WO1998013039A1 - Dfmo for the treatment or prevention of cervical intraepithelial neoplasia - Google Patents

Abstract

Methods for treating, preventing, controlling the growth of and/or reducing the risk of developing cervical cancer, particularly in patients with cervical intraepithelial neoplasia are provided employing pharmaceutically acceptable preparations of DFMO. Methods for treating a patient having cervical intraepithelial neoplasia, which methods comprise administering DFMO alone or in combination with a cytotoxic or cytostatic agent, are also provided.

Description

DFMO FOR THE TREATMENT OR PREVENTION OF CERVICAL INTRAEPITHELIAL NEOPLASIA

FIELD OF THE INVENTION

The present invention provides a pharmaceutical formulation and a method for its use in the prevention of cervical cancer. More specifically, the present invention provides a composition comprising alpha-difluoromethylornithine for treating, preventing, reducing the risk of and/or controlling the growth and progression of cervical intraepithelial neoplasia grade III into malignancy.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE PRIOR ART

Despite the advent of the Papanicolaou (Pap) smear, cervical cancers and pre-cancers remain important health problems for women, especially underobserved women in the United States (U.S.) and women in underdeveloped countries (Parkin. 1993). The incidence of both invasive cervical cancer and carcinoma-in-situ are increasing in the U.S. The reasons for this increase are unknown. In the U.S.. an estimated 2,500.000 women will have abnormal Pap smears demonstrating atypical cells of uncertain significance and low-grade intraepithelial lesions (lesions of HPV and CIN 1) annually (Kurman. 1994). The exact number of patients with high-grade squamous intraepithelial lesions (CIN II and III), not classified as carcinoma-in-situ, is unknown. A risk factor for cervical cancer is HPV. The most common types of HPV are those classified as high risk (HPV 16, 18, 45, and 56), intermediate risk (HPV 31. 33. 35, 51. 52, and 58), and low risk (HPV 6, 11). The high and intermediate risk types have been identified in 77% of high grade cervical intraepithelial neoplasia (CIN) and squamous intraepithelial lesions (SIL) and in 84% of invasive lesions. Cohort studies demonstrate that women with HPV infection have 1 1 -60 times increased risk of developing high grade CIN and 15-50 times increased risk of developing invasive cancer than do women without HPV infection for which the high-risk independent of the HPV assay method employed or epidemiologic study design (Bosch, 1995). Chemoprevention is the use of chemical agents (micro-nutrients, pharmaceuticals) to prevent or delay the development of cancer in healthy populations (Kelloff, 1994; Daly, 1993). These agents, which block the initiating and promoting events of carcinogenesis. augment the preventive strategy, which includes the avoidance of carcinogens in the environment (referred to as primary prevention) and participation in screening programs (referred to as secondary prevention), hence serving as a tertiary preventative measure. Because most current chemopreventive agents can cause side effects, they are used in individuals who have higher risk of developing cancer (for example, those with premalignant lesions). Intervention in the precancerous stage may prevent a lesion from becoming invasive (Sporn, 1993). The advantage of chemoprevention in the treatment of a pre-neoplastic condition is that the effects of chemoprevention are systemic, thus pre-neoplastic cells in all areas of the body are treated. Chemoprevention studies use surrogate endpoint biomarkers (SEB's) as intermediate measures of cancer development. These markers should be differentially expressed in normal and high-risk tissue, be measured with acceptable sensitivity and specificity, and be modulated by the chemopreventive under study. SEB's provide a glimpse of cancer biology and its modulation (Boone, 1990). There are several reasons why chemoprevention is attractive for cervical lesions. These reasons reflect the belief that pre-cancers, like cancers, represent a systemic process. Many colposcopy patients smoke, and many of these patients also have preneoplastic and neoplastic lesions of the aerodigestive tract. Infection with HPV affects the entire squamous epithelium of the female genital tract, and up to 40% of patients with CIN have multifocal lesions of the vagina, vulva, and perianal area. Polyamines (putrescine, spermidine, and spermine) and their precursors (arginine and omithine) are thought to play critical roles in cellular maintenance, proliferation, differentiation, and transformation; thus polyamines might be considered surrogate endpoint biomarkers of carcinogenesis. Polyamines are differentially expressed in normal and high-risk tissue; measured with acceptable sensitivity and specificity; and can be modulated by DFMO. Polyamines and their precursors can be measured in tissue, red blood cells, plasma, and urine.

Omithine Decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is considered a proto-oncogene that is crucial for the regulation of cellular growth and transformation and is a potent anti-proliferative chemopreventive agent and has been studied in other organ sites but has not previously been studied in the cervix (Verma, 1987; Auvinen, 1992). ODC catalyzes the first step in the biosynthesis of putrescine (a diamine), spermidine and spermine. the three major polyamines of mammalian cells. In vitro studies show that polyamines participate in nearly all aspects of DNA, RNA, and protein synthesis. Polyamine accumulation is required to maintain maximum rates of cell proliferation. Blockage of polyamine accumulation by administration of DFMO and other inhibitors during accelerated cell growth results in a significant reduction of growth in a variety of cell systems. Since the only pathway to polyamine synthesis is via omithine, synthesis depends on the activity of

ODC. ODC is present in very small amounts in resting cells but can be increased many-fold within a few hours of exposure to hormones, drugs, and growth factors. Blocking endogenous ODC has reportedly prevented transformation of rat fibroblasts by the temperature-sensitive v-src oncogene. Tumor formation in experimental animals has reportedly been prevented by inhibitors of ODC such as DFMO. DFMO has reportedly been shown to inhibit cellular replication in vitro in several malignant animal tumor cell lines, including L 1210 and L5178 Y leukemia, rat hepatoma, mouse mammary sarcoma (EMT6) and hamster pancreatic adenocarcinoma (H2T) (Mamont et al., 1978; Prakash et al., 1980; Marx et al., 1987). DFMO, either alone or in combination with other agents, has proven effective in treating and/or preventing mammary carcinomas in animal models as follows. The growth of six human tumors (three m'amrnary carcinomas, a malignant melanoma, a bladder carcinoma, and an endocervical carcinoma) was significantly decreased after DFMO treatment compared to growth in control mice. (Luk et al., 1983). In xenographs of human breast and colon carcinoma cells inoculated into nude mice, a synergistic antitumor effect was observed when

DFMO was combined with mitomycin D (Takami et al., 1989).

Studies by Nishioka et al. report increases of plasma precursor amino acids of polyamines, such as arginine and omithine, at low doses of DFMO in tissue obtained by routine cervical biopsy. DFMO was also measured as a compliance marker. Nishioka et al. proposed that polyamines and their precursor amino acids would be effective markers in analyzing the effects of DFMO, functioning as pharmacodynamic parameters, as well as biomarkers for transformation in the cervix. have been reported; most of them being considered as negative trials. A trial by Meyskens reports histological regression of CIN II cases, but not CIN III, using topical trans-retinoic acid. The rate of regression in the treated CIN II group was reported to be 43%, compared to 27% in the placebo group.

Determining the optimal dose of DFMO could be based on several parameters, including toxicity, modulation of polyamine synthesis markers, and/or response. In the Phase I study of Love, a dose of 0.5 g/m²/day was used, being based on toxicity (Love, 1993). Studies employing modulation of polyamine synthesis markers to determine optional dose of DFMO, specifically urine polyamine markers putrescine and spermidine at a DFMO dose level of 0.2 g/m²/day, have been reported in Meyskens, 1994 and Hixson, 1993. Employing polyamine synthesis markers, Meyskens, (1994) reports modulation of tissue spermidine/spermine ratio at a dose of 0.1 g/m²/day. A need continues to exist in the medical arts for methods of detecting, inhibiting and reducing the risk of developing pre-malignant tissues linked to cervical malignancies.

SUMMARY OF THE INVENTION

The present invention provides methods particularly useful in the treatment of patients with malignant cervical cancer or patients at risk of developing malignant cervical cancer. In particular applications, these methods may be employed to inhibit the progression of CIN I,

II or III to cervical cancer in a patient identified as having evidence of CIN I, II or III.

In some embodiments, the method for treating, reducing the risk, and/or inhibiting malignant cervical cancer growth and/or progression of pre-malignant tissues to a malignancy, such as a patient having evidence of cervical intraepithelial neoplasia, particularly CIN III, comprises administering to a patient in need thereof a pharmaceutically acceptable preparation comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier.

It is contemplated and within the scope of the invention that a composition as described above will be useful for the inhibition of malignant cellular proliferation and/or of CIN I, II or III progression to cervical cancer. HPV patient infection, and multifocal intraepithelial neoplasia. Hence, it is contemplated t at the present methods employing DFMO can be a treatment of particular application in women who smoke, have HPV infection, or have multifocal intraepithelial neoplasia of the cervix, vagina, or vulva. Another group for which chemoprevention with DFMO can be a choice is in women who are immunosuppressed due to HIV infection, rheumatologic disease, renal failure, or who use immunosuppressive medications, as well as in patients in whom surgical procedures are poorly tolerated. Another aspect of the present invention provides a method of preventing cervical cancer and/or pre-malignant tissue development in a patient comprising administering to a patient a pharmaceutically acceptable preparation comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier. It is contemplated that DFMO treatment of a patient having CIN III will inhibit cellular transformation against field cancerization and also remove cancerous cells, already transformed, through apoptosis. Thus, another aspect of the present invention provides a method of preventing and reducing the risk of cervical cancer in a patient comprising administering to a patient a pharmaceutical preparation comprising a pharmacologically active amount of DFMO or pharmaceutically acceptable salts thereof in a pharmaceutically acceptable carrier; and controlling the growth of cervical intraepithelial neoplasia cells. The present methods in some applications may be employed as part of an overall cancer treatment regimen for the patient that include a combination therapy with DFMO together with standard regimens. It- is expected that such will further enhance the effectiveness of the method of the invention. Standard treatment regimens for patients with cervical cancer and cervical intraepithelial neoplasia are well known to those in the medical arts of oncology, and are described in Obstet Gynechological Clin North American, 23:347-410, 1996. This reference, in so far as it provides general guidance of such routine techniques, are incorporated herein by reference for this purpose.

As used herein, a pharmacologically active amount of DFMO in the pharmaceutical preparation is defined as an amount of DFMO between about 0.01% to about 90% by weight of the preparation administered to the patient. By way of further example, a pharmacologically active amount of DFMO may be an amount that constitutes between about In some embodiments, the preparation is a liquid solution taken orally by the patient. The present invention has observed a correlation exists between the positive identification of cervical intraepithelial neoplasia (CIN) and particularly grade III (a pre-cancerous state), and the incidence of cervical cancer. The present methods therefore also advantageously employ compositions of DFMO in methods of reducing the risk of developing cervical cancer in a patient having cervical intraepithelial neoplasia comprising administering a pharmaceutical preparation comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof. In yet another aspect, the invention provides a method for inhibiting the growth of cervical intraepithelial neoplasia grade III in a patient. In some embodiments, the method comprises administering to a person a pharmaceutical preparation comprising a pharmacologically active amount of DFMO capable of inhibiting cervical intraepithelial neoplasia. or pharmaceutically acceptable salts of DFMO, and inhibiting intraepithelial neoplasia cell growth. In some embodiments, the method of the present invention provides for a DFMO dose of about 0.05 gm/m²/day to about 1.0 gm/m²/day, or about 0.10 gm/πr/day to about 0.95 gm/πr/day to about 0.50 gm/m²/day to about 0.95 gm/m²/day. The methods of the present invention also provide for the administration of a therapeutically effective amount of DFMO to be defined by reference to the plasma level of DFMO to be achieved in the patient being treated. Variations on these plasma DFMO levels as part of the presently described methods are included within the scope of the invention. Such variations may be indicated in the particular patient being-treated, as determined by the attending physician or other health care professional. The oral dosage forms may be tailored to achieve a plasma level of DFMO that provides therapeutic benefit to the patient with reasonable or tolerable toxicity to the patient. Oral dosage forms that have a reasonable therapeutic index may be further defined as between about 0.020 to about 10 g/m²/day, or of about 0.030 to about 5 g/m²/day, or between about 0.30 to about 1 g/m²/day, or about 0.03 g/m²/day to about 0.50 g/m²/day, or from about 0.03 g/m²/day to about 0.125 g/m²/day, or about 0.05 g/m²/day to about 1.0 g/m²/day, or about 0.06 to about 0.125 g/πr/day. Other dosage levels may be described as about 0.06 g/rrr/day to about 5 g/m²/day. A dose regimen of about 10 g/πr/day is yet another treatment protocol that may be used as part of the present methods. Alternatively, the patient may be provided a used at the beginning of the treatment, with the dose being adjusted upward or downward, depending on response of the patient and/or the plasma level of DFMO observed in the patient. An intravenous dose of DFMO, up to about 10 mg/kg (55 μmol/kg), may be employed in particular applications of the present methods. It is anticipated that the present preparations may also be administered orally, parenterally, or other route of administration or combination thereof, as part of the present methods.

As used herein, the terms "cervical intraepithelial neoplasia grade III" and "CIN III" refer to a premalignant state of disease wherein there is a spectrum of intraepithelial changes. These changes begin with a generally well-differentiated intraepithelial lesion, referred to as a low grade squamous intraepithelial lesion, and end with full thickness epithelial changes, previously referred to as carcinoma-in-situ, and currently referred to as high grade squamous intraepithelial lesion. The terms "high grade cervical intraepithelial neoplasia" and "cervical intraepithelial neoplasia grade III" and "CIN III" are used interchangeably herein. As used herein, the term "adsorbent" is intended to mean an agent capable of holding other molecules onto its surface by physical or chemical (chemisorption) means. Such compounds include, by way of example and without limitation, powdered and activated charcoal and the like. As used herein, the term "antioxidant" is intended to mean an agent which inhibits oxidation and thus is used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate. butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and the like. As used herein, the term "buffering agent" is intended to mean a compound used to resist change in pH upon dilution or addition of acid or alkali. Such compounds include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and the like. As used herein, the term "colorant" is intended to mean a compound used to impart color to liquid and solid (e.g., tablets and capsules) pharmaceutical preparations. Such compounds include, by way of example and without limitation, FD&C Red No. 3, FD&C Red No. 20, No. 8, caramel, and ferric oxide, red and the like.

As used herein, the term "flavorant" is intended to mean a compound used to impart a pleasant flavor and often odor to a pharmaceutical preparation. In addition to the natural flavorants, many synthetic flavorants are also used. Such compounds include, by way of example and without limitation, anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin and the like.

As used herein, the term "sweetening agent" is intended to mean a compound used to impart sweetness to a preparation. Such compounds include, by way of example and without limitation, aspartame, dextrose, glycerin, mannitol, saccharin sodium, sorbitol and sucrose and the like.

As used herein, the term "tablet antiadherents" is intended to mean agents which prevent the sticking of table formulation ingredients to punches and dies in a tableting machine during production. Such compounds include, by way of example and without limitation, magnesium stearate, talc, and the like.

As used herein, the term "tablet binders" is intended to mean substances used to cause adhesion of powder particles in table granulations. Such compounds include, by way of example and without limitation, acacia, alginic acid, carboxymethyl cellulose, sodium, compressible sugar ethylcellulose, gelatin, liquid glucose, methylcellulose, povidone and pregelatinized starch and the like.

As used herein, the term "tablet and capsule diluent" is intended to mean inert substances used as fillers to create the desired bulk, flow properties, and compression characteristics in the preparation of tablets and capsules. Such compounds include, by way of example and without limitation, dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sorbitol, and starch and the like. As used herein, the term "tablet direct compression excipient" is intended to mean a compound used in direct compression tablet formulations. Such compounds include, by way of example and without limitation, dibasic calcium phosphate (e.g., Ditab™) and the like. As used herein, the term "tablet disintegrant" is intended to mean a compound used in solid dosage forms to promote the disruption of the solid mass into smaller particles which are more readily dispersed or dissolved. Such compounds include, by way of example and without AVICEL®), polacrilin potassium (e.g., AMBERLITE®). sodium alginate. sodium starch glycolate, and starch and the like.

As used herein, the term "tablet glidant" is intended to mean agents used in tablet and capsule formulations to reduce friction during tablet compression. Such compounds include, by way of example and without limitation, colloidal silica, cornstarch. talc, and the like. As used herein, the term "tablet lubricant" is intended to mean substances used in tablet formulations to reduce friction during tablet compression. Such compounds include, by way of example and without limitation, calcium stearate, magnesium stearate, mineral oil, stearic acid, zinc stearate, and the like.

As used herein, the term "tablet/capsule opaquant" is intended to mean a compound used to render a capsule or a tablet coating opaque. An opaquant may be used alone or in combination with a colorant. Such compounds include, by way of example and without limitation, titanium dioxide and the like. As used herein, the term "tablet polishing agent" is intended to mean a compound used to impart an attractive sheen to coated tablets. Such compounds include, by way of example and without limitation, camauba wax, white wax, and the like.

It should be understood, that compounds used in the art of pharmaceutical formulation generally serve a variety of functions or purposes. Thus, if a compound named herein is mentioned only once or is used to define more than one term herein, its purpose or function should not be construed as being limited solely to that (those) named purpose(s) or function(s). As used herein, the- term DFMO is intended to mean a preparation of alpha- difiuoromethylornithine in its pharmaceutically acceptable salt and/or racemic isomeric preparations of (D) and (b) DFMO. (+)-DFMO is intended to mean alpha- difluoromethylornithine having the (D)- configuration around the alpha-carbon which is the only chiral atom present in the molecule. (-)-DFMO is intended to mean alpha- difluoromethylornithine having the (L)- configuration around the alpha-carbon. (+/-)-DFMO is intended to mean racemic alpha-difluoromethylornithine. Methods for the preparation of (+)-DFMO and (-)-DFMO are known. U.S. 4,330,559, the disclosure of which is hereby incorporated by reference in its entirety, discloses a method for the preparation of optically pure DFMO wherein racemic DFMO dihydrochloride is reacted with sodium methylate to form 3-amino-3-difIuorornethyl-2-piperidone (DFMO-pip) which yield (-)-DFMO-pip:(-)-BNPA 1 : 1 addition salt crystals leaving the (+)-DFMO-pip:(-)-BNPA addition salt in solution. Following repeated recrystallization and acidification, (-)-DFMO-pip is obtained in optically pure form. The (-)-DFMO-pip is then hydrolyzed to yield (+)-DFMO. The enantiopode (+)-DFMO may be prepared according to the above procedure by employing

(+)-BNPA to preferentially form the diastereomeric (+)-DFMO-pip:(+)-BNPA 1 :1 addition salt crystals.

Wagner, et al. (1987), the disclosure of which is hereby incorporated by reference in its entirety, discloses a reverse phase liquid chromatographic method for the resolution of racemic DFMO to yield each enantiomer of DFMO in optically pure form.

Aldous et al. (1986) discloses a gas chromatographic analytical method for the resolution of racemic DFMO to yield each enantiomer of DFMO in optically pure form.

The compounds herein described may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of olefins,

C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention unless the specific stereochemistry or isomer form is specifically indicated. It will be appreciated that certain compounds of the present invention contain an asymmetrically substituted carbon atom, and may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis, from optically active starting materials. Also, it is realized that cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. Other features, advantages and embodiments of the invention will be apparent to those skilled in the art from the following description, accompanying data and appended claims. Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this specification. The following abbreviations are used herein and are defined as follows: DFMO alpha-difluoromethylornithine

ODC omithine decarboxylase RA retinoic acid IGF-1 insulin-like growth factor 1

TGF-beta transforming growth factor beta

MX methotrexate FU 5-fluorouracil

TC tamoxifen citrate

TAM tamoxifen

DX doxorubicin

VS vincristine sulfate MNU 1 -methyl- 1 -nitrosourea

E2 estradiol

CIN III cervical intraepithelial neoplasia grade III

TGI tumor growth inhibition

DFMO-pip 3-amino-3-difluoromethyl-2-pipenidone BNPA binaphthyl phosphoric acid

MTD maximum tolerated dose

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 - Polyamine Synthesis Flow Chart.

FIG. 2 - Effects of DFMO on Polyamine Synthesis

FIG. 3A-FIG 3B - FIGURE 3A shows proportionate change and 95% confidence intervals in

SPD:SPM in abnormal tissue by dose level. FIG. 3B shows proportionate change and 95% confidence intervals in arginine. FIG. 4 - Antiproliferative effects of DFMO of the indicated concentration on cells after continuous treatment for 5 days. Results are expressed as a percentage of the respective untreated controls. The points represent mean values of 8-duplicate cultures. The standard deviation did not exceed 5%.

FIG. 5 - Shows the effect of DFMO on the growth of C33A cells. C33A cells were incubated in the absence or presence of different concentrations of DFMO for the indicated times (lμM, lOμM, 100 uM, 500μM, lOOOμM, 1400 M DFMO). carcinoma cell lines. The concentrations of 4-HPR examined were l , 5 M and 10μM. (Graph legends: C33A line, -D-; C41 1 line, — 0— ; Hela line, ~o~; ME180 line, -Δ-; C41 line, --■--; Caski line, --♦--; HT3 line, --T-; MS751 line ~v~; SiHa line, --H-). FIG 7 - 5 Days Effect of DFMO on Cervical Cancer Cell Line TCL 1

FIG 8 - 5 Days Effect of 4HPR on Cervical Cancer Cell Line TCL1 FIG 9 - 5 Days Effect of DFMO & 4HPR on Cervical Cancer Cell Line TCL1 FIG 10 - 5 Days Effect of DFMO on Cervical Cancer Cell Line Z183 FIG 1 1- 5 Days Effect of 4HPR on Cervical Cancer Cell Line Z183 FIG 12 - 5 Days Effect of DFMO & 4HPR on Cervical Cancer Cell Line Z 183

FIG 13 - Colony formation Assay (MatriGel) Cervical Carcinoma cell line Z173 FIG 14 - Growth inhibition by DFMO FIG 15- Colony formation Assay Cervical Carcinoma cell line HeLa

In some embodiments, the present invention provides a pharmaceutical composition particularly designated for use in treating, preventing, and/or controlling the growth of and/or reducing the risk of cervical cancer and solid tumor. Particular embodiments of the method comprise administering a pharmacologically active amount of DFMO, or pharmaceutically acceptable salt thereof to a patient in need or at risk of developing cervical cancer. One factor that enhances risk of cervical cancer development is a positive identification of CIN III in a patient cervical sample. In some embodiments of the method, a pharmacologically active amount of DFMO is about 0.01 % to 90% by weight of the composition or between 30% to about 85%, or about 40%, 50%, 60%, 70% or 80% by weight of the solution.

Another embodiment of the invention provides a method of treating, preventing, controlling the growth of and/or reducing the risk of developing cervical cancer and tumor in a patient comprising administering therapeutically effective amounts of a combination of a cytotoxic or cytostatic agent and DFMO, or pharmaceutically acceptable salts thereof.

In particular embodiments, the invention provides a method inhibiting cervical intraepithelial neoplasia grade III in a patient comprising administering a therapeutically effective amount of DFMO or a pharmaceutically acceptable salt thereof to the patient; and inhibiting cervical intraepithelial neoplasia cell growth. Another embodiment of the invention provides a method of reducing the risk of developing cervical cancer in a patient having cervical intraepithelial neoplasia grade III comprising administering therapeutically effective amounts of DFMO. or pharmaceutically acceptable salts thereof to the patient.

Unless otherwise indicated, all chemicals were purchased from Aldrich Chemicals (Milwaukee, WI). Racemic DFMO is available from Ilex Oncology, Inc. (San Antonio, TX).

EXAMPLE 1 HPLC METHOD FOR POLYAMINE ANALYSIS IN TISSUE AND BLOOD

Tissues and blood samples were frozen at -70 °C until analyses were performed. Each sample was analyzed in duplicate and pre- and post-treatment samples were analyzed simultaneously. To prepare samples, a 25% tissue homogenate was prepare in ODC buffer using a Polytron homogenizer (Brinkman Instruments, Westbury, NY) as described previously (15). A portion of the homogenate (20μl) was mixed with 80 μl of 15% temperature to obtain a clear supernatant for polyamine analysis. The remaining portion of the homogenate was centrifuged (700 x g) for 15 minutes at 4°C and the supernatant was analyzed for ODC activity and protein levels. Protein concentrations were determined using Bio-Rad (Richmond, CA) protein assay kits. A new procedure, using 0-phthalaldehyde in a Dionex BioLC high-performance liquid chromatograph equipped with HPLC-CS2 column and postcolumn detection system (Dionex, Inc., Sunnyvale, CA) was used for determinations of arginine, omithine, DFMO, acetylpolyamine, and free polyamine levels. Four elution buffers were prepared and filtered. Each buffer contained 1 ml phenol per liter with the following compositions: buffer A, ImM potassium citrate, pH 4.70. adjusted with HC1 ; buffer B, 0.1 M potassium citrate, pH 4.70, adjusted with HC1; buffer C, 0.2 M KC1, 10 mM KOH, 1.34 mM disodium EDTA, 1 1.7 mM HBP₃, pH 9.20; and buffer D, 1.8 M KC1, 90 mM KOH, 12.1 mM EDTA, 0.105 M HB0₃, pH 9.20. The column was equilibrated with buffer A starting at 0 min, and various buffers were introduced. A sample was injected at 20 min., and recording commenced at 30 min. Samples were injected at 20 minutes and recording commenced at 30 minutes. Plasma DFMO levels are reported in pmols/ml, plasma arginine and ornithine levels in nmol/ml, tissue polyamines in nmol/1 mg soluble protein, and RBC polyamine levels in nmol/ml packed red blood cells.

EXAMPLE 2

STUDY PROTOCOL FOR EVALUATING DFMO IN TREATING CTN ITI

Prior to enrollment, all participants underwent a complete medical history, physical examination, pelvic examination, a Pap smear, gonorrhea and chlamydia cultures, HPV testing, colposcopic examination of the vulva, vagina and cervix, risk factors and dietary interview, and counseling regarding smoking cessation, nutrition, and sunscreen use. In addition, blood samples were collected for complete blood count: serum electrolytes, chemistry, and coagulation studies; serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone, and estradiol; plasma levels of DFMO, omithine, and arginine; and red blood cell (RBC) putrescine, spermidine, and spermine.

Colposcopically directed-biopsies from normal and abnormal areas were taken for permanent section and snap-frozen for studies of polyamine synthesis including omithine decarboxylase (ODC), putrescine, spermine, and spermidine. HPV testing was performed by negative specimens were subjected to polymerase chain reaction analysis.

Patients were identified among women attending the Colposcopy Clinic of the University of Texas M. D. Anderson Cancer Center, Department of Gynecologic Oncology. Eligible patients included the following: nonpregnant women aged 18 years and older with a biopsy-confirmed diagnosis of CIN III; a lesion involving one-third the surface area of the cervix; no history of prior malignancy; a Zubrod performance status of less than or equal to 2; and normal serum chemistries, hematologic profiles and audiometry. CIN III is a precursor of invasive cervical cancer. Women were extensively counseled about contraception and were selected based on the use of hormonal contraceptive methods, having had a bilateral tubal ligation, or being postmenopausal.

Patients were assigned to each of five decreasing doses of DFMO: 1.0, 0.5, 0.25, 0.125, and 0.06 g/πr/day for 31 days. DFMO was provided by the National Cancer Institute (NCI) in elixir form in 200 mg ml vials. Patients were provided with a 5 cc syringe to assure precise dosage.

Post-treatment evaluation included the following: colposcopically directed biopsies from normal and abnormal areas for polyamine synthesis analysis, colposcopically directed loop electrosurgical excision (LEEP) of the cervix for complete histological study and definitive treatment. Blood samples were collected for determination of post-treatment plasma levels of DFMO, ODC, putrescine, spermidine, spermine, omithine and arginine and RBC levels of putrescine, spermidine, and spermine. All histological specimens were evaluated and responses were determined by histological regression of the lesion.

Baseline polyamine values between colposcopically normal and abnormal tissue areas were compared among all patients. Post-treatment DFMO values in plasma are compared by dose level. The effect of DFMO treatment on polyamine values was assessed by the differences between baseline and post-treatment polyamine values at each DFMO dose level in biopsy specimens from colposcopically abnormal tissue areas, in plasma, and in RBC. In addition, proportionate changes (and 95% confidence intervals) in polyamine tissue, plasma, and RBC, were estimated as the change due to DFMO treatment [(polyamine value post-DFMO - polyamine value at baseline) divided by polyamine value at baseline]. Because of the large variability in all polyamine measurements, data were analyzed by the Wilcoxon's matched-pairs signed-rank test. Statistical significance was set at an alpha of 0.05 based on two-sided test. PREPARATION OF SOLUTIONS CONTAINING DFMO FOR TREATMENT OF CIN III

The present example details the DFMO preparation that was used in the study of CIN

III in humans.

As will be appreciated by those of skill in the art, the preparations may be formulated according to techniques well known to those of skill in the art (Remingtons Pharmaceutical Sciences, 18 edition). A DFMO elixir (200 mg/ml) may be prepared at a concentration and provided to a patient so as to achieve a dose of between about 0.060 and about 0.125 g/m²/day for a treatment period of at least 30 days. The solution was prepared by the National Cancer Institute in elixir form and shipped to MD Anderson Cancer Center for the present studies.

EXAMPLE 4

DFMO DOSE LEVELS AND CERVICAL INTRA EPITHELIAL NEOPLASIA

Women suspected of having CIN I to CIN III were biopsied colposcopically in both normal and abnormal regions of the cervix to determine the severity or extent of disease progression (See Example 2). Thirty patients with biopsy-proven CIN III underwent a Papanicolaou (PAP) smear and colposcopically directed biopsies, using 3-6% acetic acid, for histology and for polyamine synthesis biomarkers. Patients also underwent complete medical history, nutritional survey, sexual behavior interview, physical exam, colposcopy, HPV testing, blood counts, serum chemistries, audiogram; plasma DMSO, omithine and arginine measurements, erythrocyte polyamine measurement, tissue DFMO, ODC and polyamine measurement, and smoking cessation counseling. Blood was drawn for determinations of levels of red blood cell polyamine synthesis markers and the plasma polyamine precursors omithine and arginine levels according to the method of Example 1. Patients were assigned to one of five doses of DFMO: 1.0, 0.5, 0.25, 0.125, and 0.06 g/m²/day for 31 days. Patients then underwent biopsies and repeat blood sampling for polyamine synthesis markers followed by loop electrosurgical excision of the cervix for complete histopathological study. The spermidine/spermine ratio was evaluated for tissue measurements as a way of decreasing variability and determining the pharmacological effect of DFMO. infected with high risk oncogenic HPV types. The rationale for using patients with high grade lesions is that such lesions are more likely to progress to invasive cancer; up to 36% of (CIN) lesions progressed in a series of studies in which 353 patients were followed without treatment over periods of 3 months to 30 years. The rationale for selecting patients with oncogenic HPV lesions comes from the cross-sectional and cohort studies demonstrating greatly increased risk of CIN and of cervical cancer in women who are HPV-positive compared with those who are HPV-negative.

The demographic characteristics of the study population were as follows. The median age of the study group was 27 years (range 20 to 41 years) and 70% were non-Hispanic whites, 23% Hispanics, and 7% African-Americans. No differences in age or racial/ethnic distribution were observed between DFMO dose levels. Eighty -three percent of the women were positive for HPV by dot blot hybridization and polymerase chain reaction. All patients were negative for Neisseria Gonorrhea. To determine whether polyamine levels differ between normal and abnormal tissues, baseline differences in tissue polyamines between colposcopically normal and abnormal tissue areas were measured separately and are shown in Table 1. Higher ODC, putrescine, spermidine and spermine values were observed in abnormal tissue areas compared to normal tissue areas; whereas a slightly lower spermidine:spermine (SPD:SPM) ratio was observed in abnormal tissue areas. The differences in ODC and SPD:SPM ratio between baseline normal and abnormal tissue areas were statistically significant (p < 0.05).

TABLE 1. Baseline differences in polyamine values between colposcopically normal and abnormal tissue

Normal Abnormal

Polyamine Tissue Tissue Difference p *

ODC (nmol/ml)

- mean 282.6 338.3 55.7 0.03

- std. error 48.4 64.4

- median 188.6 214.8

- minimum 26.8 65.6

- maximum 1 151.3 181 1.7 Putrescine (nmol/1 mg soluable protein)

- mean 1505.0 1664.2 184.2 0.97

- std error 245.4 328.4

- median 948.5 1462.0

- minimum 364.0 68.0

- maximum 5265.0 9144.0

Spermidine (nmol/1 mg soluable protein) - mean 5105.0 5220.4 1 14.9 0.99

- std error 735.9 657.1

- median 3562.0 3802.0

- minimum 1737.0 812.0

- maximum 19108.0 15400.0

Spermine (nmol/1 mg soluable protein)

- mean 4299.7 5426.4 1126.7 0.13

- std error 646.8 765.4

- median 3342.0 3969.0

- minimum 957.0 846.0

- maximum 18197.0 16831.0

SPD:SPM Ratio (nmol/1 mg soluable protein)

- mean 1.26 1.19 -0.065 -0.0012

- std error 0.07 0.23

- median 1.23 0.97

- minimum 0.70 0.55

- maximum 2.15 7.46

* Wilcoxon's signed-rank test 33 days). Patients missed an average of 3.6 days of DFMO (range 1 to 8 days). Post-treatment DFMO plasma levels are shown in Table 2. A large, but not statistically significant, difference in mean DFMO plasma levels between patients in the highest and lowest DFMO dose levels was detected. Although women in the highest DFMO dose level had higher DFMO plasma values and women in the lowest DFMO dose level had lower DFMO plasma levels, a large variability and overlap in DFMO plasma values was observed across dose levels. Furthermore, women in the intermediate dose groups showed little variation in DFMO plasma values. Plasma DFMO values ranged from 0 pmol/ml (below detectable values) in the dose group 0.125 g/πr/day to 621.9 pmol/ml in the dose group 1.0 g/m²/day.

Table 2. Post-treatment plasma DFMO levels (pmol/ml) DFMO Dose* n Mean(se)t Median Minimum

Maximum

1.000 6 146.3 (96.4) 73.9 1.8 621.9

0.500* 5 23.0 (8.2) 24.7 0.6 42.1

0.250 6 - 23.9 (4.6) 28.9 2.8 31.4

0.125 6 16.5 (5.3) 24.5 * *

33.2

0.060 6 5.9 (0.7) 5.4 4.3 8.8

* g/m²/day t standard error

^& One patient excluded from analysis for protocol violation

** Less than minimum value detected The median elapsed time between the final DFMO dose and the collection of post-treatment biological specimens was 3.5 hours (range 1 to 195.5 hours). In six patients the elapsed time between the final dose and specimen collection was > 5 hours (14 hours n=l, 15 hours n=2, 16 hours n=2, 196 hours n=l). The elapsed time between final dose and post-treatment collection of biological specimen appears to have an effect on the measured DFMO plasma levels but not on polyamine markers in tissue, plasma, or RBC. Lower DFMO plasma levels were observed among 5 of 6 women with elapsed time > 5 hours, ranging from 0 pmol/ml (below detectable values) in the patient with the largest elapsed time (196 hours) to 8.4 pmol/ml in a patient with an elapsed time of 15 hours.

It has now been discovered that DFMO provides a therapeutic alternative in treating CIN III. The effect of DFMO on tissue and plasma polyamine values was determined and the median values at baseline and post-treatment are presented in Table 3. Statistically significant modulation (P < 0.05) of tissue SPD:SPM ratios and plasma arginine was observed among patients in the highest DFMO dose level (1.0 g/πr/day). A modulation effect on tissue SPD:SPM ratio was detected among women receiving 0.500 g/πr/day of DFMO and on plasma arginine of women receiving 0.500 and 0.125 g/m²/day of DFMO (Figure 3). No modulation on any polyamine marker was observed among patients in the lowest DFMO dose group (0.06 g/πr/day).

Table 3. Comparison between baseline and post-treatment polyamine median values in: abnormal tissue, plasma, and RBC by dose level (n=29)

Dose ODC Putrescine Spermidine Spermine SPD:SPM Ratio*

Level median median median median median g/m2/day (range) (range) (range) (range) (range)

10

1,000

- Baseline 168 640 ' 3993 3951 1.06

(66 - 675) (496 1586) (1270- 15400) (1194-16831) (0.91 - 1.21)**

- Post-treatment 190 885 6562 6839 0.90

15 (75 - 627) (255 - 4943) (1628- 14655) (1924-21832) (0.53 - 0.97)

ro 0.500

- Baseline 182 490 2046 2199 .00

20 (103-294) (68-1925) (812- 11023) (846 - 9229)* (0.93 - .19)

- Post-treatment 63 243 3267 3763 0.87

(23 - 395) (154- 1532) (1601 - 12564) (1663- 16050) (0.61 -0.97)

25 0.250

- Baseline 155 1161 4115 4226 0.88

(81-1,812) (464-2019) (2166-5873) (2542 - 7997) (0.64-1.19)

- Post-treatment 204 822 3832 3986 1.07

30 (98 - 327) (599 - 2458) (1972-20537) (1847-13070) (0.72-1.57)

0.125

- Baseline 327 1580 3728 3777 0.87

(106-660) (590- 1867) (1932-7483) (2139-11754) (0.64 1.46)

35 - Post-treatment 236 724 2854 3019 0.99

(40-1534) (113-1119) (2120-9895) (1477-9498) (0.83-1.44)

0.60

- Baseline 331 1465 7044 6284 0.83

(134-598) (1056-1924) (2895-10513) (1410- - 14809) (0.55 - 7.46)

- Post-treatment 659 579 6413 55718 1.14

(634-1054) (130-2276) (3232-11219) (2273 - - 15992) (0.50- 1.49)

10

Plasma Plasma RBC RBC RBC Dose Omithine Arginine Putrescine Spermidine Spermine Level median median median median median

15 (g/n2/day) (range) (range) (range) (range) (range)

1,000

- Baseline 56 78 40 15660 11332

(29 - 73) (47-115)*** (26 - 90) (10877-19313) (9750 - 36194)

20 - Post-treatment 64 106 48 17084 12825 ro (30-110) (5 - 144) (0-91) (14489-21468) (8993 - 25928)

0.500

25 - Baseline 45 62 09** 17811 9773

(35-61) (51-87) (34 181) (12481 -24924) (6377-24514)

- Post-treatment 47 76 75 15980 8648

(43 - 50) (42 - 95) (0 - 88) (14022 - 25436) (3669-17428)

30

0.250

- Baseline 52 80 91 16229 16115

(35 - 76) (47-113) (63 - 205) (11076-25575) (6581-32611)

- Post-treatment 57 92 88 12383 12608

35 (39-71) (47 - 126) (0-196) (7629 - 19926) (5274-18800)

0.125

- Baseline 51 73 73 12132 25491

(27 - 63) (55- ■ 113) (29- 135) (6931 - 15003) (10340-32780)

- Post-treatment 65 81 89 16271 21391

(31-85) (40- 141) (0 - 369) (2637 - 22179) (12358-40780)

0.060

- Baseline 40 83 126 14311 10612

10 (20 - 67) (73- 100) (109-232) (7399 - 27724) (4739- 17583)

- Post-treatment 40 67 108 15823 9331

(25 - 52) (55- • 128) (74 - *305) (10601 - 20645) (6392 - 23380)

* Spermidine: Spermine ratio

15 ** Wilcoxon Matched - Pairs Signed-Ranks Test p<.05

ro co

DETERMINATION OF f+V AND f-VDFMO IN BODY FLUIDS

A partial or complete histological response was detected in 50% of the participants. Complete responses (negative histology) were detected in 5 patients and partial responses (histological regression to CIN I or II) were detected in 10 patients (Table 4). These observations were confirmed with computer-assisted quantitative histopathological analysis of feulgen stained slides. When polyamine markers were studied looking at differences between responders and non-responders, only tissue spermidine was significantly different. Observable therapeutic responses occurred at all dose levels. Thus, the present data demonstrates the utility of DFMO in preventing and reducing the risk of cervical cancer by treating and controlling the growth of CIN III cells.

Table 4 Number of histological responses by DFMO dose level

Dose Response

(g/m2/day) Complete Partial No Response Total

1.00 0 3 3 6

0.500 0 2 3 5

0.250 1 3 2 6

0.125 2 2 2 6

0.060 2 0 4 6

Total 5 10 14 29 EXAMPLE C

DOSAGE FORMS OF DFMO TN

THE TREATMENT OF CTN AND

PREVENTION/TREATMENT OV CERVICAL CANCER

The pharmaceutical composition that may be used in the methods of the present invention can be administered by a "ariety of routes such as, by way of example and without limitation: intrape- itoneal, intra-articular, intra-arterial, intracardiac, intracavity, intradermal, iiVrathecal, intrathoracic, percutaneous, intravascular. intravenous, intracoronary, inl;amuscular or subcutaneous injection; or oral, buccal. rectal or sublingual administration. Such methods of administration and others contemplated within the scope of ^f he present invention may be formulated according to techniques known to the skilled artisan. When used to prevent or reduce the risk of cervical cancer, DFMO will generally be administered chronically and at lower doses than those used for treating or controlling the growth of the cancer.

The present pharmaceutical cjmposition can be provided in a variety of dosage forms such as, by way of example r nd without limitation, solution, suspension, cream, ointment, lotion, capsule, tablet, caplet, gelcap, suppository, enema, transdermal patch, implant, gel, injectable, i.v. infvsion bag or bottle, concentrate, dressing, elixir, syrup, emulsion, film, granule, gurr . insert, jelly, foam, paste, pastille, pellet, spray, swab, tape, troche, lozenge, disk, magma, poultice, or wafer.

Methods for the preparation of the dosage forms contemplated herein are described in the included examples or in the references cited, the disclosures of which are hereby incorporated herein in their entirety. Any ingredients used in the present formulation should not degrade or decompose a significant portion of the DFMO or other therapeutic compound(s) used prior to administration.

The term "unit dosage form" is used herein to mean a single or multiple dose form containing a quantity of the formulation, said quantity being such that one or more predetermined units are normally required for a single therapeutic administration. In the case of multiple dose forms, such as scored tablets, said predetermined unit will be one fraction, such as a half or quarter of a scored tablet, of the multiple dose form.

It is contemplated that a combination of rapid-acting, short-acting, fast- releasing, long-acting, colorectal release, sustained release, controlled release. slow release dosage forms may be used in the present invention.

Pharmaceutical Formulation and Administration For injection, the pharmaceutical composition can be formulated, for reconstitution with an appropriate solution, as, for example and without limitation: freeze dried, rotary dried or spray dried powders; amorphous powders; or granules, precipitates or particulates. For injection, the composition may also be formulated as suspensions or liquids in the appropriate solutions, such as, by way of example and without limitation, water, aqueous solvents, nonprotic solvents, protic solvents, hydrophilic solvents, hydrophobic solvents, polar solvents, nonpolar solvent and/or combinations thereof, optionally containing stabilizers, pH modifiers, surfactants, bioavailability modifiers and/or combinations thereof. The pharmaceutical composition can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient. The composition can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants can employ inert materials such as biodegradable polymers or synthetic silicones, for example, SILASTIC™, silicone rubber-manufactured by the Dow-Coming Corporation.

For oral, buccal, and sublingual administration, the pharmaceutical composition of the invention may be administered as either solutions or suspensions in the form of gelcaps, caplets, tablets, capsules or powders. For rectal administration, the compounds of the invention may be administered in the form of suppositories, ointments, enemas, tablets and creams for release of compound in the intestines, sigmoid flexure and/or rectum. It is contemplated that the pharmaceutical formulation can be formulated as, for example and without limitation, freeze dried, rotary dried or spray dried powders; amorphous or crystalline powders; or granules, precipitates or particulates. The solids used can be either free-flowing or compressed. The pharmaceutical formulation can comprise by way of example and without limitation, water, aqueous solvents, nonprotic solvents, protic solvents, hydrophilic solvents, hydrophobic solvents, polar solvents, nonpolar solvent, emollients and or combinations thereof, optionally containing stabilizers, pH modifiers, surfactants, and/or combinations thereof.

The pharmaceutical composition can also be administered as liquid suspensions or solutions using a sterile liquid, such as an oil, water, an alcohol, or mixtures thereof, with or without the addition of a pharmaceutically suitable surfactants, suspending agent, or emulsifying agent for oral or parenteral administration.

For liquid preparations, the pharmaceutical composition can be formulated suitably with oils, for example, fixed oils, such as peanut oil, sesame oil, cottonseed oil, com oil and olive oil; fatty acids, such as oleic acid, stearic acid and isotearic acid; and fatty acid esters, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides; with alcohols, such as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol; with glycerol ketals, such as 2,2-dimethyl-l,3- dioxolane-4-methanol; with ethers, such as poly(ethyleneglycol) 450, with petroleum hydrocarbons, such as mineral oil and petrolatum; with water, or with mixtures thereof; with or without the addition of a pharmaceutically suitable surfactant, suspending agent or emulsifying agent.

The solid unit dosage form of the invention will comprise DFMO and can be combined with conventional carriers, for example, binders, such as acacia, com starch or gelatin; disintegrating agents, such as, com starch, guar gum, potato starch or alginic acid; lubricants, such as, stearic acid or magnesium stearate; and inert fillers, such as lactose, sucrose or com starch. The solid dosage form may comprise granules.

As used herein, the term "granule" is taken to mean particle, crystal, powder, particulate, minitablet, compact or other similar solid forms. The granules used in the invention may display diffusion and/or dissolution controlled release rate profiles according to the components from and processes by which they are made.

For gelcap preparations, the pharmaceutical formulation may include oils, for example, fixed oils, such as peanut oil, sesame oil. cottonseed oil, com oil and olive oil; fatty acids, such as oleic acid, stearic acid and isostearic acid; and fatty acid esters, such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides; with alcohols, such as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol; with glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4- methanol; with ethers, such as poly(ethylene glycol) 450, with petroleum thereof; with or without the addition of a pharmaceutically suitable surfactant, suspending agent or emulsifying agent.

Oils can also be employed in the preparation of formulations of the soft gelatin type. Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may suitably contain suspending agents, such as pectin, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives. Soaps and synthetic detergents may be employed as surfactants and as vehicles for detergent compositions. Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts. Suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene)-£/o poly(oxypropylene) copolymers; and amphoteric detergents, for example, alkyl β- aminopropionates and 2-alkylimidazoline quaternary ammonium salts; and mixtures thereof.

The formulation may also comprise adsorbents, antioxidants, buffering agents, colorants, flavorants, sweetening agents, tablet antiadherents, tablet binders, tablet and capsule diluents, tablet direct compression excipients, tablet disintegrants, tablet glidants, tablet lubricants, tablet or capsule opaquants and/or tablet polishing agents.

As used herein, the term "adsorbent" is intended to mean an agent capable of holding other molecules onto its surface by physical or chemical (chemisorption) means. Such compounds include, by way of example and without limitation, powdered and activated charcoal and the like.

As used herein, the term "antioxidant" is intended to mean an agent which inhibits oxidation and thus is used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and the like. used to resist change in pH upon dilution or addition of acid or alkali. Such compounds include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and the like.

As used herein, the term "colorant" is intended to mean a compound used to impart color to liquid and solid (e.g., tablets and capsules) pharmaceutical preparations. Such compounds include, by way of example and without limitation, FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel, and ferric oxide, red and the like.

As used herein, the term "flavorant" is intended to mean a compound used to impart a pleasant flavor and often odor to a pharmaceutical preparation. In addition to the natural flavorants, many synthetic flavorants may also be used. Such compounds include, by way of example and without limitation, anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin and the like.

EXAMPLE 7 METHOD OF TREATMENT OF CERVICAL INTRAEPITHELIAL NEOPLASIA CANCER WITH DFMO

The course and duration of administration of and the dosage requirements for the formulation of the present invention will vary according to the subject being treated, the formulation used, the method of administration used, the severity and type of cervical cancer or tumor being treated, the coadministxation of other drugs and other factors.

Although each unit dosage form contains therapeutically effective amounts of DFMO, it may be necessary to administer more than one such unit dosage form in order to obtain the full therapeutic benefit of the DFMO. More particularly, since DFMO may require moderately high doses, vide supra, it is very likely that more than one unit dosage from will need to be administered to a patient in order to obtain the full therapeutic benefit of DFMO.

For example, consider that the average 65-70 Kg human has a body surface area of about 1.65 m² to about 1.8 πr, or about 1.73 πr. If DFMO is administered at a DFMO/day, about 3 1/2 tablets containing about 0.5 g of DFMO. Correspondingly, if the dosage administered is about 0.25 g/πr/day then a patient would have to receive about 0.4 g/day, about 1 tablet containing 0.5 g of DFMO. A study of DFMO on women with CIN III has been completed. In this study,

30 patients with biopsy-proven CIN III were assigned to receive one of 5 doses of DFMO (1.0, 0.5, 0.25, 0.125, or 0.06 g/m²/day) orally for 31 days. A significant percentage (17%) of the patients expressed a complete histological response and 33% had histological regression to CIN I or II, although responses were not dose-related. These results demonstrate the utility of the present invention as a therapy for the treatment of CIN I, II or III in HIV-infected patients, as well as patients with CIN I, II or III without HIV infection.

EXAMPLE 8 GROWTH INHIBITORY EFFECTS OF -DIFLUOROMETHYLORNITHINE (OFMO, ON

HUMAN CERVICAL CARCINOMA (HCC)

CELL LINES IN VITRO

In this example the effects of DFMO on the growth of 9 HCC cell lines is demonstrated. This example demonstrates the utility of the present invention for treating and inhibiting cervical cancer and progression of CIN-characterized tissue to cervical cancer.

DFMO was found to inhibit the growth of all cell lines examined irrespective of their human papillomavirus and p53 status. The DFMO concentrations required for 50% growth inhibition on day 5 ranged from 15 μM to >5mM. These concentrations are relevant because Phase I studies of single dose pharmacokinetics reported plasma levels ranging from 14 to 50 μM. The effects were time dependent with inhibition detectable first after 2 days of treatment. These findings suggest that cervical malignancies and presumably also CIN lesions may respond to DFMO in vivo. 2-Difluoromethyl-ornithine (DFMO) was dissolved in distilled water at a concentration of 1 M and sterilized by filtration through a 0.22-um-pore membrane. Aliquots were stored at -80°C and used only once after thawing. 4-HPR was obtained from McNeil Pharmaceutical Research. It was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM, and stored in the dark at -20°C. Stock solutions were diluted to the appropriate concentrations with growth medium just prior to use.

Growth Inhibition Assays

The human cervical carcinoma cell lines C-33 A, C-4I, C-4II, Caski, HeLa, HT- 3, MS751 , and SiHa were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The ME 180 cells were obtained from Dr. Nicholas Donato (M.D. Anderson Cancer Center, Houston, TX). The details on the isolation of these cell lines and some of their properties are outlined in the ATCC catalog. All cells were grown as monolayers in a 1 : 1 mixture of Dulbecco's modified Eagle's medium: Ham's F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 u/ml penicillin and 100 ug/ml streptomycin) in a humidified incubator at 37°C in an atmosphere of 5% C02. Exponentially growing cells were seeded at densities ranging from 1000 to

3000 cells per well in 96-well cluster tissue culture plates and treated the next day with increasing concentrations of DFMO or 4-HPR for 5 - 7 days. Cell cultures grown in medium alone served as controls. Control cultures received the same amount of DMSO in 4-HPR treated cultures. Half of the growth medium with or without the supplementation of drugs was changed after 3 days of treatment. Cell numbers were estimated everyday by using the sulforhodamine B (SRB) assay with a slight modification. The medium was discarded and the adherent cells were fixed in situ by adding to each well 100 ul of cold tichloroacetic acid (TCA), 10% (w/v) and incubating for 60 minutes at 4°C. The wells were then rinsed five times with deionized water and air dried. Each well then received 50 ul of SRB (Sigma Chemical

Co., St. Louis, MO) solution (0.4% w/v in 1% acetic acid) and the plates were incubated for 10 min at room temperature. Unbound SRB was removed by rinsing five times with 1% acetic acid. After the plates were dried, the densities were read The percentage of growth inhibition (GI) was calculated by using the equation: %GI = ( 1 - Nt/Nc)* 100 where Nt and Nc represent the optical density in treated and control cultures respectively. ID50 which is the drug concentration causing cell growth inhibition by 50% was determined by interpolation from dose response curves.

(3H)Thymidine uptake and incorporation into DNA

Exponentially growing cells (3000 cells/well, 96-well plate) were incubated in the absence or presence of increasing concentration of DFMO or 4-HPR. (3H)Thymidine was added to cell cultures for the final 6 hours of incubation. The plates were frozen in -20°C and thereafter thawed and harvested by cell harvester (Cambridge Technology, Inc.). Filters were washed three times with 5% trichloroacetic acid and twice with 90% ethanol, air-dried and assayed for (3H)thymidine by liquid scintillation counting. C33A cells were plated on 10 cm diameter dishes one day before DFMO treatment. At pre-established time of treatment with 500 uM DFMO, cells were harvested by tripsinization and centrifuged at 300xg for 5 minutes, washed, and processed as below.

Two different methods will be evaluated for DNA labeling: ( 1 ) The 300xg centrifuged cell pellet was fixed in cold 70% ethanol at 4°C for 60 minutes. The cells were then centrifuged, washed in PBS and resuspended in 0.5 ml PBS. To a 0.5 ml cell sample 0.5 ml RNAse (Type I-A, Sihma, St. Louis, MO) was added, followed after gentle mixing by 1 ml propidium iodide (PI, Sigma, 100 ug/ml in PBS) solution. The mixed cells were incubated in the dark at room temperature for 15 minutes and kept at 4°C in the dark until measured.

(2) The second method was essentially that described in the protocol of APO-DIRECT. The 300xg centrifuged cell pellet was fixed in 4% cold paraformaldehyte in PBS for 15 minutes and washed in PBS, resuspended in 0.5 ml of PBS.

DNA fragmentation assay and DNA electrophoresis

C33 A cells were incubated in the absence or presence of 500 uM DFMO for the indicated times. Adherent cells were collected after trypsin treatment and pelleted (3 min, 500g) and incubated overnight at 37°C in lysis buffer (lOmM Tris/HCI, lOmM EDTA, 0.5% Triton X-100, 50 ug/ml proteinase K, 5 u ml Rnase A). DNA was extracted with equal volumes of phenol and chloroform 3-methylbutan-l-ol (24: 1, v/v) and precipitated with 0.1 vol. of 3M sodium acetate, pH 5, and 2.5 vol. of cold 100% ethanol. Pelleted DNA was resuspended in TE (lOmM Tris/HCL, ImM EDTA) and was electrophoresed on a 1.5% agarose gel in TAE buffer (40 mM Tris/acetate, ImM EDTA. The gel was stained in 1 ug/ml ethidium bromide in TAE for 30 minutes, destained in TAE and photographed under UV illumination.

RNA purification and analysis by Northern Blotting

Total cellular RNA was purified by the method of TRI Reagen (Molecular Research Center, Inc.) RNA (20 ug per lane) was fractionated on a 1.2% (w/v) agarose/formaldehyde gel and transferred to positively charged nylon filters (Amersham). The probes were labeled with 32P-dCTP to a specific activity of approximately 2x 109cpm/ug using the random hexanucleotides (Prime - It II- Kit, Stratagene, La Jolla, CA) as primers. The filters were pre-hybridized and hybridized at 65°C in Rapid-Hyb Buffer (Amersham) for 15 minutes and overnight respectively and were washed twice with 2xSSC, 0.1% SDS at room temperature for 15 minutes. The final washes were done to a stringency of 0.1 x SSC, 1% SDS at 65°C for 15 minutes for 3 times. The membranes were then placed against an Hyper-film (Amersham) for autoradiography at 70°C for 1 -2 days using intensifying screens.

These results from these studies are shown in Figures 4, 5 and 6, and in Table 5, below. Table 5. Effect of 4-HPR on the Growth of HCC Cell Lines

Cells (μM) (μM

C33A 300 <1

C-41 35 7

C-41 1 400 5

Caski 15 5

HeLa 30 Not measurable Me 180 300 6-

MS751 100 6-

SiHa 45 7+

These data demonstrate that DFMO provides an inhibitory effect on the growth of a number of diverse human tumor cell lines, and the utility of the present invention as a method for inhibiting or treating cancer, and particularly cervical cancer and CIN in humans.

EXAMPLE 9 GROWTH INHIBITORY EFFECTS OF DFMO ON HUMAN CERVICAL CARCINOMA CELL LINE IN VITRO

This example demonstrates the utility of DFMO for inhibiting and treating cervical cancer. The data here relate to DFMO and the inhibition of nine (9) human cervical carcinoma cell lines. DFMO was found to inhibit the growth of all the cell lines. The DFMO concentrations required for 50% growth inhibition on day 5 ranged from 100 μM to > 5 mM. These effects were observed to be at least in part time dependent with inhibition detectable first after 2 days of treatment. The responsiveness of the cells appeared to be independent of their papillomavirus or p53 status. These findings support the use of DFMO as an effective inhibition of cervical malignancies and lesions, as well as in the prevention of cervical cancer.

Materials and Methods α-Difluoromethyl- Omithine (DFMO) was provided by Drs. Kenjii Nishioka and A.K. Verma. It was dissolved in distilled water at a concentration of 1 M and sterilized by filtration through a 0.22-um-pore membrane. Aliquots were stored at -

80 °C and used only once after thawing.

Cell Cultures

The human cervical carcinoma cell lines C33A, c4II. Caski, HeLa, HT3, MS751, and SiHa were purchased from the American Type Culture Collection their properties are outlined in the ATCC catalog. All cells were grown as monolayers in a 1 : 1 mixture of Dulbecco's modified Eagle's medium: Ham's F12 medium (DME/F12) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin) in a humidified incubator at 37°C in an atmosphere of 5% C0₂.

Growth Inhibition Assay

Exponentially growing cells were seeded at densities ranging from 1000 to 3000 cells per well in 96- well cluster tissue culture plates and treated next day with increasing concentrations of DFMO or 4-HPR for 5 days. Cell cultures grown in medium alone served as controls. Half of the growth medium with or without the supplementation of drugs was changed after 3 days of treatment. Cell numbers were estimated everyday by using the sulforhodamine B (SRB) assay with a slight modification. Briefly, the medium was discarded and the adherent cells were fixed in situ by adding to each well 100 ul of cold trichloroacetic acid (TCA, 10%(w/v)) and incubating for 60 min at 4°C. The wells were then rinsed five times with deionized water and air dried. Each well then received 50 ul of SRB (Sigma Chemical Co., St. Louis, MO) solution (0.4% w/v in 1% acetic acid) and the plates were incubated for 10 min at room temperature. Unbound SRB was solubilized using a microtiter plate reader at 492 nm. 8 replicate wells were used for each analysis. The percentage of growth inhibition ^GI) was calculated by using the equation: %GI- (l-NtNc)* 100 where Nt and Nc represent the optical density in treated and control cultures respectively. ID_J0 which is the drug concentration causing cell growth inhibition by 50% was determined by interpolation from dose response curves.

Colony Formation Assay

The well dispersed cells were grown in 0.5% SEA PLAQUE agarose in lxDMEM/F12 growth medium with 10% FBS on top of a layer of 1 % SEA PLAQUE agarose in the same medium. Cells were grown in the gel for 2-3 weeks, until the colonies formed.

The colonies were counted under the microscope. Colony formation for the cervical carcinoma cell line HeLa was assessed in the presence of increasing decrease in colony formation of about 75% was observed (Fig. 15).

and their response to DFMO.

Cell line^a Differentiation⁶ HPV type ^c Inhibition by DFMO (IC₅₀; μM^d)

ME 180 (m) epidermoid HPV 68 200

MS 751 epidermoid HPV 18 100

C4 I epidermoid HPV 18 >5000

C4II epidermoid HPV 18 >5000

HeLa epitheloid HPV 18 200

C-33A epitheloid ND 500

CaSki (m) epidermoid HPV 16 <100

SiHa epidermoid HPV 16 120

HT-3 epitheloid ND >5000

^am, cell line derived from metastasis. ^bBased on the ATCC catalog.

The status of HPV was reported previously. ND, not detected. ^dThe IC₅₀ of growth inhibition was derived by interpolation from Fig. 14.

ANTIPROLIFERATIVE EFFECTS OF DFMO ON HUMAN CERVICAL EPITHELIAL CELLS ALONE AND IN COMBINATION WITH 4HPR

This example is provided to demonstrate the effect of DFMO on human cervical cancer. Several different cervical cancer cell lines are effectively inhibited with the techniques of the present invention.

Materials and Methods

2-Difluoromethyl-ornithine (DFMO) was provided by Drs. Kenji Nishioka and A.K. Verma. It was dissolved in distilled water at a concentration of 1 M and sterilized by filtration through a 0.22 μM pore membrane. Aliquots were stored at 80 °C and used only once after thawing. 4HPR was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM and stored in the dark at -20°C. Stock solutions were diluted to the appropriate concentrations with growth medium just prior to use.

Cell Cultures The human cervical epithelial cell lines, zl31, zl73, zl 83 and TCL1 were provided by Dr. Vittorio Defendi. These were primary cells being electroporated with cloned viral DNAs from HPV types 16, and 18. The details on the isolation of these cell lines and some of their properties are outlined in Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 563-567, 1989. All cells were grown as monolayers in a 1 : 1 mixture of Dulbecco's modified Eagle's medium: Ham's F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 u/ml penicillin and 100 ug streptomycin) in a humidified incubator at 37°C in an atmosphere of 5% C0₂.

Growth Inhibition Assay

Exponentially growing cells were seeded at densities ranging from 1000 to 3000 cells per well in 96-well cluster tissue culture plates and treated next day with increasing concentrations of DFMO or 4-HPR for 5 days. Cell cultures grown in medium alone served as controls. Half of the growth medium with or without the estimated everyday by using the sulforhodamine B (SRB) assay with a slight modification. Briefly, the medium was discarded and the adherent cells were fixed in situ by adding to each well 100 ul of cold trichloroacetic acid (TCA, 10%(w/v)) and incubating for 60 min at 4°C. The wells were then rinsed five times with deionized water and air dried. Each well then received 50 ul of SRB (Sigma Chemical Co., St. Louis, MO) solution (0.4% w/v in 1 % acetic acid) and the plates were incubated for 10 min at room temperature. Unbound SRB was solubilized using a microtiter plate reader at 492 nm. 8 replicate wells were used for each analysis. The percentage of growth inhibition (GI) was calculated by using the equation: %GI- (l-NtNc)* 100 where Nt and Nc represent the optical density in treated and control cultures respectively. ID₅₀ which is the drug concentration causing cell growth inhibition by 50%) was determined by interpolation from dose response curves.

Colony formation assay

The well dispersed cells were grown in 50% MATRIGEL in lxDMEM/F12 growth medium with 10 FBS on top of layer of SEA PLAQUER agarose in the same medium. Cells were grown in the gel for 10 days to 2 weeks, until the colonies came out. Count the colonies under the microscope.

H-Thymidine uptake and incorporation into DNA

Exponentiajly growing cells (3000 cells/well, 96-well plate) were incubated in the absence or presence of increasing concentration of DFMO. H-Thymidine was added to cell cultures for the final 4 hours of incubation. The plates were frozen in - 20°C and thereafter thawed and harvested by cell harvester (Cambridge Technology,

Inc.). Filters were washed three times with 5% richloroacetic acid and twice with 90% ethanol, air dried and assayed for H-Thymidine by liquid scintillation counting.

As demonstrated in FIG 7, DFMO present at a concentration of 100 μM provided a 50% TCL1 cell survival. Figure 8 demonstrates cell survival in the presence of 4HPR.

Figure 9 presents the combined effect of DFMO and 4HPR on TCL1 cell survival. The effect of 4HPR was examined at the two different concentrations, 2 μM and 5μM. There appeared an about 10% greater decrease in percent cell survival 4HPR was 5μM, the percent cell survival was about 75%, relative to the 85% cancer cell line TCL1 cell survival in the presence of 2μM 4HPR + DFMO (Fig 9, —O— = DFMO alone; •••<>■• = 4HPR (2μM) + DFMO; ~o~ = 4HPR (5μM) + DFMO). With the cancer cell line Zl 83, about 50% cell survival was observed in the presence of 200μM DFMO (Fig 1 1). A combination of 5μM 4HPR and 10μM DFMO resulted in a 70% cancer cell line Z183 survival (Fig 12, — °— ). The combination of 2μM 4HPR and 10μM DFMO provided about 90% cancer cell line Z183 survival (Figure 12 -0-). lOμM DFMO alone provided about 95% cell survival (Figure 12 — O— ).

The colony formation assay (Matrigel) results with the cervical carcinoma cell line Z173 is shown at Figure 13. The number of colonies observed decreased in response to increasing doses of the DFMO (lOOμM, 200μM, 500μM, l ,000μM. 5000μM). An about 50% decrease in cell colony formation was observed in the presence of about 200μM DFMO (Fig 13).

Figure 14 demonstrates the time-dependent increase in cell growth inhibition with DFMO. The doses of DFMO used were 0, 10, 1000, 200, 500, and 1000 μM. Eight (8) different human cervical carcinoma cell lines (C33A, Caski, 41 1 , S, Ha, HT3, Hela, ME180 and MS751) were examined. At each concentration, the percent growth inhibition increased. This increase in growth inhibition was also observed with increasing number of days in culture (course of five (5) days).

EXAMPLE 11 STUDY ON REGRESSION OF CIN WITH DFMO BASED ON HISTOLOGICAL

EXAMINATION OF CERVICAL TISSUE

The present example is provided to demonstrate the utility of the invention to effect a regression of CIN in a patient upon treatment with DFMO. The qualitative and quantitative toxicity of DFMO compared to a placebo will also be examined.

The following dosing regimen will be employed. Agent Dose Davs Route L?vel =1 =2

DFMO 0.5 gm/m²/day Daily p.o. 0.75 0.5

0.125 gm/nr7day Daily p.o.

Placeb Elixir Daily p.o. o

Patients that will be examined in this study are those having a CIN 3 biopsy. The patients will be followed over a period of 6 months. CIN is considered a precursor of invasive cervical cancer (ICC). Cervical intraepithelial neoplasia, called dysplasia in the past, is defined as "a spectrum of intraepithelial changes that begin as well differentiated intraepithelial neoplasm, which is classified as a mild dysplasia, and ends with invasive cancer: Neoplastic changes are confined to the squamous epithelium and include nuclear premoφhism, loss of polarity, and presence of abnormal mitoses. CIN is graded 1 to 3; the grading is based on the amount of undifferentiated cells present from the basement membrane to the surface epithelium. When one third of this distance is involved the grade is 1 , when more than one third and up to two thirds are involved the grade is 2, and when more than two thirds are involved the grade is 3. When the patient has full thickness involvement from the basement membrane to the surface epithelium with CIN III, this condition is referred to as carcinoma in situ (CIS). While the current teaching is that concerning transition from a benign to a malignant lesion have defined a separate category for CIS. The difference therefore, between CIN III and CIS is that CIN III represents two thirds involvement whereas CIS involves full thickness involvement from the basement membrane to the surface epithelium.

Over 4,000 patients have been enrolled in studies to look at the natural history of all grades of CIN in which they have been followed with biopsy only; 46% of lesions regressed, 31% persisted, and 23% progressed 91% to invasive cancer). Studies which limited enrollment to patients with carcinoma in situ (CIS), totaling 328, showed 34% progression to invasion.

The natural history of CIN comes from four sources; prospective studies of CIN in patients not treated, cervical screening program studies focusing on programs, and studies which focus on patients lost to follow-up or who recur after primary therapy. The most pertinent sources of information are prospective studies of CIN in patients not treated and data which focuses on patients who are lost to follow-up.

Drug Supply

DFMO is supplied by the Division of Cancer Prevention and Control, NCI., in Elixir containing 200 mg/m. Patent Criteria

Patients must be women with newly diagnosed CIN grade 3 (incident cases), involving at least one quadrant of the cervix without prior therapy to the cervix.

Patients must be > 18 years old, with a performance status < 2 (Zubrod Scale) and a predicted life expectancy of > 12 months. Patients must have had a bilateral tubal ligation, a Norplant, be postmenopausal, use Depo-Provera, or use a medically safe form of contraception for the duration of the study.

Patients must not have had a prior malignancy.

Laboratory data: absolute granulocyte count > 1 ,500 and platelet counts > 100,000, serum bilirubin < 1.5 mg%, serum creatinine < 1.5 mg%, and fasting triglyceride < twice normal.

All patients must complete the requirements of pretreatment evaluation, must consent to colposcopy and cervical biopsy for histologic evaluation. Colposcopy: All patients will undergo colposcopy and colpophotography with cervical biopsies and histologic assessment,

Repeat Colposcopy: After completing the treatment period (initial 6 months of study), all patients will have repeat colposcopy and colpophotography at 3, 6, 9, and 12 months; Pap smears at 3, 6, 9, and 12 months; and cervical biopsy at 6 and 12 months to assess changes, if any, in histologic status after treatment and follow-up, respectively.

Microinvasive or Invasive Squamous Carcinoma

Carcinoma discovered any time during study will result in removal from study protocol and referral for appropriate therapy. For hematologic and non-hematolopic toxicitv: Grade 0 No change

Grade 1 No change Grade 2 Halt; reduce 1 level

Grade 3 Halt; reduce 2 levels

Grade 4 Stop test medication

If > 2+ (moderate) toxicity develops at any dose level, the test medication administration will initially be halted and then restarted after recovery from toxicity at a reduced dose level. The test medication will be discontinued in event of life- threatening toxicity (Grade 4).

Patients who re-develop toxicity after dose reduction will have the test medication initially halted, then restarted at a reduced dose level (Grade 2 toxicity) or terminated ( > Grade 3 toxicity).

PRE AND POST REGISTRATION EVALUATION

Complete history and physical examination will be done, with documentation of history of: tobacco exposure, nutritional assessment, and risk factor assessment using the "Health Habit History Questionnaire" of Gladys Block.

Laboratory studies:

The following parameters will be assessed for all patients:

CBC with differential platelet count

SMA-12, electrolytes PT/PTT

Cotinine levels

DFMO serum levels

LH

FSH Progesterone

Estradiol

B-HCG pregnancy test Colposcopic examination of the cervix. Colpophotography Colposcopic directed biopsies Special Test Audiogram

EVALUATION DURING STUDY

All patients will have colposcopy, with cervical biopsies taken at 4 standardized sites, and any other colposcopically abnormal site. Each biopsy will be cut into 10 sections, the severity of dysplasia noted. A biopsy sample will be prepared separately for immunohistochemical analysis of tissue markers. Contacts (at least monthly ,-

Compliance measurements during treatment (initial 6 months or subsequent cross-over period, if applicable): Pill counts

Calendar table Additional baseline investigations

Detailed history of tobacco use At Baseline 3. 6. 9. and 12 months Papanicolaou smear

Colposcopy

Colpophotography

CBC and PLT

SMA-12 and electrolytes PT/PTT

Cotinine level

DFMO levels serum

LH

FSH Progesterone

Estradiol

B-HCG pregnancy test At 6 and 12 months Audiogram

CRITERIA FOR RESPONSE AND TOXICITY Evaluation of treatment response will be based on histologic criteria .

Histologic Response fat 6 months')

Compete Response (CK): complete reversal of CLN3 to normal epithelium. Partial Response fPRI: Improvement in severity of CIN to grade 1 or 2. No Change CNC): no change in severity of dysplasia. Progressive Disease (?O): worsening severity of CIN to carcinoma in situ

(CIS). If patients progress to microinvasive or invasive squamous carcinoma of the cervix, they will be taken off protocol. Assessment of Biomarkers

The primary marker endpoint for the reverse phase I DFMO study will be putrescine content in the cervical tissue post treatment. In addition, DFMO and ODC measurements will be made on the same tissue to determine the relationship between free ODC, covalently-bound ODC/DFMO, and putrescine levels. It is expected that the mean toxic dose of DFMO previously determined in phase I studies has already saturated the ODC/DFMO effect and that tissue putrescine levels are at their lowest point. As the DFMO dose is dropped on each consecutive group of individuals, we hope to find the minimum DFMO dose that can be given without allowing increasing putrescine levels will be identified. Parallel biomarker studies will be carried out on the tissues to determine the association of the omer endpoints with DFMO's effect on lowering polyamine levels. For example, effective DFMO doses would be expected to decrease the proliferative status of the tissue (i.e. decrease the fraction PCNA- positive cell was in the target lesion). As the DFMO dose is lowered to a less effect dose to use to determine whether proliferation is less inhibited (i.e. a higher PCNA index). Urine levels of putrescine and DFMO (expressed as per mg creatinine) will also be measured to determine whether urine levels are correlated with target tissue levels. This provides a less invasive way to assess DFMO's effect on putrescine content.

The immunocytochemically-detected biomarkers will be quantitated using our Magician Image Analysis System, using internal controls on the same slide to account blocks of cells uniformly expressing a particular marker such as PCNA, EGFR, etc. Each biopsy sample's marker expression is normalized to the same intemal control such that pre- and post- treatment biopsies can be quantitatively compared. PCNA measurements are expressed as mean normalized PCNA content per cell, mean normalized PCNA weighted index context per cell, and PCNA labeling index. EGFR measurements are expressed as normalized (compared to 886 cells) EGFR expression per unit epithelial cell area. RAS, TGFa, and TGF-α will be quantitated in a similar fashion as a normalized value normalized to intemal controls placed on the same slide.

Toxicity

Toxicities encountered during the study will be evaluated according to the grading system (0-4) (see table). Objective toxicities will be considered as the maximum numerical deviation from the normal range.

PATHOLOGY

1. Procedures Histologic Specimens: 4-6 biopsies will be taken by the colposcopist representing a wide sampling of the cervix. Each biopsy will be immediately placed into.a designated container of neutral formalin (to avoid drying), and labelled with the patient's name, hospital, number, site biopsied, date, and time of the biopsy. It will be recorded in the surgical pathology suite where it will be promptly grossed and placed in a cassette. After it is grossed, it will be processed using standard techniques and then sectioned at 4 micron thickness until 10 separate slides are prepared. It will be stained with hematoxylin and eosin, and the case will be submitted to the pathologist for blinded microscopic examination.

Criteria For Histologic Assessment

Histologic response will be assessed according to the following categories:

CJN lower one-third the distance from the basement membrane to the surface epithelium. b. A minimal degree of nuclear pleomoφhism. c. A minimal increase in the nuclearxytoplasmic ratio. d. Some minimal degree of enlargement of nuclei. e. Mild hyperchromasia, which is often minimal in CLN1 and has a finely stippled chromatin pattern.

CIN II

Some degree of nuclear abnormality that involves less than the lower two-thirds the distance from the basement membrane to the surface epithelium. b. A moderate degree of nuclear pleomoφhism. c. A moderate increase in the nuclearxytoplasmic ratio. d. Some moderate degree of enlargement of nuclei. Moderate hyper-chromasia.

CIN III a. Some degree of nuclear abnormality that involves greater than two-thirds of the distance from the basement membrane to the surface epithelium. b. A marked degree of nuclear pleomoφhism. c. A marked increase in the nuclearxytoplasmic ratio. d. Some marked degree of enlargement of nuclei. e. Marked hyperchromasia.

Polyamine and Omithine Decarboxylase (ODC) Determinations

Polyamines (blood and tissue specimens) and ODC activity (tissues) are determined as described except that in case of tissues, both parameters will be determined from the same sample which is prepared from total tissue specimen by homogenizing in buffer (50 mM Tris/HCl, pH 7.4, containing 2.5 mM dithiothreitol, 0.1 mM EDTA and freshly added 0.5 mM phenylmethylsulfonyl fluoride). The ODC assay has now been scaled down to increase sensitivity to use a total volume of 175 ml containing 125 ml of the enzyme sample along with 2.8 mM L-omithine and 0.35 blood specimens collected before initiation of treatment and periodically thereafter. These levels will be correlated with plasma levels of DFMO. We have adapted a sensitive DFMO assay procedure developed by Grove et al to our Dionex-Durrum amino acid analyzer equipped with a fluorometer. This procedure allows simultaneous determination of omithine.

TABLE 2

GOG COMMON TOXICΓΓY CRΠΈRIA

TABLE 2

TABLE 2

GOG COMMON TOXICITY CRITERIA

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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2. ALDOUS S, ET AL. Resolution of (+)- and (-1-alpha-difluoromethylomithine by capillary gas chroma tography. J. Chromatogr. (1986) 357 (2) 335-9

3. ANSEL HC, POPOVICH NG AND ALLEN LV, JR., EDS. Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. Williams & Wilkins, Baltimore, ISBN 0-683- 00193-0, 1995

4. AUVINEN M., PAASINEN A., ANDERSSON LC, HOLTTA E., Orinthine decarboxylase activity is critical for cell transformation. Nature 360:355-8, 1992.

5. BOONE C.W., KELLOFF G.J., MALONE W.E. Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res. 50:2-9, 1990.

6. BOSCH F.X., MANOS M.M., MUNOZ N„ ET AL. Prevalence of human papillomavirus in cervical cancer: A worldwide perspective. J Natl Cancer Inst 87:796- 802, 1995.

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Claims

What is claimed is:

1. A medicament for use in inhibiting the progression of cervical epithelial neoplasia to cervical cancer comprising a pharmaceutical preparation of a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier.

2. A medicament for use in reducing risk of cervical cancer in a patient with cervical epithelial neoplasia comprising administering to a patient having cervical epithelial neoplasia a pharmaceutical composition comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier.

3. A medicament for use in reducing risk of developing cervical cancer in a patient having cervical intraepithelial neoplasia comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier.

4. A medicament for use in inhibiting cervical intraepithelial neoplasia in a patient comprising a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof; in a pharmaceutically acceptable carrier.

5. The medicament of claim 1 , 2, 3, or 4 wherein the pharmacologically active amount of DFMO is between about 0.01% to about 90% by weight of the pharmaceutical preparation.

6. The medicament of claim 1, 2, 3 or 4, wherein the patient is administered a dose of between about 0.050 g/πr/day and about 1.0 g/m²/day DFMO for about 30 days.

7. The medicament of claim 3 or 4 wherein the cervical intraepithelial neoplasia is a cervical intraepithelial neoplasia grade III. positive.

9. The medicament of claim 1, 2, 3 or 4 further comprising 4HPR.

10. The medicament of claim 4 wherein the amount of 4HPR and DFMO is equimolar.

11. A medicament for providing regression of cervical cancer comprising a pharmaceutical preparation of a pharmacologically active amount of DFMO, or pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable carrier.

12. The medicament of claim 1 1 , further comprising 4HPR.

Methods for treating, preventing, controlling the growth of and/or reducing the risk of developing cervical cancer, particularly in patients with cervical intraepithelial neoplasia are provided employing pharmaceutically acceptable preparations of DFMO. Methods for treating a patient having cervical intraepithelial neoplasia, which methods comprise administering DFMO alone or in combination with a cytotoxic or cytostatic agent, are also provided.