Human Reproduction Update 1995, Vol. 1, Na 4 pp. OOO-OOO

Quinacrine rev&d

Q Oxford

Jaime Zipper Abraganly4, Alfred0 Dabancens2, Anibal Guerrero2 andValentin Trujillo3

‘Department of Physiology and Biophysics, Faculty of Medicine, University of Chile and S6tero de1 Rio Hospital, *Departmentof Experimental Medicine, Faculty of Medicine, University of Chile, and 3San JosC Hospital, Santiago, Chile

Table of Contents

introductionQuinacrine: mechanisms of actionSclerosing actionsBiochemical mechanisms leading to the

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potentiation of quinacrine fibroblastic function 000Analysis of the development of a non-surgical

technique for female sterilization withendo-uterine quinacrine plus adjuvants

Quinacrine: anticarcinogenic potentialConclusionsReferences

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Introduction

History

Quinacrine (Atebrine, mepacrine) is an a&dine derivatedescribed in 1932 by Schulemann for the treatment of ma-laria (Samuelsson and Goldyne, 1978). It was discoveredin an intensive research programme on synthetic antimal-arials carried out in the third decade of this century in theGerman laboratories of I.G. Farbenindustrie. The pro-gramme covered the preparation and trial of over 12 000compounds resulting in the identification of pamaquineand quinacrine as potential therapeutic agents.

Acridines were considered derivates of quinine, inwhich a benzene is attached to the quinoline nucleus. Eventhough quinacrine was widely used before the SecondWorld War, quinine was mainly used for the treatment ofmalaria (Green, 1932). When the supply of quinine wasinterrupted during the war for the allied nations, it becameimperative to manufacture a substitute in the USA. Techni-cal problems were soon solved, and the American substi-tute (quinacrine) turned out to be chemically andpharmacologically identical to the German chemical. Be-fore the war, the USA produced about 540 kg quinacrine/

year, but the war necessitated the production of about 900kg/day. Experience with the armed forces soon demon-strated the superiority of quinacrine for prophylaxis andquinacrine became the official medicine for the treatmentof malaria (Office of the Surgeon General, 1943).

Follow-up studies by physicians in the USA armedforces provided health professionals with de&&d in-formation on the side effects and toxicity of quinacrine,ranking it among the best studied drugs ever introduced(Joint Report of the Armored Medical Research Labora-tory, 1946; Shannon and Earle, 1944). However, the toxic-ity of long-term quinacrine administration, its relativelypoor effectiveness as a cure for malaria and its failure to actas a perfect prophylactic resulted in the search for moreactive substances.

In 1945, chloroquine replaced quinacrine in the treat-ment of malaria although quinacrine continued to be usedin the treatment of other diseases (Page, 195 1; Sorinson,1941).

Chemistry

Quinacrine is the 6-chloro-9-[[4-(diethylamino)-l-me-thylbutyl]amino]-2_methoxyacridine, with a molecularweight of 508.9. The compound is a derivative of acridine(Figure 1). It is sold as quinacrine chlorhydrate. It is avail-able as the dihydrochloride (quinacrine hydrochloride,USP) in 100 mg tablets as a bright yellow, odourless, bittercrystalline powder that is water soluble and 80% quina-crine base. Inactive ingredients include pharmaceuticalglaze, starch, talc, and stearic acid. It differs from chloro-quine in having an acridine nucleus (an extra benzene ring)instead of quinolone (Goodman and Gilman, 1992).

The LD50 of quinadrine hydrochloride for rats is 900mg/kg by oral (stomach tube) administration (Siegel andMushett, 1944). The LD50 for the i.p. route for rats has not

4To whom corrcspndcnce sould be addressed at: El Commcndador 2280, Providcncia, Santiago, Chile

C’

54 JZipper et al.

C”30

NH-C-NH-C-NH-CHI

. Chfbmggnas CH3

F’igure 1. Structural relationship of quinacrine and related compunds.

been estimated, but the experiments of Keeler and Richard-son (1966) suggest that it is -250 mgkg.

Structure-activity relationship

Quinacrine was one of the best of thousands of compoundsscreened for antimalarial activity. It is an al-quilaminacderivative of acridine. The lateral chain is equal to the oneof chloroquine. Berliner and Earle (1948) summarked thesubktitution effects in the ring and lateral chain on theactivity against experimental malaria in man and birds.

Quinacrine is usually administered by the oral routeusing tablets containing 50 or 100 mg of the drug. It hasalso been administered by i.m., i-v., i.p., intrapleural and

lectal routes (Goodman and Gilman, 1954; Gellhom andZaidenweber, 1961; Dollinger et al., 1967; Hickman andJones, 1970; Crouch et aZ., 1981). In the case of patientssuffer&g nausea and vomiting as well as in emergenciessuch as galloping malaria (Plarmodiwn falcipamn), per-nicious or cerebral malaria, coma, deliria, etc., i.m. admin-istration is preferred to treat the acute clinical attack ofindividuals who have received quinacrine. CareM study ofindividuals who received the compound by the oral routefor many months has not revealed the alteration of hepatic,renal or haematopoietic functions.

CH2CH3

Firimetamw

The usual therapeutic or suppressive doses are inocuousfor the cardiovascular system and do not alter the electro-cardiogram. Urine becomes intensely yellow-coloured onthe fourth day of treatment when it is acidified, but quina-crine does not affect the kidneys. In rare cases, quinacrinecauses visual or hearing defects. It lacks the cytotoxic ac-tion of quinine and may be employed in pregnant women(Goodman and Gilman, 1954,1992).

Quinacrine: mechanisms of action

Wallace (1989) published an extensive review on the use ofquinacrine in rheumatic diseases, mentioning all knownactions of the drug, including sclerosis of the human ut-erotubal junction (Zipper and Cole, 1987). The pharmaco-logical actions of quinacrine are numerous and will beconsidered by category. Several of the pharmaco logicalactions of quinacrine are anticarcinogenic.

Anticarcinogenic effects

Antiprostaglandin actions

One of the best studied actions of atebrine concerns its roleas a potent inhibitor of phospholipase A2. First describedby Horrobin and Manku (1977), this action occurs directly

, . .. , ‘- ;e

1. ’ -*

Quinacrine revised 55

on membrane phopholipids, especially phosphatidyl etha-nolamine. This results in the antiplatelet activity of atebrineand its inhibition of leukotrienes and cyclooxygenase(Horrobin and Ma&u, 1977; Evans and Lanham, 1986;Flynn, 1987). In platelets, the conversion of 14-C throm-boxane B2 and thromboxane A2 is suppressed. The lattersubstance is a major factor in blocking the release of ara-chidonic acid from cellular phospholipases and its conver-sion to pro-aggregatory products. Quinacrine is anon-selective antilipolytic agent that decreases prostaglan-din E(PGE) production in a dose-dependent fashion, thusblocking cycle-oxygenase.

This might contribute to its Na+, K+ ATRase inhibitoryeffects ‘stabilizing’ cell membranes. Such interference re-duces the basal rates of phopholipase methylation inmononuclear cells (Hurst and Fench, 1986).

._* DNA and RNA polymerase inhibition/suppression oflupus erythematosus cell factor

Quinacrine binds to DNA by intercalation between adjac-ent base pairs (O’Brien and Olenick, 1966; Voiculetz andSmith, 1974). This bond stabilizes DNA, inhibiting its heatdenaturation, enzymatic depolymerization, and both tran-scription and translation to RNA.

Atebrine can stimulate hydrolysis of transfer RNA bypancreatic ribonuclease A. Its high affinity for nuclear andchromosomal DNA via intercalation can be impeded bydenaturation or depurination (Summer, 1986). Quinacrinemustard fluorescent staining has been a mainstay pro-cedure in genetics laboratories where its affinity for the Ychromosome has proved to be very useful (DuPont andBerjon, 1983). Work by Dubois (1955) and Holman andKunkel (1957) showed that the binding of quinacrine tonucleoproteins can block the production of lupus erythe-matosus (LE) cell factor.

.. . .

Photochemical blockade

One of the major aggravating factors of systemic lupus isultraviolet light exposure. Like other antimalarials, quina-crine can block photodynamic actions, inhibit laser-in-duced photosensitization, and increase ultraviolet lighttolerance (Buchnicek and Turek, 1967; Bems and el-Kadi,1970).

Anti-oxidant

Superoxide anion generation is antagonized by quinacrine.The inhibition of reactive oxygen species probably is aconsequence of its effects on membrane phospholipid gen-eration (Tauber and Simons, 1983; Hurst and French, 1986;Miyachi and Yoshioka, 1986).

AntiproliferatiWantimutagenic effects

Quinacrine can block radiation-induced DNA straudbreaks and potent&s the antiproliferative effects of radi-ation. It reduces the incidence of cancer in rats given nitro-so-urea decreases the number of somatic mutationsinduced in murine leukemia cells, and reverses resistanceto vincristine (Giampeitri and Fioretti, 1980; Biller andSchachtschabel, 1982; Pfab and Schachtschabel, 1985;Inaba and Maruyama, 1988; McCormick, 1988). Radio-protective actions might be due to the scavenging of waterradicals that has been observed when quinacrine is boundto DNA (Hissung and Dertinger, 1975).

Effects on immunologic responsiveness

Quinacrine inhibits natural killer-cell cytotoxicity and en-hances the killing response of cells by X-rays (Saladinoand Ben-Hur, 1978; Fahey and Newcombe, 1979). Itblocks the primary but not the secondary proliferative re-sponse of human cytotoxic T cells to allogeneic non-T cellantigen and impedes Tut antigen (interleukin-2 receptor)expression due to its phospholipase A2-blocking actions.

Quinacrine does not interfere with the recognition ofantigens by cytotoxic T cells but suppresses the mitogenicresponse of T cells to allogenic antigen (Namiuchi andKamagai, 1984). It can impede the uptake and incorpor-ation of leucine, thymidine, and uridine in acid-insolublematerial in human lymphocytes stimulated by phytohae-maglutinin (Trist and Weatherall, 198 1).

Antimicrobial properlies

Antiparasitic, antiprotozoan, antibacterial, antiviral, andantifungal actions have been described. Quinacrine’s anti-malarial properties are based on its dose-related inhibitionof adenosine uptake into host cells of parasitized blood aswell as its effects on the RNA and DNA of the PZasmodiumorganism (Van Dyke and Lantz, 1970). Antiprotozoan ac-tions are attributable to its inhibition of succinate oxidationand interference with electron transport (Chou and Rama-nathan, 1968; Conklin and Chou, 1971).

Quinacrine can prevent bacterial resistance to variousantibiotics, (Horwitz and Eshelman, 1968; Rybinka andAstapov, 1970) and demonstrates bacteriostatic activities.Interferon production is increased by quinacrine and in-tralesional administration can treat warts (Budak, 1966;Galz and Szolgay, 1973). In-vitro antifungal propertieshave been noted (Medvedeva, 1966).

Bradykinin and histamine antagonization

As an inhibitor of phospholipase A2, quinacrine can blockthe actions of bradykinin in synovial fibroblasts and sup-press its algesic effects (Juan, 1977; Crouch et al., 1981).

56 J.Zipper et al.

Figure 2. Histological section of rat uterus treated with quinacrine ata dose of 100 mg/ml. This section at mid-level shows the uterus ob-structed by fibrotic and granulomatous tissue (original magnifica-tion x40).

Figure 3. Histological section of rat uterus treated with quinacrine ata dose of 100 mg/ml. This section at the le\,el of tubo-uterine junc-tion shows normal tube and obstruction of the uterus by fibrotic andgranulomatus tissue (original magnification x40).

Quinacrine impedes the introduction of CAMP release insynovial fibroblasts by bradykinin, which would otherwiseresult in arachidonic acid and prostaglandin E release@hey and Newcombe, 1979). Inhibition of phospholipaseA2 decreases histamine release from human basophils andanti-immunoglobulin (1g)E (Nagy and Kocsar, 1956;Mitchell, 1984; Toll and Anderson, 1986).

Sclerosing actions

Clinical and rodent data

Quinacrine is a locally effective sclerosing agent and intra-cavity administration is capable of preventing recurrent

pleural and pericardial effusions as well as pneumothom~(Cattaneo and Sirak, 1973; Bayly and Kisner, 1978; Stiskaand Korsgaard, 1979). Quinacrine pellet instiMion intothe uterus results in non-surgical sterilkation. Fibrotic andgranulomatous tissue is formed by the quinacrine-inducedepithelial fibroblastic cells which become active in thehuman tube epithelium .

Several studies on rats and rabbits (Zipper and Medel,1968; Zipper and Insunza, 1972; Zipper and F+ager, 1973)have demonstrated the granulomatous and obstructive ef-fects produced by the topical action of quinacrine in tubaland uterine epithelium (Figures 2 and 3).

This fibroblastic action of quinacrine is locally potentiatedby antiprostaglandins and the cations Cu2+ and C$+ (TableI) (Dabancens and Zipper, 1993; Zipper and Rivera, 1993).

Table I. Summary of histopathological changes observed inthe rat uterus when various combinations of quinacrine withantipros taglandins, (betamethasone, diclofenac) and cations(Cu+ Zn+) are instilled into the uterine lumen (animalssacrified 7 days after instillation)

Intensity of damage when active compounds are used alone

Quinacrine

Quinacrine

Betamethasone

Diclofenac

lndometacine

cu+

Zn+

(+++I 100 mg/ml

(++) 50 mg/ml

(-1 0.6 mg/ml

(-1 25 mg/ml

(-1 25 mg/ml

(-1 0.5 x lO-%I

(-1 0.5 x lO-*M

Changes induced by each combination employed

Combination

050 + cu

Uterus

(++++I

(-1

(+++I

(-1

(++++I

(+++I

(++I

(-1

(++I

(-1

(+-+)

+-W

(++I

Tube

(++I

(-1

(-1

(-1

(++I

(-1

(4

(-1

(-1

H

(-1

(+)

t-1

QlOO + Zn

Q50 + Beta 1.2 mg

Q50 + Beta + Zn

Q50 + Beta + Cu

Q50 + Diclo

Q50 + Diclo + Cu

Q50 + Diclo + Zn

Q50 + Diclo + Beta + Cu

Q50 + Diclo + Beta + Zn

Q50 + Diclo + Beta

Q50 + lndo

Q50 + lndo + Cu

Diclo = diclofenac; lndo = indomethacin; QlOO = quinacrine100 mg; Q50 = quinacrine 50 mg; (+) to (++++) = intensity ofdamage observed; (-) = lack of lesion or minimal lesion; (++)= moderate damage. (Some glands remain and the morph*logical recovery is complete after 30 days).

Figure 4. Examination of the rat uterus at the cervical level. Right: quinacrine given at a dose of 100 mg/ml, plus 0.5 x 1 W2M zinc, results in anormal epithelium (original magnification x40). Left: quinacrine given at a dose of 100 mg/mI, plus 0.5 x 1F2M copper, shows obstruction ofthe uterine lumen by fibrotic and granular tissue while the muscle layers remain normal (original magnification x40).

Studies of various adjuvants, quinacrine, diclofenac, be-tamethasone, and Cu2+ led to the conclusion that inhibitingthe release of arachidonic acid, as happens when phos-pholipase A2 is inhibited by quinacrine (Horrobin andMa&u, 1977), potentiates by the action of betamethasone,and in the presence of Cu 2+ cations is one of the mechan-isms that produces a rapid proliferation of fibrotic andgranulomatous tissue. In the human female, the non-steroi-dal antiprostaglandin agent, diclofenac, in the presence ofquinacrine, also activates and potentiates the fibrosing ac-tion of quinacrine (Trujillo and Zipper, 1993).

The antiprostaglandin action of quinacrine is also ex-erted by inhibition of leukotrienes and cycle-oxygenasepathways (Evans and Lanham, 1986; Flynn, 1987). In thiscondition, the presence of diclofenac and Cu2+ decreasesthe fibrosing action of quinacrine. Copper potentiates thecombination: betamethasonequinacrine. Zn2+ completelyhinders the sclerosing action of quinacrine alone or asso-ciated with antiprostaglandins (Table I; Figure 4).

Quina&ne, depending on its concentration or associationwith antiprostaglandins or Cu2+ cations, as cofactors of thesystems involved, can lead from a reversible in&mmatorystate to permanent fibrosis. The association of quina&e withCu2+ potent&es the fibroblastic action of quina&ne alone.Copper alone in the same concentrations (1 x l@M to 1 x10-3M) only produces functional changes in the endome-trium. Recently, the structure of the DNA binding domains ofglucocorticoids and oestrogen receptors have been deter-mined as well as the position of Zn atoms in them (Schwabeand Rhodes, 1991). These Zn atoms could be exchanged forCu which would modify its biochemical function.

Quinacrine sclerosing action at the ufefhe f&al level

. .“. ”:: .

Research conducted in rat and rabbit (Zipper and Medel,1968; Zipper and Insunza, 1972; Zipper and Prager, 1973)

demonstrated that instilling different pharmacological ag-ents in the uterine cavity could modify the physiology andmorphology of the endometrium, thus altering reproduc-tive processes. For example, P-adrenergic blockers pm-duced long periods of infertility (Zipper and Bruzzone,1982). The uterus becomes refractory to implantations.The same was observed with instillations of copper salts(Zipper and Medel, 1969). These substances did not alteruterine morphology and histology. Another compound in-vestigated was quinacrine; concentrations from 50-200mg/ml were instilled in a rat uterus in volume of 0.1 ml andkept in contact with the endometrium for 20-30 min. Theepithelial tissue was replaced by fibrous and granuloma-tous tissue that blocked the uterine lumen (Figures 2 and 3).

Similar experiments in rabbit showed us that both theuterine and tubal mucosa were refractory to quinacrinefibrosing action. Different studies in rabbit led to the con-clusion that the high content of Zn”+ in both organs (uterusand tube) in this species made them refractory to the actionof quinacrine (Zipper and Insunza 1972).

When the research was extended to humans, we ob-served that 250 mg of quinacrine inserted as pellets into theuterine cavity twice with an interval of one month pro-duced a bilateral tubal obstruction in 97% of cases in thefirst year (Zipper and Cole, 1987).

Biochemical mechanisms leading to thepotentation of quinacrine fibroblastic function

Quinacrine produces a granuloma in the tubal ostium be-cause a low tissue concentration of Zn exists in this area.This hypothesis was confirmed by Patek and Hagenfeldt(1974) who investigated the concentration of Cu and Zn inthe human endosalpinx and endometrium. Samples weretaken in each of the four phases of the menstrual cycle.

‘1.’:‘, ;’4::_;_.. ...:.;.-.-, ,,“4*.’

4:..

58 J.Zipper et al.

‘.. .’

.

Figure 5. Section of the uterotubal junction, after treatment with 50mg/ml quinacrine plus 1 O-* M copper. Complete obstruction and fi-brosis of the uterus and tube are shown (original magnification x40).

In the endosalpinx there was no significant change in theconcentration and relationship of Z&u in the four phasesof the cycle expressed in pg/g of dry tissue. The average forCu2+ was 5.0 pg/g and for Zn2+ 53.10 @g of dry tissue. Asignificant difference in the concentration of these cationswas found to arise from the beginning of the proliferativephase to the final secretory phase in the human endome-trium (Hagenfeldt and Plantin, 1970).

These results were expressed in pg/g of protein. At thebeginning of the cycle, Zn = 206.3 pg/g and at the end of itZn = 324.0 pg/g of protein. Cu concentration average is14.3 pg/g of protein in the proliferative phase and16.3 pg/g in the secretory phase. This high concentrationof Zn in the endometrium reduces or prevents the action ofquinacrine on this epithelium.

The series of experiments seen in Table I demonstratedthe effects produced by the associations quinacrine-be-tamethasone, diclofenac, Cu and Zn. They proved that thefibrosing and sclerosing action is highly selective and spe-cific. The study of these associations at the morphologicallevel makes us assume that the metabolic pathways of thepharmacological combinations studied are different. Thisincludes the mechanism of action of both types of antipro-staglandins, one steroidal and the other non-steroidal. Themost effective obstructive combination at uterine and tuballevel in rat, is the association of copper with quinacrine(Figure 5) and quinacrine-copper-betamethasone.

Analysis of the development of a non-surgicaltechnique for female sterilization withendo-uterine quinacrine plus adjuvants

The development of a non-surgical and ambulatory sterik-ation technique as effective as a surgical technique is a prior-

ity and a partial solution for the population explosion and itscatastmphic secondary consequences (I&se1 and Mumford,1982; Kessel, 1989). Over the next 10 years, populationgrowth is predicted to be one billion with 95% of theseindividuals coming from developing countries (Potts andRosenfield, 1990). It has been estimated that over 328 mil-lion women of reproductive age from developing countrieswill demand sterikation in the 1990s (Mumford and Kessel,1992). Since sterikation is a surgical method, only 159million (48%) will have access to this service and 169 miLlion (52%) will not. This suggests that the quinacrine pelletendo-uterine method would be the only technical solutionwhich could overcome the enormous shortfall in sterilizationservices (Mumford and Kessel, 1992). Klitsch (1982) re-viewed the available non-surgical sterihzation methods andconcluded that the only two acceptable techniques wereendo-uterine quinacrine pellets, and endo-uterine instilktions of methylcyanoacrylate (MCA).

Studies with quinacrine pellets show that one or twoinsertions of quinacrine alone obtained a 95% obstructionafter one year. The most important secondary effects werefever 4.9%, headaches 4.9%, and general malaise whichremains for a few days 6.17%. Uterine pain or uterineinfection (endometritis or anexitis) are rare complications(Agoestina and Kusuma, 1992).

The main secondary effects of MCA include abdominalpain presented after treatment (22%), with 4% of thesepatients confessing to pain even 3 months after MCA in-stillation. Fever lasting several days occurred in 5% ofpatients and 1% required hospitalization to treat pelvicinflammatory processes. Results from six different studiesin six different countries showed that tubal bilateral occlu-sion under this technique varied from 65% in thePhilippines to 80% in Brazil.

Development of non-surgical techniques

Phase 1. Use of quinacrine in an aqueous suspension

The use of quinacrine for transcervical sterilization as anaqueous suspension was validated in 1993 (Zipper et aL,1993). However, the technique produced several effectsand a cumulative bilateral obstruction of only 88.6% usingthree instillations which was not satisfactory (Zipper andPrager, 1973; Zipper and Medel, 1976).

Phase 2. Controlled-dissolution pellets

The technique was improved by forming the quinacrine intopellets with a controlled dissolution rate of 1 O-l 00 min thusreducing toxicity and avoiding rapid loss through the cervi-cal OS. Each pellet contained 36 mg of quinacrine. The dosesused for sterilkation were 180 mg (five pellets), 216 mg (sixpellets), 252 mg (seven pellets) and 288 mg (eight pellets).

Quinacrine revised 59

Table II. Pregnancy rates in women who received three administrations of quinacrine at 250 mg each

No. of women Follow-up period in years and pregnancy rates

1 3a 4 5 6 7

Zipper et al. (1980) 148 4.2 6.4 8.8 8.8 8.8 8.8 8.8+Zipbar et al. (1987) 123 3.3 6.7 6.7 6.7 7.6 7.6 7.6+Gum&n Serani (1984) 149 0.7 3.4 4.1 5.6 5.6 6.4 6.4+Bhatt et al. (1985) 81 0.0 1.2 3.7 3.7Agostina et al. (1982) 100 3.07Total (mean) 601 2.8 4.4 5.8 6.2 7.3 7.6 7.6

. .‘.’

__’ :. _.

a76% of all pregnancies occur during the first 3 years.bFamily Health International (unpublished data).

It became appaxent immediately that these pellets were a out a detailed study (Do Trong Hieu and Tran Thi Tan,major improvement over the slurries. Table II summa&s the 1993). It reports 3 1789 cases carried out in 24 provinces ofresults of five studies canied out by four researchers. Sk&f- Vietnam; the insertions were carried out f’rom January 1989fectsattributedto quinack were found but they were mostly to October 1992 (3 years and 8 months) and 252 mg quina-minor discomforts and disappeared after l-7 d;ivs. currently, crine alone was used. There were 8 18 pregnancies after thethe pelIets used have a dissolution half-life of about 30 min. procedures and 80 were caked to term.

Common side-effects include general malaise and fever.The cumulative percentage of reactions to the drug were15% (Zipper and Cole, 1987; Agoestina and Kusuma,1992). In order to reduce these side-effects we decided touse antiprostaglandins which not only reduced the afore-mentioned discomforts (fever, headaches and general mal-aise), but also reduced pregnancy rate (Trujillo and Zipper,1993). Simultaneous studies conducted in Valdivia Cityand Santiago, Chile, with a follow-up period of 8 years, ledto significant results. Three transcervical quinacrine pelletinsertions of 250 mg each were performed. The dissolutiontime of the pellets was 100 min.

One group had one insertion and the other two. At the endof the first year, 2225 cases with one insertion and 9461 withtwo insertions were analysed. The pregnancy rate for thegroup with two insertions was 2.63 per 100 woman years andfor one insertion was 5.15 per 100 woman years. There wasno maternal mortality and only eight major complicationswere reported. A surgical study would have been expected toshow 30 deaths and from 540 to 1812 serious complications.

All reported side-effects were minor and of short duration.There were 19 ectopic pregnancies, an incidence of 0.89 per1000 woman years of use. An estimated 242 maternal deathsshould be averted by these 31 789 sterilizations.

The studies summarized in Table Il were used to com-pare the clinical effectiveness of the trials. Most of thepregnancies (75%) occurred during the first 3 years; afterthis, there were few additional pregnancies.

Experience has been gained with studies of quinacrinepellets containing 252 mg with two and three insertions@.Sow personal communication). The follow-up of the fkstthree groups was 7 years, the fourth group, 4 years and the lastgroup, 1 year. The pregnancy rate stabilized at the fif?h yearand the cumulative average pregnancy rate is -5.8% at thethird year and 7.6% at the seventh year (F@re 6).

Quinacrine and antiprostaglandin studies

Our group has conducted several studies with quinacrinepellets plus adjuvants. The dose of each insertion has va-ried from 180-288 mg of quinacrine. Adjuvants in thesestudies were diclofenac, Cu and betamethasone, which im-proved the tubal obstruction and diminished the secondaryreactions such as fever, malaise and headaches in all cases.

Iatrogenic effects were present but in reduced numberand in fewer patients. Discomfort was transitory, lastingfrom hours to a few days.

In 199 1, researchers agreed that this technique can multi-ply the number of voluntary sterilizations without signifi-cant additional resourcesOne study from Indonesiadetailed the iatrogenic discomforts, i.e. headache, feverand general discomfort (Agoestina and Kusuma, 1992).Based on reported data, the Vietnam Government caked

Results of Phase 2. Adding antiprostaglandins asco-adjuvant substances to quinacrine

Due to the iatrogenic effects attributed to quinacrine alone,it was decided to add antiprostaglandins (intrauterine ori.m.) to the quinacrine pellets to minimize these effects.Two types were used: steroidal (betamethasone; Zipperand Rivera, 1993) and non-esteroidal (diclofenac; Trujilloand Zipper, 1993). The follow-up of these patients led tothe conclusions that firstly, all iatrogenic effects disappearwhen inserting quinacrine together with the antiprosta-glandin (betamethasone or diclofenac) and secondly, theaverage pregnancy rates in the years of follow-up are

60 JZipper et al.

Years of tokw-up

Figure 6. Cumulative pregnancy rates following quinacrine steril-ization. Preliminary results combined. (From David CSokal, Fami-ly Health International).

smaller in the three groups with antiprostaglandins (TableIII) when compared to quinacrine alone (Table II).

Phase 3. Quinacrine plus adjuvants antiprostaglandinand copper

It has been demonstrated that copper can stop the phos-phatidylinositol cycle and interfere with the regulation ofarachidonic acid production (Lapetina and Billah, 1981).In the binding of some hormonal receptors @estrogens,glucocorticoids) to DNA, there are three Zn2+ ions for eachbind, that are coordinated in a tetrahedric bind of cisteinligations, called ‘zinc fmgers’ (Schwabe and Rhodes,1991). Copper could displace the zinc ions altering thereceptor or subjacent gene. These two reasons and the ex-perimental observation in rabbit (endometrium with highconcentration of 2x1) where CuSO4 and Zn chelants poten-tiate the fibrosis and granuloma formation (Zipper andCole, 1987), led us to add copper to the quinacrine pellets.

A group of 118 women received 288 mg of quina&ne, 1mg betamethasone and 0.5 mg of CuSO4, all as intrauterinepellets (Zipper and Rivera, 1994; Table IV). No pregnanciesoccur at the end of 1 year follow-up. Adverse reactions weremild and not significant. One pregnancy was detected by theclose of the analysis in May, 1993. It occurred at the 19thordinal month. All these patients have accomplished afurther year of follow-up without new pregnancies.

The fibroblastic function of quinacrine is related to itsantiprostaglandin activity. The addition of compounds thatpotent&e the quinacrine antiprostaglandin function, likecopper or other antiprostaglandin agents, improves itstubal sclerosing effect. This is demonstrated in clinicaltrials by a reduction in pregnancy rates (Tables II and III).Another effect seen with the addition of antiprostaglandinsis the significant reduction in the iatrogenic reactions ob-served with quinacrine alone.

Quinacrine: anticarcinogenic potential

Prostaglandins and antiprostaglandins: their role incancer

The transformation of a normal cell to a cancer cell is acomplex process, which can be induced by viruses, radiationor chemicalsWhen a lesion causes permanent and ixrevers-ible genetic mutation, the step is called carcinogenesis initi-ation (Honn and Bockman, 1981). Oxygenation ofpolyunsaturated fatty acids by prostaglandin endoperoxidesynthetase (PES) results in the formation of prostaglandins,thromboxanes, prostacyclin, malondialdehyde (MDA), hy-droperoxy fatty acids and leukotrienes. Some of these com-pounds or intermediates in their formation are reactive andmay be carcinogenic (Schamberger and Adreone, 1974;Mukal and Goldstein, 1976; Mamet and Tuttle, 1980).

Prostaglandins may play an indirect role in carcinogene-sis by triggering the co-oxygenation of non-carcinogeniccompounds to carcinogenic derivatives. Oxygenation is animportant reaction in carcinogenesis in vivo (Heldelberger,1975; McCormick, 1988).

Table III. Quinacrine plus adjuvants: pregnancy rates in women sterilized with quinacrine pellets plus antiprostaglandins (3 yearsfollow-up data)

Regimen

ABCTotal (mean)

Research group No. of women Years of follow-up1 2_ 3

Trujillo et al (1993) 157a 2.1 3.0 3.6Zlpper eta/. (1993) 217 0.92 1.38 1.84Zipper and Rivera cm press) 114 0.0 0.87 0.87

488 (1.0) (1.75) (2.10)

Regimens were as follows:A: Quinacrine 216 mg, Mofenlac 50 mg (IU) plus diclofenlac 150 mg (i.m.) (two administrations); 6: quinacrine 250 mg, betamethasone1.2 mg (IU) (three adminktmkns); C: quinacrine 266 mg, Wasone 1.2 mg (IU), + CuSO4 0.5 mg (IU) (two adminiitions)aAll patients completed 4 years of follow-up without additional pregnancies.

:I *-*- .

a-,.

.,

.

Table IVa. Outcome in women undergoing one of threetreatment regimens

Events A B C

_.c Pregnancies 3 31. No. of women 118 95 159.

No. of administrations 232 190 318Mean age 33.1 342Live birth 3.4 3.8Women months 2700 5035 2198Ordinal months 19 132520 5,6,15Pregnancies (%) 0.8 3.15Pearl’s index 0.4 0.7 1.63Study initiation 01-1991 05-1989 -Study end 12-1991 llr1989 -Continuation rate 96.3 89.6 -Siudy analysis 06-l 993 04-1994 -

Treatment regimens were as follows: A: two administrations of288 + betamethasone 1.2 + 0.5quinacrine mg. mg. CuSO4

mg; B two intrauterine administrations of quinacrine 250 mg +1. .. :: diclofenac 75 (Sotero del Rio hospital); C: two administra-m g

tions of 216 mg quinactine pellets (iu) + 50 mg diclofenac (iu) +150 mg diilofenac (im). (San Jose hospital, Trujillo et a/. 1993).

Table IVb. Number of patients with iatrogenic symptomsoccurring in studies A and B in Table Iva. Numbers inparentheses are percentages

1 year post injection 2 years post injection

A B A B

Fever 0 (0) 2 0 (0) 1Headache 3 (2.5) 2 1 (0.87) 1Discomfort 2(1.6) 2 2 (1.7) 1Pelvic pain 2 (1.6) 1 2(1.7) 2Vaginitis 1 (0.8) 1 1 (0.8) 1Endometritis 0 (0.0) 0 1 (0.84) 0Total 8 (6.76) 8 7 (6.1) 6

Reasons for performing one administration: personal 3; medi-cal 1; total 4.

Quinacrine revised 6 1

Carcinogenesis promotion

The development of chemically-induced cancers is char-acterized by a prolonged latent period between the time ofapplication of the carcinogen and the formation of a pal-pable tumour. In animal tests, this latent period can beshortened by the repeated administration of compoundscalled promoters (Berenblum and Shublk, 1947). One ofthe most potent promoters curmntly avai&le is tetradeca-noyl phorbol acetate(TPA; Baird and Boutwell, 1971).

TPA treatment triggers a number of biochemical eventsincluding the induction of omithine decarboxylase (ODC)activity (O’Brien and Simsiman, 1975), the stimulation ofDNA synthesis (Paul and Hecker), the generation of thesuperoxide anion (Witz and Goldstein, 1980), and elev-ation of prostaglandin concentrations (PGF2). The TPAeffect is inhibited by indomethacin, indicating that the en-hanced prostaglandins concentrations are the result of de-novo synthesis. TPA would stimulate the release ofarachidonic acid from cellular lipids, which is essential forthe tumour promoter action (Levine and Hassid, 1977;Brune and Kahn, 1978).

Indomethacin, flufenamic acid and aspirin depressed theinduction of ODC activity by TPA (verma and Ashendel,1980). The inhibition of ODC induction by the PES inhibi-tors is completely overcome by treatments with PGEl orPGE2 but not PGF2; in this case the prostaglandins of theseries PGF2 do not overcome the antiprostaglandin action(Verma and Rice, 1977). This suggests that some prosta-glandins may play a role in TPA-dependent induction ofODC activity and hence tumour promotion, but they arenot themselves tumour promoters.

Table IVc. Rate of pregnancies per life accumulative table*, for women that completed the two programmed insertions ofquinacrine pellets (27 months of follow-upin study C in Table IVa

:Lx

159

ax

16

wx

48

Pregnancies

0

Quarterly AccumulativeProbability Probability

0.00 0

7-9lo -1213-1516-1814-2122-2425-27Total

9275666562575757

2 150 91 00 30 50 00 00 019 80

0.021 210.00 210.00 210.015 210.00 360.00 360.00 360.00 360.036 36x 100=3.6%

*Life table analysis achieved by Tietze method; X = quarters i.e. periods of 3 months from the start of the study; Lx = no. ofwomen included in study in period X; ax = no. of women that were absent at recording time for period X; Wx = no. of women ex-eluded from study because of lack of time; Quarterly probability = pregnancy probability per quarter year, Accumulative probablity

. = pregnancy probability per 1000 women.

62 J.Zipper et al.

The TPA-dependent induction of ODC activity is in-hibited by the PES inhibitors in the following order: in-domethacin, naproxen fltienamic acid, aspirin (venna andAshendel, 1980). The inhibition could be overcome byPGEl, PGE2 and PGD;! but not by PGFl or PGF2.

This suggests that prostaglandins El, E2, D2 and 12 mayplay a role in the induction of ODC activity by TPA and thatthis may be an important component of the mechanism oftumour promotion. These could explain the observed pro-phylactic effects of non-steroidal anti-inflammatory agentson the growth of other chemically induced and transplantedtumours (Bennet and Houghton, 1978; Lynch and Caste,1978; Lynch and Salomon, 1979; Kudo and Narisawa,1980).

Action of Cu-Z.-Co and antiprostaglandins in thepotentiation or inhibition of anticarcinogenicfunctions

Non-steroidal anti-inflammatory drugs, such as aspirin,indomethacin, piroxicam, and sulindac, inhibit the growthof colon tumours induced by chemical carcinogens in ro-dents (Kudo and Narisawa, 1980; Pollard, 1980; Pollardand Schmith, 198 1; Moorghen and Ince, 1990; Rosenbergand Palmer, 1991). The mechanism is unknown but it mayinvolve the suppression of cell proliferation or the stimula-tion of an immune response, due to an inhibitory effect onprostaglandin synthesis. A similar effect in humans hasbeen suggested in reported cases (Hen et al., 1983).

Antiprostaglandin compounds also have inhibitoryproperties on viral tumours (Ichino and Yoshinori, 1984;Gamburg, 1986). One of these antiprostaglandins, quina-crine, has multiple additional properties.

A great number of nucleoproteins related to DNA tran-scription and the role played by Zn in the replication andtranscription have been determined (Delahunty and South,1992). They have specific roles in the regulation of genes.The replacement of Zn for other metals such as Cu and Cocan lead to functional changes (Dewys and Pories, 1972;Zipper and Insunza, 1972).

Copper plays an important role in the organism. Its ex-cess displaces Zn from its various bindings, including zincfingers (Schwabe and Rhodes, 1991). Zn is not carcino-genic, mutagenic or teratogenic (Vallee and Kenneth,1993) but a relative increase in the Zn concentration mod-ifies two important properties of quinacrine; it loses itsfibroblastic and anticarcinogenic functions.

Experimental dnd clinical evidence against potentialcarcinogenicity of quinacrine

As mentioned in the first part of our paper, quinacrine canblock radiation-induced DNA strand breaks, potentiate ihe

antiproliferative effects of radiation (Hissung and Dert-inger, 1975; Biller and Schachtschabel, 1982; Pfab andSchachtschabel, 1985), decrease the number of somaticmutations induced in murine leukemia cells (Giampeitriand Fioretti, 1980), and reverse resistance to vincristine(Inaba and Maruyama, 1988).

The mechanisms of action of quinancrine have beenreviewed as an antiprostaglandin (Evans and Lanham,1986; Flynn, 1987), anti-oxidant (Disc et aZ., 1982; Miya-chi and Yoshioka, 1986), antiproliferative (McCormick,1988), antimutagenic (Hissung and Dertinger, 1975;Giampeitri and Fioretti, 1980) and DNA-RNA polymer-ase inhibitor (O’Brien and Olenick, 1966; Glaz and Szol-gay, 1973). All these mechanisms are oriented toward ananticarcinogenic function.

Since many non-steroidal antiprostaglandins such as in-domethacin piroxicam, (Fustenberger and Marks, 1978;Fustenberger and DeBravo, 1979; Lynch and Salomon,1979; Kudo and Narisawa, 1980) sulindac, aspirin, etc. actas antineoplastic agents (Bennet and Houghton, 1978;Lynch and Caste, 1978; Pollard, 1980; Pollard andSchmith, 1981; Narisawa and Hermanek, 1984; hymondand Barry, 1987; Moorghen and Ince, 1990; Baron, 1991;Rosenberg and Palmer, 1991), quinacrine, also a non-steroidal antiprostaglandin, should have a similar capacity(Horrobin and Manku, 1977).

Experimental studies in animals andepidemiological studies in humans

Experimental studies

McCormick (1988) induced mammary tumours in rat withN-methyl-nitrosourea (MNV) and found that quinacrinereduced cancer incidence and carcinoma multiplicity whenadministering MNV at 20 mg/kg body weight, but had noinhibitory activity with doses of 50 mg/kg. A culture me-dium of monoblastic leukemia cells, demonstrated thatquinacrine was active as an antileukemic agent, and chlo-roquine was not (Alade and Brown, 1992).

Quinacrine was integrated in meals having high and lowprotein content and given to rats at doses ranging from100-800 ppm for periods corresponding to the life of theanimal (Garth Fitzhugh and Nelson, 1945).

The last group with 4 mg/kg/day for 2 years, did notshow significant toxic effects. No malignant tumours weredetected in any group of animals.

Quinacrine experimental anticarcinogenic functions inmice

To study the anticarcinogenic activity of quinacrine weused three types of malignant tumours in mice: TA3,TA3-MTXR, (methotrexate resistant variant) and MMT

Quinacrinerevised 63

.

.:I.’ .), 1:

.’; ‘;

.*.

1,: I .

1;:. .1 ’

‘:;: . .,*;:,;;:” \‘.

;:9;.:yJ:: ;:St.!. ,Xi,o. t;.;.

:: _,. .

(Bittner’s retro-viral tumour from C3H strain) (Guerreroand Zipper, 1992; Guerrero and Dabances, 1993). The TA3tumour corresponds to a mammary carcinoma of asciticgrowth, maintained by transferring the same strain every 7days into the peritoneal cavity. Bittner’s tumour corre-sponds to a retrovirus (RNA) which is transmitted to theoffspring by lactation. Genetic, hormonal and viral (RNAretrovirus) factors are required in these spontaneous mam-mary tumours. They have a great malignant potential andare resistant to many chemotherapeutic agents includingvincristine, cisplatinum, adriamycin and methotrexate(TM-MTXR).

A common protocol was followed for the three experi-mental turnours. Several groups of male AJ mice wereinoculated i.m. with 1 x lo6 neoplastic cells of the threetypes of tumour in the right thigh. Each group was formedby five to 10 AJ mice each weighing 2&25 g. In order totest a correlation between the fibroblastic and anticarcino-genie actions of quinacrine we used the same pharmaco-logical compounds that improve the sclerosing propertiesof quinacrine.

There were 31 experimental groups; the control groupreceived common food and tap drinking water. The othergroups received drinking water ad Zibitum and differentcompounds that had been used as adjuvants or inhibitors ofthe fibroblastic function of quinacrine. In 19 groups weused quinacrine as the basic substance. Antiprostaglandins(betamethasone, prednisone, diclofenac, indomethacin)and cations Cu, Z-I, Co were employed in different con-centrations, alone or in association with quinacrine.

Tumoral node growth was evaluated measuring itslargest and smallest diameter every 3-4 days. The observa-tion period was one month, during which most controlanimals died. The final results of the diameters of the tu-mours were analysed using Student’s r-test. The results aresummarized in Figures 7-14 and in Table V.

The transplanted tumours TA3 TA3-MTXR had a lessdeveloped mass in animals that received quinacrine. Ani-mals that received quinacrine and ZnSO4 showed a tumo-ral development similar to the control groups. The animalshad a less developed tumoral mass when CuSO4 was addedto the quinacrine.

The best results with antiprostaglandins were obtainedwith the association of quinacrine and indomethacin whichgave 66,40 and 60% survival in TA3 tumours and 50% inMMT tumours.

Other compounds, such as indomethacin, betametha-sone, diclofenac, Cu and Co, were less active when quina-crine was not associated with them. This effect was notedin the three different types of tumours employed. Cu andCo do not have an action over tumoral growth. Although

5tI I

5i I

10 75

Time (days)

I t

20 25

Figure 7. Growth rate of TA 3 tumours in mice showing controls andmice treated with indomethacin, quinacrine and with both com-pounds. Quinacrine plus indomethacin significantly reduce the ve-locity of tumor growth, whereas quinacrine alone has no significanteffect.

35f

*-¤ Controi,-_* Quin+U

I o--a QuirWndo

, 4

4 8 12 16 2b 24 a

lime (daysj

Figure 8. Growth rate of TA 3 tumours in mice showing controls andmice treated with quinacrine and potassium, quinacrine and indo-methacin and quinacrine plus copper. Indomethacin and copper po-tent&e the antitumoral activity of the U~~YUS, while potassium doesnot. The association of quinacrine and indomethacin and quinacrineand copper gives a 60% survival at day 28.

slightly curative in action (20% survival), quinacrine is theonly one of all the compounds used, that acts as an antitu-moral (15.4 mm average tumoral diameter) and antiviral(Bittner’s tumour) compound. Considering that each ani-mal drinks an average of 5 ml/day of water with quinacrinein a concentration of 100 mg/l, i.e. 0.5 mg daily, and sinceeach mouse weighs -20 g, it could be extrapolated that for aperson weighing 50 kg, this quantity would be equivalentto 1500 mg/day or 15 times the dose used as preventive.

. . .’ I,

64 J.Zipper et al.

35r

20

-B&fWtb---alltdO- CWbCu+lndo-a Ouin+lndo+Dlcfo

Figure 9. Growth rate of TA 3 turnouts in mice given diclofenac, be-tamethasone, indomethacin, a combination of quinacrine, copperand indomethacin or a combination of quinacke, indomethacin anddiclofenac. The addition of copper to quinamine and indomethacinmaintains its antitumoral activity. Antiprostaglandins used alonehave no significant effects.

- Controlfn OuinN Quin+Dicla04 Ouin+Beto

I I L I I , I I I3 6 9 12 15 18 21 24 37

The (days)

Figure 10. Growth rate of TA 3 tumours in controls and in mice giv-en quinacrine, quinacrine plus diclofenac, and quinacrine plus beta-methasone. The antitumoral activity of qukxine increases when itis added to antiprostaglandins that have no effect alone.

However, we did not observe toxic manifestations in theanimals that survived.

Chloroquine is one of the multiple 4-aminoaquinolinesthat were tested as antimalarial agents. Its prophylacticantimalarialactionwasproventobebetterthanquinacrineand at the end of World ‘war II it replaced quina&te as anantimalarial suppressing drug. Some experiments carriedout in recent years have led to the thought that this com-pound stimulates tumoral carcinogenic growth and acti-

them with the oncogenic virus SV40 daily for a monthstarting from birth. The latency period of tumour appear-ance was 6 weeks. Maheshwaru and Srikantan (1990) in-vestigating the role of interferon as prophylactic agent inmonkeys with malaria produced by plasmodium cynomo-logy B malaria, suggested that chloquine could have in-terfered in the treatment since it inhibits interferon antiviralactivity a@ of mouse and also the polyinosinic-polycyti-dylic acid (poli 1:C) against the Semki forest virus (SFV)in mouse. Thomas and Vane (1990) associate hyperthermiawith the tumoral development of C-1300 murine neuro-blastoma. These experiments indicate that chloroquine haspharmacological actions over viral replication and can in-crease carcinogenic activity.

vates viral replication. Gamburg (1986) administered The action of primacrine and chloroquine has been in-chloroquine to 2 month old w after inoculatkg vestigated by feeding Egyptian toads (Bujb regrrlaris,

, I I.

I t J4 8 12 16 20 24 #) 32

Time (days)

F’igure 11. Growth rate of TA 3 tumours in mice given cobalt, quina-tie or both. Cobalt slightly potent&es quinac&e activity against TX3 tumour growth, whereas cobalt alone does not differ from controls.

- COMfOi

3or m Chinl - Ouin+Beta- Ouin+Beta+Cu

25 -

5

t

-_Time (days)

Figure 12. Growth rate of TA 3 tumours in mice showing controlsand mice given quinacrine, quinacrine and betamethasone or quina-crine, betamethasone and copper. The association of betamethasonewith or without copper to quinacrine improves its anticarcinogeneticactivity. Survival rates are 33 and 40% respectively.

Quinacrine revised 65

: -

-1 Quin- Chin&u

Figure 13. Growth rate of TA 3 tumours in mice showing controlsand mice given quinacrine, quinacrine and zinc or quinacrine andcopper. Only the combination of quinacrine and copper significantlyinhibits the growth of these TA 3 methotrexate-resistant tumours.The addition of zinc hinders this action..

- Control- indot-: Quino-0 Quin+lndo

I I J

6 12 16 24 30 36 42

Time (days)

Figure 14. Growth rate of Bittner tumours in mice showing controlsand mice given indomethacin, quinacrine or both. The combinationof quinacrine and indomethacin is highly active against the growthof this RNA-viral tumour. Indomethacin has the same effect as con-trol.

.

‘.:.‘_.*_ *._.: .‘.:a. :...:. .. . . ,,I. .:: ,*’ *i,

Mofty and Khudoley, 1992). Tumour formation was in-duced with primacrine in 19% of them. When chloroquineand Primacrme were given in combination, the tumourincidence increased to 23.5%.

To compare the activity of chloroquine and quinacrineover TA3 tumours, our group carried out the followingexperimental series. Three groups of AJ mice were com-pared: one group received oral quinacrine, the second oralchloroquine, and the third control group received tap drink-ing water. The animals were injected with the malignanttransplantable tumour TA3 30 days after the initiatiori of

the oral treatment. The prophylactic and/or curative actionof both compounds was evaluated.

The tumour growth curve was different in the threegroups. The control group presented a Gompertzian type ofgrowth; two of the 10 animals died at day 28. The othersdied later. All 10 animals of the chloroquine group hadrapid tumour growth and they were all dead by day 28. Ofthe 10 animals in the quinacrine group, there were no tu-mours in five of them. Tumours grew slightly in the otherfive, then regressed and the animals survived.

Conclusions

Experiments with injected tumours

The conclusions drawn are as follows (i) in our experi-ments, quinacrine used alone is the only compound thatacts as a curative agent in some cases. In the transplantabletumours TA3-TA3MTxR and MMT, it not only dimin-ishes the tumour size and growth speed, but may causesome of them to regress; (ii) the anticarcinogenic action ofquinacrine is increased when associated with an antiprosta-glandin and Cu. The most active combinations were quina-crine-indomethacin and quinacrine-Cu; (iii) all thecomplementary compounds used (antiprostaglandins andcations) required quinactine, and could be classified asanticarcinogenic quinacrine-dependent compounds for thethree types of malignant tumours tested; (iv) quinacrine isan essential substance to trigger the process of tumoraldisappearance, since none of the adjuvants used proved tobe curative when used alone. Antiprostaglandins usedalone had an effect in decreasing the tumoral size, andtumour speed of growth; (v) in all experiments where Zn isassociated to quinacrine, the antitumoral function of quina-crine is blocked; (vi) when used as a prophylactic drug,quinacrine, has a great activity against TA3 tumourgrowth.Chloroquine has the opposite action: it accelerates tumoralgrowth.

Epidemiological studies

There is no evidence that quimxine tan be carcinogenic inhumans or rodents. Bauer (1981) reported two cases ofquinacrine hydrochloride pahnar drug eruption (tropicallichenoid dermatitis) that underwent malignant transform-ation (squamous cell carcinoma). The patient group in-cluded >120 000 Australian servicemen, taking 100 or 200mg of quinacrine daily for many months, even after theonset of their drug eruption.

:..:a:, - .‘r, .,,:;. . .L*: .: : ,s .y: .

;.:.* : 4. . .

‘. ., =.. _!b :, .: . ’

,a’ 8:. . .

: ::

‘_-

(. ,

66 &Zipper et al.

Table V. Effect of oral quinacrine alone or associated with other substances upon the growth rate of TA3-TA3 MTXR-MMIVturnouts

Experimental

Qroupno.

Treatment

@xWwater)

QlOO

Q100+Cu100

CulOO

CulOO+K3

control

QlOO

control

Q100+Cu100

Q100+zn100

QlOO+BETA3

QlOO+BETA3+CuSO

QlOO+lNDQlO

QlOO+lNDQ25

QlOO+BETA3

QlOO+DlCLOlO

QlOO+INDQlO

+ DlCLOlO

control

control

INDO

QlOO+lNDQlO

INDO

Q25+lNDOS+CU25

DlCLOlO

BETA4

co100

Q100+Co100

control

QlOO

QlOO+lNDQlO

INDQlO

Q25+BETA3+Cu25

Tumour S/la ww DWW

TA3

TA3

TA3

TA3

TA3

TA3MTXR

TA3MTXR

TA3MTXR

TA3MTXFi

TA3

TA3

TA3

TA3

TA3

TA3

l/5 20 15.4

9

10

11

12

13

14

15

16

3l5 60 10.7

O/3 0 33.1

Of5 0 25.5

O/5 0 32.1

O/5 0 26.3

o/5 0 34.1

l/5 20 20.5

o/5 0 31.5

2f5 40 12.1

2/s 40 9.3

4/6 66 6.7

2/s 40 9.5

2l6 33 16.3

116 16 15.8

17

18

19

20

21

22

23

24

25

26

27

28

29

30'-_ 31

TA3 2t6 33 9.5

TA3 016 0 29.1

TA3 O/10 0 33.1

TA3 o/10 0 25.1

TA3 6/10 60 9.5

TA3 o/5 0 20.5

TA3 4110 40 11.8

TA3 015 0 22.7

TA3 o/5 0 23.4

TA3 or5 0 32.1

TA3 l/5 20 17.2

M M T O/9 0 30.6

M M T O/8 0 17.3

M M T 4/8 50 8.0

M M T O/8 0 23.5

TA3 2f5 40 112

. .

In all experiments, the oral ingestion of diierent drugs began at the same time as the injection of turnouts.S = no. of surviving healthy animals after 1 month of tumour inoculation and treatment; la, total no. of animals inoculated andtreated in each group; S (%) = survival percentage after one month of tumour inoculation and treatment; D = average growth oftumour (mm); BETA = betamethasone; INDO = indomethacin; DICLO = diclofenac.

:

The latent period between the late sequelae of the drug Intrauterine insertion of q&acheeruption (lichenoid dermatitis) and its malignant change Two large scale studies have been carried out. Dabancenscan be as late as 34 years after ingestion, and it depends and Pxuyas (1994) reported a study of a group of 1061mainly on the skin reaction and sensitivity of the patient to women in Santiago, Chile, who had requested sterilizationthe drug. No other case of cancer attributed to quinacrine since 1977. Quinacrine pellets were inserted transcervi-has apparently been &scribed (Goodman and Gilman, tally in the uterine cavity on two or three occasions at

_ 1992).

P ’

Quinacrine revised 67

. .

intervals of 30+0 days. Papanicolaou tests were carriedout before and after the sterilization. Some grade of lesionswas observed in 75 women (7.1%). In 36 women, thislesion was present before the quinacrine insertion (preva-lence 3.4%). In 39 women, lesions appeared after the qui-nacrine insertion (incidence 1.1%) in a total of 3654woman/years between the first and the last Papanicolaoutest. In this group, we found one patient with squamous cellcarcinoma surgically treated in 1986. She was alive andfree of illness at her last follow-up visit in 1990. In addition,30 patients were found with low grade intra-epithelialcervical lesions and eight women with high grade intra-epi-thelial cervical lesions (cancer in situ).

After standardizing the incidence tates, we comparedthese results with a control group of women in Santiago.The incidence rate of high grade intra-epithelial squamouscell lesions, precursors of cervical carcinoma, in patientstreated with quinacrine (2.62%) was not higher than theincidence rate in other areas of Santiago (2.56%, P > 0.05).The conclusion is that intrauterine quinacrine administra-tion does not increase the incidence of cervicHterinecancer in this population sterilized with quinacrine.

Analyses of the risk of cancer among women sterilizedwith transcervical quinacrine hydrochlorideThis study was carried out between 1977 and 1991 (Sokaland Aldrich, 1993). It was conducted as a retrospectivecohort study of 1491 women who were voluntarily steril-ized with quinacrine in Chile. From the available data ofthe whole cohort it can be reproted that 17 cancer caseswere diagnosed after quinacrine insertion, 12 cases ofcarcinoma of the cervix in situ were diagnosed, as well asseven cancers diagnosed prior to sterilization.

The cohort members provided 7941 woman-years offollow-up. The pattern of cancer occurrence among thecohort members was evaluated using conventional cohortanalyses as well as sensitive space-time cluster methods.No evidence was found of excess cancer risk associatedwith quinacrine hydrochloride transcervical sterilizationbased upon the experience of this cohort.

Research on experimental carcinogenesis related byextrapolation from in-vitro tests and in-vivo rodentcarcinogens to the human speciesSearching for additional information, a panel of expertswas commissioned by Family Health International (Re-search Triangle Park, NC, USA) to evaluate the toxicologyof quinacrine. They conchded that quinacrine was muta-genic in prokaryotes (bacteria), but that it was very unlikelythat quinacrine was a rodent or human carcinogen. Ray-

mond and Barry (1987) studying the prediction of chemi-cal carcinogenic&y in rodents from in-vitro genetic toxicityassays, demonstrated the real importance of short-termtests for genotoxic chemicals. After using 73 chemicals,they assessed the concordance of in-vitro assays as evalu-ated by mutagenesis in Salmonella and mouse lymphomacells, chromosome aberrations and sister chromatid ex-changes in Chinese hamster ovary cells. Their ability topredict the carcinogenicity of selected chemicals in rodentswas -60%. The concordance, or observed agreement, be-tween rat and mouse carcinogenicity determinations was67%. From these investigations it is clear that even with abattery of assays, not all rodent carcinogens are in-vitromutagens, nor are all in-vitro mutagens rodent carci-nogens.

In-vitro short-term tests cannot replace long-term rodentstudies for the identification of chemical carcinogens. Thereduced concordance of the short-term test results withcarcinogenicity results has increased the frequencies ofnongenotoxic carcinogens and genotoxic non-carcinogens(Raymond and Barry, 1987). Ames and Swirsky (1990)recently questioned the utility and meaning of routine ani-mal cancer tests. They suggested that mitogenesis (inducedcell division) plays a dominant role in carcinogenesis sinceit has been found that spontaneous DNA damage caused byendogenous oxidants is remarkably frequent and that inchronic testing at the maximum tolerated dose, more thanhalf of all chemicals tested (both natural and synthetic) arecarcinogens in rodents, and a high percentage of thesecarcinogens are not mutagens. Thus, any agent causingchronic mitogenesis can be indirectly mutagenic (andconsequently carcinogenic) because it increases the probability of converting endogenous DNA damage into muta-tions.

A high percentage of all chemicals might be expected tobe carcinogenic at chronic, near toxic doses, and this isexactly what is found. About half of all chemicals testedchronically at the maximun tolerated dose are carcinogens.Thus without studies of the mechanisms of carcinogenesis,the carcinogen at the maximum tolerated dose in rodentsprovides no information about low dose risk to humans.

Spingam and Weisburger (1979) used the Ames Salvo-neZZu mutagenesis assay to study the formation of mu-tagens in cooked foods. According to these tests on beef,mutagens are formed when meat is cooked by frying, boil-ing and broiling. Hamburgers from commercial franchiseswere frequently mutagenically active. Undoubtedly theseresults should be in the range of the 40% of false positiveshort-term tests described by Raymond and Barry, 1987.

.

68 J.Zipper et al.

Relationship between physical and chemical structureand possible carcinogenic activity of specific molecules

During the 1930s and 194Os, many important therapeuticcompounds derived from acridine were developed. One ofthem was quinacrine. All these compounds were submittedto carcinogenic tests (Lacassagne and Buu-Hoi, 1956),where the aim of this work was to distinguish the carcino-genic derivatives from the anti-carcinogenic ones, analys-ing the structure of the formula

Lacassagne and Buu-Hoi (1956) had intended to predictcarcinogenic activity based on the electronic charge of thenitrogen atoms in the molecule. They were searching forthe reason why benz-(c)-acridines are powerful carcino-genic substances, and benz-(a)-acridines and related mol-ecules are not. In 1956, they studied the relationshipbetween the physical and chemical properties of angularbenzacridines and possible carcinogenic activity.

A&dine, the basic molecule of the benzacridines, wasdiscovered by Graebe and Caro in 1870. It is a constantconstituent of coal and is concentrated in the anthracenicfraction when coal is fractioned by distillation. Bloch(1922), Kennaway ( 1924) and Maisin and Desmedt ( 1927)confirmed the absence of carcinogenic activity in acridine.Experiments had been carried out with 9-methylacridine,and it was found to be inactive (Lacassagne and Buu-Hoi,1956).

The research on angular monobenzacridines and diben-zacridines demonstrated their substantially different carci-nogenic activity. While linear benz-(b)-acridine and itsderivatives have no carcinogenic activity, in the angularbenzacridines and in particular in the benz-(c)-a&dineseries a considerable number of carcinogenic compoundshave been found.

The benz-(c)-acridine 7,10 and 7,9 showed greater carci-nogenic activity than 20-methylchlolanthrene. Nearly allthecarcinogenic benzacridines have a methyl group on themeso-anthracenic carbon, in position 7 and nitrogen, inposition 12. The benz-(a)-a&dines have the opposite,methyl group in position 12 and nitrogen in position 7, anddo not have carcinogenic activity.To demonstrate rodentcarcinogenesis, Lacassagne and Buu-Hoi ( 1956) used thetwo usual tests of carcinogenesis: the production of epithe-liomas (by painting the skin), and sarcomas of the subcu-taneous conjunctive tissue (by injections repeated threetimes at monthly intervals). The experiments were con-tinued up to the animal death (natural or due to cancer) orits sacrifice ‘in extremis’.

In summary, this work led to the following conclusions.Firstly, that acridine and its derivatives have no carcino-genic activity, probably due to their special configuration;methyl group in position 9 and nitrogen in position 10. The

.

CH31m:o(0

acridine(II)

3-mthylxridine

benz(a) actidine benzfc) acridine

Figure 15. Structure of acridine and its derivatives.

same situation happens with benz-(a)-acridines (Figure15). Secondly, that benz-(c)-acridines are carcinogenic,which has increased the number of carcinogenic sub-stances that permit the comparison of the electron densitytheories, and its relationship to biological experimentation.

The authors determined that the crucial point is the posi-tion of the nitrogen atom in the molecule. This positiongives the electronic charge of the nitrogen atom. When theN has the seventh position in the benz-(a)-acridine, thenthere is no carcinogenic activity. When the N is in the 12thposition, the electron density of the atom is increased andthe molecule becomes highly carcinogenic (e.g.benz-(c)-a&dine and similar molecules).

Research on acridines by the International Agency forResearch on Cancer

The International Agency for Research on Cancer (IARC)has not yet investigated the carcinogenic risk of quinacrine,but it was assessed with acridine orange (IARC, 1978).This compound has the same basic acridine structure asquinacrine but lacks the alkyl side-chain.

Employing the usual test of carcinogenicity (Lacassagneand Buu-Hoi, 1956), its possible carcinogenic activity wasstudied. It is a powerful inhibitor of DNA, RNA and pro-tein synthesis, forming non-covalent, tightly bound cornplexes with DNA by intercalation of the bases in thedouble-stranded DNA molecule.

There was no evidence of carcinogenicity due to acrid&orange using long-term carcinogenic assays in rodents.The results using short-term tests for genotoxic chemicalsdemonstrated that acridine orange could induce frameshiftmutations in replicating bacteriophage T4 and reversemutations in Escherichia cdi strain S. It also induced re-verse mutations in Salmonella typhimutium stxains TA100, TA 98 and TA 1537. No data on its embryotoxicity or

Quinacrine revised 69

teratogenicity were available to the working group. Thiswork is another example demonstrating that not all in-vitromutagens am rodent or human carcinogens.

Final conclusions

Pharmacological data

Some concentrations of quinacrine, in contact with thehuman tube uterine produce an obstructive granuloma ofits lumen at the intramural portion.

The addition of steroidal or non-steroidal antiprostaglan-dins (betamethasone, indomethacin, diclofenac) and/or theCu cation to quinacrine, potentiates the sclerosing action ofthis compound.

The association quinacrine-antiprostaglandins-cu, hasa similar action when instilled into the rat uterus. Thisexperimental model to test the sclerosing effectiveness of acompound, has been cl.inicalIy confirmed.

The combination quinacrine-betamethaon+Cu in-creases the anticarcinogenic potential of quinacrine.

The Zn2+cation antagonizes some of the C-u functions andthe fibroblastic and anticarcinogenic functions of qukcrine.

Quinacrine, like many other antiprostaglandins, is con-sidered an anticarcinogenic compound. Its pharmacologi-cal mechanisms of action are probably due to (i) inhibitionof prostaglandin synthesis by inhibiting phospholypaseand arachidonic acid synthesis, (ii) inhibition of cyclo-oxygenase, as other antiprostaglandins, (iii) inhibition ofthe synthesis of thromboxane A2-B2 and a great numberof prostaglandins.

Clinical applications

Two transcervical insertions of slow dissolution pelletscontaining 250 mg of quinacrine produce a cumulativepregnancy rate of 7% at 7 years of follow-up.

The addition of antiprostaglandins to quinacrine has re-duced the pregnancy rates in the lkst 3 years of follow-upwhen compared with quinacrine alone to 3% forquinacrine+ diclofenac and 2% for quinacrine + betamethasone.

The association quinacrine-betamethasone-Cu duringtwo insertions has produced a pregnancy rate of 4% in thefirst 3 years of follow-up. Quinacrine alone in the sameperiod produced a 5% pregnancy rate.

Any carcinogenic or toxicological assay (short-term in-vitro tests or long&m in-vivo tests) should consider thecombination of qkacrine plus the adjutants. This researchwas performed by two different centres and investigators.

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