UV light induced photodegradation of liposome encapsulated fluoroquinolones: An MS study

Abstract

Fluoroquinolone antibacterial agents are among the drugs most commonly causing phototoxic side effects. The phototoxicity may be originated in formation of reactive oxygen species upon ultraviolet exposure. Researches aiming the liposomal encapsulation of fluoroquinolones, expecting an increase in their therapeutic index, enhance the importance of studies on physicochemical properties and photostability of liposomal preparations. We studied the photodegradation of ciprofloxacin, ofloxacin and lomefloxacin by mass spectrometry upon various doses of UV irradiation. Lomefloxacin, the most phototoxic fluoroquinolone among them, was encapsulated into small unilamellar and multilamellar liposomes. Impact of vesicle structure and lipid composition – the presence of unsaturated fatty acid containing dioleoyl-phosphatidylcholine in dipalmitoyl-phosphatidylcholine liposomes – on the lomefloxacin photolysis was investigated; the structure of the main photoproducts was identified by mass spectrometry. It was found that the presence and type of lipids influence the ways of photodegradation process.

Introduction

Fluoroquinolones are the only synthetic antibacterial agents to rival β-lactams for impact and usage in the antibacterial field. In two decades, they moved from a small group of drugs used predominantly in urinal tract infection to agents with a remarkably broad spectrum of activity and excellent pharmacokinetics [1]. Among side effects induced by fluoroquinolones – in the presence of light – acute phototoxic reactions are by far the most common, and are generally characterized by erythema and oedema followed by hyperpigmentation and desquamation [2]. The fluoroquinolone antibacterials possess phototoxic properties both in human and animal subjects [3], [4]. Their phototoxicity order reported is lomefloxacin (LMFX), pefloxacin (PEFX) ≫ ciprofloxacin (CPFX) > enoxacin (ENOX), ofloxacin (OFLX) [5], while others report LMFX > OFLX > CPFX [4]. Evaluating the molecular background, the formation of reactive oxygen species (ROS) appears to be responsible for the light induced adverse effects [6], [7]. Quinolones can undergo a variety of photochemical processes such as generation of singlet oxygen, production of superoxide, decarboxylation, defluorination and oxidation of an amino substituent at C7 [8]. According to some recent studies, beside ROS, the presence of highly reactive species arising from direct photolysis of fluoroquinolones (e.g. LMFX) can justify the photogenotoxic properties associated with fluoroquinolones likely due to direct reaction of their cations with DNA [9], [10]. A notable feature, particularly of C8 fluorinated fluoroquinolones such as LMFX is its ability to undergo photolysis and defluorinate upon UV-light exposure. UVA irradiation of 10 μg/ml of LMFX in phosphate buffer (pH 7.4) lead to more than 50% decomposition within 10 min [11]. Loss of the fluorine atom at C8 and partial breakdown of the piperazine ring occur. The only two photoproducts formed under these conditions are 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminoethyl-amino)-4-oxo-3-quinolinecarboxylic acid and 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminopropyl-amino)-4-oxo-3-quinolinecarboxylic acid [11]. The mechanism of photo-induced defluorination of LMFX in aqueous media is published [11], [12], [13], [14], [15] but there are no data on the loss of fluorines in other media including the circumstances of nano-delivery systems, e.g. liposomes.

Innovative technologies aim to increase the bioavailability of fluoroquinolones by means of liposomes. It seems clear that a well-designed lipid carrier system allows increased drug concentration at the sites of action but reduces drug toxicity [16]. However, it is necessary to investigate the effects of liposomal encapsulation not only on the therapeutic efficacy of the encapsulated drug, but also on the extent of ROS generation and photodegradation of the encapsulated fluoroquinolones upon UV-irradiation. This latter informs about the photostability of liposomal fluoroquinolones in the presence of UV-exposure that is of prominent importance in case of their UV-sterilization, storage and topical administration.

In our earlier work we investigated a prominent member of the quinolones, the nalidixic acid, and observed increased free radical formation in the presence of α-l-dipalmitoyl-phosphatidylcholine (DPPC) [17]. In the present work we present a mass spectrometry (MS) study carried out on fluoroquinolone (CPFX, OFLX and LMFX) containing aqueous and liposomal samples exposed to various UVB doses. We address the question whether the presence of liposomes alters the extent and the ways of fluoroquinolone photodegradation and whether the liposome structure influences the LMFX degradation. LMFX containing small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of DPPC were examined. In order to gain further pieces of information on the role of lipid composition and the effect of unsaturated fatty acid containing lipids on the rate of LMFX photodegradation, SUVs prepared from a mixture of DPPC and dioleoyl-phosphatidylcholine (DOPC) (70/30 mol/mol%) encapsulating LMFX were also studied.

In each case the degradation constant for the encapsulated agent and the structure of photoproducts formed as a result of irradiation were determined using MS or MS–MS technique.