What happened to quinacrine non-surgical female sterilization?

Quinacrine sterilization (QS) is a nonsurgical female method used by more than 175,000 women in over 50 countries. With FDA approval, QS is expected to be used by hundreds of millions of women. The negative international health consequences of the results of a 2-year rat study in 2010 by Cancel et al. in Regulatory Toxicology and Pharmacology (RTP) (56:156-165) are incalculable. S1C(R2) was ignored in this study, including the fundamental concept of maximum tolerated dose (MTD), which resulted in the use of massive doses (up to 35 times the MTD) which killed many of the rats and destroyed the uterus of survivors. The design of this rat study was built on the false assertion that this study mimics what happens in women. Cancel et al. (2010), concludes it "seems most likely" that genotoxicity was a major factor in the carcinogenicity observed, prompting the FDA to halt further research of QS. In RTP, McConnell et al. (2010), and Haseman et al. (2015), using the authors' data, definitively determined the carcinogenicity to be secondary to necrosis and chronic inflammation. Decisions made in the design, conduct, analysis, interpretation and reporting in this study lack scientific foundation. This paper explores these decisions.

Deciphering molecular mechanisms of arginine deiminase-based therapy - Comparative response analysis in paired human primary and recurrent glioblastomas.

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (» cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations. Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

Quinacrine induces the apoptosis of human leukemia U937 cells through FOXP3/miR-183/ß-TrCP/SP1 axis-mediated BAX upregulation.

Quinacrine, which is clinically used as an antimalarial drug, has anti-cancer activity. However, mechanism underlying its cytotoxic effect remains to be completely elucidated. In the present study, we investigated the cytotoxic effect of quinacrine on human leukemia U937 cells. Quinacrine-induced apoptosis of U937 cells was accompanied with ROS generation, mitochondrial depolarization, and BAX upregulation. Quinacrine-treated U937 cells showed ROS-mediated p38 MAPK activation and ERK inactivation, which in turn upregulated FOXP3 transcription. FOXP3-mediated miR-183 expression decreased ß-TrCP mRNA stability and suppressed ß-TrCP-mediated SP1 degradation, thus increasing SP1 expression in U937 cells. Upregulated SP1 expression further increased BAX expression. BAX knock-down attenuated quinacrine-induced mitochondrial depolarization and increased the viability of quinacrine-treated cells. Together, our data indicate that quinacrine-induced apoptosis of U937 cells is mediated by mitochondrial alterations triggered by FOXP3/miR-183/ß-TrCP/SP1 axis-mediated BAX upregulation.

A critical examination of the mode of action of quinacrine in the reproductive tract in a 2-year rat cancer bioassay and its implications for human clinical use.

A rat carcinogenicity bioassay (CaBio) of quinacrine was reanalyzed to investigate its mode of tumor induction. Quinacrine's effects in the rat uterus when administered as a slurry in methylcellulose were contrasted with the human clinical experience which uses a solid form of the drug, to determine the relevance of the tumors produced in the rat to safe clinical use of quinacrine for permanent contraception (QS). A review was performed of the study report, dose feasibility studies, and clinical evaluations of women who had undergone the QS procedure. The top three doses of quinacrine in the CaBio exceeded the maximum tolerated dose, and produced chronic damage, including inflammation, resulting in reproductive tract tumors. Chronic inflammation was significantly correlated with the tumors; there was no evidence of treatment-related tumors in animals without chronic inflammation or other reproductive system toxicity. Because such permanent uterine damage and chronic toxicity have not been observed in humans under therapeutic conditions, we conclude that this mode of action for tumor production will not occur at clinically relevant doses in women who choose quinacrine for permanent contraception.

Coloración amarillenta inducida por quinacrina en una paciente con lupus eritematoso cutáneo / Yellow Skin Discoloration Induced by Quinacrine in a Patient with Cutaneous Lupus Erythematosus

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Determination of quinacrine dihydrochloride dihydrate stability and characterization of its degradants.

Although quinacrine dihydrochloride dihydrate is a widely used drug substance, a comprehensive determination of its stability profile is lacking. In this work, an integrative approach is implemented to determine the drug stability both in the solid state and aqueous solutions, identify the impurities that can be found in the active pharmaceutical ingredient, and evaluate the associated toxicity risks. Thermal analyses pointed out a two-step dehydration of the solid state. This phenomenon seems to be consistent with the organization of the water molecules in the crystal structure and results in the destruction of the lattice. Seven related compounds of quinacrine have been identified by liquid chromatography-ion trap mass spectrometry. The main thermal degradant both in the solid state and the solution corresponds to the N-deethyl compound, whereas quinacrine tertiary amine oxyde appears to be a signal impurity of oxidative stress in solution. Moreover, two photolytic impurities can be formed in solution either by aromatic amine cleavage or via O-demethylation. Additionally, using computational approaches, the analysis of the potential toxicity of the impurities compared with the parent compound one shows that ketone and O-demethyl derivatives may exhibit specific toxicity profiles.

A novel class of potential prion drugs: preliminary in vitro and in vivo data for multilayer coated gold nanoparticles.

Gold nanoparticles coated with oppositely charged polyelectrolytes, such as polyallylamine hydrochloride and polystyrenesulfonate, were examined for potential inhibition of prion protein aggregation and prion (PrPSc) conversion and replication. Different coatings, finishing with a positive or negative layer, were tested, and different numbers of layers were investigated for their ability to interact and reduce the accumulation of PrPSc in scrapie prion infected ScGT1 and ScN2a cells. The particles efficiently hampered the accumulation of PrPSc in ScN2a cells and showed curing effects on ScGT1 cells with a nanoparticle concentration in the picomolar range. Finally, incubation periods of prion-infected mice treated with nanomolar concentrations of gold nanoparticles were significantly longer compared to untreated controls.

Evaluation of phenolphthalein, diazepam and quinacrine dihydrochloride in the in vitro mammalian cell micronucleus test in Chinese hamster ovary (CHO) and TK6 cells.

The in vitro micronucleus assay has been extensively used as an in vitro screening tool to identify test articles that might have aneugenic or clastogenic potential. Currently, the Organization for Economic Co-operation and Development (OECD) is working towards a final version of the guideline for the conduct of the in vitro mammalian cell micronucleus Test (MNvit). A few questions regarding appropriate cytotoxicity measurements and cytotoxicity limits to use remain to be answered. In order to resolve the remaining questions, we compared the induction of micronuclei at the top dose (50-60% cytotoxicity) determined by either Relative Cell Counts (RCC), Relative Increase in Cell Counts (RICC), Relative Population Doublings (RPD), or Cytokinesis-Blocked Proliferating Index (CBPI) using weak and strong inducers of micronuclei in both the presence and absence of cytochalasin B (CYB) in Chinese hamster ovary (CHO) and human lymphoblastoid TK6 cells. In order to assess extensive dose-response relationships, we selected expected weak (diazepam, phenolphthalein, quinacrine dihydrochloride dihydrate) and strong (cytosine arabinoside, mitomycin C, vinblastine sulphate) inducers of micronuclei with a variety of different mechanisms of action for testing. The results clearly demonstrated that all six compounds produced positive responses using either cytotoxicity measurement. The outcome from these studies further supports the cytotoxicity measurements and cytotoxicity limits proposed in the draft OECD guideline.

An alternative interpretation of, "A lifetime cancer bioassay of quinacrine administered into the uterine horns of female rats".

This companion article offers an alternative interpretation for the quinacrine-induced uterine tumors observed in a 2-year bioassay in rats (CaBio, Cancel et al., 2010), and provides additional data from two new experiments that support a different interpretation and analysis. Our major premise is that the design of the Cancel et al. bioassay was flawed, particularly regarding dose selection that allowed for misinterpretation of carcinogenic activity. We feel the totality of the information provided herein dictates that the doses (70/70, 70/250 and 70/350 mg/kg quinacrine) causing uterine tumors in their study clearly exceeded the maximum tolerated dose (MTD) typically administered in chronic cancer studies. Our new data support this conclusion and serve to explain the development of lesions, especially the uterine tumors, they have reported. We argue that the rat uterus is not a valid surrogate for the human fallopian tube. Further, we maintain that quinacrine is not genotoxic in vivo, as suggested in their paper. In summary, we believe that quinacrine is not carcinogenic in rats at doses that do not exceed the MTD.

A lifetime cancer bioassay of quinacrine administered into the uterine horns of female rats.

This study investigated if quinacrine can induce a tumorigenic response in rats when administered in a manner similar to the intended human use for female non-surgical sterilization. Young sexually mature female rats received two doses of quinacrine (or 1% methylcellulose control) into each uterine horn approximately 21 days apart, and were observed for 23 months after the second dose administration. Dose levels were 0/0, 0/0, 10/10, 70/70, and 70/250-350 mg/kg (first dose/second dose), which represent local doses in the uterus at approximate multiples of 1x, 8x and 40x the human dose (mg quinacrine/g uterine weight) used for female non-surgical sterilization. Rats were observed for viability, clinical signs of toxicity, and changes in body weight and food consumption. At necropsy, selected organs were weighed, macroscopic observations were recorded, and tissues were collected, fixed, processed, and examined for microscopic pathologic findings. Acute quinacrine toxicity was evident during the dosing period but did not affect long-term survival. Non-neoplastic findings were more common in treated animals than controls, providing evidence of the appropriateness of the bioassay. The incidence of uncommon tumors of the reproductive tract was similar to controls at doses of 10/10mg/kg but increased with dose level and was significantly greater than controls at >or=70/70 mg/kg. We conclude that two doses of quinacrine administered approximately 21 days apart into the uterus of young sexually mature rats at a local dose approximately 8 times the human dose used for non-surgical female sterilization increased the lifetime risk of tumor development in the reproductive tract.

Variability in cytogenetic adaptive response of cultured human lymphocytes to mitomycin C, bleomycin, quinacrine dihydrochloride, Co60 gamma-rays and hyperthermia.

Adaptive response (AR) is a well-documented phenomenon by which cells or organisms exposed to low dose of a genotoxicant become less sensitive to subsequent high-dose exposure to the same or another genotoxicant. AR, if induced can modify the efficacy leading to drug or radio-resistance, during anti-neoplastic drug or radiation treatment. Contradictions exist in AR induction by different genotoxicants with respect to the biomarkers, time schedules, and inter-individual variability, reflecting the complexity of AR in eukaryotic cells. In order to further ascertain these factors, AR induced by anti-neoplastic agents mitomycin C (MMC), bleomycin (BLM) and chemosterilant quinacrine dihydrochloride was examined in different donors and time schedules using cytogenetic biomarkers chromosome aberrations, sister chromatid exchanges and micronuclei (MN). BLM- and hyperthermia (HT)-induced cross-resistance to gamma rays and MMC/BLM, respectively, was also studied. Difference between MMC- and BLM-induced protective effects in biomarkers examined in the same donors was noticed. Adaptation to BLM and HT showed cross-resistance to chromosome damage induction by gamma rays and BLM/MMC, respectively. Cell cycle analysis indicated that adaptation is not caused by change in the rate of cell proliferation after challenge dose. MN as a chromosomal biomarker in large-scale population studies on AR is advocated, based on similar AR induced in all donors by MMC/BLM and rapid assessment in binucleated cells. Influence of certain genotypes on chromosomal biomarkers used in AR studies and role of AR in radiation and chemotherapy need to be further deciphered.

A one-year neonatal mouse carcinogenesis study of quinacrine dihydrochloride.

Quinacrine is an acridine derivative under investigation for its use in nonsurgical female sterilization. Safety issues regarding the carcinogenic potential of quinacrine have been raised because it is mutagenic and clastogenic in vitro. The objective of the study was to evaluate the carcinogenic potential of quinacrine dihydrochloride (quinacrine) in neonatal mice treated with single intraperitoneal doses on postpartum days 8 and 15 and observed for 52 weeks. Neonatal Crl: CD-1 mice of each sex were randomly allocated into four treatment groups (0, 10, 50, and 150 mg/kg), dosed twice with quinacrine suspended in carboxymethylcellulose, observed for 52 weeks post dose, and then euthanized, necropsied, and subjected to a full histopathological examination. In male mice, tumor incidence was not significantly increased at any site at any dose level. In female mice, the incidence of benign uterine endometrial stromal polyps was slightly greater at the mid and high dose (> or = 50 mg/kg), as was the incidence of endometrial hyperplasia. The incidence of polyps in these groups was not significantly greater than in controls by pair-wise comparison but was significantly greater (p = .042) by the linear trend test. The authors conclude that quinacrine administered twice to neonatal mice may have enhanced or accelerated the development of endometrial hyperplasia and uterine stromal polyps at higher doses. Because uterine stromal polyps are a commonly observed benign tumor in older mice, the significance of this finding is unclear and will require a weight of evidence evaluation for a conclusion on the carcinogenic potential of quinacrine.

Photophysical and photobiological behavior of antimalarial drugs in aqueous solutions.

This article describes the results of a combined photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs quinine (Q), quinacrine (QC) and mefloquine (MQ). Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed on irradiation. The results obtained showed that fluorescence and intersystem crossing account for all the adsorbed quanta for Q and MQ (quantum yield of about 0.1 and 0.9, respectively) and only for 24% in the case of QC, which has a negligible fluorescence quantum yield (0.001). Laser flash photolysis experiments evidenced, for QC and MQ, the occurrence of photoionization processes leading to the formation of the radical cations of the drugs. The effects of tryptophan and histidine on the excited states and transient species of the three drugs were also investigated. In parallel, the photoactivity of the antimalarial drugs was investigated under UV irradiation on various biological targets through a series of in vitro assays in the presence and in the absence of oxygen. Phototoxicity on 3T3 cultured fibroblasts and lipid photoperoxidation were observed for all the drugs. The photodamage produced by the drugs was also evaluated on proteins by measuring the photosensitized cross-linking of spectrin. The combined approaches were proven to be useful for understanding the mechanism of phototoxicity induced by the antimalarial drugs.

Toxicity of quinacrine can be reduced by co-administration of P-glycoprotein inhibitor in sporadic Creutzfeldt-Jakob disease.

1. Recent publication has suggested that quinacrine may be a candidate for treatment of Creutzfeldt-Jakob disease (CJD). But serious toxicity of quinacrine to liver and hematological system has been reported. 2. We disclosed the permeability of quinacrine can be enhanced by presence of p-glycoprotein inhibitor at blood-brain barrier in vitro. Therefore, we tried the protocol of combination of quinacrine and p-glycoprotein inhibitor, verapamil for patients with CJD. 3. When compared clinical effects by quinacrine and the combination therapy, improvement of clinical findings was observed at the same level without any adverse effects. Low-dose quinacrine with verapamil can be used as safe treatment of CJD.

Frameshift mutations induced by three classes of acridines in the lacZ reversion assay in Escherichia coli: potency of responses and relationship to slipped mispairing models.

The frameshift mutagenicity of 9-aminoacridine (9AA) was compared with that of quinacrine, the acridine mustards ICR-191 and quinacrine mustard (QM), and the nitroacridine Entozon in the lacZ reversion assay in Escherichia coli. As intercalating agents, 9AA and quinacrine cause mutations through noncovalent associations with DNA. Mustards and nitroacridines form covalent adducts in DNA and give rise to different spectra of mutations. Quinacrine and 9AA most effectively induced -1 frameshifts in a run of guanine residues, with 9AA being the more potent mutagen. They also induced +G frameshifts. The acridine mustard ICR-191 was a stronger mutagen than 9AA, owing largely to its potent induction of +G frameshifts. QM induced +G frameshifts more strongly than did its nonreactive counterpart quinacrine. The nitroacridine Entozon differed from the other acridines in being a potent inducer of -2 frameshifts, but it was less effective in inducing +/-1 frameshifts. Quinacrine, although a simple intercalator, induced all five kinds of frameshift mutations detected in the assay, as did the acridine mustards. Although +A and -A frameshifts were induced, adenine runs were less susceptible to acridine mutagenesis than guanine runs. The patterns of frameshift mutagenicity in the lacZ assay are similar to those in an assay based on the reversion of mutations in the tetracycline-resistance gene of the plasmid pBR322. The similarity suggests that the responses reflect the inherent bacterial mutagenicity of the compounds in the local sequence context and are not highly dependent on the broader sequence context. The results are interpreted with respect to slipped mispairing models of frameshift mutagenesis.

Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the V79 in vitro micronucleus assay.

Clastogenicity is frequently observed following treatment of mammalian cells with new chemical entities. This clastogenicity, unless proven otherwise, is assumed to result from the imperfect repair of DNA lesions produced from covalent chemical/DNA interaction. However, clastogenicity can also arise via other mechanisms such as non-covalent chemical intercalation into DNA resulting in poisoning of cellular DNA topoisomerase II (topo II) and stabilization of DNA double strand breaks. We have recently reported modifications to the V79 in vitro micronucleus assay which allow an indirect evaluation of both the intercalative and topoisomerase-interactive activities of chemical agents. In the present studies we have used these modified assays to further assess the validity of this approach in an evaluation of a number of intercalating and non-intercalating polycyclic compounds. It is shown that intercalating agents may be catalytic topo II inhibitors (e.g. chloroquine (CHL), tacrine (TAC), 9-aminoacridine (9AA), ethidium bromide (EB)) or topo II poisons (e.g. proflavine (PROF), auramine O (AUR) and curcumin (CURC)). Still other intercalators are shown to lack detectable topo II-interactions, (e.g. imipramine (IMP), quinacrine (QUIN), 2-aminoanthracene (AA), iminostilbene (IMN) and promethazine (PHE)). It is concluded that (1) the clastogenicity of three agents, PROF (a typical DNA intercalating agent), and AUR and CURC (both structurally atypical intercalating agents, with unknown clastogenic mechanisms), may be due to topo II poisoning; (2) other intercalating agents may either act as catalytic topo II inhibitors or exhibit no functional topo II interaction; (3) The use of these cell-based approaches may provide a logical first step in determining if unexpected clastogenicity associated with test article exposure is due to a topo II interaction.

Non-surgical female sterilization with quinacrine: an update.

The intrauterine installation of quinacrine represents a simple, inexpensive, effective, and seemingly safe method of non-surgical female sterilization. Existing clinical data on its use are very encouraging: Results of a large study conducted in Vietnam with an overall sample of over 30,000 women showed high effectiveness; in addition, when a retrospective study was conducted in these women, cumulative 5-year pregnancy rates were estimated to be 13% in women younger than 35 years and 6.8% in the women older than 35. Overall, failure rates with quinacrine have been estimated, at 10 years, to be between 1.9 and 4 times higher than those obtainable with conventional surgical procedures of tubal interruption. Unfortunately, existing toxicology for topical use of quinacrine pellets is incomplete. This prompted an expert group convened by WHO, to comment, in 1994, that the toxicology of locally applied quinacrine is inadequate. To counter this statement the proponents of the method argue that it is unfair to apply the stringent pre-clinical requirements that are mandatory in the industrialized world, to methods utilized in countries plagued by both high fertility and high maternal mortality. This controversy will soon be resolved since conventional toxicological evaluation is now underway. In conclusion, the future of quinacrine for non-surgical female sterilization will depend on the results of long-term animal studies, as well as the retrospective human studies now being carried out.

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

The antimalarials quinacrine and chloroquine potentiate the transplacental carcinogenic effect of ethylnitrosourea on ependymal cells.

Quinacrine and chloroquine, two widely used antimalarials, bind strongly to deoxyribonucleic acid, thus preventing mutagenesis. We studied a possible chemoprotective effect of these substances on carcinogenesis of the nervous system induced in Wistar rats by transplacental administration of ethylnitrosourea. One experimental group consisted of rats born from mothers treated with quinacrine prior to prenatal exposure to ethylnitrosourea; a second group consisted of rats chronically treated with chloroquine after prenatal exposure to ethylnitrosourea. When compared with controls, no significant differences were observed in tumor incidence. However, early tumor growth was observed in both rats treated with quinacrine (P < 0.0004) and rats treated with chloroquine (P < 0.02). These differences were due mostly to rapid development of ependymomas of the spinal cord. Our results suggest that quinacrine and chloroquine do not prevent the structural alterations induced in DNA by ethylnitrosourea, which lead, in the long term, to a high incidence of neoplasms in the nervous system. Moreover, the antimalarials studied seem to promote the carcinogenic effects of ethylnitrosourea on ependymal cells.