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Home , Butoconazole, Candicidin, Protiofate

“Validated methods for determination of some 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 nitrate and 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 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 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 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 .

-The 38-membered ring polyenes, such as , amphotericinB and .

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 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 (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 CH COOH 3 H3C O CH3 O (a) HO H2N OH

OH OH H3C O OH HO O OH OH OH OH O CH3 COOH 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 are the largest class of antifungal agents that contain a five membered ring attached by a N-C bond to other structures. This group is divided into , 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 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 which is available for intravenous use but it is rarely administered. and are systemically active . is a with in vitro activity against Candida, while saperconazole is a triazole derivative under investigation for the treatment of systemic fungal infections .There are large number of topically active imidazoles including , nitrate ,, nitrate and seraconazole nitrate.

Voriconazole is the newest agent against fungal infections8. It is a triazole antifungal with a structure related to that of fluconazole. is effective against a number of other serious fungal pathogens. These includes infections by Fusarium spp and Scedosporium apiospermum. Voriconazole has been used to treat severe fungal corneal infection. Oxiconazole is highly effective against many dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes.9

Terconazole is a triazole antifungal agent avaliable for intra-vaginal use10.It inhibits ergosterol synthesis .Depletion of ergosterol in fungal membrane disrupts the structure and many functions of fungal membrane so leading to inhibition of fungal growth11 N N N F N N N N N N OH F N N N Cl F CH OH F 3 F N Voriconazole Clotrimazole Fluconazole

C l Cl Cl N O O N N O N Cl N Cl N N N O O H C 3 O Cl Cl Cl Cl Econazole Miconazole

N N CH3 N H3C N O N N N O N O Cl Cl Itraconazole

Figure (3): Chemical structure of Azoles

3- and related compounds.

Allylamines are synthetic antifungal agents. Their action is exerted by the inhibition of squalene epoxidase enzyme which converts squalene to squalene epoxidase resulting in the accumaltion of squalene inside the fungal cell and inhibition of ergosterol synthesis. hydrochloride is the first drug known in this class, it has a broad spectrum fungicidal activity against dermatophytes and yeast and it is approved only for topical use. is the most active , given orally and distributed in skin, sebum and nails and it is effective in treating dermatophyte infections12 CH3 CH3

CH3

C CH3 N C

Figure (4): Chemical structure of Terbinafine (Lamisil)

Tolnaftate has a fungicidal activity against dermatophytes such as Microsporum and Trichophyton species that cause superficial tinea infection4.

CH3

N O C

S

CH3

4-Fatty Acids. Figure (5): Chemical structure of

Fatty acids have fungicidal properties. The higher molecular weight members have the advantage of having lower volatility. The salts of fatty acids are also fungicidal and provide non volatile forms for topical administration. Propionic acid is an available fungicidal and its salts as sodium, potassium, calcium and zinc salts are also fungicidal. is one of the best fatty acids available as a fungicidal agent4.

O

H C 2 OH Figure (6): Chemical structure of Undecylenic acid 5-Benzofuran cyclohexane ().

Griseofulvin is natural product which is useful against dermatophytes; it is a narrow – spectrum antifungal agent isolated from cultures of Penicillium griseofluvum13. It was the first effective drug taken by mouth against superficial fungus infections14. Its mechanism of action involves an interaction with microtubules, which causes interference with spindle formation in dividing cells (mitosis). Impairment of microtubule function also interferes with the transport of material through the cytoplasm to the periphery so this action is based on the inhibition of hyphal cell wall synthesis. In addition, there is evidence that the drug binds to RNA and it inhibits nucleic acid synthesis. Selective toxicity of griseofulvin is due to lack of binding to the host cell RNA at therapeutic concentration. Griseofulvin is used in systemic treatment of fungal infections of body, nails, hair and feet.4 CH3 O O O CH3

O H3C O O

H3C Cl

Figure (7): Chemical structure of Griseofulvin

6- (5-Flurocytosine).

5- Fluorocytosine is a fluorinated pyrimidine which becomes toxic only after it enters the cell. Its mode of action takes place by deamination of 5- fluorocytosine by fungal cells to flurourical that is incorporated into RNA in place of urical base and may be converted to 5- F-2-deoxyuridylic acid which inhibits thymidine synthetase2. It is well absorbed through gastrointestinal tract. is indicated for treatment of serious systematic infection caused by susceptible strains of Candida and Cryptococcus .It’s mainly used in combination with amphotericin B for systemic infections to avoid emergence of resistance15.

NH2

F Figure (8): Chemical structure of Flucytosine N

N O H 7-Phenols.

Phenols and compounds such as phenolic groups have antifungal potency in the treatment of superficial mycoses phenol, cresol and . O

OH Figure (9): Chemical structure of Salicylic acid

OH

Haloprogin, is a halogenated phenolic ether, and it is effective in treating skin infections caused by Dermatophytes, Tinea virsicolor and cutaneous candidal lesions.

I C Cl O C

Cl Cl Figure (10): Chemical structure of

8-Ciclopirox Olamine.

It is a broad spectrum fungicidal with excellent activity against most pathogenic fungi, including dermatophytes and it has powerful sterilization effect with low toxicity and strong osmolarity. It causes keratinization of skin16. So it acts on the cell membrane of susceptible fungi at low concentrations to block the transport of amino acids into the cell. At higher concentrations, it disrupts membrane integrity thus causing loss of cellular constituents. OH N O OH H N 2

CH3 Figure (11): Chemical structure of Ciclopirox Olamine

9-Eschinocandins.

Eschinocandins are a group of cyclic peptides with long lipophilic side chain. are a new class of antifungal drugs that act by inhibition of β (1, 3)-D- glucan synthase, a key enzyme necessary for integrity of the fungal cell wall.17 , , are the three semi synthetic eschinocandins that are used for treating systematic fungal infections. They are effective against a variety of Candida species, Caspofungin is metabolized by hydrolysis into two portions of the hexapeptide ring , micafungin is metabolized by sulfotransferase and catechol –O- methyltransferase (COMT)2.

10-Other anifungal drugs.

Acrisorcin4, potassium iodide18, loflucarban , , , pyrrolnitrin are local antifungal.

Summary The content of the thesis:

Validated methods for determination of some antifungal drugs

Submitted by: Ramia Gamal El-Sayed El-Ganyany

Can be summarized in the following points This thesis is concerned with the development of simple and accurate methods for the analysis of some antifungal drugs, namely butoconazole nitrate and ciclopirox olamine. The studied drugs were analyzed in drug substance, in laboratory prepared mixtures with their degradation products and / or excipients and in drug products. The thesis consists of the following sections: Section I: Introduction This section includes an overview of antifungal drugs. A review about the activity of drugs used in the treatment of some fungal infection, their structure-activity relationship and their classification is presented followed by a detailed presentation of the investigated drugs and different methods used in the literature for their estimation. Section II: Aim and Basis of the Work In this section, the aim of this work and the basis on which the proposed methods were chosen is clarified. Section III: Experimental and Discussion This section is divided into two parts: Part I: Chromatographic and Spectrophotometric Determination of Butoconazole Nitrate in the presence of its oxidation degradation product and its excipient.

I-A- Chromatographic Determination of Butoconazole Nitrate in the presence of its oxidation degradation product.

I-A-1- Stability-Indicating RP-HPLC Method for the Determination of Butoconazole Nitrate in the Presence of its Oxidation degradation Product

Butoconazole Nitrate was analyzed in the presence of its oxidation degradation product using Reprosil-Pur C8 column and 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 and isocratic elution was applied throughout the analysis. UV spectrophotometric detection was carried out at 270 nm.

Linear relationship was obtained over the concentration range of 5-80 μg/ml. To determine the accuracy of the method, it was performed on pure samples of the intact drug, with mean percentage recovery 99.55 ± 0.360. In addition, butoconazole nitrate was determined in laboratory prepared mixtures in the presence of its oxidation degradation product with mean percentage recovery of 99.73 ± 0.534. Satisfactory results were obtained on applying the method on Butoconazole 2%® vaginal cream100.18 ± 0.99 and 100.43 ± 0.66.

I-A-2- Stability-Indicating TLC- Densitometric Method for the Determination of Butoconazole Nitrate in the Presence of its Oxidation degradation Product

A TLC- densitometric method based on the separation of butoconazole nitrate from its oxidation degradation product was described. Hexane: ethyl acetate: methanol (7: 3: 0.85, v/v/v). The scanning of the spots was performed at 276 nm.

Good linearity was obtained over the concentration range of 0.1-0.8 µg/spot, for butoconazole nitrate. The proposed method was applied for the determination of butoconazole nitrate in drug substance and in laboratory prepared mixtures with mean percentage recoveries of 100.32 ± 0.909 and 99.16, ±1.071 respectively. The proposed methods were also applied for the determination of in their pharmaceutical dosage form Butoconazole 2%® vaginal cream with mean percentage recoveries of 100.73 ± 0.985 and 99.79 ± 0.923 respectively.

I-B -Spectrophotometric Determination of Butoconazole Nitrate in the Presence of its Oxidation degradation Product and its excipient

I-B-1- Multiple Divisor Ratio Derivative Spectrophotometric Method for the Determination of Butoconazole Nitrate in the Presence of its Oxidation degradation product

A multiple divisor ratio derivative spectrophotometric method is proposed for the determination of butoconazole nitrate in the presence of its oxidation degradation product and its interfering excipient Methyl Paraben(MP) and Propyl Paraben(PP) 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 at 262 nm were selected for the determination of butoconazole nitrate. The method was successfully applied for the determination of the drug in pure form and in laboratory prepared mixture containing its oxidation degradation product and its excipient with mean percentage recoveries of 99.97 ± 0.813 and 99.79 ± 0.749, respectively. The method was successfully applied for the analysis of Butoconazole 2%® vaginal cream and its validity was further assessed by applying the standard addition technique.

I-B-2- Multiple Divisor Ratio Difference Spectrophotometric method for the Determination of Butoconazole Nitrate in the Presence of its Oxidation degradation product

A multiple divisor ratio difference spectrophotometric method is proposed for the simultaneous determination of BUT in presence of its degradation DEG1 and its interfering excipients Methyl Paraben (MP), Propyl Paraben( PP). The ratio spectra were obtained by dividing the absorption spectra of different concentrations of BUT by the sum of the absorption spectra of MP, PP and DEG1 as multiple divisor. The differences in peak amplitudes at two different wavelengths namely, 259 nm and 277 nm (P277 - 259 nm) of ratio spectra were measured. The method was successfully applied for the determination of the drug in pure form and in laboratory prepared mixture containing its oxidation degradation product and its excipient with mean percentage recoveries of 99.23 ± 0.854 and 99.17 ± 0.458, respectively. The method was successfully applied for the analysis of Butoconazole 2%® vaginal cream and its validity was further assessed by applying the standard addition technique.

I-C.Preparation of (The Major Degradation Product of Butoconazole Nitrate)

In this part, oxidation degradation product of butoconazole nitrate was prepared. This obtained degradation product was identified and confirmed by spectral data such as IR and MS.

Part II: HPLC and Spectrophotometric Determination of Ciclopirox Olamine in the Presence of its photodegradation product.

II -A-1.RP- HPLC method for analysis of Ciclopirox Olamine and its application to kinetic study of its photodegradation

The method was based on HPLC separation of CO from its photodegradation product

DEG2 using a stationary phase composed of Reprosil-Pur column (C8) .The mobile phase used was acetonitrile: water: H3PO4 (adjusted to pH 2.6 with o-phosphoric acid) (40: 60: 0.1, v/v/v). The flow rate was maintained at 2.5 ml/min. UV detection was carried out at 303 nm.

The method was successfully applied for the determination of CO in pure form with mean percentage recovery of 99.41 ± 0.834.

The method was successfully applied for the determination of CO in Batrafen cream and solution ®with mean percentage recoveries of 99.21 ± 1.057 and 99.82 ±1.443, respectively.

II-A-2 Kinetic study of photodegradation of Ciclopirox Olamine

Photodegradation of CO was carried out in methanol and the kinetic order of the degradation was evaluated. Photo degradation of CO where the drug samples were subjected to UV irradiation at 303 nm for different time intervals . Remaining concentration of CO remaining vs. time revealed good linearity (best fit line) indicating that the photo degradation of CO followed a zero order reaction under the stress conditions applied.

II-B-Spectrophotometric Determination of Ciclopirox Olamine II-B-1- Spectrophotometric determination of Ciclopirox Olamine by difference absorption (A) spectrophotometric method

Ciclopirox Olamine was determined by measuring the difference in absorbance (A) 317 nm of (CO) acidic solution in 0.1N HCl against that of the 0.1N NaOH as a blank. Linear relationship was obtained over the concentration range of 10 - 60 g/ml. To determine the accuracy of the method, it was performed on pure samples of the intact drug, with mean percentage recovery 100.35 ± 0.705.Satisfactory results were obtained on applying the method on Batrafen ®cream and solution. II-B-2. Zero Order Spectrophotometric Method for the Determination of Ciclopirox Olamine The zero order spectrum of CO was recorded and the absorbance at 303 nm was measured. Linear relationship was obtained over the concentration range of (10 - 35 g/ml). To determine the accuracy of the method, it was performed on pure samples of the intact drug; with mean percentage recovery, 99.93 ± 0.962.Satisfactory results were obtained on applying the method on Batrafen ®cream and solution. II-B-3. Spectrophotometric Determination of Ciclopirox Olamine via chelation with ferric ion. In this part, spectrophotometric method were described for the determination of ciclopirox olamine based on producing a colored complex with iron (III) CO formed orange red complex with iron (III) in the ratio of 3: 1 and the colored products were measured at 470 nm. Different parameters affecting the reaction including effect of different solvents, reagent volume, reaction time and stability of the formed complex were investigated. The suggested procedures were successfully applied for the determination of ciclopirox olamine in drug substance with mean percentage recovery 99.89 ± 0.990. The mean percentage recoveries of ciclopirox olamine in Batrafen ® cream were 99.89 ± 0.972.The validity of the suggested procedures was further assessed by applying the standard addition technique. At the end of each section, the validation parameters including the regression equation, the concentration range, interday and intraday precision of the proposed method, the limit of detection (LOD) and the limit of quantification (LOQ) were summarized. In addition, a statistical comparison between the proposed methods and the reported or pharmacopoeial methods were computed and no significant difference was found.