholipase inhibition, it is not possible to be certain that the inhibition of cellular responses by mepacrine is due to inhibition of phospholipases rather than to direct perturbation of the membrane. It is also possible that inhibition of phospholipase action by mepacrine is in part a consequence of the change in phospholipid composition. These results indicate that caution should be exercised in the interpretation of results obtained using mepacrine and that the usefulness of this compound for the investigation of the biological importance of phospholipase activation is limited.

Platelet dense bodies loaded with mepacrine. Study in chronic idiopathic thrombocytopenic purpura (ITP).

In 22 cases of chronic ITP, the platelet 5-HT storage organelles were counted by examination of platelets loaded with mepacrine and correlated with the size and volume of the platelets. Statistical analysis showed that the mean volume and the number of granules increased in ITP without increase in the mean number of granules per unit volume. A strong correlation was found between platelet long diameter and number of dense bodies in controls (44 healthy subjects) (r = 0.94; y = 2.826 x - 0.699) and in ITP (r = 0.92; y = 2.587 x + 0.06). This study demonstrated in chronic ITP the presence both of platelets without granules and others rich in granules. The anomalies were present no matter what the count of platelets and did not change the mean values for granules and for ADP in most cases. Most platelets remain morphologically normal.

Storage pool disease: comparative fluorescence microscopical, cytochemical and biochemical studies on amine-storing organelles of human blood platelets.

The platelet content of ATP, ADP, serotonin (5-HT), dopamine, noradrenaline and adrenaline as well as the net uptake of radiolabelled 5-HT and mepacrine were subnormal in six patients with Storage Pool Disease (SPD). Fewer amine-storing organelles were found by fluorescence microscopy with the fluorescent probe mepacrine and by electron microscopy with a cytochemical (uranaffin) reaction specific for 5'-phosphonucleotides. Both methods showed that SPD platelets have atypical organelles with a reduced capacity to store amines, 5'-phosphonucleotides and mepacrine. The changes were most marked in platelets of the two patients who also had oculocutaneous albinism.

Mepacrine, a tool for investigating the 5-hydroxytryptamine organelles of blood platelets by fluorescence microscopy.

In the blood platelets of various species exposed to mepacrine, the average number of green-yellow fluorescent granules (probably identical with the 5-hydroxytryptamine [5-HT] storage organelles) corresponded to that of flashes emitted by the platelets on prolonged irradiation with violet-blue light. In platelets of fawn-hooded rats the number of granules did not markedly differ from that of normal rat platelets, but the fluorescence intensity and the uptake of mepacrine in vitro showed a marked decrease and the flashes were less numerous. The heavy population of human platelets exhibited considerably more granular structures than the light population. The data suggest that (1) in normal, mepacrine-loaded platelets one flash corresponds to one 5-HT organelle and (2) mepacrine is a useful tool for investigating the number and function of the 5-HT organelles in live platelets and possibly for studying platelet age.

Uptake and liberation of mepacrine in blood platelets.

1. Isolated platelets of guinea pigs incubated in Tyrode showed a rapid accumulation of 14C-mepacrine which was less dependent on temperature than that of 14C-5-hydroxytryptamine (5HT). 2. The uptake of mepacrine was not inhibited by imipramine, cocaine, ouabain, KCN, NaF, and reserpine. 3. Various 5HT-liberating drugs, e.g. Ro 4-1284 (benzoquinolizine derivative with reserpine-like action), amphetamine, tyramine and imipramine did not markedly affect the 14C-mepacrine content of platelets previously loaded with this compound. 4. Thrombin and chlorpromazine caused a liberation of 14C-mepacrine from the platelts which was however, less pronounced than that of 14C-5HT. 5. From these and previous findings it is concluded that 14C-mepacrine accumulates in the 5HT storage organelles by a reserpine-insensitive mechanism not dependent on an active transport at the cytoplasmatic membrane level.