The photochemical stability of pharmaceutical substances is a matter of concern. Light-induced decomposition of more than 100 medicinal compounds has been reported in the literature and the European Pharmacopoeia prescribes light protection for about 250 medical drugs and a number of adjuvants. Although photostability testing has not been required by the regulating authorities up to now, this is about to change with the introduction of the ICH guidelines on photostability testing of drugs. The most obvious result of drug photodecomposition is a loss of potency of the product. In the final consequence this can result in a drug product which is therapeutically inactive. Although this is rarely the case, even less severe degradation can cause formation of (photo)toxic degradation products. The drug substance can also cause light-induced side-effects after administration to the patient by interaction with endogenous substances. This can be the case both for systemically and topically administered compounds. It is for instance, well established that parts of the sunlight spectrum (UV-A, visible light) penetrate the skin to a depth sufficient to reach the blood circulating in surface capillaries and thereby the dissolved drug molecules. Excitation of the drug molecule by absorption of irradiation in vivo may generate reactive oxygen species (ROS), free radicals or toxic photoproducts which can be biologically harmful.

It is essential to obtain information about the photoreactivity of a drug molecule as early as possible in the formulation process. Knowledge about the photochemical and photophysical properties of the compound is essential for handling, packaging and labelling of the drug substance and drug product but also in order to understand implications for lack of efficacy or possible photosensitizing activity in vivo. Several in vitro methods for phototoxicity studies are described but in many cases in vivo methods will also be required. A complete assay for photoreactivity screening is time and money consuming and requires a broad spectrum of techniques. A selection of the drugs to undergo a full screening can be made on certain criteria.1 Large variations are found among molecules that can act as photosensitizers in biological systems, and photoreactivity is difficult to predict. A photostable drug may act as an efficient in vivo photosensitizer by a series of photophysical events while a photolabile compound may cause phototoxic reactions by formation of reactive intermediates or toxic photoproducts. It is important to keep in mind that the photoreactivity of a pure compound can change when the sample is introduced into a biological system. The most frequently used antimalarial compounds are structural analogues, i.e. they belong to the group of quinolines. They fulfil almost every criteria needed to subject a drug to a complete photoassay; they are sensitive to irradiation, contain photoreactive functionalities, accumulate in tissues exposed to light, have an absorption spectrum that overlaps with the transmission spectra of the actual tissues, are administered at high accumulative dosages, and are used in climatic zones where the solar irradiance is high. On the basis of these criteria the photoreactivity of selected antimalarial compounds has been investigated according to a previously described in vitro assay. 1 The compounds discussed belong to the groups of 4-aminoquinolines (chloroquine), 8-aminoquinolines (primaquine) and 4-quinolines (mefloquine) respectively.

1.1 In vitro Screening of Compounds in Solution

1.1.1 Reaction Medium. It is difficult to predict the photoreactivity of a drug molecule from structural considerations only.2'3 Experiments have to be performed to elucidate the photochemical reaction-pattern of the substance, and the experimental design has to be planned thoroughly. In vivo conditions will always be very complex compared to what can be achieved in vitro. A certain in vitro - in vivo correlation may however be obtained by carefully designing the in vitro photoassay. It is important to mimic physiological conditions in the experimental set-up by use of aqueous solutions at physiological pH and ionic strength. Micelles, liposomes and cyclodextrines can be added to the medium to mimic the effects on membranes. Most photosensitized reactions in biological systems require the involvement of molecular oxygen. It is therefore natural to use media in equilibrium with air in photoreactivity studies. An elevated oxygen concentration or anaerobic conditions can, however, provide valuable information in mechanistic studies of photostability. An increase in oxygen concentration can lead to a decrease or an increase in degradation rate. If the drug molecule initiates the formation of singlet oxygen followed by a self-sensitized type II reaction an increase in degradation rate may be observed at higher oxygen levels. A decrease in degradation rate can be the result when quenching of the reactive triplet state of the drug molecule by oxygen is the process in favour. A change in the formation of degradation products can occur when the oxygen level is supressed. Chloroquine is demonstrated to dechlorinate during anaerobic photolysis,4 while dechlorination cannot be observed during aerobic photolysis.5 There are also some exceptions in which oxygen-independent processes have an important place in the overall picture of photosensitization.6>7 Photoreactivity studies carried out under elevated oxygen levels and under anaerobic conditions should therefore be included in the in vitro assay.

Many drug molecules, like the antimalarial compounds, are weak acids or weak bases. The pH of the reaction medium may in that case strongly influence the results obtained. The degree of protonation will affect the spectral characteristics of the drug in the medium as well as the photochemical reaction pattern. The pH of the medium can therefore have a considerable effect on the photochemical stability of a compound. It is obvious that valuable information can be lost if the experiments are carried out only in one medium, e.g. organic solvent that can not differentiate between protonated and deprotonated forms of the molecules. In mechanistic studies of drugs that are acids or bases experiments should therefore be carried out at a series of pH values covering all the protonation forms of the molecule.

1.1.2 Formation of Free Radicals and Singlet Oxygen. The photochemical decomposition of a drug substance will often involve radical processes and/or formation of singlet oxygen. The excited drug molecule can exchange electrons with another drug molecule, with oxygen or with other compounds present in the medium. Free radicals formed can subsequently react with new drug molecules, leading to degradation by several pathways. Excipients or impurities present in the drug product may also initiate radical chain reactions by absorption of radiation. The absorbed energy can further be transferred from the excited drug molecule to ground state oxygen leading to the formation of singlet oxygen which subsequently can participate in various reactions. Formation of radicals or singlet oxygen in vivo may cause damage to proteins, lipids, DNA and RNA, and/or cell membranes. Determination of the detailed photoreactivity characterisitcs of a drug molecule requires knowledge of the sensitizing and quenching properties of the molecule, e.g. quenching efficiency of the drug excited state by a substrate, the ability of the drug to transfer an electron to oxygen and the rate of reaction of the drug radical with ground state oxygen. A number of techniques have been developed to enable the detection of free radical intermediates, including electron paramagnetic resonance spectroscopy (EPR) and pulse radiolysis. An infrared luminescence technique is frequently used for determination of singlet oxygen.8 These methods require specialized equipment. A more simple approach for studying radical formation is by addition of various free radical scavengers to the medium during irradiation. The rate of disappearance of the drug and the apparence of particular products is compared with that occurring without the scavenger. Polymerization reactions (e.g. polymerization of aciylamide) can be used to detect formation of free radicals in anaerobic media.9 The photooxidation potential can be studied in terms of oxygen uptake measurements in the presence of oxidable substrates like histidine and 2,5-dimethyl furan which are substrates for singlet oxygen, and L-tryptophan which is a substrate for superoxide. The reactions should be confirmed by addition of suitable singlet oxygen quenchers and superoxide dismutase. The (lack of) specificity of the various scavengers and substrates should be taken into account, and a combination of scavengers or substrates should be used to obtain adequate information. It is important to remember that the relative reactivity of both the radicals and the scavenger will determine the outcome of the reaction. If the radical intermediates are extremely reactive, they may react with the solvent before any other reaction can occur and no change will be observed.

1.1.3 Characterization of Excited States. The lifetime of the excited singlet state of a drug molecule is generally of the order of nanoseconds. This time is normally too short for the excited molecule to react chemically even with neighbouring molecules, although photoionization can occur. Excited triplet states of drug molecules can have long lifetimes (up to several seconds). Long-lived intermediates with lifetimes around one second may diffuse between organelles or to neighbouring cells prior to a reaction with oxygen or endogenous compounds.10 Excited drug triplets formed in vivo can therefore often reach several molecular targets prior to deexcitation, implicating a high probability for phototoxic reactions. The production of singlet oxygen has been reported to occur by energy transfer both from the singlet and triplet state of a sensitizer. The singlet-triplet interaction is,however, of very low probability and formation of singlet oxygen by energy tranfer from the triplet state of the sensitizer is highly preferred. Transient species like singlet and triplet excited states can be observed by use of flash photolysis.9 Valuable information concerning the formation of singlet and triplet states can, however, also be obtained from fluorescence and phosphorescence measurements. The lifetime of a molecule in the excited singlet or triplet states is related directly to the fluorescence and phosphorescence lifetimes respectively. Information about the possible photoreactivity of the drug substance and its degradation products or metabolites can therefore be obtained from luminescence quantum yield measurements and life-time studies. Additionally, the singlet and triplet energies of the compound are of considerable interest in mechanistic studies especially with respect to energy transfer. Phosphorescence is usually too weak to be observed in solution at room temperature. The drug molecule should therefore be held in a transparent (glassy) matrix at low temperature during the experiment. Unless fluorescence is measured under the same conditions the quantum yields cannot be compared.

1.1.4 Photosensitized Damage of Endogenous Substances. A phototoxic drug can damage the body tissues by formation of radicals or by energy transfer to endogenous compounds. Oxygen is present in all body tissues, and the body therefore provides excellent conditions for formation of reactive oxygen species. Cell constituents as proteins, unsaturated fatty acids, cholesterol and DNA are likely targets for damage caused by ROS. The reactions are often leading to photomodification of the cell membranes. The tissues are normally equipped with antioxidants but accumulation of a phototoxic compound can overload the capacity of the protecting agents in a specific tissue.11' 12 Photohaemolysis of red blood cells provides a simple technique to monitor membrane photomodification. Erythrocytes have no intracellular organelles, so the oberved effects are due to effects on the membrane.13 Some of the antimalarial drugs are previously reported to accumulate in melanin-rich tissues (eye, skin, hair).14 Accumulation of drugs in light exposed areas is important for their ability to act as photosensitizers. The turnover of melanin in the body is very low, except for epidermal melanin. Compounds with high melanin affinity can be retained in melanin-containing tissues for years.15 Studies of in vitro interactions between the drug molecules and melanin is therefore of great importance in the evaluation of drug phototoxicity. The particle size and zeta potential of the melanin granules must be taken into account as the chemical composition of melanin isolated from different tissues will vary. Several of the aminoquinoline antimalarials are also known to induce ocular adverse effects. Both retina, lens and cornea are likely targets for photosensitized damage in the eye. Photosensitized reactions in the lens can lead to a modification of the amino acids in the cytosol proteins and/or a covalent binding of the sensitizer to the proteins. This leads to a change in the physical properties of the lens proteins which results in aggregation and opacification of the lens (lens cataracts).16 Isolated proteins from calf lens can be used as an in vitro biological test system for estimation of ocular phototoxicity.

1.1.5 Pharmacokinetic Parameters and Dosage Regime. Most of the adverse effects associated with the use of antimalarials are related to the eye and skin. Drugs administered systemically like the antimalarial compounds must be distributed to the actual body tissues e.g. the eye, skin, or the outermost capillaries of the skin, to act as photosensitizers. Systemical distribution of exogenous substances to the eye is however limited. Although the retina is richly supplied with blood vessels, the blood-retinal barrier is normally very tight and therefore restricts the movements of substances from the capillaries. The lens has no blood supply and drugs cannot be distributed directly from the systemic circulation to this tissue. On certain occasions the permeability of the blood-retinal barrier can be altered. Drugs will then to a larger extent enter the retinal pigmented epithelium (RPE) and retina. Drugs accumulated in the RPE can further pass through the vitreous cavity and reach the lens. Phototoxicity is generally dose dependent, i.e.dependent on the concentration of the drug sensitizer at the site of action. The pharmacokinetic properties of the compound are therefore essential factors which have to be considered when in vitro results are used to predict in vivo photoreactivity. Mefloquine is used for the prophylaxis of malaria in areas of the world where there is a high risk of chloroquine-resistant falciparium malaria. Mefloquine is usually administered as a single dose or as two divided doses and is rarely used over a long period of time in order to prevent mefloquine resistance. This is in contrast to chloroquine and sometimes also primaquine, which can be used for periods of months or even years. The dosage regime for mefloquine is, however, about to change as mefloquine now also is used in long-term prophylaxis for up to one year.17 As mentioned above most of the adverse effects reported after use of anti-malarial drugs seem to be dose-related. High accumulative dosages and long-term administration may be important factors for drug phototoxicity. The dosage regime should therefore also be taken into account when the phototoxic potential of the various drugs are to be compared.

1.2 In vitro Screening of Compounds in the Solid State

Pharmaceuticals intended for use in the tropics, like the antimalarial compounds, are required to maintain their stability under the most severe storage conditions. The bulk substance and drug product will often be distributed through local traders whom in the majority of cases are ignorant of the ideal storage requirements. The compounds may therefore be exposed to considerable amounts of humidity, heat and sunlight before use. The most frequently used dosage form for antimalarial drugs is tablets although chloroquine is also formulated as injection solutions and syrups. If the patient is seriously ill quinine should be given by long term intravenous infusion (8-12 hours).

Photodegradation of a compound in the solid state is quite different from degradation of the substance in solution. In the solid state the reactions take place only in a thin surface-layer of the compound. The particle size and shape and the structure of the crystal lattice (i.e. crystal modification) will strongly influence the photostability of the bulk material. The most obvious results of the irradiation of a solid sample is a change in appearance (i.e. change in colour) and photodegradation of the compound. Change in appearance does not necessarily relate to degradation of the compound and vice versa. This means that discolouration of e.g. a tablet in many cases not will affect the efficacy of the product but on the other hand there might be a loss in potency although the tablet looks unchanged. A discolouration of compounds stored under conditions as discussed above is often observed. It is then of great importance to evaluate the correlation between change in appearance and degradation of the parent compound. In many cases visual inspection will be the only "quality control" possible after the product has reached the local market.


The compounds discussed here represent the three main groups of quinolines used in malaria therapy, i.e. the group of 4-aminoquinolines consisting of chloroquine, hydrochloroquine and amodiaquine, the 8-aminoquinolines consisting of primaquine and the 4-quinolines consisting of quinine and mefloquine. Some of the antimalarial drugs e.g. chloroquine and hydroxychloroquine, have also proved to be useful in the treatment of other, noninfectious disorders.18 All the compounds give adverse effects which are possibly phototoxic reactions; they are known to be photolabile in solution and a discolouration is often observed in the solid state. The photoreactivity screening in the present study was performed according to the methods described above.

2.1 Chloroquine Diphosphate

2.1.1 Chloroquine in Solution. Chloroquine (CQ) can exist as a single- and double-charged cation and in a neutral form. The pKa values for CQ are reported to be 8.4 and 10.4 for the heterocyclic nitrogen atom and aliphatic nitrogen atom respectively. The pKa value for the amino group bonded to the Cio atom on the ring has not yet been determined. At physiological pH chloroquine will exist both as the dication (91%) and monocation (9%) (Figure 1). The dication of CQ is shown to exist as the imine tautomer while the monocation exists as a mixture of the amine (8.1%) and the imine (0.9%) at physiological pH (19). The compound is demonstrated to decompose on exposure to irradiation both in aqueous solution and in organic solvent, and several degradation products are isolated and identified.5'20

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