The Effect of Cicerfuran, an Arylbenzofuran from Cicer bijugum, and Related Benzofurans and Stilbenes on Leishmania aethiopica, L. tropica and L. major

• Abstract
• Introduction
• Materials and Methods
• Chemistry
• Leishmania
• Human monocytes
• Infected human monocytes
• Results and Discussion
• Acknowledgements
• References

Abstract

The effect of 3 arylbenzofurans and 7 stilbenes on the growth of Leishmania parasites and human monocytes was evaluated. Promastigotes from cultures of L. aethiopica, L. major and L. tropica were tested in the exponential phase of growth. All compounds were active at concentrations of 100 μg/mL within 6 hours. The 2-hydroxylstibene showed activity at a concentration < 1 μg/mL, with an LD₅₀ of 3 - 5 μg/mL after 48 hours of incubation. The most active compounds: cicerfuran, 2-hydroxy-2′-methyl-4′,5′-methylenedioxystilbene, 2-hydroxy-2′-methoxy-4′,5′-methylenedioxystilbene and 2-hydroxystilbene had even stronger activity against the temperature-induced amastigotes of L. aethiopica, with the latter having the highest relative potency against all three species. Leishmanicidal activity seemed to be associated with the level of oxygen substitution in each compound. The ratio between leishmanicidal activity on promastigotes and toxicity to human cells suggested that the compounds could be considered as leishmanicidal drug leads.

Introduction

Leishmaniasis affects 12 million people worldwide; 350 million are at risk and 1.5 - 2 million new cases of leishmaniasis have been estimated to occur annually [1]. Leishmaniasis comprises a group of diseases ranging from mild cutaneous lesions to fatal visceral infections. The clinical manifestation depends on the species initiating infection and also on the general health and genetic make-up of the infected individual [2]. Leishmaniasis is caused by many species/subspecies of the Leishmania protozoans, which vary in their virulence and are sensitive to different compounds [3], [4], [5].

Current pharmaceutical treatments include sodium stibugluconate and meglumine antimonite, pentamidine and amphotericin B. However, the toxicity, side effects, and expense of these drugs plus the development of resistance against them necessitate the identification and development of new therapies [6]. There has been much research on the therapeutic value of natural products, mainly plant extracts used to treat Leishmania in traditional medicine and several natural products have been tested against Leishmania [7]. Although a number of these natural products have demonstrated potential as leishmanicidal agents, most failed to meet all of the requirements needed (possibility of oral or topical administration, moderate dose effectiveness, lack of severe side effects), mainly because of their high cytotoxicity [8]. Consequently, new leishmaniasis therapies are urgently needed.

This paper describes the leishmanicidal and the cytotoxic activity of cicerfuran, two of its structural analogues and 7 stilbenes (Fig. [1]) against cutaneous Leishmania spp. and human cell lines. Cicerfuran is an antifungal defence compound produced by some wild species of chickpeas (Cicer sp.) that express resistance to fungal wilt (Fusarium oxysporum f. sp. Ciceri) [9]. It has been synthesized recently along with several benzofuran analogues and a related group of stilbene intermediates, all of which have antifungal and antibacterial activity [10].

Natural stilbenes and their synthetic analogues are known to have anti-inflammatory and antibacterial activity [11], as well as potential anticancer activities [12], [13] but their anti-parasitic activities have not been well studied. The only compounds tested in Leishmania to date are semisynthetic dihydrostilbenes, combretastatin and some heteroanalogues, in both of which groups activity has been reported [14], [15]. The antibacterial and antifungal activity of cicerfuran and related compounds, as well as the leishmanicidal activity of structurally related stilbenes suggested the possibility that these compounds might have activity against Leishmania. The aim of the present study was to determine the leishmanicidal activity of cicerfuran and related compounds on in vitro models including promastigote and amastigote stages of the parasite. Furthermore, the ability to interfere with the spread of the infection and to significantly reduce the infection ratio was evaluated. Infected macrophages were treated and the effect of the compounds on infection was established by comparing the infection rates with untreated cells.

Materials and Methods

Chemistry

Cicerfuran, 2-(2′-methoxy-4′,5′-methylenedioxyphenyl)-6-hydroxybenzofuran (3), was synthesised from sesamol (3,4-methylenedioxyphenol) and 2,4-dihydroxybenzaldehyde in ten steps via a Wittig reaction, epoxidation of the resulting stilbene and acid-catalysed cyclisation. The compound was isolated from the synthesis at > 99 % purity. The purity of the compounds was determined by high performance liquid chromatography with diode array detection (HPLC-DAD). The structure of the compound was confirmed by NMR, and its chromatographic and spectroscopic properties were identical to those of the natural product [16]. The other 9 related compounds tested were synthesised by palladium-catalysed coupling of styrenes with mono-oxygenated aryl halides, purified by HPLC and identified by NMR as 2-(3′,4′-methylenedioxyphenyl)benzofuran (1), 2-(2′-methyl-4′,5′-methylenedioxyphenyl)benzofuran (2), diphenylstilbene (4), 2-hydroxystilbene (5), 4-methoxystilbene (6), 2,4-dimethoxystilbene (7), 4-methoxy-3′,4′-methylenedioxystilbene (8) 2-hydroxy-2′-methyl-4′,5′-methylenedioxystilbene (9) and 2-hydroxy-2′-methoxy-4′,5′-methylenedioxystilbene (10) as described previously (Fig. [1]) [16].

Leishmania

The Leishmania strains were obtained from the London School of Hygiene and Tropical Medicine (London, UK) and from the Armauer Hensen Research Institute (Addis Abeba, Ethiopia).

Axenic amastigotes were obtained from promastigote cultures of L. aethiopica (MHOM/ET/72/L100). Cultures of log phase promastigotes were washed in PBS and adjusted to a concentration of 10⁶ organisms/mL in 7 mL of fresh modified JH30 medium at pH 5 [17] and incubated at 32 °C. Complete transformation into amastigotes was achieved within 10 days of incubation. The parasites were then maintained in JH30 medium at an adjusted pH of 5 for 5 months. Amastigote inhibition studies were performed on parasites which had been axenically reverted back to the promastigote stage 6 times.

Promastigote inhibition studies were performed on L. aethiopica (MHOM/ET/72/L100), L. major (MHOM/SU/73/5ASKH) and L. tropica (MHOM/SU/58/OD) grown at 22 °C in DMEM medium (SIGMA-Aldrich; Gillingham, UK), supplemented with 10 % foetal calf serum and glutamine.

Compounds were dissolved in dimethyl sulphoxide (DMSO; SIGMA-Aldrich; Gillingham, UK) 0.05 % w/v DMSO final concentration, to provide compound concentrations from 50 to 0.075 μg/mL. Parasites were plated in 96 well plates (Nunc; Lutterworth, UK) (1 × 10⁶ parasites/mL) in triplicate. Plates were incubated for 48 hours at 22 °C. Inhibition of promastigote growth was determined microscopically as previously described [18]. Amphotericin B was used as a reference drug in the statistical studies. Inhibition of amastigote growth was determined through Alamar blue (SEROTEC; Oxford, UK) assay [19], [20]. Briefly, 1 in 10 Alamar blue was added to each treated well. After one hour of incubation the plate was read at 570 nm excitation and 590 nm emission.

Human monocytes

THP-1 cells from the European Collection of Cell Cultures (ECACC; Salisbury, UK) were maintained in the same medium used for the parasites, at 37 °C and 5 % CO₂ in a humidified incubator. Cells were subcultured every second day at a starting concentration of 5 × 10⁵ cells/mL. Late log phase cells were incubated at a starting concentration of 10⁶ cell/mL in 96 well plates. Compounds were incorporated into triplicate cultures at a final DMSO concentration of 0.05 %. After 48 hours incubation at 37 °C, growth was estimated by counting viable cells, after trypan blue staining.

Infected human monocytes

THP-1 cells were transformed with retinoic acid (RA; SIGMA-Aldrich; Gillingham, UK) and infected with stationary phase promastigotes of L. aethiopica at a ratio of 10 : 1 as previously described [21]. Cicerfuran (3) and three intermediate stilbenes (5, 7 and 9) were then added at appropriate concentrations 48 hours after infection and the treated cells incubated for a further 24 hours.

The compound’s activity was determined from the percentage of infected cells and number of parasites per cell in treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. Each data set came from the average of three experiments each of which was done in triplicate. A minimum of 100 macrophages was counted for each slide.

Probit analyses with logit transformations of dose-response data [22], [23] were used to test differences between the activities of the compounds. The relative median potency as well as the lower and upper 95 % confidence limits (CL) were estimated assuming a parallel relation between the compound’s response curves. Since goodness-of-fit Chi square was significant a heterogeneity factor was used in the calculation of confidence limit values using the software package SPSS. The relative median potency (RMP) for L. aethiopica was estimated using amphotericin B as reference compound. The RMP for the other species was then estimated by comparison with the effect of the same compound in the most sensitive strain after probit analyses of each compound on the three different strains. The compounds were further tested for the overall differences in activity using the log rank test [24] and lethalities of each compound were determined.

Results and Discussion

The leishmanicidal effects on the promastigotes of different species of Leishmania were expressed as LD₅₀, which stands for lethal dose for 50 % of a given population, calculated from logit transformations of the viability values at the concentrations used. Stilbenes showed the highest activity with LD₅₀ values lower than 10 μg/mL. Stilbenes possessing hydroxy groups are already active after 6 hours at the second highest concentration tested (50 μg/mL) (results not shown). Cicerfuran is particularly active against L. tropica and it is generally more active than its analogues. The compounds showing potential as leishmanicidal agents were then tested for their toxicity in vitro towards cultured THP-1 cells and L. aethiopica infected THP-1 cells.

The leishmanicidal activities of ten compounds tested against promastigotes are shown as LD₅₀ in Table [1]. Eight out of ten compounds were active against L. aethiopica parasites at concentrations lower then 50 μg/mL. L. aethiopica was more sensitive to the action of the compounds than L. major and L. tropica, with the exception of 9. Although L. aethiopica was most sensitive to the compounds, similar results were obtained for L tropica and L. major. Six compounds were active against L. major (causing the death of at least 30 % of the population) while 5 were active against L. tropica. A stilbene (5) showed the highest relative potency against the promastigotes of all three species.

Four of the stilbenes (5, 7, 9 and 10) and one of the benzofurans (3), showed particularly potent activity against all three Leishmania species with LD₅₀ < 30 μg/mL and as low as 2.5 μg/mL (Table [1]). It is difficult to draw definitive conclusions about the structural features associated with the potency of these compounds although low activity was associated with an absence of O-substitutions. More specifically the presence of an O-substitution (either a hydroxy or a methoxy) at the C-2 (C-6 in benzofurans) appears to be associated with more potent activity since the five most active compounds were all O-substituted at this position. This is particularly well illustrated by 5, 6 and 7 in which the potently active 5 and 7 are both O-substituted at C-2 with a hydroxy and methoxy whereas 6, which showed relatively weak activity, lacks this C-2 group despite having the same C-4 methoxy as 7. Cicerfuran also contained a hydroxy group at the same equivalent position (C-6) and was more active than the other benzofurans and stilbenes which did not contain this O-substitution. However, it is not possible to conclude categorically that the activity of these compounds was simply dependent upon the presence or absence of O-substitutions at specific positions and further work on structure functions would be useful to improve efficacy and identify candidate drug leads.

The five most active compounds against all three Leishmania species were tested for their cytotoxicity on L. aethiopica amastigotes. The latter were cultured in an axenic condition (G. Getti et al., unpublished data), using a modification of the JH30 medium. Further characterisation of axenic amastigotes involved morphological study through light microscopy observations. Virulence increased with respect to the promastigote stage and ability to revert back to the promastigote form on temperature and pH shift to 22 °C and 7, respectively. With the exception of 5, five time revertant amastigotes showed an even greater sensitivity to the tested compounds than the promastigotes, with LD₅₀ values ranging from 1 to 10.8 μg/mL. Although a stilbene structure was again the most lethal against amastigote parasites, the highest activity was recorded in the presence of a methoxy group in position R¹ (10).

In order to investigate the ability of these compounds to be used as leishmaniasis drug leads, further tests were undertaken in the human cell line THP-1. Although showing cytotoxicity, the LD₅₀ values calculated for THP-1 cells by probit analysis were between 14.5 and 24.7 μg/mL: 2 - 3 times higher than the result for the promastigotes. Furthermore, infected macrophages were more sensitive to the action of the compounds than uninfected ones (Fig. [2]). Treatment caused significant decrease of the infection ratio with each compound used, with minimum decrease being from 88 % to 50 % for 5. The most active was 7 with which the percentage of infected macrophages diminished from 88 % to 18 % after 24 hours treatment with 1 μg/mL of the compound.

The present study demonstrated potent leishmanicidal activity of cicerfuran and 4 related stilbenes. The study on the effect of the compounds on THP-1 mammalian cells indicated that 3, 5, 7, 9 and 10 are differentially effective on Leishmania promastigotes and amastigotes (Table [1]) and, moreover, 3, 5, 7 and 9 significantly impaired the number of infected macrophages. Consequently, they are promising sources of new therapeutic agents and can be considered as possible leishmanicidal drug leads: the next step in the study of their pharmacological and antiprotozoal property should be in vivo assay.

Acknowledgements

The authors wish to thank Kevin Clough and Duncan Kennedy for technical support, Modupe Oriyomi Fatai-Oso for her contribution with the experiment on infected macrophages and Dr. Olivia Corcoran for critical reading of the manuscript. This work was supported by University of Greenwich HEFCE bursaries to GTMG and SNA

References

• 1 World Health Organisation division of control of tropical disease. Available at http://www.who.int/inf-fs/en/fact116.html. Accessed September 18, 2004. 
• 2 Grimaldi G Jr., Tesh R B. Leishmaniases of the New World: current concepts and implications for future research.  Clin Microbiol Rev. 1993;  6 230-50
  PubMedSearch in Google Scholar
• 3 Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P, Michel G. et al . In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis.  Antimicrob Agents Chemother. 1997;  41 827-30
  PubMedSearch in Google Scholar
• 4 Escobar P, Matu S, Marques C, Croft S L. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B.  Acta Trop. 2002;  81 151-7
  PubMedSearch in Google Scholar
• 5 Croft S L. Monitoring drug resistance in leishmaniasis.  Trop Med Int Health. 2001;  6 899-905
  CrossrefPubMedSearch in Google Scholar
• 6 Berman J. Current treatment approaches to leishmaniasis.  Curr Opin Infect Dis. 2003;  16 397-401
  PubMedSearch in Google Scholar
• 7 Chan-Bacab M J, Peña-Rodríguez L M. Plant natural products with leishmanicidal activity.  Nat Prod Rep. 2001;  18 674-88
  CrossrefPubMedSearch in Google Scholar
• 8 Tournaire C, Caujolle R, Payard M, Commenges G, Bessières M H, Bories C. et al . Synthesis and protozoocidal activities of quinones.  Eur J Med Chem. 1996;  31 507-11
  CrossrefPubMedSearch in Google Scholar
• 9 Stevenson P C, Veitch N C. A 2-arylbenzofuran from roots of Cicer bijugum associated with fusarium wilt resistance.  Phytochemistry. 1998;  48 947-51
  CrossrefPubMedSearch in Google Scholar
• 10 Aslam S N. Synthesis and biological evaluation of cicerfuran, an antifungal compound from chickpeas roots [dissertation]. Greenwich; University of Greenwich 2004
  Search in Google Scholar
• 11 Li X M, Lin M, Wang Y H. Stilbenoids from the lianas of Gnetum pendulum .  J Asian Nat Prod Res. 2003;  5 113-9
  CrossrefPubMedSearch in Google Scholar
• 12 Waffo-Teguo P, Hawthorne M E, Cuendet M, Merillon J M, Kinghorn A D, Pezzuto J M. et al . Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures.  Nutr Cancer. 2001;  40 173-9
  PubMedSearch in Google Scholar
• 13 Kinghorn A D, Su B N, Jang D S, Chang L C, Lee D, Gu J Q. et al . Natural inhibitors of carcinogenesis.  Planta Med. 2004;  70 691-705
  Thieme ConnectPubMedSearch in Google Scholar
• 14 del Olmo E, García Armas M, López-Pérez J L, Muñoz V, Deharo E, San Feliciano A. Leishmanicidal activity of some stilbenoids and related heterocyclic compounds.  Bioorganic Med Chem Lett. 2001;  11 2123-6
  PubMedSearch in Google Scholar
• 15 del Rey B, Ramos A C, Caballero E, Inchaustti A, Yaluff G, Medarde M. et al . Leishmanicidal activity of combretastatin analogues and heteroanalogues.  Bioorg Med Chem Lett. 1999;  9 2711-4
  CrossrefPubMedSearch in Google Scholar
• 16 Aslam S N, Stevenson P C, Phythian S J, Veitch N CV, Hall D R. Synthesis of cicerfuran, an antifungal hydroxylated benzofuran from chickpea roots and its analogues. Tetrahedron, in press
• 17 Pan A A. LeishmaniaLeishmania mexicana: serial cultivation of intracellular stages in a cell-free medium.  Exp Parasitol. 1984;  58 2-80
  PubMedSearch in Google Scholar
• 18 Escobar P, Matu S, Marques C, Croft S L. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B.  Acta Trop.. 2002;  81 51-7
  PubMedSearch in Google Scholar
• 19 Mikus J, Steverding D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue.  Parasitol Int. 2000;  48 265-9
  CrossrefPubMedSearch in Google Scholar
• 20 Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R. The Alamar blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro .  Acta Trop. 1997;  68 139-47
  CrossrefPubMedSearch in Google Scholar
• 21 Ogunkolade B W, Colomb-Valet I, Monjour L, Rhodes-Feuillette A, Abita J P, Frommel D. Interactions between the human monocytic leukaemia THP-1 cell line and Old and New World species of Leishmania .  Acta Trop.. 1990;  47 71-6
  PubMedSearch in Google Scholar
• 22 Finney D J. Probit Analysis. Cambridge; Cambridge University Press 1971
  Search in Google Scholar
• 23 Finney D J. Statistical method in biological assay. 3rd edition High Wycombe. Charles Griffin & Co. 1978: p 501
  Search in Google Scholar
• 24 Peto R, Pike M C, Armitage P, Breslow N E, Cox D R, Howard S V. et al . Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introduction and design.  Br J Cancer. 1976;  34 585-612
  PubMedSearch in Google Scholar

Giulia Getti

School of Health and Bioscience

University of East London

Stratford Campus

Romford Road

London E15 4LZ

UK

Phone: +44-208-223-4037

Email: g.getti@uel.ac.uk

References

• 1 World Health Organisation division of control of tropical disease. Available at http://www.who.int/inf-fs/en/fact116.html. Accessed September 18, 2004. 
• 2 Grimaldi G Jr., Tesh R B. Leishmaniases of the New World: current concepts and implications for future research.  Clin Microbiol Rev. 1993;  6 230-50
  PubMedSearch in Google Scholar
• 3 Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P, Michel G. et al . In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis.  Antimicrob Agents Chemother. 1997;  41 827-30
  PubMedSearch in Google Scholar
• 4 Escobar P, Matu S, Marques C, Croft S L. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B.  Acta Trop. 2002;  81 151-7
  PubMedSearch in Google Scholar
• 5 Croft S L. Monitoring drug resistance in leishmaniasis.  Trop Med Int Health. 2001;  6 899-905
  CrossrefPubMedSearch in Google Scholar
• 6 Berman J. Current treatment approaches to leishmaniasis.  Curr Opin Infect Dis. 2003;  16 397-401
  PubMedSearch in Google Scholar
• 7 Chan-Bacab M J, Peña-Rodríguez L M. Plant natural products with leishmanicidal activity.  Nat Prod Rep. 2001;  18 674-88
  CrossrefPubMedSearch in Google Scholar
• 8 Tournaire C, Caujolle R, Payard M, Commenges G, Bessières M H, Bories C. et al . Synthesis and protozoocidal activities of quinones.  Eur J Med Chem. 1996;  31 507-11
  CrossrefPubMedSearch in Google Scholar
• 9 Stevenson P C, Veitch N C. A 2-arylbenzofuran from roots of Cicer bijugum associated with fusarium wilt resistance.  Phytochemistry. 1998;  48 947-51
  CrossrefPubMedSearch in Google Scholar
• 10 Aslam S N. Synthesis and biological evaluation of cicerfuran, an antifungal compound from chickpeas roots [dissertation]. Greenwich; University of Greenwich 2004
  Search in Google Scholar
• 11 Li X M, Lin M, Wang Y H. Stilbenoids from the lianas of Gnetum pendulum .  J Asian Nat Prod Res. 2003;  5 113-9
  CrossrefPubMedSearch in Google Scholar
• 12 Waffo-Teguo P, Hawthorne M E, Cuendet M, Merillon J M, Kinghorn A D, Pezzuto J M. et al . Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures.  Nutr Cancer. 2001;  40 173-9
  PubMedSearch in Google Scholar
• 13 Kinghorn A D, Su B N, Jang D S, Chang L C, Lee D, Gu J Q. et al . Natural inhibitors of carcinogenesis.  Planta Med. 2004;  70 691-705
  Thieme ConnectPubMedSearch in Google Scholar
• 14 del Olmo E, García Armas M, López-Pérez J L, Muñoz V, Deharo E, San Feliciano A. Leishmanicidal activity of some stilbenoids and related heterocyclic compounds.  Bioorganic Med Chem Lett. 2001;  11 2123-6
  PubMedSearch in Google Scholar
• 15 del Rey B, Ramos A C, Caballero E, Inchaustti A, Yaluff G, Medarde M. et al . Leishmanicidal activity of combretastatin analogues and heteroanalogues.  Bioorg Med Chem Lett. 1999;  9 2711-4
  CrossrefPubMedSearch in Google Scholar
• 16 Aslam S N, Stevenson P C, Phythian S J, Veitch N CV, Hall D R. Synthesis of cicerfuran, an antifungal hydroxylated benzofuran from chickpea roots and its analogues. Tetrahedron, in press
• 17 Pan A A. LeishmaniaLeishmania mexicana: serial cultivation of intracellular stages in a cell-free medium.  Exp Parasitol. 1984;  58 2-80
  PubMedSearch in Google Scholar
• 18 Escobar P, Matu S, Marques C, Croft S L. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B.  Acta Trop.. 2002;  81 51-7
  PubMedSearch in Google Scholar
• 19 Mikus J, Steverding D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue.  Parasitol Int. 2000;  48 265-9
  CrossrefPubMedSearch in Google Scholar
• 20 Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R. The Alamar blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro .  Acta Trop. 1997;  68 139-47
  CrossrefPubMedSearch in Google Scholar
• 21 Ogunkolade B W, Colomb-Valet I, Monjour L, Rhodes-Feuillette A, Abita J P, Frommel D. Interactions between the human monocytic leukaemia THP-1 cell line and Old and New World species of Leishmania .  Acta Trop.. 1990;  47 71-6
  PubMedSearch in Google Scholar
• 22 Finney D J. Probit Analysis. Cambridge; Cambridge University Press 1971
  Search in Google Scholar
• 23 Finney D J. Statistical method in biological assay. 3rd edition High Wycombe. Charles Griffin & Co. 1978: p 501
  Search in Google Scholar
• 24 Peto R, Pike M C, Armitage P, Breslow N E, Cox D R, Howard S V. et al . Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introduction and design.  Br J Cancer. 1976;  34 585-612
  PubMedSearch in Google Scholar

Giulia Getti

School of Health and Bioscience

University of East London

Stratford Campus

Romford Road

London E15 4LZ

UK

Phone: +44-208-223-4037

Email: g.getti@uel.ac.uk

• Supplementary Material