Evaluation of Anti-inflammatory Activity of Novel HDAC Inhibitors using in vitro Methods

 

Swaliha C K¹*, Jayachandran T P², Shadiya C K³

¹Senghundhar College of Pharmacy, Kumaramangalam (PO), Tiruchengode - 637205,

Namakkal Dt,  Tamil Nadu.

²Division of Pharmacology, Department of Pharmaceutical Science, CPAS,

Cheruvandoor Kottayam, under KUHS Kerala 686631.

³National College of Pharmacy,  Manassery P.O, KMCT Medical College Road, Mukkam,

Kozhikode - 673602, Kerala.

 

ABSTRACT:

Inflammation is a neighborhood reaction to cell injury that is set apart by fine enlargement, leukocytic invasion, redness, heat, agony, expanding and loss of function. HDAC inhibitors play crucial role in regulation of inflammatory gene expression. The point of the investigation was to assess the anti-inflammatory activity of novel HDAC inhibitors having cinnamyl moiety. In the present study, two cinnamyl sulfonamide hydroxamate (CSH) derivatives (4a and 4b) were evaluated for their anti-inflammatory activity by in vitro methods, includes COX, LOX inhibitory assay, iNOS, MPO and determination of cellular nitrite level. The present study revealed that the newly derived compound 4a have potential anti-inflammatory activity and can serves as lead molecule in drug discovery.

 

 

INTRODUCTION:

The term inflammation is taken from the Latin word "inflammare" (to burn) (de oliveira). Inflammation is one of the most focal cycles needed with regards to animal cells against specific wounds or microbial infections¹.

 

Inflammation is characterized as the local response of living mammalian tissues to injury from any agent². It is a body guard response to kill or restrict the spread of damaging specialist, trailed by evacuation of the necrosed cells and tissue^3,4. Cells of immune system travel to site of damage or infection and cause inflammation⁵. It is believed that current drugs available such as opioids and non-steroidal anti-inflammatory drugs (NSAIDs) are not beneficial in all cases of inflammatory conditions, because of their side effects and potency⁶.

 

Traditional anti-inflammatory treatments, for example, steroids focused on fiery cycles at numerous levels, while later medication advancement has zeroed in rather on focusing on individual inflammatory mediators along a particular incendiary pathway⁷. Arising new mediation techniques include focusing on a particular quality class that controls different inflammatory pathways, and a significant new class of such unbridled controllers of downstream inflammatory pathways is the enzyme group known as histone deacetylases (HDACs)^8,9.

 

HDAC inhibitors (HDACi) at first viewed as "epigenetic modifiers" acting prevalently through histone acetylation, it is then again alluded to as lysine deacetylase or basically deacetylase^10,11,12. HDAC inhibitors (HDACi) can adjust articulation of various genes, including inflammatory gene, and meddle with a wide assortment of cellular function^8,11. Various investigations have shown that different wide acting HDACi's, including FR901228, phenylbutyrate and trichostatin A (TSA), stifle sickness action in animal models of incendiary joint pain¹³.

 

The potential of cinnamic acid as anti-inflammatory agent has been studied previously. In recent studies cinnamic acid derivatives indicated anti-inflammatory activity by means of HDAC inhibition^14,15.

 

In the present study, chloro and fluoro aniline derivatives of cinnamyl sulfonamide hydroxamate were evaluated for their anti-inflammatory activity via in vitro methods.

 

MATERIALS AND METHODS:

All chemicals and reagents used for the study were of analytical grade procured from approved organization.

 

In vitro anti-inflammatory methods:

Cell culture:

RAW 264.7 cells was procured from National Centre for Cell Sciences (NCCS), Pune, India and maintained Dulbecco’s modified Eagles medium, (DMEM) supplemented with 10% FBS and grown to confluence at 37^oC at 5% CO₂ in a CO₂ incubator.

 

The cells were grown to 60% confluency followed by activation with 1μL lipopolysaccharide (LPS: 1μg/mL). LPS stimulated RAW cells were exposed with different concentration (25,50, 100μg/mL) of 4a, 4b, Indomethacin and incubated for 24hours. After incubation the assays were performed using the cell lysate ¹⁶.

 

1. Cyclooxygenase (COX) inhibitory assay:

The COX activity was assayed by the method of Walker and Gierse.

100μl cell lysate was incubated with Tris-HCl buffer (pH 8), glutathione 5mM/L, and hemoglobin 5 mM/L for 1 minute at 25°C. The reaction was initiated by the addition of arachidonic acid 200 mM/L and terminated after 20 minutes incubation at 37°C, by the addition 200μL of 10% trichloroacetic acid in 1 N hydrochloric acid. After the centrifugal separation and the addition of 200μL of 1% thiobarbiturate, the tubes were boiled for 20 minutes. After cooling, the tubes were centrifuged for three minutes. COX activity was determined by reading absorbance at 632 nm^17,18.

 

Percentage inhibition of the enzyme was calculated as,

 

% Inhibition = [Absorbance of control-Absorbance of test/ Absorbance of control] X100

 

2. Lox inhibitory assay:

The determination of LOX activity was done as per Axelrod et al.

Briefly, the reaction mixture (2mL final volume) contained Tris-HCl buffer (pH 7.4), 50μL of cell lysate, and sodium linoleate (200μL). The LOX activity was monitored as an increase of absorbance at 234nm, which reflects the formation of 5-hydroxyeicosatetraenoic acid^8,19.

Percentage inhibition = [Absorbance of control – Absorbance of test/Absorbance of control] X100

3. Inducible nitric oxide synthase (iNOS) level estimation:

Nitric oxide synthase was determined by the method described by Salter et.al 1997.

Cell lysate was homogenized in 2mL of HEPES buffer. The assay system contained substrate 0.1mL L-Arginine, 0.1mL manganese chloride, 0.1mL 30μg dithiothreitol (DTT), 0.1mL NADPH, 0.1mL tetrahydropterin, 0.1mL oxygenated haemoglobin and 0.1mL enzyme (sample). Increase in absorbance was recorded at 401nm^20,21.

 

Percentage inhibition of the enzyme was calculated as,

% Inhibition = ((Absorbance of control-Absorbance of test)/Absorbance of control) × 100

 

4. Estimation of cellular nitrite level:

The nitrite level was estimated by the method of Lepoivre et al. (1990).

To 0.5mL of cell lysate, 0.1mL of sulphosalicylic acid was added and vortexed well for 30minutes. The samples were then centrifuged at 5,000rpm for 15 minutes. The protein-free supernatant was used for the estimation of nitrite levels. To 200μL of the supernatant, 30μL of 10% NaOH was added, followed by 300μL of Tris-HCl buffer and mixed well. To this, 530μL of Griess reagent was added and incubated in the dark for 10–15 minutes, and the absorbance was read at 540nm against a Griess reagent blank^16,22.

 

5. Myeloperoxidase (MPO) activity:

Cell lysate was homogenized in a solution containing 50 mM potassium phosphate buffer and 0.57% hexadecyl trimethyl ammonium bromide (HTAB). The samples were centrifuged at 2000 g for 30 minutes at 4°C, and supernatant was assayed for MPO activity. MPO in the sample was activated by the addition of 50 mM phosphate buffer (pH 6) containing 1.67 mg/mL guaiacol and 0.0005% H2O. The change in absorbance at 460 nm was measured. MPO activity was presented as units per mL of cell lysate. One unit of MPO activity was defined as that degrading 1 μM of peroxide per minute at 25°C ^16,23.

 

           ΔOD · 4 · Vt · dilution factor

U = –––––––––––––––––––––––––––––––

                    L · £470 ·Δt · Vs

 

ΔOD = density change

Vt = total volume (mL) (1.1 mL )

L = light path (1 cm)

£470 = extinction coefficient for tetraguaiacol (26.6 mM-1·cm-1)

 

Statistical analysis:

All the experiments were carried out in triplicate. Data expressed are mean ± SD. IC₅₀ values were calculated using MS Excel 2007 and sigma plot version 12.2 software.

 

RESULTS:

Two derivatives of cinnamyl sulfonamide hydroxamate (CSH) (E)-N-hydroxy-3-(4-(N-(phenyl chloro) sulfamoyl) phenyl) acrylamide (4a) and (E)-N- hdroxy-3-(4-(N-(phenyl fluoro) sulfamoyl) phenyl) acrylamide (4b) were evaluated for their anti-inflammatory activity using several In vitro methods.

 

In vitro anti-inflammatory studies:

Cyclooxygenase (COX) inhibitory assay:

The percentage inhibition of standard Indomethacin increased with increasing concentration and showed maximum of 88.253% at a concentration of 100μg/mL. Similarly, the synthesized compounds showed a concentration dependent increase in the percentage inhibition. IC₅₀ value of 4a, 4b and standard were found to be 67.16 μg/mL, 50.47μg/mL and 50.49 μg/mL respectively. The percentage inhibitions obtained from different concentration were tabulated in table 1.

 

Table: 1 Effect of 4a, 4b and Indomethacin on COX activity in RAW 264.7 cell line

Values were expressed as Mean ± SD of triplicate values

 

Lipooxygenase (LOX) inhibitory assay:

The percentage inhibition increased with increasing concentration and Indomethacin showed maximum of 87.428% at a concentration of 100μg/mL. Similarly, the synthesized compounds showed a concentration dependent increase in the percentage inhibition. The percentage inhibition obtained from different concentration was tabulated in Table 2.

 

Table: 2  Effect of 4a, 4b and Indomethacin on LOX activity in RAW 264.7 cell line

 

Inducible nitric oxide synthase (iNOS) level estimation:

The compounds 4a, 4b and standard showed reduction in the inducible nitric oxide synthase level in a dose dependant manner. IC₅₀ value of 4a, 4b and standard were found to be 77.57μg/mL, 95.48μg/mL  and 63.76 μg/mL respectively. The percentage inhibition obtained from different concentration was tabulated in Table 3.

 

Table: 3 Effect of 4a, 4b and Indomethacin on iNOS level

Data were represented as Mean ± S.D of triplicate determination.

 

 

Estimation of nitrite level:

It was observed that there was dose dependent decrease in the nitrite level in RAW 264.7 medium. Decreased cellular nitrite level is an indication of the capacity to inhibit nitric oxide synthase, thus inhibiting the production of nitric oxide. The absorbance of these compounds was measured at 540nm. The concentration of nitrite produced in different sample concentration was given in Table 4.

 

 

Table: 4 Cellular nitrite level estimation of 4a, 4b and Indomethacin on RAW 264.7 cell line

The values are expressed as Mean ± SD of triplicate values

 

 

Myeloperoxidase (MPO) enzyme activity:

The synthesized compounds 4a and 4b was found to be effective in inhibiting myeloperoxidase. Inhibition of MPO, which is a naturally occurring constituent of neutrophil, showed that the test drugs are able to prevent the accumulation of neutrophils and thus inhibits inflammation. The reduction in the myeloperoxidase activity by 4a, 4b and Indomethacin on comparison with control are tabulated in table 5.

Table:5 Estimation of Myeloperoxidase (MPO) activity

Values were expressed as Mean ± SD of triplicate values

 

DISCUSSION:

Inflammation is the response of the body to injury or infection. The purpose of the inflammatory response is to protect the tissue from injury and to contain any infectious process.

 

Two derivatives of CSH 4a and 4b were screened for their in vitro anti-inflammatory activity by COX and LOX inhibitory assay, myeloperoxidase (MPO) assay, iNOS level estimation and estimation of cellular nitrite. LPS stimulated RAW 264.7 cells were treated with different concentration of 4a and 4b showed inhibitory activity on reactive oxygen species and mediators of inflammation.

 

In COX and LOX inhibitory assays, both 4a and 4b showed anti-inflammatory activity which is comparable to standard drug indomethacin. They inhibit the formation of prostaglandin and leukotrienes. A dose dependent increase in the inhibition of myeloperoxidase activity was exhibited by both standard and test compounds 4a and 4b. Inhibition of MPO, which is a naturally occurring constituent of neutrophil, showed that the test drugs are able to prevent the accumulation of neutrophils and thus inhibits inflammation. The expression of inducible nitric oxide synthase (iNOS) is one of the direct consequences of inflammatory processes. Here Both standard and test drugs showed a marked reduction in the inducible nitric oxide synthase level in a dose dependant manner. Nitric oxide (NO) can induce tissue damage, especially after reaction with superoxide anion O2- . Decreased cellular nitrite level is an indication of the capacity to inhibit nitric oxide synthase, thus inhibiting the production of nitric oxide.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Principal, Department of Pharmaceutical Science, CPAS, cheruvandoor campus, Kottayam, under KUHS for providing all the facilities to conduct this research work.

 

The author is thankful to Kerala State Council for Science, Technology and Environment (KSCSTE) for availing the finance assistance (SRS grant) for this research work.

 

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Accepted on 22.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5455-5458.