Introduction of antineoplastic drug NSC631570 in an inpatient and outpatient setting: Comparative evaluation of biological effects

Mrii Ruyk*,Olexner Feorhuk,Yroslv Susk, Yroslv Nowiky,Lrys Skivk

aMicrobiology and General Immunology Department,ESC“Institute of Biology”,Taras Shevchenko National University of Kyiv,Ukraine

bR.E.Kavetsky Institute of Experimental Pathology,Oncology and Radiobiology,National Academy of Sciences of Ukraine,Ukraine

cO.O Bogomolets National Medical University,Kyiv,Ukraine

dUkrainian Anticancer Institute,Vienna,Austria

Introduction of antineoplastic drug NSC631570 in an inpatient and outpatient setting: Comparative evaluation of biological effects

Mariia Rudyka,*,Olexander Fedorchukb,Yaroslav Susakc, Yaroslav Nowickyd,Larysa Skivkaa

aMicrobiology and General Immunology Department,ESC“Institute of Biology”,Taras Shevchenko National University of Kyiv,Ukraine

bR.E.Kavetsky Institute of Experimental Pathology,Oncology and Radiobiology,National Academy of Sciences of Ukraine,Ukraine

cO.O Bogomolets National Medical University,Kyiv,Ukraine

dUkrainian Anticancer Institute,Vienna,Austria

A R T I C L EI N F O

Article history:

Available online 10 February 2016

NSC631570

The aim of this study is to evaluate the effect of moderate physical exercise and treatment time on the organism’s response to NSC631570.The sensitivity of circulating phagocytes to the drug at different times of day was estimated inin vitroexperiments.NSC631570 was administered intravenously to healthy volunteers(eleven men,23±2 years)in a single therapeutic dose in an inpatient and an outpatient setting.Blood samples were obtained before the drug administration,30 min after the drug injection and every fourth hour throughout the 24 hour period.Biochemical parameters were determined using the hematological analyzer.Flow cytometry was used to evaluate phagocyte metabolism.Treatment of circulating phagocytes with NSC631570in vitroresulted in an increase in ROS production along with a decrease in their phagocytic activity,most expressed in the morning time.Drug injection to sedentary persons resulted in pro-infammatory metabolic polarization of circulating phagocytes.Introduction of NSC631570 to active persons was accompanied by a signifcant increase in phagocyte endocytosis along with a decrease in the daily mean of ROS generation.Signifcant oscillation(but in the normal ranges)of urea,creatinine,alanine aminotransferase and aspartate aminotransferase after NSC631570 introduction in the outpatient setting was shown during the day.Physical activity interferes with immunomodulatory action of NSC631570 and abrogates pro-infammatory shift of circulating phagocytes.Biochemical parameters of blood from patients treated with NSC631570 in the outpatient setting must be interpreted cautiously considering the effect of physical activity on some metabolic biomarkers.

?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

NSC631570 is a derivative of the extract of the plantChelidonium majusL.and thiophosphoric acid[1–5].The drug consists of a mixture of eightC.majusalkaloids(chelidonine,sanguinarine,chelerythrine,protopine,allocryptopine,homochelidonie, berberine,and coptisine)that reacted with an alkylating agent (preferably thiotepa)in organic solvent.The preparation has the ability to be selectively accumulated in tumor tissue and activate apoptosis only in tumor cells and not in normal cells, probably due to increased consumption of the drug by tumor cells[5–8].In addition,the therapeutic effect of NSC631570 is always accompanied by the stimulation of immune responses. The preparation shows an ability to modulate immunologic reactivityin vivoand to alter functions of immunocytes including phagocytesin vitro[9–11].

In accordance with their functions and metabolism,phagocytes have been classifed into two subpopulations.Classically, activated cells(M1 macrophages or N1 neutrophils)exhibit proinfammatory phenotype that is characterized by increased production of reactive oxygen species,pro-infammatory cytokines and chemokines.Alternatively,activated M2 macrophages or N2 neutrophils are described as cells with antiinfammatory phenotype with increased phagocytic activity and regulatory cytokine expression[12,13].In our previous investigation,we observed the ability of NSC631570 to cause an infux of macrophages into the tumor growth area after intravenous administration[9].The previous result from our laboratory also revealed that NSC631570 induces M1 functional polarization of peritoneal macrophages and circulating phagocytes in tumor-bearing mice and can restore pro-infammatory functions of macrophages,alternatively polarized by hypoxiain vitro[14].Thus,the modulation of phagocyte functions can be considered as an important component of the immunomodulatory effect of NSC631570.

The effcacy of an immunomodulating preparation depends on the reaction of immune cells to the given drug.This reaction of immune cells substantially depends on their initial functional state[15].The initial functional state of immune cells in turn depends on many factors that are rarely appreciated in the treatment regimen such as treatment time and condition of the patients(inpatient vs outpatient setting).Numerous studies have demonstrated that even moderate physical exercise modulates the functional response of immune cells, including monocytes and granulocytes and affects cytokine generation,reactive oxygen(ROS)and nitrogen species production, and chemotaxis and phagocytosis of these cells[16–20].It is widely reported that therapeutic effects can be markedly improved by administering a drug taking into account the rhythm of cell functions[21–25].Treatment with NSC631570 is carried out in the morning in both inpatient(associated with limited excursion)and outpatient settings(associated with moderate physical activity).Clinical observations suggest that the therapeutic effcacy of the drug is lower when treatment is carried out in an outpatient setting.

Traditionally,biochemical parameters have been used as markers to assess the infuence of exercise on different systems, organs and tissues[26–28].Many biochemical parameters also exhibit diurnal variations that must be considered in the clinical setting,when concentration changes in the parameters are evaluated.

The aim of this study was to evaluate the effect of treatment time and moderate physical exercise on the response of circulating phagocytes to NSC631570 as well as on daily oscillations of various biochemical blood parameters after drug administration.

2.Materials and methods

2.1.Subjects

Eleven healthy adult men aged 23±2 years were recruited to participate in the study.Exclusion criteria included a history of somatic disease and a physically active lifestyle.Approval was obtained from the Ethics Committee of the City Clinical Emergency Hospital of Kyiv,and consent was obtained from all subjects before the commencement of the study.

2.2.Study design

The studies were carried out in three stages.The aim of the frst stage was to investigate the effect of treatment time on the sensitivity of circulating phagocytes to NSC631570in vitro. For this purpose,blood samples of 5 healthy volunteers in the inpatient setting were collected at different times of day(starting at 8:00 h then every 4 h over the next 24 h).Blood samples from each point in time were immediately treated with NSC631570(Nowicky Pharma,Austria)at a concentration of 20 μg/ml.Phagocytic activity and ROS generation of circulating monocytes and granulocytes were analyzed in treated and untreated samples.The aim of the second stage was to imitate administration of the drug in the inpatient setting(inpatient setting model).For this purpose,11 volunteers spent most of their time in bed.NSC631570 was administered i.v.at 8:00 h at a volume of 20 ml(a single therapeutic dose in clinical practice).Blood samples were collected before and 30 min after drug administration(at 8:30 h),as well as every 4 h over the next 16–24 h.The aim of the third stage was to imitate administration of the drug in the outpatient setting(outpatient setting model).For this purpose,11 volunteers were allowed to move and in addition they were asked to take part in one session of standardized moderate physical exercise.NSC631570 was administered i.v.at 8:00 h at a volume of 20 ml.The physical exercise program(at 12:00 h)included 50 slow squats.Blood samples were collected before and 30 minutes after drug administration,before standardized moderate physical exercise, immediately after it,and every 4 h over the next 16–24 h.Phagocytic activity and ROS generation of circulating monocytes and granulocytes as well as biochemical parameters were analyzed in all collected blood samples.

2.3.Intracellular ROS assay

ROS levels were measured using 2′7′-dichlorodihydro-fuorescein diacetate(carboxy-H2DCFDA,Invitrogen),which is converted into a non-fuorescent derivative(carboxy-H2DCF)by intracellular esterases as described earlier[29].Carboxy-H2DCF ismembrane impermeable and is oxidized to fuorescent derivative carboxy-DCF by intracellular ROS.Two hundred microliters of heparinized whole blood was incubated with 4.3 μl of PBS containing 10 μM carboxy-H2DCFDA for 30 min at 37°C. A short recovery time was allowed for the cellular esterases to hydrolyze the acetoxymethyl ester or acetate groups and render the dye responsive to oxidation.Erythrocytes were lysed with lysis buffer.The cells were then transferred to polystyrene tubes with cell-strainer caps(Falcon,Becton Dickinson) and analyzed with fow cytometry(excitation:488 nm,emission:525 nm).Only living cells,gated according to scatter parameters,were used for the analysis.

The modulating effect of the drugin vitrowas characterized by the modulation coeffcient that was calculated by the formula 100?(S?100/B),where S is the index value after treatment with NSC631570in vitro;B is the index value of untreated cells(basal value).

The modulating effect forin vivoexperiment was represented as a percentage of daily mean value.

2.4.Phagocytosis assay

The fow cytometry phagocytosis assay was performed as described above[29].Staphylococcus aureusCowan I cells(collection of the Department of Microbiology and General Immunology of Taras Shevchenko National University of Kyiv)were grown on beef-extract agar and subsequently were heat inactivated and fuorescein isothiocyanate(FITC)labeled.

The stock of FITC-labeledS.aureusat a concentration of 1×107cells/ml in a volume of 5 μl was added to 200 μl of heparinized whole blood.A tube with whole blood only served as a negative control.All samples were incubated at 37°C for 30 min.At the end of the assay,phagocytosis was arrested by the addition of cold stop solution(PBS with 0.02%EDTA and 0.04%paraformaldehyde).Erythrocytes were lysed with lysis buffer.Fluorescence of phagocytes with ingested bacteria was determined by fow cytometry.The results were registered as phagocytosis index(PhI)that was calculated with the following formula:[Gmeanpos/Ppos]?[Gmeanneg/Pneg],where Ppos is the percent of positive cells,Gmeanpos is the mean channel fuorescence,Pneg is the percent of positive cells in the negative control,and Gmeanneg is the mean channel fuorescence of the negative control.The modulating effect of the drugin vitrowas evaluated as mentioned above.

2.5.Evaluation of biochemical parameters

The concentrations of glucose,urea,creatinine,sodium,chloride,amylase,phosphorus,cholesterol,triglycerides,direct highdensity lipoprotein cholesterol(HDLC),uric acid,total protein, albumin,aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALKP),gammaglutamyl transferase(GGT),and total bilirubin(TOTAL BILI)were determined using the hematological analyzer Particle counter model PCE 210(ERMA,Japan).

2.6.Statistical analysis

Data are reported as mean±SD.To perform statistical analysis,daily mean values of all investigated indices were calculated for each individual.PhI and ROS generation values were then normalized to daily mean and used for calculation of means±SD.Means were compared using the Student’s t-test, and P values of 0.05 or less were considered signifcant.

3.Results and discussion

3.1.The treatment time dependence of circulating phagocytes’sensitivity to NSC631570in vitro

The effect of treatment time on the sensitivity of circulating phagocytes to the antineoplastic drug NSC631570 was evaluated inin vitroexperiments.The functional state of phagocytes that was characterized by increased phagocytic activity along with decreased ROS generation was considered as antiinfammatory or alternative polarization.In contrast,the functional state of phagocytes that was characterized by decreased phagocytic activity along with increased ROS generation was considered as pro-infammatory or classic polarization [12,14,30].

Functional polarization of circulating monocytes depended on the time of day(Fig.1).Pro-infammatory metabolic profle in circulating monocytes at 4 pm was identifed by a 72%increase(as compared with daily mean value)in ROS generation accompanied by a 46%reduction in phagocytosis. Anti-infammatory polarization of these cells characterized by a 54%increase in phagocytic activity along with a 15% decrease in ROS production was registered at midnight.Metabolic polarization of circulating phagocytes at the other time points was absent or could be considered as a neutral functional state.Treatment of blood samples with NSC631570in vitroat all time points resulted in an increase in ROS generation and a decrease in phagocytic activity in monocytes indicating the pro-infammatory modulating effect of the drug. The most expressed pro-infammatory effect of the drug on metabolic polarization of circulating monocytes in treated samples was detected at 8 h.In this time point,untreated monocytes were characterized by a neutral functional profle. This time point is closest to the time point of NSC631570 introduction in clinical practice.

Functional polarization of circulating granulocytes also showed time-dependent variations over the period of observation(Fig.2).Similarly to monocytes,a signifcant increase (110%from daily mean value)in ROS production accompanied by relatively low phagocytic activity was observed in circulating granulocytes at 16:00 h indicating their classical(N1) polarization at this time point.Moderate anti-infammatory(N2) polarization of these cells,characterized by the enhanced phagocytic properties(80%from daily mean value),was registered at 20:00 h.At the other time points,circulating granulocytes did not display distinct metabolic polarization and their functional state was considered as neutral.Treatment of blood samples with NSC631570in vitroat different time points over the day caused an increase in ROS generation in circulating granulocytes,most expressed at noon.In this time point, untreated granulocytes were characterized by a neutral metabolic profle.

Fig.1–Functional polarization of circulating monocytes before and after treatment with NSC631570in vitro.(A)Functional polarization of circulating monocytes from healthy volunteers;(B)functional polarization of circulating monocytes from healthy volunteers after treatment with NSC631570in vitro.Each value represents the mean±SD of 5 healthy volunteers. *P＜0.05 was considered signifcant compared to phagocytic index value.

3.2.Circulating phagocytes’polarization after the introduction of NSC631570 in inpatient and outpatient settings

Clinical investigations revealed that the therapeutic effect of NSC631570 is greater when treatment is provided in the inpatient setting associated with limited physical exercise.Thus, the second research task was to investigate the effect of moderate physical exercise(that is characteristic for patients in an outpatient setting)on the sensitivity of circulating phagocytes to NSC631570.Physical activity is a systemic phenomenon with impact on immune cell function.The effect of physical activity on immune cells depends on its intensity and regimen as well as participant age[17,20].Of innate immune system cells,neutrophils and macrophages appear to be most responsive to the infuence of physical activity,both in terms of numbers and function.

In vivointroduction of NSC631570 in an inpatient setting caused pro-infammatory polarization of circulating monocytes 4 h after drug injection(Fig.3).This functional state of cells was registered for the following 8 h,whereas introduction of the drug in an outpatient setting was associated with moderate anti-infammatory polarization of monocytes,which in this case was characterized by signifcantly increased phagocytic activity(by 76%at 12:00 h)and moderate oxidative metabolism.Phagocytic function of these cells showed a further 20%increase immediately after physical exercise as determined at 12:30 h.Starting from 16:00 h(4 h after the bout of physical activity),both oxidative metabolism and phagosytosis in circulating monocytes were signifcantly decreased.Slight features of the anti-infammatory metabolic profle of these cells were observed.Interestingly,phagocytic activity of monocytes before drug introduction was slightly higher in sedentary volunteers than that in an active state.It is a well-known fact that moderate physical activity promotes alternative polarization of mononuclear phagocytes [8,9,18].Acute moderate physical activity led to further alternative polarization of monocyte metabolism although it was not defned as prolonged.

Intravenous administration of NSC631570 also resulted in pro-infammatory polarization of circulating granulocytes in sedentary persons 30 minutes after drug introduction(Fig.4). These cells were characterized by signifcantly increased oxidative metabolism(by 66%as compared with daily mean)along with decreased phagocytic activity(by 27%).Drug injection to active volunteers did not change their polarization.Slightly expressed features of anti-infammatory metabolic polarization of these cells were registered 4 h after the bout of physicalexercise(at 16:00 h)and over the next 12 h.These results suggest that physical activity interferes with the infuence of NSC631570 on circulation granulocytes of healthy volunteers and abrogates the pro-infammatory effect of the drug on these cells.

Fig.2–Functional polarization of circulating granulocytes before and after treatment with NSC631570in vitro. (A)Functional polarization of circulating granulocytes from healthy volunteers;(B)functional polarization of circulating granulocytes from healthy volunteers after treatment with NSC631570in vitro.Each value represents the mean±SD of 5 healthy volunteers.*P＜0.05 was considered signifcant compared to phagocytic index value.

3.3.Patterns of biochemical parameter oscillations after NSC631570 introductionin vivoin an inpatient and an

outpatient setting

Clear time-of-day-dependent changes have been shown to be relevant for some biochemical parameters of the human blood. Biochemical markers are known to oscillate in response to a number of environmental agents and physiological activity. Acute bouts and chronic exercises have an effect on metabolic processes and might serve as the underlying reason for signifcant alterations in biochemical and hematological measurements[26,27].

Administration of NSC631570 in the early morning(at 8:00 h) did not cause signifcant changes in levels of biochemical markers measured in blood samples of healthy men in an inpatient setting 30 min after injection(Table 1).All of the biochemical markers corresponded to the normal range of values for healthy individuals of the same age.Meanwhile,total bilirubin concentration showed a transient increase immediately after injection followed by a fall at noon to normal(daily mean)values determined for this parameter.It may refect an overlapping of the effect of the drug and the peak value of total bilirubin that occurred in the morning as described for diurnal variation in this biochemical marker of blood[27].Signifcantly higher concentrations than daily mean value was registered in amylase,cholesterol,and triglycerides with peaks for these measures at 20:00 h,16:00 h and at noon,respectively.The lowest values,that were signifcant as compared with daily mean,were observed for cholesterol at noon and for highdensity lipoprotein cholesterol at midnight.We suppose that this showed that the trend was linked to normal daily fuctuation of the mentioned biochemical parameters.Moreover, food intake might have a major effect on the extent of daily fuctuation of such parameters as triglycerides,cholesterol and high-density lipoprotein cholesterol,levels of which can increase rapidly after a high-fat meal[31–35].

In active volunteers(Table 2),administration of NSC631570 led to a sharp increase in the concentration of gammaglutamyl transferase(by 67%from basal level)in 30 min after drug introduction(at 8:30 h).Also,the comparison of daily mean values of biochemical measures from active and sedentaryvolunteers showed signifcant differences in levels of glucose (lower by 20%in outpatient setting),creatinine(higher by 11% in outpatient setting),alanine aminotransferase(higher by 61% in outpatient setting)and alkaline phosphatase(lower by 75% in outpatient setting).Some of the observed alterations,in particular the increase in glucose intake and increase in creatinine as well as alanine aminotransferase and aspartate aminotransferase(peaked in the evening),refect the metabolic outcome of muscle activity and energy output[31].This is the main reason why taking moderate-intensity physical exercise was associated with more variable biochemical parameters in healthy active volunteers who were given NSC631570 injection.In an outpatient setting,of the measured parameters, urea,amylase,and triglycerides showed signifcant increases 30 min after a single bout of physical activity(at 12:30 h).Although increased urea,marker of protein catabolism,refects the process of protein utilization as an energy substrate during acute bouts[26],variations of amylase and triglyceride concentrations might occur as a result of exercises on gastrointestinal tract(including liver)and enzyme synthesis.Also, some changes in blood parameters and immune cells activity can be infuenced by stress hormones and cytokines.

Fig.3–Functional polarization of circulating monocytes after a single injection of NSC631570 in inpatient and outpatient settings.(A)Functional polarization of circulating monocytes in healthy volunteers after the introduction of NSC631570 in the inpatient setting;(B)functional polarization of circulating monocytes after the introduction of NSC631570 in the outpatient setting.Each value represents the mean±SD of 11 healthy volunteers.*P＜0.05 was considered signifcant compared to phagocytic index value.

4.Conclusion

The present data show that both treatment time and moderate physical activity infuence the immunomodulating effect of the antineoplastic drug NSC631570.Treatment timedependency of the sensitivity of circulating phagocytes to NSC631570 was revealed.The drug caused most expressed proinfammatory stimulation of these cells during the early morning when the metabolic profle of investigated phagocytes can be considered as neutral.Physical activity interferes with the immunomodulatory action of NSC631570 and abrogates M1(N1)shift of circulating phagocytes.Administration of NSC631570 in an inpatient setting did not cause any signifcant alterations in blood biochemical parameters.Increased values of urea,creatinine,alanine aminotransferase andaspartate aminotransferase concentrations after NSC631570 introduction in an outpatient setting can be considered as an outcome of the synergetic effect of the drug and physical exercise and could potentially affect the treatment effcacy of the drug.Thus,consequences of treatment with NSC631570 against the background of physical activity have to be monitored and interpreted cautiously,taking into account the possible modulatory effect of physically active states on different metabolic parameters and functions of immune system cells.

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Fig.4–Functional polarization of circulating granulocytes after a single injection of NSC631570 in inpatient and outpatient settings.(A)Functional polarization of circulating granulocytes after the introduction of NSC631570 in the inpatient setting. (B)functional polarization of circulating granulocytes after the introduction of NSC631570 in the outpatient setting.Each value represents the mean±SD of 11 healthy volunteers.*P＜0.05 was considered signifcant compared to phagocytic index value.

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*< class="emphasis_italic">Corresponding author.

.Microbiology and General Immunology Department,ESC“Institute of Biology”,Taras Shevchenko Kyiv National University,Dniprovs’ka Str.,9,ap.38,Vyshgorod,Kyiv reg.07300,Ukraine.Tel.:+380504438775;fax:+380445253841.

E-mail address:rosiente@gmail.com(M.Rudyk).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2016.02.004

1818-0876/?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Physical exercise

Blood biochemical parameters

Phagocytes functional polarization