Stability kinetics of 4-dedimethylamino sancycline,a new anti-tumor drug, in aqueous solutions

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Authors Pinsuwan, Sirirat, 1961-

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STABILITY KINETICS OF 4-DEDIMETHYLAMINO SANCYCLINE, A NEW ANTI-TUMOR DRUG, IN AQUEOUS SOLUTIONS

by

SIrirat Pinsuwan

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF PHARMACY PRACTICE AND SCIENCE

In Partial fulfillment of the requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN PHARMACEUTICAL SCIENCES

in the Graduate College

THE UNIVERSITY OF ARIZONA

1 9 9 8

UHI Number: 9906522

UMl Microfonn 9906522 Copyright 1998, by UMI Company. All rights reserved.

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THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE

As members of che Final Examlnacion Comoiccee, ve certify that ve have

read the dissertation prepared by Sirirst PinSUWBn

entitled Stability Kinetica of 4-Dedimethylamino Sancydirr,

A New Antitumor Drug, in Aqueous Solutiona

and recommend that it be accepted as fulfilling the dissertation

requirement for the Degree of DoCtOr Of Philosophy

Date amuel Hyraikowslcy, Ph. L Michael Mayersohn,(9h.D. Date

Sherry Chow. Ph.D. Date

^ Srini Raghavan, Ph.D. Date

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Dissert-aLtion^lrector Date

3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited In the University Library to be made available to borrowers under rules of the library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when In his or her judgment the proposed use of the materials is in the Interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED;

4

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Dr. Samuel H.

Yalkowsky, my mentor, for his invaluable guidance, support, generous time and

effort he has given me throughout my entire study period.

I wish to acknowledge the National Cancer Institute (NCI) for the

financial support, as well as Mr. Fernando A. Alvarez Nianez, Ms. Purvi Shah,

Mr. Chuck C.K. Leung, and Mr. Nirav Patel for their work on this project.

I would also extend my thanks to all my Thai friends in Tucson and all

my friends in the pharmaceutical group for the wonderful years spent together.

I would like to give my special thanks to my parents for their love,

understanding and support. Without them, I would never reach my dreams.

Finally, the financial support from the Thai Government is gratefully

acknowledged.

TO MY PARENTS

6

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS 9

LIST OF TABLES 11

ABSTRACT 12

CHAPTER I. INTRODUCTION 14 Cancer Chemotherapy 14 New Drug Development -.15 Aims of the study 16 4-Declimethylamino Sancycline (Col-3) 17

Drug Description 17 Synthesis 18

Synthesis of Sancycline methiodide 18 Synthesis of Col-3 18

Pharmacology 19 Stability 19

CHAPTER 11. DRUG CHARACTERIZATION 27 Introduction 27 Experimental 27

Materials 27 Melting Point Determination 28 Ultraviolet Spectrum 28 Mass Spectrum 28 Determination of Dissociation Constants 29 Solubility 29

Solubility In Pure Solvents 29 pH-solubility 30

Results and Discussion 31 Melting Point Determination 31 Ultraviolet Spectrum 31 Mass Spectrum 32 Determination of Dissociation Constants 33 Solubility 36

Solubility In Pure Solvents 36 pH-solubility 36

7

TABLE OF CONTENTS - Continued

Page

CHAPTER III. STABILITY-INDICATING ASSAY OF COL-3 USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 52

Introduction 52 Experimental 53

Materials 53 Instrumentation 53 Chromatographic Conditions 53 Standard Solution 54 Standard Calibration Curves 54 Method Validation 54 Stability of Col-3 under Acidic and Basic Conditions 55

Results 55 Chromatography 55 Standard Calibration Curves 56 Method Validation 56 Stability of Col-3 under Acidic and Basic Conditions 56

Discussion 57

CHAPTER IV. STABILITY KINETICS OF COL-3 IN AQUEOUS SOLUTIONS 66

Introduction 66 Experimental 66

Materials 66 Instrumentation 67 Buffer Solutions 68 Kinetic Measurements 68 Effect of Drug Concentration 68 Effect of Buffer Concentration 69 Effect of Light 69 Effect of Additives 69 Effect of Temperature 70 Statistical Analysis 70

Results and Discussion 71 Reaction Order and Reaction Rate Constants 71 Effect of Drug Concentration 71 Effect of Buffer Concentration 72 pH-rate Profile 74 Effect of Light 76 Effect of Additives 77 Effect of Temperature 78

8

TABLE OF CONTENTS - Continued

Page

CHAPTER V. SEPARATION AND UV SPECTROPHOTOMETRIC IDENTIFICATION OF DEGRADATION PRODUCTS 93

Introduction.... 93 Experimental 93

Materials 93 Generation of Degradation Products 94 Liquid-liquid Extraction 94 Spectrometric Identification of Degradation Products 95

Results and Discussion 95 Separation of Degradation Products 95 Spectrometric Identification of Degradation Products 96

SUMMARY 102

REFERENCES 105

9

LIST OF ILLUSTRATIONS

Page

Figure 1 -1. Chemical synthesis of col-3 24

Figure 1 -2. Degradation pathways of tetracycline 25

Figure 1-3. Photo-oxidation of tetracycline 26

Figure 2-1. DSC scan of col-3 41

Figure 2-2. TGA scan of col-3 42

Figure 2-3. UV spectra of col-3 in different solvents 43

Figure 2-4. Chromophoric regions in col-3 molecule 44

Figure 2-5. Mass spectrum of col-3 45

Figure 2-6. Structural groupings and pl values of the tetracyclines 46

Figure 2-7. UV spectra of col-3 at different pH values 47

Figure 2-8. Resonance stabilization of col-3 anions 48

Figure 2-9. pH-absorbance profile of col-3 at 286 nm 49

Figure 2-10. pH-absorbance profile of col-3 at 380 nm 50

Figure 2-11. pH-solubility profile of col-3 51

Figure 3-1. Representative HPLC chromatogram of a standard solution of col-3 64

Figure 3-2. Representative HPLC chromatograms of degraded col-3 under basic conditions 65

Figure 4-1. Apparent first-order plots for the degradation of col-3 in aqueous solutions at various pH values 85

10

LIST OF ILLUSTRATIONS - Continued

Page

Figure 4-2. Plots illustrating first-order dependence in col-3 concentration at pH 4.0 86

Figure 4-3. Plots illustrating first-order dependence in col-3 concentration at pH 9.0 87

Figure 4-4. Plots of the apparent first-order rate constants of col-3 as a function of phosphate buffer concentration and pH 88

Figure 4-5. pH-rate profile of col-3 in aqueous solutions at zero buffer concentration (25°C) 89

Figure 4-6. Degradation profiles of col-3 in basic solutions with and without additives 90

Figure 4-7. Degradation profile of col-3 under basic conditions illustrating the initial lag time 91

Figure 4-8. Arrhenius plots for the apparent first-order rate constant of col-3 at pH 3.0 and 8.0 92

Figure 5-1. Liquid-liquid extraction procedure for partial separation of col-3 and its degradation products 98

Figure 5-2. Representative HPLC chromatograms illustrating the separation of col-3 and its degradation products before and after extraction 99

Figure 5-3. Representative HPLC chromatogram illustrating the UV spectra of col-3 and its degradation products detected by the photo diode array detector 100

Figure 5-4. UV spectrum changes for the degradation of col-3 under basic conditions 101

11

LIST OF TABLES

Page

Table 1 -1. Essential drugs for oncology and their Indications 22

Table 2-1. Spectrophotometric pKai detemnination of col-3 38

Table 2-2. Spectrophotometric pKaa determination of col-3 39

Table 2-3. Solubility of col-3 in various solvents at 25°C 40

Table 3-1. Intra-day reproducibility of the analysis of col-3 60

Table 3-2. Inter-day reproducibility of the analysis of col-3 61

Table 3-3. Intra-day reproducibility of retention times of col-3 and its four major degradation products 62

Table 3-4. Inter-day reproducibility of retention times of col-3 and its four major degradation products 63

Table 4-1. Observed apparent first- order rate constant for the degradation of col-3 in phosphate buffer solutions at various pH values and buffer concentrations 80

Table 4-2. Half-life of col-3 in phosphate buffer solutions as a function of pH and initial drug concentration 81

Table 4-3. Observed buffer-catalytic rate constant for the decomposition of col-3 as a function of pH and fraction of the individual buffer species 82

Table 4-4. Macroscopic rate constants and ionization constants obtained from a fit of the experimental data to the kinetic model 83

Table 4-5. Effect of light on degradation rate constant of col-3 in aqueous solutions at various pH-values 84

12

ABSTRACT

4-Dedimethylamino sancycline (col-3) is a new antitumor antibiotic of the

tetracycline family. Preformulation studies have indicated that col-3 Is not

stable in aqueous solutions. The overall purpose of this research project is to

investigate the stability kinetics of this drug in aqueous solutions.

The physicochemical properties of col-3, including melting point, UV

spectrum, mass spectrum, dissociation constants and solubility were

determined. Col-3 is an acidic compound with two pKa values of 5.9 (pKai) and

8.1 (pKa2). It is slightly soluble in water (0.01 mg/mL) and readily soluble in

organic solvents such as polyethylene glycol and benzyl alcohol. Although the

solubility of col-3 increases with increasing pH, its stability decreases with

increasing pH.

A HPLC assay was developed to quantitate col-3 and separate its

degradation products. Four major degradation products of col-3 were detected

under alkaline conditions. These degradates were identified by their elution

times and their UV-absorption spectra.

The kinetics of degradation of col-3 in aqueous solution at 25''C were

investigated by HPLC over the pH-range of 2-10. The Influence of pH, buffer

concentration, light, temperature and some additives on the degradation rate

were studied. The degradation of col-3 was found to follow first-order kinetics

13

at 25°C. A rate expression covering the degradation of the various ionic forms

of the drug was derived and shown to account for the shape of the

experimental pH-rate profile. Under basic conditions, the degradation of col-3

involves oxidation, which is catalyzed by metal ions.

The separation of the four initial degradation products of col-3 was

investigated. Partial separation of these compounds is achieved by liquid-

liquid extraction. However, due to the instability of these compounds, their

complete isolation cannot be successful. The UV spectroscopic analysis of

these compounds shows that an absorbance at 360 nm is partially decreased

in degradates I and II and totally absent in degradates III and IV. These results

suggest that the phenolic diketone moiety, which produces this absorption

band, has been altered upon degradation.

14

CHAPTER I. INTRODUCTION

Cancer Chemotherapy

Cancer is one of the major diseases for winich a definite cure has not

been established. Many treatments including surgery, radiotherapy, chemo­

therapy, and immunotherapy have been developed for this disease. Recently,

cancer chemotherapy has become increasingly important and has greatly

progressed with occasional healings of neoplastic diseases.

Modern cancer chemotherapy began with the discovery of antileukemia

effect of nitrogen mustard during World War II, followed by similar finding on

antimetabolites: methotrexate in 1949, 6-mercaptopurine in 1952, and 5-fluoro-

urecil in 1957 (Remers, 1991; Calabresi and Chabner, 1992; Foye and

Sengupta, 1995). Subsequently, several potential antineoplatic agents have

been screened from natural products of both plant and microbial origin.

Examples of these compounds include actinomycin, mitomycin, mithramycin,

doxorubicin, and the vinca alkaloids. In recent years, more than 80 cytotoxic

drugs have been developed. An overview of the drugs, which play a major role

in current oncologic practice, is given in Table 1-1 (Stjernsward, 1995).

Antineoplastic drugs are highly toxic and must be administered with

extreme caution. The toxicity of these compounds usually involves rapid

proliferating tissue such as bone marrow and the gastrointestinal epithelium.

15

Some of them also show toxic effects on kidneys, heart, lungs and other vital

organs (Remers, 1991). Because of its toxicity, chemotherapy is seldom the

initial treatment used against cancer. Usually, it is used to treat malignancy

when it is most curable and when the patients are best able to tolerate

treatment.

Due to toxicities and low efficacy, most antineoplastic drugs are limited

for their long-term use. Therefore, the research for new cytotoxic agents, which

possess a more favorable therapeutic index, needs to be continued.

New Drug Development

In general, two strategies of drug development are usually followed.

The first is the development of analogs of compounds of proven usefulness

and second is the investigation of new structures obtained by chemical

synthesis or by isolation from natural products. The leadership and financial

support for development of antineoplastic drugs in the United States is mainly

derived from the National Cancer Institute (NCI).

A National Cancer Chemotherapy program to discover and develop new

antitumor agents was established by the NCI in 1955. The system begins with

the acquisition of new compounds, which are then screened in animals,

produced and formulated, toxicologically evaluated, and then phased into a

series of human clinical trials. Several types of compounds have been

16

investigated including synthetic compounds, fermentation products, plant

products, marine animal products, as well as biological response modifiers.

Between 1955 and 1982 well over 700,000 substance have been screened for

antitumor activity (Gringauz, 1997; Schepartz, 1978).

The antitumor antibiotics rank as one of the most important chemo-

theraputic drug groups and have been the object of intensive research and

development. A number of antibiotics have been tested and reached clinical

trials. Recently, the NCI has reported a new synthetic antibiotic, 4-dedimethyl-

amino sancycline (col-3), which belongs to the family of tetracyclines to have

significant antitumor activities. Currently, it is in Phase I clinical trial at the NCI

and has been selected for further dosage form development.

Aims of the study

The alms of this research are to investigate the physicochemical

properties and stability kinetics of col-3. This knowledge is essential in order to

establish the precautions required to maintain the stability of dosage forms as

well as to ensure the efficacy of the pharmacological, pharmacokinetics, and

toxicological tests performed during dmg development.

17

4-Dedimethylamino Sancycline (Col-3)

Drug Description

Chemical name: 4-Dedimethylamino 6-demethyl 6-dehydroxy

tetracycline

Formula: CigHigNOg

Molecular weight: 371.35

Chemical Structure:

OH

12a 11a JOa

•CONH2 OH

Ri R2 R3 Tetracycline N(CH3)2 OH CH3 Sancycline N(CH3)2 H H Col-3 H H H

Appearance: A yellow crystalline power, odorless

18

Synthesis

Col-3 was chemically synthesized from Sancycline methiodide as shown

in Figure 1-1. The synthesis consists of two steps as follows (information from

NCI):

1.1. Synthesis of Sancycline methiodide

Sancycline methiodide was prepared by stirring a slurry of sancycline

(500 gm) in a mixture of 1:1 (v/v) acetone and tetrahydrofuran (15L), containing

methyl iodide (1L) at room temperature for 48 hours. The solution was then

stripped to dryness on a rotovap.

1.2. Synthesis of Col-3

Zinc dust (500 mg) was added to a solution of sancycline methiodide in

aqueous acetic acid (50%, 10L). The green colored suspension was stirred at

room temperature for 30 min. Then, acetone (10L) was added and suction

filtered to remove the zinc residues. The solid powder was washed again with

acetone (3L), and then, the total filtrate was concentrated on a rotovap to a

volume of approximately 12L The yellow suspension resulting from this step

was then extracted three times with methylene chloride (5L). The combined

extracts were washed with water, dried over anhydrous magnesium sulfate, and

stripped to dryness. In the last step, the product was purified by dissolving in

19

hot 50% aqueous acetone (10L), followed by hot acetone-hexane mixture (1:2,

12L). This process yielded about 250-mg col-3 powder.

Pharmacology

Col-3 exhibits in vitro and in vivo activity as an inhibitor of matrix

metalloproteinases, tumor invasion, and metastasis of a variety of tumor types

(personal communications from NCI). There Is currently no specific information

for the mechanism of action in the literature. However, as most of the

tetracyclines, it is believed that the antitumor effect of col-3 may be due to the

inhibition of mitochondrial protein synthesis (Kroon etai., 1984; Van den Bogert

et al., 1981). This effect has also been found in doxycycline and oxytetra-

cycline, other tetracycline analogues, which have significant antitumor activities

in several tumor systems (Van den Bogert et a!., 1988, 1985, 1983). The

antiproliferative effects resulting from prolonged inhibition of mitochondrial

protein synthesis have been demonstrated in several tumor model systems

(Van den Bogert ef a/., 1987,1986).

Stability

Preliminary investigations indicate that col-3 is not stable in aqueous

solutions (Pinsuwan et al., 1997). The degradation of col-3 is indicated by the

color change of the solution from yellow to pink, red and brown depending

20

upon the extent of degradation. To date, no details of the degradation process

of col-3 are available in the literature.

In general, the tetracycline group of antibiotics is known to possess

limited stability (Ali, 1984; Mitscher, 1978; Vej-Hansen et a/., 1987, 1979,

1978). The most common of the degradation pathways are shown in Figure

2-2. Epimerfzation of the dimethylamino group at the C4 position produces the

4-epitetracyclines, which exhibits much less activity than the natural isomers

(Hussar, 1968; Blackwood and Stephens, 1965; Remers etal., 1963; Kaplan et

ai, 1957; McCormick ef a/., 1957, 1956; Sterphens ef a/., 1956; Doerschuk et

al., 1955). This reaction takes place most rapidly between pH 2-6 in aqueous

solutions (Sokoloski et al., 1977; Schlecht ef al., 1973). In strong acid

solutions, tetracyclines undergo dehydration at the C5a-6 position to form the

inactive 5,6-anhydrotetracyclines (Miller et al., 1962; Conover et al., 1953;

Boothe etal., 1953; Waller ef a/., 1952; HIavka and Krazinski, 1963; Green et

a/.,1960). Another degradation reaction takes place in basic solutions, which

involves a reaction between the 6-hydroxy and 11-ketone group (Ali, 1984;

Mitscher, 1978). This reaction causes the bond between C11 and CI la to

cleave and to form the lactone ring, found in the inactive isotetracyclines.

Since col-3 differs structurally from the tetracyclines by the absence of the 4-

dimethylamino group and the 6-hydroxyl group, these degradation pathways

cannot be applied for col-3.

21

An alternative pathway for the degradation of tetracycline-type drugs is

photo-oxidation (Leeson and Weidenheimer, 1969; Wiebe and Moore, 1977;

Moore et al., 1983; Sanniez and Pilpel, 1980). This degradation pathway

occurs in alkaline media. As shown in Figure 1-3, the reaction involves the

loss of the 4-dimethyamino group due to the photodissociation, followed by

oxidation and dehydration to form a quinone type compound (IV). The

occurrence of this compound is found to be accompanied by color change of

the solution from yellow to red (Moore etaL, 1983). This oxidative degradation

could also be envisaged for col-3, although no specific information is available

for this compound.

22

Table 1-1. Essential drugs for oncology and their Indications.

Class Drug Disease

Alkylating gents Cyclophosphamide Acute and chronic lymphocytic leukemia, multiple myeloma, soft-tissue sarcomas

Decarbazine Malignant melanoma, Hodgkin's disease, soft-tissue sarcomas

Antimetabolites Methrotrexate Acute lymphotic leukemia. Choriocarcinoma, breast, lung, osteogenic sarcoma

Fluorouacil Breast, colon, stomach, pancreas, ovary, bladder, skin, head and neck

Cytarabine Acute granulocyctic and acute lymphocytic leukemia

Mercaptopurine Acute lymphocytic, acute granulocytic leukemia

Vinca Alkaloids Vinblastine Hodgkin's disease, non-Hodgkin's lymphomas, breast, and testis

Vincristine Acute lymphotic leukemia, neuroblastoma, Wilm's tumor, small-cell lung

Antibiotics Dactinomycin (actinomycin D)

Choriocarcinoma, Wilm's tumor, rhabdomyosarcoma, testis

Daunorbicin Acute granulocytic and acute lymphocytic leukemia

23

Table 1-1. Essential drugs for oncology and their Indications - Continued.

Class Drug Disease

Antibiotics (cont)

Doxorubicin Soft-tissue, osteogenic, and other sarcomas, acute lymphocytic leukemia, breast, genitourinary, thyroid, lung, and stomach

Antibiotics (cont)

Bleomycin Testis, head and neck, skin, lung, esophagus, genitourinary tract

Antibiotics (cont)

Mitomycin (mitomycin C)

Stomach, cervix, colon, breast, pancreas, bladder, head and neck

Enzymes L-Asparaginase Acute lymphotic leukemia

Hormones and Antagonist

Prednisolone Acute and chronic lymphotic leukemia, non Hodgkin's lympho­mas, Hodgkin's disease, breast

Hormones and Antagonist

Tamoxifen Breast

Hormones and Antagonist

Leuprolide Prostate

Miscellaneous agents

Cisplatin Testis, ovary, bladder, lung, cervix, head and neck, osteogenic sarcoma

Miscellaneous agents

Hydroxyurea Chronic granulocytic leukemia, essential thrombocytosis, malignant melanoma

Miscellaneous agents

Procarbazine Hodgkin's disease

24

f N{CH3)3 N(CH3)2

•k. Acetone/THF CONH2 2 days

N(CH3)2

CONH2

Zn ACOH/H2P 50:50 30 min

CONH2

in

Figure 1-1. Chemical synthesis of col-3- Keys: Sancycline (1), Sancycline methiodide (II), and col-3 (111).

25

A. Epimerization

H3C ^OH ,..N(CH3)2 "• -OH

CONH2 OH 6 OH" O

I

B. Acid-base degradation

H3C., ^OH H ..N(CH3)2 -OH

OH O OH O

ketq

CONH2 enol

H^ ..N(CH3)2 OH

CONH2

H3C.. ^QH K, .N(CH3)2 -OH

CONH2

H3C,. OH H. ..N(CH3)2 -OH

CONH2

H. .N(CH3)2 OH

CONH2

Figure 1-2. Degradation pathways of tetracycline. Keys: Tetracycline (I), 4-Epitetracycline (II), 5-6-Anhydrotetracycline (III), and Isotetracycline (IV).

26

HsQ, ^OH ..N(CH3)2 ,0* hu

OH O OH O CONH2

H3C. ^OH H, „00H

OH O OH O

H3C.. .OH

H,0

CONH2

H3C OH H,

OH O OH O

H3K OH H

CONH2

CONH2

OH O OH O

H3C.. ^QH

OH O OH' ' 0

m

C0NH2

-HjO

(okT OH O OH O

CONH2

Figure 1-3. Photo-oxidation of tetracycline.

27

CHAPTER II. DRUG CHARACTERIZATION

Introduction

Prior to the dosage form development of a new drug, it is essential that

certain fundamental physical and chemical properties of the drug molecule are

determined. This information often leads to a better understanding of the

interrelationship between molecular structure and drug action. In this study,

the following physicochemical properties of col-3 were determined; melting

point, ultraviolet spectrum, mass spectrum, dissociation constants, and

solubility.

Experimental

Materials

Col-3 was generously supplied by the National Cancer Institute

(Bethesda, MD). Methanol (HPLC grade) was purchased from J.T. Baker

(Phillipburg, NJ). N/10 hydrochloric acid and sodium hydroxide solutions

(certified grade) were purchased from Fisher Scientific Company (Santa Clara,

CA). Phosphoric acid, potassium phosphate (monobasic), potassium

phosphate (dibasic) and potassium chloride were purchased from Sigma

Chemical Company (St. Louis, MO). All chemicals were used as received

without further purification.

28

Melting Point Detennination

The melting point of col-3 was detennined using a DuPont Instruments

910 Differential Scanning Calorimeter; DSC (TA Instrument Inc., New Castal,

DE). Small quantities (approximately 2-5 mg) of col-3 were weighed and

placed in a 3.5-mm diameter aluminum pan. In order to minimize thermal

gradients throughout the sample and to ensure good thermal contact between

the sample and detection system, the sample pan was sealed with an inverted

lid, which had been punctured to form a pin hole. The DSC equipment was

operated at a heating rate of 10 degree per minute and 1 atmosphere pressure.

An empty pan sealed with an inverted lid was used as a reference.

Ultraviolet Spectrum

The UV spectra of CGI-3 were determined in different solvents including

methanol, N/1 hydrochloric acid, and N/1 sodium hydroxide solution. The UV

scan was performed using a Beckman DU-640 UV spectrophotometer

(Beckman Instruments, Fullerton, CA).

Mass Spectrum

The low-resolution electron impact mass spectrum of col-3 was recorded

on a HX 11 OA SEOL Sector Instrument via direct inlet. The ion potential was

70 eV.

29

Determination of Dissociation Constants

The macroscopic acid dissociation constants of col-3 were determined

spectroscopicaily. Phosphate buffer solutions were prepared in a pH range of

2-10 and adjusted to an ionic strength of 0.2. A 4.0 mL of each buffer solution

was spiked with 20 fiL of a stock solution of col-3 (1.0 mg/mL in methanol) to

produce 5.0 |ig/mL (1.3 x 10'® M) solutions with a final methanol concentration

of 0.5%. The absorbance of the resulting solution was immediately assessed

at the predetermined analytical wavelengths of 286 and 380 nm. All

determinations were performed In duplicate at room temperature.

Solubility

Solubility in Pure Solvents

The solubilities of col-3 in different solvents were determined in

duplicate. Suspensions of col-3 in screw-capped vials were placed in a

mechanical rotator at room temperature for 24-48 hours. After equilibration,

each suspension was filtered through a 0.5-mm filter (Millex-LRCi3, Millipore

Corporation, Bedford, MA). An aliquot of the filtrate was diluted and analyzed

using HPLC. No degradation of col-3 was observed in any of the solvents

during the time needed to establish the equilibrium solubility.

The HPLC system consisted of a Beckman 126 solvent delivery system,

equipped with a Beckman 168 diode array detector (Beckman Instruments,

30

Fullerton, CA). Sample of 100 (il were Injected with a Rheodyne 710 manual

injector (Rheodyne, Cotati, CA). The analytical column was a 4.6 x 150 mm,

Mixed-mode column (Alltech Associates, Deerfield, IL) packed with 5 i m,

C-8/cation bonded silica. The mobile phase consisted of 0.1 M acetic acid with

0.05% EDTA and acetronltrile (65:35 by volume). The column eluent was

monitored at a wavelength of 268 nm, with a flow rate of 1 mL/min.

pH-solubility

The pH-solubility profile of col-3 was performed in Mcllvaine buffer

(Diem and Lentner, 1974) ranging in pH from 2 to 8. Excess col-3 was

equilibrated in the buffer solutions at 25''C in the dark. Preliminary experiments

with periodic sample analysis indicated that 10 hours was sufficient for

equilibration. This short time was chosen to minimize the possible effects of

degradation products on solubility. The final pH was measured after the

solution was saturated. The apparent solubility was determined from HPLC

analysis of the filtrate as described previously.

31

Results and Discussion

Melting Point Detemiination

As shown in Figure 2-1, two endotherms are observed in the DSC scan

of col-3. The first endotherm indicates the melting point where, the onset

temperature occurs at 173®C and the maximum rate of phase transition occurs

at ISS^C. The second endotherm is seen at about 190°C. The basis for this

endotherm was further investigated by using Thermal Gravimetric Analysis

(TGA). The TGA scan shown in Figure 2-2 indicates a weight loss of about

15% over the temperature range of 190 - 250°C. The fact that only a fraction of

the drug is lost indicates that the second endotherm is not due to boiling, but

rather, due to drug decomposition with the evaporation of gas.

Ultraviolet Spectrum

The UV-absorption spectra along with the list of the absorption maxima

and the molecular extinction coefficients of col-3 in methanol, 0.1 N hydrochloric

acid and 0.1 N sodium hydroxide solution are shown in Figure 2-3.

As most of the tetracyclines, col-3 contains two distinct ultraviolet

absorbing chromophoric regions. It has been determined that the first

absorption band at about 260 nm is produced by the p-tricarbonyl system

composing CI, C2, and C3 of ring A (see Figure 2-4), while the second visible

band at about 360 nm is produced by the conjugated system composing the

32

aromatic ring D and the p-diketone moiety in ring B and C (Ali, 1984;

McCormick ef a/., 1957; Sterphens eta!., 1954; Regna etal., 1951).

Mass Spectrum

Figure 2-5 illustrates the mass spectrum of col-3. Due to its non-

volatility and thermal instability, the electron-impact mass spectrum of col-3 is

rich in fragment ions and weak in molecular ion intensity. In the highest mass

region, the m/e 371 is observed for the molecular peak. The presence of the

carboxamide group (CONH2) is suggested by the presence of the peaks at m/e

354, 337, and 327 for the loss of NH3 (m/e 17), NH3 plus OH (m/e 34) and

CONH2 (m/e 44), respectively. Hoffman (1969) indicated that most of the

fragments seen in the spectra of most tetracycline derivatives involved ring A

and B. For col-3, the cleavages of ring A led to the intense peaks at m/e 284

and 243, due to a loss of 0H-CH=CH-C0NH2 (m/e 87) and CH3-

C(0H)=C(C0NH2) (m/e 128), respectively. The most intensive peak at

m/e 187 (C11H7O3) and m/e 161 (C10H9O2) are the fragments of ring C and D

after ring B was cleaved. The analog of these peaks were also found in other

tetracycline derivatives (Hoffman, 1966).

33

Determination of Dissociation Constants

It has been detemnined that the tetracycline antibiotics contain three

ionizable groups (Martin, 1991; Rigler, 1965; Stephens eta/., 1956) as shown

in Figure 2-6. The first dissociation constant is due to the tricarbonyl system,

the second due to the phenolic diketone system, and the third due to the

dimethylammonium functional group at C4 (Lien, 1979; Rigler, 1965; Stephen

et al., 1956). The approximate pKa values for each of these groups in some

tetracycline derivatives are list in Figure 2-6 (Martin, 1991; Lien, 1979; Stephen

etai, 1956).

Col-3 can be seen to have the first two of the acidic groupings of the

tetracyclines. However, due to the absence of the dimethyammonium

functional group at C4, the third pl does not exist for col-3. The two pKa

values suggest that this compound can exist as the unionized species, a

monovalent anion, or a divalent anion depending on the pH. The UV-visible

absorption spectra of col-3 in aqueous solutions at various pH values are

plotted in Figure 2-7.

Notice that the first absorption band at about 260 nm is produced by the

3-tricarbonyl system where the first deprotonation occurs. Similarly, the

second visible band at about 360 nm is produced by the phenolic diketone

moiety where the second deprotonation occurs (Ali, 1984; McCormick et al.,

1957; Regma etai, 1951; Sterphen efa/.,1954). Both anionic sites of col-3 are

34

Stabilized by resonance (see Figure 2-8) and they are separated from each

other by carbons 4a and 12a. These sp^ hybrid atoms isolate the two light

absorbing regions and make it possible to determine their pKi values

independently by spectrophotometry.

Spectrophotometric techniques are ideal procedures for the evaluation

of acidity constants when a substance is too insoluble for potentiometry or

when the pka value is particularly low or high (Cookson, 1974; Albert and

Serjent, 1971). These techniques are applicable if (i) the compound shows

appreciable absorption at the working wavelength, (ii) the site of protonation or

deprotonation is conjugated with or is an inherent part of a chromophoric group

in the compound, and (iii) the conjugate acid-base species have different

absorption spectra.

In our experiment, absorption at 286 nm of the first band and at 380 nm

of the second band were chosen as the analytical wavelengths for determining

the first and the second pKa values, respectively. These two wavelengths were

chosen due to their maximum absorbance changes with pH.

The absorbance-pH curve of col-3 at 286 nm is shown in Figure 2-9.

The profile shows that the absorbance of the drug increases in the pH range of

4 to 7, and is constant below pH 4 and above pH 7. It Is indicated at this

wavelength that the absorbance of the ionized species is higher than that of the

35

unionized species, while the mono- and divalent species cannot be

differentiated. The first pKa of col-3 was calculated at 286 nm using;

pKa = pH + log ~ ^ Eq. 2-1 A — AY

where Aj and Au are the absorbance of the ionized and the unionized species,

respectively. The results are shown in Table 2-1. The calculated pKai (5.86) is

approximately equal to the phQ determined using the derivative graph shown in

Figure 2-9(B).

Similarly, for the second pKg determination, the absorbance changes at

380 nm over the pH range of 2 to 10 were potted as in Figure 2-10. At this

wavelength, the absorbance of divalent species is higher than that of the

monovalent species, while the unionized and monovalent species cannot be

differentiated. Equation 2-1 can be used to calculated the second pKa as

previously. The results are shown in Table 2-2. The calculated value of 8.09

is again in good agreement with the value determined from the derivative graph

shown in Figure 2-10(B).

From the results, the spectroscopically determined pKa values of col-3

are 5.86 (pl i) and 8.09 (pKaa). Notice that its second pKa value which

corresponds to the loss of a proton from the phenolic diketone moiety is close

to those of the tetracyclines (-7.8). However, the first ptQ value which is due to

the loss of a proton from the p-tricarbonyl system, is shown to be over two pH

units higher than that those of the tetracyclines (-3.3). This difference is due to

36

the absence of the electron withdrawing dimethylamino group at C4. A similar

increase of the pKa value of this system due to the absence of the

dimethylamino group is found in desdedimethylamino tetracycline, which has a

pKa value of 5.94 (Stephens, 1956).

Solubility

Solubility in Pure Solvents

Col-3 is slightly soluble in water, however, it is more soluble in some

organic solvents such as methanol, benzyl alcohol, and PEG 400. The

solubility of col-3 in different solvents is listed in Table 2-3.

pH-solubility

The solubility-pH profile of col-3 is shown in Figure 2-11. Since col-3 is

an ionizable compound, its solubility varies with pH. The minimum solubility is

that of the unionized species (known as the intrinsic solubility) and increases

with ionization.

The total solubility of col-3 can be expressed as the sum of the

concentration (C) of all species:

where subscripts A, B, and C denote a neutral, monovalent and divalent anion,

respectively. For saturated solutions, where the solid drug is in equilibrium

37

with the drug In the solution, the concentration of the unionized species can be

substituted by the intrinsic solubility, Su. The concentration of the ionized

species can be calculated from the Handerson-Hasselbalch equation (Albert

and Serjent, 1971), which relates concentration with pH and pKa. The

incorporation of these substitutes and rearrangement of equation 2-2 gives the

following expression for the total solubility of col-3:

St = SO [1 + 2(1 ) +1 -pK-z ] Eq. 2-3

The theoretical line shown In Figure 2-11 was calculated from equation

2-3, where the intrinsic solubility of col-3 is equal to 0.007 mg/ml and pKai and

pKa2 are equal to 5.86 and 8.09, respectively. The solubility data of col-3 from

pH 2 to 7 fit well with the theoretical line, however, negative deviation of the

data was observed at higher pH, which may be due to the degradation of col-3

In basic solutions.

Table 2-1. Spectrophotometric pKii determination of col-3.

PH Absorbance at 286nm®

Ar'^-A l o g - i

pKai value calculated from Eq. 2-1

1.93 0.086

4.06 0.087" - -

5.01 0.095 1.073 6.08

5.53 0.114 0.426 5.96

6.06 0.147 -0.210 5.85

6.20 0.156 -0.415 5.79

6.45 0.168 -0.725 5.73

6.75 0.173 -0.928 5.82

7.00 0.177 -1.221 5.78

7.39 0.183® - -

8.00 0.182

8.37 0.183

average pKai = 5.86 ± 0.12

® mean from two determinations "Au (absorbance of the unionized species) ° Ai*^ (absorbance of the monovalent anion)

Table 2-2. Spectrophotometric pKaa determination of col-3.

PH Absorbance at 286nm®

A r ^ - A log-^ r

A - A f ^

pKa2 value calculated from Eq. 2-1

5.01 0.077

6.06 0.078" - -

6.20 0.082 1.651 7.85

6.75 0.087 1.287 8.04

7.00 0.091 1.123 8.12

7.60 0.122 0.485 8.08

8.00 0.147 0.188 8.19

8.37 0.174 -0.085 8.28

9.08 0.242 -1.172 7.91

10.08 0.253^

10.55 0.251

average pKaa = 8.09 ± 0.15

® mean from two determinations (absorbance of the monovalent anion)

° Af^ (absorbance of the divalent anion)

40

Table 2-3. Solubility of col-3 in various solvents at 25°C.

Solvents Approximate solubility (mg/mL)

Water (final pH 4.3) 0.01

Ethanol 3.2

Propylene glycol (PG) 5.3

Benzyl alcohol 30.0

Polyethylene glycol (PEG 400) 50.0

Triacetin 8.5

Tributyrin 7.1

Benzyl benzoate 6.1

20% Cyclodextrin solution 0.8

10% Gentisic acid solution 0.2

2% Tween 80 solution 1.7

S«*pla: col3 Siza; 2.7000 ag Method: C0L3 CoMwnt: 760 HMg

0.1

-0.1-

-0.3 200 100 120 140 160 160 220

Taaparatura (*C)

Figure 2-1. DSC scan of col-3.

100'

95-

V 5

200 150 250 100

Temperature (°C)

Figure 2-2. TGA scan of col-3.

43

— MeOH -- HCI - - NaOH 0.4 --

0.3 --

0.1

380 430 480 230 280 330

X (nm)

Solvents (nm) e

Methanol 262,355 18151, 15503

0-1NHCI 265,350 16326,12514

O.INNaOH 265,380 16653,18793

Figure 2-3. UV spectra of col-3 in different solvents.

44

Tricarbonyl system Phenolic diketone system

HO. 0.5 T

0.4-- OH OH

0)

^ 0.3

o CO V

^ 0.2--

0.1 --

480 430 380 330 280 230

X(nm)

Figure 2-4. Chromophoric regions in col-3 molecule illustrating that absorption bands at 260 and 360 nm are produced from the tricarbonyl system and the phenolic diketone system, respectively.

45

File >SP1 Bpk flb 1134.

1200;

iiooi

100>

90(>

80>

700

600

500

400

300

200

100

C0L3-1 SUB

187

DIP / EI

161

55

115 /

44 /

40

243

\

197

/

120 200

310 /

284 258 / /

337 /

Se*n 427 7 .68 nin.

rilO

100

90

80

70

60

50

^0

30

20

10

371 /

1 ' I I ' r 240 280 320 360

^0

Figure 2-5. Mass spectrum of col-3.

46

Dimethylamonium group

; I pK^

R4 ; NH(CH3)2 :

OH

OH 0 ^ OH: pKaS

_ _1

Phenolic diketone system

CONH2

P pKal

Triciarbonyj system

Tetracycline H CH3 OH H 3.3 7.7 9.5 Chlorteracydine CI CH3 OH H 3.3 7.4 9.3 Demedocycline CI H OH H 3.3 7.2 9.1 Oxytetracydine H CH3 OH OH 3.3 7.3 9.7 Doxycycline H CH3 H OH 3.4 7.7 9.7 Minocydine N(CH3)2 H H H 2.8 7.8 9.3

Figure 2-6. Structural groupings and pKa values of the tetracyclines.

47

0.25

0.20 pH7.0-9.5

pH9.5

S I 0.15

o 05 jQ <

0.10

pH8.8 pH6.0

pH8.5

pH 1.0-5.0

0.05 pH 1.0-7.0

0.00 480 380 430 330 230 280

A.(nm)

Figure 2-7. UV spectra of col-3 at different pH values.

48

A. Tricarbonyl anion

OH

CONH2

-H+

O

CONHz

«0

XONht O

B. Phenolic diketone anion

OH 0 OH OH O O OH O O

Figure 2-8. Resonance stabilization of the tricarbonyl anion (A) and the phenolic diketone anion (B).

49

0.25

0.20

0.15

Abs 0.10

0.05

0.00

0.10

AAbS 0-°®

0.02

-0.02

0 2 4 6 8 10 12

pH

Figure 2-9. pH-absorbance profile of col-3 at 286 nm (A) and the first derivative of absorbance with respect to pH (B).

50

0.30

Abs 0.15

AAbs ApH

pKa2«5 8

6

pH

8 10 12

Figure 2-10. pH-absorbance profile of col-3 at 380 nm (A) and the first derivative of absorbance with respect to pH (B).

51

2 -

1 - -

-3 0 2 4 6 8 10

pH

Figure 2-11. pH-solubliity profile of col-3. The solid line represents the theoretical line where the Intrinsic solubility of col-3 is equal to 0.007 mg/mL and pkai and pKa2 are equal to 5.86 and 8.09, repectively.

52

CHAPTER III. STABILITY- INDICATING ASSAY OF COL-3 USING

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Introduction

In order to examine the stability of col-3, a stability-indicating assay has

to be developed. Several techniques have been reported for the analysis of

tetracycline-like compounds, including titrimetric (Yokoyama and Chatten,

1958), polarographic (Chatten et al., 1976; Caplis et al., 1965; Doan and

Riedel, 1963), chromatographic (Leenheer and Nelis, 1977; Butterfield et al.,

1975; TsujI et aL, 1974; Fernandez e^ a/., 1969; Simmons et al., 1969, 1966;

Ascione et al., 1967a, 1967b), and spectrophotometric techniques (Lever,

1972; Wilson et al., 1972, 1971; Chartten and Kranse, 1971; Pernarowski

et al., 1969; Kelly eta!., 1969; Kohn, 1961; Chiccarelli et al., 1959). Among

these techniques, high performance liquid chromatography (HPLC) combined

with UV detection is the most frequently employed method to separate and

identify these compounds and their degradation products (Ray and Harris,

1989; Yasin and Jefferies, 1988; Hermansson and Andersson, 1982; Sharma

and Bevill, 1978; Ali and Strittmatter, 1978; Tsuji and Robertson, 1976;

Butterfield etaL, 1973).

The purpose of this study was to develop a HPLC assay to quantitate

col-3 as well as to detect and separate its degradation products.

53

Experimental

Materials

Col-3 was supplied by the National Cancer Institute (Bethesda, MD).

Methanol and glacial acetic acid (HPLC grade) were purchased from J.T. Baker

(Phillipburg, NJ). Spectrophotometric grade acetronitrile was purchased from

Baxter (Muskegon, Ml). N/10 hydrochloric acid (HCI) and sodium hydroxide

(NaOH) solutions (certified grade) were purchased from Fisher Scientific

(Fairlawn, NJ). Edetic acid (EDTA, disodium salt) was purchased from Sigma

Chemical Company (St. Louis, MO).

Instrumentation

The chromatographic equipment consisted of a Beckman 126 solvent

delivery system, a Beckman 168 diode array detector (Beckman Instruments,

Fullerton, CA) and a Rheodyne 710 manual injector (Rheodyne, Cotati, CA)

with 100-^L loop. Data analysis was performed using System Gold Chromato­

graphic Acquisition Software (Beckman Instruments).

Chromatographic Conditions

Separations were performed at room temperature on a Mixed-Mode RP-

C8/cation (4.6 x 150 mm, 5 nm) column equipped with a Mixed-mode RP-C8/cation

(7 nm) guard column (Alltech, Deerfield IL). The aqueous and organic phases

54

were pumped separately and mixed in a ratio of 65:35 with a gradient controller.

The aqueous phase consisted of 0.1 M acetic acid with 0.005% disodium EDTA

and was filtered through a NYLON66 membrane, 0.45 [im filter (Alltech, Deerfield

IL) prior to use. The organic phase was acetonitrile. The column eluent was

monitored at a wavelength of 268 nm, with a flow rate of 1 mUmin.

Standard Solution

Stock solutions of col-3 (0.1 mg/mL) were prepared in methanol. Standard

solutions were freshly prepared before use by diluting the stock solution with

methanol.

Standard Calibration Curves

Calibration curves were determined from five standard solutions of col-3,

which covered a concentration range of 0.5-10 jig/mL. The standard curve was

constructed by plotting peak area versus col-3 concentration.

Method Validation

Standard solutions of 0.5,1.0, 2.0, 5.0 and 10.0 lag/mL of col-3 were used to

determine the reproducibility of the method. For each day, six replicate samples

were prepared for each concentration and assayed using the HPLC method

described above. This procedure was repeated over three separate days. Intra-

55

day variability was determined from the six replicate samples for each day (n = 6).

The concentrations determined over the 3 day assay were pooled and used to

calculate the inter-day variation (n = 18). The variability in each case is reported

as percent relative standard deviation (%RSD).

Stability of Col-3 under Acidic and Basic Conditions

A 20-(iL solution of 250 |ig/mL of col-3 in methanol was diluted to 1.0 mL

with either 0.005 M hydrochloric acid or 0.005 M sodium hydroxide solution.

These test solutions were exposed to ambient light at room temperature for 3

days. Before analysis, the solutions were evaporated and reconstituted with

200 (xL of the mobile phase.

Results

Chromatography

The HPLC system developed in this study can detect col-3 and its major

degradation products. A typical chromatogram of a standard solution of col-3 is

shown in Figure 3-1. The retention time of the drug is about 6.3 minutes. The

peaks of unidentified impurities were observed at 2.2,2.4, and 8.6 minutes.

Figures 3-2 (A) and (B) represent chromatograms obtained from degraded

col-3 in basic solutions at time 0 and 48 hours, respectively. The major

degradation peaks appear at about 3.0, 4.0, 8.0, and 10.0 minutes.

56

Standard Calibration Curves

Each standard calibration curve of col-3 was found to be linear over the

2 range of concentrations used in the study. The correlation coefficient (r) values

were in the range of 0.9992 to 0.9999. The mean slope was 3.534 m\J\xg (SD =

0.087, n = 18), and the mean intercept was 0.0839 (SD = 0.017, n = 18).

Method Validation

Table 3-1 shows the intra-day reproducibility of the col-3 assay. At each

concentration, the intra-day relative standard deviation varies between 0.53 and

3.15%. The accuracy of the average predicted concentration for each day,

reported in terms of percent bias was less than 2.5%. The inter-day reproducibility

of the method, given in Table 3-2, shows that the relative standard deviation varies

between 0.7 to 2.7% and the bias is less than 2.5%.

Stability of Col-3 under Acidic and Basic Conditions

In acid solutions, no degradation of col-3 was observed within 3 days.

However, in basic solutions, the degradation of col-3 was evidenced by color

change of the solution from yellow to pink, red and brown depending on the

extent of degradation. Four major degradation products were detected by

HPLC; however, they were formed in very small quantities (as indicated by

peak area). The quantitative detection of these compounds was, therefore,

57

carried out in more concentrated solutions (> 40 [ig/mL). Identification of these

compounds was based on their elution orders, I (3.0 min), II (4.0 min), col-3

(6.3 min), III (8.0 min) and IV (10.0 min), as shown in Figure 3-2 (B). The intra-

day and inter-day variations of their retention times are listed in Table 3-3 and

3-4, respectively. For all compounds, the relative standard deviation of the

retention time was less than 3% for both intra-day and inter-day assays. The

resolution was greater than 1.7 for the poorest separation of compounds I and

II.

Discussion

As mentioned above, there are a number of HPLC methods that have

been reported for the determination of tetracycline group of compounds.

Unfortunately, due to the absence of the dimethylamino functional group at the

C4 position and the hydroxyl and methyl groups at the C6 position, the

physicochemical properties of col-3 are significantly different from most

tetracycline analogues, and the previously determined chromatographic

conditions could not be applied to col-3. Consequently, a new HPLC method

had to be developed.

Col-3 is a hydrophobic compound, and therefore, a reversed-phase

chromatographic system was chosen for its assay development In the initial

attempt, a reversed-phase Ca column was applied. Very strong retention of

58

col-3 was observed; and high concentrations of organic solvents (up to 90% of

acetonitrile) were necessary in order to completely elute the drug. As this

result, a mix-mode column, which is more hydrophilic, was used in this study.

EDTA was found to be required for the elution of col-3. Without EDTA,

severe tailing of the peak (peak tailing factor > 2.5) was observed. Similar

phenomena have been reported in the assay of most tetracycline analogues

(Yasin and Jefferles, 1988; Leenheer and Nelis, 1977; Tsuji and Robertson,

1976; Butterfield et al., 1973). The tailing is believed to be the results of

complex formation between these compounds and metals or divalent cations,

such as calcium, which are present In the column (Leenheer and Nelis, 1977).

Therefore, a small amount of metal chelating agent, e.g., EDTA (0.002-0.005

M) is often added to the mobile phase to minimize the peak tailing (Yasin and

Jefferies, 1988; Tsuji and Robertson, 1976; Butterfield eta/., 1973).

The HPLC method developed allows the quantitative and reproducible

analysis of col-3 in aqueous solutions. The detection limit of col-3 was found to

be 25 ng/mL or 2.5 ng on column. The standard curve reproduced well from

day-to-day. The accuracy and precision of the method are reproducible in both

intra-day and inter-day validation.

The assay also allows the detection of the major degradation products

simultaneously with the parent compound. Baseline separation of col-3 and its

degradation products was achieved as shown in the chromatogram (Figure 3-

59

2). The retention times of each moiety are very reproducible. In addition, the

diode array detector connected to the HPLC provides the verification of the

peak homogeneity. This HPLC method is suitable for determining the

degradation kinetics of col-3.

60

Table 3-1. Intra-day reproducibility of the analysis of col-3

Cone. Days Intra-day Reproducibility (lag/mL) Mean Cone.'

(uQ/mL) Relative std

deviation (%) % Bias'

0.5 1 0.51 0.63 1.14 2 0.50 0.80 -0.08 3 0.50 0.94 0.31

1.0 1 1.02 2.27 2.27 2 1.01 2.17 2.17 3 1.00 1.74 1.74

2.0 1 2.01 0.58 0.48 2 2.05 2.61 2.41 3 2.08 3.15 4.24

5.0 1 4.99 0.53 -0.08 2 5.01 1.01 0.19 3 5.08 1.10 1.73

10.0 1 10.07 0.56 0.78 2 10.03 0.93 0.39 3 10.04 0.61 0.40

®Mean of 6 samples " % bias = (Found - Added)/ Added x 100

61

Table 3.2. Inter-day reproducibility of the analysis of col-3.

Cone. Inter-day Reproducibility (ng/mL) Mean Cone. ®

(ptg/mL) Relative std deviation (%)

%Bias''

0.5 0.50 0.92 0.45

1.0 1.02 2.20 2.08

2.0 2.04 2.74 2.37

5.0 5.03 1.19 0.61

10.0 10.05 0.70 0.53

®Mean of 18 samples " % bias = (Found - Added)/ Added x 100

62

Table 3-3. Intra-day reproducibility of retention times of col-3 and its four major degradation products.

Compounds Days Intra-day Reproducibility Retention time®

(min) Relative std devlation(%)

Col-3 1 6.34 0.86 2 6.37 0.39 3 6.31 0.43

1 1 3.01 0.83 2 3.03 0.80 3 2.99 0.72

II 1 4.11 1.88 2 4.06 2.73 3 4.02 2.02

III 1 8.04 0.88 2 8.06 0.58 3 8.18 0.47

IV 1 10.04 0.37 2 10.19 0.98 3 9.95 1.16

®Mean of 6 samples

63

Table 3.4. Inter-day reproducibility of retention times of col-3 and its four major degradation products

Compounds Inter-day Reproducibility Retention time® (min) Relative std deviation (%)

Col-3 6.34 1.02

1 3.01 0.92

II 4.06 2.30

III 8.09 1.01

IV 10.08 1.43

®Mean of 18 samples

64

o inH col-3

S.

o. w a

o.oo —r— 5.00 10.00

Time (min)

Figure 3-2. Representative HPLC chromatogram of a standard solution of col-3.

65

col-3 A

1 1 . . 1 1

B

•

I

1 ° 1 m IV

—yv <100 5^00 10.00

Time (min)

Figure 3-2. Representative HPLC chromatograms of degraded col-3 under basic conditions at t = 0 hr (A) and 48 hr (B). Keys: I, II, III, and IV = major degradates, x = minor degradates.

66

CHAPTER IV. STABILITY KINETICS OF COL-3 IN AQUEOUS SOLUTIONS

Introduction

Like most tetracycline derivatives, col-3 is not stable in aqueous

solution (Pinsuwan et a!., 1997). Although the degradation kinetics of the

tetracyclines are well known (Ali, 1984; Vej-Hansen et aL, 1987, 1979, 1978;

Mitscher, 1978), they cannot be applied to col-3. Due to the absence of the

4-dimethylamino group and the 6-hydroxy group, col-3 cannot undergo the

epimerization and dehydration reactions of tetracyclines. To date, no reports on

the degradation kinetics of col-3 are available in the literature.

In this study, the stability kinetics of col-3 were examined. The

degradation was studied as a function of pH, buffer concentrations, light, and

the presence of some additives. The purpose of this work is to describe the

kinetics of degradation of this compound in aqueous solutions and to provide

Information on various factors affecting overall stability.

Experimental

Materials

Col-3 was obtained from the National Cancer Institute (Bethesda, MD).

Methanol and glacial acetic acid (HPLC grade) were purchased from J.T. Baker

(Phillipburg, NJ). Spectrophotometric grade acetronitrile was purchased from

67

Baxter (Muskegon, Ml). N/10 hydrochloric acid (HCI) and sodium hydroxide

(NaOH) solutions (certified grade) were purchased from Fisher Scientific

(Fairlawn, NJ). Phosphoric acid, potassium chloride, potassium phosphate

(monobasic), potassium phosphate (dibasic), and Edetic acid (EDTA, disodium

salt) was purchased from Sigma Chemical Company (St. Louis, MO). All

reagents were used as received without further purification.

Instrumentation

All pH measurements were made at room temperature using a Corning pH

meter (model# 140) which was standardized with buffer solutions (VWR

Scientific company. West Chester, PA). High-performance liquid chromato­

graphy (HPLC) was carried out on an instrument composed of a Beckman 126

solvent delivery system, a Beckman 168 diode array detector (Beckman

Instruments, Fullerton, CA) and a Rheodyne 710 manual injector (Rheodyne,

CotatI, CA), with a 100 |iL loop. The column was a 4.6 x 150-mm, Mixed-mode

column packed with 5 |im, C-8/catlon bonded silica (Alltech Associates, Deerfield,

IL). Data analysis was performed using System Gold Chromatographic Acquisition

Software (Beckman Instruments).

68

Buffer Solutions

Phosphate buffer solutions ranging In pH from 2 to 10 were used for the

kinetic studies. The buffer concentrations were 0.01 M except for the

experiments where the influence of the buffer concentration was tested. A

constant ionic strength of 0.2 was maintained by adding of an appropriate

amount of potassium chloride.

Kinetic Measurements

Solution kinetic studies were performed by adding 200 |iL of a stock

solution of col-3 (0.5 mg/mL in methanol) to 20 mL buffer solutions, to achieve

the initial drug concentration of 5 |ig/mL All sample solutions were sealed in

clear glass ampules. Degradation was carried out in a thermostatically

controlled water-bath, protected from light. All kinetic studies were conducted

at 25 ± 0.2''C, unless stated otherwise. The reaction samples were withdrawn

at suitable time intervals. The concentration of residual compound was then

determined using the HPLC method as described in Chapter 3.

Effect of Drug Concentration

The influence of the col-3 concentration on Its degradation rate constant

was Investigated in the range of 5-40 (ig/mL in basic conditions (pHs 9 and 10).

However, due to its limited solubility («=10 ixg/mL), only the concentrations of

69

3 and 6 |j.g/mL were studied in acidic (pH 4) and neutral (pH 7) conditions. The

experiments were conducted in the same manner as described above.

Effect of Buffer Concentration

The effect of buffer concentration on the degradation rate constant of

col-3 was determined by varying concentration of the phosphate buffer from

0.01 to 0.05 M, while the ionic strength of the solution was kept constant at 0.2.

Effect of Light

The influence of light on the degradation of col-3 in aqueous solutions

was studied. The experiments were conducted in the same manner as

previously described in the kinetic measurements; except all test solutions

were left in the presence of daylight to assess stability.

Effect of Additives

The effect of antioxidants (e.g. ascorbic acid and sodium bisulfite) and

chelating agent (EDTA) on the degradation of col-3 were investigated. The

experiments were performed by determining the stability of col-3 in 0.05M

phosphate buffer pH 10 =0.2) with and without 0.005% of either ascorbic

acid, sodium bisulfite, or EDTA.

70

Effect of Temperature

The temperature-dependence of the degradation rate of col-3 was

examined at pHs 3 and 8 (0.05 M phosphate buffer, ^=0.2) in the temperature

range of 25 to 72°C. Temperature was controlled by immersing the test

solutions into a temperature-controlled water-bath.

Statistical Analysis

Analysis of variance (ANOVA) and the student's t-test were used to

determine statistical significance among groups and between two groups,

respectively. A p value of 0.05 or less was considered significant. The

correlation between the rate constants as a function of buffer concentration

was determined by testing the hypothesis that the slope of the regression line

Is equal to zero.

71

Results and Discussion

Reaction Order and Reaction Rate Constants

At any pH, the overall ISX loss of col-3 displayed an apparent first-

order kinetic behavior. Figure 4-1 shows typical first-order plots for the

degradation of col-3 at different pH values. The observed first-order rate

constants (kobs) for the overall degradation were calculated from the slope of

linear plots of the natural logarithm of the residual drug concentration versus

time:

ln[C] = ln[Co ] - /Coijsf Eq. 4-1

where [Co] and [C] are the initial and time (t)-dependent concentrations of col-3,

respectively. Values of kobs for col-3 degradation at several pHs and buffer

concentrations are listed in Table 4-1.

EfTect of Drug Concentration

Figure 4-2 and 4-3 illustrate the plots of log concentration versus time at

different Initial concentrations of col-3 at pHs 4 and 9, respectively. In both

cases, the plots were linear and parallel for each concentration of col-3

studied. Similar results were also observed at pHs 7 and 10. In addition, as

shown in Table 4-2, the half-life (Un) determined at each pH value is

independent of concentration. The fact that the half-life is independent of the

72

initial concentration of the drug indicates that the degradation of col-3 is first-

order over the pH range of 4-10. At lower pH values (pH 2 and 3), where the

degradation of col-3 was very slow, the kinetics were analyzed by the initial

rates assuming first-order kinetics.

Effect of Buffer Concentration

When the degradation of solutes in buffered media Is studied, a

combination of hydronium-, hydroxyl-, solvent-, and specific buffer-catalyzed

reactions should be considered. The contribution of the buffer at a given pH

can be calculated from a series of measurements at constant pH, solvent

composition, and ionic strength; but varying only buffer concentrations

(Carstensen, 1995; Connors etal., 1986):

^obs ~ i^o-^kt,uf{buf\ Eq. 4-2

According to this equation the observed rate constant, kobs, plotted against the

buffer concentration, [buf], yields a straight line with a slope equal to the

contribution of the buffer-catalyzed reaction (kbuf). The intercept corresponds to

the intrinsic (hydronium-, hydro)yl-, and solvent-catalyzed) reaction (/Cq ).

The dependence of the apparent first-order degradation rate of col-3

upon phosphate buffer concentration at several pH values is shown in Figure

4-4. Significant buffer catalysis was observed in the pH range of 4 to 10

73

(p < 0.05), whereas no significant buffer effect was observed at pHs 2 and 3.

This Indicates that the degradation of col-3 is catalyzed not only by specific

hydroxide ions but also by general bases, including the ionized buffer species,

H2PO4', HP04^* and P04^.

In order to understand the contribution of each buffer species, the

observed buffer-catalytic rate constants (kbuti) were calculated from the slope of

linear plots of k<,bs versus buffer concentration (Figure 4-4). These values were

then considered along with the fraction of the individual buffer species {f{)

present at each pH value. For phosphate buffer, f,- can be calculated as

follows:

Eq. 4-3 +[H^]K'\K2 +K^K2K2

f = i-" i ' 1 H,POi +[H+]K,K2 + K,K2K3

Eq. 4-4

Eq. 4-5

por ^ + [H^]K-IK2 + K^K2K2 K^K2K2 Eq. 4-6

where Ki, K2 and K3 are the first-, second-, and third ionization constants of

phosphate, respectively.

74

The catalytic components of the phosphate buffer can be described as:

Values for the observed buffer catalytic rate constants of col-3 and the fraction

of buffer species at different pH values are listed In Table 4-3. The highest

value of the observed catalytic rate constants is seen between about pHs 4 and

6. This suggests that H2PO4* species is the most catalytic since the highest

catalytic rate constant Is associated with the greatest proportion of H2PO4*.

pH-rate Profile

The pH-rate profile of col-3 at ZS'C is shown in Figure 4-5. The data

points of this plot represent the intrinsic rate constants (ko) obtained from the

apparent first-order rate constant (kobs) extrapolated to zero buffer con­

centration (Figure 4-4), while the curve drawn through the data points

represents the mathematical model developed In the following discussion.

As seen In Figure 4-5, the degradation rate constant of col-3 increases

In a non-linear manner as pH Increases. The pH of maximum stability is

^obs~ ^0 + kphosphatelPhOSPh3te]

+ „po5.[HPOf-l + /Cpo^[PO|-l

Eq. 4-7

Eq. 4-8

^obs = ^0 + ^H3P04 H^PO^ "t" ^H^POj,

^ H P o f - H P o f - ^ p d t P d t Eq. 4-9

75

between pHs 2 and 3. The degradation rate increases with pH between 3 and

6, reaching a plateau between about pHs 6 and 7, after which the rate

increases again until minimum stability is observed at about pH 10. This shape

of the profile suggests that the dissociation equilibria of the drug and the

hydroxide ion concentration are the major factors, which influence the

degradation rate. The mathematical model was, therefore, developed by

considering the degradation rates of various ionic species of the drug whose

concentrations change with pH.

In aqueous solution, col-3 exists as three ionic species, the unionized

species, the monovalent anion and the divalent anion, depending on the pH of

the solution. The total rate equation for col-3 can be written as:

where the subscripts A, B and C denote the unionized species, and the

monovalent- and divalent anions; k and f are the macroscopic rate constant

and the fraction of the species in the solution, respectively.

The fraction of individual species can be calculated as a function of pH

and the ionization constants (Ki and Kz) of the drug:

ko = + /csfg + kQfc Eq. 4-10

[H*f Eq.4-11

Eq. 4-12

76

Eq. 4-13

Substituting tlie appropriate fractional compositions into Eq 4-10 yields:

The ionization constants and the macroscopic rate constants can be

obtained from the fit of this model to the experimental data. The calculated

values of these parameters are shown in Table 4-4. The pKa values

determined from this kinetic model are 5.41 for pKai and 8.70 for pKaz- These

values are comparable to the values determined by the spectrophotometric

method (5.89 for pKai and 8.09 for pKaz). This supports the contention that this

model is consistent with experimental data and can be used to explain the pH-

rate profile of col-3.

Effect of Light

The effect of light on the degradation of col-3 in aqueous solutions was

investigated at several pH values. The results shown in Table 4-5 indicate that

light has a significant effect on the degradation of col-3 only in basic conditions

(pH > 8).

The degradation of col-3 in basic solutions is evidenced by the change

of color from yellow to red, which is more rapid in the presence of light. With

kA[H-^l2+kBKi[H-^l + kcKiK2 •^obs ~ r~o 1

[H-^]2+Ki[H-^] + KIK2 Eq. 4-14

77

additional acid, the solution reverts to the original yellow color, even though all

degradation products are still detected by HPLC analysis. These results

suggest the formation of a metastable red product in the degradation of col-3

under alkaline conditions. This is consistent with the fact that a metastable red

product has been reported for the oxidative degradation of tetracyclines, which

occurs in alkaline media (Wieb and Moore, 1989 ).

Effect of Additives

In order to confirm whether or not oxidation is involved in the

degradation of col-3, the effect of antioxidants (e.g. ascorbic acid and sodium

bisulfite) were Investigated. Additionally, the effect of a chelating agent, EDTA,

was studied because It can complex metal ions that may catalyze oxidation.

The experiments were performed by determining the stability of col-3 in 0.05 M

phosphate buffer at pH 10 with and without 0.005% of either ascorbic acid,

sodium bisulfite, or disodium EDTA. The solutions were placed in the presence

of light to assess stability. The results are graphically presented in Figure 4-6.

In all cases, col-3 was more stable in the solution with the additives.

The greatest stability was observed with the solution containing EDTA,

suggesting that the degradation of col-3 in basic solutions is due to oxidation

which is catalyzed by metal ions. Ascorbic acid showed the smallest effect on

78

increasing stability of col-3 because it itself Is not stable In aqueous solutions

at high pH values (Carstensen, 1995).

As described previously in Chapter 1, the postulated mechanism for the

photo-oxidation of tetracycline derivatives is primarily due to the reduction of

the 4-dimethylamlno group by photo-reaction, resulting in a formation of free

radicals which consequently undergo oxidation and hydroxyl-catalyzed

dehydration (Davies et al, 1979). This oxidative degradation pathway could be

envisioned for coi-3. The initial lag time observed in the degradation profile of

col-3 (see Figure 4-7) is suggestive of a free radical reaction. The presence of

lag time coincides with the assumption that metal-catalyzed oxidation is

involved in the degradation of col-3 under basic conditions.

Effect of Temperature

The quantitative relationship of the reaction rate and temperature can be

expressed by the Arrhenius equation (Carstensen, 1995):

Eq.4-15

where A is the pre-exponential factor which is a constant associated with the

entropy of the reaction and/or collision factors, Ea is defined as the activation

energy and R is the gas constant. Equation 4-15 is usually employed in the

logarithmic form:

79

-E logk = — + logA Eq. 4-16

^ 2.303RT

The temperature dependence of the degradation of coi-3 was examined

in phosphate buffer pHs 3 and 8 over the temperature range of 25-72''C. In

Figure 4-7, the observed rate constants were plotted according to the

Arrhenius equation (Eq. 4-16). The apparent activation energy (Ea) obtained

from the slope of the linear lines were 17.33 kcal mol"^ and 11.49 kcal mol"^ at

pHs 3 and 8, respectively.

80

Table 4-1. Observed apparent first-order rate constant (k<jbs) for the degradation of col-3 in phosphate buffer solutions of various pH values and buffer concentrations (25''C, \i = 0.2).

Buffer conc. (M) PH^ Kobs'Cday-^) SD' r ''

0.01 2.0 0.006 0.001 0.899 3.0 0.006 0.002 0.991 4.0 0.046 0.006 0.952 5.0 0.055 0.008 0.995 5.8 0.068 0.006 0.988 6.5 0.074 0.013 0.968 7.2 0.087 0.007 0.972 8.2 0.101 0.010 0.984 9.0 0.155 0.005 0.993

10.0 0.198 0.011 0.997 0.03 2.0 0.015 0.003 0.910

3.0 0.021 0.004 0.978 4.0 0.128 0.007 0.982 5.0 0.141 0.008 0.991 5.8 0.161 0.018 0.974 6.5 0.156 0-006 0.998 7.2 0.129 0.009 0.976 8.2 0.172 0.004 0.994 9.0 0.236 0.008 0.992

10.0 0.262 0.009 0.995 0.05 2.0 0.021 0.003 0-937

3.0 0.031 0-002 0.969 4.0 0.197 0-007 0.994 5.0 0.205 0.009 0-995 5.8 0.221 0.019 0.938 6.5 0.209 0.012 0.988 7.2 0.199 0-006 0-979 8.2 0.209 0-007 0-988 9-0 0.265 0.010 0-996

10-0 0.289 0-004 0-995

® pH within 0.1 units of the target values " average value (n = 3)

Standard deviation

81

Table 4-2. Half-life (T1/2) for the decomposition of col-3 in phosphate buffer solutions ( 0.01 M, |i = 0.2, 25°C) at different pH values and Initial drug concentrations.

pH' Col-3 concentration. T ^ 11/2 (^ig/mL) days (SD)

4.0 3.0 15.16 (0.42) 6.0 14.83 (0.20)

7.0 3.0 9.01 (0.10) 6.0 8.86 (0.25)

9.0 5.0 4.54 (0.08) 10.0 4.47 (0.10) 20.0 4.25 (0.14) 40.0 4.51 (0.09)

10 5.0 3.51 (0.10) 10.0 3.46 (0.22) 20.0 3.26 (0.12) 40.0 3.45 (0.18)

® pH within 0.1 units of the target values " n=3, SD = standard deviation

82

Table 4-3. Observed buffer-catalytic rate constant® (kbuf) for the decomposition of col-3 in phosphate buffer solutions (fi = 0.2, 25°C) as a function of pH and fraction (f,) of the individual buffer species.

pH^ Kbof' fi (%) (day' M-^)

H3PO4 H2PO4" HP04 ' PO4'*

2.0 0.425 58.79 41.21 - -

3.0 0.617 12.48 87.51 0.01 -

4.0 3.775 1.40 98.51 0.08 -

5.0 3.824 0.14 99.07 0.79 -

5.8 3.826 0.01 92.59 7.39 -

6.5 3.401 - 79.83 20.17 -

7.2 2.973 - 55.58 44.41 -

8.2 2.725 - 11.13 88.87 -

9.0 2.732 - 1.23 98.72 0.05

10.0 2.305 0.12 99.40 0.47

' pH within 0.1 unit ofthe target values " obatained from the slope of the plot of kobs versus buffer concentration

83

Table 4-4. Macroscopic rate constants for the decomposition of each ionic species of col-3 and the ionization constants obtained from non­linear fit' of the experimental data to the kinetic model (Eq. 4-10).

Macroscopic rate constants (day"^) Ionization constants

KA KB kc Ki K2

6.07x10"^ 4.33x10' 1.99 x10'' 3.94x10"® 1.69x10'®

1.30x10-^ 20.8x10"^ 1.49x10"^ 1.01x10"® 3.86x10"^°

2.14 4.86 0.75 25.5 2.28

values

SE"

%C\A

V = 0.99 " Standard error " % coefficient of variation

84

Table 4-5. Effect of light on the degradation rate constants of col-3 in phosphate buffer solutions (0.01 M, ^ = 0.2, 25"C) of various pH values.

pi? kobs'. day-^ (SD)

dark light

2.0 0.006 (0.001) 0.005 (0.003)

3.0 0.006 (0.002) 0.008 (0.001)

4.0 0.046 (0.006) 0.040 (0.009)

5.0 0.055 (0.008) 0.054 (0.008)

5.8 0.068 (0.006) 0.061 (0.010)

6.5 0.074 (0.013) 0.074 (0.009)

7.2 0.087 (0.007) 0.083 (0.012)

8.2'= 0.101 (0.010) 0.189 (0.015)

9.0^ 0.154 (0.005) 0.245 0.009)

10.0° 0.198 (0.011) 0.498 (0.005)

^ pH within 0.1 unit of the target values '' average value (n = 3), SD = standard deviation significant difference between light and dark (p< 0.05)

85

5

f4 (0 E 0) i-

"O

3

2 15 10 5 0

time (days)

Figure 4-1. Apparent first-order plots for the degradation of col-3 in aqueous solutions at various pH values (0.01 M phosphate buffer, |j. = 0.2, 25°C, dark). Key: (•) pH 2.0, (•) pH 4.3, (A) pH 7.2, (•) pH 9.0, and (*) pH 10.0. The solid lines represent the linear regression fit to the data.

86

2.0

c CD £ 0 t—

C —I 0.5

0.0

-0.5 30 40 20 10

Time (days)

Figure 4-2. Plots illustrating first-order dependence in col-3 concentration at pH 4.0 (0.01 M phosphate buffer, |j. = 0.2, 25°C). Key: (•) 6, (•) 3 |ig/ml. The solid lines represent the linear regression fit of the data.

Figure 4-3. Plots illustrating first-order dependence in coi-3 concentration at pH 9.0 (0.01 M phosphate buffer, ^ = 0.2, 25°C). Key: (•) 40, (•) 20, (A) 10, (•) 5 p.g/mL The solid lines represent the linear regression fit of the data.

88

0.4

0.3

2. 0.2

0.04 0.06 0.02 0

Buffer conc. (M)

Figure 4-4. Plots of the observed first-order rate constants of col-3 as a function of phosphate buffer concentration at various pH values. Key: (•) pH 10.0, (•) pH 5.2, (A) pH 4.0, and (•) pH 2.0. The solid lines represent the linear regression fit to the data.

89

>» (0 x>

Oi o

6

PH

8 10 12

Figure 4-5. pH-rate profile of col-3 In aqueous solutions (zero buffer con­centration, n = 0.2, 25°C). The SGlid line represents the non-linear fit of the experimental data (•) to the kinetic model (Eq. 4-10).

90

I

3

days

Figure 4-6. Stability profiles of col-3 in basic conditions (0.05 M phosphate buffer, pH 10.0 ji. = 0.2, 25''C, light) with and without additives. Key: (•) 0.05% EDTA, (•) 0.05% sodium bisulfite, (A) 0.05% ascorbic acid, and (•) without additive. The solid lines represent the linear regression fit to the data.

91

0 - » ^ ^ ^

0 2 4 6 8 Time (hours)

Figure 4-7. Degradation profile of col-3 in 0.01 M phosphate buffer (pH 9.0, 11=0.2, 25''C, light) illustrating the initial lag time.

92

1 1

-3 J ^

2.8 3.0 3.2 3.4 3.6

[1/T](x10^)

Figure 4-8. Arrhenius plots for the apparent first-order rate constant of col-3 in aqueous solutions (0.05 M phosphate buffer, (a =0.2, dark) at pH 8.0 (•) and pH 3.0 (•). The solid lines represent the linear regression fit to the data.

93

CHAPTER V. SEPARATION AND UV SPECTROPHOTOMETRIC

IDENTIFICATION OF DEGRADATION PRODUCTS

Introduction

Significant changes in color are seen in the solution of col-3 under

basic conditions. As described in Chapter 2, the degradation of col-3 is

indicated by the color changes of the solution from yellow to pink, to red and

finally to brown. Four major degradation products are found in the partially

degraded red solutions. However, these degradation products are not stable

and tend to further degrade, to form several minor degradation products.

In this study, the separation of these four initial degradation products

from col-3 was investigated and the UV spectrum of each compound was

determined.

Experimental

Materials

Col-3 was obtained from the National Cancer Institute (Bethesda, MD).

Dichloromethane and ethylacetate (analytical grade) were purchased from

Mallinckrodt Baker Inc. (Paris, KY). Spectrophotometric grade acetronitrile was

purchased from Baxter (Muskegon, Ml). Glacial acetic acid (HPLC grade) was

purchased from JT. Baker (Phillipburg, NJ). N/10 hydrochloric acid (HCI) and

94

sodium hydroxide (NaOH) solutions (certified grade) were purchased from Fisher

Scientific (Fairlawn, NJ). Edetic acid (EDTA, disodium salt) was purchased

from Sigma Chemical Company (St. Louis, MO). All reagents were used as

received without further purification.

Generation of Degradation Products

A 10 mL solution of col-3 (0.5 mg/mL) in 0.005 M NaOH was prepared

and kept in the presence of light at room temperature for 2-3 days until partial

degradation of the drug was obtained as indicated by the color change from

yellow to red. The test solution was analyzed periodically, using the HPLC

system described in Chapter 3, until maximum amounts of the four degradation

products were obtained as indicated by their peak areas.

Liquid-liquid Extraction

The major degradation products of col-3 were separated using the

liquid-liquid extraction procedure shown in Figure 5-1. The test solution was

first adjusted to pH 5 with 5% HOI solution and then extracted twice with 5.0-mL

dlchloromethane by shaking the mixture for 10 min and then centrifuging at

lOOOxg for 5 min at room temperature. The dlchloromethane layer was then

separated and evaporated to dryness under N2 gas. The further extraction of

compounds remaining the aqueous phase was perfomned by first adjusting the

95

aqueous pH to 3 and then extracting with ethylacetate. The extraction

procedure was conducted in the same manner as described above. The

ethylacetate was separated and evaporated to dryness under Na gas.

In order to monitor the presence or absence of the degradates, the dried

residue from each phase was reconstituted with the mobile phase and injected

into the HPLC system.

Spectrometric Identification of Degradation Products

The UV-spectrum of each degradation products of col-3 was recorded

using a photo diode array detector connected to the HPLC.

Results and Discussion

Separation of Degradation Products

Partial separation of col-3 and its four degradation products was

achieved with the liquid-liquid extraction procedure used in this study. Both

col-3 and degradates 11 and 111 were extracted into the dichloromethane layer,

while degradates 1 and IV remained in the aqueous layer. The chromatograms

of these two fractions are shown in Figure 5-2 (B) and (C).

In an attempt to further separate these degradates, a number of

additional procedures were used such as fraction collection from the HPLC

mobile phase, thin-layer chromatography, and precipitation techniques.

96

However, none of these procedures were successful due to the fact that these

degradation products are both unstable and present in very small amounts. In

addition, an equilibrium between the parent compound and the degradates was

observed as indicted by reversal of the degradates to the parent compound,

i.e., col-3.

The potential use of liquid chromatographic-mass spectrometry (LC-MS)

for identification of these compounds was also investigated. However, due to

the need for EDTA in the mobile phase, this method could not be used for peak

identification.

Spectrometric identification of Degradation Products

UV spectrophotometric analysis of col-3 and its degradation products

was performed using the photo diode array detector connected to the HPLC

system. As shown along with the chromatogram in Figure 5-3, the first

absorption band at about 260 nm is seen In the parent drug and ail four

degradation products. However, the second absorption band at about 360 nm

is decreased in degradates I and II and is totally absent in degradates HI and

IV. In addition, in both compound 111 and IV, there exists a new absorption

band in the 400-500 nm region.

The absence of the second absorption peak in the degradates III and IV

suggests that the chromophore responsible for this peak was destroyed in the

degradation process. As mentioned in Chapter 2, for tetracycline-type

compounds, an absorption at 360 nm is produced by the conjugated system

composed of the aromatic ring D and the diketone moiety (see Figure 2-4).

This suggests that it is this moiety has been altered in degradates III and IV.

The disappearance of the second absorption band was also observed on

recording the UV spectrum of col-3 in basic solutions at different periods of

time as shown in Figure 5-4. This result is consistent with the change in the

UV spectra of the degradation products.

98

adjust to pH 5 extract with DCM

adjust to pH 3 extract with EtAC

and col-3

Aqueous phase

Aqueous phase

Organic phase

Organic phase

Degraded col-3 in basic solution, pH 10

I and IV

Figure 5-1. Liquid-liquid extraction procedure for partial separation of col-3 and its degradation products.

99

O </> A <

aoo 5,00 iaoo

Time (min)

15.00

Ffgure 5-2. Representative HPLC chromatograms of degraded col-3 under basic conditions (pH 10) after 48 hr (A) and after extraction: in dichloromethane layer (B) and ethylacetate layer (C).

100

q_

col-3

m

o

Q.OO S.00 10.00 Time (min)

Figure 5-3. Chromatogram of degraded col-3 under basic conditions illustrating the UV spectra of col-3 and its degradation products which were detected by the photo diode array detector connected to the HPLC system.

101

0.30

0.25

0 hr

0.20 2 hr

ID O c ni .a w

4 hr

o in

< 6 hr

10 hr 0.10

0.05 24 hr

0.00

260 360 410 310

X (nm)

Figure 5-4. UV spectrum changes for the degradation of col-3 under basic conditions (pH 10.0, light).

102

SUMMARY

Since the use of available anticancer drugs is limited due to serious

side-effects and low efficacy, there is a need for new cytotoxic agents which

possess a more favorable therapeutic index. The antitumor antibiotics rank as

one of the most important classes of chemotherapeutic drugs, and these types

of compounds have been the object of intensive research and development.

Recently, 4-dedimethylamino sancycline (col-3), a new synthetic antibiotic of

the tetracycline family, was reported to have significant antitumor activity.

Col-3 is currently in Phase I clinical trial at the National Cancer Institute (NCI)

and has been selected for further development.

Like most tetracycline derivatives, col-3 is not stable in aqueous

solutions. However, due to the absence of the dimethylamino functional group

at the C4 position and the hydroxyl and methyl groups at the C6 position, col-3

cannot undergo the epimerization and dehydration reactions typically seen in

the tetracyclines. To date, no details of the degradation of col-3 are available

in the literature.

In this research project, the physicochemical properties and stability

kinetics of col-3 are investigated. The purpose is to describe the kinetics of

this compound in aqueous solutions and provide information on various factors

that affect the overall stability.

103

Col-3 is an acidic compound with two pKa values of 5.9 (pKai) and 8.1

(pKaa). It is slightly soluble in water (approx. 0.01 mg/mL) and readily soluble in

some pharmaceutically acceptable solvents such as ethanol, polyethylene

glycol, and benzyl alcohol.

While the aqueous solubility of col-3 increases as pH increases, its

stability decreases. Under alkaline conditions, the degradation of col-3 is

evidenced by color change of the solution from yellow to pink, red, and brown

depending on extends of degradation.

The pH-rate profile indicates that the rate of col-3 decomposition is pH

dependent and increases with increasing hydroxyl ion concentration. The

maximum stability is between pH 2 - 3 and the least stability is at about pH 10.

Incandescent light also increases degradation but only under basic conditions.

In a pH range of 4 -10, phosphate buffer significantly catalyzes degradation of

col-3. The largest catalytic effect is found for H2PO4* species. The metal

chelating agent (EDTA) and the antioxidant (sodium bisulfite) significantly

increase the stability of col-3 under basic conditions. These results indicate

that col-3 undergoes oxidation which is catalyzed by both alkaline and metal

ions.

Four major degradation products are found in the partially degraded red

solutions. Partial separation of these compounds can be achieved by the

liquid-liquid extraction described in Chapter 5. However, these products are

104

not stable and tend to further degrade, to form several minor degradation

products. Because of these results, the complete isolation of these compounds

was not accomplished.

Although the degradation products of col-3 cannot be isolated, they can

be detected and separated using the HPLC method described in Chapter 2.

Using the HPLC photo diode array detector allows the UV spectra of the four

major degradation products to be determined. The spectrophotometric analysis

showed that the absorption band at 360 nm was decreased in degradates I and

II and the totally absent in degradates III and IV. This suggests that the

phenolic diketone moiety of col-3, which produces this absorption band, is

altered by base-catalyzed degradation. However, for the establishment of the

mechanism of degradation of col-3, the identification of the degradation

products of col-3 must await an isolation procedure.

105

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