6. Merged formatting files-IJMPS final

International Journal of Medicine and Pharmaceutical Sciences (IJMPS) ISSN 2250-0049 Vol.2, Issue 3, Dec 2012 41-49 © TJPRC Pvt. Ltd.,

PROTECTIVE ROLE OF Β CYCLODEXTRIN AGAINST PROTEIN DEGRADATION INDUCED BY ANTIBIOTICS 1

OMALI Y. EL-KHAWAGA, 2I. H. EL-SAYED, 1M.M. EL-NAGGAR, 1I.S. SHEHATA 1

2

Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt.

Molecular Biology Department, Genetic Engineering and Biotechnology Institute, Minofua University, Sadat City, Egypt.

ABSTRACT Cyclodextrin has a wide range of applications in different areas of drug delivery and pharmaceutical industry due to its complexation ability and other versatile characteristics. The most common pharmaceutical application of cyclodextrin is to enhance the solubility, stability, safety and bioavailability of drug molecules. The purpose of the present study was to investigate the protective activity of β cyclodextrin against the degradation of bovine serum albumin (BSA) induced by some antibiotics as amoxycilin, cloxacillin-Na and sulphamethoxazole as indicated by the estimation of some markers of protein oxidation. BSA was incubated with different concentrations of the antibiotics under investigation. An aliquot from this mixture was used for sodium dodecyl sulphate/ polyacrylamide gel electrophoresis (SDS-PAGE).

Oxidative damage of protein was assessed as a generation of carbonyl, quenone and advanced oxidation protein products (AOPP). All the antibiotics of this study had a degradation effect on BSA but sulphamethoxazole showed the highest oxidant activity. The increase in protein oxidation products was concentration dependent. Moreover, this effect of antibiotics was ameliorated by β cyclodextrin. The carbonyl, quenone and AOPP contents were highly significantly elevated in sulphamethoxazole, amoxycilin and cloxacillin-Na -treated protein respectively when compared to the control and β cyclodextrin protein samples. The present results confirm the oxidant activities of the above antibiotics which may be attributed to the generation of unstable radicals.

KEY WORDS:Cyclodextrins,Proteins Degradations, Antibiotics, SDS-PAGE, Protein Oxidation Products INTRODUCTION Aggregation of macromolecules, such as proteins and peptides happens mainly by the interaction of hydrophobic residues in the molecules and is often accompanied by drastic reduction of biological potency in formulating drug delivery systems 1. Proteins are distributed in all cellular compartments as well as forming key components of the extracellular matrix and maintaining the osmotic pressure of plasma which are sensitive to the noxious effect of the reactive oxygen species 2. β Cyclodextrins (CD) are a family of cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity 3. They are widely used as molecular cages in the pharmaceutical, agrochemical, food and cosmetic industries 4-5 In aqueous solutions, β cyclodextrins are able to form inclusion complexes with many drugs by taking up the drug molecule or some of its lipophilic moiety into the central cavity

6-7

. In the pharmaceutical industry, they are used as

complexing agents to increase the aqueous solubility of poorly soluble drugs and to increase their bioavailability and stability 8. Cyclodextrins were reported enhance the antiviral activity of ganciclovir on human cytomegalovirus and reduce the drug toxicity 9-10. Moreover, they have been used to ameliorate the irritation caused by drugs 11.

Sulphamethoxazole is an antibacterial drug which has been used in the treatment of various systemic infections in humans

12

. Amoxicillin and cloxacillin are broad-spectrum antibiotics that are used in the treatment of various bacterial

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Omali Y. El-Khawaga, I. H. El-Sayed, M.M. El-Naggar, I.S. Shehata

infections 13. It was reported that amoxicillin induced DNA damage through the generation of free radicals investigators showed that many antibiotics induced protein oxidations

16-18

14-15

. Several

. Since there is no published data on the the

protection of β cyclodextrin against the behavior of antibiotics on bovine serum albumin (BSA) in vitro, so the present study was designed to investigate the protective effect of β cyclodextrin on antibiotics- induced BSA degradation..

MATERIALS AND METHODS 1.

Chemicals: All chemicals used in this study were of analytical grade and were purchased from Sigma (USA).

2.

Cleavage of BSA by antibiotics: It was carried out in a mixture total volume 40 µl: 0.5 mg BSA, 10 mM Tris buffer pH 7.0 and 10- 40 µM antibiotic and 20- 40 µM β-cyclodextrin. After the incubation for 2h at 37Co the reaction was stopped by addition of 4 X SDS sample buffer followed by analysis of the damage in BSA by SDS PAGE 19. The gel was stained with coomasie brilliant blue R 250.

PROTEIN OXIDATION MARKERS. Protein-catechol (DOPA)/quenone content Quenones were detected by nitroblue tetrazolium (NBT) reduction as described by Paz et al

20

. The protein-

antibiotics mixture was incubated with NBT / glycine in the dark for 1 h before the absorbance at 530 nm was measured.

Protein-carbonyl content Protein carbonyl content was measured by reaction with 2,4-dinitrophenyl hydrazine(DNPH) as described by Reznik and Packer 21. The difference in absorbance between the DNPH treated samples vs the HCl blank was determined at 390 nm. The results are expressed as nmoles of carbonyl groups/mg protein using a molar extinction coefficient of 22, 000 Cm-1 for the DNPH derivatives.

Advanced Oxidation Protein Products(AOPP) AOPP was determined according to the method described by Abou-Seif and Youssef

22

. 200 µl of antibiotics-

treated protein diluted 1: 5 with PBS (pH 7.4) to which 10 µl of 1.16 M KI and 20 µl of acetic acid were added and absorbance was immediately measured at wavelength 340 nm against a blank consisting of 200 µl PBS treated in the same manner as a sample. The concentration of AOPP is expressed in chloramin T unit ( µmole /l).

RESULTS SDS-PAGE of BSA treated with the specified antibiotics reveals the generation of specific fragments of the protein treated with 40µM amoxycilin and 40 µM cloxacilin-Na after 2 hrs incubation (Lanes 9, 10 respectively, Fig. 1).

However, the degradation of BSA by 10, 20, 40µM of either amoxycilin or cloxacilin-Na is eliminated by the addition of 40µM β cyclodextrin to the reaction mixture. Moreover, the protection of BSA against damage induced by 40µM amoxycilin is increased with the increase of β cyclodextrin concentration (Fig. 2). High concentration of cloxacilin-Na (40 µM) induces more protein degradation. On the other hand, addition of 20 or 40 µM β cyclodextrin to the reaction mixture inhibites the degradation of BSA by Cloxacilin-Na (Fig. 3). Furthermore, specific BSA fragments were generated by treating the protein with sulphamethoxazole for 2 hrs (Fig. 4). The formation of these new fragments induced by treatment of BSA with sulphamethoxazole is inhibited by the addition to the reaction mixture of 10, 20, 30 or 40 µM β cyclodextrin (Fig. 5). Fig. 6 shows that treatment of BSA with amoxycilin ,cloxacilin-Na or sulphamethoxazole high significantly reduces more NBT than either the control or β cyclodextrin but the highest effect is apparent in the case of sulphamethoxazole. These results refers to the presence of catechol (DOPA)/quenone compounds on the oxidized protein.

The generation of carbonyl during incubation of protein with amoxycilin ,cloxacilin-Na and sulphamethoxazole are

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Protective Role of Β Cyclodextrin against Protein Degradation Induced by Antibiotics

observed in Fig.7. The carbonyl groups increased progressively with increasing the concentrations of the studied antibiotics. The mean concentration values of advanced oxidation protein products (AOPP) are highly significantly elevated when BSA was incubated with sulphamethoxazole , amoxycilin and cloxacilin-Na respectively in comparison to the control or β cyclodextrin –treated BSAs (Fig.8).

DISCUSSIONS Cyclodextrins are useful functional excipients that have widespread attention and use in the pharmaceutical industry. Studies in both humans and animals have shown that cyclodextrins can be used to improve the drug delivery from almost any type of drug formulations

11,23

. The reactive oxidant species with different concentrations of chloramphenicol

increased the carbonyl residues and advanced oxidation protein products (AOPP) as consequence of oxidative stress24.

Moreover, the effect of stabilization produced by β cyclodextrin on carbon centered radicals afforded by some mercptopyridine congeners were shown to possess strong anticancer activity in vitro 25. Many investigators showed that the formation of complexes between rifampicin and cyclodextrin resulted in an increase in rifampicin apparent solubility 2627

. The present study revealed the degradation of BSA by amoxycilin, cloxacillin-Na and sulphamethoxazole and this

degradation was inhibited by β cyclodextrin. This effect stimulated BSA damage at the highest concentrations used. Also, these data show that amoxycilin, cloxacillin-Na and sulphamethoxazole have the ability to reduce nitroblue tetrazolium (NBT) (Fig 6). The protein-bound catechol/quinone compound, which might be produced on proteins damaged by ˙OH radicals due to the attack of tyrosine residues in BSA

28

. The results presented here (Fig 7) show that carbonyl groups

increased progressively with increasing the studied antibiotics concentrations, confirming that the oxidation effects of these antibiotics might be mainly attributed to the generation of ˙OH radicals

29-31

. The elevation of carbonyls in the studied

antibiotics -treated BSA might also be attributed to the oxidation of amino acid residues in BSA 32. The results presented in Fig(8) show that the levels of AOPP in BSA increased with increasing the antibiotics concentrations. The elevation of AOPP in amoxycilin, cloxacillin-Na and sulphamethoxazole -treated BSA might be attributed to: [1] the reactive oxygen species generated by antibiotics. This argument is strengthened by the finding that AOPP, as terminal products of protein exposure to free radicals, has been considered as a reliable marker of the degree of oxidative protein damage by free radicals

33-35

. [2] The increased AOPP levels might be due to the increased oxidation products such as quenone and

carbonyl contents which results from the aromatic amino acid residues of BSA. The activity of β cyclodextrin may be attributed to its nature as having a cage responsible for preventing the interaction of radicals produced from the interaction of antibiotics with the medium causing the damage of protein. Furthermore, the presence of β cyclodextrin has been shown to delay the generation of radicals in water due to a possible interaction between cyclodextrin cavity and carbonyl moiety of antibiotics.

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Figure 1: SDS-PAGE Of BSA Treated with Different Concentrations of Amoxycillin and Cloxacillin-Na after 2 Hrs Incubation Lane 1: Control BSA Lane 2 : BSA treated with 40µM β cyclodextrin Lanes (3-5): BSA treated with 10, 20, 40µM amoxycillin with 40µM β cyclodextrin Lanes (6- 8): BSA treated with 10, 20, 40µM cloxacillin-Na with 40µM β cyclodextrin Lane 9: BSA treated with 40µM amoxycillin Lane 10: BSA treated with 40µM cloxacillin-Na

Figure 2: SDS-PAGE of BSA Treated with 40 µm Amoxicillin and Either 20 or 40 µm Β Cyclodextrin after 2 Hrs Incubation

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Omali Y. El-Khawaga, I. H. El-Sayed, M.M. El-Naggar, I.S. Shehata

Lane 1: Control BSA Lane 2: BSA treated with 40 µM β cyclodextrin Lane 3: BSA treated with 40 µM amoxycillin Lane 4: BSA treated with 40 µM β cyclodextrin and 40 µM amoxycillin Lane 5: BSA treated with 20 µM β cyclodextrin and 40 µM amoxycillin

Figure 3: SDS-PAGE Of BSA Treated with 40 µm Cloxacillin-Na and Either 20 or 40 µm Β Cyclodextrin after 2 Hrs Incubation Lane 1: Control BSA Lane 2: BSA treated with 40 µM β cyclodextrin Lane 3: BSA treated with 40 µM cloxacillin-Na Lane 4: BSA treated with 40 µM β cyclodextrin and 40 µM cloxacillin-Na Lane 5: BSA treated with 20 µM β cyclodextrin and 40 µM cloxacillin-Na

Figure 4: SDS- PAGE of BSA Treated with either 20 or 40sulphamethoxazole and 40µm Β Cyclodextrin after 2hrs Incubation

Protective Role of Β Cyclodextrin against Protein Degradation Induced by Antibiotics

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Lane 1: control BSA Lane 2: BSA treated with 20 µM sulphamethoxazole and 40 µM β cyclodextrin Lane 3: BSA treated with 40 µM sulphamethoxazole and 40 µM β cyclodextrin Lane 4: BSA treated with 40 µM sulphamethoxazole

Figure 5: SDS-PAGE of BSA treated with 40 µM sulphamethoxazole and different concentration of β cyclodextrin after 2 hrs incubation : Lane 1: control BSA Lane 2: BSA treated with 10µM β cyclodextrin and 40 µM sulphamethoxazole Lane 3: BSA treated with 20 µM β cyclodextrin and 40 µM sulphamethoxazole Lane 4: BSA treated with 30 µM β cyclodextrin and 40 µM sulphamethoxazole Lane 5: BSA treated with 40 µM β cyclodextrin and 40 µM sulphamethoxazole Lane 6: BSA treated with 40 µM sulphamethoxazole

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Omali Y. El-Khawaga, I. H. El-Sayed, M.M. El-Naggar, I.S. Shehata

Protective Role of Β Cyclodextrin against Protein Degradation Induced by Antibiotics

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Figures Legends: Figure 1:

SDS-PAGE of BSA treated with different concentrations of amoxycillin and cloxacillin-Na after 2 hrs

incubation: Figure 2: SDS-PAGE of BSA treated with 40 µM amoxicillin and either 20 or 40 µM β cyclodextrin after 2 hrs incubation: Figure 3: SDS-PAGE of BSA treated with 40 µM cloxacillin-Na and either 20 or 40 µM β cyclodextrin after 2 hrs incubation : Figure 4: SDS- PAGE of BSA treated with

either 20 or 40sulphamethoxazole and 40µM β cyclodextrin after 2hrs

incubation: Figure 5: SDS-PAGE of BSA treated with 40 µM sulphamethoxazole and different concentration of β cyclodextrin after 2 hrs incubation : Fig ( 6): Detection of protein- bound Catechol/ quinone in BSA after its incubation

with different concentrations of

antibiotics Data are means ± SD of 5 experiments Fig ( 7): carbonyl contentof BSA after its incubation with different concentrations of antibiotics Data are means ± SD of 5 experiments Fig (8): Advanced oxidation protein products (µmole/mg protein