Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7): 1033-1039, October 2002
In Vitro Chloroquine Resistance Modulation Study on Fresh
Isolates of Brazilian Plasmodium falciparum: Intrinsic
Antimalarial Activity of Phenothiazine Drugs
Carla MS Menezes, Karin Kirchgatter*, Sílvia M Di Santi*, Carine Savalli**,
Fabiola G Monteiro**, Gilberto A Paula**, Elizabeth I Ferreira/+
Faculdade de Ciências Farmacêuticas **Instituto de Matemática e Estatística, Universidade de São Paulo, Av. Prof. Lineu Prestes
580, Bl. 13, 05508-900 São Paulo, SP, Brasil *Divisão de Programas Especiais, Superintendência de Controle de Endemias,
Phenothiazine drugs – fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine – were evaluatedas modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming tosimulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum ma-laria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-foldserial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, thephenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodialactivity in concentrations lower than those described in the literature. In addition, IC estimates have been shownto be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increasein the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when usedin combination.
Key words: antimalarial - chloroquine - multidrug resistance - phenothiazines - modulating agents - chemosensitizer agents
The global situation of malaria is claiming attention
we evaluated the potential effect of the phenothiazine
more than ever. It has been estimated that about 300 mil-
drugs – fluphenazine, chlorpromazine, methotrimeprazine
lion acute clinical cases are reported each year, with at
and trifluoperazine (Fig. 1) –, on modulating the chloro-
least one million deaths (WHO 2000). Antimalarial drug
quine resistance of Brazilian P. falciparum fresh isolates.
resistance is among the principal factors responsible for
MATERIALS AND METHODS
this serious public health problem. Resistance has beenassociated with the parasite’s natural biological mecha-
The drugs used were chloroquine diphosphate
nisms in response to uncontrolled and unregulated drug
(Fundação para o Remédio Popular), chlorpromazine hy-
distribution programs, resulting in genetic mutations
drochloride, fluphenazine dihydrochloride and me-
thotrimeprazine maleate (Cristália Produtos Químicos e
Many efforts have been made in the search for a new
Farmacêuticos Ltda.), and trifluoperazine dihydrochloride
and effective antimalarial agent. However, little success
has been achieved and, therefore, the restoration of cur-
Two fresh isolates of P. falciparum were used: Isolate
rent available drugs becomes an important alternative.
1 (Sucen 198/94) and Isolate 2 (Sucen 206/94). Isolate 1
Combination therapy is particularly studied concerning
was collected from a 37-year-old woman in her third infec-
chloroquine (Fig. 1), although the major Plasmodium
tion (6,600 asexual parasites per mm3) and Isolate 2 from
falciparum strains and the emergent P. vivax are resis-
a 21 year-old-man in his second infection (7,500 asexual
tant, this drug remains the most important antimalarial
parasites per mm3). The infections occurred in the North
agent. Good pharmacokinectic features, easy use and low
of Brazil and the individuals had not been submitted to
cost are its principal properties (Ward & Bray 2001).
antimalarial treatment in the previous 28 days (Bruce-
With the purpose of restoring chloroquine efficacy
Chwatt 1986). Blood samples were collected after formal
and considering the reported reversal effect of tricyclic
drugs on resistant malaria (Bitonti et al. 1988, Basco & Le
Assays were performed in microplates with 96 flat-
Bras 1990, 1991, Kyle et al. 1990, 1993, Peters et al. 1990,
bottomed wells. The culture medium was RPMI-1640,
Basco & Le Bras 1992, Miki et al. 1992, Oduola et al. 1998),
HEPES buffer, gentamicine sulphate, glucose, hypoxan-thine, sodium bicarbonate and human type A serum.
The statistical analysis was carried out using the soft-
ware S-Plus, version 4.5, and the Microsoft Excel for Win-dows, version 5.0. In vitro assays - The biological assays were carried
out based on the in vitro microtechnique for drug suscep-
Corresponding author. Fax: +55-11-3815-4418. E-mail:
tibility (Rieckmann et al. 1978). The microplates were ti-
trated with two-fold serial dilutions of fluphenazine (0.06-
4 µg/l), chlorpromazine (3.75-240 µg/l), methotrimeprazine
In Vitro Phenothiazine Antimalarial Activity Carla MS Menezes et al.
Day 1980, Collett 1991). Linear and quadratic logistic mod-
els and the log-log complement model were considered. According to these, coincident and separated, parallel and
concurrent (one or two intercepts) lines were fitted. The10% significance level was adopted for the likelihood ra-tio statistic which, in this case, corresponds to the differ-
ence between two goodness-of-fit statistics. In addition,
the 50% inhibitory concentration, IC , of each drug and
its combination with chloroquine was estimated.
The susceptibility of the isolates to chloroquine, phe-
nothiazine drugs and their combinations can be seen in
the descriptive analysis lines (Figs 2 to 6 show the linescalculated for Isolate 1). A decrease in the parasitaemiarate can be observed along the drug concentrations, mark-
The inferential analysis suggested the concurrent lines
(two intercepts) as the best fitted model for fluphenazine,chlorpromazine (at the 5% significance level), andmethotrimeprazine for Isolate 1. The coincident lines werefitted to trifluoperazine in the same isolate (Table I). For
Isolate 2 the coincident lines model was the best fit for all
the phenothiazines, with the exception of methotrime-prazine. The concurrent lines model (two intercepts) wasthe best fit for this drug (Table II).
The IC estimates are presented in the natural loga-
Fig. 1: chemical structures of the phenothiazines and chloroquine. Parasitaemia rate
(5-320 µg/l), and trifluoperazine (0.31-20 µg/l). In each se-ries, the intermediate value corresponded to the usual anti-
ln(concentration)
psychotic therapeutic concentration (Clarke 1986, Benetet al. 1996). Chloroquine (30 µg/l) was added along the
series (Tracy & Webster 1996). The phenothiazine series
were also assayed alone with the purpose of evaluating
the intrinsic antiplasmodial effect. The chloroquine sus-ceptibility was tested in a range from 3.75 to 240 µg/l.
A 10% haematocrit solution of infected blood was
added to the plates. These were incubated according to
the candle jar method (Trager & Jensen 1976) at 37°C for
Parasitaemia rate
40 h (Isolate 1) and for 46 h (Isolate 2). Schizonts withthree or more nuclei in 200 parasites were counted. Statistical analysis - Lines for the parasitaemia rate –
number of parasites in each concentration/number of para-
ln(concentration)
sites in the control – as a function of the chloroquine andphenothiazine drug concentrations and the respective
Fig. 2: ratios of the number of Plasmodium falciparum parasites in
combinations with chloroquine were constructed and sub-
drug-treated cultures to the number in control cultures (parasitaemia
mitted to descriptive and inferential analyses (Breslow &
rate) after exposure to chloroquine (A: Isolate 1; B: Isolate 2).
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7), October 2002
Parasitaemia rate Parasitaemia rate ln(concentration) ln(concentration) Parasitaemia rate Parasitaemia rate ln(concentration) ln(concentration)
Fig. 3: ratios of the number of Plasmodium falciparum parasites in
Fig. 4: ratios of the number of Plasmodium falciparum parasites in
drug-treated cultures to the number in control cultures (parasitaemia
drug-treated cultures to the number in control cultures (parasitaemia
rate) after exposure to fluphenazine (▲) and fluphenazine plus
rate) after exposure to chlorpromazine (▲) and chlorpromazine
chloroquine (■) (A: Isolate 1; B: Isolate 2).
plus chloroquine (■) (A: Isolate 1; B: Isolate 2).
Fitted linear logistic model for the phenothiazine drugs and their combinations with chloroquine (Isolate 1, Sucen 198/94)
In Vitro Phenothiazine Antimalarial Activity Carla MS Menezes et al. Parasitaemia rate ln(concentration) ln(concentration) Parasitaemia rate 0,2 Parasitaemia rate ln(concentration) ln(concentration)
Fig. 5: ratios of the number of Plasmodium falciparum parasites in
Fig. 6: ratios of the number of Plasmodium falciparum parasites in
drug-treated cultures to the number in control cultures (parasitaemia
drug-treated cultures to the number in control cultures (parasitaemia
rate) after exposure to methotrimeprazine (▲) and metho-
rate) after exposure to trifluoperazine (▲) and trifluoperazine plus
trimeprazine plus chloroquine (■) (A: Isolate 1; B: Isolate 2).
chloroquine (■) (A: Isolate 1; B: Isolate 2).
Fitted linear logistic model for the phenothiazine drugs and their combinations with chloroquine (Isolate 2, Sucen 206/94)
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7), October 2002
Estimated median inhibitory concentrations (IC ) of chloroquine, the phenothiazine drugs and the respective combinations on
a: Log 50% inhibitory concentration; b: same values (coincident lines model)
DISCUSSION
trimeprazine, and trifluoperazine. Other tricyclic com-
Many hypotheses have been advanced to explain the
pounds, e.g., desipramine, cyproheptadine, and their ana-
P. falciparum resistance to chloroquine. The reversal of
logues (Bitonti et al. 1988, Basco & Le Bras 1990, Peters et
chloroquine resistance by verapamil suggested a similar
al. 1990, Basco et al. 1991) were early reported as modulat-
mammalian tumor cells multidrug-resistant phenotype in
ing agents on resistant malaria. Previous studies with
malaria. Furthermore, many different drugs and com-
chlorpromazine, trifluoperazine and promethazine have
pounds have been assayed for their modulating effect in
also demonstrated the in vitro and in vivo reversal of chlo-
restoring antimalarial drugs effectiveness (Ward & Bray
roquine resistance (Kyle et al. 1990, 1993, Basco & Le
Bras 1992, Miki et al. 1992, Oduola etal. 1998).
In our study, a series of drugs reported as modulat-
However, different results were observed in our study.
ing agents in resistant malaria and/or neoplastic
The phenothiazines drugs did not modulate chloroquine
multidrug-resistant cell lines were evaluated in Brazil-
resistance. Similar behavior was observed for verapamil
ian chloroquine-resistant fresh isolates of P.
(Menezes et al. unpublished data), imipramine (Menezes
falciparum. The in vitro microtechnique (Rieckmann et
et al. 1997) and most of the other modulating agents as-
al. 1978) was chosen as it matches closely with
sessed. These results corroborated recent observations
scintillometric measurements (Le Bras et al. 1984), and
when Brazilian chloroquine-resistant strains demonstrated
it presents great feasibility in field studies (Yang et al.
a lesser degree of susceptibility to verapamil when com-
1997, Philipps et al. 1998, Arez et al. 1999, Warsame et
pared to strains from Africa and Southeast Asia (Mehlotra
al. 1999). The chloroquine IC estimates indicated that
the isolates were resistant, according to WHO state-
It is also important to note that, in our study, all the
ments (Bruce-Chwatt 1986). Moreover, these concen-
phenothiazine drugs demonstrated intrinsic antiplasmodial
trations are among those reported in previous studies
effects at concentrations inferior to those described as
on chloroquine P. falciparum resistance reversal
sub-inhibitory in modulating the antimalarial resistance
(Krogstad et al. 1987, Martin et al. 1987, Bitonti & Mc
(as example, 625 nM, that corresponds to 199.30 µg/l, for
Cann 1989, Basco & Le Bras 1990, Kyle et al. 1990).
chlorpromazine) (Kyle et al. 1990, Basco & Le Bras 1992).
With the purpose of simulating therapeutic schedules,
However, in spite of the common phenothiazine chemical
a particular drug combination was adopted. The interme-
structure (Fig. 1), distinct behaviors were observed in the
diate concentration in the two-fold serial dilutions corre-
combinations with chloroquine. For Isolate 1, the concur-
sponded to the usual therapeutic concentration of the
rent lines model (two intercepts) was the best fit for me-
assessed modulating agent. A fixed concentration of chlo-
thotrimeprazine, fluphenazine and chlorpromazine while
roquine (30 µg/l) was employed in the combinations. This
the coincident lines model was the best model for triflu-
concentration is capable of clearance of parasitaemia in
operazine. The latter model was also the best fit for all
sensitive P. falciparum infections (Tracy & Webster 1996).
drugs except methotrimeprazine, in the case of Isolate 2.
Once the modulating effect was observed, chloroquine
The distinct fitted statistical lines models may be related
would return its effectiveness. A fixed concentration of
to peculiarities of the isolates. To date, these results are
chloroquine was also used to determine the antimalarial
response modification index throughout the combination
Inhibition of falcipain and the interaction with heme
of modulating agents (Kyle et al. 1990, Oduola et al. 1998).
moiety (ferriprotoporfyrin IX) have been associated to
This study reports the evaluation of the phenothiaz-
the antiplasmodial activity of phenothiazine compounds
ine drugs: fluphenazine, chlorpromazine, metho-
(Panijpan & Kantakanit 1983, Domínguez et al. 1997). Dur-
In Vitro Phenothiazine Antimalarial Activity Carla MS Menezes et al.
ing the intraerythrocytic stage, inside the food vacuole,
Basco LK, Ringwald P, Le Bras J 1991. Chloroquine-potentiat-
specific parasite proteases, such as falcipain, degrade the
ing action of antihistaminics in Plasmodium falciparum in
host hemoglobin in order to generate amino acids for pro-
vitro.Ann Trop Med Parasitol85: 223-228.
tein synthesis. The resulting heme is detoxified by a poly-
Benet LZ, Øie S, Schwartz JB 1996. Design and optimization
merization process to form the malarial pigment, hemozoin.
of dosage regimens pharmacokinetic data. In JG Hardman,LE Limbird, PB Molinoff, RW Ruddon, AG Gilmann (eds),
Thus, inhibition of falcipain would precede the interfer-
Goodman & Gilman’s: the Pharmacological Basis of Thera-
ence in the heme polymerization process, the most ac-
peutics, 9th ed., Pergamon-Press, New York, p. 1707-1792.
cepted hypothesis regarding the mechanism of chloro-
Bitonti AJ, McCann PP 1989. Desipramine and cyprohepta-
quine action (Padmanaban & Rangarajan 2000).
dine for reversal of chloroquine resistance in Plasmodium
Our results should be contemplated taking into ac-
falciparum. Lancet2: 1282-1283.
count the phases in which chloroquine and phenothiaz-
Bitonti AJ, Sjoerdsma A, Mccann PP, Kyle DE, Oduola AMJ,
ine compounds are believed to interfere with the Plasmo-
Rossan RN, Milhous WK, Davidson Jr DE 1988. Reversal
dium hemoglobin metabolism. Inhibition of falcipain and/
of chloroquine resistance in malaria parasite Plasmodium
or a stronger interaction of the phenothiazine drugs with
falciparum by desipramine. Science242: 1301-1303.
heme moiety compared to that of chloroquine could be
Breslow NE, Day NE 1980. Statistical Methods in Cancer Re-
responsible for the predominant antiplasmodial effects of
search. 1. The Analysis ofCase Control Studies, Interna-tional Agency for Research on Cancer, Lyon, 350 pp.
these compounds over chloroquine. This hypothesis may
Bruce-Chwatt LJ 1986. Chemotherapy of Malaria, 2nd ed.,
be supported by the coincident lines model. Conversely,
the competition for the heme moiety could explain the
Clarke EC 1986. Isolation and Identification of Drugs in Phar-
concurrent lines model. In this situation, the addition of
maceuticals, Body Fluids and Post-mortem Material, 2nd
chloroquine caused an increase in the parasitaemia rate
ed., Pharmaceutical Press, London, 1223 pp.
for most phenothiazine concentrations.
Collett D 1991. Modelling Binary Data, Chapman & Hall, Lon-
Although further assays are needed to check the pro-
posed mechanistic hypothesis, we believe our findings
Domínguez JN, López S, Charris J, Iarruso L, Lobo G, Semenov
are important to demonstrate the antiplasmodial effect of
A, Olson JE, Rosenthal PJ 1997. Synthesis and antimalarial
phenothiazine drugs at concentrations lower than those
effects of phenothiazine inhibitors of a Plasmodium
employed in anti-psychotic therapy, as observed by the
falciparum cysteine protease. J Med Chem 40: 2726-2732.
Geary TG, Divo AA, Jensen JB 1986. Effect of calmodulin
estimated IC values. In addition, those concentrations
inhibitors on viability and mitochondrial potential of Plas-
showed to be lower than those previously related to anti-
modium falciparum in culture. Antimicrob Ag Chemother
malarial activity. Falcipain inhibition as well as the inter-
action with heme moiety (Panijpan & Kantakanit 1983,
Kristiansen JE, Jepsen S 1985. The susceptibility of Plasmo-
Domínguez et al. 1997) and other antiplasmodial activities
dium falciparumin vitro to chlorpromazine and the stereo-
of phenothiazines, such as calmodulin antagonism
isomeric compounds cis(Z)- and trans(E)-clopenthixol. Acta
(Scheibel et al. 1987), membrane stabilization (Kristiansen
Pathol Microb Immunol Scand (Section B) 93: 249-251.
& Jepsen 1985), disturbance in mitochondrial function
Krogstad DJ, Gluzman IY, Kyle DE, Oduola AMJ, Martin SK,
(Geary et al. 1986) and the inhibition of glutathione reduc-
Milhous WK, Schlesinger PH 1987. Efflux of chloroquine
tase (Luond et al. 1998) were all observed in higher and, in
from Plasmodium falciparum: mechanism of chloroquine
resistance. Science238: 1283-1285.
In resume, the results here presented corroborate pre-
Kyle DE, Milhous WK, Rossan RN 1993. Reversal of Plasmo-diumfalciparum resistance to chloroquine in Panamanian
vious findings in considering phenothiazine derivatives
Aotus monkeys. Am J Trop Med Hyg48: 126-133.
as interesting lead compounds in the design of new anti-
Kyle DE, Oduola AMJ, Martin SK, Milhous WK 1990. Plas-
malarial agents (Vennerstrom et al.1995, Atamna et al. modiumfalciparum: modulation by calcium antagonists of
resistance to chloroquine, desethylchloroquine, quinine, and
REFERENCES
quinidine in vitro. Trans R Soc Trop Med Hyg84: 474-478.
Le Bras J, Andrieu B, Hatin I, Savel J, Coulaud JP 1984. Plas-
Arez AP, Snounou G, Pinho J, Sousa CA, Modiano D, Ribeiro
modium falciparum: interprétation du semi-microtest de
H, Franco AS, Alves J, Rosario VE 1999. A clonal Plasmo-
sensibilité in vitro par incorporation de 3H-hypoxanthine. dium falciparum population in an isolated outbreak of ma-
Pathol Biol32: 463-466.
laria in the Republic of Cabo Verde. Parasitology118: 347-
Luond RM, McKie JH, Douglas KT, Dascombe MJ, Vale J
1998. Inhibitors of glutathione reductase as potential anti-
Atamna H, Krugliak M, Shalmiev G, Deharo E, Pescarmona G,
malarial drugs. Kinetic cooperativity and effect of dimethyl
Ginsburg H 1996. Mode of antimalarial effect of methylene
sulphoxide on inhibiton kinetics. J Enzyme Inhib 13: 327-
blue and some of its analogues on Plasmodium falciparum
in culture and their inhibition of P. vinckei petteri and P.
Martin SK, Oduola AMJ, Milhous WK 1987. Reversal of chlo-
yoelii nigeriensis in vivo. Biochem Pharmacol51: 693-700.
roquine resistance in Plasmodium falciparum by verapamil.
Basco LK, Le Bras J 1990. Reversal of chloroquine resistance
with desipramine in isolates of Plasmodium falciparum from
Mehlotra RK, Fujioka H, Roepe PD, Janneh O, Ursos LMB,
Central and West Africa. Trans R Soc Trop Med Hyg84:
Jacobs-Lorena V, Mcnamara DT, Bockarie MJ, Kazura JW,
Basco LK, Le Bras J 1992. In vitro activities of chloroquine in
Kyle DE, Fidock DA, Zimmerman PA 2001. Evolution of a
combination with chlorpromazine and prochlorperazine
unique Plasmodium falciparum chloroquine-resistance phe-
against isolates of Plasmodium falciparum. Antimicrob. Ag
notype in Papua New Guinea and South America. ProcChemother36: 209-213. Natl Acad Sci USA98: 12689-12694.
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7), October 2002
Menezes CMS, Kirchgatter K, Di Santi SMF, Savalli C,
nists inhibit human malaria parasites (Plasmodium
Monteiro FG, Paula GA, Ferreira EI 1997. Antimalarial
falciparum): implications for drug design. Proc Natl Acad
effect in vitro and lack of modulating effect of desipramine
Sci USA84: 7310-7314.
and imipramine. Trans R Soc Trop Med Hyg91: 697-700.
Tracy JW, Webster Jr LT 1996. Drugs used in the chemotherapy
Miki A, Tanabe K, Nakayama T, Kiryon C, Ohsawa K 1992.
of protozoa infections: malaria. In JG Hardman, LE Limbird,
Plasmodium chabaudi: association of reversal of chloro-
PB Molinoff, RW Ruddon, AG Gilmann (eds), Goodman
quine resistance with increased accumulation of chloroquine
& Gilman’s: the Pharmacological Basis of Therapeutics,
in resistant parasites. Exp Parasitol74: 134-142.
9th ed., Pergamon-Press, New York, p. 965-985.
Oduola AMJ, Sowunmi A, Milhous WK, Brewer TG, Kyle
Trager W, Jensen BB 1976. Human malaria parasites in con-
DE, Gerena L, Rossan RN, Salako LA, Schuster BG 1998.
tinuous culture. Science193: 673-675. In vitro and in vivo reversal of chloroquine resistance in
Vennerstrom JL, Makler MT, Angerhofer CCK, Williams JA
Plasmodium falciparum with promethazine. Am J Trop Med
1995. Antimalarials dyes revisited: xanthenes, azines, ox-
azines, and thiazines. Antimicrob Ag Chemother39: 2671-
Padmanaban G, Rangarajan PN 2000. Heme metabolism of Plas-modium is a major antimalarial target. Biochem Biophys Res
Warsame M, Kilimali AEB, Wernsdorfer WH, Lebbad M, Rutta
Panijpan B, Kantakanit N 1983. Chlorpromazine enhances
AS, Ericsson O 1999. Resistance to chloroquine and
haemolysis induced by haemin. J Pharm Pharmacol35:
sulfadoxine-pyrimethamine in Plasmodium falciparum in
Muheza district, Tanzania. Trans R Soc Trop Med Hyg93:
Peters W, Ekong R, Robinson BL, Warhurst DC, Xing-Qing P
1990. The chemotherapy of rodent malaria. XLV. Reversal
Ward SA, Bray PG 2001. Is reversal of chloroquine ready for
of chloroquine resistance in rodent and human Plasmodium
clinic? Lancet357: 904.
by antihistaminic agents. Ann Trop Med Parasitol84: 541-
Wellems TE, Plowe CV 2001. Chloroquine-resistant malaria. J Infect Dis184: 770-776.
Philipps J, Radloff PD, Wernsdorfer W, Kremsner PG 1998.
WHO-World Health Organization 2000. Malaria, a Global
Follow-up of the susceptibility of Plasmodium falciparumCrisis. Fact sheet 1. The problem, WHO 03/06/00. Avail-
to antimalarials in Gabon. Am J Trop Med Hyg58: 612-618.
Rieckmann KH, Sax LJ, Campbell GH, Mrema JE 1978. Drug
Yang HL, Liu DQ, Yang YM, Huang KG, Dong Y, Yang PF, Liao
sensitivity of Plasmodium falciparum. An in vitro micro-
MZ, Zhang CY 1997. In vitro sensitivity of Plasmodium
technique. LancetI: 22-23. falciparum to eight antimalarials in China-Myanmar and
Scheibel LW, Colombani PM, Hess AD, Aikawa M, Atkinson
China-Lao PDR border areas. South Asian J Trop Med Pub
CT, Milhous WK 1987. Calcium and calmodulin antago-
In Vitro Phenothiazine Antimalarial Activity Carla MS Menezes et al.
KOMMUNKANSLIET/TILLVÄXT AVESTA Datum Avgiftsfri kollektivtrafik Sammanfattning En utredning har gjorts av kommunkansliet för att beräkna kostnader för avgiftsfri kollektivtrafik för barn och ungdom i Avesta kommun. Fyra alternativ presenteras: oförändrad trafik, avgiftsfri kollektivtrafik för barn och ungdom 9 månader/år, avgiftsfri kollektivtrafik för barn och