“Validated Methods for Determination of Some Antifungal Drugs”
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“Validated methods for determination of some antifungal drugs” A thesis presented by R a m i a G a m a l E l - S a y e d E l - G a n y a n y B.Sc. Pharm. Sci. Faculty of Pharmacy, MUST University 2004 Submitted in the partial fulfillment for The degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry) Supervised by Prof. Dr. Ramzia Ismail El-Bagary Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University Head of Pharmaceutical Chemistry Department Faculty of Pharmacy- Future University Dr. Marianne Alphonse Mahrouse Associate Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University Dr. Maha Medhat El-Hakeem Lecturer of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University Faculty of Pharmacy Cairo University Abstract Butoconazole nitrate and ciclopirox olamine are topical antifungal, (HPLC) method for the analysis of Butoconazole nitrate BUT in drug substance and in drug product was developed and validated. The method is based on isocratic HPLC separation of BUT from its oxidation degradation product DEG1 using a mobile phase consisting of methanol: phosphate buffer (20 mM adjusted to pH 6.5 with o-phosphoric acid): triethylamine (80: 20: 0.1, v/v /v). The flow rate was maintained at 1.5 ml/min .UV spectrophotometric detection was carried out at 270 nm. TLC- densitometric method was described for the determination of Butoconazole nitrate BUT in presence of its oxidation degradation product DEG1, in drug substance and in drug product. The suitable developing system to achieve the best separation was hexane: ethyl acetate: methanol (7: 3: 0.85, v/v/v). The scanning of the spots was performed at 276 nm. A multiple divisor ratio difference spectrophotometric method is proposed for the simultaneous determination of BUT The differences in peak amplitudes at two different wavelengths namely, 259 nm and 277 nm (P277 - 259 nm) of ratio spectra were measured. A muliple divisor ratio spectrophotometric method is proposed for the simultaneous determination of Butoconazole nitrate BUT . The ratio spectra were obtained by dividing the absorption spectra of different concentrations of the spectrum of BUT by the sum of the absorption spectra of MP, PP and DEG1 as a multiple divisor. The first derivative of the obtained ratio spectrum of BUT was then recorded. The amplitude of the maximum at 262 nm . HPLC method was developed for the separation of Ciclopirox Olamine (CO) and Photodegradtion (DEG2) in drug substance and pharmaceutical dosage form using a stationary phase composed of Reprosil-Pur column (C8) The mobile phase used was acetonitrile: water: H3PO4 (pH 2.6) (40: 60: 0.1, v/v/v). The flow rate at 2.5 ml/min .UV detection was carried out at 303 nm. Photodegradation of CO was carried out in methanol and the kinetic order of the degradation was evaluated. The method presented in this section is based on measuring the difference in absorbance (A) at 317 nm o f (CO) a c i d i c solution in 0.1N HCl against that of the 0.1N NaOH as a blank. A zero order spectrophotometric method is proposed for the determination of Ciclopirox Olamine (CO). The zero order spectrum of CO was recorded and the absorbance at 303 nm was measured. spectrophotometric method is proposed for the determination of Ciclopirox Olamine (CO). The method is based on chelation between iron (III) and CO producing a colored complex. The absorbance of the developed orange red colored complex was measured at 470 nm. I.1.Introduction Fungi are heterotrophic, eukaryotic microorganisms that are distinguished from algae by lack of photosynthetic ability. Fungi grow in either colonies of single cells (yeast) such as pathogenic Candida albicans and Saccharomyces cerevisiae or in filamentous multicellular aggregates (molds) such as Trichophyton rumbrum and Aspergillus fumigans1. Most fungi live as saprophytes in soil or dead plant material and are very important in the mineralization of organic matter2. Cells of fungi pathogenic for animals have a rigid cell wall containing chitin and polysaccharide. The true fungi are grouped into four classes: Phycomyceters (algaelike), Ascomycetes (Sac- fungi), Basidiomyctes (mushrooms), and Deuteromycetes (imperfect fungi)3. Fungal infections may be classified as: 1-Superficial mycoses: -Superficial mycoses affecting only hair and most superficial layer of epidermis .The most common are black piedra, tinea nigra, tinea versicolor and white piedra. 2-Cutaneous mycoses: -The cutaneous mycoses, infect only epidermis and its appendages (hair and nail) producing a condition known tinea or ringworm. These infections are caused by fungi known as dermatophytes, as infection of feet (Tinea pedis), of body (Tinea corporis) and of the scalp (Tinea capitis). 3-Subcutaneous mycoses: -The subcutaneous mycoses include skin and subcutaneous tissues. 4-Systemic mycoses: -Systemic mycoses involve skin and deep organs as lungs, lymphatic tissues and various organs, for example actinomycoses (tumors in jaw and tongue), blast mycoses (initial pulmonary infection followed by progressive dissemination to most organs) and cryptococcosis (CNS infections). Fungal infections may also be described as local when they are restricted to one body area or when the infection spread from the primary site to other organs of the body. Treatment of fungal infections is more difficult than that of bacterial infection as many fungal infections occur in poorly vascularised or vascular tissues.3 In the last 30 years, there was a steady increase in systematic fungal infections because of wide spread use of broad-spectrum antibiotics and increase of number of individuals with reduced immune response or due to administration of immunosuppressant drugs. So antifungal treatment should be chosen after the infecting organisms have been identified but treatment may be started before the pathogen can be cultured and identified. Not all antifungal agents are fungicidal, many are only fungistatic and certain topical antifungal drugs may owe their efficacy due to keratolytic action3. Classification of antifungal drugs: 1-Polyenes. The polyenes are a number of structurally complex antifungal isolated from soil bacteria .They contain a conjugated system of double bonds in large lactone rings. Polyenes fall into two groups based on the size of the macrolide ring: -The 26-membered ring polyenes, such as natamycin. -The 38-membered ring polyenes, such as nystatin, amphotericinB and candicidin. They also differ in the number of double bonds present in the lactone ring ,for example natamycin is a tetraene , nystatin is a hexaene while both amphotericin B and candicidin are heptaene4. The mechanism of action of polyenes is by binding tightly to sterols present in cell membrane. The resulting deformity of the membrane allows leakage of intracellular ions and enzymes, causing cell death5. Polyenes have an affinity for sterol-containing membranes, insert into the membranes, and disrupt membrane functions. The cells treated with polyenes die because of the loss of essential cell constituents, such as ions and small organic molecules. The basis for selective toxicity of ployene to fungi is due to drug binding to ergosterol (the main sterol in fungi membrane) which is at least tenfold stronger than binding to cholesterol (the main sterol in plasma membrane of animal cells).3 The usefulness of polyenes for the treatment of systemic infections is limited by their toxicities, low water solubility and poor chemical stability. However, amphotericin B is the only polyene available for the treatment of serious systematic infections but it must be solubilized with the aid of an emulsifying agent. On other hand, nystatin is a useful polyene compound and an effective topical antifungal against a wide variety of organisms.Nystatin is too toxic to be used systemically, because very little drug is absorbed but it may be administered by mouth to treat fungal infections of the mouth and gastrointestinal tract. OH OH OH H3C O OH HO O OH OH OH O CH3 COOH H3C O CH3 O (a) HO H2N OH OH OH H3C O OH HO O OH OH OH OH O CH COOH 3 H (b) H3C O CH3 O HO H2N OH O OH O OH H C O OH O 3 COOH (c) O CH3 O HO H2N OH Figure (1): Chemical structure of (a) Nystatin (b) Amphotericin B (c) Natamycin 2-Azoles. Azoles are the largest class of antifungal agents that contain a five membered azole ring attached by a N-C bond to other structures. This group is divided into imidazoles, and mechanism of action of azoles is by inhibition of synthesis of ergosterol from lanosterol, which is an important component of the cytoplasmic membrane in fungal cells. One of the nitrogen atoms of the azole ring binds to the iron atom of cytochrome P450 and inhibit its activation and as a result its production is impaired. The imidazoles also damage the cytoplasmic membrane directly but this is only achieved at higher concentrations. The imidazoles have a broad spectrum action including most yeasts and yeast-like fungi, the dimorphic fungi and the dermatophytes. Filamentous fungi causing systemic infection are variable in sensitivity. Butoconazole Nitrate is an imidazole derivative with potent antifungal activity. Butoconazole is commonly used in treating gynecological fungal disease . It exhibit a fungicidal activity against fungus genera Candida ,Trychophyton, Microsportum and Epidermaphyton and several gram –positive bacteria 6 . It is thought to alter the permeability of the cell by attacking the cell membrane, thus causing decrease in osmotic resistance. It is also found to interfere with the biosynthesis of lipid and ergosterol , so it disrupts replication and inhibits growth of cell and causes lysis of fungal cell.7 Butoconazole is fungistaic at low therapeutic concentration. Cl HNO Cl S 3 N Cl N Figure (2): Chemical structure of Butoconazole Nitrate Other imidazoles include Ketconazole, which is a systemically active imidazole and miconazole which is available for intravenous use but it is rarely administered.