JOURNAL OF EXPLORATORY RESEARCH IN PHARMACOLOGY

CONTENTS 2018 3(3):71–91

Original Articles Induction of Amodiaquine Metabolism by Rifampicin Following Concurrent Administration in Healthy Volunteers Adebusuyi Akande Ademisoye, Julius Olugbenga Soyinka, Samuel Olanrewaju Olawoye, Sharon Iyobor Igbinoba, Samuel Anu Olowookere, Adelola Taiwo Ademisoye, Cyprian Ogbona Onyeji 71 Evaluation of Diagnostic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bacteriuria Among Pregnant Women in Ashanti Region, Ghana Desmond O. Acheampong, Michael K. Afoakwah, Alex Boye, Richard Opoku, Godwin Kwakye-Nuako, Christian K. Adokoh, Samuel A. Baafi, Daniel Somuah 78

Review Article Luteolin: Anti-breast Cancer Effects and Mechanisms Ying Wang, Jing Wang, Xing Gong, Xinxuan Wen, Xinsheng Gu ...... 85

Letter to the Editor Re: Evaluation of Diagnostic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bacteriuria among Pregnant Women in Ashanti Region, Ghana Chenglong Wang, Jiaming Wang, Yingdan Gu, Danqing Pang, Dong Yin 91 Original Article

Induction of Amodiaquine Metabolism by Rifampicin Following Concurrent Administration in Healthy Volunteers

Adebusuyi Akande Ademisoye1, Julius Olugbenga Soyinka1*, Samuel Olanrewaju Olawoye1, Sharon Iyobor Igbinoba2, Samuel Anu Olowookere3, Adelola Taiwo Ademisoye1 and Cyprian Ogbona Onyeji1

1Department of Pharmaceutical Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria; 2Department of Clinical Pharmacy and Pharmacy Administration, Obafemi Awolowo University, Ile-Ife, Nigeria; 3Department of Community Health, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

Abstract

Background and objective: Malaria and tuberculosis remain endemic in tropical regions and most often coexist, increasing the burden of malaria mortality due to unintended interactions of the co-administered drugs employed in the management of the infections. Rifampicin (RIF) and amodiaquine (ADQ) are likely to be administered con- currently in the treatment of patients with tuberculosis and malaria. The metabolism of ADQ is mediated princi- pally by CYP2C8, while RIF is a known inducer of this enzyme. This study, therefore, investigated the effect of RIF on the disposition of ADQ, a major partner drug in the first-line treatment against uncomplicated malaria in line with the World Health Organization recommendations.

Methods: Sixteen healthy volunteers received oral 150 mg dose of RIF daily for 5 days with or without oral 600 mg single dose of ADQ on the fifth day (5th dose) with a 4-week wash out period, in a crossover fashion. Blood samples were collected at predetermined time intervals of 0, 1, 2, 4, 6, 8, 12, 24, 36 and 48 h. Plasma samples were analyzed for ADQ and its major metabolite desethylamodiaquine using a validated RP-high-performance liquid chromatography. Pharmacokinetic parameters were obtained using appropriate software and subjected to appropriate statistical analysis.

Results: Coadministration of ADQ and RIF resulted in significant decreases in the critical pharmacokinetic param- eters of ADQ, such as area under the curve (AUC0–∞) of about 66%, time to peak plasma concentration (Tmax) of about 10%, maximum plasma concentration of about 44%, and elimination half-life of about 55%, while the AUC0–∞ and Tmax of the main metabolite desethylamodiaquine increased about 2-fold and 3-fold respectively during the coadministration of RIF with ADQ. The metabolic ratio increased significantly, from 1.55 to 2.68. The AUC0–∞ and Tmax of the drug ADQ, as well as the maximum concentration of both the drug and its metabolite fell outside the point of estimates of the test/reference ratio of the geometric means of 80–125% of bioequivalence range.

Conclusions: An interaction was established during coadministration of RIF and ADQ, confirming RIF as a strong inducer of CYP2C8 in vivo, which may lead to malaria therapeutic failure or adverse drug reactions with ADQ Keywords: Amodiaquine; CYP2C8; Desethylamodiaquine; Induction; Rifampicin. and contribute to the rate at which resistance to ADQ Abbreviations: AUCT, area under plasma concentration-time curve; BE, bioequiva- lence; CL/F, oral clearance; Cmax, maximum concentration; CYP, cytochrome P450; develops. DEAQ, desethylamodiaquine; HPLC, high-performance liquid chromatography; RIF, rifampicin; T½, elimination half-life; T½b, terminal elimination half-life; TB, tuber- culosis; Tmax, time to reach maximum concentration. Received: November 21, 2017; Revised: March 23, 2018; Accepted: March 27, 2018 Introduction *Correspondence to: Julius O. Soyinka, Department of Pharmaceutical Chem- istry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. Tel: +2348035822785; E-mail: [email protected] Between 2010 and 2015, concerted efforts aimed at controlling ma- How to cite this article: Ademisoye AA, Soyinka JO, Olawoye SO, Igbinoba SI, Olowookere SA, Ademisoye AT, Onyeji CO. Induction of Amodiaquine Metabolism laria infection globally reduced the incidence of malaria by 21%, by Rifampicin Following Concurrent Administration in Healthy Volunteers. J Explor its mortality rates by 29% among at-risk populations and by 35% Res Pharmacol 2018;3(3):71–77. doi: 10.14218/JERP.2017.00024. among children under 5 years of age.1 In spite of these statistics,

Journal of Exploratory Research in Pharmacology 2018 vol. 3 | 71–77

Copyright: © 2018 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. J Explor Res Pharmacol Ademisoye AA. et al: Induction of amodiaquine by rifampicin malaria remains one of the most serious health problems world- capsules of 150–300 mg, and the recommended dose is 10 mg/kg/ wide.2 Plasmodium falciparum, which causes the most severe form day; the dose should not exceed 600 mg/day. The drug is usually of malaria, is predominant in Africa; and, in 2015, about 212 million given in combination with other anti TB drugs, such as isoniazid. cases of malaria occurred worldwide, mostly in Africans (90%).3 In- Concurrent infections with malaria and TB may occasion the need cidentally, in regions with high incidence of malaria infections, such for ADQ and RIF coadministration,7,9 and such practice may give as sub-Saharan Africa, malaria may coexist with other infections.4 rise to clinically important drug-drug interactions that may compro- Tuberculosis (TB), an infection caused by Mycobacterium tu- mise the therapeutic outcome. RIF and lopinavir/ritonavir,21,22 in berculosis, is one of the top 10 leading tropical diseases that re- separate studies, enhanced the therapeutic activity of ADQ in mice quire long-term therapies. TB and malaria are the most prevalent infected with chloroquine-resistant Plasmodium berghei. bacterial and parasitic infections that coexist frequently in hu- It has been reported that withdrawal of RIF from the medication mans.5 This can potentially contribute to morbidity and mortality regimen of a TB patient on atenolol, amlodipine and ramipril (ACE in impoverished populations in the tropics.6 Moreover, concurrent inhibitor) in the management of essential hypertension brought the infections involving TB and malaria have been reported to modify fatal consequence of hypertension.23 Since ADQ is mainly metabo- immune response in the coinfection situation and to increase anti- lized by CYP2C8 and activity of this isozyme has been demon- bacterial intolerance,7 which may be detrimental to the prognosis strated to be modulated by RIF,21 theoretically, RIF is expected to of TB.6 In addition, malaria infection can result in rapid increase induce the metabolism of ADQ. The extent of interaction between in parasite burden if there is a delay in or application of ineffective RIF and ADQ when used concurrently in man is not known. Since 8 treatment, with reported case fatality rates of 10% to 20%. effective malaria treatment is important in TB patients and RIF may Therefore, a treatment goal for TB patients with malaria will be reduce the overall exposure to ADQ, this study set out to determine the prevention or effective treatment of malaria in TB patients in how RIF influences the disposition of ADQ in man. order to reduce overall morbidity and mortality.6,9–11 For instance, induction of CYP3A4-mediated quinine metabolism in a patient infected with the human immunodeficiency virus by the antiretro- Methods viral drug nevirapine worsened malaria symptoms, and the clinical deterioration was only abated when quinine was substituted with proguanil/atovaquone, a non-CYP3A4 substrate.12 Concurrent sul- Materials phamethoxazole and trimethoprim combination with amodiaquine (ADQ) has been found to increase pharmacokinetic parameters of Amodiaquine hydrochloride dihydrate (Sigma, St. Louis, MO, ADQ, due to inhibition of its metabolism when concurrently ad- USA). Desethylamodiaquine (TLC PharmaChem, Ontario, ministered.13 Canada). Hydroxychloroquine sulphate (AK Scientific Inc., San ADQ is a frontline antimalarial recommended by the World Francisco, CA, USA). High-performance liquid chromatography Health Organization for artemisinin derivative-based combination (HPLC)-grade methanol and triethylamine (Scharlau Chemie, therapy.14 It is available in tablets of 150–600 mg, and the recom- Barcelona, Spain). Analytical grade diethyl ether (Lobal Chemie, mended dose is 10 mg/kg of ADQ base once daily for 3 days, usual- Mumbai, India). Orthophosphoric acid (Aldrich, Hamburg, Ger- ly in combination with other antimalarial, such as artesunate. ADQ many). Amodiaquine tablets (Camoquine®) (Parke-Davis, Pfizer, is almost entirely metabolized by the polymorphic cytochrome Detroit, MI, USA). Rifampicin (Rifabon) (Bond Pharmaceuticals, P450 (CYP) isoform 2C8 to the pharmacologically active desethyl- Nigeria). Hydrochloric acid (Sigma-Aldrich, UK). amodiaquine (DEAQ).15 Expression of each CYP is influenced by a unique combination of mechanisms and factors, including genetic polymorphisms, induction by xenobiotics, regulation by cytokines Instrumentation hormones and during disease states, as well as sex, age and others.16 Multiallelic genetic polymorphisms, which strongly depend on The HPLC system (Agilent 1200 series; Agilent Technologies, ethnicity, play a major role in the function of CYPs 2D6, 2C8, 2C9 Frankfurt, Germany) consisted of a binary pump with a degas- and 2B6, and lead to distinct pharmacogenetic phenotypes, termed ser and isocratic gradient mixer, reverse-phase C18 column (150 as poor, intermediate, extensive and ultra-rapid metabolizers.17 For mm × 4.6 mm i.d. × 5 µm) (Agilent Eclipse Plus), rheodyne valve these CYPs, the evidence for clinical significance regarding adverse injector loop (20 µL), variable wavelength ultraviolet detector, drug reactions, drug efficacy and dose requirement is rapidly grow- Chromjet data integrator and Hewlett-Packard Chemstation re- ing. Knowledge of the intrinsic and extrinsic factors that influence corder. Other equipment included a vortex mixer (XHC, Nanjing, expression and function of the responsible enzymes is, thus, a pre- China), centrifuge (800-CE; Jiangsu, China), vacuum filter (Su- requisite for predicting variable pharmacokinetics and drug response. pelco, Bellefonte, PA, USA), pH meter (Hanna Instruments, Wilm- The human CYP2C subfamily consists of the four highly ho- ington, NC, USA), and micropipettes (Microlax, Milan, Italy). mologous genes, CYP2C18-CYP2C19-CYP2C9-CYP2C8, which are localized in this order (from centromere to telomere) in a 390 kb gene cluster on chromosome 10q23.3.18 Although CYP2C18 Ethical clearance mRNA is highly expressed in liver, the transcript is not efficiently translated into protein and does not make significant contributions Ethical clearance for the study was obtained from the Health Re- to drug metabolism.18 CYP2C9 is the highest expressed member, search Ethics Committee of the Institute of Public Health, Obafemi being expressed at similar or even higher protein levels than CY- Awolowo University, Ile-Ife, Nigeria (Reference Number: IPH/ P3A4, while CYP2C8 and CYP2C19 are expressed at about 2-fold OAU/12/539; 15th March, 2016). and 10-fold lower levels.18 Similarly, rifampicin (RIF) is an important drug in the treatment of TB and is used extensively despite its broad effects on drug-drug Study population interactions arising from its activities on most CYP and/or P-glyco- proteins in the gastrointestinal tract and liver.19,20 It is available in Healthy nonsmoking men and women who were within 20%

72 DOI: 10.14218/JERP.2017.00024 | Volume 3 Issue 3, August 2018 Ademisoye AA. et al: Induction of amodiaquine by rifampicin J Explor Res Pharmacol of their ideal bodyweight for height and sex (Metropolitan Life to give concentration ranges of 100–1,000 ng/mL for both ADQ Scale) were deemed eligible to participate in the study. Subjects and DEAQ. The samples were taken through the extraction proce- were excluded if they met one of the following criteria: pregnancy; dure described above. The mobile phase, consisting of 2 % TEA breastfeeding; history of hypersensitivity reactions to RIF, ADQ or buffer solution (pH 2.2): methanol (80:20), was prepared and similar agents (mefloquine, chloroquine, quinidine, quinine); se- pumped through the column at 1.0 mL/min. The column was set rum creatinine >1.5 times the upper limit of normal; liver function at 30 °C, while the column effluent was monitored with a variable test >3 times the upper limit of normal; or evidence by history or wavelength ultraviolet detector set at 340 nm. physical examination of gastrointestinal, psychiatric, cardiovascu- lar or neurological disorder. Electrocardiogram findings were also Data and statistical analyses recorded. Nine men and seven women, who had all been certified healthy by a doctor in the team, were recruited as well, but only fourteen volunteers completed the study. The mean ± standard The peak plasma concentrations (Cmax) and the time to reach peak deviation age and weight of the subjects was 24.75 ± 2.8 years concentration (Tmax) were noted directly from the concentration- (range: 18–43) and 57.88 ± 4.5 kg (range: 40–80) respectively. time profiles. Other pharmacokinetic parameters, such as terminal None of them had received any other drugs for at least 1 month elimination half-life (T½b) and oral clearance (CL/F) were cal- before the study. All subjects gave written informed consent before culated from individual plasma concentration-time profiles, using participation in the study. standard noncompartmental methods.25 For example, the total area under the plasma concentration-time curve (AUCT) was deter- mined using the linear trapezoidal rule to the last datum and ex- Study design and drug administration trapolation to infinity. The area from the last datum points (Ct) to infinity was obtained as Ct/b. The elimination rate constant, b, was The study was an open-label, randomized, two-period crossover calculated by linear regression analysis of the terminal phase of the pharmacokinetic study. The subjects were randomized into two log concentration-time profile. T½b was calculated from 0.693/b groups (Groups A and B). After an overnight fast, Group A sub- and CL/F was determined from dose/AUCT. Pharmacokinetic jects received a single oral 600 mg dose of ADQ, while Group B calculations were carried out using the pharmacokinetic program received oral 150 mg dose of RIF daily for 5 days and 600 mg dose WinNonlin (standard edition, version 1.5; Scientific Consultant of ADQ with the fifth RIF dose. After allowing 4-weeks wash-out Inc., Apex, NC, USA). In the model option for the noncompart- period, Group A subjects received the same drug treatment that had mental analysis, the linear trapezoidal rule was used for calculation been given earlier to group B, and vice versa. No other drugs or of the AUC. The Wilcoxon matched pairs signed-ranked test was alcohol were permitted during the study. used to evaluate the difference between pairs of data; a p-value below 0.05 was considered significant. Sample collection Results Before drug administration, venous blood samples (5 mL) were collected from all subjects for determination of serum chemistry and hematological screening. Thereafter, blood samples (5 mL) The retention times of internal standard, DEAQ and ADQ were were drawn by venipuncture into heparinized tubes from the fore- 3.5, 5.2 and 6.4 m, respectively. The coefficients of variation for arm of each subject before ADQ administration and at 1, 2, 4, 6, 8, both the intraday and interday analyses at 150, 300 and 900 ng/ 12, 24, 36 and 48 h afterwards. The samples were centrifuged im- mL ranged from 3.10–6.86% for ADQ, and from 4.68–8.98% for mediately (3,000 ×g for 15 m) to separate the plasma. The plasma DEAQ. The mean extraction recoveries of ADQ and DEAQ were aliquots were stored at −20 °C until analysis. 80.0% and 78.9%, respectively, at concentrations at the lower and upper limits of their calibration curves. The mean ADQ and DEAQ plasma concentrations versus time Drug analysis profiles in the 14 healthy subjects who completed the study, fol- lowing oral administration of single doses of ADQ (600 mg) alone Plasma samples were analyzed for ADQ and DEAQ using the and coadministration of ADQ with RIF (150 mg daily for 5 days) HPLC method reported by Adedeji et al.24 Sample extraction in- are illustrated in Figures 1 and 2, respectively. Tables 1 and 2 sum- volved precipitation of the plasma proteins, followed by extraction marize the pharmacokinetic parameters (mean ± standard devia- as follows: in a 15 mL extraction tube, 1 mL of plasma, 1 mL of tion) for ADQ and DEAQ following a single oral administration acetonitrile and 2 µL of 50 µg/mL of hydroxychloroquine (internal of 600 mg dose of ADQ to each of the subjects before and after standard) were added and centrifuged for 20 min at 3,000 ×g to concurrent administration of RIF (150 mg daily for 5 days), re- precipitate the plasma protein. The supernatant was then separated spectively. The demographic features of the study participants are into clean and dry extraction tubes, to which 2 mL of diethyl ether presented in Table 3. was added. The content was vortexed for 2 m and centrifuged for The Cmax of ADQ when administered separately was 47.43 ± 20 m at 3,000 ×g. The organic layer was transferred into taper-end 6.20 ng/mL, but decreased significantly to 26.49 ± 4.39 ng/mL (p tubes. The exercise was repeated with another 2 mL diethyl ether < 0.05) when co-administered with RIF, resulting in about a 44% and the combined organic layers in the tubes were evaporated un- decrease, whereas the Cmax of the metabolite was 54.42 ± 3.56 der stream of warm air. The residue was reconstituted with 1 mL and 59.94 ± 3.8 ng/mL for ADQ and its concurrent administration of 0.1M hydrochloric acid. with RIF, respectively, and was comparable to the baseline (p > A 20 µL aliquot was injected onto the HPLC column by using 0.05). The Tmax was 3.43 ± 0.98 and 2.75 ± 0.97 h (p > 0.05) for an autosampler. The calibration procedures were as reported.24 A ADQ alone and coadministration with RIF, respectively, but there calibration curve, based on peak area ratio, was prepared by spik- was a 3-fold increase in the Tmax of the metabolite after single 600 ing drug-free plasma with standard solutions of ADQ and DEAQ mg dose of ADQ and its coadministration with RIF (150 mg daily

DOI: 10.14218/JERP.2017.00024 | Volume 3 Issue 3, August 2018 73 J Explor Res Pharmacol Ademisoye AA. et al: Induction of amodiaquine by rifampicin

Fig. 1. Mean ADQ plasma concentrations versus time profiles in 14 Fig. 2. Mean DEAQ concentrations versus time profile in 14 healthy -sub healthy subjects following oral administration of single dose of ADQ at jects following oral administration of single dose of ADQ at 600 mg alone 600 mg alone and coadministration of ADQ with RIF at 150 mg daily for 5 and coadministration of ADQ with RIF at 150 mg daily for 5 days. Abbre- days. Abbreviations: ADQ, amodiaquine; RIF, rifampicin. viations: ADQ, amodiaquine; DEAQ, desethylamodiaquine; RIF, rifampicin. for 5 days), specifically 2.01 ± 0.05 and 6.01 ± 1.2). The Cmax of 205.4 L/h to 3,183 ± 588.48 L/h (p < 0.05). The oral clearance of both the drug and its metabolite fell outside the bioequivalence the metabolite decreased by about 44%, from 778.18 ± 172.0 L/h range of 80–125%, being −58.2 (49.2, 68.1) and 9.7 (6.1, 13.6), to 436.78 ± 125.17 L/h (p < 0.05). respectively. The metabolic ratio, which is the AUC(metabolite)/AUC(drug), The Tmax of the drug also fell outside the range, being 19.1 that is, the ratio of AUC of the metabolite to that of the unchanged (9.2, 30.4), whereas the Tmax of the metabolite was bioequivalent. drug, and is a known measure of extent of metabolism of a drug, There was an almost 66% fall in ADQ exposure, as depicted by was found to increase significantly, from 1.55 ± 0.53 to 2.68 ± 1.85 the AUC0–∞ after single dose of ADQ, which was 572.53 ± 121.96 before and after RIF coadministration, respectively (p < 0.05). ng/L*h and 196.42 ± 77.39 ng/L*h (p < 0.05) when it was coad- ministered with RIF, with corresponding increase by about 90% of the AUC0–∞ of the metabolite DEAQ (795.53 ± 193.84 vs. 1,510 Discussion ± 942.60, p < 0.05) during single oral dose of ADQ and concur- rent administration with RIF. Only the AUC of the metabolite fell outside the 90% confidence interval range of 64.1 (43.6, 77.2), This study was designed to evaluate the plausible interaction be- whereas the AUC for the drug was bioequivalent, being 107 (94.1, tween ADQ and RIF. Only 2 out of the 16 recruited volunteers, 120.5). a male and a female, withdrew from the study after complaining There was a 52% decrease in the mean residence time of ADQ about dizziness and pain at the site of blood collection, so that a when taken alone and when RIF was coadministered, with results total of 14 volunteers completed the study. Their withdrawal, how- being 17.92 ± 10.16 h and 8.52 ± 5.98 h, respectively (p < 0.05). ever, did not in any way affect the conduct, the goal nor the find- There was equally a 55% decrease in the elimination half-life (T1/2) ings of the study. of ADQ when it was administered alone and when coadministered There is a constant stream of new information about drug in- with RIF, being 12.35 ± 7.27 h and 5.5 ± 5.85 h, respectively, with teractions in patients with malarial infection, who may also need a corresponding significant elevation of its CL/F, from 1,087 ± to take antimalarial drugs together with drugs for other ailments.

Table 1. Derived pharmacokinetic parameters of ADQ following oral administration of single 600 mg dose of the drug to each of 14 volunteers and when coadministered with multiple doses of RIF at 150 mg daily for 5 days PK parameters ADQ alone ADQ+RIF Statistics with 90% CI Cmax, ng/mL 47.43 ± 6.20 26.49 ± 4.40 p < 0.05 −58.2 (49.2, 68.1)* Tmax, h 3.43 ± 0.98 2.75 ± 0.97 p > 0.05 19.1 (9.2, 30.4)*

AUC0–∞, ng/mL*h 575.53 ± 121.96 196.42 ± 77.39 p < 0.05 107 (94.1, 120.5) T1/2, h 12.35 ± 7.27 5.60 ± 5.85 p < 0.05 MRT, h 17.92 ± 10.16 8.52 ± 5.98 p < 0.05 CL/F, L/h 1,087 ± 205.4 3,183 ± 588.48 p < 0.05 Vd/f, L 73.04 ± 27.96 473.7 ± 177.28 p < 0.05

*Values within the 80–125% bioequivalence range. Abbreviations: ADQ, amodiaquine; CI, confidence interval; CL/F, oral clearance; MRT, mean residence time; PK, pharmacoki- netic; RIF, rifampicin.

74 DOI: 10.14218/JERP.2017.00024 | Volume 3 Issue 3, August 2018 Ademisoye AA. et al: Induction of amodiaquine by rifampicin J Explor Res Pharmacol

Table 2. Derived pharmacokinetic parameters of desethylamodiaquine following oral administration of single 600 mg dose of ADQ to each of 14 volun- teers and when coadministered with multiple doses of RIF at 150 mg daily for 5 days PK parameters ADQ alone ADQ+RIF Statistics with 90% CI Cmax, ng/mL 54.42 ± 3.56 59.94 ± 2.38 p > 0.05 9.7 (6.1, 13.6)* Tmax, h 2.01 ± 0.05 6.01 ± 1.20 p < 0.05 111.0 (99, 123.1)

AUC0–∞, ng/mL*h 795.53 ± 193.84 1,510.63 ± 942.60 p < 0.05 64.1 (43.6, 77.2)* Metabolic ratio 1.55 ± 0.53 2.68 ± 1.85 p < 0.05 MRT, h 6.30 ± 1.12 12.55 ± 1.03 p < 0.05 CL/F, L/h 778.18 ± 172.0 436.78 ± 125.17 p < 0.05 Vd/f, L 192.11 43.47 ± 12.42 p < 0.05

*Values within the 80–125% bioequivalence range. Abbreviations: ADQ, amodiaquine; CI, confidence interval; CL/F, oral clearance; MRT, mean residence time; PK, pharmacoki- netic; RIF, rifampicin.

RIF has been shown to have the potential to interact with a range narrow therapeutic window, the significant elevation of the Cmax of other drugs,20,21,23 and healthcare providers need to be aware of may explain some of the observed adverse reactions, such as the basis of these interactions and the potential impact on therapy. agranulocytosis and hepatitis.27 Clinical situations that require antibiotic therapy are prevalent in Drug toxicities must be acceptable and cause less harm than the many parts of the world, including sub-Saharan Africa, where ma- malaria itself, in order to encourage compliance and reduce mor- 26 28 laria is endemic. Moreover, the comorbidities increase the poten- tality. The AUC0–α after single dose of ADQ alone was 572.53 ± tial for drug interactions during poly therapeutics. 121.9644 ng/L*h, while the value of 196.42 ± 77.39 ng/L*h (66% Information on interactions between antimalarial and antibiot- decrease in exposure to ADQ) was obtained when it was coadmin- ics are limited. Combining antibiotic medications with an antima- istered with RIF. Also, the AUC0–∞ values of DEAQ were 795.53 larial can result in treatment failure when plasma concentrations ± 193.84 and 1,510.63 ± 942.60 ng/mL*h (representing 90% in- fall below therapeutic levels or toxicity resulting from increased crease in values) for ADQ and pretreatment with RIF. The appar- plasma drug levels. Our study evaluated the possible interaction ent CL/F for ADQ was 1,087 ± 205.4 L/h and 3,183 ± 588.48 L/h between ADQ and RIF, when coadministered in healthy human (about 200% increase), while the DEAQ clearance was 778.18 ± subjects. The results from the present study indicated that ADQ 172 L/h and 436.78 ± 125.17 L/h (about 44% decrease). The ap- was rapidly absorbed after oral administration in all the subjects. parent fall in the exposure to ADQ could be a result of significant The Tmaxs of ADQ were 2.0 ± 0.05 and 6.01 ± 1.2 h for single oral clearance, amounting to about 200% when ADQ was coad- dose ADQ and coadministration with RIF, respectively, which ministered with RIF. revealed enhanced exposure of the drug to the body, with cor- RIF is a major player in induction of many enzymes, including responding values for the metabolite DEAQ of 3.43 ± 0.98 versus CYP2C8,20,21 a notable enzyme in the metabolism of ADQ,14,22 2.75 ± 0.97, respectively. which may have clinical significance during antimalarial therapy. While the Tmax for the metabolite did not translate to clinical Therefore, the observed decrease in plasma ADQ exposure found relevance, as assessed by 90% confidence intervals for its mean in this study, seen as significant decreases in Cmax, T½ and AUCT ratios that are bioequivalent, the Tmax of the unchanged drug had of the drug following concurrent administration of RIF, is most value of 19.1 (9.2, 30.4) that are not within the range of 80–125%. probably attributable to induction of CYP2C8 by RIF, the enzyme The point estimates of the test/reference ratio of the geometric that catalyzes formation of DEAQ. This assertion is supported by means for the Cmax of the unchanged drug and its metabolite were the observed corresponding increase in the Cmax and AUC of the 58.2 (49.2, 68.1) and 9.7 (6.1, 13.6), respectively, falling outside metabolite. the bioequivalence range. Although ADQ is not known to have a The stimulatory effects on the metabolism of ADQ can allow the body to detoxify as the plasma concentration falls below thera- Table 3. Demographic features of the study participants peutic levels, conferring advantage of the enhanced drug’s action Demographic features through increased activity of the metabolite DEAQ. For instance, in a 3-day dosage, blood concentrations higher than breakpoints of Participants, total 16 135 ng/mL was associated with treatment success in the manage- Participants, completed the study 14 ment of uncomplicated Plasmodium falciparum.29 In addition, the extra-hepatic metabolism by CYP1A1 and 1B1 reported by some Sex, female/male 6/8 researchers may constitute less significance in the overall metabo- 30 a lism of ADQ, which may have contributed to the fall. Mean ± SD The 90% confidence intervals computed for the ratios of the Age, years 24.75 ± 2.80 geometric means for ADQ and the metabolite clearly showed that there was interaction for the point of estimates for AUC0–∞ of the Weight, kg 57.88 ± 4.50 metabolite which fell outside the 80–125% range for ADQ and Height, m 1.57 ± 0.12 coadministration with RIF. AUC is known to be directly propor- tional to dose and may be disproportional if there are saturable BMI, kg/m2 24.80 ± 4.20 pathways for some routes of elimination. The enhanced AUC of aMean ± the standard deviation of demographic features. Abbreviations: BMI, body the metabolite may increase as dosage increases and will worsen if mass index; SD, standard deviation. coadministered with inducers such as RIF, due to formation of the

DOI: 10.14218/JERP.2017.00024 | Volume 3 Issue 3, August 2018 75 J Explor Res Pharmacol Ademisoye AA. et al: Induction of amodiaquine by rifampicin toxic metabolite amodiaquine quinoneimine, known for causing Conclusions adverse effects of ADQ.31,32 The mean residence time of ADQ also showed a significant de- This study evaluated, for the first time, the effect of RIF- coad crease in values by about 52% when taken alone and when RIF was ministration on the pharmacokinetics of ADQ in healthy adult vol- coadministered with it (17.92 ± 10.16 vs. 8.52 ± 5.98 h). Similarly, unteers. The study demonstrated that concurrent administration of the T1/2 for ADQ showed a significant decrease in the presence of RIF, a known inducer of CYP2C8, with ADQ, a substrate of the RIF, resulting in more than 50% shortening of the half-life (12.35 isoenzyme, results in a significant reduction in the plasma levels of ± 7.27 h vs. 5.5 ± 5.85 h) with wide interindividual variation, as the antimalarial drug. Plasma levels of DEAQ, the major metabo- observed in the deviations from the mean values. It is known that lite of ADQ, are elevated in the presence of RIF. 33 ADQ exhibits interindividual variations in its pharmacokinetics. Adjustment of the ADQ dose may be necessary when the drug This is especially true because genetic polymorphisms can charac- is coadministered with RIF, but this should be balanced against the terize CYP2C8 in the metabolism of drugs and provide basis for potential increase in toxicity that may be associated with elevated pharmacokinetic variations in individuals as well as responses to plasma levels of the metabolite. There is a need to further deter- drug actions.34 mine the clinical implications for the extensive formation of the There was occurrence of a pronounced increase in the metabolic metabolite in this interaction, since DEAQ has potential for further ratio. This parameter, AUC (metabolite)/AUC (drug), the ratio of metabolism to toxic metabolites when there is elevated plasma AUC of the metabolite to that of the unchanged drug, is a known level, and this may be associated with increased toxicity. measure of the extent of metabolism of a drug. This was found to increase from 1.55 ± 0.53 to 2.68 ± 1.85 in the absence and pres- ence of RIF, respectively. The marked increase in the metabolic Acknowledgments ratio of ADQ further strengthens the point that a metabolic interac- tion occurs between ADQ and RIF, and the hypothesis that RIF The authors appreciate the support of Sam Ace Pharmaceuti- induces the metabolism of ADQ. cal Company Nigeria Limited, for providing the HPLC facility Since antimalarial activity is partly dependent on the metabolite used in the analysis of drugs in the plasma samples of this study. DEAQ, it is evident that ADQ had been rapidly metabolized to its The authors are also sincerely grateful to the commitment of the metabolite and this can enhance the synergistic activity of ADQ subjects who participated in the study and the assistance of the and DEAQ. On the other hand, with the increased levels of the me- technical staff of the Department of Pharmaceutical Chemistry, tabolite, there is a higher possibility of the formation of the toxic Obafemi Awolowo University, Nigeria, in making some materials ADQ metabolite quinoneimine. This toxic metabolite is a product and equipment available during the research study. This work was of conversion from DEAQ by enzymes other than CYP2C8 and is conducted using personal funds of the researchers, and no external reported to be responsible for some of the reported adverse drug funding or grant was received. effects of ADQ.31,32 The question remains whether the efficacy of ADQ is affected by a gene polymorphism. Genotype-inferred low metabolizers are Conflict of interest found in 1–4% of African populations, which corresponds to mil- lions of expected exposures to the drug.35 It is necessary to in- vestigate the possible relevance, or lack of relevance, of CYP2C8 The authors have no conflict of interests related to this publication. polymorphisms in the present and future efficacy of ADQ. To date, several nucleotide sequence variations have been identified Author contributions in CYP2C8. CYP2C8*3 (c.416G > A, p.Arg139Lys and c.1196A > G, p.Lys399Arg) is the most frequent variant allele changing the amino acid sequence of CYP2C8 in the Caucasian population, Designing the research (JOS, COO, AAA, SII), recruiting volun- with an allele frequency of about 10–20%.36 On the other hand, the teers (JOS, AAA, ATA, SOO, SII, SAO), certifying medical fitness most common amino acid changing polymorphism in black popu- of volunteers (SAO), analyzing samples (JOS, AAA, ATA, SOO, lations is CYP2C8*2 (c.805A > T, p.Ile269Phe), but it is very rare SII), preparing the manuscript (JOS, AAA, ATA, SOO, SII, SAO, in Caucasians.36 In vitro studies have suggested that CYP2C8*3 COO). has reduced activity for metabolizing paclitaxel and ADQ,37,38 but amiodarone N-demethylation was not affected by this polymor- phism.39 It is therefore necessary to evaluate the in vivo effect of References CYP2C8*3 on the pharmacokinetics of ADQ. [1] World Health Organization WHO. Available from: http://www.who. int/mediacentre/factsheets/fs094/en/ accessed 8/04/17. Accessed Future research directions 2015. [2] World Health Organization WHO. Available from: http://www.who.int/ mediacentre/factsheets/fs104/en/ accessed 8/4/17. Accessed 2017. The clinical significance of this study would have been enhanced [3] World Health Organization (WHO). World Malaria Report. Geneve, by also investigating the effect of ADQ coadministration on the Switzerland. 2016. disposition kinetics of RIF, and by carrying out the bidirectional [4] Gwer S, Newton CRJC, Berkley JA. Over-diagnosis and co-morbidity pharmacokinetic interaction studies on patients with malaria. This of severe malaria in African children: A guide for clinicians. Am J Trop Med Hyg 2007;77(Suppl 6):6–13. is because there is a possibility of changes occurring in the phar- [5] Etiaba E, Onwujekwe O, Uzochukwu B, Uguru N, Okoronkwo I, Ad- macokinetic profiles of these drugs due to malaria infection. More jagba A. What co-morbidities do people with malaria have and what studies are needed to further evaluate the impact of this interaction are their patterns of health seeking in Nigeria? Niger J Clin Pract and of CYP2C8 genetic polymorphisms on the clinical outcomes 2015;18(1):22–26. doi:10.4103/1119-3077.146974. of treatment. [6] Scott JA, Berkley JA, Mwangi I, Ochola L, Uyoga S, Macharia A, et

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DOI: 10.14218/JERP.2017.00024 | Volume 3 Issue 3, August 2018 77 Original Article

Evaluation of Diagnostic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bacteriuria Among Pregnant Women in Ashanti Region, Ghana

Desmond O. Acheampong1*, Michael K. Afoakwah1, Alex Boye2, Richard Opoku1, Godwin Kwakye-Nuako1, Christian K. Adokoh1, Samuel A. Baafi3 and Daniel Somuah4

1Department of Biomedical Sciences, University of Cape Coast, Cape Coast, Ghana; 2Department of Medical Laboratory Technology, University of Cape Coast, Cape Coast, Ghana; 3Ellolab Diagnostic Centre, Kumasi, Ghana; 4Anglogold Ashanti Health Foundation Hospital, Obuasi, Ghana

Abstract

Background and objective: Asymptomatic bacteriuria (ASB) poses a serious health problem to pregnant women and fetuses. However, in most developing countries, routine screening for ASB and antimicrobial sensitivity test are rarely performed. This study, therefore, aimed to determine the best diagnostic method for routine screen- ing of ASB and antimicrobial susceptibility pattern among pregnant women attending an antenatal clinic in the Ashanti Region of Ghana.

Methods: Urine samples from 412 pregnant women between the ages of 16 and 45 years-old attending antenatal clinic at Anglogold Ashanti Health Foundation Hospital and Ellolab Diagnostic Centre were screened for ASB by microscopy, dipstick urinalysis and bacteria culture. Susceptibility of the positive isolates were assessed against commonly used antimicrobial agents, adopting the disc diffusion test method.

Results: Of the 412 pregnant women screened, 72 tested positive for ASB by the urine culture method, which translates into an overall prevalence of 17.5%. There was no association between age, marital status, occupation, parity, educational background nor duration of pregnancy with ASB (p > 0.05). Additionally, dipstick urinalysis was found to be a better diagnostic method than microscopy. The most isolated bacteria wereEscherichia coli (62.5%) and Klebsiella pneumoniae (30.6%), and nitrofurantoin and nalidixic acid were the most effective antimicrobial agents.

Conclusions: Routine urine culture and antimicrobial susceptibility test should be carried out on all pregnant women attending antenatal clinic to detect possible ASB and prescribe appropriate drugs, such as nitrofurantoin and nalidixic acid, to prevent any related complications. However, in health centers that lack bacterial culture facilities, dipstick urinalysis should be the preferred screening option.

Introduction

Keywords: Asymptomatic; Bacteriuria; Antimicrobial agent; Diagnostic methods. Abbreviations: ASB, asymptomatic bacteriuria; UTI, urinary tract infection; PPV, Pregnancy is commonly associated with urinary tract infection positive predictive value; NPV, negative predictive value. (UTI), and has been attributed to hormonal and physiological Received: January 19, 2018; Revised: April 13, 2018; Accepted: April 16, 2018 changes that occur during pregnancy.1 At week 6 of most preg- *Correspondence to: Desmond O. Acheampong, Department of Biomedical Scienc- es, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana. nancies, the ureters begin to dilate as a result of the physiological Tel: +233 543710234, E-mail: [email protected]; do.acheampong@gmail. changes, a condition commonly referred to as hydronephrosis of com pregnancy.2 This condition peaks at 22–26 weeks and persists until How to cite this article: Acheampong DO, Afoakwah MK, Boye A, Opoku R, delivery.2,3 Interestingly, both progesterone and estrogen levels in- Kwakye-Nuako G, Adokoh CK, Baafi SA, Somuah D. Evaluation of Diagnos- crease during pregnancy and lead to decreases urethral and bladder tic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bacteriu- ria Among Pregnant Women in Ashanti Region, Ghana. J Explor Res Pharmacol tone. Increased bladder volume and decreased bladder tone along 2018;3(3):78–84. doi: 10.14218/JERP.2018.00003. with decreased urethral tone contribute to increased urinary stasis

Journal of Exploratory Research in Pharmacology 2018 vol. 3 | 78–84

Copyright: © 2018 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Acheampong DO. et al: Asymptomatic bacteriuria in pregnant women J Explor Res Pharmacol and ureterovesical reflux, which could result in asymptomatic bac- the Anglogold Ashanti Health Foundation Hospital in Obuasi and teriuria (ASB). Additionally, the uterus sits directly on top of the Ellolab Diagnostic Centre in Kumasi, both located in the Ashanti urinary bladder, and as the resultant weight of the uterus increases, region of Ghana. it blocks the drainage of urine from the bladder, thus causing uri- nary stasis that might lead to infection of the urinary tract.1 Questionnaire administration Moreover, the immune system of pregnant women becomes compromised, rendering the pregnant woman more susceptible to both pathogenic and nonpathogenic microorganisms. Another im- Questionnaires were administered to pregnant women attending an- portant predisposing factor is the female sex itself, due to the rela- tenatal clinic at these hospitals during the study period. However, tively shorter urethra. Thus, proximity of the vagina to the anus, pregnant women with clinical signs and symptoms of UTI and those coupled with the inability of pregnant women to completely empty who were on antimicrobial treatment prior to sample collection were their bladder, predispose them to UTI.4 Finally, the anatomical link excluded. Respondents were briefed about the study and their consent between the urethra and vagina makes it liable to trauma during sought before administering the questionnaires. A total of 412 ques- sexual intercourse, lending further susceptibility to infections.5 tionnaires were administered to access certain vital sociodemographic Untreated ASB during pregnancy can adversely affect both data from the study subjects. Each interview lasted for 5 to 10 m. mother and fetus, and is related to a greater chance of progression to pyelonephritis and possibly an increased susceptibility to pre- Collection and processing of urine samples eclampsia, premature birth and low neonatal birth weight.6 It is, therefore, imperative to routinely screen pregnant women attend- ing antenatal clinic for ASB, to be able to initiate remedial meas- The women were educated about the correct procedures for collect- ures early enough to curb any related complications, should ASB ing a urine sample for clinical purposes. Subsequently, sterile urine be detected. The diagnostic methods commonly used for this pur- containers were given to them to collect a clean-catch midstream pose are the diagnostic dipstick, microscopy and urine culture; the urine sample for the test. Urine samples were preliminarily analyzed latter being the most reliable.7 Urine culture is the gold standard for ASB by Multistix 10SG urine test strips (Siemens Healthcare Di- against which the other two diagnostic techniques are evaluated.8 agnostics, Tarrytown, NY, USA). This was done within 2 h of sample Gram-negative bacteria, such as Escherichia coli, Klebsiella collection. A Multistix 10SG urine strip (Siemens Healthcare Diag- and Proteus, and Gram-positive bacteria, such as hemolytic Strep- nostics) was dipped in the urine sample and analyzed according to the tococci and Staphylococcus saprophyticus, are the most implicated manufacturer’s instructions. Subsequently, microscopic analysis was bacteria species for ASB in pregnancy.9 Earlier studies conducted performed (Model CX22; Olympus, Tokyo, Japan). The urine sam- in Ghana found UTI prevalence of 7.3%,10,11 with E. coli being the ples were cultured within 2 h of sample collection. Using a calibrated dominant bacteria isolated from the pregnant women who attended wire inoculating loop (0.001 mL), urine samples were inoculated into antenatal care during the study period. These high prevalence rates cystine lactose electrolyte-deficient medium (Liofichem, Ltd., Piane in the earlier studies paint a gloomy picture that requires immedi- Vomano, Italy) and incubated at 37 °C for 24 h. Colonies were count- ate and appropriate interventions.10,11 Unlike the bacteria culture ed to check the presence of significant bacteriuria. method, the dipstick and microscopy diagnostic methods readily Colony count yielding bacterial growth of 105 CFU/mL of urine produce results in a short space of time, which is an important was regarded as significant bacteriuria. Subsequent to this, the requirement in the routine diagnostic method. Therefore, any of specificity, sensitivity, positive predictive value (PPV) and nega- these two diagnostic methods that proves efficient can be recom- tive predictive value (NPV) of microscopy and dipstick diagnostic mended to be used to screen pregnant women for ASB. methods were calculated using the following formulae: sensitivity The use of antibiotics is, to a greater extent, the only impor- = number of true positives ÷ (number of true positives + number of tant modifiable risk factor for antibiotic resistance.12 Most often, false negatives); specificity = number of true negative ÷ (number treatment of UTI is started empirically and it is based on the prior of true negatives + number of false positives); PPV = number of information physicians have on the antimicrobial resistance pat- true positive ÷ (number of true positives + number of false posi- terns of the urinary pathogens.13 However, in recent times many tives); NPV = number of true negative ÷ (number of true negative reports have been published on increased antimicrobial resistance + number of false negatives). among urinary tract isolates, from all over the globe.14,15 In Afri- Bacterial isolates were then identified based on their colonial can countries, including Ghana, the use of drugs that are not up to morphology, the Gram reaction and pattern of biochemical reac- standard, indiscriminate use of antibiotics and erratic prescription tions. The disc diffusion antimicrobial sensitivity test was subse- by unqualified drug peddlers have been identified as contributing quently performed for the positive cultures using antimicrobial sen- to the surge in antimicrobial resistance.16 sitivity discs on Mueller-Hinton agar (Oxoid Ltd., Hampshire, UK) The current study aimed to assess the effectiveness of microsco- as instructed by the standardized table published by the Clinical and py and dipstick diagnostic methods for diagnosing ASB compared Laboratory Standards Institute (2014). Antimicrobial discs tested to the bacteria culture method (as gold standard), estimating the against the positive isolates included ampicillin (10 µg), cefuroxime prevalence of ASB and investigating the antimicrobial suscepti- (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), tetracycline (30 bility pattern of the bacteria responsible for ASB among pregnant µg), nalidixic acid (30 µg), nitrofurantoin (300 µg), pipemidic acid women attending antenatal care in the Ashanti region, Ghana. (20 µg) and meropenem (10 µg) (Oxoid Ltd.). Reference strains of E. coli (No. 25922; American Type Culture Collection, Manassas, VA, USA) and S. aureus (No. 25923; American Type Culture Col- Methods lection) preserved in our laboratory were used as controls. The protocol was approved and ethically cleared by the Depart- ment of Biomedical Sciences, College of Health and Allied Sci- Study site ences, University of Cape Coast (Ghana), and written informed consent was obtained from all patients prior to enrollment. Ethical This study was undertaken from January 2017 to May 2017 at approval was also obtained from the Anglogold Ashanti Health

DOI: 10.14218/JERP.2018.00003 | Volume 3 Issue 3, August 2018 79 J Explor Res Pharmacol Acheampong DO. et al: Asymptomatic bacteriuria in pregnant women

Table 1. Prevalence of asymptomatic bacteriuria based on sociodemographic characteristics determined by the culture method Characteristic Total Number, n (%) Tested Negative, n (%) Tested Positive, n (%) Chi-Square Value, X2 p-value Age 16–20 26 (6.3) 23 (88.5) 3 (11.5) 3.146 0.678 21–25 86 (20.9) 74 (86.0) 12 (14.0) 26–30 154 (37.4) 125 (81.2) 29 (18.8) 31–35 77 (18.7) 64 (83.1) 13 (16.9) 36–40 47 (11.4) 38 (80.9) 9 (19.1) 41–45 22 (5.3) 16 (72.7) 6 (27.3) Marital status Single 73 (17.7) 58 (79.5) 15 (20.5) 0.581 0.272 Married 339 (82.3) 282 (83.2) 57 (16.8) Educational background Illiterate 22 (5.3) 20 (90.9) 2 (9.1) 11.651 0.109 Primary 30 (7.3) 26 (86.7) 4 (13.3) Secondary 229 (55.6) 198 (86.5) 31 (13.5) Higher learning 131 (31.8) 96 (73.3) 35 (26.7) Occupation Student 13 (3.2) 11 (84.6) 2 (15.4) 3.959 0.138 Housewife 66 (16.0) 60 (90.9) 6 (9.1) Employed 333 (80.8) 269 (80.8) 64 (19.2) Parity <1 149 (36.2) 119 (79.9) 30 (20.1) 1.636 0.802 1 103 (25.0) 86 (83.5) 217 (23.6) 2 68 (16.5) 56 (82.4) 12 (17.6) 3 54 (13.1) 47 (87.0) 7 (13.0) ≥4 38 (9.2) 32 (84.2) 6 (15.8) Duration of pregnancy First trimester 34 (8.3) 31 (91.2) 3 (8.8) 2.891 0.236 Second trimester 117 (28.4) 99 (84.6) 18 (15.4) Third trimester 261 (63.3) 210 (80.5) 51 (19.5)

Foundation Hospital and Ellolab Diagnostic Center. All proce- the majority (n = 154, 37.4%) belonging to the age group of 26–30 dures were performed in accordance with the ethical standards years (Table 1). The mean and median ages were 29.27 and 29 outlined in the Helsinki Declaration of 1975, as revised in 2013. years respectively, whereas the modal age was 28 years. Educa- tional status of participants included illiterate (n = 22, 5.3%), pri- Statistical analysis mary school level (n = 30, 7.3%), secondary school level (n = 229, 55.6%) and tertiary level (n = 131, 31.8%). Among the 412 study participants, 34 (8.3%), 117 (28.4%) and 261 (63.3%) were in the Data was analyzed using the SPSS software package version 21 first, second and third trimesters respectively (Table 1). A total of (IBM Corp., Armonk, NY, USA). The data were analyzed using chi- 73 (17.7%) of the women were single (unmarried), whereas the 2 square (χ ) test, to test whether differences between values are signifi- remaining 339 (82.3%) were married (Table 1). cant. p-values less than 0.05 were considered statistically significant. Of the 412 pregnant women, 72 were found to be positive for ASB by the urine culture method (Table 2), which translates into an overall prevalence of 17.5%. The age group of 41–45 years Results presented the highest prevalence, being 27.3% (Table 1). Also, as shown in Table 1, the pregnant women in the third trimester The ages of the study subjects ranged from 16 to 45 years, with showed the highest prevalence, being 19.5%. Prevalence of ASB

80 DOI: 10.14218/JERP.2018.00003 | Volume 3 Issue 3, August 2018 Acheampong DO. et al: Asymptomatic bacteriuria in pregnant women J Explor Res Pharmacol

Table 2. Overall prevalence of symptomatic bacteriuria based on micro- Table 4. Comparison between dipstick urinalysis and microscopy using bial culture and microscopy bacteria culture as standard Negative, n (%) Positive, n (%) Dipstick Microscopy Microbial culture 340 (82.5) 72 (17.5) Positive Negative Positive Negative Microscopy 341 (82.8) 71 (17.2) Positive 71 1 54 18 Dipstick 338 (82.0) 74 (18.0) Culture 3 337 17 323 Negative 74 338 71 341 among pregnant women based on microscopy was 17.2% (Table 2). Also, presented in Table 3 is prevalence of symptomatic bac- inferred from the above results that the prevalence of ASB var- teriuria using various dipstick parameters, and prevalence of ASB ies from one geographical location to another, which can possibly based on dipstick diagnostic method was 18.0% (Table 2). The be attributed to differences in the mode of screening and/or com- sensitivity, specificity, PPV and NPV of microscopy as calculated pounding risk factors such as age, parity, educational level, envi- from Table 4 were 74.32%, 94.9%, 76.3% and 94.4% respectively. ronmental and personal hygiene.11,24 Notwithstanding, the 17.5% The sensitivity, specificity, PPV and NPV of dipstick urinalysis prevalence recorded in this study is relatively high, and consider- as calculated from Table 4 were 95.9%, 99.7%, 98.6% and 99.1% ing the demography of study locations, it could possibly represent respectively. These were determined based on leukocyte esterase the national prevalence of Ghana. It, therefore, calls for concerted and nitrite parameters. efforts among all interested parties to formulate a national policy Among the isolated bacteria species (n = 72, 17.5%), the Gram- negative bacteria E. coli, K. pneumoniae and P. mirabilis were the to help curb this situation. most prevalent (n = 71, 98.6%) compared to the Gram-positive The current study did not find any association between age, Enterococcus fecalis (n = 1, 1.4%; Table 5). The most dominant marital status, occupation, parity, educational background and du- bacteria isolated was E. coli (n = 45, 62.5%), followed by K. pneu- ration of pregnancy (p > 0.05) for positive results of ASB, and moniae (n = 22, 30.6%). As shown in Table 6, E. coli was relatively implies that the incidence of ASB among pregnant women might sensitive to nitrofurantoin (75.6%) and nalidixic Acid (57.8). K. not be dependent on these demographic parameters and therefore might not be considered risk factors. This was consistent with pneumoniae also demonstrated a similar trend to nalidixic acid 25,26 (72.7%) and nitrofurantoin (63.6%). P. mirabilis, on the other similar studies conducted in Ethiopia. The highest prevalence hand, exhibited 50% sensitivity to both nalidixic acid and mero- of this study was recorded for the age group of 41–45 years and penem. E. fecalis, the only Gram-positive strain, was sensitive to the lowest for the age group of 16–20 years. The finding of the only meropenem (100%). highest and lowest prevalence rates being represented by the oldest and youngest age groups, respectively, was very instructive. This relatively high prevalence among the older women might be due Discussion to a compromised immune system, and also, during pregnancy, the lining of the tissues around the vagina becomes more fragile, which get worse with age, making older pregnant women more Pregnant women at any stage of pregnancy are at increased risk susceptible and younger ones less susceptible to the infections.27 17,18 of UTI, although such infections are usually asymptomatic. In This study also recorded high ASB prevalence among pregnant this study of the prevalence of ASB in pregnant women, suitable women in the third trimester, which was followed by the second routine screening methods and antimicrobial susceptibility pat- trimester of pregnancy. This was consistent with the outcome terns of the isolates were investigated. In the quest to recommend of earlier studies in Nigeria.28,29 Possibly, susceptibility to ASB an efficient routine diagnostic method that could produce results in at this stage of pregnancy could be due to dilatation of the ure- relatively shorter time for possible ASB, the sensitivity and speci- ters, which starts around 6 weeks and reaches the peak at weeks ficity of the microscopic and dipstick diagnostic methods were 22–24.30 It could also be due to poor personal hygiene since most also determined and compared. The diagnosis of ASB in this study pregnant women at this stage find it difficult to clean thoroughly. was based on a single urine sample from each study participant. This was, however, inconsistent with the findings of Obirikorang Based on bacterial culture method, the gold standard diagnostic et al.,24 which indicated highest prevalence occurring in the second method, the overall prevalence of ASB among pregnant women in trimester, whereas Boye et al.11 and Turpin et al.10 recorded high- this study was 17.5%. This prevalence was relatively higher than est prevalence in the first trimester in Ghana. those reported in earlier studies, including those from Ethiopia This study showed that the dipstick diagnostic test has higher 19,20 21 (3.3% and 11.6%), Iran (6.1%), Ghana (7.3%) and Mexico sensitivity and specificity than the urine microscopy test, indicat- 10,22 (8.4%). It was however lower than in other studies carried out ing that dipstick urinalysis was a better diagnostic method for ASB in Nigeria (22.2%) and Ghana (56.5%).11,23 It can, therefore, be Table 5. Frequency of isolated bacteria species Table 3. Prevalence of symptomatic bacteriuria using various dipstick parameters Bacterial Species Frequency, n (%) Negative, n (%) Positive, n (%) E. coli 45 (62.5) Leukocytes 352 (85.4) 60 (14.6) Klebsiella pneumoniae 22 (30.6) Nitrites 341 (82.8) 71 (17.2) Enterococcus fecalis 1 (1.4) Proteins 400 (97.1) 12 (2.9) Proteus mirabilis 4 (5.6) Blood 404 (98.1) 8 (1.9) Total 72

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Table 6. Susceptibility pattern of isolates to commonly used antimicrobial agents Antibiotic Sensitivity E. coli, n (%) Klebsiella pneumoniae, n (%) Proteus mirabilis, n (%) Streptococcus fecalis, n (%) Ampicillin Resistant 45 (100.0) 22 (100.0) 4 (100.0) 1 (100.0) Sensitive 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Cefuroxime Resistant 45 (100.0) 22 (100.0) 4 (100.0) 1 (100.0) Sensitive 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Cotrimoxazole Resistant 45 (100.0) 22 (100.0) 3 (75.0) 1 (100.0) Sensitive 0 (0.0) 0 (0.0) 1 (25.0) 0 (0.0) Gentamicin Resistant 38 (84.4) 18 (81.8) 3 (75.0) 1 (100.0) Sensitive 7 (15.6) 4 (18.2) 1 (25.0) 0 (0.0) Tetracyclin Resistant 45 (100.0) 22 (100.0) 4 (100.0) 1 (100.0) Sensitive 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Nalidixic acid Resistant 19 (42.2) 6 (27.3) 2 (50.0) 1 (100.0) Sensitive 26 (57.8) 16 (72.7) 2 (50.0) 0 (0.0) Nitrofurantoin Resistant 11 (24.4) 8 (36.4) 4 (100.0) 1 (100.0) Sensitive 34 (75.6) 14 (63.6) 0 (0.0) 0 (0.0) Pipemidic acid Resistant 44 (97.8) 19 (86.4) 4 (100.0) 1 (100.0) Sensitive 1 (2.2) 3 (13.6) 0 (0.0) 0 (0.0) Meropenem Resistant 22 (57.9) 9 (50.0) 2 (50.0) 0 (0.0) Sensitive 16 (42.1) 9 (50.0) 2 (50.0) 1 (100.0) than microscopy. This finding was consistent with an earlier study E. coli the most commonly isolated bacteria in this study and by Glissmeyer et al.31 Among the five parameters considered un- was highly susceptible to nitrofurantoin and nalidixic acid but der dipstick analysis, nitrite was the most sensitive and, therefore, demonstrated slight sensitivity to meropenem and a relatively low could be the appropriate parameter for the dipstick diagnostic sensitivity to gentamicin and pipemidic acid. It was, however, re- method. The nitrite test precisely predicted 71 out of the 72 posi- sistant to ampicillin, cefuroxime, tetracycline and cotrimoxazole; tive cases, where all of the 71 cases represented enterobacteriaceae as such, these drugs would not be suitable for empirical treatment colonization. The nitrite test is based on the ability of the bacte- of ASB in these pregnant women. K. pneumoniae on the other ria to reduce nitrate to nitrite in the bladder urine.32 Reduction of hand was highly sensitive to nalidixic acid and nitrofurantoin. It nitrate to nitrite by bacteria is dependent on time, and a positive also demonstrated 50% sensitivity to meropenem and was slightly result requires a prolonged bladder incubation period of more than sensitive to gentamicin and pipemidic acid. However, just as E. 4 h for significant residual urine.33 The nitrite test was unable to coli, it demonstrated resistance to ampicillin, cefuroxime, tetracy- predict the case of the Enterococcus colonization because the or- cline and cotrimoxazole. E. fecalis was sensitive to meropenem ganism cannot reduce nitrate to nitrite, thereby accounting for the only. P. mirabilis demonstrated 50% susceptibility to nalidixic acid negative result.34 Both dipstick and microscopy had high NPV and and meropenem, 25% sensitivity to cotrimoxazole and gentamicin, PPVs in screening for possible ASB in pregnant women. However, and was also resistant to ampicillin, cefuroxime, tetracycline, ni- the dipstick method had a higher sensitivity, specificity, PPV and trofurantoin and pipemidic acid. NPV than microscopy. As such, the dipstick diagnostic method is Evidently, the level of resistance against antimicrobial agents apparently a better diagnostic method than microscopy. However, has increased over time.37,38 This corroborates earlier reports about carrying out a urine culture as a confirmatory test is imperative for the increasing resistance to potent antimicrobials by bacteria re- appropriate diagnosis. sponsible for UTI and can be attributed many factors,14,39 which This study has also demonstrated that E. coli is the dominant include improper use of antimicrobial agents. In view of this de- bacteria responsible for ASB in pregnant women, accounting for velopment, antimicrobial sensitivity test is strongly recommended 62.5% of the ASB cases. This was corroborated by earlier studies before a drug is prescribed to control this unpleasant occurrence. by Boye et al.11 and Turpin et al.10 in Ghana. The high prevalence In situations where empirical treatment is unavoidable, potent anti- of E. coli might be due to a number of virulence factors specific microbial agents, such as nitrofurantoin and nalidixic acid, should for colonization and invasion of the urinary epithelium, such as the be considered. This study, therefore, supports the use of nitrofuran- P-fimbria and S-fimbria adhesions associated with E. coli.35 Addi- toin and nalidixic acid for empirical treatment of ASB in pregnant tionally, this could be due to urinary stasis common in pregnancy, women. Nitrofurantoin works by destroying the DNA of bacteria because most E. coli strains thrive well in this environment and, during a highly reactive state at its reduced form. Nitrofurantoin is therefore, are capable of producing ASB.36 The second dominant reduced rapidly by flavoproteins found in the bacterial cell to many bacteria K. pneumoniae accounted for 30.6% of the ASB cases. reactive intermediates that react with DNA, ribosomal proteins and This was also consistent with the findings by Boye et al.11 These other important macromolecules within the cells.40 Nalidixic acid bacteria are, therefore, the major cause of ASB in pregnant women. is a synthetic quinolone that works by inhibiting replication of

82 DOI: 10.14218/JERP.2018.00003 | Volume 3 Issue 3, August 2018 Acheampong DO. et al: Asymptomatic bacteriuria in pregnant women J Explor Res Pharmacol

DNA and transcription in the bacteria.41 References

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We appreciate the valuable contribution of the management and [13] Wilson ML, Gaido L. Laboratory diagnosis of urinary tract infec- staff of Anglogold Ashanti Health Foundation Hospital and Ellolab tions in adult patients. Clin Infect Dis 2004;38(8):1150–1158. doi:10.1086/383029. Diagnostic Center to this study. [14] Zorc JJ, Kiddoo DA, Shaw KN. Diagnosis and management of pediat- ric urinary tract infections. Clin Microbiol Rev 2005;18(2):417–422. doi:10.1128/CMR.18.2.417-422.2005. Conflict of interest [15] Acheampong DO, Boamponsem LK, Feglo PK. Occurrence and spe- cies distribution of Klebsiella isolates: A case study at Komfo Anokye The authors have no conflict of interest related to this publication. Teaching Hospital (KATH) in Ghana. Adv Appl Sci Res 2011;2(4):187– 193. [16] Chinedum IE. Microbial resistance to antibiotics. Afr J Biotechnol 2005;4(13):1606–1611. Author contributions [17] Schneeberger C, Kazemier BM, Geerlings SE. Asymptomatic bacte- riuria and urinary tract infections in special patient groups: women This study was carried out in collaboration between all authors. with diabetes mellitus and pregnant women. Curr Opin Infect Dis Authors DOA, MKF and RO designed the study, performed the 2014;27(1):108–114. doi:10.1097/QCO.0000000000000028. [18] Angelescu K, Nussbaumer-Streit B, Sieben W, Scheibler F, Gartlehner statistical analysis, wrote the protocol, and wrote the first draft of G. Benefits and harms of screening for and treatment of asympto- the manuscript. Authors AB, SAB and DS managed the analyses matic bacteriuria in pregnancy: a systematic review. BMC Pregnancy of the study and edited the final manuscript for intellectual content. Childbirth 2016;16(1):336. doi:10.1186/s12884-016-1128-0. Authors CKA and GKN managed the literature searches. All au- [19] Demilie T, Beyene G, Melaku S, Tsegaye W. 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84 DOI: 10.14218/JERP.2018.00003 | Volume 3 Issue 3, August 2018 Review Article

Luteolin: Anti-breast Cancer Effects and Mechanisms

Ying Wang, Jing Wang, Xing Gong, Xinxuan Wen and Xinsheng Gu*

Department of Oncology and Central Laboratory in Xiangyang No. 1 People’s Hospital, and Department of Pharmacology in School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, Hubei, China

Abstract

Luteolin is a flavonoid compound and exhibits antioxidant, antiinflammatory, antibacterial, antidiabetic and antipro- liferative properties. Studies have shown that luteolin may inhibit cell proliferation, metastasis, and angiogenesis of numerous types of cancers, including breast cancer, through inducing cell cycle arrest and apoptosis and by modulat- ing cell signaling. In this review, we have summarized the recent studies on inhibitory effects and underlying mecha- nisms of luteolin in breast cancer. These studies support that luteolin is a promising drug to treat breast cancer.

Introduction molecular markers, such as estrogen receptor (ER), PR, Her2, and Ki67, breast cancer is classified into the following five sub- + − low Luteolin is the nickname of 3′, 4′, 5, 7-tetrahydroxyflavone. It be- types: luminal A (ER/PR HER2 Ki-67 ), luminal B (ER/ PR+HER2−/+Ki-67high), Her-2-enriched (ER/PR−HER2+Ki-67high), longs to biologically active flavonoids that are widely found in − − high the plant kingdom.1 Dietary intake of flavonoids is inversely as- triple negative/basal-like (ER/PR HER2 Ki-67 ), and “other” (including all those that cannot be attributed to the four subtypes). sociated with risk of lung, prostate, stomach and breast cancers in This molecular classification predicts cancer progression and di- humans.2–4 Luteolin contains three rings in its molecule, including rects the choice of therapeutic modality.24–29 two benzene rings (A, B) and an oxygen-containing (C) ring, which Patients with high expression of HER-2 and Ki-67 were found is called the C6-C3-C6 structure (Fig. 1). There is a C2-3 double to have a poor prognosis.30,31 The luminal A subtype is more re- bond in the oxygen-containing (C) ring, and two hydroxyl groups sponsive to endocrine therapy than other subtypes. The luminal in each benzene ring.5 Luteolin does not have an –OH substitution B subtype can be treated by chemotherapy. The Her-2-enriched on C3.6 The C2-3 double bond and C3′ and C4′ hydroxyl groups of 7 subtype is treated by HER-2-targeted therapy, and anthracycline- luteolin are essential for its biochemical and biological activities, based chemotherapy. The triple negative/basal-like subtype is including antioxidant, antiinflammatory, antibacterial, antidiabetic 24–29 8,9 treated by platinum-based chemotherapy and PARP inhibitors. and antiproliferative actions. In the past decades, the anticancer Although therapeutic approaches for breast cancer have been de- effect of luteolin has been demonstrated on many kinds of cancers, veloped, the cure of breast cancer is still unsatisfactory because of such as hepatocellular carcinoma, melanoma, and gastric carcino- 10–22 the heterogeneity and recalcitrant nature towards various drug thera- ma, ovarian cancer, colorectal cancer, and breast cancers. pies.24–29,32 To control proliferation and metastasis during breast can- Breast cancer is the most common and life-threatening cancer 23 cer therapy, novel drugs are needed. In this paper, we have summa- diagnosed among women worldwide. According to genomic rized the studies on the anticancer effects of luteolin on breast cancer. expression profiles and immunohistochemical staining of a few

Anticancer effects of luteolin on breast cancer Keywords: Luteolin; Breast Cancer; Mechanisms; Inhibitory Effects. Abbreviations: AEG-1, astrocyte elevated gene 1; AKT, protein kinase B; AP-1, acti- vator protein-1; CAT, catalase; DR5, death receptor 5; EGFR/HER, epidermal growth The anticancer effects of luteolin on breast cancer have been investi- factor receptor; EMT, epithelial mesenchymal transition; ER, estrogen Receptor; gated using pertinent cell lines which are relevant to molecular sub- ERK, extracellular signal-regulated kinase; ESP, estrogen signaling pathway; FAS, 33 fatty acid synthase; JNK, Jun N-terminal kinase; KDR, vascular endothelial growth types of breast cancer. Luteolin was shown to effectively block the factor receptor 2; MAPK, mitogen-activated protein kinase; MMP, matrix metallopro- IGF-1-stimulated luminal A subtype ERα-positive MCF-7 cell pro- teinase; NF-κB, nuclear factor-kappa B; PARP, poly ADP-ribose polymerase; PI3K, liferation in a dose- and time-dependent manner34 and to suppress tri- phosphatidylinositol 3-kinase; PKC, protein kinase C; PLK-1, polo-like kinase 1; PR, ple negative/basal-like ERα-negative MDA-MB-231 cell growth.35 progesterone receptor; ROS, reactive oxygen species; SOD, superoxide dismutase; STAT, signal transducers and activators of transcription; TNBC, triple-negative breast Luteolin was also shown to decrease the viability of breast cancer 36 cancer; TNFα, tumor necrosis factor alpha; TPL2, tumor progression locus 2; VEGF, cells MCF7/6 and MDA-MB231-1833. Luteolin-supplementation vascular endothelial growth factor; VRK1, vaccinia-related kinase 1. at 0.01% or 0.05% significantly reduced tumor burden in nude mice Received: April 9, 2018; Revised: July 01, 2018; Accepted: July 27, 2018 inoculated with MDA-MB-231 cells.35 This was confirmed by stud- *Correspondence to: Xinsheng Gu, 30 South Renmin Road, Shiyan, Hubei Province, ies that showed dose-dependent inhibitory effects of luteolin on the 442000, China. E-mail: [email protected] proliferation of MCF-7 cells and MDA-MB-231 cells.37,38 How to cite this article: Wang Y, Wang J, Gong X, Wen X, Gu X. Luteolin: Anti- breast Cancer Effects and Mechanisms. J Explor Res Pharmacol 2018;3(3):85–90. Extensive studies have shown that luteolin significantly inhibits doi: 10.14218/JERP.2018.00011. cell cycle of MCF-7 and MDA-MB-231 cells, resulting in cell cy-

Journal of Exploratory Research in Pharmacology 2018 vol. 3 | 85–90

Copyright: © 2018 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. J Explor Res Pharmacol Wang Y. et al: Luteolin: Anti-breast Cancer Effects and Mechanisms

generation, DNA damage, activation of the ATR→Chk2→p53 signaling pathway, inhibition of the nuclear factor (NF)-κB signal- ing pathway, activation of the p38 pathway and depletion of antia- poptotic proteins.46 In MCF-7 cells, luteolin enhanced the expres- sion of death receptors, such as DR5, and enhanced the activities of caspase-8 and -9, which in turn induced caspase-3 activity in a dose-dependent manner, followed by inactivation of PARP. Luteo- lin also induced mitochondrial membrane potential collapse and cytochrome c release, and increased Bax expression by inhibiting Fig. 1. Luteolin molecular structure. The C2-3 double bond and C3′ and C4′ hydroxyl groups of luteolin are essential for the biochemical and bio- expression of Bcl-2. Therefore, luteolin induces apoptosis by acti- 47 logical activities. vating the extrinsic and intrinsic pathways. cle arrest at the G2/M and S stages.34,38–40 Luteolin can also induce PI3K/Akt apoptosis in breast cancer cells.34–36 These studies support that lu- teolin inhibits the proliferation of breast cancer cells of various Luteolin markedly decreases IGF-1-dependent IGF-1R and Akt subtypes. In an epidemiological study on dietary intake of selected phosphorylation without affecting extracellular signal-regulated ki- flavonols, flavones and flavonoids, luteolin in food was shown to nase (ERK)1/2 phosphorylation in MCF-7 cells, and the inhibiting have no anticancer effect.41 This might be due to low daily uptake IGF-1-mediated PI3K-Akt pathway is dependent on ERα expres- of luteolin (<2.0 mg/d). The therapeutic doses or concentration of sion, resulting in suppression of MCF-7 cells.34 Luteolin induces luteolin are much higher and could be 10–30 mg/mL according to cell cycle arrest and apoptosis in breast cancer cells through in- the experimental research findings. hibition of PI3K/Akt activation and increase of FOXO3a activa- The formation of new blood vessels from the preexisting vas- tion.40 Inhibiting the PI3K/Akt signaling pathways also mediated culature (angiogenesis) is a crucial stage in cancer progression. In- suppression of the epidermal growth factor receptor (EGFR) signal- deed, studies showed that luteolin exerted strong antiangiogenesis 38,42 ing pathway and the proliferation of ERα-positive MCF-7 cells and activity in chick chorioallantoic membrane. Metastases are the ERα-negative MDA-MB-231 cells by luteolin in a dose-dependent primary cause of human cancer deaths. Most breast cancer-related manner.37 deaths from triple negative breast cancer (TNBC) occur following metastasis of cancer cells and development of tumors at second- ary sites. Because TNBCs lack the three receptors (ER, PR, and EGFR HER2) targeted by current chemotherapeutic regimens, they are typically treated with extremely aggressive and highly toxic non- EGFRs/HERs are implicated in pathogenesis of breast cancer and targeted treatment strategies. their over-expression is associated with poorer prognosis and out- Luteolin can efficiently suppress migration and invasion of the comes of breast cancer.48–50 Luteolin was shown to down-regulate highly metastatic TNBC cell lines MDA-MB-231 and BT-549, EGFR expression and inhibit EGF-induced mitogen-activated and inhibit the lung metastases of breast cancer xenograft tumors protein kinase (MAPK) activation in a dose-dependent manner, 38,42–44 derived from MDA-MD-231 cells. Luteolin can also signifi- including the phosphorylation of ERK, p38 and AKT, and to sup- 38 cantly inhibit migration of ERα-positive MCF-7, inhibit cell mi- press the proliferation of MDA-MB-231 ER-negative breast can- gration and viability of human MDA-MB-435 and MDA-MB-231 cer cells and ERα-positive MCF-7 cells.35,37 (4175) LM2 TNBC cells in vitro, and suppress metastasis of these cells to the lungs in a mouse metastasis model.44 Luteolin may inhibit breast cancer intravasation through the lymphatic barrier, Polo-like kinase 1 (PLK-1) as revealed by an in vitro assay that showed luteolin exerted an antiintravasative effect of MDA-MB231 breast cancer spheroids PLK-1 is a mitotic kinase and actively regulates the G2/M transi- through the lymph endothelial barrier.45 Luteolin can efficiently tion, mitosis, mitotic exit, and cytokinesis.51 The over-expression suppress the migration and invasion of MDA-MB-231 and MCF-7 of PLK-1 is strongly correlated with a wide spectrum of human cells as well.38,42–45 Epithelial mesenchymal transition (EMT) is cancers and their poor prognosis.52 Luteolin inhibits PLK1 gene also a key event in metastasis. Luteolin can reverse EMT of MDA- expression in MCF-7 breast cancer cells53 and MDA-MB-231 ER- MB-231 and BT5-49 breast cancer cells, which may be mediated negative breast cancer cells.35 This inhibition is probably caused by down-regulation of β-catenin.43 by decreased acetylation of histone H4 associated with the PLK1 All these studies support that luteolin may induce cell cycle ar- gene promoter.53 rest at the G2/M and S stages, as well as apoptosis, and inhibit mi- gration and epithelial mesenchymal trans-differentiation of breast Estrogen signaling cancer cells, thereby inhibiting the development, progression and metastasis of breast cancer. It has been shown that luteolin possesses estrogen agonist activ- ity.54 However, luteolin exerts its antiestrogen activity by regu- Mechanism involved in anticancer effects of luteolin on breast lating many estrogen signaling pathway (ESP) genes, including cancer GTF2H2, NCOR1, TAF9, NRAS, NRIP1, POLR2A, DDX5 and NCOA3, in MCF-7 breast cancer cells probably through epige- netic mechanism.53 Some breast cancer subtypes, such as luminal Apoptotic pathways A, are considered to be primarily regulated by ESPs, and hormone therapy using antiestrogen drugs, such as tamoxifen, is effective Luteolin-induced apoptosis involves reactive oxygen species (ROS) for breast cancers expressing ERα.55–57 Therefore, luteolin could

86 DOI: 10.14218/JERP.2018.00011 | Volume 3 Issue 3, August 2018 Wang Y. et al: Luteolin: Anti-breast Cancer Effects and Mechanisms J Explor Res Pharmacol

Fig. 2. Luteolin modulates the signaling pathways and inhibits the growth and migration as well as promotes apoptosis of breast cancer cells. be an effective drug for breast cancer therapy. suppresses the metastasis of TNBC by reversing EMT via down- regulation of beta-catenin expression.43 Notch and microRNA Luteolin directly inhibits kinases protein kinase C (PKC), VRK1 Luteolin inhibits Notch signaling and regulates miRNAs. Treat- and TLP2 in breast cancer cells ment of MCF-7 and MDA-MB-231 cells with luteolin results in decreasing expression levels of Notch-1, Hes-1, Hey-1, Hey-2 as As reported as early as 1989, luteolin is a PKC inhibitor. It com- well as vascular endothelial growth factor (VEGF), cyclin D1 and petitively blocks the ATP binding site on the catalytic unit of matrix metalloproteinase (MMP) expression in breast cancer cells. PKC.59 TPL2 is a protooncogene that is over-expressed in various Luteolin induces miR-181a, miR-139-5p, miR-224 and miR-246 cancer types, including breast cancer.60 Luteolin may directly bind expression levels and decreases the miR-155 level in both MCF-7 TPL2 and inhibit TPL2 activity in an ATP-competitive manner,58 and MDA-MB-231 cells, and increases miR-34a level in MDA- leading to similar effects of luteolin on TNF-α-mediated signaling MB-231 cells. MiR-34a and miR-224 regulates Notch signaling pathways and COX-2 expression.58 Recently, luteolin was found and inhibits breast cancer cell survival migration and angiogen- to significantly and directly interact with the catalytic domain of esis.38 vaccinia-related kinase 1 (VRK1), a mitotic kinase that functions in cell cycle regulation, and to reduce VRK1-mediated phospho- rylation of the cell cycle-related substrates BAF and histone H3.61 Inflammation-related signaling Therefore, luteolin is a direct inhibitor for kinases such as PKC, VRK1, and TLP2. Luteolin down-regulates the transactivity of NF-κB and activator protein-1 (AP-1), resulting in inhibition of TNFα-induced COX-2 expression. Luteolin also inhibits TNFα-induced phosphorylation Luteolin inhibits fatty acid synthase in breast cancer cells of MAPK/ERK kinase 1/ERK/p90RSK, MAPK kinase 4/Jun N- terminal kinase (c-JNK)/c-Jun, and Akt/p70S6K. These effects of Luteolin inhibits fatty acid synthase (FAS), which is a key lipo- luteolin are probably due to blocking tumor progression via locus genic enzyme over-expressed in many human cancers, resulting 2 serine/threonine kinase (TPL2).58 in inhibition of cell growth and induction of apoptosis of prostate and breast cancer cells.39,62 Although the underlying mechanism of FAS inhibition by luteolin is not completely clear, the presence Metastasis-associated signaling of a C-2,3 double bond, a 4-ketone function and hydroxyl groups on positions 5, 7, 3′, and 4′ plays a favorable role to inhibit lipid Luteolin down-regulates the expression of astrocyte elevated gene synthesis in intact cells.39 1 (AEG-1), a novel oncoprotein, and MMP-2.42 Relatively low Overall, luteolin may modulate multiple cell signaling pathways levels of luteolin significantly inhibit VEGF secretion in MDA- and target genes in breast cancer cells. These altered pathways lead MB-231 (4175) LM2 cells, suggesting that it has the ability to to cell cycle arrest, apoptosis, and inhibition of cell growth, migra- suppress a potent angiogenic and cell survival factor.44 Luteolin tion, and epithelial mesenchymal trans-differentiation (Fig. 2).

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Luteolin affects the effects of therapeutic drugs on breast on breast cancer can be investigated when combined with tradi- cancer tional endocrine therapy, chemotherapy, and/or targeted therapy for the current and new molecular subtypes. The efficacy of luteo- lin in clinical treatment of breast cancer remains to be determined. Luteolin enhances the effects of therapeutic drugs on breast can- It is also worthy of investigating whether luteolin is useful in male cer breast cancer and functional in novel personalized therapeutic ap- proaches such as genomics-based therapies and immunity-based Several studies have provided evidence that luteolin can be used therapies.76–78 as a chemosensitizer for antitumor agents.63–68 Luteolin adminis- tered at doses less than 100 µM reverses tumor resistance of 4T1 and MCF-7 cells to doxorubicin and promotes death of tumor cells Conclusion under hypoxia in vitro, although it has only slight effect on cell growth and cytotoxicity of doxorubicin under normoxia. The same Luteolin is active in inhibiting the proliferation, progression and 64 was found in 4T1 and MCF-7 bearing mice. In these models, metastasis of breast cancer in a dose-dependent manner through luteolin suppressed glycolytic flux but did not affect glucose up- suppressing cell cycles, inducing apoptosis, and inhibiting migra- take, the P-glycoprotein, antioxidative enzymes under hypoxia in tion and EMT of breast cancer cells. These effects of luteolin are vitro, or the intratumor doxorubicin level in vivo associated with achieved through multiple signaling pathways, such as Notch, increased and decreased activity of SOD and CAT in serum and in PI3K/Akt, MAPK/ERK1/2, EGFR, and NF-κB (Fig. 2). tumor, respectively.64 The combination of celecoxib and luteolin induced synergistic and inhibitory effects on the growth of cancer cells in vivo via Akt inactivation and ERK signaling inhibition in Future research directions MCF-7 and MCF7/HER18 cells and via Akt inactivation and ERK signaling activation in MDA-MB-231 and SkBr3 cells.65,66 Numerous studies have supported that luteolin is a promising drug Luteolin reduces drug-resistance of human breast cancer cells 69 for breast cancer therapy. To push its clinical use, several issues to tamoxifen via the inhibition of cyclin E2 expression. Luteo- need to be addressed and solved in the future, including (1) de- lin enhances paclitaxel-induced apoptosis in human breast cancer 70 termining the efficacy of luteolin combined with traditional en- MDA-MB-231 cells by blocking STAT3. These studies suggest- docrine therapy, chemotherapy, and /or targeted therapy for the ed that luteolin may enhance the effect of chemotherapeutic drugs current and new molecular subtypes on breast cancer, (2) clinical on breast cancer by reducing drug resistance and promoting apop- research on the efficacy of luteolin in treatment of several subtypes totic effects and inhibition of breast cancer cell growth. of breast cancer, (3) determining whether luteolin is useful in male breast cancer, and (4) determining if luteolin is functional in novel Low doses of luteolin protect breast cancer cells from cytotoxic- personalized therapeutic approaches such as genomics-based ther- ity of therapeutic drugs apies and immunity-based therapies.

71 In a study by Sato et al. luteolin was found to have biphasic Acknowledgments effects on viability of the human breast cancer cell line MCF-7. Luteolin at low concentrations, as low as 10 µM, was shown to protect the MCF-7 cells from doxorubicin toxicity, while in con- This work was supported by the Natural Science Foundation of trast, it inhibited cell viability at higher concentrations (>30 µM). Hubei Province, China (Grant# 2016CFB530), the Faculty De- Luteolin at 10 µM also attenuated doxorubicin-induced cytotoxic- velopment Foundation of Hubei University of Medicine (Grant# ity in the presence of the ER antagonist ICI 182 780 and in the 2014QDJZR01), the Science and Technology Research Project MDA-MB-453 human breast cancer cell line. Luteolin reduced of Health and Family Planning Commission of Hubei Province doxorubicin-induced ROS generation and increased the antiapo- (WJ2017Q038), and the Hubei Provincial Department of Educa- ptotic protein Bcl-2 levels in MCF-7 cells treated with doxoru- tion (Q20172104). bicin. This may contribute to the ameliorating effects of luteolin on doxorubicin-induced cytotoxicity of MCF-7 cells.71 Therefore, the dosage of luteolin is a critical factor for determining its effects Conflict of interest on breast cancer. The authors have no conflict of interest related to this publication.

Current challenges and future research directions Author contributions Breast cancer is a complex disease with a large degree of inter- and intratumoral heterogeneity and personalized therapy should be Wrote the manuscript and provided funds (YW, JW, and XGu); assumed for cure.72–74 Although ER, PR, HER-2 and Ki-67 have wrote the manuscript (XGo and XW); revised the manuscript for been used for molecular classification of breast cancer and have important intellectual content (XGu); contributed equally to the achieved good outcomes, numerous subtypes of breast cancer have work and should be regarded as co-first authors (YW, JW). been identified based on novel molecular biomarkers,75 this surely will lead to better understanding and cure of breast cancer in the future. On the other hand, the corresponding therapeutic drugs References should also be developed. The inhibitory effects and underlying mechanisms of luteolin [1] Kocic B, Kitic D, Brankovic S. Dietary flavonoid intake and colorec-

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90 DOI: 10.14218/JERP.2018.00011 | Volume 3 Issue 3, August 2018 Letter to the Editor

Re: Evaluation of Diagnostic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bacteriuria among Pregnant Women in Ashanti Region, Ghana

Chenglong Wang1, Jiaming Wang2, Yingdan Gu3, Danqing Pang1 and Dong Yin4*

1Faculty of Graduate Studies, Guangxi University of Chinese Medicine, No. 179 Mingxiu Dong Road, Nanning 530001, Guangxi Zhuang Autonomous Region, China; 2Health School, Zhoushan Tourism and Health College, No. 99 Xueyuan Road, Zhoushan 316000, Zhejiang Province, China; 3Faculty of International Education, Guangxi University of Chinese Medicine, No. 179 Mingxiu Dong Road, Nanning 530001, Guangxi Zhuang Autonomous Region, China; 4Department of Orthopedics, The People’s Hospital of Guangxi Province, No. 6 Taoyuan Road, Nanning 530001, Guangxi Zhuang Autonomous Region, China

Dear Sirs, ranted.

We read with great interest the article by Acheampong et al.1 regarding evaluation of the diagnostic methods and antimicrobial Acknowledgments susceptibility pattern of asymptomatic bacteriuria (ASB) among pregnant women in developing countries. This well-written arti- No source of funding was used for this study. This article does not cle topic provides very important for women suffering from ASB contain any protocols or procedures with human participants or and who are living in the developing world. The incidence of animals performed by any of the authors. ASB was relatively high in the study; therefore, the authors call for screening every pregnant woman and concerting efforts of all stakeholders to formulate a national policy to help curb this Conflict of interest situation in the developing world. We praise the authors for their having carried out serious analytical work to obtain the finding that the dipstick diagnostic method is more efficient than micros- The authors have no conflict of interests related to this publication. copy. However, we would like to communicate some ideas to the authors. Author contributions 1. The limitation of the study was that the diagnosis of ASB relied on a single urine sample. This is not entirely in ac- Drafted or revised the manuscript (CW, JW, YG, DP), approved cordance with the Infectious Diseases Society of America the final version (DY, CW). guidelines2 for the diagnosis of ASB in adults, and two consecutive urine specimens are required with isolation of the same bacterial strain in women. References 2. These are results from a small study, and larger, prospec- tive studies are needed to learn more about the topic. A [1] Acheampong DO, Afoakwah MK, Boye A, Opoku R, Kwakye-Nuako long-term and further study should be conducted to help us G, Adokoh CK, et al. Evaluation of diagnostic methods and antimi- understand the subject comprehensively and objectively. crobial susceptibility pattern of asymptomatic bacteriuria among pregnant women in Ashanti region, Ghana. J Explor Res Pharmacol 2018;3(3):78–84. doi:10.14218/JERP.2018.00003. The authors have made a great contribution towards this topic [2] Nicolle LE, Bradley S, Colgan R, Rice JC, Schaeffer A, Hooton TM. related to the welfare of the pregnant women living in the devel- Infectious Diseases Society of America guidelines for the diagnosis oping countries. It could be a valuable reference for clinicians; and treatment of asymptomatic bacteriuria in adults. Clin Infect Dis however, further strict studies with large sample sizes are war- 2005;40(5):643–654. doi:10.1086/427507.

Keywords: Asymptomatic bacteriuria; Antimicrobial agent; Diagnostic methods. Abbreviations: ASB, asymptomatic bacteriuria. Received: June 23, 2018; Accepted: June 29, 2018 *Correspondence to: Dong Yin, Department of Orthopedics, The People’s Hospi- tal of Guangxi Province, No. 6 Taoyuan Road, Nanning 530001, Guangxi Zhuang Autonomous Region, China. Tel: +8607712186970; E-mail: [email protected] How to cite this article: Wang C, Wang J, Gu Y, Pang D, Yin D. Re: Evaluation of Diagnostic Methods and Antimicrobial Susceptibility Pattern of Asymptomatic Bac- teriuria among Pregnant Women in Ashanti Region, Ghana. J Explor Res Pharmacol 2018;3(3):91. doi: 10.14218/JERP.2018.00015.

Journal of Exploratory Research in Pharmacology 2018 vol. 3 | 91

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