Journal of Pharmacy and Pharmacology

Journal of Pharmacy and Pharmacology Volume 4, Number 5, May 2016 (Serial Number 30)

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Publication Information Journal of Pharmacy and Pharmacology is published monthly in hard copy (ISSN 2328-2150) by David Publishing Company located at 616 Corporate Way, Suite 2-4876, Valley Cottage, NY 10989, USA. Aims and Scope Journal of Pharmacy and Pharmacology, a monthly professional academic journal, covers all sorts of researches on Pharmacokinetics, Biopharmaceutics, Pharmaceutical Analysis, Pharmaceutical Biotechnology and Drug Delivery, Pharmaceutical Outcomes and Policy, Pharmacy Administration, Advanced Pharmacology, Experimental Method and Technique of Pharmacology, Clinical Pharmacology, Medical Statistics, Pathophysiology, and Medicinal Chemistry, as well as other issues related to Pharmacy and Pharmacology. Editorial Board Members Dr. Jinhua Zhang (Canada), Dr. Preetpal Singh Sidhu (USA), Dr. Xiaoming Xie (China), Dr. Young Jin Chun (Korea), Dr. Sumio Chono (Japan), Dr. İnci Selin (Zorkun) DOĞAN (Turkey), Dr. Katarzyna Kieć-Kononowicz (Poland), Dr. Horng-Jyh Harn (Taiwan), Dr. Michele Navarra (Italy), Dr. Jordi Caballé Serrano (Spain), Dr. Leonardo Luiz Gomes Ferreira (Brazil), Dr. Qiliang Cai (China), Dr. Susruta Majumdar (India), Dr. Swati Misra (India), Dr. Junyan Liu (China), Dr. Andre Filipe de Barros Vieira (Portugal), Dr. Beom-Jin Lee (Korea), Dr. Farzin Roohvand (France), Dr. Yuanye (Vickie) Zhang (China), Dr. Shayli Varasteh Moradi (Iran), Dr. Haibin Zhou (China). Manuscripts and correspondence are invited for publication. You can submit your papers via E-mail to pharmacy@ davidpublishing.com or [email protected]. Submission guidelines are available at http://www.davidpublisher.com. Editorial Office 616 Corporate Way, Suite 2-4876, Valley Cottage, NY 10989, USA Tel: 1-323-984-7526, 323-410-1082; Fax: 1-323-984-7374, 323-908-0457 E-mail: [email protected], [email protected] Copyright©2015 by David Publishing Company and individual contributors. All rights reserved. David Publishing Company holds the exclusive copyright of all the contents of this journal. In accordance with the international convention, no part of this journal may be reproduced or transmitted by any media or publishing organs (including various websites) without the written permission of the copyright holder. Otherwise, any conduct would be considered as the violation of the copyright. The contents of this journal are available for any citation. However, all the citations should be clearly indicated with the title of this journal, serial number and the name of the author. Abstracted / Indexed in Database of EBSCO, Massachusetts, USA InfoBase Index Universe Digital Library S/B, ProQuest Summon Serials Solutions, USA Google Scholar (scholar.google.com) Chinese Database of CEPS, American Federal Computer Library Center (OCLC), USA Universe Digital Library Sdn Bhd (UDLSB), Malaysia China National Knowledge Infrastructure (CNKI), China Subscription Information Price (per year): Print $520, Online $320, Print and Online $600. David Publishing Company 616 Corporate Way, Suite 2-4876, Valley Cottage, NY 10989, USA. Tel: 1-323-984-7526, 323-410-1082; Fax: 1-323-984-7374, 323-908-0457 E-mail: [email protected] Digital Cooperative Company: www.bookan.com.cn

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Journal of

Pharmacy and Pharmacology Volume 4, Number 5, May 2016 (Serial Number 30)

Contents Original Articles 183

Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease Phuong Kim Huynh, Lap Vu Cong, Xuan Quang Truong and Canh Nguyen Xuan

192

Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy? Jakobsons Eriks, Cakstina Inese, Erglis Kristaps, Erglis Martins, Pirtniece Liene, Elina Krumina, Narbute Inga, Briede Ieva , Jegere Sanda, Smolova Rita, Muiznieks Indrikis and Erglis Andrejs

199

The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage Molaba Gloria Mmadira and Motaung Shirley Keolebogile

212

Computational Calculations of Molecular Properties and Molecular Docking of New and Reference Cephalosporins on Penicillin Binding Proteins and Various β-Lactamases Shakir Mahmood Alwan

226

Biodegradable Protection for Medical Devices with Medical Drugs Controlled Separation Milentina V. Seregina, Evgeny А.Nemets, Alina A. Akhmedova, Pavel B. Kurapov and Elena Yu. Bachtenko

231

The Effect of Gnaphalium oxyphyllum var. natalie on Cancer and Blood Cells Elisa Vega-Avila, Shindu Irais Gómez-Covarrubias, Adolfo Espejo-Serna, Elizabeth Hernández-Pérez and María de los Angeles Aguilar-Santamaría

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Journal of Pharmacy and Pharmacology 4 (2016) 183-191 doi: 10.17265/2328-2150/2016.05.001

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Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease Phuong Kim Huynh, Lap Vu Cong, Xuan Quang Truong and Canh Nguyen Xuan, Cho Ray Hospital, 201B Nguyen Chi Thanh Street, District 5, Ho Chi Minh city (+084), Viet Nam Abstract: To investigate diagnostic accuracy of 2-hour protocol of rest thallium-201/stress technetium-99m sestamibi dual-isotope myocardial perfusion SPECT in chronic coronary artery disease. Sixty-seven patients with suspected CAD were enrolled in the prospective study. All patients underwent myocardial perfusion scintigraphy in SPECT with dual isotopes of Tl-201 and dipyridamole stress Tc-99m sestamibi. Rest and stress imaging protocol were performed in 2 hours by dose of 3 mCi Tl-201 and 25 mCi Tc-99m sestamibi. The acquisition parameters includes LEHR collimator, energy peak of 72 and 167 keV for Tl-201 and 140 keV for Tc-99m, 180-degree rotation from RAO to LPO, matrix size 6464, and 25second/frame/64 frames. The 20-segment model of left ventricle was used in automatic quantitation software. Coronary angiography was used as gold standard. CAD was defined as 50% of lumen stenosis on coronary angiography. Rest Tl-201/stress tc-99m sestamibi dual-isotope SPECT demonstrated a sensitivity of 94.59% and specificity of 70%, positive predictive value of 79.54% and negative predictive value of 91.3% in detection of coronary artery disease. Sensitivity and specificity for detecting multi-vessel coronary artery disease were 82.75% and 81.57% for the left anterior descending, 77.77% and 91.83% for left circumflex and 94.11% and 82% for right coronary artery. 2-hour protocol of rest thallium-201/stress technetium-99m sestamibi dual-isotope myocardial perfusion SPECT has high sensitivity, specificity, positive predictive value and negative predictive value in detecting chronic coronary artery disease with greater than 50% stenosis assessed by coronary angiography. Moreover, this imaging protocol gives high imaging quality, time-saving and convenience. Key words: MPS (myocardial perfusion scintigarphy), SPECT (single proton emission computed tomography), CAD (coronary artery disease), CA (coronary angiography).

1. Introduction Although there was advanced treatment for coronary artery disease, this disease is still one of main mortality causes in the world. Therefore, continuously improved diagnostic methods for detecting early ischemic heart disease are necessary. Coronary angiography is gold standard for diagnosis of ischemic heart disease, but it is an invasive method. Now, there are many non-invasive diagnostic methods for detecting ischemic heart disease such as: stress electrocardiography, stress echocardiogram or Corresponding author: Phuong Kim Huynh, M.D., Ph.D., research fields: coronary artery disease, coronary angiography. Email: [email protected].

nuclear methods such as myocardial perfusion SPECT (single photon emission computed tomography) or PET (positron emission tomography) with different isotopes. Myocardial perfusion SPECT is advantage non- invasive study that not only accessing perfusion, but also accessing heart function, viability and prognostic as well. In recent 20 years, nuclear cardiology has been developed strongly and there are many researches about using myocardial SPECT for detecting ischemic heart disease. Routine imaging procedure of myocardial perfusion SPECT can be acquired with thallium or with any 99mTc-labeled radiopharmaceutical. In more than recent 10 years,

184 Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease

dual-isotope technetium-99m and Thalium-201 imaging procedure lasted 2 hours demonstrates high diagnostic values for detecting ischemic heart disease. Dual-isotope SPECT myocardial perfusion scintigraphy was introduced in 1993 with Tl-201 at rest and Tc-99m sestamibi (MIBI) at stress. Since that time, this protocol has become widely used for the diagnosis and assessment of prognosis in patients with known or suspected CA (coronary artery) disease in a wide variety of clinical settings. We conducted research “Rest thallium-201/stress technetium-99m sestamibi dual-isotope myocardial perfusion

single-photon

emission

computed

tomography in detecting of chronic coronary artery disease” with purposes as followings below: Investigate

sensitivity,

specificity,

positive

predictive value, negative predictive value of 2-hour protocol of rest thallium-201/stress technetium-99m sestamibi dual-isotope myocardial perfusion SPECT in chronic coronary artery disease, and diagnose stenosed coronary artery as coronary angiographic stenosis on PTCA is gold standard (CA stenosis criteria ≥ 50%) Investigate

correlation

between

angiographic

stenosis on PTCA and segments of myocardial perfusion

defect

thallium-201/stress

on

2-hour

protocol

technetium-99m

of

rest

sestamibi

dual-isotope SPECT (CA stenosis criteria ≥ 50%) This research was the first study about role of myocardial perfusion SPECT with 2-hour protocol of rest thallium-201/stress technetium-99m sestamibi Table 1

2. Patient and Methods 2.1 Patient Population From a consecutive series of patients who underwent a study of myocardial perfusion SPECT with Rest thallium-201/stress technetium-99m sestamibi dual-isotope imaging procedure and coronary artery angiography, 67 were selected and represent the population of this study. The criteria to enter the study were as follows: (1) suspected coronary artery disease (2) and agree to attend this research. The criteria to exclude the study were as follows: (1) recent myocardial infarction (< 1 month), (2) unstable angina, (3) congestive heart failure occurring 1 month before the study, (4) valve heart disease, (5) arrythmiasm (block AV II or III, sinus node disfunction), (6) unstable hypertension, (7) scleroderma, (8) hyperthyroid, (9) severe fever, (10) severe underlying diseases, (11) chronic obstructive pulmonary disease. All patients were in stable hemodynamic condition over the study period. 2.2 Study Protocol Baseline studies included 2-hour protocol of rest thallium-201/stress technetium-99m sestamibi dual-isotope myocardial perfusion SPECT and coronary angiography. The rest study is usually performed first. 3milicuries

Imaging parameters based on SPECT imaging’s SPECT imaging of Americant Society of Nuclear Cardiology [3-6].

Isotope dose Collimator Detector Energy Window Turn angle Picture number 2D Matrix

dual-isotope in chronic coronary artery disease in Viet Nam.

Rest T1- 201: 3 mCi LEHR 400mm 72 KeV W: 30% 167 KeV W: 20% 1800 (-450 RAO-> 1350 LPO) 64 pictures and 25 second/picture 64 x 64

Stress Tc-99m mibi: 25 mCi LEHR 400mm 140 KeV 0

W: 15% 0

180 (-45 RAO-> 1350 LPO) 64 pictures and 25 second/picture 64 x 64

Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon 185 Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease

of thallium was administered at rest. The acquisition can begin as soon as 15 minutes after injection. After the completion of rest study acquisition, a stress study is performed using Dipyridamole with dose 0,568 mg/kg/4 min. for stress pharmaceutical test and a technetium-labeled myocardial perfusion imaging agent with dose 25-30 milicuries of Tc-99mibi injected as soon as 7 minutes after starting of stress test by Dipyridamole. Imaging can be 30 minutes after injection of Tc-99 mibi. The images should be reviewed before the patient is released. In all patients, calcium antagonist, digoxin and oral nitrates had been withdrawn for at least 48 hours, β-blockers for at least 72 hours, and transdermal nitrates for at least 12 hours before the protocol study. Theophylline and caffeine also had been withdrawn for at least 24 hours before the protocol study. Informed consent was obtained form each patient before the protocol study (which was approved by the institutional ethical committee) [17-20].

2.4 Dual Isotopes Imaging All patients underwent rest Tl-201/stress Tc-99m sestamibi tomography as previously described. In each patient, 8 consecutive midventricular slices of 3 sections including short axis, vertical long axis, horizontal long axis for both rest and stress phage. Tomogram were then divided into 6 sectors of equal arc, representing segments such as: 1: antero (apex), 2: anteroseptal (apex), 3: posteroseptal (apex), 4: inferior (apex), 5: posterolateral (apex), 6: anterolateral (apex), 7: antero (mid), 8: anteroseptal (mid), 9: posteroseptal (mid), 10: inferior (mid), 11: posterolateral (mid), 12: anterolateral (mid), 13: antero (base), 14: anteroseptal (base), 15: posteroseptal (base), 16: inferior (base), 17: posterolateral (base), 18: anterolateral (base), 19: apex (anterior), 20: apex (posterior). Thus 20 myocardial regions were analyzed for each patient. Of these segments, 9 segments (number 1, 2, 3, 7, 8, 13, 14, 19, 20) were assigned to the territories of the left anterior descending coronary artery, 6 segments

2.3 Coronary Angiography

(number 5, 6, 11, 12, 17, 18) were assigned to the

All angiograms were analysed by an investigator blinded to dual isotopes perfusion SPECT data. Coronary angiographic result was gold standard of chronic coronary artery disease. Vessel stenosis was defined in left main, LAD (left anterior descending coronary), RCA (right coronary artery), LXC (left circumflex artery) and stenosis diameter ≥ 50% was significant stenosis and positive result. Collateral circulation was also defined dominantly in right or left circulation [2, 8, 9].

territories of the left circumflex coronary artery, and 5 segments (number 4, 9, 10, 15, 16) were assigned to the territories of the right coronary artery (Fig. 1). Alignment and analysis of the study were visually made by operator who was unaware of the echocardiography result and coronary angiography result. 5-point scoring system (0, normal uptake; 1, mild defect; 2, moderate defect; 3, severe defect; and 4, absent tracer uptake) was used to compute a summed STRESS Inject 20 – 25mCi Tc – 99m

REST Injest 3mCi Tl - 201

O Fig. 1

REST SPECT

15

STRESS STRESS

40

Rest Tl-201/stress Tc-99m sestamibi dual-isotope study protocol.

SPECT

60

90

186 Rest Th hallium-201/S Stress Techne etium-99m Se estamibi Dua al-Isotope Myo ocardial Perffusion Single-Photon Emission Computed Tom mography in Detecting of Chronic Coro onary Artery Disease

Fig. 2 Chartts showing divvision of left ventricle into 20 segments in sh hort axis, long vertical axis aand long horizo ontal axis. Red d segments werre assigned to LAD, L pink segm ments were assigned to LCx,, white segmen nts were assign ned to RCA.

stress scoree, a summedd rest score, and a sum mmed difference score, as described d prreviously. Thhese summed scoores were connverted into the t percentagge of myocardium m that was abnormal, fixedd, and reversible, respectively, by use of a standard appproach [11,, 12, 14-16]. 2.5 Statisticaal Methods Statisticall analysis waas performedd with the SPSS S 10.5 statisttical packagge. Continuous data ware w expressed as mean ± SD D and categgorical data as a a percentage. The unpaiired t-test (for continuuous variables) and a the χ2 -teest (for categgorical variabbles) were used as appropriiate. Value P < 0.05 was considered statistically significant. Sensitivity and specificity of SPECT T results versus v coronnary angiographyy were calcullated with the usual form mulas with the associated 95% confiddence intervvals. Correlationss between continuous variables were w assessed by regression annalysis. The anallysis was performed p inn patients who underwent myocardial perfusion scintigraphy by SPECT, percutaneouss translum minal coronnary angiographyy, in order to access sensittivity, specificity, negative preedict value, positive p predict value of dual isotopes SPE ECT as coronnary angiograaphy was deffined gold standarrd for diagnoosis of ischem mic heart disease and LAD, LCx, L RCA corronary artery lesion ≥ 50% %.

The T correlation analysis was conduccted betweenn myo ocardial terriitories assignned by LAD, LCx, RCA A and d culprit coronnary artery.

3. Result R This T study waas comprised of 67 patien nts underwentt duaal isotopes myocardial perfusion scintigraphyy SPE ECT and coroonary angiogrraphy. Tab ble 2

Patient characteristics c s.

All Age (y) 57 (40-70 0) Sex x: M/F 49/18 Rissk factors for CA AD Hyp percholesteroleemia 59 (88,1% %) Hyp pertension 46 (68,7% %) Sm moking 20 (29,9% %) Diaabetes 15 (22,4% %) Obesity 15 (22,4% %) milial history foor CAD 21 (31,3% %) Fam LV VEF (%) < 50% 5 %) 19 (28,4% ≥ 50% 5 48 (71,6% %) Streess ECG (+) 19 (28,4% %) Streess ECG (-) 35 (52,2% %) Streess EHO (+) 31 (46,3% %) Streess ECHO (-) 23 (34,3% %) Dual-isotope SPEC CT (+) 44 (65,7% %) Dual-isotope SPEC CT (-) 23 (34,3% %) CA A (+) 37 (55,2% %) CA A (-) 30 (44,8% %) CA: cardial angioggraphy; CAD, ccoronary artery y disease; ECG,, electrocardiographyy; ECHO, elecctrocardiograph hy; LVEF, leftt ECT: single prroton emissionn venttricular ejectionn fraction; SPE com mputed tomograpphy. Valu ues represent median m (25th-755th percentile), frequency (N),, or mean m ± SD.

Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon 187 Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease Table 3

Dual-isotope myocardial perfusion SPECT.

Perfusion defect level on SPECT Reversibility Partial reversibility Fixed Table 4

Percent %

8 12 17

21,6 32,4 46

Segments of perfusion defect on Dual-isotope myocardial perfusion SPECT scintigraphy relating to stenosed CA.

Segments of perfusion defect Table 5

Case number

LAD 135

LCx 65

RCA 71

Total number 271

Result of PTCA. All cases PTCA (+) n = 37 cases n = 67 CA

Percent %

PTCA PTCA (+) 37 PTCA (-) 30 1-vessel CAD 16 2-vessel CAD 13 3-vessel CAD 7 4-vessel CAD 1 LAD stenosis 8 LCx stenosis 4 RCA stenosis 4 LAD + LCx stenosis 6 LAD + RCA stenosis 7 LAD + LCx + RCA stenosis 5 LAD + LCx + LM stenosis 2 LAD + LCx + RCA + LM stenosis 1 LAD, left anterior descending; LCx, left circumflex artery; PTCA, percutaneous transluminal coronary artery. Table 6

55,2 44,8 43,2 35,1 19 2,7 21,6 10,8 10,8 16,2 18,9 13,5 5,4 2,7 coronary angiography RCA; right

CA stenosis diagnosed by PTCA. All CA (n = 67) (LAD, RCA, LCx, LM) 25 39

CA stenosis ≥50% 27. So that, this resu ult should bee con nsidered and 5 cases of poositive SPECT T myocardiall perffusion was faalse or true. The T mechaniisms underlyying the prroduction off perffusion defectts in dilated cardiomyopaathy or heartt failu ure remain unclear. u Prevvious studies showed thatt non nhomogeneouus perfusion defects werre associatedd with h myocardiall fibrosis. In n 9 cases of o false posiitive SPECT T myocardiall perffusion, there were 4 cases of hypertensiion. This wass

190 Rest Thallium-201/Stress Technetium-99m Sestamibi Dual-Isotope Myocardial Perfusion Single-Photon Emission Computed Tomography in Detecting of Chronic Coronary Artery Disease

explained by abnormally blood retension of CA in hypertension patients causing perfusion defecting on SPECT as CA stenosis ≤ 50%. Our study had 23 cases of negative SPECT myocardial perfusion, in which 2 cases is false negative SPECT myocardial perfusion and true negative SPECT myocardial perfusion. 2 cases of false negative happened on female patients with BMI ≥ 35. This was explained by obesity of patients in our study. Furthermore, our study also demonstrated sensitivity for diagnosing stenosis of CA. The result showed 94.1% for RCA, 82.8% for LAD and 77.8% for LCx. Our study’s result was differed from the result of Wacker Fran J. TH et al. [24, 25] represented that sensitivity for detecting stenosis of LAD is higher this of LCx and RCA (80%, 70% and 63% respectively). Similarly, study of Nishu et al. revealed that sensitivity for diagnosing stenosis of LAD and RCA is same (75%, CA stenosis ≥ 50%) and higher than this of LCx (60%) [21]. However, result of Van Train KF et al. showed that sensitivity for detecting stenosis of RCA is higher than this of LAD and LCx (77%, 69% and 70%, respectively) [24]. So that, majority of results showed that the sensitivity for detecting LAD stenosis was highest. The discrepancy may be explained by cases of false positive SPECT myocardial perfusion regarding to LAD, so that sensitivity for diagnosing LAD decreased subsequently. In our study, the sensitivity for diagnosing stenosis of LCX is lowest. The detecting perfusion defect segments relating to LCx is more difficulty than these to LAD and RCA, even as using qualitative analysis. This may be explained myocardial segments perfused by LCx is posterior and far from imaging detector and artifacts caused by diaphragm and breast. In our study, the specificity for diagnosing LCX stenosis was higher than this LAD and RCA stenosis (91.8%, 81.6% and 82%, respectively). The study of Nishu et al. on 101 patients also revealed result same as our study [22].

Sensitivity and specificity in our study were same as or higher than these in studies used mono-isotope SPECT myocardial perfusion scintigraphy [11, 12, 14, 15, 19, 22, 24, 25]. Imaging protocol of rest Tl-201 and stress Tc-99m mibi dual-isotope SPECT myocardial perfusion scintigraphy in our study was conducted among only 2 hours with optimal picture quality, which is significant for solving patient congestion at public urban hospitals in Vietnam.

5. Study Limitations The generalizability of our findings is a potential limitation, as it is a single-site study. Refferal bias may be effect on specificity in our study. Our study was also limited because the number of patients was not too many.

6. Conclusion Our study was the first study in Vietnam about 2-hour dual-isotope imaging protocol (rest Tl-201/stress Tc-99m mibi) myocardial perfusion SPECT in detecting of chronic coronary artery disease. The quality SPECT image, perfect imaging parameter as well as short imaging duration lasted only 2 hours is meaningful for solving overcrowded patients in Vietnam’s central hospitals. These results of our study suggest potentially complementary roles of non-invasive technique as dual-isotope myocardial perfusion SPECT scintigraphy in the detection of patients with suspected CAD.

Acknowledgment There are no financial conflicts of interest for our study.

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Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy? Jakobsons Eriks2,3, Cakstina Inese2, Erglis Kristaps2, Erglis Martins2, Pirtniece Liene2, Elina Krumina2, Narbute Inga1,3, Briede Ieva1 , Jegere Sanda1,3 , Smolova Rita1, Muiznieks Indrikis4 and Erglis Andrejs1,3 1. Latvian Centre of Cardiology, Pauls Stradins Clinical University Hospital Riga, LV-1002, Latvia 2. Cell Transplantation Centre, Pauls Stradins Clinical University Hospita,l Riga, LV-1002, Latvia 3. Research Institute of Cardiology, University of Latvia, Riga, LV-1586, Latvia 4. Department of Biology, Division of Microbiology and Biotechnology, University of Latvia, Riga, LV-1586, Latvia Abstract: Different cell populations from bone marrow are used in various clinical trials for cardiac diseases during last decade. Four clinical studies are on going in our institution and enrol patients with cardiac diseases, coronary disease and type 2 diabetes, patients with osteoarthritis. Density gradient is used to separate bone marrow mononuclear cells. Cell processing looses are significant. To find out critical control points we analysed processing process and differences in cell yields between operators performing cell extraction. Bone marrow mononuclear cells were isolated using Ficoll density gradient centrifugation. Cells were counted using flow cytometry for mononuclear cell total counts, CD34+ population count and viability analysis. The patients who underwent bone marrow aspiration followed by cell isolation received cell suspension for transplantation. Two cells processing for separate patients were performed at once. Same standard operation procedures were applied. Processing looses between operators performing cell extraction were analysed. Bone marrow samples from eight patients were processed. Mononuclear cells were extracted. Operator performances were compared. Operator A average bone marrow mononuclear cell yield in starting material was 9,97 ± 9,98 %, CD34+ population yield 75,46±79,67%. Operator B average bone marrow mononuclear cell yield in starting material was 24,68 ± 14,8 %, CD34+ population yield 70,42 ± 44.84%. Operator A average cell viability in starting material was 45,24 ± 9,55%, after cell processing 42,96 ± 23,66 %. Operator B average cell viability in starting material was 49,85 ± 5,48%, after cell processing 69,52 ± 6,65 %. Key words: Bone marrow, mononuclear cells, cell yield, cell extraction, cell count

Abbreviations BM MNC

bone marrow; mononuclear cell

1. Introduction Different cell types are evaluated for their regenerative potential and therapeutic applicability for cardiac tissue regeneration. Studies are focusing on the use of cells isolated from bone marrow, peripheral blood, skeletal muscle, adipose tissue and umbilical cord, all of which have shown to improve cardiac function in animal models [1-3]. Amongst possible cell sources autologous bone marrow derived

Corresponding author: Erglis Andrejs, Prof., M.D., Ph.D., research fields: biology, microbiology and biotechnology.

mononuclear cells (BM-MNC) and their subpopulations are most extensively being tested in clinical trials [4-6]. Many unresolved questions concerning cell transplantation still exist including the exact mechanism of the beneficial effect of cell transplantation, the optimal transport of cells into the myocardium, the type and amount of cells implanted, the timing of transplantation, the assessment of responsiveness of individual patients to the cell therapy, and the amount of transplanted cells required to achieve a significant myocardial regeneration [7]. BM-MNC isolation methods are well established; manual or automated processing is performed to produce cell suspensions for transplantation. Currently different cell delivery methods are employed in clinical

Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy? 193

praxis, including intracoronary injection and intramyocardial injection. The questions remaining unanswered yet are effective dose of transplanted cells and best timing for cell transplantation [8-10]. Numbers of transplanted BM-MNCs varies among clinical trials [11, 12] and there is little known about cell isolation and CD34+ enrichment efficacy during processing of bone marrow aspirate. In this study we compared BM-MNCs yields between operators performing cell processing and if it has effect on cell isolation efficiency.

2. Materials and Methods 2.1 Bone Marrow Harvesting and Cell Preparation Bone marrow mononuclear cells were harvested by iliac crest puncture performed under local anaesthesia. For adult patients a 38 to 45 ml of bone marrow was aspirated into heparin pre-filled syringes (500 U/ml of bone marrow aspirate). The bone marrow aspirate was shipped at room temperature to the central cell-processing laboratory and further processed under Good Manufacturing Practice. In short, aspirate was diluted with sterile 0.9% NaCl (1:5) (BBraun), filtrated through 100 µm cell strainer (BD Biosciences), and bone marrow mononuclear cells (BM-MNCs) were isolated and enriched by density gradient with the use of Ficoll-Paque Premium (GE Healthcare Ltd.) according to manufacturers instruction, with minor protocol modifications. Two biotechnologists at least to minimise risks of faults and ensure minimal processing time perform bone marrow processing. All activities, timing, used reagents, batch numbers, shelf life and personnel involved recorded to bone marrow processing file. Each processing is documented in detail and is coded with specific identifier to ensure processing traceability. Processing starts with workplace preparation. Two laminar hoods are switched on. Laminar hood sash closed and UV sterilisation for 10 min. applied. Laminary hood air cascade switched on and after

airflow stabilisation wiped with disinfectant agent. Sterile instruments, disposable materials, racks and liquid waste containers placed in hood. Reagents before placing in laminary hood are checked if shelf life is valid and if container sealing is intact. For each separate bone marrow processing intact reagent containers are used. Centrifuges are wiped with disinfectant agent and switched on. Senior biotechnologist make certain if sterile room ventilation cascade is working according to present parameters. Air microbiological conditions are checked by routine microbiological air monitoring program. Head of laboratory make certain if procedures of work place preparation are performed according to standard operation procedure and authorize processing. Bone marrow aspirate is shipped at room temperature in marked transport container. By receiving BM sample biotechnologist check sample coding and record incoming biological material file. BM sample is forwarded to laminar hood using incoming material route. Before placing BM and reagents container in laminar biotechnologist wipes containers with antibacterial agent. Special attention should be applied to container lids. Prepare 1 L 0,9% sodium chloride and heparin solution, end concentration 10 U/ml of heparin. Mark container with container opening date and heparin concentration. Divide BM sample in 6 separate 50 ml Falcon centrifugation tubes. Samples transfer with 10 ml sterile serological pipette. Add sodium chloride and heparin solution to reach 30ml volume in each Falcon tube. Place in laminar 6 separate 50 ml Falcon centrifugation tubes marked with „F” for filtration. Samples filtrate using 100 µm cell strainers. Place the cell strainer in the Falcon tube. Use sterile 25 ml serological pipette to place BM solution on strainer. Used cell strainers remove to solid waste container. If filtration is interfered by blood cloths or bone debris, replace cell strainer with new.

194 Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy?

For initial cell count analysis 10 μl of filtrated solution was diluted in 990 μl 0,9% sodium chloride solution. Sterile filter containing micropipette tip was used to take sample. Fill 15 ml of Ficoll-Paque Premium in 6 separate 50 ml Falcon centrifugation tubes. To remove excess ficol drops on tube walls do centrifugation of tubes for 1 min at 600 x g. The 25 ml serological pipette used to pour BM solution on density gradient layer. Falcon centrifugation tube placed in 45-degree angle to laminar work place surface. The slowest discharge speed applied to minimise risks of density gradient and BM solution mixing. BM solution discharged on Falcon tube wall approximately 5 cm from ficoll surface. When 15 ml of solution discharged Falcon tube is slowly turned to 90-degree angle position. Pouring of BM solution on density gradient should not exceed 20min for all tubes. Present centrifugation speed 800 x g, 25 minutes, + 21 oC temperature, turn off centrifuge brake mode. Make sure if all tubes contain the same volume of sample, place tubes symmetric and start centrifugation. After centrifugation 3 fractions were formed-top plasma phase, interphase containing mononuclear cells and lower phase containing mostly erythrocytes and thrombocytes. Part of plasma phase was slowly removed by serological pipette. Pipette tip was dipped in top layer of plasma phase and followed as level drops. Slowest suction mode was applied to minimise liquid turbulences that can disturb integrity of buffy coat. Approximately 7 ml of plasma phase was left in tube. Removed plasma was discharged to liquid waste container. The 10 ml serological pipette was used to remove buffy coat. Slowest suction mode was applied. Pipette tip was dipped down to ficol layer and slowly moved over ficol surface like carpet vacuuming. Up to 7ml of cell containing fraction form each tube was transferred to sterile tube. Tubes containing cell fraction were

filled with 0,9% sodium chloride and heparin solution up to 50 ml total volume. Sample tubes were closed and mixed by few times gently turning upside down. Centrifugation followed by 800 x g, 20 min, temperature + 21 oC, centrifugation starting speed and brake on maximum. After centrifugation supernatant was removed and pellet resuspended in 0,9% sodium chloride and heparin solution up to 50 ml total volume. Centrifugation followed by 600 x g, 20 min, temperature + 21 oC, centrifugation starting speed and brake on maximum. After centrifugation supernatant was removed and pellet resuspended in 0,9% sodium chloride solution without heparin up to 50 ml total volume. Centrifugation followed by 600 x g, 20 min, temperature + 21 oC, centrifugation starting speed and brake on maximum. After centrifugation supernatant was removed and 1ml left in each centrifugation tube. Pellet was resuspended in left supernatant. The 10ml sterile serological pipette was used to unite all samples in one and add 0,9% sodium chloride solution up to 15 ml. Mark tube as final material. For final cell count analysis 250 μl of cell solution was aliquoted. Sterile filter containing micropipette tip was used to take sample. Final cell solution was filtrated using 100 µm cell strainers. After filtration solution was filled in sterile container and packed in sterile secondary package and marked. Facility authorised person make sure if processing comply with standard operation procedure and sign permit to release product for application. Biotechnologist forward cell count samples for testing. 2.2 Flow Cytometry Samples from BM and final product (BM-MNCs) were counted and used for flow cytometric analysis within 2h after preparation. Stem kit from Beckman


Coulter was used for cell labeling with CD45-FITC,

4. Discussion

CD34-PE, 7-AAD and Stem-Count fluorospheres.

Due to ease of harvesting bone marrow is most convenient source of MNCs applicable for both acute and chronic diseases. Processing of bone marrow in order to isolate the mononuclear cell fraction is associated with cell loss. Some studies, investigating human bone marrow processing, show BMNC recovery rates between 15 to 30% after ficoll density gradient centrifugation. Processing has a major impact on the cell counts, viability and functional activity of bone marrow derived progenitor cells. The assessment of cell counts and viability may not entirely reflect the functional quality of cells in clinical application. Some groups suggested that controversial clinical effects in large-scale clinical trials are due to technological differences in cell processing and BMNC composition [13]. In fact, it has been proven that efficacy and functionality of BMNCs are significantly influenced by red blood cell by the contamination, the content of apoptotic cells, differences in washing steps and centrifugation speed. In this study we focus on bone marrow derived mononuclear cell yields obtained from eight BM processing that were performed using same methodology for all patients. Loses of therapeutically effective cell populations during all steps of density gradient separation negatively correlates with red blood cell and granulocyte

Cells were analysed using FC-500 (Beckman Coulter). Analysis protocol was developed manually. Stem CXP program was used for MNC, CD34+ cell count and cell viability detection. Gating was performed according to ISHAGE

protocol

according

to

manufacturer

suggestion. Cell viability was obtained using 7AAD method that is included in ISHAGE protocol (Keeney et al., 1998). Each measurement contained at least 50,000 events. Maximum number of events was 100, 000. Obtained numbers of cells/µL were calculated for total number of MNC and CD34+ cells within transplantation material. Measurements with less than 50,000 events were excluded.

3. Results Bone marrow mononuclear cells were harvested by iliac crest puncture performed under local anaesthesia. All samples contained 43 ml of bone marrow solution. Four pairs of BM processing were performed. Separate work places and equipment were used. Same standard operation procedures were applied for all processed bone marrow samples. Each final cell solution was released for clinical application. Start and final product cell counts were analysed by flow cytometry (see Table 1). Table 1 No. 1.

2.

3.

4.

Cell counts starting BM processing and after processing. Comparison of operator’s performance.

Operator Operator MNC CD34+ Viability % MNC CD34+ Viability % MNC CD34+ Viability % MNC CD34+ Viability %

Start cell count mln A 551.00 1.03 53.49 485.00 0.21 40.60 255.00 0.21 34.00 96.30 1.14 52.85

Final cell count mln A 80.56 1.88 70.01 0.80 0.00 12.70 8.61 0.07 40.90 20.90 0.98 48.23

Yield % 14.62 182.52 0.16 0.00 3.38 33.33 21.70 85.96

Start cell count mln B 55.00 0.25 41.86 138.00 1.46 50.73 253.00 1.61 52.94 163.00 0.82 53.85

Final cell count mln B 23.50 0.29 65.98 13.40 0.32 62.58 40.40 0.71 76.92 49.40 0.82 72.60

Yield % 42.73 116.00 9.71 21.58 15.97 44.10 30.31 100.00

196 Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy?

contamination that probably can cause unwanted clinical effects in order to get higher cell yields. Studies describing the BMNC clinical applications use BMNC processing methods vary considerably from study to study and are not fully described. General standard operation procedures for cell extraction are not established till now. These differences may result in inconsistencies that may affect study clinical outcomes [7]. Many factors can influence material quantitative and functional quality and the study result comparisons are difficult. Factors that could affect the cell processing outcome is associated with bone marrow extraction: instruments used - aspiration needle specification (side

holes, diameter), aspiration needle placement (depth, angle, radio control used), the applied negative pressure, administration of anticoagulation agents, peripheral blood volume in the sample, as well as the patient individual specificities – iliac crest thickness and internal lumens (varies depending on age, gender and health conditions). Despite factors mentioned above each individual operator performance can influence quantitative and functional quality of cell preparation for clinical application. Results obtained in this study clearly show differences in processing efficacy (Figs. 1 and 2.) Both operators are experienced-operator A have processed more than 140 BM samples in period of seven

Fig. 1

CD34+ population extraction yield. Operator A and B performance comparison.

Fig. 2

Mononuclear cell population extraction yield. Operator A and B performance comparison.


Table 2

Average MNC and CD34+ population yields and standard deviation.

Operator A Operator B Table 3

MNC 9.97 24.68

StDev 9.98 14.80

Start 45.24 9.55

Operator A Final 42.96 23.66

years. Operator B has performed more than 50 BM processing during two years. Comparison of average yields despite BM differences of each individual patient, show that stability of operator performance varies significantly (see Tables 2 and 3.)

5. Conclusion Operator performance seems can affect cell and tissue processing yields. Processing performance analysis show - no significant differences between operators were found, except minor differences in buffy coat transfer. Operator B transfer nearly all plasma phase, operator A leave approximately 1 ml of plasma phase over density gradient layer. Sample size is too small to prove statistical significance of finding, more data is needed to study buffy coat transfer impact on cell extraction efficacy.

Acknowledgements This study was supported in part by Latvian National Research Program "Biomedicine for Public Health" (BIOMEDICINE) and by grant from corporation “Sistemu Inovacijas”.

References

[2]

StDev 79.67 44.84

Average cell viability of starting /final material and standard deviation.

Average viability StDev

[1]

CD34+ 75.46 70.42

Giuseppe, A., Sabrina, S., Viviana, L. C., Francesco, S., Daniel, S., Lucia, T., Gianni, S., Tiziano, M. 2010. “Bone Marrow Derived Stem Cells in Regenerative Medicine as Advanced Therapy Medicinal Products.” Am. J. Transl. Res. 2 (3): 285-95. Lachtermacher, S., Esporcatte, B. L., da Silva de Azevedo, F., Rocha, N. N., Montalvao, F., et al. 2012. Functional and Transcriptomic Recovery of Infracted Mouse Myocardium Treated with Bone Marrow Mononuclear Cells.” Stem Cell Rev. 8: 251-61.

Start 49.85 5.48 [3]

Operator B Final 69.52 6.45

Korf-Klingebiel, M., Kempf, T., Sauer, T., Brinkmann, E., Fischer, P., et al. 2008. “Bone Marrow Cells Are a Rich Source of Growth Factors and Cytokines: Implications for Cell TherapyTrials after Myocardial Infarction.” Eur. Heart J. 29: 2851-8. [4] Lunde, K., Solheim, S., Aakhus, S., Arnesen, H., Abdelnoor, M., et al. 2006. “Intracoronary Injection of Mononuclear Bone Marrow Cells in Acute Myocardial Infarction.” N. Engl. J. Med. 355: 1199-209. [5] Schachinger, V., Erbs, S., Elsasser, A., Haberbosch, W., Hambrecht, R., et al. 2006. “Intracoronary Bone Marrow-Derived Progenitor Cells in Acute Myocardial Infarction.” N. Engl. J. Med. 355: 1210-21. [6] Strauer, B. E., Brehm, M., Zeus, T., Kostering, M., Hernandez, A., et al. 2002. “Repair of Infarcted Myocardium by Autologous Intracoronary Mononuclear Bone Marrow Cell Transplantation in Humans.” Circulation 106: 1913-8. [7] Clifford, D. M., Fisher, S. A.,Brunskill, S. J., Doree, C., Mathur, A.,Watt, S., and Martin-Rendon, E. 2012. “Stem Cell Treatment for Acutemyocardial Infarction.” Cochrane Database of Systematic Reviews 2. [8] Matoba, S., Tatsumi, T., Murohara, T., Imaizumi, T., Katsuda, Y., et al. 2008. “Longterm Clinical Outcome after Intramuscular Implantation of Bone Marrow Mononuclear Cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] Trial) in Patients with Chronic Limb Ischemia.” Am. Heart J. 156: 1010-8. [9] Leri, A., Kajstura, J., Anversa, P., and Frishman, W. H. 2009. “Myocardial Regeneration and Stem Cell Repair.” Current Problem in Cardiology 33: 91-153. [10] Bartunek, J., Vanderheyden, M., Hill, J., and Terzic, A. 2010. “Cells as Biologics for Cardiac Repair in Ischaemic Heart Failure.” Heart 96: 792-800. [11] Aktas, M., Radke, T. F., Strauer, B. E., Wernet, P., and Kogler, G. 2008. “Separation of Adult Bone Marrow Mononuclear Cells Using the Automated Closed Separation System Sepax.” Cytotherapy 10: 203-11. [12] Florian, H. S., Torsten, T., Nicola, K., Andreas, M. Z., and Stefanie, D. 2007. “Cell Isolation Procedures Matter: A Comparison of Different Isolation Protocols of Bone

198 Bone Marrow Mononuclear Cell Extraction. Does the Operator Performance Affect Processing Efficacy? Marrow Mononuclear Cells Used for Cell Therapy in Patients with Acute Myocardial Infarction.” European Heart Journal 28: 766-72 [13] Van Beem, R. T., Hirsch, A., Lommerse, I. M., Zwaginga, J. J., Noort, W. A., et al. 2008. “Recovery and

Functional Activity of Mononuclear Bone Marrow and Peripheral Blood Cells after Different Cell Isolation Protocols Used in Clinical Trials for Cell Therapy after Acute Myocardial Infarction.” Euro. Intervention 4: 133-8.


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The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage Molaba Gloria Mmadira and Motaung Shirley Keolebogile Tshwane University of Technology, Faculty of Science, Department of Biomedical Sciences, Private Bag X 680, Pretoria, South Africa, 0001 Abstract: This study aimed to investigate the effects of resveratrol and bone morphogenetic protein 7 on type II collagen from superficial and middle zone of porcine articular chondrocytes. Articular cartilage was isolated from dissected porcine knee joint n = 12. Isolated cells were plated as monolayers at a density of 1 × 105 cells/well in 12-well culture plates and incubated at 37°C in a humid atmosphere of 5% carbon dioxide and 95% air. Cell cultures were treated for four days with various concentrations of bone morphogenetic protein-7 and resveratrol. Cells were then collected and analysed for collagen type II expression by real time polymerase chain reaction and protein level quantification by enzyme-linked immunosorbent assay. Cartilage tissue sections were localised for collagen type II by immunohistochemistry. Moreover, resveratrol and bone morphogenetic protein-7 effects on cartilage matrix contents were analysed by histology. Resveratrol and bone morphogenetic protein-7 stimulates expression of collagen type II mRNA and protein level accumulation in the surface zone and middle zone at 50 µM + 300 ng/ml (RSV + BMP-7). Immunohistochemistry results confirmed the presence of collagen type II on articular cartilage. Histological tissue sections confirmed that chondrocytes were obtained from different zones of articular cartilage. The study suggests that a combination of bone morphogenetic protein-7 and resveratrol up-regulate the expression and synthesis of collagen type II. Key words: Articular cartilage, osteoarthritis, collagen type II, resveratrol, bone morphogenetic protein-7.

1. Introduction Articular cartilage is critical to the normal function of human and animal joints, providing lubrication and load-bearing to allow locomotion and movement. It is a uniquely avascular, aneural, and alymphatic tissue comprised of an ECM (extensive extracellular matrix) with very few cells [1]. Cartilage has a defined stratified structure composed of superficial, middle, deep, and calcified zones [2, 3], each with distinct cell densities and phenotypes, molecular architecture, and mechanical properties [4, 5]. It is composed of ECM macromolecules such as type II collagen, aggrecan, hyaluronan, chondroitin sulfate, and decorin. Collagen type II fibrils provide the tensile strength and maintain the integrity of mammalian articular cartilage by forming a network that resists the swelling pressure resulting from the hydration of the polyanionic Corresponding author: Dr SCKM Motaung, D. Tech. biomedical technology, research field: tissue engineering.

proteoglycan aggregates in the extracellular matrix. Damage to this fibrillar meshwork, made up of primarily type II collagen may be a critical event in the pathology of many arthritides, due in part to the very slow rate of collagen turnover within the cartilage [6-9]. On a microscopic scale, the collagen and proteoglycan content in the tissue varies with depth from the articulating surface. Collagen is the major organic constituent of cartilage and accounts for 15-22 % of the wet tissue weight [10-12]. Collagen content is highest in the surface zone and decreases by approximately 15 % in the middle and deep zones of the tissue [1, 4, 6-8]. Proteoglycan accounts for 4-7 % of the wet tissue weight [1, 13-15]. In contrast to the distribution of collagen content, proteoglycan content is lowest in the surface zone and increases by approximately 15 % in the middle and deep zones [6-8]. Water distribution throughout the tissue is similar to

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The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage

that of collagen, accounting for more than 80 % of the wet tissue weight at the surface and 65 % in the deep zone [8, 16]. The surface zone of the articular cartilage has flattened discoid cells that are aligned tangentially to the surface and secrete a proteoglycan called SZP (surface zone protein) [17, 18]. The surface zone of articular cartilage is responsible for appositional growth, and appears to contain progenitor cells [19]. The middle zone consists of spherical cells arranged in perpendicular columns and is characterised by type II collagen, aggrecan, and CILP (cartilage intermediate layer protein) [20]. The deep zone includes the calcified area of cartilage the tidemark distinguishes between the noncalcified and calcified areas [6, 7, 13]. The characteristic zonal architecture is intimately linked to the biological function [9, 15, 21]. The ECM (extracellular matrix) of cartilage contains numerous non-collagenous proteins, collagens and proteoglycans [22, 23]. The main type of proteoglycan found in cartilage is aggrecan, which provides the compressive strength of cartilage [24]. Others include decorin, biglycan and fibromodulin. One of the more prominent non-collagenous proteins is COMP (cartilage oligomeric matrix protein) [25-27]. Cartilage repair is dependent on the production of matrix by chondrocytes, which are stimulated by anabolic morphogenetic proteins, such as BMPs (bone morphogenetic proteins), TGF-β (transforming growth factor), and IGF- (1insulin-like growth factor-1), and the actions of catabolic cytokines such as IL-1β (Interleukin-1 beta), and TNF-α (tumour necrosis factor alpha). Changes in the cartilage homeostasis are thought to precede the shift to OA (osteoarthritis). It is well established that morphogens and growth factors play an important role in cartilage homeostasis [28-34]. Bone morphogenetic proteins are pleiotropic regulators of the cartilage and bone differentiation cascade including chemotaxis of progenitor cells, mitosis of mesenchymal stem cells, and differentiation of cartilage and bone. It has been shown that BMPs

induce new bone and cartilage formation in vitro and in vivo [28]. BMP-7 (bone morphogenetic protein-7), also called human OP-1 (osteogenic protein-1), plays an important role in human and bovine cartilage homeostasis and repair [30, 35-37]. Many studies have shown that BMP-7 and TGF-β1 (transforming growth factor-beta 1) can synergistically promote increased survival and matrix synthesis by normal and osteoarthritic human articular chondrocytes [31, 38-41]. Other growth factors, such as basic FGF-2 (fibroblast growth factor-2), IGF-1 (insulin-like growth factor-1) [42, 43] and PDGF (platelet-derived growth factor), have all been shown to be anabolic for cartilage and chondrocytes [41, 44]. RSV (resveratrol) is isolated from the roots of white hellebore. Resveratrol (3,5,4-trihydroxystilbene) is a polyphenolic phytoalexin compound found in various plants, such as grape vines, berries, peanuts, seeds and roots; the highest concentration is in the skin of red grapes [45-48]. This component of red wine has potent anti- inflammatory properties and may reduce the side effects of non-steroidal anti-inflammatory drugs that are currently used and may thus offer new opportunities for the treatment of OA. The anti-inflammatory effects of resveratrol have been shown in several animal model studies [49]. Therefore, resveratrol might be the relevant compound for potential use in osteoarthritis therapy. There is therefore a dire need to detect cartilage loss before it is severe. Natural products such as RSV do not have the disadvantages mentioned above like the non-steroidal anti-inflammatory drugs. This offers novel and alternative treatment opportunities for OA. The existence of traditional and complementary medicine is known to be a fertile ground source of western medicine. Both of these molecules RSV and BMP-7 are involved in bone and cartilage regeneration [50-52]. However, the regulation of collagen type II by RSV and BMP-7 in the different zones of porcine articular cartilage has not been fully investigated.


2. Materials and Methods 2.1 Tissue Acquisition for Cell Culture Total of 12 different porcine stifle (knee joint) from 3 month-old pigs, representing a sample size of 12 experiments were obtained from local abattoir and dissected under aseptic conditions to expose the femoral condyles as described by [53]. The superficial zone cartilage (100 μm) of the femoral condyles was harvested using a dermatome (Phoenix Surgical, Cape Town, SA). Osteochondral plugs were excavated from the femoral condyles using 3 mm diameter steel dermal puncher (Thermo Scientific, Waltham, MA) and the middle zone was removed from each plug using a custom cutting jig. 2.2 Isolation and Monolayer Culture The superficial zone was digested with 0.2% collagenase- P (Roche Pharmaceuticals, Nutley, NJ, USA) for 45 minutes and the middle zone for 1hour. Chondrocytes were plated as monolayers at a density of 1 x 105 cells/well in 12-well culture plates and incubated at 37 0C in a humid atmosphere of 5% carbon dioxide and 95% air. Chondrocytes were cultured overnight in DMEM/F-12 medium (Life Technologies, Carlsbad, CA, USA) containing 1% fetal bovine serum (FBS; Thermo Scientific). The next day the medium was changed to serum-free DMEM/F-12 medium with insulin transferrin selenium (ITS) + Premix (Life Technologies). Cells were then treated with different concentrations of RSV (20 µM, 50 µM and 100 µM) [54] and BMP-7 (100 ng/ml and 300 ng/ml) [53] and incubated for 4 days. Untreated control was assigned for all the experiments. 2.3 RT-PCR (Real Time Polymerase Chain Reaction) Analysis Total RNA was extracted from cultured monolayer cells using RNeasy mini kit (Qiagen, Valencia, CA, USA) with on-membrane DNase 1 (Qiagen) digestion to avoid genomic DNA contamination. Extracted RNA

201

quality and quantity were checked by Qubit 2.0 fluorometer instrument (Life Technologies) accordingly. Total RNA was reverse-transcribed into single- stranded cDNA, using Veriti® Thermal Cycler (Life Technologies). Real time quantitative PCR was performed in triplicate on cDNA with StepOne plus Real-Time PCR sequence detector system and Tag-Man gene expression reagents (Life Technologies), following the recommended protocols. Collagen type II mRNA levels were normalized to GAPDH (glyceraldehydes 3-phosphate dehydrogenase) levels and expressed relative to the control (untreated) culture levels (∆CT methods; Life Technologies). The primers for collagen type II (Ss 03373345 g1) and GAPDH (Ss03375435 µL) were predesigned by Life Technologies. 2.4 ELISA (Enzyme-Linked Immunosorbent Assay) Analysis of Collagen Type II Protein An ELISA kit was used for quantitative determination of collagen type II (MD Biosciences, St. Paul, MN, USA) levels in monolayer cell culture. The assay is based on colorimetric based immunoassay. The collagen type II ELISA is a heterogeneous sandwich ELISA. A monoclonal anti-collagen type II antibody is pre-coated onto a 96-well microplate and any collagen type II present in the sample will be bound to the microplate by the antibody. A secondary biotinylated antibody is then added and will bind to the immobilized antigen forming the antibody-antigen complex. Substrate is then added and converted by the enzyme to produce a quantifiable coloured product that is in direct proportion to the amount of antigen bound in the initial reaction. 2.5 Immunolocalization of Collagen Type II Full-thickness articular cartilage sections were dissected from porcine femoral chondyles. The specimens were fixed with 10% neutral buffered formalin (Sigma-Aldrich) and embedded in paraffin. Tissue sections (5 μm thick) were prepared, sectioned

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vertically from the surface to the bottom and collagen type II was localised using novalink min polymer detection system kit (Leica Microsystems, Newcastle, UK). The sections were deparaffinised and endogenous peroxidase was blocked with 1 % hydrogen peroxidase. The sections were then incubated over night at 40C with a 1:1000 dilution of rabbit polyclonal collagen type II antibody (Leica Microsystems). The sections were incubated with biotinylated link antibody. No primary antibody was applied in the control experiment. Visualisation was achieved using DAB chromagen resulting in a brown precipitate evaluated microscopically. Control experiments were incubated with PBS only. Images were obtained on Leica DMIL LED biological microscope with appropriate photographic software (Leica Microsystems) at 10X magnification. 2.6 Histological Analysis To confirm that cartilage slices had been acquired from the specific zones of articular cartilage, consecutive formalin-fixed and paraffin-embedded tissue sections were stained with 1% toluidine blue, safranin

O,

Masson’s

trichrome

and

haematoxylin-eosin staining for histologic evaluation using standard procedure. Each experiment was assigned a set of control and treated sections with RSV (20 µM, 50 µM and 100 µM) and BMP-7 (100 ng/ml and 300 ng/ml). Images were obtained on a Leica DMIL LED biological microscope with appropriate photographic software (Leica Microsystems) at 10 X magnification 2.7 Statistical Analysis The sample size for all experiments was twelve (n = 12), which represent twelve different animals. All the quantitative data were presented as means ± standard deviations. ANOVA (A one-way analysis of variance) with Tukey’s HSD (Honestly Significant Differences) to account for multiple comparisons was used to determine the effects of RSV and BMP-7 on collagen

type II expression in different zone of articular cartilage. A paired t-test was performed to determine the difference between the control and treated cells. P-values less than 0.05 were considered significant for all comparisons.

3. Results 3.1 Chondrocytes Morphology Analysis Articular chondrocytes from the superficial and middle zones were cultured as monolayers. Before treatment, the chondrocytes morphology is similar in both surface and middle zone cultures (Figs. 1A-1D and 1E-1H). Both cultures were then treated with various concentrations of RSV (20 µM, 50 µM and 100 µM) and BMP-7 (100 ng/ml and 300 ng/ml) and incubated. After day one of treatment, there is a slight change in cell morphology with chondrocytes differentiating and moving away from a round shape nature (Figs. 2B-2D and 3B-3D). Control cell morphology after day one treatment remains the same as cells before treatment (Figs. 2 A and 3.A). The effect of RSV and BMP-7 was successfully observed through transition of chondrocytes from round to an elongated polygonal morphology on day four of incubation (Figs. 2E-2H and 3E-3H). It was observed that there was more cell development on a concentration of RSV (50 µM) + BMP-7 (300ng/ml) in both surface and middle zone cultures (Figs. 2C and 3C) compared to control and other treatment concentrations on day four. 3.2 Influence of RSV and BMP-7 on Collagen Type II mRNA and Protein Level Articular chondrocytes obtained from the surface and middle zone were cultured for four days as monolayers and treated with different concentrations of RSV+BMP-7 (20 µM + 100 ng/ml, 50 µM + 300 ng/ml and 100 µM + 100 ng/ml) and TGF-β1 (3 ng/ml). Collagen type II mRNA expression was assessed by means of RT-PCR as shown in Figs. 4A and 4C) and collagen type II protein level was

The Use U of Bone Morphogenet M tic Protein-7 and a Resverattrol in Collagen Type II of Articular Carrtilage

quantified by b means of ELISA E as shhown in Figs. 4B and 4D). Thhe insignificaant in collageen type II mR RNA expression and proteinn levels byy surface zone z

(a)

Fig. 1

(b)

2033

ondrocytes was observed on TGF-β1 as comparedd cho to RSV R + BMP--7 (50 µM + 300 ng/ml), Figs. F 4A andd 4B)). Treatment with w RSV andd BMP-7 at 20 2 µM + 100

(c)

(d)

(e) (f) (g) (h) A-D Represents R sup perficial zone chondrocytes c b before treatment and E-H miiddle zone chondrocytes befo ore treatment.

(a)

(b)

(c)

(d)

(e) (f) (g) (h) CONTROL 20µM RS SV+100ng/ml BMP-7 B 50µM M RSV+300ng/m ml BMP-7 1000µM RSV+100ng/ml BMP-7 R sup perficial zone chondrocytes c a after treatmentt by different concentrations c s of RSV: 20 µM, 50 µM and d Fig. 2 A-D Represents 100 µM and BMP-7: B 100 ngg/ml and 300 ng/ml n on Day 1 and E-H reprresents superfi ficial zone chon ndrocytes afterr treatment byy different conccentrations of RSV: R 20 µM, 50 5 µM and 1000 µM and BMP P-7: 100 ng/mll and 300 ng/m ml on Day 4.

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(a)

(b)

(c)

(d)

(g) (e) (f) (h) 20µM RS ONTROL SV+100ng/ml BMP-7 B 50µM RSV+300ng/m ml BMP-7 1000µM RSV+100n ng/ml BMP-7 CO Fig. 3 A-D Represents miiddle zone choondrocytes afteer treatment by b different cooncentrations oof RSV: 20 µM M, 50 µM and d 100 µM and BMP-7: 100 ng/ml n and 300 ng/ml on Dayy 1 and E-H represents r mid ddle zone chon ndrocytes afterr treatment byy different conccentrations of RSV: R 20 µM, 50 5 µM and 1000 µM and BMP P-7: 100 ng/mll and 300 ng/m ml on Day 4.

ng/ml for 4 days d significaantly enhanceed the expresssion of Collagen type II mRN NA at 1.27 fold f increase and collagen typpe II protein level at the concentration of 1.30 ng/ml in i the surfacee zone as in Figs. 4A andd 4B compared too the middle zone where the t expressioon of collagen typpe II mRNA A expressionn was 1.64 fold increase andd protein conncentration was w 1.32 ng/m ml as in Figs. 4C and a D. Treatment with RSV and BMP-7 at 50 µM + 300 ng/ml for four days significantlyy enhanced the expression of o Collagen type II mRN NA at 1.50 fold increase (Figg. 4A) and coollagen type II I protein levvel at the concenttration of 1..49 ng/ml (F Fig. 4B) in the superficial zone z compareed to the middle zone where w the expressiion of collagen type II mRNA m expresssion was 2.53 fold increase (Fig. 4C C) and prootein concentratioon was 1.56 ng/ml (Fig. 4D). Howeever, treatment with w RSV andd BMP-7 at 100 µM + 100 ng/ml for foour days signnificantly deecreased collaagen type II exprression relatiive to the coontrol had a 0.58

fold d increase and 1.19 nng/ml conceentration forr superficial zonee (Figs. 4A and 4B) an nd 1.09 foldd incrrease and 1.15 ng/ml for m middle zone (Figs. ( 4C andd 4D)). A higher increase i in thhe expression n of collagenn typee II mRNA and a protein leevel was obseerved at RSV V (50 µM) + BMP P-7 (300 ng/m ml concentratiion as shownn in Fig. 4B coompared to the controll and otherr con ncentrations (F Figs. 4B and 4D) 3.3 Immunohistoochemistry Annalysis Negative N conntrol (withoutt primary an nti-body) andd positive controll (with prim mary anti-bod dy) untreatedd carttilage tissue sections s prepaared were ob bserved underr a light l microsccope for coollagen type II stainingg (Fig g. 5). Posittive stainingg was iden ntified by a brow wnish colourr with round chondrocyte morphologyy (Fig gs. 5B and 5D). Negaative (withou ut anti-bodyy incu ubation) andd positive (inncubated witth anti-body)) treaated with RS SV (50 µM) and BMP-7 (300 ng/ml)) prep pared cartilaage tissue secctions were also a observedd


Fold Increase

Four‐day Surface Zone RT‐PCR

**

*

** **

Treatment groups (a)

ng/ml collagen type Il

Four‐day Surface Zone ELISA

**

*

*

Treatment groups (b)

Four‐day Middle Zone RT‐PCR Fold Increase

**

*

*

Treatmnet groups (c)

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ng/ml collagen type II

Four‐day Middle Zone ELISA

*

*

**

Treatment groups (d) Fig. 4 (A) Indicates the effects of RSV and BMP-7 on collagen type II mRNA expression on the surface zone. (B) Indicates the effects of RSV and BMP-7 on collagen type II protein level on the surface zone. (C) Indicates the effects of RSV and BMP-7 on collagen type II mRNA in the middle zone and (D) Indicates the effects of RSV and BMP-7 on collagen type II protein level in the middle zone. Articular cartilage surface and middle zone chondrocytes were treated with different concentrations of RSV and BMP-7 (20 μM+100 ng/ml, 50 μM+300 ng/ml and 100 μM+100 ng/ml). Untreated cells and TGFβ1 (3 ng/ml) served as positive control. A significance change of * represents a p value = 0.05 and ** p value = 0.01.

under the light microscope for collagen type II staining (Figs. 5C and 5D). Sections were no primary antibody was added, showed no staining demonstrating the reliability and validity of the method (Figs. 5A and 5C). Larger chondrocytes emerging from the deeper zone of the RSV 50 μM and BMP-7 300 ng/ml treated cartilage section (Figs. 5C and 5D) compared to the control sections (Figs. 5A and 5B) were observed. The staining was strong in the superficial zone, mild in the middle zone and decreasing further in the deep zone. 3.4 Histology Analysis Histological evaluation confirmed that cartilage tissue sections had been obtained from the superficial and middle zones of articular cartilage. The upper layer had small cells with flattened or ellipsoidal cellular morphology, whereas the middle and lower layers had large cells with oval or round cellular morphology. The upper zone had the highest cellularity, followed by the middle zone and lower zone. Cells of the superficial zone were smaller than cells of the middle and deep zones (Fig. 6). Tissue sections’ staining was positive

for articular cartilage matrix components. The intensity of toluidine blue was highest in the lower zone of the cartilage (Figs. 6A and 6E). Safranin O staining, demonstrating proteoglycan content was highest in the lower zone, followed by the middle zone and upper zone (Figs. 6B and 6F). The intensity of Masson’s trichrome staining was most noteworthy in the middle zone, followed by the upper zone and lower zone, which directly relates to collagen cartilage matrix distribution (Figs. 6D and 6H). The purpose of the Masson’s trichrome stain is the demonstration of collagen and muscle in normal tissue and also to differentiate collagen and muscle in tumors. The middle zone had the highest collagen type II content, which was in line with the highest collagen content observed in Masson’s trichrome staining. Lastly, haematoxylin-eosin stained the cartilage matrix positive pink and the nucleus an orange to green colour (Figs. 6C and 6G). Control tissue sections (Figs. 6A-6D) showed smaller round, flattened chondrocytes when compared to larger chondrocytes emerging from the deeper zone of RSV: 50 µM and BMP-7: 300 ng/ml treated sections (Figs. 6E-6H).

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Control sections


Fig. 5 Untreeated collagen type II negativve control with hout primary antibody a (A) and a positive control with prim mary antibodyy (B) tissue. Trreated (RSV: 50 5 µM and BM MP-7: 300 ng/m ml) collagen typ pe II negative control c withou ut primary anttibody (C) and d positive contrrol with primary antibody (D D) tissue section ns, observed att 10X magnificcation. Scale bar = 200 µM.

Fig. 6 Reprresentation of cartilage c tissue staining. Control tissue seections stained with Toluidin ne blue (A), Sa afranin O (B),, Haematoxylin n & Eosin (C) and Masson’s trichrome (D)) and Treated tissue sections E-H (RSV: 500 µM and BMP P-7: 300 ng/mll) observed at 10X Magnificattion. Scale barr, 200 µm.

4. Discussiion Cartilage is an avascuular tissue, whhich is unablle to self-heal or regenerate after injury [20, 53, 56--60]. Three types of cartilage are a hyaline (aarticular), fibrous and elastic cartilage c [61] Articular carrtilage consists of

fourr different zones: z superfficial, middlle, deep andd calccified zones, which diffeer in matrix composition,, morrphology, meechanical andd metabolic prroperties [56,, 61]. Study of the t differentt cartilage zones aim too perm mit a bettter understaanding of stimuli thatt up-rregulates bioosynthesis off collagen ty ype II in thee

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chondrocytes subpopulation. Resveratrol has also proved to have chondro-protective effects on articular cartilage chondrocytes with concentrations between 25 μM and 50 μM [62]. The present study demonstrates that collagen type II mRNA and protein level is expressed in surface and middle zone chondrocytes culture (Figs. 4A-4D). However, the middle zone culture expressed more collagen type II mRNA and protein level as compared to the surface zone. Possible explanation might be that collagen type II protein is highly expressed in the middle zone of articular cartilage [55,63]. Although all the concentrations showed expression, the highest expression was shown on the 50 µM + 300 ng/ml (RSV + BMP-7) for both surface and middle zone of RT-PCR and ELISA (Figs. 4A-4D). BMP-7 has the ability to reduce the progression of OA. The expression of anabolic genes, aggrecan and collagen type II is increased when cartilage is treated with BMP-7 [64]. Resveratrol has extensive biological properties including anti-inflammatory, cardiovascular, anti-carcinogen and anti-aging effects [65, 66]. Immunohistochemistry confirms the presence of

of RSV and BMP-7 may stimulate the production of collagen type II in articular cartilage zones. Collagen type II was expressed in both the surface and middle zone after being treated with different concentrations of RSV (20 µM, 50 µM and 100 µM) and BMP-7 (100 ng/ml and 300 ng/ml). This study has provided new information about the synthesis and secretion of collagen type II in different zones of articular cartilage when treated with RSV and BMP-7. Furthermore, regulation of collagen type II will be of great importance for tissue engineering to review different zones of articular cartilage. In conclusion we can safely conclude that the results support our hypothesis that the in vitro administration of RSV and BMP-7 to cultures of articular chondrocytes up-regulate the expression and synthesis of collagen type II. Further research is needed to focus on exploring the mechanism of action of RSV in combination to BMP-7 and other growth factors on other articular cartilage matrix proteins such as collagen type I, X, aggrecan and SOX 9 before in vivo studies.

collagen type II. The staining was strong in the superficial zone, mild in the middle zone and decreasing further in the deep zone (Fig. 5D). This localisation of collagen type II confirms the observations reported in other studies [67]. Histological tissue sections confirmed that cartilage

The authors wish to acknowledge the financial support of the National Research Foundation of South Africa and the Department of Biomedical Sciences, Tshwane University of Technology for financial and technical support.

slices used had been obtained from different zones of articular cartilage. We also observed increased intensity of the stains on treated tissue sections which suggests that RSV (50 μM) + BMP-7 (300 ng/ml) stimulate synthesis of the cartilage collagen matrix (Figs. 6E-6H). These results on cell distribution and

6. Funding

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The data of this study confirm that the combination

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Computational Calculations of Molecular Properties and Molecular Docking of New and Reference Cephalosporins on Penicillin Binding Proteins and Various β-Lactamases Shakir Mahmood Alwan Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Bab Al-Moadham, P. O. Box 14026, Iraq Abstract: An approach of using molinspiration calculations and molecular docking on PBPs (penicillin-binding proteins) and certain β-lactamases is employed to predict the molecular properties, bioactivity and resistance of newer and reference cephalosporins. The previously synthesized cephalosporins 1-8 and reference cephalosporins were subjected to extensive evaluations by calculating the molecular properties, drug-likeness scores on the bases of Lipinski’s rule and bioactivity prediction using the method of molinspiration web-based software. The TPSA (topological polar surface area), OH-NH interactions, n-violation and the molinspiration Log partition coefficient (miLogP) values were also calculated. The investigated cephalosporins were subjected to molecular docking study on PBPs (1pyy) and on β-lactamases produced by S. aureus, K. pneumonia, E. coli and P. auroginosa using 1-click-docking website. Molecular properties of 1-8 recorded higher TPSA than cephalexin and were lower than the reference cephalosporins and do not fulfill the requirements for Lipinski’s rule. Bioactivities of 1-8 were predicted to be less and their docking scores on PBPs were comparable to those of the reference cephalosporins, particularly ceftobiprole. The references recorded various docking scores on the above β-lactamases and as expected, ceftobiprole recorded the lowest scores on all β-lactamases. Cephalosporins 1-8 recorded various docking scores on β-lactamases. Molecular docking studies on PBPs and β-lactamases are considered as very useful, reliable and practical approach for predicting the bioactivity scores and to afford some information about the stability and selectivity of the newly proposed cephalosporins against β-lactamases of certain pathogenic microbes, such as P. auroginosa and MRSA, by recording the relative docking scores in comparison with those of reference cephalosporins. Key words: Cephalosporins, Molinspiration, Molecular docking, β-lactamases, Lipinski’s rule.

1. Introduction Drug resistance is a serious situation that limits the treatment choices of infections caused by MRSA (methicillin-resistant Staphylococcus aureus). MRSA strains have spread worldwide and are considered as the most dangerous threat to humans [1-4] and the outbreaks of MRSA in the community have increased alarmingly. The widespread resistance of MRSA to β-lactam antibiotics has made treatment of infections by these antibiotics extremely difficult [5]. Besides, Corresponding author: Shakir Mahmood Alwan, Ph.D. medicinal chemistry, assistant professor, chemical synthesis of new antimicrobial agents and prodrugs.

strains of S. aureus have also become resistant to so-called “drugs of last choice” including vancomycin, linezolid and daptomycin [6-8]. The development of potent new drugs is one of the most difficult and complicated processes in pharmaceutical industry. Cephalosporins are the most widely used β-lactam antibiotics for treatment of bacterial infections and perform their action through covalent binding with PBPs, thus inhibiting the final step in cell wall biosynthesis. The development of bacterial resistance is a major concern that encourages the development of new resistant antibiotics towards bacterial β-lactamases. Cephalosporins have different

Computational Calculations of Molecular Properties and Molecular Docking of New and Reference Cephalosporins on Penicillin Binding Proteins and Various β-Lactamases

antibacterial spectra, β-lactamase sensitivity/ resistance and pharmacokinetic properties [9]. The fifth generation cephalosporins, ceftobiprole [10] and ceftaroline [11] are characterized by having unique spectrum on G (-) bacteria (powerful anti-Pseudomonal activity) and an expanded spectrum against G (+) bacteria include MRSA. Their activities are beyond all other cephalosporins and appear to be less susceptible to develop resistance. Ceftobiprole exhibits a high level of affinity for PBPs of MRSA [12]. CADD (computer-aided drug design) approach aims to shorten the time and tedious procedures and provide better efficiency in the processes of drug discovery. This approach provides more details and aid to coordinate the information to make the drug design more rational [13-15]. Rational drug design helps to facilitate and fasten the procedures of drug designing process, which includes various methods to identify and select the novel potent compounds. Molecular docking of a drug molecule with a certain receptor is one of these approaches [16].Docking is the binding or interaction of a ligand with targeted receptor in the 3-D (three dimensional structure) spaces in order to study the molecular properties and degree of binding that reflect its bioactivity [17-19]. There is an increasing interest and potential application of this approach in the field of drug design and discovery. The antibacterial activity of novel α-amino acid functionalized fluoroquinolones is validated by molecular docking studies and is in good correlation with the experimental results [20]. Docking study of polycyclic quinolone-based molecules revealed that these compounds recorded weak cytotoxic effects and poor binding affinity to human topoisomerase [21]. Results of docking paclitaxel and its analogues on target proteins tubulin B-1 chain and B-c1-2 showed various docking scores and predicted only two analogues as more suitable than paclitaxel [22]. Molecular docking studies of cyclic octapeptide (D-proline-incorporated wainunuamide) revealed more potent affinity for HPV18-2IOI (HeLa cancer cell lines) and exhibited

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better antitumor activity [23]. In view of the cumulative information, it was considered that CADD may be useful to design new cephalosporins of great potential and have better activities and properties, particularly β-lactamase resistant and antipseudomonal activity. Molecular docking as one of the CADD strategies was used for providing extensive molecular modeling calculations, bioactivity

prediction

and

docking

scores

of

cephalosporins to PBPs and β-lactamases from different sources. This approach may aid in the discovery of novel potent cephalosporins that are resistant to β-lactamases. Based on the author’s knowledge, no docking study arising from the use of cephalosporins on β-lactamases have been reported, so far. Application of this approach was attempted on the previously synthesized cephalosporins [24], and selected reference cephalosporins representing the five generations that are susceptible or resistant to β-lactamases to confirm its validity.

2. Experimental 2.1 The Investigated Cephalosporins Cephalosporins 1-8 were previously synthesized, characterized and identified and evaluated for their antimicrobial activities [24].The chemical structures of 1-8 are given as supplementary information on Fig. 1. Reference cephalosporins selected from the five generations, that are of various degree of stability against β-lactamases were used, such as, cephalexin and cefuroxime (susceptible to hydrolysis by β-lactamases) and ceftazidime, ceftriaxone, cefozopran, and ceftobiprole (resistant to β-lactamases). Using one of the chemoffice softwares (ChemDraw Ultra 10.0 program), the chemical structures of all the investigated cephalosporins and their SMILES notation were obtained. Their chemical structures are illustrated on Fig. 1 and chemical nomenclature are as follows; compounds 1-4 [Substitiuted-3-(acetoxymethyl)-7-(2-(5-benzylidenea


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N

N

R

R

N

S

H N

S O

N

S

N N

O OH

mino)-1,3,4-thiadiazol-2-yl-thio)-acetamido)-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid] and compounds 5-8 [Substituted-3-(acetoxymethyl)-7-(2-((5-benzylidenea mino)-1,3,4-thiadiazol-2-yl)-disulfanyl)acetamido)-8oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid]. Molecular

Properties

and

2.3 Docking Study on PBPs (Penicillin Binding Proteins) docking

study

S

O O

CH3

O

N OH

O

R= Cl, cephalosporins 2 and 6 R= NO2, cephalosporins 4 and 8

confirm and support the antimicrobial activities of cephalosporins 1-8 [24] and to be compared with reference cephalosporins of various activities to validate this approach. This study was conducted using 1-click-docking

software

(mcule.com)

and

the

chemical structures of PBPs were retrieved from the protein

data

bank

(PDB,

www.rcsb.org

(DOI:10.2210/pdb3b60/pdb). 2.4 Docking Study on Certain β-Lactamases

Lipinski’s rule of five [25, 26] was used to evaluate the drug-likeness and calculate the molecular properties that are essential factors for a drug pharmacokinetics, including ADME (absorption, distribution, metabolism and excretion). Molinspiration website-based software (www.molinspiration.com) was employed to obtain certain molecular parameters. The values of miLogP, as (octanol/water partition coefficient) and TPSA of the investigated cephalosporins were determined using the method developed by molinspiration [27]. Drug-likeness scores were calculated to represent the amount of fragments based on contributions and correction factors [28]. The prediction of bioactivity scores of these cephalosporins were calculated by recording the activity scores of GPCR (G-protein coupled receptors ligand), KI (kinase inhibitor), PI (protease inhibitor), EI (enzyme inhibitor), ICM (ion channel modulator) and NRL (nuclear receptor ligand).

The

H N

S

O

R= H, cephalosporins 1-5 R= Br, cephalosporins 3 and 7 Fig. 1 General structures of the investigated cephalosporins 1-8.

2.2 Calculation of Bioactivity Scores

S O

CH3

O

N O

S

of

the

investigated

cephalosporins on PBP2 (1pyy) was conducted to

The structures of the bacterial β-lactamases were retrieved from PDB. Different β-lactamases of various bacteria were used for docking study of these cephalosporins to calculate the docking scores of the binding energies and consequently, investigate the possibility of resistance toward the above types of β-lactamases. These types of β-lactamases are; PDB (ID: 1xgj) of E. coli, PDB (ID: 3q6x) of K. Pneumonia, PDB (ID: 1ome) of S. aureus and PDB (ID: 2wzz) of P. auroginosa

3. Results and Discussion 3.1 Molinspiration Calculations Molecular properties were calculated on the bases of Lipinski's rule and its components. The cephalosporins 1-8 have higher TPSA than cephalexin, particularly, 4 and 8 (196.98), which do not comply with Lipinski’s rule (Table 1). Furthermore, TPSA values of the reference cephalosporins are higher (except for cephalexin) and increasing with advancing in generations, particularly, ceftriaxone (221.61) and ceftobiprole (203.18) and these do not comply with


Lipinski’s rule (Table 1). These cephalosporins largely differ in their ADME properties. The values of OH-NH polar fragments representing the proton donors and proton acceptors of 1-8 and the reference cephalosporins were 2 and 3 to 6 respectively (Table 1). The OH-NH values centered polar fragments should be