In vivo and in vitro study on potential combination therapy of COL 3 and tamoxifen in breast cancer a pilot study

The method was successfully applied to the routine analysis of COL-3 in the biological samples to study the pharmacokinetics of COL-3 following its oral and intravenous administration in

Chapter 1 Introduction

1.1 BACKGROUND

1.1.1 Overview of Current Breast Cancer Therapy

The current treatment of breast cancer is a multidisciplinary effort, with goals dependent on the specific clinical situation Various clinical breast cancer treatment are listed in Table 1.1 [1-4]

For patients who have not actually developed breast cancer but at high risk for the development of the disease, prevention of the breast cancer with a selective estrogen receptor modulator (tamoxifen) is more frequently used to treat established disease[5, 6]

Patients with non-invasive disease (intraductal carcinoma or ductal carcinoma in situ) are now routinely treated with surgery (mastectomy in some cases, breast conservation with or without radiation therapy in others) followed by a treatment with tamoxifen[7-9]

Patients with invasive breast cancer are generally treated with surgery followed by adjuvant systemic therapy tailored to their level of risk At the same time, it is common to see patients treat with preoperative (‘neo-adjuvant’) therapy For them, interpretation of operative findings can be a challenge[10-12]

Patients with hormone receptor-positive early-stage tumors -almost without regard to

tumor size or other risk factors—are routinely offered adjuvant hormonal treatment

Tamoxifen remains a gold standard for patients of all ages, but there is increasing

evidence regarding the role that aromatase inhibitors may play in postmenopausal patients[13] At present, we know that an average planned course of 5 years of tamoxifen is probably appropriate for most patients Longer or shorter durations might

be optimal for selected patients, there is even more reason to carefully evaluate the

risks and benefits of ovarian ablation as a component of systemic treatment[14-16]

It is apparent that treatment of breast cancer is evolving rapidly and has changed dramatically over the past decade Given the understanding of the basic biology of

breast cancer, the pending revolution in our ability to finely subtype it, and the availability of hundreds of novel agents, it seems reasonable to expect the improvements of the past decades to accelerate in the future[17]

Table 1.1 Summary of breast cancer treatment

Doxorubicin Cyclophosphamide

methotrexate use of anti-cancer drugs that go throughout the entire body

6-mercaptopuri

Vincristin

kill tumor cells that may be living in lymph nodes[2-4] Hormonal therapy Tamoxifen reduce the risk of recurrence

Aromatas inhibitors (Letrozole) if tumor expresses estrogen receptors[2-4]

Biologic therapy Herceptin (or Trastuzumab) block receptor HER-2/neu[3]

1.1.2 The Role of Angiogenesis in Breast Cancer

Angiogenesis, the process of new blood vessel formation, plays a crucial role in local tumor growth and distant metastasis in breast cancer[18] Angiogenesis precedes transformation of mammary hyperplasia to malignancy Transfection of tumor cells with angiogenic stimulatory peptides increases tumor growth, invasiveness, and metastasis Conversely, inhibitors of angiogenesis will decrease growth and metastasis of tumor cells[19]

Tamoxifen, initially believed to be a competitive inhibitor of estradiol, may have estrogen-independent mechanisms of action[20] It has been reported that tamoxifen has antiangiogenic activity (Table 1.2)

Other agents used in breast cancer, which have antiangiogenic activity, include several chemotherapeutic agents (paclitaxel[21], cyclophosphamide[22], methotrexate[23], etc.), protease inhibitors[24], growth factor/receptor antagonists[25] and endothelial toxins[26]

Table 1.2 Antiangiogenic activity of tamoxifen

Reference Antiangiogenic activity of tamoxifen

[27, 28] Inhibits VEGF (vascular endothelial growth factor) and fibroblast growth factor (FGF)-simulated embryonic angiogenesis [29] Decrease endothelial density and increases the extent of necrosis in MCF-7 tumors growing in nude mice [30] Inhibition of angiogenesis was detected before measurable effects on tumor volume [31] Down-regulation of CD36, a glycoprotein recptor for matrix proteins, thrombospondin-1, and collagen types I and IV

1.1.3 Overview of Combination Therapy in Cancer Treatment

Sometimes, more than one type of treatment is used to treat breast cancer Combination therapy may be adjuvant therapy - two types of treatment such as surgery followed by radiation or concurrent therapy - two types of treatment given during the same period of time With surgery becoming more conservative in the amount of tissue that is removed, it is common for even those with early stage cancers

to receive combination therapy Examples for combination therapy in breast cancer treatment are shown in Table 1.3

Early in the 1950’s, some encouraging results already showed that combined drug therapy improved the treatment of cancer, even though most patients treated had advanced solid tumors[32, 33] In the past few decades, concept of “synergism” has been developed, that in the combination therapy, chemotherapeutic agents interfere with either differing metabolic pathways or act at different sites in the same pathway This concept of synergism suggested that drugs might be used together to block the formation of essential cellular components, resulting in tumor cell death[34] Several mechanistic concepts evolved, including sequential blockade, or the inhibition of two

or more enzyme-mediated steps in the production of a necessary metabolite; concurrent blockade, or the inhibition of two or more parallel pathways in the synthesis of the necessary metabolite; and complementary inhibition, or the interference with different but related biochemical processes[35-37]

Table 1.3 Combination Regimens in breast cancer treatment [38]

Cyclophosphamide 200 mg/m 2 /d PO days 3-6 CAF Cyclophosphamide 100 mg/m 2 /d PO days 1-14 Every 4 weeks until 450mg/m 2

Doxorubicin 30 mg/m 2 /d IV day1,8 of doxorubicin then start metho-

Fluorouracil 500 mg/m 2 /d IV days 1,8 trexate 40 mg/m 2 IV and increase

5-FU to 600mg/m 2 IV CMF Cyclophosphamide 100 mg/m 2 /d PO days 1-14 Every 4 weeks

Methotrexate 40-60 mg/m 2 /d IV days 1,8

Fluorouracil 600 mg/m 2 /d IV days 1,8 CMFP Cyclophosphamide 100 mg/m 2 /d PO days 1-14 Every 4 weeks

Methotrexate 60 mg/m 2 /d IV days 1,8

Fluorouracil 700 mg/m 2 /d IV days 1,8 Prednisone 400 mg/m 2 /d PO days 1-14 CMFVP Cyclophosphamide 2.5mg/kg PO daily Every week for 8 weeks followed

Methotrexate 25-50 mg IV weekly by reduced therapy for maintenance Fluorouracil 12 mg/kg/d IV days 1-4, then 500mg

IV weekly Vincristine 0.035 mg/kg IV weekly (max dose 2 mg)

Prednisone 0.75 mg/kg PO daily FAC Fluorouracil 500 mg/m 2 /d IV days 1,8 Every 3 weeks

Doxorubicin 50 mg/m 2 /d IV day1

Cyclophosphamide 500 mg/m 2 IV days 1

Vinblastine 6 mg/m 2 IV/d days 1,21

Tamoxifen citrate has a molecular weight of 563.62, the pKa' is 8.85, the equilibrium solubility in water at 37°C is 0.5 mg/ml and in 0.02 N HCl at 37°C, it is 0.2 mg/ml

1.2.2 Pharmacology

Tamoxifen is one of the Selective estrogen receptor modulators (SERMs), which are a class of compounds with interesting pharmacology[39] They have the capability of acting as estrogen receptor (ER) agonists in some tissues and as antagonists in others[40-43] Tamoxifen is a potent ER antagonist, and its major antitumour activity

is achieved by competitively inhibiting estradiol binding at the estrogen receptor[40, 44] Therefore, patients with ER positive cancers respond best to tamoxifen treatment [45]

1.2.3 Clinical Use

Tamoxifen (Nolvadex) has been the standard hormonal agent used for breast cancer

It is the prototype for a growing class of compounds called selective estrogen receptor- modulators (SERMs) SERMs chemically resemble estrogen and trick the breast cancer cells into accepting it in place of estrogen Unlike estrogen, however, they do not stimulate breast cancer cell growth Other SERMs being studied for breast cancer include toremifene (which is very similar to tamoxifen), idoxifene, and droloxifene[39]

The antiestrogenic effects of tamoxifen are manifest over a wide range of dose A dose of 10 to 20 mg, twice daily, was used in early clinical trials The dose-response effect of tamoxifen was tested over a range of 2 to 100 mg/m2 body surface area, twice daily No clear increase in antitumor activity of tamoxifen was shown with the larger doses[46]

Tolerance to tamoxifen is good The most frequent side effect has been hot flushes, which are tolerable in most patients Approximately 10% patients have mild nausea and vomiting that can be quite severe and require interrupting treatment A less frequent side effect is bone pain[47] Other rare side effects include vaginal bleeding, thrombophlebitis, and ocular toxicity[48-50]

Tamoxifen can be used in the treatment of metastatic breast cancer in postmenopausal women (Table 1.4) as well as in premenopausal women (adjuvant trials) (Table 1.5)

Tamoxifen as adjuvant treatment in postmenopausal has been established as useful, either alone or combined with chemotherapy

Table1.4 Tamoxifen in Premenopausal Patients (Phase II studies)

[51] 20 mg twice daily 10 First trial to show antitumor activity

[52] 20 mg twice daily 11 Response observed despite incomplete

suppression of ovarian function [53],[54] 20 mg twice daily 74 Median duration of response, 13 months [55] 10 mg twice daily 21 Response duration, 3 to 18 months

[56] 10 to 20 mg twice daily 26 No evidence of dose-response relationship [57] 10 mg twice daily 38 Median duration of response, 9 months [58] 10 mg twice daily 44 Response duration, 3 months

[59] 20 mg twice daily 43 Median response duration, 20 months

Table 1.5 Adjuvant Trials with Tamoxifen in Postmenopausal Patients

Reference Patients Dose of Tamoxifen Remarks

[60] 588 20 mg/day Trend toward increase in survival

but not significant

[62] 1135 10 mg twice daily Significant increase in survival of

patients treated with tamoxifen [63] 503 20 mg/day Survial similar; estrogen receptors

unknown in 52% of patients [64] 1650 10 mg twice daily Trend toward improved survival

with tamoxifen; estrogen receptors unknown in 80% patients

[65] 170 10 mg twice daily Survial unchanged; estrogen receptors

positive in 85 % [66] 400 10 mg twice daily Survival unchanged except for a trend

in progesterone-receptor-positive tumors

[67] 1312 20 mg/day Significant increase in survival of

all patients [68] 179 40 mg/day Significant improvement in 5-year

survival of patients with receptor- positive tumors

1.2.4 Pharmacokinetics

1.2.4.1 Human Study

Absorption Tamoxifen is rapidly absorbed from the gastrointestinal tract[69] Its

bioavailability is high (approximately 100%) and independent of dose, suggesting minimal first-pass metabolism[70, 71] Following single-dose administration of tamoxifen 40 mg, peak plasma concentration (Cmax) was approximately 65 μg/L and time to Cmax (tmax) was reached in 3-4 hours in healthy subjects[72] Cmax was dose dependent[73]

Distribution Tamoxifen is highly lipophilic agents, resulting in extensive plasma

protein binding (>95%), with the majority bound to albumin[44, 71, 74] Complete tissue distribution studies have been conducted in animals using [14C]tamoxifen High concentrations of radioactivity were found in the breast tissue, uterus, liver, kidney, lung and pancreas[75] A human study of tamoxifen distribution was conducted by Lien et al[76] in 14 patients ranged in age from 28 to 89 years of age, and biopsies were recovered during surgery or autopsy over the course of 3 years High concentrations of tamoxifen were found in liver, lung, pancreas, brain, ovaries and breast tissue Distribution of tamoxifen in the human uterus was studied by Fromason and Sharp[77] in women prior to hysterectomy Tamoxifen has a high affinity for the endometrium, as tamoxifen concentrations were found to be 2-3 times higher in the uterus than in plasma[77, 78] In humans, the apparent volume of distribution was approximately 50-60 L/kg[79]

Metabolism Tamoxifen undergoes phase I metabolism in the liver by microsomal

cytochrome P450 (CYP) enzymes[80, 81] Tamoxifen is mostly metabolized by CYP3A and CYP2C isoforms[82], but CYP2D6 may also be involved The major metabolites of tamoxifen are N-desmethyltamoxifen (resulting from N-demethylation) and 4-hydroxytamoxifen (resulting from 4-hydroxylation)[83-85]

Excretion Tamoxifen is excreted in the bile and eliminated through the faces, with

small amounts eliminated in the urine[44, 78, 83] Following oral administration of tamoxifen, elimination is biphasic and is dependent on the cumulative dose The terminal elimination half-life (t1/2β) of tamoxifen is 5-7 days, which may be due to enterohepatic circulation, plasma protein binding and autoinhibition of metabolism[44, 71, 81, 83, 86, 87]

Steady-state pharmacokinetics Steady-state serum concentrations of tamoxifen are

usually achieved within 3-4 weeks of daily tamoxifen administration at dosage ranging between 20 and 40 mg/day[44, 74, 83] The average steady-state concentrations for dosage of 20mg/day (long-term treatment) and 40 mg/day (2-month treatment) ranged from 164-494 and 186-214 μg/L, respectively[74, 88, 89] The area under the concentration-time curve (AUC) for tamoxifen following a regimen of 10 mg tamoxifen twice daily for 21 days was 1597μg·h/L in women with advanced breast cancer[90]

1.2.4.2 Animal Study

Distribution of tamoxifen and metabolites into tissues of rats:

In all rat tissues except fat, essentially the same amounts of drugs were found after 3 days and 14 days of treatment of tamoxifen, suggesting that steady-state is obtained within 3 days The content of tamoxifen and its metabolites in tissues was orders of magnitude higher than in serum The demethylated and hydroxylated metabolites were abundant in most tissues, except in fat tissue, where tamoxifen was the predomination species The concentrations of the hydroxylated metabolite and the demethylated metabolite were especially high in lung and liver and kidney

In rat adipose tissue, the fluctuation in the tamoxifen and metabolite concentrations during one dosing interval were less than those observed in other tissues of the rat Tamoxifen was the predominating species; only small amounts of the demethylated metabolites and the hydroxylated metabolite were observed These findings may be explained by slow distribution of tamoxifen into fat tissue, where this lipophilic drug partitions into lipid droplets and is preserved due to low activity of drug metabolizing enzymes

Tissue kinetics in rat and similar metabolite profiles in most tissues suggest an exchange of tamoxifen and metabolites between most tissues, and between serum and tissues, including brain In contrast, fat tissue contains low levels of metabolites and seems to sequester tamoxifen; it may function as a “deep” compartment[76]

Some studies also showed that rapid N-desmethylation and 4-hydroxylation of tamoxifen resulting in similar serum levels of N-desmethyltamoxifen and 4-

hydroxytamoxifen to the parent compound in the mature mouse and, hence, similar AUCs throughout a 96-hr study In contrast, the immature rat has a less dominant 4-

hydroxylation pathway, but AUC for tamoxifen and N-desmethyltamoxifen was in a

similar ratio in the rat to that of the mouse The rate of elimination from the serum

following a single large oral dose of tamoxifen is similar for tamoxifen,

N-desmethyltamoxifen, and 4-hydroxytamoxifen in the rat and the mouse[91]

Figure 1.2 Chemical structure of COL-3

COL-3, the simplest tetracycline, differs from tetracycline by the absence of the dimethylamino, 6-hydroxyl, and 6-methyl groups (Figure 1.2) COL-3 is the first non-antimicrobial, chemically modified tetracycline to be assessed for anticancer effects in humans Due to its physiochemical properties, it is anticipated that COL-3 would have

4-a longer h4-alf-life (t1/2) 4-and l4-arger volume of distribution (Vd) th4-an tetr4-acycline 4-and doxycyline[92] COL-3 is a yellow, odorless crystalline compound with a molecular

weight of 371.35 Although the solubility of COL-3 increases with increasing pH, its stability decreases with increasing pH Due to the absence of the 4-dimethylamino group, COL-3 cannot exist as a zwitterions and, therefore, differs from the tetracyclines in its acid-base properties, which leads to its poor solubility (0.01mg/ml

in water at pH 4.3)[93] It is readily soluble in organic solvents such as methanol, polyethylene glycol, and benzyl alcohol

1.3.2 Pharmacology

COL-3 is a non-antimicrobial chemically modified tetracycline (CMT) Since the first CMT was described in 1987[94], more than 30 different CMTs, in which the 4-dimethylamino group is removed., have been developed CMTs lose the antibacterial activity of tetracycline but retain or even enhance the inhibition activity of matrix metalloproteinases (MMPs)

The MMPs are a family of ECM (extracellular matrix)-modifying enzymes associated with the malignant phenotype, and studies with natural or synthetic MMP inhibitors demonstrated that MMP activity is required for tumor progression and metastasis in several model systems[95] Inhibition of MMPs is obtained through protease inhibitors such as α2-macroglobulin and by a group of specific tissue inhibitors of metalloproteinases (TIMPs) It is thought that an imbalance between the activation and inhibition of MMP activity in favor of the MMP activity plays an important role

in the pathophysiology of cancer by facilitating the invasion of tumor cells through the ECM[96]

Unlike other MMPIs, tetracycline derivatives not only inhibit collagenase activity but also downregulate its production, inhibit its activation, and increase the degradation of the proenzyme[96] COL-3 potentially inhibits MMPs (i.e., MMP-2, MMP-9, and MT1-MMP) [97-100], which are believed to be positive contributors in tumor growth and metastasis With the development of second generation inhibitors with specificity for individual MMP family member, one may be able to target specific events in breast tumor progression associated with specific MMPs[95]

1.3.3 Clinical Use

Recently, CollaGenex Pharmaceuticals, Inc carried out some studies, which showed positive results of phase II clinical study evaluating effects of COL-3 for treating rosacea presented at North Carolina Dermatology Association The study achieved its primary endpoint, demonstrating a greater reduction in inflammatory lesion count from baseline for the COL-3 treated patients compared to the patients on placebo At endpoint (Day 42), COL-3 patients had a mean reduction of 12.8 lesions while placebo patients showed an increase of 2.3 lesions This difference was statistically significant (p=0.0213) Importantly, the onset of action was very rapid, with approximately 80% of the reduction in lesion count observed at Day 42 already present at Day 14 At endpoint, 75% of all COL-3 treated patients were clear or near-clear of disease symptoms as measured by the Investigators Global Assessment score Erythema showed a slightly better improvement in the COL-3 group, with a 2.5-point reduction in the clinician's erythema assessment score for the COL-3 group compared

to a 1.7-point reduction for the placebo group COL-3 was well tolerated and the adverse event profile was unremarkable

Based on some promising preclinical studies, COL-3 has been evaluated in clinical trials in patients with solid tumors who receive the agent by oral administration in a continuous dosing schedule 70 mg/m2/day administered orally and the dose limiting toxicity was cutaneous photosensitivity, which was observed in 69% of patients Other side effect observed were generally mild, and included anemia, anorexia, constipation, dizziness, elevated bilirubin and transaminases, fatigue, fever, headache, hearburn, nausea, vomiting, neurotoxicities, and three cases of drug-induced lupus erythematosus[101]

1.4 COMBINATION OF TAMOXIFEN WITH COL-3 AND DRUG-DRUG INTERACTION

The combination of tamoxifen and COL-3 is rational, based on the marked antitumor activity of both agents against a variety of solid tumors, such as breast cancer, prostate cancer, lung cancer, colon cancer, etc, and their different mechanism The difference

in the mechanism of action between the two drugs has led to this preclinical study combining the two agents

The inability to control metastasis is the leading cause of death in patients with cancer Control of metastasis, therefore, represents an important therapeutic target[102] Observations of some studies suggested that specific CMTs could potentially be used to suppress the formation and magnitude of metastases associated with certain cancers, and used in conjunction with other cancer treatment regimes, lead to a more efficacious treatment of those metastatic diseases[100]

In several studies, MMP-2 has been shown to be expressed in breast 109] In limited series, MMP-2 positivity is associated with unfavourable prognosis in both premenopausal and postmenopausal node-positive breast carcinoma patient[110-112] Some studies also show that MMP-2 immunoreactive protein is an independent prognostic indicator that might prove valuable in certain subgroups, such as patients with a receptor-negative breast carcinoma MMP-2 negativity was proved to serve as

carcinoma[103-a mcarcinoma[103-arker for distinctly fcarcinoma[103-avourcarcinoma[103-able prognosis in brecarcinoma[103-ast ccarcinoma[103-arcinomcarcinoma[103-a pcarcinoma[103-atients carcinoma[103-and MMP-2 positivity is also shown to correlate to poor survival in node-negative breast carcinoma[113] COL-3 is devoid of antimicrobial properties and is a competitive and selective inhibitor of MMP-2 and MMP-9 isoenzymes, making it an attractive candidate for clinical breast cancer treatment

MMPIs should be regarded as cytostatic drugs that inhibit tumor growth It is theoretically attractive to combine MMPIs with some hormone therapy like using tamoxifen to treat breast cancer so that their effectiveness can be augmented Tamoxifen is considered the golden standard for the hormonal therapy of breast cancer in early stage Several cased of drug-drug interaction have been reported about tamoxifen (Table 1.6)

Medicines are often used concomitantly with other drugs, and some degree of drug interaction occurs with concomitant use Although only a small proportion of this interaction is clinically significant, it sometimes causes serious adverse reactions For example, drug interactions, particularly with drugs having a narrow therapeutic range, may have serious adverse consequences Therefore, in the evaluation and clinical application of drugs, appropriate efforts should be made to predict the nature

drug-and degree of drug interactions so that patients will not be adversely affected A careful evaluation of the pharmacokinetic and pharmacodynamic profiles of drugs in animal models is of primary importance for safety and effectiveness assessment prior

to clinical trails

Several mechanisms may account for the drug-drug interaction Pharmacokinetic interaction, like drug-drug and nutrient-drug interaction at the absorption site is one of the explanations to the mechanism of drug interaction[114] The influence of wide-ranging factors such as the effect of fluid volume of gastrointestinal physiology, splanchnic blood flow, passive diffusion, gastric emptying time, pH of the intestinal contents, intestinal motility, ionic content of foods, dietary fat, and gastrointestinal disease can have considerable effect on the absorption of drugs and they are mechanisms common to both drug-drug and drug-food interactions in the gut Another mechanism of drug-drug interaction is the interactions at plasma- and tissue-binding sites When a displacing agent interacts with a primary drug the result is an increase in the free concentration of the displaced drug in the plasma And the increased free drug in the plasma quickly distributes throughout the body and may localize in the tissue Many interactions can also be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues Induction or inhibition of the collection of isoenzymes, such as cytochrome P450 enzymes,are mechanisms that have been shown to underlie some of the more serious drug-drug interactions Interactions involving renal excretory mechanisms can have important clinical implications in terms of patient mortality and morbidity Molecular mechanism of renal drug transport has allowed predictions to be made of potential drug interactions in early phase drug development[114]

Pharmacodynamic drug interaction, for instance, interaction at the receptor and other active sites is one of the most common mechanisms by which pharmacodynamic interactions occur Not all drug-drug interactions are hazardous, and some synergistic interactions when they occur can be of clinical value in therapy Indeed some food-drug interactions can be valuable especially when food improves the bioavailability of the active drug substance In other samples, the inhibition of the oxidative phase of hepatic drug metabolism by, for example, cimetidine may potentiate the effect and/or duration of a variety of drugs Increasing the plasma concentration of a primary drug may well also increase the probability of enhanced toxicity[115]

In current medical practice there are a number of examples of combined formulations and/or co-prescribing of active ingredients on the basis of their believed synergistic action For many of these the evidence for efficacy may not stand up to critical analysis

Much of the drug interaction in vitro in the past has been interactions between drug and drug, and drug and fluid in intravenous infusions Newer work has concentrated

on interactions between specific drugs, notably chloroquine, cyclosporin insulin and vasodilator nitrates, and pharmaceutical packaging materials (glass and plastics), and the mechanisms by which these in vitro interactions occur[115]

Frequently the influence of medication on the reliability of subsequent laboratory tests

is not realized or merely overlooked The mechanisms of such interactions can be generally classified into two areas: pharmacological and methodological interferences Whenever a drug-laboratory test interaction is established it should be

communicated to the medical community so that they may avoid possible errors in diagnosis[115]

The present study was performed to investigate potential drug-drug interaction between COL-3 and tamoxifen in female rats and further to examine the underlying

mechanism by some in vitro experiments

Table 1.6 Reported interactions of tamoxifen

Rifampicin (rifampin) plasma concentration of tamoxifen

Aminoglutethimide Decreased serum concentrations of tamoxifen and its metabolites [118]

Letrozole Decreased plasma concentrations of letrozole [119],[120]

Chapter 2 Aims and Objectives

The novel anti-cancer agent COL-3 is one of the most potent CMTs (chemically modified tetracyclines) studied to date and has demonstrated antitumor activity both

in vitro and in vivo[24] Clinically, some of the patients with refractory metastatic

cancer showed some degree of clinical benefit from COL-3[121] In a phase I study, COL-3 was well tolerated and had moderate antitumor activity in patients with AIDS-related Kaposi’s Sarcoma[122] However, very limited data has been reported about combination use of COL-3, except recently, it was found that acute doxorubicin administration decreased COL-3 oral bioavailability and prolonged COL-3 elimination in rats[123] In the present study, a potential combination therapy of

COL-3 and tamoxifen will be evaluated using some in vivo and in vitro models

The main purpose of this research work is to assess the feasibility of coadministering

of tamoxifen, a nonsteroidal antiestrogen with inhibitory effect on estrogen-dependent growth-stimulatory mRNA synthesis, and COL-3, an oral chemically modified tetracycline derivative with potent inhibitory effect on matrix metalloproteinases

activity and production, against breast cancer using animal model and some in vitro

models

The primary objective is to investigate the effect of tamoxifen on the pharmacokinetic (PK) aspects of COL-3 in female rats The PK profile of COL-3 would be examined after its oral/intravenous administration given alone and with low-dose/high-dose tamoxifen in fed/fasted female rats

In addition, the effect of tamoxifen on in vitro serum protein binding of COL-3 would

be explored to investigate if protein displacement would be a source of the PK interaction between COL-3 and tamoxifen in the animal study

To evaluate whether these two drugs produce synergistic, additive, or antagonistic

effects on human cancer cell lines when given together, an in vitro investigation

would be conducted to examine the effects of COL-3 and tamoxifen, in single agent

or in combination against human breast cancer cell lines, MDA-MB-231 and MCF-7

Finally, attempt would be made to account for an irregular absorption profile of

COL-3 with double- or plateau- peak concentration following oral administration of COL-COL-3

in fed/fasted rats Two-site absorption model[124], discontinuous absorption model[125, 126] and two enterohepatic recirculation models[127] would be evaluated

to see which one is better with some meaningful PK parameters to describe COL-3 serum concentration-time profile after its oral administration with low/high dose tamoxifen in fed/fasted rats

Chapter 3 Analytical Methods

3.1 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF TAMOXIFEN IN BIOLOGICAL SAMPLES

3.1.1 Introduction

Tamoxifen is an anti-estrogenic agent widely used in the treatment of breast cancer and many researchers have reported methods to determine tamoxifen and its metabolites Many of these methods involved conversion of tamoxifen and its metabolites to their fluorescent phenanthrene derivatives followed by HPLC[128] or TLC[129] This reaction was carried out both pre- [128] and post- [130] column Gas liquid chromatography-mass spectrometry has also been used[131, 132]

There were also methods that using capillary electrophoresis[133].Also, methods that involved solid-phase extraction had been reported However, some of these methods required tedious sample preparation procedure and some assays needs complicated equipments such as photochemical reaction unit to convert tamoxifen to its fluorescent derivative Thus, a simple and sensitive method is desirable in our study where a large number of serum samples need to be analyzed

In the present study, a simple HPLC method with short retention time and good separation was developed to analyze tamoxifen and its major metabolite, 4-OH-tamoxifen, in rat serum

3.1.2 Materials and Methods

Tamoxifen and its metabolite (4-OH-tamoxifen) were purchased from Sigma (St Louis, MO, USA) Methanol and acetonitrile, both were of high performance liquid chromatography (HPLC) grade, were purchased from Tedia Company Inc (USA) and Lab Scan (Thailand), respectively Ammonium acetate was also purchased from Sigma

To determine the concentrations of tamoxifen and one of its major metabolites, tamoxifen, in rat serum, 100μl of serum was deproteinized by adding 100μl acetonitrile and allowed to stand at room temperature for 10 min Then samples were centrifuged at 10000g, 40C for 15 min Supernatant was collected and an aliquot of 20μl was injected into the HPLC with UV detector Shimadzu HPLC system (LC 2010A) (Shimadzu, Kyoto, Japan) was used Chromatographic separation was conducted on a XterraTM RP18 column (150 × 4.6 mm I.D., particle size 5μm) with SentryTM guard column (XterraTM RP18, 20 × 3.9 mm I.D., particle size 5μm) (Waters, Milford, MA, USA) The mobile phase consisted of 50mM ammonium acetate (pH 8.0) and methanol (25:75, v/v)[134] The mobile phase was degassed by ultrasonication and was delivered isocratically at a flow-rate of 0.5ml/min The column was maintained at 250C The eluent was monitored at a wavelength of 265

4-OH-nm

3.1.3 Results

Figure 3.1 shows a chromatogram of a rat serum sample 24 hours after oral

administration of high-dose tamoxifen (30mg/kg) The entire running time for one sample is within 20min 4-OH-tamoxifen was eluted first at around 10min and tamoxifen gave its peak at around 15min

Table 3.1 lists the intra- and inter-day precision and accuracy for 4-OH-tamoxifen (at

3 concentrations of 50, 100, 200ng/ml) and tamoxifen (at 3 concentrations of 100,

Figure 3.2 Chromatogram of a rat serum sample 24 hr after oral administration of

high-dose tamoxifen (30mg/kg) in female rat

Table 3.1 Intra- and inter-day precision and accuracy of the assay for determination

of tamoxifen and its metabolite 4-OH-tamoxifen

3.1.4 Discussion

In summary, a simple and reliable HPLC method was developed and validated for the simultaneous quantification of high-dose tamoxifen and 4-OH-tamoxifen in rat serum Numerous sample preparations, such as liquid-liquid extraction, were attempted to resolve the interferences with no success In the present study, direct deproteinization was involved for the sample preparation and was sufficient enough to produce clean chromatograms The intra- and inter-day coefficients of variation were all less than 8% at the three concentrations of both 4-OH-tamoxifen and tamoxifen with the accuracy varied from –8.1 to 1.1% for 4-OH-tamoxifen and –11.0 to 3.3% for tamoxifen

The assay was possible to detect tamoxifen and 4-OH-tamoxifen in all serum samples

of rats after oral administration of high-dose tamoxifen in our in vivo study, thus

offering an ideal chromatographic method for a routine measurement of tamoxifen and 4-OH-tamoxifen in rat serum However, as for the rats under treatment of low-dose tamoxifen, the assay is not sensitive enough to detect tamoxifen and its metabolite It has been reported that with a post-column photoreactor included in the HPLC system, assay could permit a sensitive, fast and reproducible determination of tamoxifen and its metabolite in rat plasma down to 100 pg/ml concentration[135] Since the photochemical reactor is not available in our research lab, the HPLC system could not be developed to be sensitive enough to detector the low concentration of tamoxifen and its metabolites in rat serum after administration of low-dose tamoxifen

A more sensitive and simple method would be required to detect the low

concentration of tamoxifen, for example, LC/MS or GC/MS, to give a more sensitive and accurate measurement

3.2 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF COL-3 IN BIOLOGICAL SAMPLES

In the present study, an established method [136] using reversed-phase performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed to measure COL-3 concentration in biological samples like serum, urine and feces The method was successfully applied to the routine analysis of COL-3 in the biological samples to study the pharmacokinetics of COL-3 following its oral and intravenous administration in rats Briefly, the compound was detected at wavelength

high-of 350 nm, where no interferences occurred at the same retention time as COL-3 in the serum, urine and feces samples, giving very clear chromatograms The sample treatment procedure is simple and efficient, with one-step deproteinization by adding

a mixture of acetonitrile-methanol-oxalic acid Oxalic acid in the sample treatment was crucial to retain good peak shape It was not only functioning as a metal blocking agent to release COL-3 from its chelate complex with polyvalent metallic ions in biological samples, but also adjusted the pH to less than 2 at which COL-3 is quite stable and exists as un-ionized form so that it can be easily distributed into the organic phase from serum protein[136] This method could also be applied to the analysis of COL-3 in other rat biological samples, such as rat bile samples

Chapter 4 Animal Study

4.1 INTRODUCTION

COL-3, the oral matrix metalloproteinases (MMP) inhibitor, has already undergone phase I clinical trials in patients with refractory metastatic cancer[121] and AIDS-related Kaposi’s sarcoma[122] A random phase II trial reported that COL-3 administered orally once daily, is reasonably well tolerated and demonstrates antitumor activity in patients with AIDS-related Kaposi’s saroma[137] Phase III clinical trials of MMP inhibitors (MMPIs) initiated in 1997-98 using marimastat (AG3340), and BAY 12-9566 in patients with advanced cancers such as lung, brain,

GI tract and prostate cancer [102, 138, 139] However, results of the phase III trials revealed a lack of efficacy of MMPIs in these patients Moreover, a recent study showed that BMS-275291 failed to improve patients’ outcome when added to systemic chemotherapy in advanced NSCLC (Non–Small-Cell Lung Cancer)[140] Nevertheless, the concept of MMPIs is too appealing to completely reject their development MMP expression is closely related to tumor development in patients Serum MMP-2 levels were increased in patients with prostate cancer compared with healthy patients[141] It was also found that high MMP-1 expression within the colon tumor correlated with hematogenous metastasis[142] And this kind of relationship between increased MMP expression and clinical results was also found in gastric cancer[143], small cell lung cancer[144] and breast cancer[145] Although the results

of phase III trials with MMPI have been disappointing, more preclinical research could be done to have a better understanding of the role of MMPs in tumor

progression so that more insight in the development of drugs could be provided for future clinical trials

In breast cancer, estrogen deprivation is the primary mechanism of action of hormonal therapies[119] Tamoxifen is one of the most widely used hormonal drugs Combining COL-3 and tamoxifen in the breast cancer treatment would be compelling, since both COL-3 and tamoxifen have been shown to have antitumor activity as single agents but have different mechanisms of action It is possible that their different mechanisms of action would be complementary, with COL-3 inhibits the MMP in the process of tumor progression and tamoxifen function effectively competes with estrogen for the estrogen receptor So far no clinical or preclinical data have been reported about this combination regimen We therefore propose the combination therapy of tamoxifen and COL-3 and perform a preclinical study using animal model

to provide more information for the future clinical study The main purpose of this investigation was to examine the influence of tamoxifen on the pharmacokinetics of COL-3 administered orally/intravenously to rats

4.2 MATERIALS AND METHODS

4.2.1 Chemicals and Reagents

COL-3 was a gift from CollaGenex Pharmaceuticals, Inc (Newtown, PA, USA) Tamoxifen and its metabolite (4-OH-tamoxifen) were purchased from Sigma (St Louis, MO, USA)

Carboxylmethyl cellulose sodium (CMC) was purchased from Sigma Chemical Co (Louis, MO, USA) All other chemicals and solvents used in this study were of analytical grade or high performance liquid chromatography (HPLC) quality

COL-3 suspension was freshly prepared by suspending COL-3 in 2% (w/v) CMC to obtain COL-3 concentration of 5 mg/mL prior to its oral administration to the rats A COL-3 solution was freshly prepared prior to its intravenous injection to the rats: 15

mg of COL-3 was dissolved in 2.4 mL of PEG-400, followed by mixing with 3.6 mL

of phosphate buffer (0.1 M, pH 7.6) A crystalline yellow solution with COL-3 concentration of 2.5 mg/mL was obtained

Tamoxifen, supplied as tamoxifen citrate (Sigma, USA), was suspended in 1% CMC

to a concentration of 0.5 mg/ml for low-dose-tamoxifen group and 5mg/ml for dose-tamoxifen group

high-4.2.2 Animals

Female Sprague-Dawley rats (200-250 g) were obtained from Laboratory Animal Center (National University of Singapore, Singapore), and housed separately in metabolic cages in temperature-controlled room (25 ± 10ºC) with a 12-h light-dark cycle The animals had free access to food (standard mouse pellets) and water ad libitum during the experiment, unless stated otherwise Potential pharmacokinetic interaction between COL-3 and tamoxifen was examined in two separate experiments:

I and II In experiment I, oral administration of COL-3 (20mg/kg) was combined with oral administration of high-dose (30mg/kg) or low-dose (3 mg/kg) of tamoxifen Rats

were randomly divided into 5 groups with 5 rats each group (Table 4.1)

Table 4.1 Rats under different dosing regimen (COL-3 given orally)

Drug administration Fasted / Fed

Group 3 COL-3 and high-dose tamoxifen fasted

Group 4 COL-3 and high-dose tamoxifen fed

Group 5 COL-3 and low-dose tamoxifen fed

In experiment II, intravenous injection of COL-3 was given via tail vein while tamoxifen was given orally in high-dose (30mg/kg) They were randomly assigned into 4 groups with 3 rats each group (Table 4.2)

Table 4.2 Rats under different dosing regimen (COL-3 given intravenously)

Drug administration Fasted / Fed

Group 3 COL-3 and high-dose tamoxifen fasted

Group 4 COL-3 and high-dose tamoxifen fed

For the rats in the fasted groups, they were fasted overnight prior to drug administration and allowed access to the standard pellet food and water ad libitum 1 h postdosing

At the end of experiment the rats were given euthanasia in a CO2 chamber The research adhered to the principles of laboratory animal care (NIH publication #85-23, revised 1985)

4.2.3 Sample Collection

To analyze COL-3 concentration in rat serum, blood samples were taken from rats’ tail veins, prior to and at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 30, 36, 48 and 60 hours after

COL-3 administration About 200 μl of blood obtained without anticoagulation was centrifuged (10 min at 1000g, at 100C) to separate serum fraction Serum samples obtained were then stored at –200C until analyzed

For analysis of tamoxifen level in rat serum, blood samples were taken every 12 hours after tamoxifen administration

Urine and feces were collected up to 72 h after COL-3 administration, and stored at –

200C until analysis

4.2.4 Determination of Tamoxifen and Its Major Metabolite in Rat Serum

A reversed-phase high-performance liquid chromatographic method was used to determine the concentration of tamoxifen and its major metabolite, 4-OH-tamoxifen,

in rat serum as described in Chapter 3, 3.1 High-performance liquid chromatographic method for the determination of tamoxifen in biological samples

4.2.5 Determination of COL-3 in Rat Serum, Urine and Feces

A reversed-phase high-performance liquid chromatographic method[136] was used to

determine COL-3 concentration in rat serum, urine and feces as described in Chapter

3, 3.2 High-performance liquid chromatographic method for the determination of COL-3 in biological samples

4.2.6 Pharmacokinetic Calculation

Pharmacokinetic analyses were performed using the WinNonlin iterative curve-fitting program (Standard edition, 5.0, Scientific Consulting Inc., Lexington, KY, USA) based on nonlinear regression analysis

The single-dose intravenous and oral pharmacokinetics of COL-3 was analyzed using model-independent noncompartmental analysis The peak serum concentration(s) (Cmax1, Cmax2) and the time(s) of occurrence for peak concentration (Tmax1, Tmax2) after oral administration were obtained by visual inspection of the serum concentration-time curve The initial concentration (C0) after intravenous injection was determined

by extrapolating the curve to t = 0 obtained through linear regression on the logarithmic transformation using the first several data points The area under the serum concentration-time curve (AUC0-last) was calculated using the mixed linear (for prepeak area) and the log-linear (for postpeak area) trapezoidal rule and the log-linear trapezoidal rule for the oral and intravenous data of COL-3, respectively The total area under the serum concentration-time curve from time zero to infinity (AUC0-∞) as well as the area under the first moment curve (AUMC0-t) from time zero to the last measurable time (tz) point was calculated as the sum of AUC0-t and the extrapolated area, which was estimated as the last measurable serum concentration (Cz) divided by the terminal rate constant (λz), where λz was estimated using the terminal log-linear phase of the serum concentration-time curve The area under the first moment curve from time zero to infinity (AUMC0-∞) was calculated as the sum of AUMC0-t and the

extrapolated area, which was estimated as (tz×Cz/λz + Cz/λz2) Terminal serum life (t1/2, λz) was calculated as 0.693/λz The serum clearance for intravenous dose (CL)

half-or the apparent serum clearance fhalf-or half-oral dose (CL/F) was calculated as dose/AUC0-∞ The mean residence time for intravenous dose (MRTiv) and oral dose (MRToral) was determined as AUMC0-∞/AUC0-∞ The volume of distribution during the terminal phase (Vz ) was estimated as CL/λz The volume of distribution at steadystate (Vss) was

estimated as CL×MRT

4.2.7 Statistical Analyses

Statistical analyses were performed using SPSS 13.0 (SPSS Inc., USA) Data were expressed as mean ± standard deviation (SD) Comparisons of means for each of the pharmacokinetic parameters among all the groups were performed using one-way analysis of variance (ANOVA) A P-value of less than 0.05 was considered as statistically significant

4.3 RESULTS

4.3.1 Gastrointestinal Absorption of COL-3

An erratic or irregular absorption profile of COL-3 with either a double- or plateau- peak concentration was observed in each of the rats studied, which was consistent with the previous study [146] The first peak was achieved at 2-8 h, while the second one occurred at 6-12 h after COL-3 administration The concentration of the two peaks had a similar mean value (Table 4.3)

4.3.1.1 Effect of Food on the Serum Concentration-time Profile of COL-3

When COL-3 was administered to the rats alone, the mean COL-3 serum concentrations in the fed rats (Experiment I, Group2) tended to be higher than those in the fasted rats (Experiment II, Group1) at all time points, especially during the elimination phase (Figure 4.1), although the difference was not statistical significant (p > 0.05)

COL-3 alone,with food

COL-3 and high dose tamoxifen,without food

COL-3 and high-dose tamoxifen, with food

COL-3 and low-dose tamoxifen,withfood

Figure 4.1 The mean serum concentration-time profiles of 3 following: 1)

COL-3 given alone to fasted rats; 2) COL-COL-3 given alone to fed rats; COL-3) COL-COL-3 given with high-dose tamoxifen to fasted rats; 4) COL-3 given with high-dose tamoxifen in fed rats; and 5) COL-3 given alone with low-dose tamoxifen in fed rats COL-3 was given

at dose of 20 mg/kg for oral administration Tamoxifen was given at 3 mg/kg as dose and 30 mg/kg as high-dose Each data point represents the mean (+SD) of 5 rats

low-4.3.1.2 The Effect of Tamoxifen on COL-3 pharmacokinetics and Disposition in Vivo

COL-3 pharmacokinetic profile was not significantly altered by low-dose tamoxifen when fed rats were given COL-3 and low-dose tamoxifen concurrently COL-3 was eliminated slightly slower when it was administered with low-dose tamoxifen (p>0.05) (Table 4.3, Figure 4.1) There were no significant differences in the serum concentrations of COL-3 at all sampling time points between the two groups (Experiment I, Group2 and Group5) (p>0.05)

When tamoxifen was given as a high dose, the extent of oral absorption of COL-3 was not affected by tamoxifen when the rats were fasted overnight before COL-3 administration And the elimination phase of COL-3 serum concentration-time profile was also slightly altered by tamoxifen in the same way as in the low-dose-tamoxifen group (p>0.05) (Table 4.3, Figure 4.1)

However, when the rats were not fasted overnight before COL-3 administration, COL-3 pharmacokinetic profile was markedly altered by high-dose tamoxifen (Figure 4.1)

We compared: a) 1 COL-3 alone, fed and COL-3 with high-dose tamoxifen, fed

rats group:

High-dose tamoxifen didn’t alter the extent of absorption of COL-3’s PK profile of fed rats, since Cmax1 and Cmax2 has no significant difference compared to the control

group when COL-3 was given alone to the fed rats (p>0.05) However, high-dose tamoxifen markedly reduced the AUC0-∞ and AUClast of COL-3 while the mean CL/F was increased in the combination group ( p< 0.05) Terminal half-life, λz, Vz/F and Tmax wasn’t changed by high-dose tamoxifen in fed rats Nevertheless, the obvious reduction of AUC of COL-3 in the fed rats group of high-dose tamoxifen was related

to a trend of increasing clearance (Table 4.3)

b) COL-3 with high-dose tamoxifen, fasted and COL-3 with high-dose tamoxifen, fed rats group:

When COL-3 and high-dose tamoxifen both given to the rats, compared the fasted and fed rats group, no apparent difference was found in PK parameters except for CL/F and λz were increased when COL-3 was coadministered with tamoxifen to fed rats (p < 0.05)

In summary, the pretreatment of fed rats with high-dose tamoxifen for 3 days resulted

in a notable decrease in area under plasma concentration-time curve of COL-3, compared to other 4 groups So we attributed this interesting observation to some interaction within high-dose tamoxifen, COL-3 and food, most possibly is due to some change of the physiological environment More work will be done to confirm this hypothesis

The renal route of elimination was negligible in the overall excretion of COL-3 Tamoxifen had no apparent influence on total COL-3 (unchanged drug and its glucuronide conjugate) urinary excretion following oral administration of COL-3 When rats were fasted overnight, unchanged COL-3 urinary excretion was not

affected by high-dose tamoxifen (p > 0.05) However, for those fed rats, unchanged COL-3 recovered in rat urine after its oral administration was significantly decreased

in both low-dose and high-dose tamoxifen combination groups compared to control group (COL-3 alone, fed rats) (p < 0.05) And when rats were both given COL-3 and high-dose tamoxifen, unchanged COL-3 urinary recovery is lower in the fed rats than that of fasted ones (p < 0.05)

COL-3, when given as single oral dose, exhibited a significant fecal excretion with 48.1 ± 17.8 and 26.0 ± 16.8 of unchanged drug recovered in rat feces over 0-48 h in fasted and fed rats groups, respectively Coadministration of low-dose/high-dose of tamoxifen didn’t significantly alter the COL-3 fecal excretion in fasted/fed rats (Table 4.4)

Table 4.3 Pharmacokinetic parameters of COL-3 following its single oral dose (20

mg/kg) when given alone and in combination with tamoxifen (3mg/kg daily as

low-dose and 30 mg/kg daily as high-low-dose) in female rats

COL-3 alone COL-3 alone COL-3 and high-dose tamoxifen COL-3 and high-dose tamoxifen COL-3 and low-dose tamoxifen

Values are given as the mean ± SD of 5 rats

Table 4.4 Urinary and fecal recovery of COL-3, when it was given orally alone (20

mg/kg) and in combination with tamoxifen (3 mg/kg daily as low-dose and 30 mg/kg

daily as high-dose) in female rats, within 48 h postdose

COL-3 alone COL-3 alone COL-3 and high-dose tamoxifen

COL-3 and high-dose tamoxifen COL-3 and low-dose tamoxifen

Urine collected within 60h (ml) 16.4 ± 4.3 11.3 ± 3.1 14.5 ± 5.0 10.6 ± 5.2 10.0 ± 6.9

(12.2-23.2) (6.5-14.2) (10.4-21.9) (5.2-18.8) (7.0-22.1) Urinary recovery of unchanged 0.064±0.012 0.055±0.017 0.053±0.010 0.032±0.009 0.038±0.015

COL-3 (% dose) (0.054-0.085) (0.034-0.080) (0.041-0.067) (0.019-0.042) (0.020-0.059)

Urinary recovery of the total 0.396±0.190 0.453±0.196 0.437±0.077 0.481±0.058 0.358±0.158

of COL-3 and its glucuronide (0.223-0.694) (0.263-0.761) (0.330-0.545) (0.428-0.568) (0.262-0.638)

conjugate (%dose)

Feces collected within 48 h (g) 7.6 ± 1.2 4.4 ± 2.1 3.1 ± 1.8 5.9 ± 2.0 3.0 ± 1.2

(5.9-8.9) (2.0-6.8) (0.9-6.0) (4.3-9.2) (1.7-4.9)

Fecal recovery of 48.1 ± 17.8 26.0 ± 16.8 37.1 ± 13.5 48.0 ± 22.2 27.1 ± 17.7

unchanged COL-3 (%dose) (24.8-73.7) (9.4-48.8) (16.8-48.3) (24.9-77.8) (7.8-54.7)

Values are given as the mean ± SD (range) of 5 rats