Doses Of Quinacrine Research Articles

ATRT-05. REPURPOSED ANTI-MALARIAL QUINACRINE ACTIVATES P53 AND INHIBITS ATRT TUMORIGENICITY

Atypical teratoid rhabdoid tumors (ATRTs) are fatal pediatric brain tumors that warrant improved therapies urgently. ATRTs are characterized by loss of INI1, a subunit of the SWI/SNF chromatin-remodeling complex. ATRTs grow aggressively despite majority of primary tumors expressing p53, suggesting inactivation of this tumor suppressor pathway. Reactivation of p53 could be a potential therapeutic strategy for inhibiting ATRT growth. Our laboratory specializes in researching mechanisms contributing to ATRT pathogenesis and potential therapies. In line with this, we investigated an anti-malarial drug called quinacrine that has been safely used in children for decades and can induce p53 in renal cell carcinoma. We used 5 patient-derived ATRT cell lines (BT-37, BT-12, CHLA-06, CHLA-266, CHLA-05) for our studies. We show that ATRT cell lines treated with quinacrine for 6 hours show increased expression of p53, suggesting its activation. Treatment of ATRT cell lines with increasing doses of quinacrine for 24 hours showed dose-dependent decrease in cell growth and proliferation (assessed by MTS assay and BrdU incorporation, P<0.05) and increase in apoptotic cell death (CC-3 and cleaved PARP expression). Nude mice harboring flank tumors of ATRT cell lines and treated with quinacrine for 3 weeks showed significant reduction in tumor growth compared to control animals (P<0.05). Since quinacrine is a substrate for the drug-efflux proteins P-gp/BCRP, we used quinacrine in combination with elacridar (Pgp/BCRP inhibitor) in our intracranial xenograft experiments to increase quinacrine’s retention in the brain. Mice harboring intracranial xenografts of ATRT cells showed increased survival when treated with quinacrine and elacridar (median survival 46 days) compared to control animals (median survival 25 days). These results suggest that quinacrine inhibits ATRT growth, partly by activating p53. Our studies are the first to show quinacrine’s effect on ATRTs and our current experiments include further investigation of quinacrine’s mechanism.

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes.

Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

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.

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 1×, 8× and 40× 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 10/10 mg/kg but increased with dose level and was significantly greater than controls at ⩾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.

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 μM, 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 μM, 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. This work was supported by a grant from the National Institutes of Health (U01AI5385802) and U.S. Army (DAMD170210699). Jason E. Comer, a predoctoral fellow, was supported by National Institutes of Health T32 Predoctoral Training Grants in Emerging and Tropical Infectious Disease (2T32A8007526) and in Biodefense (1T32AI060549). Bacillus anthracis Ames strain was generously provided by C. Richard Lyons at the University of New Mexico Health Science Center, Albuquerque, NM, and duly registered with the Centers for Disease Control (CDC). The authors appreciate the editorial assistance of Mardelle Susman.

Morphometric and kinetic studies on the changes induced in the intestinal mucosa of rats by intraperitoneal administration of quinacrine.

The intraperitoneal administration of large doses of quinacrine in rats results in a state of enteromegaly affecting mainly the distal small bowel, caecum and proximal colon. This enteromegaly is associated with mucosal crypt hyperplasia, and hypertrophy and hyperplasia of the Muscularis propria. In order to investigate the changes in the intestinal mucosal crypts, morphometry and a metaphase-arrest experiment with vincristine were undertaken on a group of rats given 12 mg of quinacrine hydrochloride by intraperitoneal injection daily for 5 days 2 weeks previously, and comparisons drawn with a group of control animals. In the quinacrine-treated animals there was marked enteromegaly affecting the distal small bowel, caecum and proximal colon, and in these segments there was clear crypt hyperplasia. Proximal and distal to the dilated bowel hyperplasia was not seen. No consistent pattern of change in crypt-cell birth rate was evident. The mechanisms by which quinacrine may effect kinetic and morphometric changes in the intestinal crypts are considered.

Biologic Changes Induced by Unilateral Intrauterine Instillation of Quinacrine in the Rat and Their Reversal by Either Estradiol or Progesterone

Quinacrine had previously been found to induce alterations in the en dometrium of the rat and also had been safely employed in humans for its carcinostatic effect. Single injections of from 5 to 40 mg in a .2 ml aqueous suspension were instilled into the right uterine horn of each rat. The animals were mated at different intervals daily vaginal smears made and implantation sites and corpora lutea recorded on Days 9 or 10 after coitus. In another group a search for blastocysts was made on Day 5 after injections of 20 mg or 40 mg. In a third group either estradiol benzoate 50 mcg or progestogen 500 mcg was injected at varying times from 24 hours to 28 days after the quinacrine. Animals were sacrificed 10 days after the last hormone injection. Doses of quinacrine below 20 mg did not alter the fertility of the rats. With larger doses there was a gradual time-related recovery with regard to the number of implantations. This recovery paralleled restoration of uterine patency. Another antimalarial drug chloroquine had no effect on implantation rates. Microscopic studies of some uterine horns of rats treated with 20 mg of quinacrine showed a proliferative reaction and complete obstruction of the lumen. Quinacrine crystals were observed within the mass of foreign-body giant cells and stroma cells for as long as 90 days. These changes after 20 mg reversed spontaneously after 3 months but those after 40 mg doses persisted. Use of talc powder as a particulate matter did not produce any tissue reaction. This action of quinacrine was prevented by either estrogen or progestogen when injected within a short time after quinacrine instillation. In these cases the uterine lumina were patent and the uterine epithelium showed typical changes induced by either estrogen or progestogen. Also the luminal patency was restored when the hormones were administered when obstructed uteri were present.

Psychotic Reactions to the Ingestion of Large Doses of Quinacrine in Normal Subjects

Psychic disturbances following the treatment of malaria with quinacrine have been reported by a number of authors.1–4 In such instances it has usually been impossible to exclude as the cause of the mental disturbance the malarial fever itself or some hypothetical toxic substance resulting from the rapid destruction of a large number of parasites by quinacrine. In the present report malaria could be positively excluded as a factor in a number of individuals who developed severe toxic mental reactions following the ingestion of large doses of quinacrine.PROCEDUREThirty-one medical, dental and medical administrative corps officers of a United States Army General Hospital volunteered to take quinacrine at first in suppressive and then in therapeutic dosage. None of these officers gave a history of having had malaria, and all of them had been stationed both before and throughout the observation period in a non-endemic area.The dosage schedule was as follows: Quinacrine hydrochloride 0.1 gram daily for 1–2 weeks,0.1 gram twice daily for one week, and concluded by 0.4 gram three times the next day, 0.3 gram threetimesthe following day, and 0.1 gram thrice daily for the remaining four days.