ADAPTIVE REACTIONS INVESTIGATION UNDER EXPERIMENTAL HYPOXIA CONDITIONS WITH THE PRIOR TREATMENT OF THE 4-THIAZOLIDINONE DERIVATIVE

Received on 27 September, 2013; received in revised form, 29 October, 2013; accepted, 16 January, 2014; published 01 February, 2014

ADAPTIVEREACTIONSINVESTIGATION UNDER EXPERIMENTALHYPOXIACONDITIONSWITHTHEPRIORTREATMENTOFTHE4-THIAZOLIDINONEDERIVATIVE

Оlha І. Antoniv¹, Svitlana М. Kovalchuk ², Оxana І. Terletska², Danylo V. Kaminskyy ³, Roman B. Lesyk ³, Оleh R. Pinyazhko*¹

Department of Pharmacology ¹, Department of Normal Physiology ², Department of Pharmaceutical, Organic and Bioorganic Chemistry ³, Danylo HalytskyLvivNational Medical University, Lviv, Ukraine

ABSTRACT: The effects of 4-thiazolidinone-derivative – Les-589 as perspective anti-hypoxic agent under the experimental hypoxic hypoxia actions were studied. The values of peripheral blood and hematological indexes as well as parameters of cellular oxidative metabolism (level of lipoperoxidation products, activity superoxide dismutase and catalase, levels and ratio of lactic and pyruvic acids in the blood and liver tissue) of the experimental animals were investigated. The presented data confirm the anti-hypoxic properties of the Les-589 under study conditions and are the strong argument for further Les-589 study as perspective antihypoxant with preventative effect.

4-Thiazolidinones, Antihypoxic agents,Hematologicalindexes, Adaptivereactions, Oxygen-dependentmetabolism

INTRODUCTION:Optimalflowofallreactionsintheorganismrequiresenoughenergy, whichissuppliedbytheoxygendependentmetabolism. A lot of diseases can cause the phenomenon of hypoxia that considerably affects the energy balance of the body.

Hypoxia,aswellasanyotherextremefactors,leadstoformationofdifferentadaptivereactions types: compensation, super-compensation and decompensation reactions ^1-3.

Versatility of hypoxic syndrome needs permanent extension of the range of antihypoxic agents which must initiate the adequate anti-stress reactions. 

Atthesametimeeffectivenessofanti-hypoxictreatmentdependsonpathogenesis oftheoxygendeficiency and degree of its manifestation^{4, 5}. Definingtheprotectivepropertiesofthepotentialanti-hypoxic agentsneedsthemostsensitiveandavailableteststoassessadaptive functions of the body and predict the adaptive function type.

Blood systemandhematopoieticcells, in particular, show highsensitivitytohypoxia, thus, assessmentoftheperipheralbloodstateisanadequatetestfordynamiccontrolofthe post hypoxic adaptationquality. At the same time, little research is carried out to study the state and direction of adaptive-compensative reactions based on the defining of the peripheralbloodstate with the anti-hypoxic agents application.

One of the promising groups of 4-thiazolidinones are 4-thiazolidinone-3-carboxylic acidsextensively explored as compounds with anti-inflammatory, anti-diabetic, antibacterial and antitumor activities ^6,7.

A majority of the biologically active 4-thiazolidinone-derived compounds are 5-ylidene derivatives, probably because of the substituents at the C5 position of basic heterocycle which is crucial for the pharmacological effects ^8-10. On the other hand, ylidene fragment conjugation to the C4 carbonyl group makes compounds to be electrophilic and potentially reactive due to possible Michael addition of the nucleophilic protein residues to the exocyclic double bond. These Michael acceptors are one of the most efficient activators of Nrf2 through the modification of Keap1 ¹¹ and new highly specificand potent inhibitors of the mitochondrial pyruvate carrier¹².

Thus, 5-ylidene-thiazolidinones can be treated as main building blocks for various derivatives series synthesis in so-called privileged substructure-based diversity oriented synthesis strategy ¹³, which has proven to be a fruitful tool to rapidly discover biologically active lead compounds.

Modification of the oxygen homeostasis/oxidative stress and/or ROS inhibition is considered to be possible modes of action of 4-thiazolidinone-3-carboxylic acids derivatives ^{14, 15}.Importantinthiscontextisthefactofcombinationofanti-oxidant properties with anti-inflammatory, immunomo-dulatory and antitumor pharmacological profiles ¹⁶, specifyingthedirectionofnovel 4-thiazolidinone-3-carboxylic acids study in the spirit of modern triad: stress-inflammation-cancer.

Informationaboutnewanti-hypoxicagentssearchamongdiscussedcompoundsisscrimpy, althoughsomeanti-hypoxicagentswithantioxidantpropertiesareidentified¹⁷.

Thus, theaimofthepresented manuscript wastodefinethetypesoftheadaptivereactionsastheresponseto the experimental hypoxic hypoxia actions withthepriortreatmentofthe5-ylidene-4-thiazolidinone-3-carboxylic acidderivative (Les-589).

Forthispurpose,the valuesofperipheralbloodandhematologicalindexeswereassessed, aswellassomeoxidative metabolism parameters.

MATERIALS AND METHODS: The studies have been performed on the white rats (male, 180-200 g), within the general ethical principles of humane treatment to experimental animals ¹⁸. Animals were divided into next groups: 1^st - control, ratsfrom the 2^ndandthe 3^d group were given thiotriazoline (morpholin-4-ium (3-methyl-4H-[1, 2, 4]triazol-5-ylsulfanyl)-acetate) (2 mg/kg, ip, 2.5 % sol.),animalsofthe 4^thandthe 5^thgroupweregivenLes-589 (100 mg/kg,ip, dissolvedin Polysorbate 80). Animalsofthe 3^dandthe 5^thgroups in 45 minafter treatment with thiotriazoline and Les-589 wereexposed to the hypoxic hypoxia(identicaltothe heightof 6000 meters above sea level) during 1 hour.

Les-589 (potassiumsaltof 3-[5-(3-phenylallylidene)rhodanin-3-yl]-propionic acid) was selected from department of Pharmaceutical, Organic and Bioorganic Chemistry in home library of 4-thiazolidinone derivatives, following our previous investigations ^{6, 10, 17} and primary data. Les-589 prolongedthesurvivaltimeof the experimental animals underthehypoxiacondition (identicaltothe heightof11000 meters above sea level)forthelongerperiod (from (3.5±1.2) min. (control group) to (7.8±1.4) min.). The level of TBA-reactive products (TBARs) ¹⁹, activity of superoxide dismutase (SOD) ²⁰ and catalase (CAT) ²¹ were estimated in blood and liver of the experimental animals for the study of the lipid peroxidation – anti-oxidation activity (LPO - AOA) system changes.

Hypoxia level was controlled by the levels and relation of lactic and pyruvic acids in blood and liver tissue ^{22, 23}. Blood parameters were defined 24 hours after hypoxia at the COULTER-T840 analyzer.

Based on the hematological parameters the following hematological indexes were calculated: adaptation index (І_А); granulocyte-agranulocyte index (GAI); lymphocyte-granulocyte index (LGI); relation of neutrophils and lymphocytes index (NLCI) ^{24, 25}. Statistical analysis of the obtained results was carried out (STATISTICA).

RESULTS AND DISCUSSION: The hypoxic exposition led to erythrocyte level decrease by 5,4% comparing to the control (Table 1).

TABLE 1: CHANGES IN ERYTHROCYTES AND THEIR PARAMETERS UNDER THE STUDIED CONDITIONS (M±m, n=10)

* – р < 0,05 to “control” group; ● – р < 0,05 to “hypoxia” group

At the same time, the tendency of erythrocyte and hemoglobin increase was observed in the experimental animals that were given thiotriazoline and Les-589, as well as groups with combination of hypoxia and tested compounds. The level of erythrocytes increased by the 5.7% and 9.1% in the 2^nd and 4^th group correspondingly comparing to the control group. Effects of tested compounds introduction showed the significant increase of erythrocyte level by 13.7% and 12.1% correspondingly (р < 0,05) in comparison with the groups only under the hypoxia (2^nd and 4^th groups). Analogicalchangesofhematocritwere observed. Thus, Les-589 and thiotriazoline, in case of their previous application before the hypoxia action, increased the oxygen capacity of blood following the increase of the erythrocyte and hemoglobin levels.

Under the influence of hypoxia the decrease of leucocytes by 32 % (р < 0,05) was detected (Table 2).

ТABLE 2: CHANGES IN LEUCOGRAM UNDER THE STUDIED CONDITIONS (M±m, n=10)

* – р < 0,05 to “control” group, ● – р < 0,05 to “hypoxia” group

Intense leukocytosis was observed in studied groups under the tested compounds treatment. The level of leukocytosis was almost the same in all groups. While, both thiotriazoline usage and hypoxia following the thiotriazoline treatment provided the increase of the percent of eosinophiles by 33% and 145% correspondingly, comparing to the control group. The trend of lymphocytes level increasing was observed, however its values were remained within the limits of maximum allowable upper limit. According to the literature, these findings indicate the mineralocorticoid production activation ^24-26. Modeling of hypoxic hypoxia following thiotriazoline usage led to the increase of the monocytes levels to 3.3%, comparing to 2.0% in the control group (remains within the norm limits), while the drug’s introduction itself was accompanied with significant monocytosis (up to 4.5%). Effect of thiotriazoline introduction before the hypoxic exposition in comparison to the hypoxia modelling without protective agents was characterized in almost double increase of monocytes level (р < 0.05). Rats, which were treated with Les-589, had the following changes in leucogram: lymphocytosis – to 66% and 65% correspondingly, absence of eosinophiles, level of monocytes – within the norm limits, which certifies about the production of glucocorticoids at the same level as mineralocorticoids. In the group with hypoxia action under the above mentioned leukopenia, the increase of the lymphocytes level (by17 %), decrease of segmented neutrophils (by 24 %), eosinophiles (by 62 %) and monocytes (by 43 %) compared to the control were observed.Based on the hematologic parameters the following relation indexes were calculated for estimation of the adaptive reactions types: adaptation index (І_А) (ratio of lymphocytes tosegmented neutrophils); granulocyte-agranulocyte index (GAI) - ratio of the sum of band neutrophils, segmented neutrophilsand eosinophils to the sum of monocytes and lymphocytes; lymphocyte-granulocyte index (LGI) – ratio of lymphocytes to sum of band segmentedneutrophils, and eosinophils; index of the ratio of neutrophils and lymphocytes (NLCI)^{24, 25}.According to thestudiedhypoxia conditionstherelative increaseof І_А (from 2.21±0.09 (control) to 3.37±0.12) and LGI, as well as decrease of GAI and NLCI were detected (Table 3).

TABLE 3: CHANGES IN HEMATOLOGICAL INDEXES UNDER THE STUDIED CONDITIONS (M±m, n=10)

* – р < 0,05 to “control” group, ● – р < 0,05 to “hypoxia” group..

TheincreaseofI_Aby29 % (againstthecontrol) in the group which were treated with thiotriazoline before the hypoxic exposition, while the absence of significant changes in the case of the drug introduction itself was shown. The increase of I_A (on 33 %) was also detected in the group where Les-589 was used, as well as in animals previously treated with Les-589 (by 16 %). LGIvalueschangedanalogicallytoI_A, thefirstincreasedin all groups, but was the most expressed under the influence of hypoxia following the Les-589 application.

The changes of GAI and NLCI were opposite to I_A and LGI. As the result of the research, the relative decrease (comparing to control) of these indexes under Les-589 action and their renovation during combination of Les-589 and hypoxia almost to normal values were discovered. Thiotriazoline effect was characterized by the opposite direction of changes: increase of these indexes under thiotriazoline treatment and their relative decrease under the hypoxia conditions.

Based on the reactions of hematopoiesis, especially leukopoiesis, incomplexwiththestateofimmuneorgans, adrenals, thefollowingtypesofadaptivereactionsaredifferentiated: orientation, activation (quiet and raised), over-activation, deficient activation, stress ^{24, 25}. The complexofchangesinleucogram, especiallylymphocytosis, monocytosis, I_Aincrease, detectedunder hypoxiafollowing thethiotriazolineindicated thedevelopmentoftheraisedactivationtype of adaptive reaction, whichbelongstothepositiveprognostic adaptive reactions.

Lymphocytosis, eosinopeniain 24 hoursafterLes-589 introductionandatthefirst 24 hoursofpost-hypoxicperiodfollowing the compound usage, could indicatetheincreaseofmineralocorticoidsandglucocorticoidssecretion. Together with the I_A increase it indicated the successful flow of the adaptive reactions at the optimal hormone status, which equally to the thiotriazoline effect, was realized via the reaction of the raised activation.

Worth noting is that based on the same parameters and hematological indexes the adaptive reaction to the hypoxic exposition was realized in form of the deficient type of adaptation. This was confirmed by leucopenia, decrease of the relative levels of monocytes, eosinophiles, significant increase of adaptation index.

Summarizing the presented results we can conclude the positive effect of the Les-589. The protective usage of which modified the adverse type of the adaptive reaction into the prognostically favorable anti-stress reaction. To confirm this finding, the study of the oxidative metabolism was the following step of our research. It is known that modificationofoxygendependentmetabolism caused byhypoxia canformthebasisforwiderangeofmetabolicdisordersaccompaniedbythe formationofdesadaptationstates^{4, 5}, which requires timely identification.

Receiveddatashowed the activationoffree radical peroxidation processes in blood and liver tissue of the experimental animals under hypoxic exposition. This is reflected in the increase of TBARs level together with the decrease of catalases and SOD activity (fig. 1).

FIGURE 1: CHANGES OF TBARS AND ANTIOXIDANT ENZYMES ACTIVITIESLEVELS IN THE BLOOD AND LIVER OF EXPERIMENTAL ANIMALS

Analysis of the literature and our data certified that the coefficient of ratio of AOD (antioxidant defense) parameters to the lipid peroxidation (LPO) activity (K=АОA/LPO) ⁵ is the most informative for estimation of pro/antioxidant system.

Our findings showed thatthevalueofsuch coefficient (K = 100% in thenormal condition) under thehypoxiaexposition was drasticallydecreasedboth inthe bloodandliveroftheexperimentalrats (fig. 2).

FIGURE 2: CHANGES OF COEFFICIENT (K = AOA/LPO) IN THE BLOOD AND LIVER OF EXPERIMENTAL ANIMALS

The coefficient value decreased by 70% in blood and in the liver tissue by 53 % (р< 0.05) correspondingly.

ApplicationofLes-589inthecorresponding groupled to theincreaseofthecoefficientvaluesinthebloodin 2,1 times (р< 0.01), in the liver - in 1.3 times (р < 0.05). Thiswasmainlyprovidedbythe increaseofthecatalaseactivityandsuppression of LPO. This significant increase of the coefficient’s values could indicate the restoring of the balance in the system of pro- and anti-oxidant processes and favorable type of the adaptive reaction. The hypoxicexpositioncausedtherelativeincreaseofthelactatelevelandincreaseinlactate/pyruvateratio bothinbloodandlivertissue (fig. 3).

FIGURE 3: CHANGES OF LACTATE AND PYRUVATE LEVELS IN THE BLOOD AND LIVER OF EXPERIMENTAL ANIMALS

The preventative introduction of Les-589 led to decrease of the lactic acid level in blood by 70 % (р < 0.05) in comparison to the group without correction. While thepyruvicacidlevel intheblood increased slightly. The lactate/pyruvate ratio which was used as balance index of aerobic and anaerobic processes, decreased in the blood by 88 % (р < 0.05). The same character of changes was observed in the liver tissue under compound treatment.

However, the value of these changes was lower (decrease by 53 %, almost reaching the normal value). This data, in generally, showed the decrease of the post-hypoxic effects expression level and clearly confirmed the anti-hypoxic properties of the Les-589.

Also,the mentioned indexes(K = AOA/LPO and K = lactate/pyruvate) had been considered as the most prominent diagnosticparameters. Presented data demonstrated the necessity of the complex assessment of the states of pro- and anti-oxidant processes, aerobic and anaerobic reactions for defining the efficiency and quality of the adaptive reaction. Moreover, it grounded the validity of the hematological parameters and indexes for the control of adaptive reaction under hypoxic syndrome and its correction.

Ingeneral, theanti-hypoxicpropertiesofLes-589 were confirmed andpresented data arethestrongargumentfor its further investigation as potential antihypoxic agent with preventative effect.

CONCLUSION: The decrease of the degree of post-hypoxic effects manifestation and the resumption of the pro- and antioxidant system balance under Les-589 treatment were shown. In accordance to the hematological and biochemical parameters analysis the protective usage of Les-589 before hypoxic hypoxia action converts adverse type of adaptive reaction (inferior adaptation type) in prognostically favorable type (increased activation).

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    How to cite this article:

    Оlha IA, Svitlana KМ, Оxana TІ, Danylo TV, Roman LB and Оleh PR: Adaptive reactions investigation under experimental hypoxia conditions with the prior treatment of the 4-thiazolidinone derivative. Int J Pharm Sci Res 2014; 5(2): 361-67.doi: 10.13040/IJPSR.0975-8232.5(2).361-67

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