INTRODUCTION:

At preclinical stages of drug development it’s important to have information about the degree, type and mechanism of drug transport through biological membranes. The chemical structure of a drug molecule determines its mechanism and rate of transport through biological membranes^1,2,3. Xenobiotics pass into the cells only by passive diffusion^2,3,4 in the direction of concentration gradient at a rate dependent on their lipophilicity⁵. Distribution of a compound by passive diffusion may lead to its increased accumulation in cell organelles, depending on their pH. For example, cationic amphiphilic compounds may accumulate in lysosomes with acidic pH⁶.

Some diseases may change the physiological properties of a cell and change its permeability to xenobiotics, for example, pH change to alkaline in cells accelerates the elimination and reduces the efficacy of compounds with weak base pH⁷.

Pharmacokinetics of a compound is heavily dependent on its binding to blood plasma proteins^8,9. Only the free, unbound fraction of a compound exerts a pharmacodynamic effect and is eliminated, while protein-bound fraction is less available not only to receptors but also to the elimination processes. On one hand, high plasma protein binding protects a compound from biological breakdown and prolongs its exposure time, on the other hand, potential displacement of the compound from protein by another compound with higher lipophilicity may lead to adverse effects. Also, many pathologies can affect protein binding; for example, hypoalbuminemia (arising from burns, kidney failure, liver disease, starvation, etc.) may require dose correction to ensure adequate treatment^10,11. Due to the aforementioned factors, studying the distribution and transmembrane transport is very important in drug development.

The new antiaggregant drug (codenamed GRS), examined in the present study, is an indolinone derivative. It’s the first antiplatelet drug that is a stimulator of cytosolic (soluble) guanylate cyclase^12,13.

The goal of the present study: assess the distribution and transmembrane transport of an antiaggregant drug GRS of indolinone series.

MATERIAL AND METHODS:

Chemical and Reagents:

GRS is a 2-[2-[(5RS)-5-(hydroxymethyl)-3-methyl-1,3-oxazolidine-2-yliden]-2-cyanoethylidene]-1H-indol-3(2H)-one (Fig. 1), synthesized in «Tomskaya Farmacevticheskaya Fabrika» (Russia, Tomsk). LD₅₀ of GRS > 5000mg/kg for male and female rats.

GRS

Fig. 1 Chemical structure of GRS

Verapamil (Sigma-Aldrich, USA), which accumulates mostly in the plasma, and elsulfavirin (IICR, Russian Federation), which accumulates mostly in blood cells, were used as reference compounds to study the blood to plasma ratio.

Studies of GRS transport through cell monolayer used its aqueous solutions in 1 and 10mcM concentrations or aqueous solutions of ranitidine (Santa Cruz, USA) (a compound with low cell monolayer permeability), propranolol (Santa Cruz, USA) (a compound with high permeability) and rhodamine 123 (Sigma-Aldrich, USA) (a P glycoprotein substrate). Cyclosporin A (Sigma-Aldrich, USA) was used as a Pgp inhibitor. Warfarin (Sigma-Aldrich, USA) was used as a control compound with a high degree of binding to plasma proteins.

Animals:

Studies were performed on 90 male Sprague Dawley rats of SPF status with body mass 300±30g, aged 2.5 – 3 months. Animals were kept in plastic cages at air temperature 18-26°С, relative air humidity 45-65%, air exchange 10–11 changes per hour and lighting mode 12:12. Before test compound administration the animals were deprived of food for 16 hours. Research adhered to the «Principles of Laboratory Animal Care» (NIH publication #85-23, revised in 1985). As well studies were approved the Institutional review board and was performed in accordance with the rules of European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986).

Tissue Distribution after Single oral administration:

GRS was administered in 20mg/kg dose to rats once orally as a suspension in 0.5% aqueous solution of carboxymethyl cellulose. Animal blood and organs were collected before GRS administration (blank samples) and 0.5; 1; 2; 4; 8; 16 and 24 h after administration. The blood was stabilized with sodium heparin (B. Braun Medical Inc., Germany), plasma was separated and stored with the organs at -70°С until analysis. Concentration dynamics was used to determine the pharmacokinetic parameters of GRS.

Blood cell/plasma distribution and plasma protein binding:

To assess the blood to plasma ratio, GRS was administered to animals in 20mg/kg dose once orally, the blood was collected and stabilized with sodium heparin, plasma was separated. GRS distribution was assessed based on the concentration differences of GRS in whole blood and plasma.

All in vitro studies used plasma from 10 intact rats. Studies of GRS binding with plasma proteins used its aqueous solution in 1 mcM concentration or an aqueous solution of warfarin (Sigma-Aldrich, USA) in 1 mM concentration.

A Thermo Scientific Pierce dialysis system with Teflon plate was used to assess plasma protein binding. Pooled rat blood plasma samples (from 5 animals), diluted with phosphate buffer by 2 times, were used as the model medium. Testing was performed in a 48-well plate with Teflon coating, each well had two chambers separated by a vertical semipermeable dialysis membrane with pores, permeable to compounds with maximal mass of 8 kDa. Plasma sample containing GRS was put into one chamber, phosphate buffer solution with pH 7.2 was put into another chamber. After some time, passive diffusion of the unbound compound took place and equilibrium between the two chambers was reached. Unbound fraction of GRS was assessed quantitatively using chromato-mass spectrometry.

Transmembrane transport:

A MultiScreen Caco-2 test system (Millipore Corp., USA) was used to access transport through cell monolayer. GRS transport from apical to basolateral membrane (А-В) and in the reverse direction (В-А) was studied in a 21-day old colorectal adenocarcinoma cell culture Caco-2 (RRID CVCL_0025) for 2 hours. Monolayer integrity was confirmed by measuring its electric resistance (acceptable value was at least 3 kOhm per well). After incubation, GRS quantity in the corresponding wells was studied.

Quantification of GRS in Tissue by HPLC/МS:

Quantitative assay of GRS was performed by an HPLC/МS method (Agilent 1260 Infinity (USA) liquid chromatograph, AB Sciex QTRAP 4500 (USA) mass spectrometer. GRS extraction was performed by liquid-liquid extraction with acetonitrile. The developed bioanalytical method was validated in advance^14,15.

Statistical Analysis:

Statistical processing of data was performed using «Statistica 8.0» software. The data was presented as mean values and mean standard deviation (M ± m). Significance of difference (р<0.05) between the tests was assessed using Mann–Whitney U test.

RESULTS AND DISCUSSION:

The new antiaggregant drug GRS, an indolinone derivative, was quickly absorbed after single oral administration in 20mg/kg dose to rats, reaching maximal concentration after 1 hour (73.7 ng/ml). GRS distribution in rat organs was uneven. It accumulated most in the liver (f_t = 3.87±1.30, K_d = 3.22±1.15). Its exposition in the heart was close to plasma levels (f_t = 0.78±0.19, K_d = 1.16±0.35). GRS had less exposition in kidneys (f_t=0.43±0.06, K_d=0.29±0.05). (Table 1) and didn’t cross into the brain and skeletal muscle. Pharmacokinetic profile of mean GRS concentration in each studied tissue type is presented in Fig. 2. Maximal GRS concentration in the liver (239.1 ng/ml) was achieved after 1 h, and after 1.6 and 3.5 h in the kidneys and liver, respectively.

Table 1: Pharmacokinetic parameters of GRS in rat organs after single oral administration in 20 mg/kg dose

Organ	С_max, ng/ml	T_max, h	AUC_0-t, ng×h/ml	k_el, h^-1	f_t	K_d
Liver	237.1 ± 37.0	1.0 ± 0.5	1652.8 ± 1754.3	0.27 ± 0.05	3.87 ± 1.30	3.22 ± 1.15
Heart	25.4 ± 6.7	1.1 ± 0.2	296.2 ± 87.2	0.13 ± 0.03	0.78 ± 0.19	1.16 ± 0.35
Kidney	27.0 ± 6.8	1.2 ± 0.2	200.0 ± 57.6	0.07 ± 0.01	0.43 ± 0.06	0.29 ± 0.05

GRS maximal concentration was reached faster in the liver (T_max = 1.0±0.5) and elimination there was also faster (kel = 0.30±0.14) compared to elimination in the heart and kidneys. After 3-6 h GRS concentration in all organs fell by over 2 times, showing the absence of its accumulation in the organism.

Dialysis study has shown that GRS has low binding with rat blood plasma proteins – 55,1% (Table 1). Reference compound warfarin had high plasma binding (97,2%).

Blood to plasma ratio study has shown that GRS concentration is 1.6 times higher in blood cells than in blood plasma (Table 2).

Fig. 2. Pharmacokinetic profile of GRS in rat organs and plasma after single oral administration in 20 mg/kg dose

Table 2: Distribution of the test compound and reference compounds between blood cells and plasma

Compounds	Blood to plasma ratio
GRS	1.6 ± 0.1
Verapamil	0.73 ± 0.0
Elsulfavirin	53.8 ± 1.9

Reference compounds propranolol and ranitidine cross through the cell monolayer in A-B direction at a rate of 22×10^-6 and 1.9×10^-6 cm/sec, respectively. GRS in 1 mcmol concentration had low permeability from apical to basolateral membrane (A-B). This is due to the fact that GRS is highly hydrophobic and has difficulty passing through the aqueous layer on cellular model of gastrointestinal tract mucosa. GRS has 8.8 times higher permeability in the reverse direction (B-A). Adding cyclosporin A (a Pgp inhibitor) to the model medium considerably inhibits the transport of GRS (by 5 times) in B-A direction, while the transport efficiency of rhodamine 123 (a Pgp substrate) falls by 3.6 times (Table 3). Cyclosporin A doesn’t change the rate of A-B transport. Increasing GRS concentration gradient leads to a corresponding increase in GRS transport in A-B direction.

Table 3: GRS membrane permeability and transport efficiency of GRS and reference compounds through Сасо-2 cell monolayer

Compound, concentration	Papp A-B, 10^-6 cm/sec	Papp B-A, 10^-6 cm/sec
GRS 1 mcmol	1.4 ± 0.3	7.9 ± 1.1*
GRS 1 mcmol + cyclosporin A	1.2 ± 0.7	1.6 ± 1.0
GRS 10 mcmol	7.8 ± 1.4	8.4 ± 0.7
GRS 10 mcmol + cyclosporin A	7.3 ± 1.2	1.7 ± 1.0**
Ranitidine	1.9 ± 0.2	– #
Propranolol	22 ± 1.2	– #
Rhodamine 123	0.5 ± 0.0	3.6 ± 0.4
Rhodamine 123 + cyclosporin A	0.7 ± 0.1	1.0 ± 0.1

* - Differences with the group GRS 1 mcmol (A-B) are statistically significant, p ≤ 0.05

** - Differences with the group GRS 10 mcmol (B-A) are statistically significant, p ≤ 0.05

Note - # not tested, since B-A transport of these compounds is negligible

High liver accumulation of GRS may lead to its high interaction with biotransformation enzymes and intense elimination by biological breakdown. Relatively low GRS accumulation in the kidneys shows the insignificance of renal elimination, at least in unchanged form. GRS distribution in the liver and kidneys would potentially require the monitoring of their function in patients taking the new drug. Absence of GRS in the brain shows that it doesn’t cross the blood-brain barrier.

Low plasma protein binding of GRS makes it safer and less likely to lead to dangerous drug interaction in combined therapy, caused by protein binding competition with other drugs.

Higher GRS distribution in blood cells creates high localized concentration of the active agent near the target – soluble guanylate cyclase. GRS doesn’t produce high toxic concentration in the plasma, which is especially important for a compound with low binding to plasma proteins.

Passive diffusion plays an important role in transmembrane transport from apical to basolateral membrane, while transport in the reverse direction is heavily dependent on P-glycoprotein. Proportional increase in transmembrane transport efficiency in A-B direction when increasing GRS concentration from 1 to 10 mcmol and absence of cyclosporin A effect on A-B transport of GRS shows that transmembrane transport of GRS relies mainly on passive diffusion (for example, during absorption). Higher intensity (by 8.8 times) of B-A transport compared to A-B transport of GRS shows the presence of a transporter protein. Considerable decrease in transport efficiency in B-A direction after the addition of cyclosporin A shows that P glycoprotein is involved in the transport of GRS. Increasing the concentration gradient of GRS from 1 to 10 mcmol in B-A direction doesn’t lead to a proportional increase in transport efficiency, showing the saturation of transporters (including Pgp) and insignificant involvement of passive diffusion in its B-A transport.

CONCLUSION:

Distribution of an innovative antiaggregant drug GRS, a soluble guanylate cyclase inhibitor, in rat organs and tissues was studied, its transmembrane transport mechanisms were identified using cell model of Caco-2 human adenocarcinoma. After oral administration to rats, GRS is quickly absorbed from the gastrointestinal tract, reaching maximal concentration after 1 hour. GRS has uneven organ distribution: its liver concentration is over 3 times higher than its plasma concentration.

This may accelerate the liver metabolism of GRS. GRS isn’t transformed by P450 enzymes as the authors have shown earlier in¹⁶. GRS accumulation in the heart shows that it may be used as an antiaggregant drug for preventing cardiovascular events. GRS has low distribution in kidneys and has very poor excretion with urine, at least in unchanged form, it’s possible that it’s metabolites may be excreted using this pathway. Renal elimination assessment is very important for patients with impaired renal clearance. GRS doesn’t cross the blood-brain barrier, although some indolinone derivatives are known to have psychotropic action^17-20. Low plasma protein binding of GRS reduces the risk of accumulation and dangerous drug interaction caused by protein binding competition with other drugs. At the same time, high GRS distribution in the blood cells prevents the formation of dangerously high concentrations in the plasma. GRS relies mainly on passive diffusion for its transmembrane transport, showing the predictability of its pharmacokinetics, however, the involvement of P glycoprotein in GRS transport requires the monitoring of P glycoprotein activity in combined therapy.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 20.12.2020 Modified on 25.02.2021

Research J. Pharm.and Tech 2022; 15(3):1241-1244.

DOI: 10.52711/0974-360X.2022.00207