SPUTUM INDUCTION AND ANALYSIS OF SPUTUM TO

DETECT CANCER ASSOCIATED SUBSTANCES

METHOD OF EARLY LUNG CANCER DETECTION VIA SPUTUM

INDUCTION AND ANALYSIS OF SPUTUM TO DETECT

CANCER ASSOCIATED SUBSTANCES

INTRODUCTION

This application is a continuation-in-part application of U.S. Serial No. 08/725,943, filed October 8, 1996, which is incorporated herein by reference.

Technical Field

This invention relates to the diagnosis of cancer, specifically, to a method of sputum induction and cytologic assay for the early detection of lung cancer.

Background of the Invention

Lung cancer is the most common fatal malignant neoplasm of both men and women in the United States, with an approximate 13% to 15% survival rate 5 years after diagnosis (T. Petty, Med. Clin. North Amer. 80(3), 645-55 (1996)). Lung cancer typically occurs after a prolonged latent period of several years to several decades, during which the normal airway epithelium undergoes cellular changes which progressively become more severe until ultimately they progress to carcinoma in situ and to invasive carcinoma. However, if detected in its early stages, i.e., carcinoma in situ or microinvasive cancer, the potential for cure is essentially 100% (S. Lam, abstract presented at the Annual Respiratory Disease Symposium, Vancouver, B.C., October 27-28, 1995). Therapeutic measures in these early stages include photodynamic therapy, cryotherapy, or surgery. Premalignant lesions can be treated with chemopreventative agents such as 13- cis-retinoic acid to suppress and even reverse the carcinogenic process.

Currently, the prevailing method of lung cancer diagnosis involves a conventional chest roentgenogram following the appearance of symptoms such as chronic cough or hemoptysis. Unfortunately, this approach usually only detects lung cancer in its advanced stages, when regional and distal metastases

have often already occurred (J. Mulshine, et al., /. Clin. Oncol. 4, 1704-15 (1986)). Thus, chest roentgenograms cannot be relied upon to detect lung cancer in its early and more curable stages.

The present invention discloses an effective means of early lung cancer detection by means of inducing expectoration of deep lung sputum with nucleoside phosphates, preferably uridine 5'-triphosphate (UTP) or P ¹ , P ⁴ - di(uridine 5'-)tetraphosphate (U2P4), and then analyzing the sputum for the presence of labeled reagents selectively bound to substances in the sputum whose presence correlates with the development of lung cancer. Such substances are commonly referred to as biomarkers of lung cancer. Such biomarkers can be, for example, cellular proteins whose enhanced presence correlates with the development of lung cancer, or mutated gene sequences which also correlate with the development of lung cancer.

Normal exfoliation of epithelial cells in the tracheobronchial tree results in .an abundance of epithelial cells in sputum. The field carcinogenesis concept, proposed by Slaughter four decades ago, holds that exposure of the entire lung epithelium to inhaled carcinogens may transform cells throughout the lung field (D. Slaughter, et al., Cancer 6, 963-68 (1953)). Thus, a deep lung sputum sample from a patient with a radiologically occult cancerous growth may contain many cells showing neoplastic or pre-malignant ch.anges. This idea of sputum cytologic analysis as a method of early lung cancer detection was first proposed nearly two decades ago (G. Saccomanno, Lab. Med. 10, 523-527 9(1979)). From 1979 to 1984, the National Cancer Institute (NCI) sponsored a large clinical study in which 30,000 volunteers at high risk for lung cancer (i.e., chronic smokers) underwent periodic chest radiography and sputum cell cytology in order to evaluate the cost-effectiveness of these techniques as a means of early lung cancer detection. Using then state-of-the-art cytology methods, i.e., visualization of morphological changes via conventional light microscopy, only 10% of

subsequently developing lung cancers were detectable by sputum cell analysis (M. Tockman, et al., Chest 106, 385S-390S (1994)). Thus, the conclusion of the study was that sputum cytology was not yet a cost-effective means of early lung cancer detection. However, recent advances in immuno-staining using monoclonal antibodies (Mabs) have sparked renewed interest in the prognostic value of sputum cytology. Mabs have been generated against two biomarker antigens which appear on the surface of both lung tumor cells and premalignant, atypical cells from sputum. By biom.arkers, it is meant that the enhanced presence of these antigens on the surface of bronchial epithelial cells has been shown to be correlated with the later development of lung cancer. Using these Mabs, Drs. Tockman and Erozan at John Hopkins Hospital have re-tested sputum samples from the NCI study, and have been able to successfully correlate 91% of subsequently developing lung cancers with Mab-reactive sputum samples taken approximately two years before these cancers became clinically evident (M. Tockman, et al., supra). Use of these Mabs in sputum cytology for the early detection of lung cancer is described in U.S. Patent No. 5,455,159 (applicant intends this and all other patent references be specifically incorporated herein). Before this immuno-staining technique can be applied to a sputum sample, however, a sufficient sputum specimen must be induced from the lungs of an at-risk patient. A sputum specimen not originating from ϊdeep in the lungsϊ, e.g., nasal aspirates or saliva, are of little use because they typically do not contain exfoliated cells from the lung. Twenty percent of current smokers and 30% of former smokers do not produce a satisfactory sputum specimen for examination despite inhalation of hypertonic saline followed by a rigorous sputum induction procedure (S. Lam, supra). The current invention discloses a method of facilitating induction of deep lung sputum for the purpose of early lung cancer detection.

Researchers have also identified specific gene mutations which correlate with the subsequent development of lung cancer. Mutations of either the ras or p53 genes detected in sputum samples taken during the John Hopkins Lung Project were also detected in subsequently developing lung cancers in 8 of 10 patients (L. Mao, et al., Cancer Res. 54, 1634-37 (1994)). It is suspected that additional gene mutations are involved in the development of lung cancer. However, this preliminary study clearly demonstrates the prognostic potential of PCR analysis of sputum samples.

It is important to keep in mind that the first step in any lung cancer detection method based upon sputum analysis is the collection of an adequate sample of deep lung sputum. The ability to expectorate sputum relies on the coordinated function of the mucociliary escalator and the cough mechanism. The mucociliary escalator relies on the integrated action of three mech.anisms: 1) mucus section by goblet cells and submucosal glands; 2) cilia beating to propel the mucus out of the lungs; and 3) epithelial ion transport systems which maintain the ionic milieu of, and hence, the viscosity of airway surface liquid. It is now known the UTP and related nucleotide compounds modulate all three components of the mucociliary escalator. First, UTP has been shown to increase both the rate and total amount of mucin secretion by goblet cells in vitro (M. Lethem, et al., Am J. Respir. Cell Mol. BioL 9, 315-22 (1993)). Second, UTP has been shown to increase cilia beat frequency in human airway epithelial cells in vitro (D. Drutz, et al., Drug Development Research, 37(3), 185 (1996)). And third, UTP has been shown to increase intracellular Ca ⁺⁺ concentration due to stimulation of phospholipase C as a result of initial occupation of the P ₂ γ2 _T receptor by UTP (H. Brown, et al., Mol. Pharmocol. 40, 648-55 (1991)), and UTP has been shown to increase Cl secretion, and hence, water secretion from airway epithelial cells in vitro (S. Mason, et al, Br. }. Pharmacol. 103, 1649-56 (1991)); M. Knowles, et al., N. Engl. J. Med. 325, 533-38 (1991)). UTP's modulation of all three components of the

mucociliary escalator system results in at least a 2.5-fold improvement in mucociliary clearance (MCC) without any significant side-effects in normal volunteers (as measured by radiolabel techniques and administered on an acute basis — long-term therapy has not yet been tested) (K. Olivier, et al, Am. J. Respir. Crit. Care Med. 154, 217-23 (1996)). It should be noted that in some disease states the cilia are not fully operational, e.g., primary ciliary dyskinesia, chronic bronchitis and the flu. UTP can still increase secretion clearance in patients with PCD via increased mucin production and hydration of the mucus (P. Noone, et al, Am. J. Respir. Crit. Care Med. 153(4), A530 (1996)). Because of UTP's demonstrated effectiveness in improving mucociliary clearance, applicant postulates that aerosolized UTP or U2P4 inhaled into the lungs can be more effective sputum induction agents than the current agents in use ~ hypertonic saline, isotonic saline or distilled water. Sputum induction using nebulized hypertonic saline is traumatizing to a patient because of its foul taste and because of its tendency to irritate the lining of the lungs, bronchi and trachea after the required prolonged inhalation period (C. Parry, Tubercle Lung Dis. 76, 72-76 (1995)). Also, hypertonic saline is usually administered by a specially trained respiratory therapist, and many smaller hospitals and clinics do not have these personnel on staff. Additionally, hypertonic saline is only effective about 70-80% of the time in inducing a sufficient sputum specimen from the lower lung (S. Lam, supra). Because of hypertonic salineϊs shortcomings as a sputum induction agent, many hospitals no longer perform sputum cytology; instead they perform bronchoscopy in an attempt to locate and biopsy the neoplasm. Bronchoscopy is a much more expensive procedure, averaging about $2,000 per procedure, including physicianis fees. Another drawback of bronchoscopy is that it is difficult and time consuming to adequately disinfect the device after every use; this has contributed to the recent rise in nosocomial infections (D. Reeves and N. Brown, Arch. Intern. Med. 155(19) 2050-54 (1995)).

Sputum induction using UTP or U2P4 is expected to be safer, faster, less traumatizing to the patient, requiring less expertise to administer, and may be more reliable in inducing a sufficient quantity and quality of lower lung sputum than hypertonic saline or other sputum induction agents currently in use. Because of these advantages, it is believed that use of UTP or U2P4 as a sputum induction agent will lower health care costs by avoiding the need for bonchoscopy. In combination with the powerful detection methods of Mab immuno-staining or PCR analysis, applicants believe that health care costs can be even more substantially lowered by providing a means of detecting lung cancer in its early and more curable stages. The method of the present invention could be incorporated into a large-scale routine screening program (i.e., every 6 months to 1 year) for the general population or for populations at risk of developing lung cancer, so that lung cancer is detected as early as possible.

SUMMARY OF THE INVENTION A novel method of early detection of lung cancer is disclosed. The method comprises using uridine 5'- triphosphate (UTP), P ¹ , P ⁴ -(uridine 5'-) tetraphosphate (U2P4) or related compounds to induce a sputum sample from the lower lungs of an individual, then analyzing this sputum for the presence of labeled reagents selectively bound to substances in the sputum sample whose enhanced presence correlates with the development of lung cancer. The method of the present invention is particularly useful for individuals who are at high risk for lung cancer, including cigarette smokers or people who have been chronically exposed to airborne carcinogens such as asbestos, coal dust, tin ores, pesticides, fungicides, etc., or groups of people subject to occupational or environmental exposures such as steel workers, blast furnace operators, crop dusters, etc. The method of the present invention could also be incorporated into a routine, periodic screening program (i.e., every 6-12 months) for the general population or high risk population. The method involves: first, administrating a

pharmaceutically effective amount of aerosolized UTP or U2P4 to the lungs of a subject at high risk for lung cancer to induce expectoration of sputum suitable for cytologic analysis; and second, analyzing this sputum for the presence of labeled reagents selectively bound to substances in the sputum sample whose presence correlates with the development of lung cancer. A preferred method of sputum analysis entails Mab immunostaining to determine the enhanced presence of cell surface antigens which have been found to correlate with the development of lung cancer. An additionally effective method of sputum analysis entails PCR analysis to detect gene mutations which correlate with the subsequent development of lung cancer.

Sputum is induced by administering to the patient a compound of Formula I, II, III or IV, or a pharmaceutically acceptable salt thereof, in an amount effective to allow the patient to expectorate or produce an amount of sputum from the lower lung sufficient for subsequent cytologic analysis:

Formula I

wherein:

Xi, X2, and X3 are each independently either O" or S-. Preferably, X2 and X3 are O".

Ri is O, imido, methylene, or dihalomethylene (e.g., dichloromethylene, diflouromethylene). Preferably, Ri is oxygen or difluoromethylene.

R2 is H or Br. Preferably, R2 is H. Particularly preferred compounds of Formula I are uridine 5'-triphosphate [UTP] and uridine 5'-0-(3- thiotriphosphate) [UTPγS].

In addition to Formula I, Formula II, i.e., P ¹ , P ⁴ -di(uridine-5') tetraphosphate [U2P4] is also a preferred embodiment of the invention. Another compound of Formula II is P ¹ , P ⁴ -di(adenosine-5') tetraphosphate [A2P4]. The method of the present invention can also include administering a compound of Formula III (adenosine 5 ¹ triphosphate [ATP] or l,N ⁶ -ethenoadenosine 5'- triphosphate or adenosine 1-oxine 5'-triphosphate), or Formula IV (cytidine 5'- triphosphate [CTP] or 3,N ⁴ -ethenocytidine 5'-triphosphate).

Formula II

wherein:

B is uracil or adenine or l,N ⁶ -ethenoadenine, attached as in Formulae I and III.

Formula III

wherein:

Ri, Xi, X ₂ , and X3 are defined as in Formula I.

R3 and R4 are H while R2 is nothing and there is a double bond between N- 1 and C-6 (adenine), or

R3, .and R4 are H while R ₂ is O and there is a double bond between N-l and C-6 (adeninel -oxide), or

R3, R4 and R2 taken together are -CH=CH-, forming a ring from N-6 to N-l with a double bond between N-6 and C-6 (l,N ⁶ -ethenoadenine).

Formula IV

wherein:

Ri, Xi, X2, and X3 are defined as in Formula I.

R5 and R ₆ are H while R7 is nothing and there is a double bond between N- 3 and C-4 (cytosine), or,

R5, Re and R7 taken together are -CH=CH-, forming a ring from N-3 to-N-4 with a double bond between N-4 and C-4 (3,N ⁴ -ethenocytosine).

After an adequate sputum sample is obtained, an optional first step is to cytologically analyze the sputum to confirm that the sputum originated in the deep lung. This is typically done by looking for the presence of alveolar macrophages which localize in the deep lung and are found in sputum originating from the deep lung. Labeled reagents are then applied to the sputum to determine the presence of tumor-associated substances. One such labeled

reagent is a monoclonal antibody (Mab) used to detect cell surface proteins associated with the development of lung cancer. Another such labeled reagent is an oligonucleotide probe which selectively hybridizes with mutated gene sequences amplified through PCR, such mutated gene sequences correlating with the development of lung cancer.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS The method of the present invention may be used to detect lung cancer at an early stage, while it is typically still surgically rescectable and potentially curable. The method comprises using nebulized uridine 5'-triphosphate (UTP) (or related compounds) which is inhaled into the airways by a subject at risk for lung cancer in order to induce expectoration of sputum. The method of the present invention further comprises analyzing exfoliated lung epithelial cells contained in the sputum specimen by means of immunocytochemical staining with monoclonal antibodies (Mabs) reactive to specific cancer-associated antigens.

UTP is well suited as a sputum induction agent because of its ability to increase mucociliary clearance (MCC). UTP increases MCC in three ways: (1) by increasing the ciliary beat frequency of cilia on the surface of luminal epithelia cells, (2) by increasing the secretions of mucirts by goblet cells, .and (3) by increasing the chloride ion secretion and simultaneously increasing the secretion of water into the periciliary liquid layer by luminal epithelial cells, which would tend to lower the viscosity of the mucus. In disease states where the cilia are not fully operational, e.g., primary ciliary dyskinesia, chronic bronchitis and the flu, UTP can still increase clearance of mucus in these patients by increasing mucin production and hydration of the mucus, therefore making the sputum easier to expectorate.

The compounds of the present invention are illustrated in Formulae I - IV. The preferred embodiments are formula I--UTP, where Xi, X2 and X3=OH, and formula II--U2P4, where B = uracil.

Compounds illustrative of the compounds of Formula I above include: (a) uridine 5'-triphosphate (UTP); (b) uridine 5'-0-(3-thiotriphosphate) (UTPγS); and (c) 5-bromo-uridine 5'-triphosphate (5-BrUTP). These compounds are known or may be made in accordance with known procedures, or variations thereof which will be apparent to those skilled in the art. See generally N. Cusack and S. Hourani, Annals N.Y. Acad. Sci. 603, 172-81 (entitled "Biological Actions of Extracellular ATP"). For example, UTP may be made in the manner described in Kenner, et al., /. Chem. Soc. 1954, 2288; or Hall and Khorana, /. Am. Chem. Soc. 76, 5056 (1954). See Merck Index, Monograph No. 9795 (11th Ed. 1989). UTPγS may be made in the manner described in R. S. Goody and F. Eckstein, /. Am. Chem. Soc. 93, 6252 (1971). For simplicity, Formulae I-IV herein illustrate the active compounds in the naturally occurring D-conf iguration of the sugar, but the present invention also encompasses compounds in the L-configuration, and mixtures of compounds in the D- and L- configurations, unless otherwise specified. The naturally occurring D-conf iguration is preferred. Compounds illustrative of the compounds of Formula II include P ¹ ,? ⁴ - di(uridine-5') tetraphosphate (U2P4) or P ¹ , P ⁴ -di(adenosine-5') tetraphosphate (A2P4). These compounds can be made in accordance with known procedures, or variations thereof which will be described by: P. Zamecnik, et al., Proc. Natl. Acad. Sci. USA 89, 838-42 (1981); and K. Ng and L. E. Orgel, Nucleic Acids Res. 15 (8), 3572-80 (1987). U2P4 can be prepared by methods similar to that described in C. Vallejo, et al., Biochem. Biophys. Ada 438, 304-09 (1976).

Compounds illustrative of the compounds of Formula III above include (a) adenosine 5'-triphosphate (ATP) and (b) l,N ⁶ -ethenoadenosine 5'-triphosphate.

Compounds illustrative of the compounds of Formula IV above include (a) cytidine 5 '-triphosphate and (b) 3,N ⁴ -ethenocytidine 5 '-triphosphate. These compounds can be made in accordance with known procedures, or variations thereof which will be apparent to those skilled in the art. For example, phosphorylation of nucleosides by standard methods such as D. Hoard and D. Ott, /. Am. Chem. Soc. 87, 1785-1788 (1965); M. Yoshikawa, et al., Tetrahedron Lett. 5065-68 (1967) and idem.. Bull. Chem. Soc. (Jpn) 42, 3505-08 (1969); J. Moffatt and H. Khorana, /. Am. Chem. Soc. 83, 649-59 (1961); and B. Fischer, et al., /. Med. Chem. 36, 3937-46 (1993) and references therein. Etheno derivatives of cytidine and adenosine are prepared by known methods such as: N. Kotchetkov, et al.,

Tetrahedron Lett. 1993 (1971); J. Barrio, et al., Biochem. Biophys. Res. Commun. 46, 597 (1972); J. Secrist, et al., Biochemistry 11, 3499 (1972); J. Bierndt, et al., Nucleic Acids Res. 5, 789 (1978); K. Koyasuga-Mikado, et al., Chem. Pharm. Bull. (Tokyo) 28, 932 (1980). Derivatives with alpha, beta and gamma thiophosphorus groups can be derived by the following or by adapting methods of: J. Ludwig and F.

Eckstein, /. Org. Chem. 54, 631-35 (1989); F. Eckstein and R. Goody, Biochemistry 15, 1685 (1976); R. Goody and F. Eckstein, /. Am. Chem. Soc. 93, 6252 (1971).

Compounds of Formulas I, III, or IV where Ri is CCI2 and CF2 can be prepared by methods similar to that described in G. Blackburn, et al., }. Chem. Soc. Perkin Trans. 1, 1119-25 (1984). Compounds of Formula I, II, III where Ri is CH ₂ can be prepared by methods similar to that described in T. Myers, et al., /. Am. Chem. Soc. 85, 3292-95 (1963).

In addition, UTP, ATP, CTP, A ₂ P ₄ , 3,N ⁴ -ethenocytidine triphosphate, 1,N ⁶ - ethenoadenine 5'-triphosphate, adenosine 1-oxide 5'-triphosphate, ATPγS, ATPβS, ATPαS, AMPPCH2P, AMPPNHP, N ⁴ - ethenocytidine and W- ethenoadenosine are commercially available, for example, from Sigma Chemical Company, PO Box 14508, St. Louis, MO 63178.

The active compounds of Formulae I - IV may be administered by themselves or in the form of their pharmaceutically acceptable salts, e.g., an alkali metal salt such as sodium or potassium, an alkaline earth salt, or an ammonium and tetraalkyl ammonium salts, NX4+ (wherein X is O-4 alkyl). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

The sputum induction agent of the present invention may be administered to the airways by a variety of suitable means, but is preferably administered by means of nebulizing a liquid/ liquid suspension via any of several commercially available nebulizer (e.g., jet, ultrasound, etc.). One such nebulizer is the Pari LC Jet Plus™ .

The dosage of active compound to hydrate mucous secretions and stimulate ciliary beat frequency in the airways will vary depending on the state of the subject, but generally an effective amount is the amount sufficient to achieve concentrations of active compound on the airway passages of the subject of from about 10 - to about 10 moles/liter (e.g., for UTP, from about 0.00005 mg/mL to about 50 mg/mL), and more preferably from about 10 - ⁶ to about 10 ^Λ moles/liter (e.g., for UTP, from about 0.0005 mg/mL to about 50 mg/mL).

After a sufficient sputum sample is obtained, it may be possible to directly split the sputum into aliquots, if not, the sputum may be homogenized, concentrated, and placed in Saccomannois preservative solution (SPS, 2% polyethylene glycol in 50% ethanol) using standard methods as described in Saccomanno, et al., Ada Cytol. 2, 305-10 (1963). An optional next step is to cytologically analyze the sputum to confirm that the sputum originated in the deep lung. This is typically done by looking for the presence of alveolar macrophages which usually localize in the deep lung and which are usually found in sputum originating in the deep lung. Next, in the current best mode contemplated, immunocytochemical staining is performed on aliquots of the

preserved samples to determine the presence of tumor-associated biomarker antigens on the surface of exfoliated lung epithelial cells contained in the sputum. A description of the immunostaining procedure, the two monoclonal antibodies used, and the computer-assisted cytometry employed is contained in M. Tockman, et al., Diagnostic Cytopathology 9, 615-622 (1993). In this method, sputum samples may be incubated with murine nomonclonal antibodies 624H12 (American Type Culture Collection Accession Number HB10479, Rockville, MD) and 703D4 (ATCC Ace. No. HB8301), developed against small cell and non-small cell lung cancer, respectively (J. Mulshine, et al., /. Immunol. 131, 497-502 (1983)). These antibodies and the immunostaining procedure are described in U.S. Patent No. 4,569,788 (applicant intends this .and all other patent references be specifically incorporated herein). The immunostaining procedure involves incubation of the sputum with a marker-specific primary antibody, followed by incubation with a biotinylated secondary antibody (directed against the specific immunoglobulin in which the primary antibody was raised), followed by incubation with a biotinylated tertiary antibody (directed against the secondary antibody).

Mab 703DR, a murine I G2b monoclonal antibody, targets an antigen associated with small cell lung cancer; this antigen is a 31 kD protein which is homologous to the ot2βι splice variant of the RNA-binding domain of the pre- mRNA-binding protein, called hnRNP. The antigen detected by Mab 624H12, a rat IgM monoclonal antibody, targets an antigen associated with non-small cell lung cancer; this antigen is a difucosylated ceramide related to the Lewis-X family of antigens. The immunostained tumor-associated antigens may be detected by any suitable screening technique, such as immunoassay, immunoprecipitation assay, or immunohistochemistry assays. A preferred embodiment entails image analysis of the immunostained sputum samples by means of a computer-assisted

imaging program. The intensity of light transmitted through the immunostained sputum specimens is evaluated by a computer program at two frequencies of light. Positive and negative controls .are stained and anaylzed with each run. As a result of immunostaining, the cytoplasm of each cell acquires a brown (diaminobenzidine, i.e., DAB) color, while the nucleus has received a blue

(hematoxylin) counterstain. The DAB stain has a maximum transmission at 600 nanometers (nm), and a transmission minimum at 570 nm. At 600 nm, the cytoplasm of a positively staining cell appears relatively translucent, while at 510 nm the cytoplasm appears opaque. Each image should be calibrated and corrected for shading.

An alternative method of sputum analysis involves PCR amplification and detection of mutated gene sequences, the presence of which correlates with the development of lung cancer. One such PCR analysis is described in L. Mao, et al., Cancer Res. 54, 1634-37 (1994). Sputum DNA is amplified by PCR with primers for K-ras and p53 that contain EcoRI sites to facilitate cloning. Following 35 cycles of amplification, products are cleaved with EcoRI and ligated to Lambda Zap II (Stratagene, La Jolla, CA). XLI-blue cells infected with bacteriophage are plated on L-Agar at a density of 500-3000 plaques/ plate, transferred to nylon membranes, and hybridized with oligonucleotides specific for wild type or mutant K-ras and p53. The oligonucleotides used for hybridizations are labeled with [γ- ³² P] ATP and hybridized according to the method of D. Sidransky, et al., Science 252, 706-709 (1991). Oligonucleotides used for detection include:

WT rus: 5'-GGAGCTGGTGGCGTAGGCAA-3'

Val ⁱ² mutant: 5'-GGAGCTGTTGGCGTAGGCAA-3'

Asp ¹² mutant: 5'-GGAGCTGATGGCGTAGGCAA-3'

Ser ¹² mutant: 5'-GGAGCTAGTGGCGTAGGCAA-3' Cys ¹² mutant: 5'-GGAGCTTGTGGCGTAGGCAA-3'

WT p53: 5'-ATGGGCGCCATGAACCGG-3'

His ²⁷³ mutant: 5'- i GAGGTGCATGTTTGTG-3'

Gly ²⁸¹ mutant: 5'-TGGGAGAGGCCGGCGCA-3'

The foregoing description of the specific embodiments reveals the general nature of the invention that others may readily modify by applying current knowledge without departing from the general concept disclosed herein; therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. The present invention is further explained in the examples which follow. These examples are intended as illustrative of the invention, and are not to be taken as limiting thereof.

EXPERIMENTAL Example 1 Trachael Mucus Study in Sheep

The effects of UTP and U2P4 on trachael mucus velocity (TMV) were studied using the following procedures: The nasal passages of conscious adult ewes were anesthetized with a 2% lidocaine solution. After local anesthesia was produced, a modified endotracheal tube 7.5 mm was placed such that the cuff was just below the vocal cords (verified by fluoroscopy). Inspired air was warmed and humidified. The cuff of the endotracheal tube was inflated only during administration of the test compound to minimize possible impairment of TMV by the cuff. Test compounds were administered by nebulization in a volume of 4 mL over a period of 10-12 min. TMV was measured by fluoroscopy. Ten to twenty radiopaque disks

(Teflon ^® /bismuth trioxide; 1 mm diameter, 0.8 mm thich, weighing 1.8 mg) were introduced into the trachea through a modified suction catheter with a puff of compressed air (3-4 L/min). Velocities of the individual disks were recorded on

videotape from a portable image intensifier unit. Individual disk velocities were calculated by measuring the distance traveled by each disk during a 1 min observation period. Values reported are the means of the individual disk velocities. A collar was worn by the sheep which was used as a standard to correct for magnification errors inherent in the fluoroscope.

Both UTP and U2P4 produced significant dose-related effects on tracheal mucus velocity. The doses ranged from 4 to 400 μmole. Both compounds had their maximal effects at a dose of 400 μmole (4 ml of lO^M). UTP produced a maximal effect of 125 + 7% of baseline (mean + standard error, n = 6). U2P4 produced a maximal effect of 144 + 9% of baseline (n = 6). Both compounds produced their maximal effects 15 min after administration. The highest dose of

UTP produced significant effects on TMV up to 4 h after administration. The effects of U2P4 were significant out to 2 h after administration. Results are shown in Figures 1 - 3.

Example 2

Mucociliary Clearance Study in Sheep

In this study healthy adult ewes were given ^99m Tc-labeled human serum albumin (99m Tc-HSA) via a nebulized aerosol. The ^99m TC-HSA (20mCi) was administered over 5 min through a nasotracheal tube introduced under local anesthesia with 2% lidocaine. After administration of the ^99m Tc-HSA, the animals were given a test compound: either UTP or U2P4. Test compounds were administered by nebulization in a volume of 4 mL over a period of 10-12 min. The test compounds were given at a dose of 400 μmole. After the administration of the test compound, the animals were extubated. Clearance of the radiolabeled particles was monitored with a gamma camera. Measurements were made at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 76, 90, 105 and 120 min. Initial results (n

= 7) have shown that both test compounds promote clearance of the radiolabeled particles (compared to the saline control). Results are shown in Figure 4.

The results of the studies in sheep on tracheal mucus velocity (TMV) and whole lung mucociliary clearance (WLC) demonstrated that UTP and U ₂ P ₄ can enhance mucociliary clearance, resulting in quality sputum samples for subsequent cytological analysis. UTP and U2P4 were able to produce an enhancement of TMV. These data strongly suggest that UTP and U ₂ P ₄ will enhance both TMV and mucociliary clearance, as evidenced by the human clinical data described below.

Example 3 Clinical Study in Normal Healthy Volunteers (Non-Smokers)

- Study Design

This single-center, Phase I Unit study, was a randomized, double-blind, evaluation of escalating, single doses of aerosolized UTP in 48 healthy male volunteers. Four successive groups of 12 volunteers were enrolled at each dose level and were randomized in a 2:1 fashion to receive UTP or placebo. Placebo was normal saline and the four dose levels of UTP evaluated in this study were: 0.5 mg/mL, 5 mg/mL, 25 mg/mL and 45 mg/mL. At the time of dosing, 4 mL of placebo or 4mL of the appropriate UTP solutions were placed in a nebulizer for aerosolization. The purpose of this study was to determine safety and tolerability of UTP at various dose levels and to determine if UTP could enhance the ability of normal, healthy subjects to expectorate sputum containing alveolar macrophages, cells indicative of a deep lung sample, when compared to placebo (normal saline).

- Treatment Assignments and Administration of Study Drug

Each subject was randomly assigned to receive a single dose of either UTP or placebo. Each dose consisted of 4mL of placebo or the appropriate solution of

UTP (0.5, 5, 25 or 45 mg/mL) and was administered using a jet nebulizer (Pari LC PLUS™) powered by a portable compressor set at a flow rate of 14 L/min. Inhalation of placebo or UTP took approximately 8-15 minutes. - Efficacy Results Sputum was collected for the purpose of cytological examination to determine whether the sample contained alveolar macrophages. The presence of alveolar macrophages in a sputum sample indicates that the sample is a quality specimen arising from deep within the lungs (not simply salivary secretions). For this study, sputum was collected for cytological examination at the following times: baseline (pre-dose), immediately post-dosing, post-dosing to 4 hours, 4 hours to 8 hours post-dosing, 8 hours to 12 hours post-dosing and upon rising the following day.

For purposes of data analysis, all patients receiving placebo were combined (n=16) and patients receiving UTP (n=32, all four dose levels) were combined. Data from all 48 subjects were combined for the baseline sputum sample timepoint. Figure 5 illustrates the timecouse for the effect of placebo and UTP on the percentage of sputum samples determined to be positive for alveolar macrophages (AM). Although the majority of the subjects (pre-dose period) could produce a sputum sample at baseline (44/48 subjects spontaneously and 47/48 subjects produced a sample following a simple deep breath and cough maneuver); only -30% of these samples were considered to be a quality sputum sample as evidenced by cytological examination (presence of AMs). Aerosolization of single dose of placebo (normal saline) in 16 subjects failed to improve the percentage of samples determined to contain alveolar macrophages at the immediately post-dosing time point compared to baseline. However, in the UTP combined group (n=32), over 80% of the sputum samples were considered positive for alveolar macrophages, representing a 2.5 fold improvement over baseline and placebo. The effect of UTP to improve the

percentage of samples positive for alveolar macrophages was also evident at the second time point (end of dosing to 4 hours post-dosing); 37% of samples were positive following administration of placebo versus 57% following UTP. The differences observed between UTP and placebo were no longer evident by the 4 hour to 8 hour timepoint at which point both the placebo and UTP groups were essentially the same as the pre-dose values. This is consistent with the relatively short duration of action of UTP as well as findings in a previous study indicating that single, inhaled doses of UTP enhance whole long mucociliary clearance for -1.5 hours.

Example 4 Clinical Studies in Smokers - Study Design

This initial study in smokers was a single-center, randomized, double- blind, evaluation of escalating, single doses of aerosolized UTP. A total of 48 male smokers were enrolled in this study. In order to qualify for the study, the smokers had to be currently smoking .and have relatively normal lung function (forced expiratory volume in 1 second of >80% of predicted). Four successive groups of 12 smokers were enrolled at each of the four dose levels of UTP; subjects were randomized in a 2:1 fashion to receive UTP or placebo. The four dose levels of UTP evaluated in this study were: 0.5 mg/mL, 5 mg/mL, 25 mg/mL and 45 mg/mL. The doses of placebo and UTP were administered as 4 mL of the appropriate solution placed into a nebulizer for aerosolization. The purpose of this study was to determine if UTP could enhance the ability of smokers to expectorate sputum over placebo. Chronic smokers are at significant risk to develop lung cancer. In clinical settings to screen for lung cancer, one agent frequently used for inducing sputum induction is saline; therefore, the placebo chosen for this study was normal saline.

- Treatment Assignments and Administration of Study Drug

Each subject was randomly assigned to receive a single dose of either UTP (one of four doses) or placebo. Each dose consisted of 4mL of the appropriate solution (0.5, 25 or 45 mg/mL) and was administered using a jet nebulizer (Pari LC PLUS™) powered by a portable compressor set at a flow rate of 14 L/min. Inhalation of placebo or UTP took approximately 8-15 minutes.

- Efficacy Results

The amount of sputum expectorated (weight in grams) was collected at baseline (pre-dose), immediately post-dosing, post-dosing to 4 hours, 4 hours to 8 hours post-dosing, 8 hours to 12 hours post-dosing and upon arising the following day.

For purposes of data analysis, all patients receiving placebo were combined (n=16) and patients receiving UTP (n=32, all four dose levels) were combined. All patients (n=48) were included in the baseline (pre-dose) period. As shown in Figure 6, UTP consistently increased the amount of sputum expectorated over placebo (an increase in sputum weight of -70%) at the immediately post-dosing and post-dosing to 4 hour time points. By the 8 hour time point, there was no substantial difference in sputum weights between the UTP and placebo groups. This is consistent with the relatively short duration of action of UTP and the timecourse in the previous study described in healthy volunteers (non-smokers)

- Study Design

This second study in smokers was a single-center, randomized, double- blind, cross-over evaluation of multiple daily doses of the highest dose level of aerosolized UTP evaluated in the previous study. Fifteen smokers were enrolled in this study. Subjects had to be currently smoking and to have relatively normal lung function (forced expiratory volume in 1 second of >80% of predicted) at study entry. The subjects received both UTP (45 mg/ml) and placebo (normal

saline) three times a day for three consecutive days, with a 1 week wash-out between periods. The purpose of this study was to determine if the highest dose of UTP could be effective in enhancing the amount of sputum expectorated over several consecutive days. This mimics the collection of sputum for the purpose of lung cancer screening which is often done by asking patients to collect their sputum each morning for several consecutive days.

- Treatment Assignments and Administration of Study Drug

Each subject was randomly assigned to receive multiple daily doses (three times a day for three consecutive days) of placebo and UTP with at least 1 week between the two dosing periods. Each dose consisted of 4mL of the 45 mg/mL solution or placebo (normal saline) administered using a jet nebulizer (Pari LC PLUS™) powered by a portable compressor set at 14 L/min. Inhalation of placebo or UTP took approximately 8-15 minutes.

- Efficacy Results The amount of sputum expectorated (weight in grams) was collected pre- and post-dosing of UTP and placebo at various time points across the three days of dosing in each study period. Sputum was also evaluated by cytological examination to determine whether the sputum samples contained alveolar macrophages; indicative of a deep-lung sample. Figure 7 illustrates the sputum weights (g) at pre-dosing and post-dosing of placebo and UTP over the 3 days of treatment. As shown in Figure 7, inhalation of placebo (normal saline) did not increase the amount of sputum expectorated immediately post-dosing during any of the three days of dosing (comparison of sputum weights at pre-dosing to immediately post-dosing). In contrast, inhalation of UTP consistently increased the amount of sputum expectorated immediately post-dosing on all three days of dosing (comparison of sputum weights at pre-dosing to immediately post-dosing); the magnitude of the increase in the amount of sputum expectorated (pre to post dosing of UTP) was

consistent on each of the three days for the UTP dosing period. Figure 8 illustrates the cytology data for the sputum expectorated by smokers receiving UTP versus placebo. As shown in Figure 8, smokers receiving UTP were more likely to produce a sputum sample containing alveolar macrophages (AM) on any of the 3 days than smokers receiving placebo (the difference between placebo and UTP was most pronounced on Day 2). There actually appeared to be a trend for placebo to decrease the likelihood of producing a specimen containing alveolar macrophages (comparison of pre-dosing to post-dosing in the placebo group). This study demonstrated that UTP consistently increases the amount of sputum expectorated (that contains alveolar macrophages) over that produced by placebo. This effect of UTP was demonstrated on three consecutive treatment days.

Example 5

Clinical Study in Patients with Chronic Bronchitis

This single center study, conducted at a major academic center in the US, was a randomized, double-blind, cross-over evaluation of escalating, single inhaled doses of UTP in patients with chronic bronchitis. Patients enrolled in this study had to meet the American Thoracic Society definition of chronic bronchitis (excessive mucous production over 3 months of the year, for at least 2 successive years). Patients were included that had mild to moderate airflow obstruction (forced expiratory volume over 1 second >65% of predicted at study entry). A total of 26 (14 females and 12 males) patients were enrolled in this study and the majority were currently smoking. Five successive groups of five subjects (an additional patient was added in one cohort due to a drop-out) received a single dose of placebo and the appropriate dose of UTP (2.5, 5, 15, 25 and 45 mg/mL) in a randomized order. The dose of UTP and placebo were separated by at least 24

hours. The purpose of this study was to more carefully define the timecourse by which UTP enhances the expectoration of sputum in a patient population known to be at high risk for developing lung cancer. In contrast to the previous studies in smokers, the cytologic examination included identification and quantification of both alveolar macrophages as well as respiratory ciliated epithelial cells in the sputum samples.

- Treatment Assignments and Administration of Study Drug Each subject was randomly assigned to receive single inhaled dose of placebo (normal saline) or one of five doses of UTP on two separate days. For dosing, each dose consisted of 4mL of 2.5, 5, 15, 25, and 45 mg/mL solution or placebo (normal saline) administered using a jet nebulizer (Pari LC PLUS) powered by a portable compressor set at 14 L/min. Inhalation of placebo or UTP took approximately 8-15 minutes.

- Efficacy Results The amount of sputum expectorated (weight in grams) was collected at baseline and at various time points post-dosing of UTP and placebo. The timepoints were: immediately to 5 minutes post-dosing, 6 minutes to 30 minutes post-dosing, and 31 minutes post-dosing to discharge (within several hours of post-dosing). For the purposes of analysis, patients receiving placebo across all dose groups were combined (n=25); patients receiving UTP (all doses) were combined; and patients receiving the three highest dose levels (15, 25 and 45 mg/mL) [n=15] were combined. Figure 9 illustrates the effect of placebo versus UTP on the amount of sputum expectorated (weight in grams) at two time points: baseline (spontaneous expectoration) versus immediately to 5 minute post-dosing. UTP (all doses combined) significantly enhanced the amount of sputum expectorated compared to baseline and placebo (all doses combined). The effect of UTP was even more pronounced when comparing the three highest dose levels of UTP

(n=15) to the placebo group. The ability of UTP to enhance the amount of sputum expectorated over placebo and baseline was also quite evident at the later timepoint of 6 minute - 30 minute post-dosing, as shown in Figure 10. Figure 11 shows that at the 31 minute to discharge time point there was essentially no difference between the effect of UTP and placebo on the amount of sputum expectorated indicating that the effect of UTP is manifest over a short timeframe, consistent with the previous studies.

The cytology results from sputum samples collected at the 6-30 minute post-dosing time point are shown in Figures 12 (sputum containing alveolar macrophages) and Figure 13 (sputum containing respiratory ciliated epithelial cells). UTP (all doses combined) and UTP (three highest dose levels combined) significantly improved the percentage of patients producing a sputum sample containing alveolar macrophages (AM) compared to placebo (Figure 12). UTP (all doses combined) and UTP (three highest dose levels combined) also significantly improved the percentage of patients producing a sputum sample containing respiratory ciliated epithelial cells, when compared to placebo (Figure 13). It is noteworthy in Figure 13 that only 8% of the patients were able to produce a sample containing ciliated epithelial cells at baseline or after aerosolization of placebo; whereas, 73% of patients could produce such a sample in the UTP (3 highest dose level) group. These effects of UTP were also observed at the immediately to 5-minute post-dosing timepoint for alveolar ciliated epithelial cells (baseline/ placebo = 8%/4%; UTP (all doses) = 32%; UTP (3 highest doses) = 50%). The effect of UTP had returned to close to the baseline/ placebo values at the 31-minute to discharge timepoint, consistent with the findings on sputum weight.

This study clearly demonstrates that aerosolized doses of UTP (particularly the three highest dose levels of 15, 25 .and 45 mg/mL) are more effective than placebo (normal saline) in eliciting the production of deep sputum

samples containing cells that could be evaluated for pre-cancerous lesions/ changes. Further, UTP can induce the production of a quality sputum sample (containing both alveolar macrophages and ciliated epithelial cells) within a short time frame (within 30 post-dosing). Data from the 4 studies described supports the concept that aerosolized

UTP represents a safe, rapid and non-invasive approach to obtaining a quality sputum sample for the purposes of screening for abnormal cells.

The invention and the manner and process of making and using it, are now which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.