Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

Quinacrine directly dissociates amyloid plaques in the brain of 5XFAD transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia characterized by the abnormal accumulation of amyloid-ß (Aß) in the brain. Aß misfolding is associated with neuroinflammation and synaptic dysfunction, leading to learning and memory deficits. Therefore, Aß production and aggregation have been one of the most popular drug targets for AD. Failures of drug candidates regulating the aforementioned Aß cascade stimulated development of immunotherapy agents for clearance of accumulated Aß in the brain. Here, we report that quinacrine, a blood-brain barrier penetrating antimalarial chemical drug, dissociates Aß plaques in the brain of AD transgenic mice. When co-incubated with pre-formed Aß fibrils, quinacrine decreased thioflavin T-positive ß-sheets in vitro, on top of its inhibitory function on the fibril formation. We confirmed that quinacrine induced dissociation of high-molecular-weight Aß aggregates into low-molecular-weight species by dot blots in association with size cut-off filtrations. Quinacrine was then administered to adult 5XFAD transgenic mice via weekly intravenous injections for 6 weeks, and we found a significant reduction of Aß plaques and astrocytosis in their cortex and hippocampus. In western blots of quinacrine-administered mouse brains, amelioration of AD-related biomarkers, glial fibrillary acidic protein, postsynaptic protein 95, phosphorylated cAMP response element-binding protein, phosphorylated c-Jun N-terminal kinase were observed. Lastly, quinacrine-stimulated dissociation of misfolded aggregates induced recovery of synaptic function associated with Aß in excitatory post-synaptic current recordings of primary rat cortical neurons treated with Aß aggregates and quinacrine. Collectively, quinacrine can directly dissociate Aß fibrils and alleviate decreased synaptic functions.

DNA intercalation by quinacrine and methylene blue: a comparative binding and thermodynamic characterization study.

There is compelling evidence that cellular DNA is the target of many anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA-binding agents, quinacrine (QNA) and methylene blue (MB), with calf thymus (CT) DNA. The study revealed noncooperative binding phenomena for both the drugs to DNA with an affinity one order higher for QNA compared to MB as observed from diverse techniques, but both bindings obeyed neighbor exclusion principle. The data of the salt dependence of QNA and MB from the plot of log K versus log [Na+] revealed a slope of 1.06 and 0.93 consistent with the values predicted by theories for the binding of monovalent cations, and have been analyzed for contributions from polyelectrolytic and nonpolyelectrolytic forces. The binding of both drugs was further characterized by strong stabilization of DNA against thermal strand separation in both optical melting and differential scanning calorimetry studies. The binding data analyzed from the thermal denaturation and from isothermal titration calorimetry (ITC) were in close proximity to those obtained from spectral titration data. ITC results revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. The heat capacity changes obtained from temperature dependence of enthalpy indicated -146 and -78 cal/(mol.K), respectively, for the binding of QNA and MB to CT DNA. Circular dichroism study further characterized the structural changes on DNA upon intercalation of these molecules. Molecular aspects of interaction of these molecules to DNA are discussed.

Evaluation of the protective effects of quinacrine against Bacillus anthracis Ames.

Bacillus anthracis has gained notoriety as a dangerous biological weapon because of its virulence and ability to produce highly resistant spores. In addition, the ability of this organism to produce plasmid-encoded edema toxin (EdTx) and lethal toxin (LeTx) plays a pivotal role in the pathogenesis of anthrax. In this study, the efficacy of quinacrine was evaluated against the effects of anthrax toxins in vitro and its ability to provide protection against challenge with B. anthracis Ames strain spores in an intranasal mouse and guinea pig model. Quinacrine protected murine macrophages in vitro against cytotoxicity and cAMP production induced by LeTx and EdTx, respectively, at concentrations of 40-80 microM, most likely by preventing acidification of the endosomes. However, animals dosed with human equivalent doses of quinacrine were not protected against respiratory spore challenge. The failure of quinacrine to provide protection against inhalation anthrax was attributed to our inability to attain inhibitory concentrations of the drug in the serum or tissues. After daily administration of 43.3 mg quinacrine to guinea pigs (300 g), serum levels after 96 h were only 9.9 microM, a concentration not sufficient to protect macrophages in vitro. Administration of high doses of quinacrine (86.6 mg/kg) was toxic to the animals. These results illustrate some of the difficulties in developing protective therapeutic strategies against inhalation anthrax even when antitoxic drugs appear effective in vitro.

A possible pharmacological explanation for quinacrine failure to treat prion diseases: pharmacokinetic investigations in a ovine model of scrapie.

Quinacrine was reported to have a marked in vitro antiprion action in mouse neuroblastoma cells. On compassionate grounds, quinacrine was administered to Creutzfeldt-Jakob disease patients, despite the absence of preclinical in vivo studies to evaluate efficacy. Quinacrine failed to provide therapeutic benefit. The aim of the study was to investigate possible pharmacokinetic and/or pharmacodynamic explanations for the discrepancy between the proven action of quinacrine in vitro and its lack of clinical efficacy. We conducted in vitro experiments reproducing the culture conditions in which antiprion effects had been previously observed and recalculated the EC(50) by determining the actual extracellular (120 nM) and intracellular (6713 nM) quinacrine neuroblastoma concentrations with the reported quinacrine EC(50) (300 nM). A randomized clinical trial in scrapie-affected ewes confirmed the absence of therapeutic benefit of quinacrine. The in vivo quinacrine exposure was evaluated in a pharmacokinetic investigation in healthy ewes. Cerebrospinal fluid concentrations (<10.6 and 55 nM after administration of therapeutic and toxic quinacrine doses, respectively) were much lower than the quinacrine extracellular neuroblastoma concentrations corresponding to the reported EC(50). The total brain tissue concentrations (3556 nM) obtained after a repeated therapeutic dosage regimen were within the range of the intracellular neuroblastoma quinacrine concentrations. In conclusion, in order to avoid in vivo trials for which failure can be predicted, the measurement in vitro of the antiprion EC(50) in both intra- and extracellular biophases should be determined. It can then be established if these in vitro antiprion concentrations are achievable in vivo.

Antimalarial activity of optical isomers of quinacrine dihydrochloride against chloroquine-sensitive and -resistant Plasmodium falciparum in vitro.

Both enantiomers of quinacrine and the racemic form of the drug showed equal activity in vitro against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, without detectable stereoselectivity. This contrasts with observations on chloroquine, where a similar lack of stereoselectivity in vitro is accompanied by a 10-fold loss of activity against the resistant strain. The observed in vivo differences reported for the enantiomers of chloroquine and the observations on the optically active metabolites of chloroquine and quinacrine may therefore be ascribed to a difference in the pharmacokinetics of their enantiomers.

The use of quinacrine (Atabrine) in rheumatic diseases: a reexamination.

Atabrine has been available for nearly 60 years. It has a variety of actions and has been administered to millions of individuals. Its antirheumatic properties have been well documented but have not been exploited optimally for a variety of reasons. The drug is generally quite safe and could be used in low doses in lupus and rheumatoid arthritis patients as a steroid-sparing agent or synergistically with hydroxychloroquine. Its bothersome side effects should not deter the clinician from using it, because they are easy to deal with or prevent (Table 5). Future studies should attempt to better characterize the immunosuppressive actions of this powerful drug, particularly in the treatment of lupus erythematosus and rheumatoid arthritis. Studies of the role of combination or single-agent antimalarial therapy in combination with other "remittive" drugs could be of great potential benefit.

Interstitial quinacrine for elimination of abnormal tissue; therapy of experimental glioma.

BACKGROUND: When quinacrine is injected interstitially, an intense migration of leukocytes and accumulation of various lymphokines is obtained locally, and the reaction is followed by cicatricial fibrosis. This property has been used in humans to induce tubal fibrosis in women and pleurodesis in patients with pleural effusion. METHODS: In a controlled study, a single dose of 150 mg of quinacrine was injected interstitially into a C6 glioma implanted in the subcutaneous tissue of Wistar rats. Changes in size, histologic variations, and microscopic characteristics of leukocyte subpopulations infiltrating the tumor were studied by immunohistochemistry. Tumor necrosis factor and interleukin-1 beta were measured at different times in tumor homogenates. RESULTS: The day after the injection of quinacrine, infiltration of leukocytes and macrophages was observed, accompanied by an accumulation of proinflammatory endogenous cytokines. Tumoral necrosis soon ensued; complete tumor disappearance was obtained in 72% of the animals. Cicatrization proceeded without injury of perilesional structures. In all controls injected with the vehicle, a large tumor developed (P <.0001). CONCLUSIONS: Quinacrine, when administered interstitially in a single dose, elicits an intense local recruitment and proliferation of activated immune cells that, at the dose used in this study, induces tissue necrosis within a radius of 1 cm around the site of quinacrine injection, leaving the surrounding tissue unharmed.

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.

Phase I prehysterectomy studies of the transcervical administration of quinacrine pellets.

To determine the safety of transcervical administration of quinacrine pellets as a method of voluntary female sterilization, three noncomparative Phase I clinical trials of the administration of 250 mg quinacrine were carried out in 21 women who were scheduled to undergo hysterectomy 24 h or one month later. Detailed results are presented for one of the trials using 10-min pellets. Six of 10 women had minor transitory complaints during the postinsertion 24-h follow-up period. Five women reported pelvic/abdominal cramping, one experienced headache, and one experienced dizziness. Blood chemistry values were not adversely influenced by the quinacrine. The average plasma level of quinacrine peaked at 3 h, 36.1 ng/ml, slightly lower than the value observed 4 h after oral administration of 200 mg in a previous study. An average of 27% of the administered dose was recovered in tampons. Quinacrine was detected in the plasma of two women at the four/six-week visit. Selected results are presented from two other trials that were halted because of slow recruitment. The transcervical administration of 250 mg of 10-min quinacrine pellets was well tolerated. However, based on recent mutagenicity testing and meetings with regulatory officials, it appears unlikely that the use of quinacrine for nonsurgical sterilization could be approved in the United States or Europe.

Pharmacokinetics of quinacrine after intrapleural instillation in rabbits and man.

Quinacrine was given by intrapleural instillation or intravenous infusion to 10 rabbits. The uptake of quinacrine from the pleural space was rapid and complete. The mean absorption half-life was approximately 7 min and the mean bioavailability was slightly in excess of 100%. Similar absorption characteristics generally applied in man, in a pilot study on four patients. In three of them, peak quinacrine plasma concentrations were reached that were far above the normal therapeutic range. Known systemic side-effects of quinacrine comprise CNS stimulation, toxic psychosis and convulsions. In view of the high bioavailability and the large doses used for pleural sclerosing (pleurodesis) in patients, neurological disease and psychiatric disturbances that predispose to CNS toxicity should be considered as contraindications to intrapleural quinacrine.

Location of quinacrine pellets in different positions of non-surgical female sterilization.

OBJECTIVE: To compare quinacrine pellets distribution in the uterine cavity between women standing up promptly after insertion and those lying down for a further 30-minutes. DESIGNS: Randomized controlled trial MATERIAL AND METHOD: Twenty women who, desired interval female sterilization, were equally randomized into 2 groups. Quinacrine pellets were inserted twice, one month apart in both groups. Transvaginal ultrasonography (TVS) was performed after insertion in order to locate the position of the quinacrine pellets. In group I, the TVS was performed after the women stood up promptly. In group II, the TVS was performed after the women lay down for 30-minute. The distribution of quinacrine pellets, measured from the uterine fundus to the lowest pellet, were compared. RESULTS: There was no difference at the baseline and 30 minutes measurement of both groups. In the first insertion, there was statistical difference of the distance of the quinacrine pellets when compared in the same group immediately and the 30-minute measurement (Gr 1: 19.10+/-3.28 mm vs 22.30+/-3.50 mm); (Gr 2: 18.70+/-3.40 mm vs 24.40+/-5.95 mm). In the same manner, the authors found statistical difference in the same group of the second insertion (Gr 1: 21.80+/-5.39 mm vs 24.70+/-7.24 mm); (Gr 2 : 20.89+/-4.78 mm vs 28.30+/-7.59 mm) CONCLUSIONS: There was statistical difference of quinacrine pellet distribution in uterine cavity after time. However, body movement did not effect the position of the pellets. The failure rate of quinacrine pellet insertion for non-surgical female sterilization may not be explained by the changes of position after insertion.

[Prospects of the therapeutic approaches to Creutzfeldt-Jakob disease: a clinical trial of antimalarial, quinacrine].

Prion diseases are lethal transmissible neurodegenerative illnesses that affect humans and many other animals. Since the accumulation of the pathogenic form of prion protein is a pivotal event in prion diseases, most of the therapeutic strategies are designed to prevent the conformational change of normal prion protein to that of the pathogenic form or to remove the accumulated prion protein. Quinacrine is one of the compounds that can inhibit the accumulation of pathogenic prion protein in cultured neuroblastoma cells. Here we report the results of clinical trials of quinacrine administration to the patients of Creutzfeldt-Jakob disease. In some patients, response to visual and auditory stimulations improved transiently. Lemon-yellow discolorization of skin and liver dysfunction were common side effects by quinacrine.

Multifunctional targeting daunorubicin plus quinacrine liposomes, modified by wheat germ agglutinin and tamoxifen, for treating brain glioma and glioma stem cells.

Most anticancer drugs are not able to cross the blood-brain barrier (BBB) effectively while surgery and radiation therapy cannot eradicate brain glioma cells and glioma stem cells (GSCs), hence resulting in poor prognosis with high recurrence rates. In the present study, a kind of multifunctional targeting daunorubicin plus quinacrine liposomes was developed for treating brain glioma and GSCs. Evaluations were performed on in-vitro BBB model, murine glioma cells, GSCs, and GSCs bearing mice. Results showed that the multifunctional targeting daunorubicin plus quinacrine liposomes exhibited evident capabilities in crossing the BBB, in killing glioma cells and GSCs and in diminishing brain glioma in mice. Action mechanism studies indicated that the enhanced efficacy of the multifunctional targeting drugs-loaded liposomes could be due to the following aspects: evading the rapid elimination from blood circulation; crossing the BBB effectively; improving drug uptake by glioma cells and GSCs; down-regulating the overexpressed ABC transporters; inducing apoptosis of GSCs via up-regulating apoptotic receptor/ligand (Fas/Fasl), activating apoptotic enzymes (caspases 8, 9 and 3), activating pro-apoptotic proteins (Bax and Bok), activating tumor suppressor protein (P53) and suppressing anti-apoptotic proteins (Bcl-2 and Mcl-1). In conclusion, the multifunctional targeting daunorubicin plus quinacrine liposomes could be used as a potential therapy for treating brain glioma and GSCs.

Pharmacokinetics of quinacrine efflux from mouse brain via the P-glycoprotein efflux transporter.

The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp-deficient Mdr1(0/0) mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr1(0/0) brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS.

Mitochondrial targeting liposomes incorporating daunorubicin and quinacrine for treatment of relapsed breast cancer arising from cancer stem cells.

Breast cancer stem cells play a crucial role in the relapse of breast cancers because they are resistant to a standard chemotherapy and the residual cancer stem cells are able to proliferate indefinitely. The objectives of present study were to construct a kind of mitochondrial targeting daunorubicin plus quinacrine liposomes for treating and for preventing the recurrence of breast cancer arising from the cancer stem cells. MCF-7 cancer stem cells were identified as CD44(+)/CD24(-) cells and cultured in free-serum medium. Evaluations were performed on MCF-7 cancer stem cells, MCF-7 cancer stem cell mammospheres, and the relapsed tumor by xenografting MCF-7 cancer stem cells into female NOD/SCID mice. The particle size of mitochondrial targeting daunorubicin plus quinacrine liposomes was approximately 98 nm. The mitochondrial targeting liposomes evidently increased the mitochondrial uptake of drugs, were selectively accumulated into mitochondria, activated the pro-apoptotic Bax protein, dissipated the mitochondrial membrane potential, opened the mitochondrial permeability transition pores, released cytochrome C by translocation, and initiated a cascade of caspase 9 and 3 reactions, thereby inducing apoptosis of MCF-7 cancer stem cells. The mitochondrial targeting liposomes showed the strongest efficacy in treating MCF-7 cancer cells in vitro, in treating MCF-7 cancer stem cells in vitro, and in treating the relapsed tumor in mice. Mitochondrial targeting daunorubicin plus quinacrine liposomes would provide a new strategy for treating and preventing the relapse of breast cancers arising from cancer stem cells.

Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents.

Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10-20 mM for 1-2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3-4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo.

Nanokinetics of drug molecule transport into a single cell.

AIMS: To analyze drug transport at a single cell level, a mast cell line, RBL-2H3, was treated with cell-permeable fluorescent compounds, such as quinacrine, and was monitored by a fluorescence video microscope. METHODS: Small areas in the video that corresponded to granules and part of the cytosol in a cell were chosen and the signal intensity in these areas was monitored sequentially. RESULTS: The initial rate of quinacrine uptake through the cell membrane calculated from the fluorescent signal was correlated with quinacrine concentration, and it decreased at a lower temperature, showing that the transport was an energy-requiring process, such as active transport. The kinetics of the transport through the microgranular membrane did not depend on the temperature but the pH in the cytosol, therefore this process should be passive transport by pH gradient. CONCLUSION: These data indicate that the observation of video microscope-mediated drug transport using fluorescent dye is useful in kinetic analysis at the nanometer scale.

Quinacrine is mainly metabolized to mono-desethyl quinacrine by CYP3A4/5 and its brain accumulation is limited by P-glycoprotein.

Quinacrine (QA), an antimalarial drug used for over seven decades, has been found to have potent antiprion activity in vitro. To determine whether QA can be used to treat prion diseases, we investigated its metabolism and ability to traverse the blood-brain barrier in mice. In vitro and in vivo, we identified by liquid chromatography-tandem mass spectrometry the major metabolic pathway of QA as N-desethylation and compared our results with an authentic reference compound. The major human cytochrome (P450) isoforms involved in QA mono-desethylation were identified as CYP3A4/5 by using specific chemical and antibody inhibition as well as cDNA-expressed P450 studies. QA transport from the basolateral to apical side in multidrug resistance protein 1 gene (MDR1)-transfected Madin-Darby canine kidney (MDCK) cells was markedly greater than in control MDCK cells and was inhibited by the potent P-glycoprotein (P-gp) inhibitor GG918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-iso-1-quinolynyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). In MDR1-knockout (KO) mice, QA brain levels were 6 to 9 times higher after a single i.v. dose of 2 mg/kg QA and 49 times higher after multiple oral doses of 10 mg/kg/day QA for 7 days, compared with those in wild-type (WT) FVB mice. In contrast, the QA levels in plasma, liver, spleen, and kidney were similar after a single 2 mg/kg i.v. dose and <2 times greater after 10 mg/kg oral doses in MDR1-KO mice compared with WT mice. These results indicate that P-gp plays a critical role in transporting QA from the brain.

A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict.

Many weakly basic drugs incubated with cells have been shown to specifically accumulate in lysosomes. The mechanistic basis and substrate specificity for this sequestration have not been rigorously evaluated; however, conditions are favorable for a pH-partitioning type accumulation. In some circumstances, this compartmentalization can be very extensive, which can impact the therapeutic efficacy of a drug. Despite the pharmaceutical importance, direct quantitative assessments of drug accumulation in lysosomes have not been previously described. We report here a novel magnetic capture technique that allows for quick and efficient isolation of lysosomes from cultured HL-60 cells that have been preincubated with model compounds. The amount of compound associated with the isolated fraction is determined by HPLC. Extensive biochemical and morphological characterizations of isolated lysosomes, together with HPLC data, allowed for estimates to be made regarding the concentration of model compounds in lysosomes. The corresponding theoretically determined concentration values, based on pH-partitioning theory, were also calculated for comparison purposes. Interestingly, experimentally determined values were approximately 3-15 times higher than theoretically predicted values. This finding suggests that mechanisms, in addition to pH-partitioning, may play a significant role in the accumulation of drugs in lysosomes.