CZ9903637A3 - Identification method of compounds inhibiting tumor process and preparations containing such compounds - Google Patents

Abstract

The solution provides pharmaceutical compositions for the treatment of tumor, consisting of a pharmaceutically acceptable carrier and a compound, which was selected on the basis of activity determination, inhibiting cyclooxygenase, PDE inhibitory activity, inhibitory activity tumor cell growth and PDEZ inhibiting activity. Furthermore, they are isolated phosphodiesterase, pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound selected on the basis of an antitumor evaluation activity of the compound in terms of its effect on activity enhancement PKG, to reduce β-catenin, to phosphorylate β-catenin. Further is providing a method of selecting a compound for treating a tumor, the method identifying the compound for tumor treatment, the method of testing compounds effective in treating a tumor.

Description

Technical field

This invention relates to the use of one or more forms of phosphodiesterase type 2 ("PDE2"), phosphodiesterase type 5 ("PDE5") and / or protein kinase G for the identification of compounds useful for treating and preventing precancerous and tumor processes in mammals, for pharmaceutical compositions. containing these compounds as well as for therapeutic methods of treating tumors with these compounds.

BACKGROUND OF THE INVENTION

Currently, non-surgical treatment of a malignant tumor in those patients whose malignant tumor has developed to the point where the therapeutic benefits of chemotherapy / hormone therapy outweigh their very serious side effects, the administration of one or more highly toxic chemotherapeutic agents or hormone therapy. These side effects are well known to all oncologists and vary from medicine to medicine. However, standard chemotherapeutic agents are mostly used only for short periods of time, often alternating chemotherapy with periods without treatment, thus avoiding excessive burdening of the patient with side effects of drugs. Because of the risk of change, they usually eliminate the side effects of initiating chemotherapy in those patients who experience pre-cancerous processes or who are not continuing long-term chemotherapy or hormone therapy after the tumor has been removed to try to prevent it from recurring.

About ten years ago, there were signs of hope from unexpected sources: non-steroidal anti-inflammatory drugs (“NSAIDs”). The field of cancer and precarcinoma research is crowded with publications describing various biochemical molecules that are overexpressed in tumor tissue. This has led a number of research groups to investigate whether specific, overexpressed molecules are responsible for the disease and whether the tumor process can be attenuated by inhibiting these overexpressions. For example, in the case of familial adenomatous polyposis ("FAP"), Waddell hypothesized in 1983 (Waddel, WR et al., "Sulindac for Polyposis of the Colon", Journal of Scientific Oncology, 24: 83-87, 1983) that while prostaglandins have been overexpressed in these polyps, non-steroidal anti-inflammatory drugs ("NSAIDs") should alleviate the disease state by inhibiting prostaglandin synthetase (PGE2) activity. Therefore, the author administered a non-steroidal anti-inflammatory drug (“NSAID”) sulindac (a PGE2 inhibitor) to several patients with FAP. Waddel found that the polyps had retreated and did not recur after this therapy. Inhibition of PGE2 results from inhibition of cyclooxygenase (COX) by NSAIDs. The success of Wadell with the sulindac compound and the PGE2 / COX relationship seemingly confirmed the role of the other two biochemical targets - PGE2 and COX - in carcinogenesis, and the subsequent literature has reinforced these findings.

An indication of hope for cancer patients was that sulindac undoubtedly exhibited much milder side effects than conventional chemotherapies or hormones, opening up the possibility of treating malignant tumors in the early stages of the disease and compared to conventional chemotherapies for extended periods of time. However, this hope had to be alleviated by the question of whether a compound such as sulindac can be used to treat a detected malignant tumor. This question arose because Waddell only administered sulindac to patients with precancerous conditions, FAP.

This hope was also mitigated by a number of side effects of the NSAIDs themselves. Sulindak and other NSAIDs, when applied for a long time, worsen the digestive tract, where PGE2 plays a protective role. Further, when taken for a long time, they show side effects affecting the kidneys and interfere with normal blood clotting. Unfortunately, during his study, Waddell found that some of his sulindac-treated patients stopped taking the drug because of side effects (see Waddell, WR et al., "Sulindac for Polyposis of the Colon", The American Journal of Surgery, 157: 175-179 (1989)) and most of them have returned to other surgical procedures to control polyps. Therefore, these drugs do not represent for patients with tumors such as FAP, patients with sporadic polyps or for men after prostatectomy with elevated PSA (an increase in PSA in these men indicates a recurrence of a disease that may not yet be present as a detectable, visible malignant tumor) , practical long-term treatment. These side effects also limit the use of NSAIDs in any further indication of a tumor process requiring long-term drug administration. Some scientists have suggested that COX-specific NSAIDs, such as celecoxib, will be used. However, renal and other side effects of these compounds are expected to limit the dosage and duration of treatment with these compounds during long-term anti-tumor indications. Furthermore, recent published data suggest that very high doses of drugs such as celecoxib are required to achieve minimal effect on colon polyps only in predefined colorectal areas. For the treatment of malignant colon cancer, it may be more important that certain colon tumors (eg HCT-116) do not express COX-2 and therefore these inhibitors are not effective against these tumors (see Sheng et al., "Inhibition of Human Colon Cancer Cell Growth By Selective Inhibition Of Cyclooxygenase-2 (J. Clin. Invest., 99 (9): 2254-2259, 1997).

Recent discoveries are further away from COX / PGE2 targets, as these targets may not be primary (or possibly secondary) to successful long-term treatment of cancer patients. Pamukcu et al., U.S. Pat. U.S. Patent No. 5,401,774 revealed that sulfonyl compounds, which were reported to be practically non-PGE2 and COX-inhibiting (and therefore are not NSAIDs or anti-inflammatory compounds), unexpectedly inhibited the growth of a variety of tumor cells including colon polyp cells. These sulfonyl derivatives have been shown to be effective in model colon carcinogenesis in rats and one variant (now referred to as exisulind) has been shown to be effective in clinical trials with FAP patients. Even more significant was the effect in the established malignant tumor: the prostate malignant tumor, which was found in the clinical study below. Recent research has also convincingly established that COX I and / or COX II are not expressed in all tumors, reducing the chance that specific inhibitors of COX I and COX II could be widely therapeutically useful in the treatment of tumor processes (see Lim et al., " Sulindac Derivatives Inhibit Growth and Induction Apoptosis in Human Prostate Cancer Cell Lines (Biochem. Pharmacology, Vol. 58, pp. 1097-1107 (1999) in press).

As with many other proteins overexpressed in tumors, overexpression of PGE2 / COX may not be the cause of some tumors, but rather the result of some. However, by combining these findings the question arose how compounds such as exisulind (which is effective against both COX-expressing and non-COX-expressing tumors) work? What do these compounds do to tumor cells?

Piazza et al., (In US Patent Application Nos. 08/866027 and 09/046739) discovered that compounds (such as exisulind) inhibited GMP-specific cyclic phosphodiesterase (eg PDE5) and that other similar compounds could be via this enzyme, which could lead to the discovery of other compounds that could be developed and formulated into anticancer pharmaceutical compositions.

These pharmaceutical compositions can be highly antitumor and practically do not have the side effects associated with conventional chemotherapeutic agents. It may also not have side effects associated with inhibition of COX or PGE2 if these side effects should be avoided. Anti-tumor compounds that inhibit cGMP-specific PDE can induce apoptosis (a form of programmed cell death) in tumor cells, but not in normal cells. These new compounds are therefore beginning to be referred to as a new class of anticancer agents known as selective apoptotic anticancer drugs ("SAANDs"). Thus, SAANDs contradicted several points of common knowledge '(1) that antitumor compounds cannot be effective without

simultaneous killing of normal cells; (2) that COXs are responsible for tumor processes; and (3) that the prevention of colon tumors by NSAIDs is mediated by inhibition of one or both types of COX.

New research, however, has shown that not all compounds showing classical PDE5 inhibition induce apoptosis in tumor cells. For example, the well-known PDE5 inhibitors, zaprinast and sildenafil, do not induce apoptosis by themselves or inhibit tumor cell growth. However, since proapoptotic PDE5 inhibitors induced apoptosis selectively (i.e. only in tumor cells, not normal ones) and could do so without substantial inhibition of COX, the utility of PDE5 as a tool for testing desirable antitumor compounds is undisputed.

However, it is desirable to adapt the PDE5 assay method to find antitumor, pro-apoptotic but safe compounds so that new pharmaceutical compositions for therapeutic use in the treatment of tumors, including precarcinomas and malignant tumors, can be formulated.

SUMMARY OF THE INVENTION

In researching why some PDE5 inhibitors themselves induced apoptosis while others did not, we discovered a form of GMP-specific cyclic phosphodiesterase activity that has not been previously described. This new phosphodiesterase activity has not been characterized until then. Notwithstanding any specific theory, it is believed that this new PDE activity may be a new PDE2 conformation that substantially lacks cAMP-hydrolytic activity, i. is cGMP-specific. Classical PDE2 is not cGMP-specific (also hydrolyzes cAMP) and is also found in tumor cells. These new PDEs and PDE2 are useful in testing the desired antitumor properties of pharmaceutical compounds. Briefly, apoptosis occurs in tumor cells where PDE5-inhibiting antitumor compounds inhibit PDE5 and PDE2 activities (in its new and conventional conformations). If only PDE5 is inhibited (but not several forms of PDE2), apoptosis does not occur.

Broadly speaking, this new PDE conformation has activity characterized by:

(a) higher specificity for cGMP than for cAMP;

(b) positive cooperative kinetic behavior in the presence of a cGMP substrate;

(c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase.

Other characteristics of the new PDE include: it has reduced susceptibility to zaprinast inhibition and E4021, can be separated from classical PDE5 activity by anion exchange ion exchange chromatography, is not activated by the calcium / calmodulin system, and is insensitive to crolipram, vinpocetin and indolidan.

Another embodiment of the present invention involves assessing whether the compound causes an increase in cGMP-dependent protein kinase G ("PKG") activity and / or a decrease in β-catenin in tumor cells. Unexpected characteristics of SAAND compounds have been found to increase PKG activity and decrease β-catenin in tumor cells exposed to SAAND compounds. We believe that the increase in PKG activity occurs at least in part due to an increase in cGMP caused by inhibition of the corresponding PDEs by SAANDs as described above.

Other characteristics of SAAND are (1) inhibition of PDE5, reported in the aforementioned '694 patent, (2) inhibition of a new conformation of cGMP-specific PDE, (3) inhibition of PDE2; (4) the fact that SAANDs increase intracellular cGMP in tumor cells, and (5) the fact that they decrease cAMP levels in some types of tumor cells.

One embodiment of the novel method of the present invention is to evaluate whether the compound causes an increase in PKG activity in tumor cells and whether the compound inhibits PDE5. Another embodiment of the novel assay method of the present invention is to evaluate whether the compound causes an increase in PKG activity in tumor cells and whether the compound inhibits the novel cGMP-specific PDE described above and / or PDE2. A third embodiment is to assess whether the compound causes an increase in PKG activity in tumor cells and whether the compound causes an increase in cGMP in tumor cells and / or whether it causes a decrease in cAMP levels. The compounds successfully evaluated in these procedures can be applied as SAAND.

Among other things, the present invention relates to novel in vitro and in vivo methods for selecting compounds capable of safely treating tumors and preventing tumor processes, particularly precancerous processes. In particular, the present invention is a method of selecting compounds that can be used to treat and prevent tumors, including precancerous processes. The compounds thus identified may have minimal side effects attributable to COX inhibition and other non-specific interactions associated with conventional chemotherapeutic agents. The compounds of interest may be tested by exposing the above-described new PDEs to the compounds and, if the compound inhibits this new PDE, is further evaluated for its antitumor properties (eg, by in vitro or in vivo model testing or animal or human testing) .

One approach of the present invention therefore includes a test / selection method for identifying compounds effective in the treatment of tumor, which comprises detecting inhibition of this new PDE and / or

* * · · · · · · · · ·

PDE2 by the compound of interest and its inhibition by COX. The assay and selection methods of the present invention further preferably include determining whether the compound inhibits tumor cell growth in vitro or in vivo.

For the development of pharmaceutical compositions and therapeutic treatment of tumor processes, the selection of compounds thus effected can identify potentially beneficial and improved compounds for the treatment of tumor processes more rapidly and with greater accuracy than previously possible. Further benefits are apparent from the following detailed description.

The present invention also encompasses pharmaceutical compositions comprising the compounds and therapeutic methods comprising the compounds.

Overview of the drawings

Figure 1 is a graph of cGMP activities of cGMP phosphodiesterases derived from SW480 tumor cells as measured in the eluent after chromatography on a DEAE Trisacryl M column.

Figure 2 is a graph of cGMP activities of reintroduced cGMP phosphodiesterases derived from SW480 tumor cells as measured in the eluent after chromatography on a DEAE Trisacryl M column.

Figure 3 is a graph of the kinetic behavior of the new PDE of the present invention.

Figure 4 shows the effect of sulfide and sulfone derivatives of sulindac (exisulind) on purified cyclooxygenase activity.

Figure 5 shows the effect of test compounds B and E on COX inhibition.

Figure 6 shows the inhibitory effects of sulindacic sulphide and exisulind on PDE4 and PDE5 purified from tumor cell culture.

Figure 7 shows the effects of sulindacic sulfide on cyclic nucleotide levels in HT-29 cells.

Figure 8 shows the phosphodiesterase inhibitory activity of compound B.

Figure 9 shows the phosphodiesterase inhibitory activity of compound E.

Figure 10 shows the effects of sulindacic sulphide and exisulind on apoptosis and cell necrosis

HT-29.

Figure 11 shows the effects of sulindacic sulfide and exisulind on the inhibition of HT29 cell growth and induction of apoptosis as determined by DNA fragmentation.

Figure 12 shows apoptosis inducing properties of Compound E.

Figure 13 shows apoptosis inducing properties of Compound B.

Figure 14 shows the effects of sulindacic sulphide and exisulind on tumor cell growth.

Ί

Figure 15 shows growth inhibitory activity and apoptosis inducing activity, sulindaculphide and control (DMSO).

Figure 16 shows the growth inhibitory activity of compound E

Figure 17 shows the inhibition of pre-malignant tumor lesions in mouse mammary gland culture by sulindac metabolites.

Figure 18A is an SDS gel of lysates of SW480 cell lysates proteins exposed to drug in the absence of added cGMP. Cells were exposed for 48 h to DMSO (0.03%, lanes 1 and 2), exisulind (200, 400 and 600 μΜ; lanes 3,4.5) and E4021 (0.1, 1 and 10 μΜ; lanes 6,7,8).

Figure 18B is an SDS gel (after exposure to film) from PKG assays in SW480 cell lysates from drug-treated cell lysates in the presence of added cGMP. Cells were exposed for 48 h to DMSO (0.03%, lanes 1 and 2), exisulind (200, 400 and 600 μΜ; lanes 3,4.5) and E4021 (0.1, 1 and 10 μΜ; lanes 6,7,8).

Figure 19 is a bar graph of Western blot results showing the effect of exisulind on β-catenin and PKG levels in tumor cells compared to control.

Figure 20 is a graph of cGMP activities of cGMP phosphodiesterases derived from HTB-26 tumor cells as determined in the eluent after column chromatography on DEAE-Trisacryl M.

Figure 21 is a graph of cGMP activities of cGMP phosphodiesterases derived from HTB-26 tumor cells as determined in the eluent after chromatography on a DEAE-Trisacryl M column with low and high substrate concentrations.

Figure 22 is a graph of cGMP activity of cGMP phosphodiesterases derived from LnCAP tumor cells as determined in the eluent after chromatography on a DEAE-Trisacryl M column.

Figure 23 is a graph of cGMP activities of cGMP phosphodiesterases derived from LnCAP tumor cells as determined in the eluent after chromatography on a DEAE-Trisacryl M column with low and high substrate concentrations.

Figure 24 is a bar graph depicting the binding specificity of non-catalytic cGMP PDE5 binding sites for cyclic nucleotide analogs and selected PDE5 inhibitors.

Figure 25 is a graph of cGMP activities of cGMP phosphodiesterases derived from SW480 tumor cells as determined in the eluent after DEAE-Trisacryl M column chromatography using ethylene glycol in buffer.

Figure 26 is a graph of cGMP activities of cGMP phosphodiesterases derived from SW480 tumor cells grown in rotary culture flasks as determined in the eluent after chromatography on a DEAE-Trisacryl M column.

Figure 27A shows the time dependence of the increase in the amount of histone-linked fragmented DNA in LNCaP cell culture following treatment with 50 µ 50 Compound I.

Figure 27B shows the course of action of Compound I (50 μΜ) on PrEC prostate cells with no effect on DNA fragmentation for up to 4 days.

Detailed Description of Preferred Embodiments

I. New cGMP-specific phosphodiesterase and PDE2 from tumor cells

A. Isolation of the new PDE conformation

Isolated cGMP-specific phosphodiesterase (which appears to be a new conformation of PDE2) was first prepared from a human carcinoma cell line, commonly referred to as SW480, available from the American Tissue Type Collection in Rockville, Maryland, USA. SW480 is a human colon cell line that originates from a slightly different epithelial adenocarcinoma. As discussed below, a similar conformation was also isolated from breast tumors (i.e., linie-26 cell lines) and prostate (LNCAP cell lines).

By "isolated" we mean (as understood in the art) not only isolated from tumor cells, but also produced by recombinant methods (eg, expressed in cell lines of bacterial or other non-human host vectors). However, we now believe that isolation from human tumor cell lines is preferred because we assume that such isolated protein targets have a structure (ie conformation or topography) that is similar, if not the same, as one of the possible native conformations in the tumor cell. . This conformation assists in selecting anti-tumor compounds that will inhibit the target enzyme (s) in vivo.

The activity of the new PDE was first detected in the SW480 colon tumor cell line. To isolate the new phosphodiesterase from SW480, approximately 400 million SW480 cells were cultured to confluent state and after washing twice with 10 ml cold PBS, cells were wiped from a 150 cm ² tissue culture dish area and centrifuged. The pellet cells were resuspended in homogenization buffer (20 ml TMPI-EDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc 2, 0.1 mM EDTA, 0.8% Triton-100, 10 μΜ benzamidine, 10 μΜ TLCK, 2000 U / ml aprotinin, 2 μΜ leupeptin, 2 μΜ pepstatin A) and homogenized in an ice bath (3 times, 20 s / pulse). The homogenized material was centrifuged at 105,000 xg for 60 • 9

9 9 9 <· ·· min. at 4 ° C in a Beckmann L8 centrifuge and the supernatant was diluted with TMPI-EDTA (60 mL) and applied to a 10 ml DEAE-Trisacryl M column, previously equilibrated with TMPI-EDTA buffer. The loaded column was washed with 60 mL (TM) -EDTA and PDE activities were eluted with a 120 mL linear NaOAC (0-0.5 M) gradient in TM-EDTA, at a flow rate of 0.95 mL / min, 1.4 mL / fraction. In 80 fractions that were collected, cGMP hydrolysis was determined immediately (i.e. within minutes). Figure 1 shows the elution profile of a column showing two initial peaks of cGMP PDE activity, peak A and B, which were eluted from 40 to 50 mM and 10 mM respectively. 70 to 80 mM NaOAC. As explained below, peak A is PDE5, while peak B is a novel cGMP-specific phosphodiesterase activity.

PDE cGMP activity was determined in each fraction by the modified two-step radioisotope method of Thomson et al. (Thomson W. J. et al., Adv. Cyclic Nucleotide Res. 10: 69-92, 1979), which is described below. The reaction was run in a 400 µl reaction mixture containing Tris-HCl (40 mM; pH 8.0), MgCl 2 (5 mM), 2-mercaptoethanol (4 mM), bovine serum albumin (30 µg), cGMP (0 , 25 μΜ to 5 μΜ) with a tritium-labeled substrate (200,000 cpm). The incubation time was adjusted to cause less than 15% hydrolysis. The mixture was incubated at 30 ° C, then the reaction was stopped by boiling for 45 s. The mixture was then cooled, snake venom (50 µg) was added and the mixture was incubated for 10 min. at 30 ° C. MeOH (1 mL) was added to stop the reaction and the mixture was transferred to an anion exchange column (Dowex 1-X8, 0.25 mL resin). A second mL of MeOH was added to the eluent and the sample was applied to the resin and after 6 mL of scintillation liquid was added for 1 min. measured on a Beckmann LS 6500 tritium activity.

In order to distribute the cGMP hydrolytic activities of peak A and B, fractions 15 to 30 of the original 80 were further loaded onto a DEAE-Trisacryl M column and eluted with a linear gradient of NaOAC (0 to 0.5 M) in TM-EDTA. For the fractions, cGMP hydrolysis was immediately determined (using the 0.2, 2.5 μΜ substrate described above). The results of the assay are shown graphically in Fig. 2. One observation regarding peak B shown in Fig. 2 is that, in contrast to peak A, increasing concentrations of cGMP substrate significantly increased activity. While this is consistent with being PDE2, the fact that the enzyme characterized in Figure 2 is cGMP-specific (see below) assumes a new conformation compared to the classical PDE2 reported in the literature. Peak A activity shows a clear saturation with the substrate of the high affinity catalytic sites.

«9 · · ♦ 99» 9 9 · 9 9 · 9 9 · 9 99

B. Isolation of classic PDE2 from SW480

Two procedures were found which allowed the isolation of the "peak B" from SW480 cells so that the enzyme had classical PDE2 activity (i.e., it was not cGMP-specific but was stimulated by cGMP). The first procedure involves the growth of SW480 in 850 cm ² rotating culture flasks instead of 150 cm ² tissue culture flasks. SW480 were grown in rotating flasks at 0.5 rpm, each flask containing 200 ml RPMI 1640, 2 mM glutamine and 25 mM HEPES. Cells were harvested as follows. The PBS medium was heated for at least 15 min. at 37 ° C. 200 ml of a solution containing 5% FBS / complete RPMI 1640 medium was prepared and 5 ml of glutamine was added. 5 ml of antibiotic / antifungal mixture was also added.

To 10 ml of 4X Pancreatin solution was added 70 ml of PBS solution. The mixture was left at room temperature. The medium was removed and the flask was rinsed with 4 ml PBS to ensure that the bottom of the flask was washed. The solution was removed by pipette. 4 ml of diluted Pancreatin was added to the flask and the flask was shaken to cover the bottom of the solution. The flask was incubated for 8-10 min. at 37 ° C. Then it was quickly verified under a microscope that all cells were spherical in shape. For better cell separation, the flask was gently turned to the side several times. 10 ml of chilled complete medium was added to the flask to stop the proteolysis of Pancreatin. In order to collect the cells together, the solution in the flask was stirred in a swirling motion. The medium was removed with a 25 ml pipette and the cells were placed on ice in 50 ml centrifuge tubes. The tubes were centrifuged in a clinical centrifuge for 5 min. at 1000 rpm and at 4 ° C and the cells were obtained as a pellet. The supernatant was discarded and each pellet was frozen with liquid nitrogen for 15 s. Harvested cells can be stored at -70 ° C.

PDEs from harvested SW480 cells were isolated by FPLC. A Pharmacia AKTA FPLC was used to control sample loading and elution on a 18 ml DEAE TrisAcryl M column. About 600 million SW480 cells were used for the chromatography resulting in the elution profiles. After resuspension of cells in homogenization buffer (20 ml TMPIEDTA-Triton pH 7.4: 20 mM Tris-HOAc, 5 mM MgAc ₂ , 0.1 mM EDTA, 0.8% Triton-100, 10 μΜ benzamidine, 10 μΜ TLCK, 2000 U / ml aprotinin, 2 μΜ leupeptin, 2 μΜ pepstatin A) samples were manually homogenized. Buffer A contained 8 mM Tris-acetate, 5 mM magnesium acetate, 0.1 mM EDTA, pH 7.5, and Buffer B contained 8 mM Tris-acetate, 5 mM magnesium acetate, 0.1 mM EDTA, 1 M sodium acetate, pH 7.5. The supernatants were applied to the column at a flow rate of 1 ml per minute and the column was then washed at 60 ml of buffer A at a flow rate of 1 ml per minute.

the gradient was 0 to 15% buffer B in 60 ml, 15 to 50% buffer B in 60 ml and 50 to 100% buffer B in 16 ml. Fractions of 1.5 ml were collected during gradient elution.

The elution profile obtained was similar (Fig. 26) to that obtained above for the new PDE activity (see, eg, Fig. 1) except that peak B isolated by this procedure showed cAMP hydrolytic activity at substrate concentration of 0.25 μΜ and which could be activated 2 to 3 times by 5 μΜ cGMP.

The second procedure for isolation of classical PDE2 from SW480 cells involved DEAE column chromatography but not in FPLC (see section IA) and with the adjustment that the buffers contained 30% ethylene glycol, 10 mM TLCK and 3.6 mM β-mercaptoethanol. The addition of these reagents to the buffers caused a change in the elution profile (see Fig. 25), which shifted from the low sodium acetate concentration to the high sodium acetate concentration, so that peak A shifted from 40 mM sodium acetate to 150 mM, peak B from 75 mM to 280 mM and peak C from 200 mM to 500 mM (see Figure 25). Peak B in Fig. 25 was determined with a 2 μΜ cAMP substrate and showed 2-fold activation with a 5 μΜ cGMP solution (see Fig. -Y). Selective PDE2 inhibitor, EHNA, inhibited PDE activity at this peak B with 2 μΜ cGMP with an IC50 of 1.6 μΜ and PDE activity peak B with 2.0 μΜ cAMP with an IC50 of 3.8 μΜ (and an IC50 of 2.5 μΜ after addition of 10 μΜ rolipram).

C. cGMP-specificity of PDE from peak A and new activity from peak B

CGMP-hydrolytic activity (0.25 μΜ cGMP) in the presence or absence of Ca ⁺⁺ or Ca ⁺⁺ CaM and / or EGTA, and cAMP-hydrolytic activity (0.25 µg) were also determined for each fraction after DEAE column chromatography from section IA. μΜ cAMP) in the presence or absence of 5 μΜ cGMP. Neither peak A or peak PDE (fractions 5 to 22; see Fig. 1) significantly hydrolyzed cAMP, meaning that none of them showed activity as a cAMP-hydrolyzing classic family of PDEs (i.e. PDE 1,2,3).

Ca ⁺⁺ (with or without calmodulin) failed to activate cAMP or cGMP hydrolytic activity of both A and B peaks and cGMP failed to activate or inhibit cAMP hydrolysis. These results determined that peaks A and B formed cGMP-specific PDE but not classical or previously known PDE1, PDE2, PDE3 or PDE4.

In the case of a new PDE peak B, as discussed below, the cyclic GMP activated cGMP hydrolytic activity of the enzyme but did not activate any cAMP hydrolytic activity (as opposed to peak B from section IB above). This has shown that the new peak B PDE - the new phosphodiesterase of the present invention - either does not show cGMP-stimulated cAMP hydrolysis * 9 9 9 9 ("cGS") or does not belong to classical or previously known families of PDE2 activities, as known PDE2 isoforms hydrolyze both cGMP and cAMP.

D. Peak A is a classical PDE5, but the new peak B - a new cGMP-specific PDE - is not

To characterize any PDE isoform, kinetic behavior and substrate specificity were determined.

Peak A exhibited typical "PDE5" characteristics. For example, K _m of the enzyme for cGMP was 1.07 μΜ and V _ma x was 0.16 nmol / min / mg. Further, as discussed below, zaprinast (IC 50-1.37 µΜ), E4021 (IC 50 = 3 nM) and sildenafil inhibited peak A activity. Zaprinast further showed inhibition of cGMP hydrolytic activity of peak A, consistent with the results reported in literature.

The peak B PDE of section IA exhibited markedly different kinetic properties from the peak A PDE. For example, using the Eadie-Hofstee yield for peak A, cGMP hydrolysis data provided a simple line that decreased with increasing substrate concentration, suggesting kinetic behavior according to Michaelis-Menten. Using EadieHotfstee yields, peak B in the absence of cAMP showed a new cGMP hydrolysis property with increasing substrate concentration of cGMP having a decreasing pattern (apparent K _m = 8.4) and then an increasing pattern (K _m <1) (see Figure 3). This determined the submicromolar peak B affinity for cGMP (i.e., where K _m <1).

Increased cGMP hydrolytic activity in the presence of increasing concentrations of cGMP substrate was consistent with kinetic studies (i.e., Figure 3) and positive-cooperative kinetic behavior in the presence of cGMP substrate. This was discovered by comparing 0.25 μΜ, 2 μΜ and 5 μΜ cGMP concentrations in the presence of PDE peak B after the second separation at DEAE. This was done to avoid cAMP hydrolysis and to exclude this new enzyme, previously identified as PDE5. In the case of PDE peak B, higher concentrations of cGMP caused a disproportionately higher cGMP hydrolysis, as shown in Figure 2.

These observations indicated that binding of cGMP to the peak B enzyme causes conformational changes in the enzyme. While this confirms the advantage of using the native tumor cell enzyme, the present invention is not limited to the native form of the enzyme having the above characteristics.

9 '· 9 • »·» »· · · ·

Ε. Insensitivity of PDE peak vrcholu to Zaprinast and Sildenafil compared to peak A and their effects on other PDE inhibitors

Different PDE inhibitors were studied using twelve different drug concentrations ranging from 0.01 μΜ to 100 μΜ and using 0.25 μΜ of ³ H-cGMP substrate. IC 50 values were calculated from different slope sigmoid curves obtained using Prism 2.01 (GrapgPad). The results are shown in vtab. While compounds E4021 and zaprinast inhibited peak A, IC 50 values (with high affinities), calculated for new peak B PDE activity (section IA), are significantly elevated (> 50-fold). This confirms that A peak is PDE5. These data further show that the activity of the new PDE of the present invention is insensitive to zaprinast and E4021, which is important for practical purposes.

Table 1

Comparison of PDE inhibitors for peak A and peak B of section IA (cGMP hydrolysis)

Compound PDE inhibitor families IC _5O IC ₅₀ peak A (μΜ) peak Β (μΜ) Ratio (IC50 peak A / peak B E4021 5 0.003 8.4 0.0004 Zaprinast 5 1.4 > 30 <0.05 Compound E 5 and others 0.38 0.37 1.0 Sulindaku sulphide 5 and others 50 50 1.0 Vinpocetin 1 > 100 > 100 EHNA 2.5 > 100 3.7 Indolidan 3 31 > 100 <0.31 Rolipram 4 > 100 > 100 Sildenafil 5 0.0003 > 10 <0.00003

In contrast, sulindacide sulfide and Compound E competitively inhibited peak A and B phosphodiesterase activity (IC 50 = 0.38 µΜ for PDE peak A; 0.37 µΜ for PDE peak B).

For the treatment of tumors and for the selection of useful compounds for this treatment, it is important that peak B (or its form) is insensitive to zaprinast, while peaks A and B are sensitive to sulindaculphide and Compound E. We tested zaprinast, E4021 and sildenafil to found out

4 * ·· * · ** ♦ · ♦ ** * · if it induces apoptosis or inhibits tumor cell growth and the same was found for Compound E. As will be explained below, zaprinast itself did not have significant induction properties apoptosis or growth inhibition, while sulindaculphide and Compound E do. In other words, the ability of a compound to inhibit both peak A and B PDEs is related to its ability to induce apoptosis in tumor cells, whereas if the compound (eg, zaprinast) is specific only to peak A PDEs, the compound will not itself induce apoptosis.

F. Insensitivity of new peak B PDEs to incubation with cGMP-dependent protein kinase G

Additional differences between peak A PDE and new peak B PDE (section IA) were observed in their corresponding cGMP-hydrolytic activities in the presence of different concentrations of cGMP-dependent protein kinase G (which phosphorylates typical PDE5). Peak fractions A and B from section IA were incubated with various concentrations of protein kinase G at 30 ° C for 30 min. After the phosphorylation attempt, cGMP hydrolysis of both peaks was determined. Peak A, in accordance with published information on PDE5, showed increased cGMP hydrolysis in response to incubation with protein kinase G, suggesting that peak A was phosphorylated. However, peak B remained unchanged (i.e., it was not phosphorylated and did not respond to incubation with cGMP-dependent protein kinase G). These data coincide with data for peak A, an isoform, consistent with the known PDE5 family, and peak B of section IA is the activity of a new cGMP-specific PDE.

G. New B peak in prostate and breast cancer cell line

The new peak B was isolated from two other tumor cell lines, the breast tumor cell line, HTB-26, and the prostate tumor cell line, LnCAP, following a procedure similar to that used for its isolation from SW480 above. The protocol has been modified in several ways. For even greater reproducibility when comparing different cell lines, a Pharmacia AKTA FPLC device was used to control sample loading and elution from a DEAE TrisAcryl M column of 18 ml. To obtain reference peak B, SW480 were applied the same procedure several times. 200-400 million SW480 cells were used for elution profiles. 70 million LnCAP cells (see Figs. 22 and 23) were used for the elution profile and 32 million HTB-26 cells (see Figs. 20 and 21) were used in another experiment. After resuspending the cells in homogenization buffer, the samples were manually homogenized. Buffer A contained 8 mM TRIS acetate, 5 mM magnesium acetate, 0.1 mM EDTA, pH 7.5 and buffer B contained 8 mM TRIS acetate, 5 mM magnesium acetate, 0.1 mM EDTA, 1 M sodium acetate, pH 7 , 5. The supernatants were at

»F · ··» «9 ·»

9 9 9 9

9 9

9 9

9 columns applied at a flow rate of 1 ml / min. and then the column was washed with 60 ml of buffer A at a flow rate of 1 ml / min. The gradient used was 0 to 15% buffer B in 60 ml, 60 ml of 15 to 50% buffer B and 50 to 100% buffer B in 16 ml. 1.5 ml fractions were collected during the gradient. Peaks corresponding to cGMP PDE activity were eluted around fraction 65, corresponding to a sodium acetate concentration of 400 mM (see Figures 20-23). This activity was determined at a cGMP concentration of 0.25 μΜ (indicating a submicromolar affinity for cGMP).

Rolipram, a PDE4-specific drug, inhibited most of the cAMP activity of PDE (i.e., cAMP activity was due to PDE4), suggesting that cGMP activities of peak B were more specific for cGMP than for cAMP. All three B peaks (from SW480, HTB-26 and LnCAP) showed no Ca / calmodulin stimulation and were resistant to 100 nM E4021, a specific PDE5 inhibitor such as zaprinast (see Figures 20 and 22). Peaks B also showed a large increase in activity when the cGMP substrate concentration was increased from 0.25 μΜ to 5 μΜ (indicating positive-cooperative kinetics) (see Figures 21 and 23). These three peaks also showed similar inhibition by the exisulind and Compound I below.

II. Inclusion of protein kinase G and β-catenin - in general

To determine what effect, if any, an antitumor cGMP-specific PDE inhibitor, such as exisulind, has on cGMP-dependent protein kinase G ("PKG") in tumor cells containing either a defect in the adenomatous poles of the intestinal pose ("APC") gene ') or a defect in the gene that encodes β-catenin, a number of experiments have been performed. As explained below, this inhibitor causes an increase in PKG activity in these tumor cells. This increase in activity occurs not only due to increased PKG activation in cells containing both defects, but also due to increased PKG expression in cells containing an APC defect. Further, when PKG is immunoprecipitated from tumor cells with both defects, it precipitates with β-catenin.

β-catenin has been shown in a number of different malignant tumors, by researchers finding high levels of β-catenin in cancer patients with tumor suppressing APC gene mutations. In humans with mutations of this gene, thousands of small tumors often appear in the lining of the colon from birth. If the APC gene works properly, it encodes a normal APC protein that is believed to bind to and regulate β-catenin. The discovery that PKG is bound to β-catenin in tumor cells containing either an APC gene defect or β-catenin actually demonstrates that PKG is incorporated into one of the major cellular pathways that leads to a malignant tumor. Furthermore, due to the relationship between cGMP-specific inhibition and PKG increase during φ φ φ · φ · · · · · φ φ φ There is an association between cGMP and PKG / β-catenin / APC defect in these cells with SAAND treatment.

The existence of this association is further supported by the observation that β-catenin itself is reduced when tumor cells containing an APC defect or a β-catenin defect are exposed to SAANDs. This β-catenin reduction is initiated by PKG itself. PKG phosphorylates β-catenin - another new observation associated with this invention. Phosphorylation of β-catenin allows degradation of β-catenin by the ubiquitin-proteasomal system.

Phosphorylation of β-catenin by the PKG enzyme is important in tumor cells because it excludes the effect of APC and β-catenin mutations. The mutated APC protein negatively affects the binding of β-catenin bound to the mutant APC protein, and this change in binding was intended to prevent β-catenin phosphorylation by GSK-3b kinase. In the case of mutant β-catenin, increasing PKG activity also allows phosphorylation of the mutant β-catenin. In tumor cells containing both types of mutation, inhibition of cGMP-PDE and β-catenin phosphorylation (resulting in its degradation) are expected to increase with PKG activity in the tumor.

Briefly, these findings not only lead to new pharmaceutical assay methods for identifying other SAAND-type compounds, but also support the role of inhibiting cGMP-specific PDE in therapeutic approaches to tumor processes. Unexpectedly a wide range of inhibitory effects of SAAND compounds on tumor processes can also be explained by this observation, because, as explained below, both tumors with or without APC defect can be treated.

III. PDE testing of pharmaceuticals

A. General

The novel PDEs of the present invention and PDE2 with or without PDE5 are useful for identifying compounds that can be used to treat or prevent tumors and which are not characterized by a number of side effects.

Malignant tumor and precarcinoma can be considered as a disease involving unregulated cell growth. Cell growth consists of a number of different factors. One factor is how fast cells multiply and another is how fast cells die. Cell death can occur either by necrosis or apoptosis, depending on external conditions. Cell differentiation is another factor that affects the kinetics of tumor growth. In order to find an important target for pharmaceutical therapy, it is important to decide which of many aspects of cell growth is influenced by the compound.

• • * * * * 9 9 9 9 9 9 9 9 9 9 9 9

9

.......

In selecting compounds that inhibit growth and have proapoptotic activity, assay assays based on this technology can be combined with other methods.

The present invention is the result of several important discoveries. First, the inventors have found that desirable inhibitors of tumor cell growth induce apoptotic death of immature malignant tumor cells (see Piazza, G.A., et al., Cancer Research, 55 (14), 3110-3116, 1995). Secondly, several inventors have unexpectedly discovered compounds that selectively induce apoptosis without simultaneous inhibition of COX and also inhibit PDE5. In particular, and in contrast to significant scientific studies, compounds desirable for the treatment of tumor conditions inhibit PDE5 (EC 3.1.4.17). PDE5 is one of at least ten phosphodiesterase gene families. PDE5 and the new PDEs of the present invention are unique in that they selectively degrade cyclic GMP and do not degrade cAMP, while other PDE families selectively degrade / hydrolyze cAMP rather than cGMP or degrade selectively both cGMP and cAMP. Desirably, the desired compounds used to treat cancer processes almost do not inhibit non-selective or cAMP degrading phosphodiesterase types.

B. COX testing

A preferred embodiment of the present invention includes determining the cyclooxygenase inhibitory activity of a compound as well as determining its cGMP-specific PDE inhibitory activity. Test compounds are evaluated for their ability to treat either directly or indirectly tumor processes by comparing their activities against known compounds useful for treating tumor processes. A standard compound known to be effective in the treatment of cancer processes without causing gastric irritation is 5-fluoro-2-methyl-1- (p-methylsulfonylbenzylidene) -3-indenylacetic acid ("exisulind"). Another group of compounds useful for comparable purposes are those known to inhibit COX, such as indomethacin and sulindacic sulfide metabolites: 5-fluoro-2-methyl-1- (p-methylsulfinylbenzylidene) 3-indenylacetic acid (" sulindac sulfide '). Other useful compounds include those known to inhibit cGMP-specific PDEs, such as 1- (3-chloroanilino) -4-phenylphthalazine ("MY5445").

As used herein, the term "precancerous processes" includes syndromes that are represented by abnormal tissue changes, including dysplastic. Examples include dysplastic growths in the colon, breast, prostate or lung tissues or conditions such as dysplastic nevi syndrome, polyposis syndromes, colon polyps, precancerous cervix damage (i.e., cervical dysplasia), esophagus, lung, dyspeoplasia ·· · · Φ φ ♦ · φ φ φ φ φ • • • • φ φ φ φ φ

..............

prostate, breast and / or skin and associated conditions (eg, actinic keratosis), while the processes are or are not clinically identifiable.

As used herein, the term "cancer" or "malignant tumor" refers to lesions that are malignant. Examples include malignant melanomas, malignant breast, prostate, and colon cancer. The terms "tumor process" and "tumors" as used herein refer to both malignant and precancerous processes.

As used herein, PG stands for prostaglandin; PS means prostaglandin synthetase; PGE2 is prostaglandin E2; PDE means phosphodiesterase; COX means cyclooxygenase; cyclic nucleotide, RIA stands for radioimmunomethod.

COX inhibition by a compound can be determined in two ways. One procedure involves measuring PGE2 secretion by intact HL-60 cells following exposure of the cells to the test compound. Another method involves measuring the activity of purified cyclooxygenases (COX) in the presence of a test compound. Both procedures include protocols previously described in the literature, but preferred protocols are listed below.

Compounds can be evaluated for inhibition of prostaglandin E2 ("PGE2") production. This can be determined by measuring PGE2 with the PGE2 enzyme immunoassay kit (EIA) such as the commercially available kit from Amersham, Arlington Heights, IL USA. Suitable cells are those that produce large amounts of PG such as HL-60 cells. HL-60 cells are human promyelocytes that are distributed into mature granulocytes by DMSO (see Collins, SJ, Ruscetti, FW, Gallagher, RE, and Gallo, RC, "Normal Functional Characteristics of Cultured Human Promyelocytic Leukemia Cells (HL-60) After Induction of Differentiation By Dimethylsulfoxide (J. Exp. Med., 149: 969-974, 1979). These differentiated cells produce PGE2 upon stimulation with a calcium ionophore, A23187 (see Kargman, S., Prasit, P. and Evans, JF, Translocation of HL-60 Cell 5-Lipoxygenase, J. Biol. Chem., 266: 23745-237752). (1991). HL-60 are available from ATCC (ATCC: CCL240). They can be cultured in an atmosphere of 5% CO 2 at 37 ° C in RPMI 1640 medium, which is enriched with 20% heat-inactivated fetal bovine serum, 50 U / ml penicillin and a streptomycin solution of 50 pg / ml. To induce myeloid differentiation, cells are exposed to 1.3% DMSO for 9 days, then washed and resuspended in phosphate buffered saline (Dulbecco) to a cell concentration of 3 x 10 ⁶ cells / ml.

Differentiated HL-60 cells (3 × 10 ⁶ cells / ml) are incubated for 15 min. at 37 ° C in the presence of the desired concentrations of test compounds. The cells are then stimulated

with A23187 (5 χ ΙΟ ' ⁶ Μ) 15 min. PGE ₂ secreted into the medium is measured as described above.

As indicated above, the second procedure for determining COX inhibition by a compound of interest consists of measuring COX activity in the presence of a test compound. Two different forms of cyclooxygenase (COX-I and COX-2) have been reported in the literature that regulate prostaglandin synthesis. COX-2 represents an inducible form of COX, while COX-I is a constitutive form. COX-I activity can be measured by the method of Mitchell et al. ("Selectivity of Nonsteroidal Antiinflammatory Drugs as Inhibitors of Constitutive and Inducible Cyclooxygenase," Proc. Natl. Acad. Sci. USA, 90: 11693-11697, which is incorporated herein by reference) that uses COX-I purified from rams. seminal vesicle as described by Boopathy and Balasubramanian, "Purification and Characterization of Sheep Platelet Cyclooxygenase" (Biochem. J., 239: 371-377, 1988, which is incorporated herein by reference). COX-2 activity can be measured using COX-2 purified from sheep placenta as described by Mitchell et al., 1993.

Cyclooxygenase inhibitory activity of a drug can be determined by methods known in the art. For example, Boopathy and Balasubramanian, 1988, described a procedure in which prostaglandin H synthetase 1 (Cayman Chemical, Ann Arbor, Michigan) is incubated at 37 ° C for 20 min. with 100 μΜ arachidonic acid (Sigma Chemical Co.), cofactors (such as 1.0 mM glutathione, 1.0 mM hydroquinone, 0.625 μΜ hemoglobin and 1.25 mM CaCl ₂ in 100 mM TrisHCl, pH 7.4) and tested medicine. After incubation, the reaction can be stopped with trichloroacetic acid. After stopping the reaction by addition of thiobarbituric acid and malonaldehyde, enzyme activity can be measured spectrophotometrically at 530 nm.

A compound that exhibits lower COX-I or COX-2 inhibitory activity compared to its higher combined PDE5 / new PDE / PDE2 inhibitory activities may be a desired compound.

The amount of COX inhibition is determined by comparing cyclooxygenase activity in the presence and absence of the test compound. Residual (i.e. less than about 25%) or no COX inhibitory activity at a concentration of about 100 μΜ is an indication that the compound should be further evaluated for its usefulness in the treatment of cancer processes.

C. Determination of phosphodiesterase inhibitory activity

The compounds may be tested for their inhibitory effect on the activity of the novel phosphodiesterase of the invention using either an enzyme isolated as described above, a recombinant version, or a novel PDE and / or PDE2 together with PDE5. Cyclic nucleotide levels

• · · · · · · · · Whole cells are measured by the RIA method and compared to levels in untreated or zaprinast treated cells.

Phosphodiesterase activity can be determined by methods known in the art, such as, for example, a method using radioactive H cyclic GMP (cGMP) (cyclic 3 ', 5'guanosine monophosphate) as a substrate for the PDE enzyme. (Thomson, WJ, Teraski, WL, Epstein, PM, Strada, SJ, Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which is incorporated by reference). Briefly, a solution of H-cGMP substrate of defined specific activity (0.2 μΜ; 100,000 cpm; containing 40 mM Tris-HCl (pH 8.0), 5 mM MgCl ₂ and 1 mg / ml BSA) is mixed with the test total volume of 400 μΐ. The mixture is incubated for 10 min. at 30 ° C with the isolated PDE of the present invention. The reaction is stopped, for example, by boiling the reaction mixture for 75 s. After cooling in ice, 100 μΐ of a 0.5 mg / ml snake venom solution (O. Hannah poison, available from Sigma) is added and incubated for 10 min. at 30 ° C. This reaction is then quenched by the addition of an alcohol such as 1 ml of 100% methanol. The assay samples are applied to a 1 ml Dowex 1-X8 column and the column is washed with 1 ml 100% methanol. The amount of radioactivity is measured by scintillation counting in a volume corresponding to the dead volume of the column and in the eluate. The degree of phosphodiesterase inhibition is calculated from the amount of radioactivity in the reactions in which the drug was present and by comparing the results obtained with the control (reaction mixture without test compound but drug solution).

The ability of the desired compounds to inhibit the phosphodiesterases of the present invention manifests as an increase in cGMP in tumor cells exposed to the test compound. The amount of PDE activity can be determined by measuring the amount of cyclic GMP in the extract of the treated cells using radioimmunoassay (RIA). In this procedure, HT-29 or SW480 cells were plated and grown to confluent phase. As indicated above, SW-480 cells contained both PDE5 and the novel PDE of the present invention, so that when PDE activity is increased, the combined cGMP hydrolytic activity is simultaneously measured. The test compound at concentrations of about 200 μΜ to about 200 pM is then incubated with the cell culture. After about 24 to 48 hours, the medium is separated from the cells and the cells are dissolved. The reaction is stopped by the addition of 0.2 N HCl / 50% MeOH. A protein sample is taken. The cyclic GMP is purified from an acid / alcohol cell extract by anion exchange chromatography such as a Dowex column. cGMP is dried, acetylated according to a published procedure such as a solution of acetic anhydride in triethylamine (Steiner, AL, Parker, CW, Kipnis, DM, J. Biol. Chem., 247 (4): 1106-1113, 1971). incorporated by reference). Acetylated cGMP is quantified by radioimmunoassay (Harper, J., Brooker, G., Advances in Nucleotide Research, W: 1-33, 1979, incorporated herein by reference). Iodinated ligands (methyl ester of tyrosine) of derivatized cyclic GMP are incubated with standards or unknown samples in the presence of antiserum and appropriate buffers. The antiserum can be prepared by controlled techniques using a cyclic nucleotide-hapten system. Antiserum is from sheep that have been applied with succinyl-cGMP-albumin conjugates and is diluted 1/20,000. Dose interpolation and deviation from standard curves are applied as described previously (Seibert, AF, Thompson, WJ, Taylor, A. , Wilbourn, WH, Barnard, J. and Haynes, J., J. Applied Physiol., 72: 389-395, 1992, incorporated herein by reference).

Culture media can be acidified, frozen (-70 ° C) and analyzed for cGMP and cAMP content.

In addition to the observed increases in cGMP levels in tumor cells due to the action of the desired compounds, a decrease in cAMP content was also observed. It has been observed that the specifically desired compound (i.e., that selectively induces apoptosis only in tumor cells but almost not in normal cells) has the same time course as inhibition of cGMP-specific PDE as an initial action that occurs within minutes with increased cGMP content. Second, treatment of the tumor cells with the desired antitumor compound leads to a decrease in cAMP content within 24 hours. Intracellular drug targets are studied below, but recent data supports the idea that an initial increase in cGMP content and a subsequent decrease in cAMP prevent apoptosis in tumor cells exposed to the desired compounds.

Changing the ratio of two cyclic nucleotides may be a more accurate means for determining the desired cGMP-specific phosphodiesterase inhibitory activity of the test compounds rather than just measuring absolute cGMP, inhibiting cGMP specific phosphodiesterase, or determining only the level of cGMP hydrolysis. In tumor cells that have not been treated with anti-tumor compounds, the cGMP / cAMP ratio is in the range of 0.03 to 0.05 (i.e., 300 to 500 fmol / mg protein for cGMP to 6000 to 8000 fmol / mg protein for cAMP). After exposure of the cells to the desired antitumor compounds, the ratio is increased several-fold (preferably at least a 3-fold increase) resulting from an initial increase in cyclic GMP and a later decrease in cyclic AMP.

Particularly desirable compounds have been observed to achieve an initial increase in cGMP content in treated tumor cells to a cGMP level greater than about 500 fmol / mg protein. Further, a particularly desirable compound causes a later decrease in cAMP content in the treated tumor cells to a cAMP level of less than about 4000 fmol / mg protein.

Radioimmunoassays similar to those described above for cGMP were used to determine the cyclic AMP content. Briefly, cyclic nucleotides are from acid / alcohol • 4 9 Φ φ φ • Φ 9 4 9

9 9 4 4 5

. 4 9 4 9

94494 44

9 4

4 cell extracts purified by anion exchange resin, are dried, acetylated according to published procedures and quantified by radioimmunoassay. The iodinated ligands of derivatized cyclic AMP and cyclic GMP are incubated with standards or unknown samples in the presence of specific antisera and appropriate buffers.

The content of the cyclic nucleotide can be verified by determining the change or accumulation of the cyclic nucleotides in intact cells. Pre-labeled ³ H-adenine (Whalin, ME, Garrett Jr., RL, Thompon, WJ, and Strada, SJ) is used to measure intact cell cAMP according to a published procedure. in intact brain slices (Sec. Mess. and Phos. Protein Research, 12: 311-325, 1989, which is incorporated herein by reference). This procedure measures the conversion of labeled ATP to cyclic AMP and, depending on the specific protocol, the procedure can be used to estimate adenylate cyclase or cyclic nucleotide phosphodiesterase activities in intact cells. Accumulation of cyclic GMP was very low and therefore could not be used to study intact pre-labeled cells according to published procedures (Reynolds, PE, Strada, SJ, and Thompson, WJ). "Cyclic GMP Accumulation in Pulmonary Microvascular Endothelial Cells "Life Sci., 60: 909-918, 1997, which is incorporated herein by reference).

The effect of a compound on PDE inhibitory activity can also be determined in a tissue sample. Tissue biopsies in humans or tissue from anesthetized animals are taken from subjects exposed to the test compound. The tissue sample is homogenized in 500 μΐ 6% TCA. A known amount of homogenate is taken for protein determination. The remaining homogenate is left for 20 min. in ice to precipitate proteins. Next, the homogenate is centrifuged for 30 min. at 15,000 g and at 4 ° C. A supernatant and a pellet are thus obtained. The supernatant is washed 4 times with 5 volumes of water saturated with diethyl ether. The top ether layer is removed after each wash. The aqueous ether extract is dried. The dried sample may be frozen for further use or may be used immediately. The dried extract is dissolved in 500 μΐ of assay buffer. The amount of cGMP-specific inhibition is determined by measuring the amount of cyclic nucleotides by the RIA method described above.

The amount of inhibition is determined by comparing the activity of the new PDE (or PDE2) in the presence and absence of the compound. Inhibition of the activity of a new PDE (or PDE2) is an indication that the compound is useful for the treatment of tumor processes. Significant inhibitory activity, greater than that of exisulind, more preferably greater than 50% at a concentration of 10 μΜ or less, is an indication that the compound could be further evaluated for its antitumor

4 · ··

44

4 4 4

4 4 4

4 4 4

4 4 4

44 properties. More preferably, the IC50 of the inhibition of the new PDE for the compound for which further use is considered should be less than 50 μΜ.

D. Determining whether the compound prevents tumor cell growth

In an alternative embodiment, the method of the present invention further comprises determining whether the compound prevents tumor cell growth.

Different cell lines may be used in the sample, depending on the tissue being tested. For example, these cell lines include: SW-480 - colon adenocarcinoma; HT-29 - colon adenocarcinoma, A-427 - lung adenocarcinoma; MCF-7 - breast adenocarcinoma and UACC-375 melanoma line; and DUI45- prostate cancer. Cytotoxicity data obtained using these cell lines indicate the inhibitory effect of tumor lesions. These cell lines are well characterized and are used by the United States National Cancer Institute as a test program for new cancer drugs.

The ability of compounds to inhibit tumor cell growth can be measured using the human colon carcinoma cell line HT-29 obtained from ATCC. HT-29 cells have been characterized as an important model of colon cancer cell culture (Fogh, J., and Trempe, G. In: Human Tumor Cells in Vitro, J. Fogh (eds.), Plenary Lecture, New York, p. 115-159, 1975). HT-29 cells are maintained in a humidified atmosphere of 95% air and 5% CO 2, at 37 ° C, in RPMI medium supplemented with 5% fetal calf serum (Gemini Bioproducts, Inc., Carlsbad, CA), 2 mM glutamine and 1% antibiotic / antifungal solution. Briefly, HT-29 cells are dispensed into 96-well microtiter plates so that there are 500 cells per well and the plates are incubated for 24 h at 37 ° C. The compound is then added. Each cell count is performed 6 times. After 6 days, cells are fixed by the addition of chilled trichloroacetic acid such that the final concentration is 10% and the protein content is measured by a colorimetric method for protein determination using sulforhodamine B (SRB) described in the literature by Skehan, P., Storeng, R. Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, JT, Bokesch, H., Kenney, S., and Boyd, MR. "New Colorimetric Assay For Anticancer Drug Screening," J. Nati Cancer Inst. 82: 1107 to 1112, 1990, which is incorporated herein by reference.

In addition to SRB assays, a variety of other growth inhibition assays are available that can replace SRB assays. These include counting viable cells after fixation with trypan blue solution, labeling cells capable of DNA synthesis, BrdU or radiolabeled thymidine, fixating viable cells neutral red, or fixating viable MTT cells.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Significant inhibition of tumor cell growth, ie, greater than 50% at doses of 100 μΜ or less, is another indication that the compound is useful for the treatment of tumors. More preferably, the IC 50 value that is used for comparison is determined. This value is the concentration of drug that is required for 50% inhibition of tumor cell growth compared to control. For a compound further considered for use in the treatment of cancer processes, the IC50 should preferably be less than 100 μΜ.

E. Determining whether the compound induces apoptosis

In a second alternative embodiment, the assay procedure of the present invention further comprises determining whether the compound induces apoptosis in tumor cell cultures.

Two different forms of cell death can be described using morphological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased plasma membrane permeability; the cells swell and the plasma membrane is broken within minutes. Apoptosis is characterized by membrane blistering, cytoplasmic condensation, and activation of endogenous endonucleases.

Apoptosis naturally occurs during normal tissue transformation and during embryonic development of organs and limbs. Apoptosis is also induced by cytotoxic T lymphocytes and natural cell killer, ionizing radiation and reliable chemotherapeutic drugs. It is believed that inappropriate regulation of apoptosis plays an important role in a number of pathological conditions including cancer, AIDS, Alzheimer's disease, and the like. Compounds can be tested for induction of apoptosis by tumor cell cultures maintained under the conditions described above. The treatment of cells with the test compounds involves either pre-confluent or post-confluent cultures and treatment for 2 to 7 days at various concentrations. Apoptotic cells are measured in both infected and "floating" parts of cultures. After a centrifugation wash step (10 min, 2000 rpm), both parts are collected by removing the supernatant, trypsinizing the infected cells and joining the two preparations. A protocol for treating tumor cells with sulindac and related compounds to obtain a significant degree of apoptosis has been described in the literature (see Piazza, GA, et al., Cancer Research, 55: 3110-3116, 1995, which is incorporated herein by reference). . New significant features include collecting both floating and attacked cells, determining an optimal duration of action and dosing such that apoptosis is observed, and identification of cell culture-optimal conditions.

After the compounds are treated with the cultures, apoptosis and necrosis can be determined by fluorescence microscopy after previous labeling with acridine orange and ethidium

bromide. A method for measuring the number of apoptotic cells has already been described by Duke and Cohen, "Morphological And Biochemical Assays Of Apoptosis," Current Protocols In Immunology, Coligan et al., Eds., 3.17.1 to 3.17.16 (1992, incorporated by reference).

For example, floating and infested cells can be harvested by trypsinization and washed three times with PBS. Aliquots of cells can be centrifuged. The pellet can then be resuspended in medium and a mixture of dyes containing acridine orange and ethidium bromide, which is prepared in buffer and mixed gently. The mixture can then be placed on a slide and the major morphological features of apoptosis can be determined.

Apoptosis can also be quantified by measuring the increase in DNA fragmentation in cells treated with test compounds. For the quantitative in vitro determination of cytoplasmic histone-linked DNA fragments (mono and oligonucleosomes), a commercial photometric EIA (Cell Death Detection ELISA ^Oxygen , Cat. No. 1774425, Boehringer Mannheim) is available. This assay is based on the principle of sandwich enzyme immunoassay using murine monoclonal antibodies against DNA and histones. This allows specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates.

In addition to assaying apoptosis with a kit, apoptosis can be measured as follows. The sample (cell lysate) is placed in a microtiter plate coated with streptavidin ("MTP"). Furthermore, a mixture of anti-histone-biotin antibody and anti-DNA peroxidase antibody conjugates is added and incubated for 2 hours. During incubation, the anti-histone antibody binds to the histone nucleosome component while fixing the immunocomplex to streptavidin-coated MTP via biotinylation. The peroxidase-labeled anti-DNA antibody reacts with the DNA component of the nucleosomes. After removal of unbound antibodies in the washing step, the amount of nucleosomes is quantified by peroxidase retained in the immunocomplex. Peroxidase is determined photometrically with ABTS7 (2,2'-Azido- [3-ethylbenzthiazoline sulfonate]) as a substrate.

For example, SW-480 colon adenocarcinoma cells were dispensed into a 96-well MTP microtiter plate such that the cell density was 10,000 cells / well. Cells were then exposed to the test compound and allowed to incubate at 37 ° C for 48 h. After incubation, the MTP was centrifuged and the supernatant was discarded. The cell pellet in each well was resuspended in lysis buffer for 30 min. Lysates were centrifuged and aliquots of the supernatant (i.e., cytoplasmic fractions) were transferred to streptavidin-coated MTPs. Care should be taken that lysed pellets (ie cell nuclei containing high molecular unfragmented DNA) are not mixed in the MTP. The samples were then analyzed.

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 · 9

The fold stimulation (FS = OD _{m and} x / OD _in h), as an indicator of apoptotic response, is determined at given concentrations for each test compound. EC 50 values can also be determined by evaluating a number of concentrations of the test compound.

A statistically significant increase in apoptosis (i.e., greater than 2-fold stimulation at 100 μΜ) is another indication that the compound is useful for the treatment of tumors. More preferably, the EC50 for apoptotic activity for a compound that is further considered for use in the treatment of cancer is less than 100 μΜ. EC ₅₀ is defined herein as the concentration that causes 50% induction of apoptosis compared to vehicle treatment.

F. Model tests of organ culture of mammary gland

Test compounds identified by the above procedures can be tested for anti-tumor activity based on their ability to inhibit the progression of tumor processes in the mammary organ culture system. This mouse mammary organ culture technique has been successfully used by other scientists to study the effects of known anti-tumor agents such as certain NSAIDs, retinoids, tamoxifen, selenium, and certain natural products and can be used to verify the accuracy of the assay methods of the present invention.

For example, female BALB / c mice can be treated with a combination of estradiol and progesterone, which are administered daily to induce a hormone response in vitro in the gland. Animals are sacrificed and the mammary glands are aseptically excised and incubated for 10 days in a growth medium supplemented with insulin, prolactin, hydrocortisone and aldosterone. DMBA (7,12-dimethylbenz (a) anthracene) is added to the medium to induce the formation of premalignant processes. The fully developed glands are then deprived of prolactin, hydrocortisone, and aldosterone, which arise in the regression of the glands, but not in premalignant processes.

The test compound is dissolved in DMSO and added to the culture. At the end of the culture period, the glands are fixed in 10% formalin, fixed with salum carmine and placed on glass slides. The occurrence of mammary gland damage is the ratio of damaged mammary glands to glands without damage. The incidence of mammary gland involvement in the glands that were under the influence of the test compound is compared with those that were not treated with the compound.

The area occupied by mammary gland damage can be quantified by projecting the gland image into digital form. The area corresponding to the gland is found in the digital image and considered 100%. The digitized image also outlines a computer-quantified space occupied by any structure without regression.

• *

DETAILED DESCRIPTION OF THE INVENTION

Experimental results

In a variety of protocols, a number of compounds have been tested for use in cancer treatment. The results of these tests are shown below. The test compounds are further indicated by the following pi exchange code:

A. rac-threo- (E) -1- (N, N'-diethylaminoethanethio) -1- (butane-1 ', 4'-olido) - [3', 4 ': 1,2] -6-fluoro -2-methyl-3- (p-methylsulfonylbenzylidene) -indane;

B. (Z) -5-Fluoro-2-methyl-1- (3,4,5-trimethoxybenzylidene) -3-acetic acid;

C. (Z) -5-fluoro-2-methyl-1- (p-chlorobenzylidene) -3-acetic acid;

D. rac- (E) -1- (Butane-1 ', 4'-olido) - [3', 4 ': 1,2] -6-fluoro-2-methyl-3- (p-methylsulfonylbenzylidene) - 1-Indanyl-N-acetylcysteine;

E. (Z) -5-Fluoro-2-methyl-1- (3,4,5-trimethoxybenzylidene) -3-indenylacetamide, N-benzyl;

F. (Z) -5-Fluoro-2-methyl-1- (p-methylsulfonylbenzylidene) -3-indenylacetamide,

N, N'-dicyclohexyl;

G. ribo- (E) -1-triazolo- [2 ', 3': Γ ', 3] -1- (butane-1', 4'-olido) - [3 ', 4': 1,2] -6-fluoro-2-methyl-3- (p-methylsulfonylbenzylidene) -indane;

H. rac- (E) -1- (Butane-1 ', 4'-olido) - [3', 4 ': 1,2] -6-fluoro-2-methyl-3- (p-methylsulfonylbenzylidene) - (Indanyl-glutathione).

Example 1 - Determination of COX inhibition

The COX inhibitory activity of the reference and test compounds was analyzed according to the COX assay protocol. Giant. 4 shows the effect of different concentrations of sulindaculphide or exisulind on the activity of purified cyclooxygenase (Type 1). Activity

The cyclooxygenase was determined using purified cyclooxygenase from the ram seminal vesicles as described previously (Mitchell et al.) The IC ₅₀ calculated for sulindaculphide was about 1.76 μΜ while for exisulind it was higher than 10,000 μΜ. These data show that sulindacic sulphide is a COX-1 inhibitor, unlike exisulind. Similar data was obtained for the COX-2 isoenzyme (Thompson et al., Journal of the National Cancer Institute, 87: 1259-1260, 1995).

Figure 5 shows the effect of test compounds B and E on COX inhibition. The COX activity was determined as in the case of the compounds shown in FIG. 4. The data show that neither test compound B nor E significantly inhibited COX-1.

Table 2

Cyclooxygenase inhibitory activity in a number of compounds

Reference compounds % inhibition at 100 μΜ Indomethacin 95 MY5445 94 Sulindaku sulphide 97 Exisulind <25 Test compounds % inhibition at 100 μΜ AND <25 (B) <25 C 87 D <25 E <25

In accordance with the protocol described above, COX inhibitory activity was evaluated for compounds A to E, as shown in Tab. 2. Compound C at 100 μΜ inhibited COX by more than 25% and was therefore not selected for further testing.

Example 2- Determination of cGMP PDE inhibition

The reference and test compounds were analyzed for their cGMP PDE inhibitory activity in accordance with the assay protocol described above. Giant. 6 shows the effect of different concentrations of sulindacic sulphide and exisulind on PDE4 or cGMP PDE activity, purified from HT- cell culture cells. 29 have been described previously (WJ Thompson et al.). The IC50 of sulindacide for PDE4 inhibition was 41 μΜ, and for cGMP PDE inhibition was 17 μΜ. The IC50 of exisulind for inhibition of PDE4 was 181 μΜ, and for inhibition of cGMP PDE was 56 μΜ. These data show that both sulindacic sulfide and exisulind inhibit phosphodiesterase activity. Both compounds showed higher selectivity for the cGMP PDE isoenzyme form than for the PDE4 isoform.

Giant. 7 shows the effect of sulindaculphide on cGMP or cAMP production, which was determined in HT-29 cell culture according to the assay described. HT-29 cells were exposed to sulindac sulphide for 30 min. and cGMP or cAMP was measured by a suitable radioimmunoassay. As indicated by sulindac sulphide, cGMP levels increase by more than 50% with an EC50 of 7.3 μΜ (Fig. 7A). The cAMP levels were not altered by the compound, although the known PDE4 inhibitor rolipram increased cAMP (Fig. 7B). The data demonstrate the pharmacological importance of inhibiting cGMP PDE relative to PDE4.

Giant. 8 shows the effect of the indicated dose of test compound B on cGMP PDE or PDE4 phosphodiesterase isoenzymes. The calculated IC50 was 18 μΜ for cGMP PDE and 58 μΜ for PDE4.

Giant. 9 shows the effect of the indicated dose of test compound E on PDE4 or cGMP PDE. The calculated IC50 was 0.08 μΜ for cGMP PDE and greater than 25 μΜ for PDE4.

Table 3

CGMP PDE inhibitory activity found in a number of compounds

Reference compounds % inhibition at 10 μΜ Indomethacin 34 MY5445 86 Sulindaku sulphide 97 Exisulind 39 Test compounds % inhibition at 10 μΜ AND <25 (B) <25 C <25 D 36 E 75

For compounds listed in Tab. 3, PDE inhibitory activity was evaluated according to the above protocol. Of the compounds that did not inhibit COX, only Compound E was found to cause greater than 50% inhibition at a concentration of 10 μΜ. As shown in Figure 8, Compound B exhibits greater than 50% inhibition at 20 μΜ. For this reason, depending on the dose level used in the single dose test, compounds that may be active at slightly higher doses may be captured. The dosage used is individual and may be reduced if certain levels of the compound at which the compound is active are detected. Thus, even more potent compounds can be identified.

Example 3 - Apoptosis Assay

For the reference and test compounds, the inhibitory activity of the new PDE was determined in accordance with assay protocols. In accordance with these protocols, FIG. 10 shows the effect of sulindacic sulphide and exisulind on apoptotic and necrotic cell death. HT-29 cells were exposed to indicated doses of sulindac or exisulind for 6 days. Apoptotic and necrotic cell death was determined first (Duke and Cohen, V: Current Protocols in Immunology, 3.17.1 to 3.17.16, New York, John Wiley and Sons, 1992). The data show that both sulindacide and exisulind are capable of causing apoptotic cell death without inducing necrosis. All data was obtained from the same experiment.

Figure 11 shows the effect of sulindacide and exisulind sulphide on tumor growth inhibition and apoptosis induction as determined by DNA fragmentation. The upper figure (1 IA) is the inhibition of growth (open symbols, left axis) and DNA fragmentation (closed symbols, right axis) by exisulind. The bottom figure (11B) is the inhibition of growth (open symbols) and DNA fragmentation (closed symbols) with sulindacide sulphide. Growth inhibition was determined by SRB after 6 days of treatment. DNA fragmentation was determined after 48 h treatment. All data was obtained from the same experiment.

Figure 12 shows properties of Compound E that induce apoptosis. HT-29 colon adenocarcinoma cells were treated with the indicated concentration of compound E for 48 h and then apoptosis was determined by DNA fragmentation. The calculated EC50 was 0.05 μΜ.

Figure 13 shows properties of Compound B that induce apoptosis. HT-29 colon adenocarcinoma cells were treated with the indicated concentration of compound B for 48 h and apoptosis was determined by DNA fragmentation method. The calculated EC50 was 175 μΜ.

»Ό 1 1 1 1 1 1 1 1 1 1 · 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 · ·· · «· ·· ··

Table 4

Apoptosis inducing activity of many compounds

Reference compounds times the induction at 100 μΜ Indomethacin <2.0 MY5445 4.7 Sulindaku sulphide 7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0 Sildenafil <2.0 EHNA <2.0 Test compounds times the induction at 100 μΜ AND <2.0 (B) 3.4 C 5.6 D <2.0 E 4.6

In accordance with the multiple induction protocol, apoptosis-inducing activity, shown in Tab. 4. Compounds B, C and E exhibit significantly more than twice the apoptosis inducing activity at a dose of 100 μΜ. Of these three compounds, only compound B did not inhibit COX at given doses, but inhibited cGMPspecific PDE.

Apoptosis inducing activity has been determined in a number of phosphodiesterase inhibitors. The data are shown in Tab. 5, see below. HT-29 cells were treated with various phosphodiesterase inhibitors for 6 days. Thereafter, apoptosis and necrosis were morphologically determined according to the described procedure, after labeling with acridine orange and ethidium bromide. The data show that new cGMPspecific PDE is useful for testing compounds that induce apoptosis of HT-29 cells.

.:

• • · · · · · · · · ·

Table 5

Data ο induction of apoptosis in PDE inhibitors

Inhibitor reported selectivity % apoptosis % necrosis Vehicle 8 6 8-Methoxy-IBMX PDE1 2 1 Milrinon PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445 PDE5 80 5 IBMX non-selective 4 13

Example 4 - Determination of Growth Inhibition

The reference and test compounds were analyzed for their PDE5 inhibitory activity in accordance with the assay protocol. Figure 14 shows the inhibitory effect of different concentrations of sulindaculphide and exisulind on HT-29 cell growth. HT-29 cells were exposed to different doses of exisulind (triangles) or sulindac sulphide (squares) for 6 days. Cell counts were measured by the sulforhodamine method (SRB method) described previously (Piazza et al., Cancer Research, 55: 3110-3116, 1995). The IC50 for sulindacide was approximately 45 μΜ and for exisulind 200 μΜ. The data show that both sulindacic sulphide and exisulind are capable of inhibiting tumor cell growth.

Figure 15 shows growth inhibitory activity and apoptosis inducing activity for sulindaculphide. The time course of the experiment is shown for HT-29 cells treated with vehicle, 0.1% DMSO (open symbols) or sulindac sulphide at 120 μΜ (closed symbols). Growth inhibition (15A above) was measured by counting viable cells after fixation with trypan blue solution. Apoptosis (15B below) was detected by morphological determination after fixation with acridine orange and ethidium bromide solution as described above (Duke and Cohen, in: Current Protocols in Immunology, 3.17.1 to 3.17.16, New York, John Wiley and Sons, 1992). The data show that sulindacic sulphide is able to inhibit tumor cell growth and that the effect is associated with an increase in apoptosis. All data is from the same experiment.

Figure 16 shows the growth inhibitory activity of test compound E. HT-29 colon adenocarcinoma cells were exposed to the indicated concentration of compound E for 6 days and the cell number was determined by the SRB method. The calculated IC50 was 0.04 μΜ.

Table 6

Growth inducing activity found for many compounds

Reference compounds % inhibition at 100 μΜ Indomethacin 75 MY5445 88 Sulindaku sulphide 88 Exisulind <50 E4021 <50 Sildenafil <50 Zaprinast <50 Test compounds % inhibition of concentration at 100 μΜ AND 68 (B) 77 C 80 D 78 E 62 Compounds A to E were tested in accordance with the above test protocol growth inhibitory activity, results are shown all test compounds activity. vtab. 6. At a single dose of 100 μΜ they showed A number of phosphodiesterase inhibitors have been evaluated for their growth inhibitory activity cells. Data are shown in Table. 7. HT-29 cells were exposed to various inhibitors phosphodiesterase for 6 days. Cell growth was determined by the SRB method described. Below together, the data presented above shows that inhibitors of the new PDE effectively inhibited tumor cell growth. Table 7 Data indicating cell growth inhibition exhibited by PDE inhibitors Inhibitor reported selectivity growth inhibition (IC50, μΜ) 8-Methoxy-IBMX PDE1 > 200 μΜ

9-9 9-9 9 9 9 9 9 9 9 9

Table 7-continued

Inhibitor known selectivity growth inhibition (IC50, μΜ) Milrinon i PDE3 > 200 μΜ RO-20-1724 PDE4 > 200 μΜ MY5445 5 PDE5 5 μΜ IBMX non-selective > 100 μΜ Zaprinast PDE5 > 100 μΜ Sildenafil PDE5 > 100 μΜ E4021 PDE5 > 100 μΜ

In order to show the effectiveness of this method of testing various forms of tumor processes, the compounds were tested on a number of cell lines. The effects of sulindacic sulphide and exisulind on various cell lines were determined. The data are shown in Tab. 8. IC ₅₀ values were determined by the SRB method. The data show the broad efficacy of these compounds, whose doses are comparable, to a wide array of tumor processes. Therefore, the compounds thus identified and selected in the present invention should be useful for the treatment of multiple forms of cancer.

Table 8

Growth inhibition data for different cell lines

Cell type / Tissue specificity IC50 (μΜ) sulindaku sulphide exisulind Compound E * HT-29, large intestine 60 120 0.10 HCT116, large intestine 45 90 MCF7 / S, breast 30 90 UACC375, melanoma 50 100 ALIGN! A-427, lungs 90 130 Bronchial epithelial cells 30 90 NRK, kidney (not ras-transformed) 50 180 KNRK, kidney (ras-transformed) 60 240 PC3 Human prostate cancer 82 0.90

Table 8-continued

Cell type / Tissue specificity IC ₅₀ (μΜ) sulindaku sulphide exisulind Compound E * Colo205 1.62 DU-145 0.10 HCT-15 0.60 MDA-MB-231 0.08 MDA-MB-435 0.04

* Determined by the neutral red method as described by Schmid et al. In Proc. AACR Vol

39, p. 195 (1998).

Example 5 - Activity in a model organ culture of the mammary gland

Giant. 17 shows the inhibition of premalignant processes in the mammary organ culture caused by sulindac metabolites. The mammary organ culture experiment was performed according to the previously described procedure (Mehta and Moon, Cancer Research, 46: 5832-5835, 1986). The results show that sulindac and exisulind effectively inhibit the formation of premalignant lesions, while sulindac sulphide was inactive. The data support the hypothesis that inhibition of cyclooxygenase is not necessary for the antitumor properties of the desired compounds.

Analysis

For the selection of compounds for the treatment of tumor processes, the present invention provides the principle of comparing experimental data of test compounds. Within the scope of the present invention, the test compounds may be classified according to their potential use in the treatment of human tumors. These compounds having the desired effects can be selected for further testing and subsequent use in humans.

See below in Tab. 9 shows the quality data of various test compounds along with several protocols. The data show that exisulind, Compound B and Compound E exhibit the activity required to pass the 4 point assay: do not inhibit COX, effectively inhibit cGMP-specific PDE, inhibit growth, and induce apoptosis. The activity of these 9 9 9 9 9 9 9 9 9

9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9 ·

9 9 · · · · · · · ·

999 9 9 99 9 99 99 99 compounds in the mammary organ culture confirm the efficacy of the invention. Based on the evaluation of the quality of the test protocols, Compound E was ranked best, followed by Compound B and exisulind.

Table 9

Activity profile of various compounds

Compound Inhibition COX Inhibition PDE Inhibition Orgánová culture mammary glands growth Apoptosis Exisulind - ++ ++ ++ +++ Sulindaku sulphide ++++ +++ +++ +++ - MY5445 ++++ +++ +++ +++ + AND - - +++ ++ ++ (B) - +++ +++ +++ ++ D - - ++ - - E - ++++ ++++ ++++ ++++ F - - ++ + - G - - +++ +++ H ++

Explanatory notes to tab. 9: Activity of compounds based on evaluation of a number of experiments including maximum activity and potency assays.

- inactive + low active ++ medium active +++ strong active ++++ highly active

A new PKG activity assay has also been disclosed which is used in the assay methods of the present invention, but which also has more general efficacy in determining PKG activity for other purposes (e.g., to study the role of PKG in normal cell function). For explanation, this is useful in describing the PKG assay before explaining how PKG activity can be.

useful in evaluating drugs to determine whether the compound may be useful in the treatment of tumors.

Re-determination of PKG

The new PKG assay of the present invention involves solid phase binding with amino acid sequences, each of which comprises at least a cGMP binding domain and a phosphodiesterase type 5 ("PDE5") phosphorylation site. This sequence is known and described in the literature. More preferably, the bound PDE5 sequence does not comprise the catalytic domain of PDE5 as described below. One way to bind the PDE5 sequences to the solid phase is to express these sequences as a fusion protein comprising the PDE5 sequences and one member of the amino acid binding pair and chemically attach the other member of the amino acid binding pair to the solid phase (e.g., beads). The binding pair that can be used is glutathione S-transferase ("GST"), which is expressed as a fusion protein with the PDE5 sequence, and glutathione ("GSH"), which is covalently bound to the solid phase. In this case, the PDE5 sequence / GST fusion protein can be bound to the solid phase by pushing the solution containing the fusion protein through the solid phase as described below.

The RT-PCR method is used to obtain the cDNA domain of PDE5 with forward and upstream primers designed from the cDNA sequence of bovine PDE5A (McAllister-Lucas LM et al., J. Biol. Chem. 268: 22863-22873, 1993) and to choose between PDE families 1-10. For HT-29 cells, 5'-3'Inc are used. Total RNA and mRNA purification kits on oligo (dT). A direct primer (GAA-TTC-TGT-TAG-AAA-AGC-) is used to synthesize a 1484 bp fragment encoding the phosphorylation site and cGMP binding sites of human PDE5A with both low and high affinity (203-1686 bp, cGB-PDE5). CAC-CAG-AGA-AAT-G, 203-227) and upstream primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG, 1664-1686). The synthesized cGB-PDE5 nucleotide fragment encodes 494 amino acids with 97% similarity to bovine PDE5A. The fragment is then cloned in the pGEX-5X-3 glutathione-S-transferase (GST) fusion vector (Pharmacia Biotech.) With the tac promoter and EcoRI and XhoI restriction sites. The fusion vector is then transfected in E. coli BL21 (DE3) (Invitrogen). The transfected BL21 is grown to the log phase and then IPTG is added as an inducer. Induction takes place at 20 ° C for 24 h. Bacteria are harvested and lysed. The soluble cell lysate is incubated with Sepharose 4B and GSH conjugate (GSHSepharose 4B). The GST-cGB-PDE5 fusion protein can bind to GSH-Sepharose beads while the other proteins are eluted from the beads by excess cold PBS.

9 9 9

I · «

The expressed GST-cGB-PDE5 fusion protein is shown on a 7.5% polyacrylamide gel after SDS electrophoresis as a 85 kD protein. It is characterized by binding cGMP and phosphorylated by protein kinase G and A. It has two binding sites for cGMP and K _d is 1.6 ± 0.2 μΜ, close to a Ka = 1.3 μΜ for native bovine PDE5. GST-cGB-PDE5 on GSH-conjugated Sepharose beads can be phosphorylated in vitro with cGMP-dependent protein kinase and cAMP-dependent protein kinase A. K _m phosphorylation of GST-cGB-PDE5 by PKG is 2.7 μΜ and V _max is 2.8 μΜ, while K _m phosphorylation of BPDEtide is 68 μΜ. PKG phosphorylation shows that the ratio between the incorporated molecular phosphate and GSTcGB-PDE5 is 1 to 1.

To determine the liquid phase in which the PKG is predicted by PDE5 bound to the solid phase, the sample and the solid phase are mixed with a phosphorylation buffer containing ³² -γATP. The solution is incubated for 30 min. at 30 ° C to achieve phosphorylation of the PDE5 sequence by the PKG enzyme to determine if PKG is present. The solid phase is then separated from the solution (eg by centrifugation or filtration) and washed with phosphate buffered saline ("PBS") to remove any remaining solution and any unreacted ³² Ρ-γATP.

The solid phase can then be directly tested (eg, by liquid scintillation equipment) to determine if P is incorporated. If so, it means that the sample contained PKG because PKG phosphorylates PDE5. If PDE5 is bound by a fusion protein as described above, then the fusion protein containing PDE5 may be eluted by the solid phase with SDS buffer and then the ³² P content may be determined in the eluent. that other proteins are present. The eluent can then be processed (e.g., by gel separation) to separate the various proteins so that the fraction containing the fusion protein can be determined by incorporating P. The phosphorylated fusion protein can be eluted from the solid phase by SDS buffer and further redissolved at electrophoresis. When separation takes place in the gel, the proteins can be stained to see the position of the proteins and ³² P phosphorylation of the PDE5 fraction of the fusion protein due to PKG can be measured by exposing the gel to the film. If ³² P is visible on the film, this means that PKG was present in the original sample containing PKG, which phosphorylated the PDE5 fraction of the fusion protein eluted from the solid phase.

In the assay, an excess (eg, 100-fold) of a protein kinase inhibitor ("PKP") that specifically and strongly inhibits protein kinase A ("PKA") and does not inhibit PKG may be added to the assay buffer. Inhibiting PKA is desirable because PKA may contribute to substrate phosphorylation

PKG (eg PDE5). The addition of PKI eliminates any contribution of PKA to phosphorylation and the detected phosphorylation is due only to PKG itself.

• 9 9 99 99 9 9

The kit that can be used for the assay of the present invention comprises the following pre-prepared reactants distributed in separate containers:

1. Cell Lysis Buffer: 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM

NA3VO4, 1 mM NaF, 500 μΜ IBMX, proteinase inhibitors.

2. Solid-phase protein kinase G substrate: recombinant GST-cGB-PDE5, bound to

Sepharose 4B (50% suspension).

3. 2x phosphorylation buffer: ³² Ρ-γ-ΑΤΡ (3000 mCi / mmol, 5-10 pCi / assay), 10 mM KH ₂ PO ₄ , mM K ₂ HPO ₄ , 200 µΜ ATP, 5 mM MgCl ₂ .

4. Protein kinase I inhibitor PKA

Disposable containers and accessories in which the above reactions are performed may also be provided in the kit.

Based on the above findings, one of ordinary skill in the art of analytics will readily anticipate various ways to adapt the assay described. Briefly, using at least a proportion of PDE5 (or any other protein that can be selectively phosphorylated by PKG), the presence and relative amount (relative to control) of PKG can be ascertained by assessing the phosphorylation of a protein that can be phosphorylated with a labeled phosphorylating agent.

SAANDs increase PKG activity in tumor cells

Using the above PKG assays, the following experiments were performed to determine whether SAAND compounds increased PKG activity either because they increased PKG expression or because they increased cGMP levels (or both) in tumor cells exposed to SAAND.

Testing procedures

Two different types of PDE inhibitors were evaluated for effect on PKG in tumor cells. SAAND-exisulind, has been evaluated because it is antitumor. Also, the classical PDE5 inhibitor, which is not a SAAND agent, E4021, has been found to increase PKG only as a result of classical PDE5 inhibition, or if PKG increase is part of the proapoptotic effect of SAAND, which includes PDE5 inhibition and new PDE inhibition. , disclosed in US Patent Application No. 09/173375 to Liu et al., filed Oct. 15, 1998.

φφ

Φ φ φ · * φ • Φφ · φ · φφ

9

9 9 9 9 9 9 9 9 9 9

999 9 9 99 ♦ «· Φ Φ Φ

9 9 9

9 9 9

9 9 ♦

99

SW480 malignant colon tumor cells were used to test the effect of inhibition of cGMP-specific PDE on tumors containing the APC mutation. SW480 are known as cells that contain an APC mutation. About 5 million SW480 cells in RPMI with 5% serum were placed on each of the 8 plates.

- 10 cm dish - 30 μΐ DMSO control vehicle (no drug),

- 10 cm dishes - 200 μΜ, 400 μΜ, 600 μΜ exisulind in DMSO and

- 10 cm dish - E4021; Ο, ΙμΜ, 1 μΜ and 10 μΜ in DMSO.

The plates were incubated for 48 h at 37 ° C in 5% CO ₂ .

The liquid media was aspirated from the dishes (the cells themselves held on the dish). The adherent cells were washed in each well with cold PBS and 200 μΐ of cell lysis buffer (i.e. 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na ₂ VO4, was added to each well, mM NaF, 500 μΜ IBMX with proteinase inhibitors). Immediately after addition of the cell lysis buffer, lysed cells from each well were wiped. The cell lysate from each plate was transferred to a microtube which was incubated for 15 min. at 4 ° C. During this time, the tubes were mixed gently to complete cell lysis. After lysis was complete, the tubes were centrifuged (14,000 rpm) for 15 min. The supernatant from each tube was transferred to a new microtube.

Protein content was determined in each tube because, if the drug inhibited cell growth, the total amount of protein would be higher in control than in the drug treated samples. Indeed, if the drug is effective, the total protein content of the drug-treated samples should of course be the same as that of the control. In the above situation, control tubes and E4021 had to be diluted to standardize with samples treated with high doses of exisulind (the group with lower doses of exisulind must be standardized to the highest doses of exisulind). After measuring the protein content, the total protein concentration of the various samples must be standardized (eg by dilution).

For each drug concentration and control, 2 PKG assays were performed, one with cGMP addition and one without cGMP addition, as described in more detail below. The reason why these 2 different PKG assays have been made is because cGMP specifically activates PKG. When PKG activity is determined by a new PKG assay of the present invention, it is not possible to determine if the increase in PKG activity is due to increased cGMP in cells (which may be due to inhibition of cGMP-specific PDE) or if the level of PKG activity is due to increased protein expression PKG. By determining PKG activity in a sample with and without cGMP addition, it can be determined whether PKG activity is increased and whether this is due to increased PKG expression. Thus, if an anticancer drug increases PKG activity compared to control, it can be estimated if it is an increase.

· Φ »♦ · · · · · ·

Drug-induced φ φ • φ φ due to overexpression of PKG protein (as opposed to activation) in the drug-treated sample if (1) the drug-treated sample with extra cGMP shows higher PKG activity compared to the control sample with extra cGMP and (2) drug treated sample without extra cGMP showed higher PKG activity compared to control.

After preparing parallel samples with and without cGMP addition, 50 μΐ of each cell lysate was added to the 20 μΐ suspension of the PDE5 / GST solid phase substrate described above. For each control and drug cell lysate sample, the reaction is started by adding a phosphorylation buffer containing a solution of 10 pCi ³² Pγ-ΤΡΤΡ (200 μΜ ΤΡΤΡ, 4.5 mM MgCl 2; 5 mM KH 2 PO 4; 5 mM K 2 HPO 4;) to each mixture. . The resulting mixtures are incubated for 30 min. at 30 ° C. The mixtures are then centrifuged to remove the solid phase and the supernatant is no longer used. The solid phase in each tube was washed with 700 μΐ cold PBS. Laemmli sample buffer (30 μΐ) (Bio-Rad) was added to the solid phase. The mixtures were boiled for 5 min. and loaded onto a 7.5% SDS polyacrylamide gel. Electrophoresis was carried out for 1 hour at 150 V. The obtained bands were fixed with commassie blue solution to visualize them, and thus found if bands of 85 kD were present which correspond to the GST-PDE5 fusion protein. The gel is dried and placed on a film which, when PDE5 is phosphorylated, shows a corresponding dark bond. The darkness of each bond corresponds to the degree of phosphorylation.

As shown in Figures 18A and 18B, SAAND exisulind causes dose-dependent increase in PKG activity in both samples, ie with and without cGMP added, compared to controls with no extra cGMP. This is confirmed by the darker appearance of the 85 kD bonds in each drug treated sample. In addition, exisulind-treated SW480 samples have higher PKG phosphorylation activity when cGMP was added to the assay as compared to samples treated with exisulind alone (i.e., without the addition of cGMP). Thus, an increase in PKG activity in drug-treated samples is not only due to PKG activation by increasing cellular cGMP when SAAND inhibits cGMP-specific PDE, but an increase in PKG activity in an APC mutant tumor is also due to increased PKG expression.

Also, the fact that SW480 samples treated with E4021 do not show PKG activation compared to control (see Figs. 18A and 18B) shows that increased PKG activation caused by SAAND in tumor processes containing an APC mutation is not merely due to inhibition of classical PDE5. .

As an analytical technique for evaluating PKG activation, the method of determining the degree of · ♦ • · may be used in the 85 kD SDS gel binding site, as described above, as described above.

9 9 9 • 9 9 9

9 9 9

9 9 9

99 phosphorous, so that the band is excised from the gel and ³² P, incorporated in the excised bond, can be detected in a ³² P window scintillation (beta) device.

To test the effect of inhibiting cGMP-specific PDE in β-catenin mutant tumors, HCT116 malignant colon tumor cells were used. HCT116 is known to contain a β-catenin mutation but not an APC mutation.

For the growth of HCT116 cells, the same procedure as for SW480 described above was used. Only exisulind and controls were used in this experiment. Exisulind-treated cells produce PKG that has been phosphorylated to a higher degree than the corresponding controls, suggesting that drug treated cells activate PKG that is independent of the APC mutation.

For purposes of the present invention, the term "reduced β-catenin" is used in the claims and is intended to mean natural β-catenin and / or a mutant form of the protein.

Confirmation of PKG overexpression and decrease of β-catenin in SW480 cells by Western blot

As shown above, SAANDs cause an increase in PKG expression and an increase in cGMP levels, both resulting in an increase in PKG activity in SAAND-treated tumor cells. This overexpression of PKG protein was further verified by a relatively quantitative Western blotting method as described below.

Exisulind-treated SW480 cells were first rinsed from the microtube with ice-cold PBS. Cells were lysed with gentle agitation for 15 min. in a modified RIPA buffer. The cell lysate was centrifuged in a refrigerated room. The supernatants were transferred to new microtubes immediately after centrifugation. Protein concentrations were determined in the samples using BioRad DC Protein Assay (Temecula, CA). Samples were standardized for protein concentration, see above.

pg of each sample was loaded on a 10% SDS gel. SDS electrophoresis was performed and then the proteins were transferred to a nitrocellulose membrane. The blotted nitrocellulose membrane was blocked in freshly prepared TBST containing 5% nonfat dry milk with stirring for 1 hour at room temperature.

Primary goat anti-PKG antibody is diluted to the desired concentration / dilution with fresh TBST / 5% non-fat milk powder. The nitrocellulose membrane is placed in the primary antibody solution and incubated for 1 hour at room temperature with stirring. The nitrocellulose membrane is then washed 3x10 min. with a new TBST solution. The nitrocellulose membrane is incubated with stirring for 1 hour at room temperature in the solution containing it

43 * «4 ·· * ·

4 4 · 4 · 4 · 4

444 44 4 4445

445 4 · 9 9 9 9 ·

4 4 4 4 4 4 4 4

444 44 4 · 444 99 44 secondary rabbit anti-goat antibody conjugated to POD. The nitrocellulose membrane is then washed 3 x 10 min. with a new TBST solution. Detection is performed with a substrate for POD BM blue (Boehringer Mannheim).

As shown graphically in Fig. 19, exisulind causes a decrease in β-catenin and an increase in PKG, the data of which were obtained by western blot. SW480 cells were exposed to exisulind or vehicle (0.1% DMFO) for 48 h. 50 µg of each cell lysate was loaded on a 10% SDS gel and blotted onto a nitrocellulose membrane, incubated with rabbit anti-β-catenin and rabbit antibody. anti-PKG antibody.

SAANDs reduce β-catenin levels in tumor cells

This was done when culturing SW480 cells with 200, 400 or 600 μΜ exisulind or vehicle (0.1% DMSO). Cells were harvested 48 h after test substance addition and immunoblotting was performed. The immunoreactive protein can be detected by Western blot. Western blot analysis showed that β-catenin expression was reduced by 50% compared to the control for exisulind-treated cells. These results indicate that the β-catenin content is reduced by the action of SAAND substances. The results obtained above, which indicate that PKG activity increases with this action, and, together with the results below, which indicate that β-catenin is phosphorylated by PKG, suggest that β-catenin reduction in tumor cells is initiated by PKG activation. The use of PKG activity in tumors is therefore useful as a means of selecting anti-tumor compounds.

Phosphorylation of β-catenin by PKG

PKG phosphorylates β-catenin in vitro. Experimentally this was determined by immunoprecipitation of the complex from SW480 cells (not treated with any drug) containing β-catenin. The precipitation was performed as described below under the term "β-catenin immunoprecipitation". The immunoprecipitated complex still retained on the solid phase (e.g., beads) is mixed with ³² Ρ-γ-ΑΤΡ and pure PKG (100 U). Appropriate controls without PKG are also prepared.

The protein is released from the solid phase by SDS buffer and the protein-containing composition is subjected to SDS electrophoresis in a 7.5% gel. Electrophoresis removes excess ³² Ρ-γ-ΑΤΡ from the mixture. Therefore, any ³² Ρ-γ-ΑΤΡ detected in the 93 kD band of β-catenin is due to phosphorylation of β-catenin. Any increase in Ρ-γ-ΑΤΡ detected in the β44 band

93kD extra PKG treated catenin compared to a control that does not contain extra PKG is due to phosphorylation of β-catenin in the extra PKG treated band.

The results obtained showed an increase in phosphorylation of the PKG-treated band compared to the control showing minimal, almost undetectable phosphorylation. This result suggests that β-catenin may be phosphorylated by PKG.

Phosphorylation of mutant β-catenin PKG

The same procedure as described in the immediately preceding section was performed with HCT116 cells that do not contain an APC mutation but a β-catenin mutation. The results of these experiments suggest that mutant β-catenin is also phosphorylated by PKG.

For the purposes of the present invention, β-catenin phosphorylation is claimed in the claims such as phosphorylation of the wild-type and / or mutant forms of the protein.

β-catenin precipitates with PKG

Cell lysates supernatants of both SW480 and HCT116 cells were prepared in the same way as in western blot experiments. The cell lysate is first clarified by adding 150 μ 150 of a suspension of 50% protein A Sepharose beads per 500 µg cell lysate and incubated for 10 min. at 4 ° C on a test tube. The beads were removed by centrifugation at 14,000 x g, 10 min. The supernatants were transferred to a new centrifuge tube. 10 µg of rabbit polyclonal anti-β-catenin antibody (Upstate Biotechnology, Lake Placid, New York) was added to 500 µg of cell lysate. The cell lysate / antibody mixture was gently stirred at 4 ° C for 2 h. The immunocomplex was captured by adding 150 μΐ of protein A Sepharose beads suspension (75 μΐ of prepared beads) and gently stirring the mixture overnight at 4 ° C. Sepharose beads were collected by pulse centrifugation (5 sec in a microcentrifuge at 14,000 rpm). The supernatant fraction was discarded and the beads washed with 3 x 800 μΐ ice-cold PBS buffer. Sepharose beads were resuspended in 150 μΐ of sample buffer and mixed gently. Sepharose beads were boiled for 5 min to dissociate the immunocomplex from the beads. The beads were centrifuged and the supernatant was subjected to SDS electrophoresis.

The supernatants were subjected to Western blotting and the membrane was then incubated with rabbit anti-β-catenin antibody. Then the membrane was washed 3 x 10 min. TBST to remove excess anti-β-catenin antibody. A goat anti-rabbit antibody has been added. 99 · 99 · · · · · · · · · · · · · · ·

9 9 9 9 9 9 9 9 9 9

9 9 9 9 9

9 99 99 999 99 99 horseradish peroxidase conjugated antibody followed by incubation for 1 h at room temperature. Upon termination, the presence of β-catenin can be visualized using HRPO substrate. In this experiment, β-catenin was clearly visualized.

To detect PKG on the same membrane, the anti-β-catenin antibody conjugate was first removed from the membrane with 62 mM Tris-HCl buffer (pH 7.6) with 2% SDS and 100 μΜ 2β-mercaptoethanol in a water bath at 55 ° C for 0, The clean membrane was then blocked in TBST solution with 5% non-fat milk powder for 1 hour at room temperature with gentle stirring. The blocked clear membrane was then incubated with rabbit polyclonal anti-PKG antibody (Calbiochem, LaJolla, CA), which is further detected with a secondary goat anti-rabbit antibody-HRPO conjugate. The presence of PKG on the blotted membrane is visualized with the HRPO substrate. In this experiment, PKG was visualized. Since only proteins that immuniprecipitated with β-catenin in cell supernatants are present on the membrane, this result clearly states that PKG was physically associated with β-catenin-containing protein complex in the cell supernatants.

The same membrane after Western blot was also incubated with an anti-GSK3-β antibody to see if it co-precipitated with β-catenin. In this experiment, we also detected GSK3β on the membrane, suggesting that GSKJ-β precipitates with GSKJ-β and PKG, suggesting that the three proteins may be part of the same complex. Since GSKS-β and the β-catenin form in normal cells form part of the APC complex, PKG may be part of the same complex and may be incorporated into β-catenin phosphorylation as part of this complex.

Anti-tumor pharmaceutical compositions comprising cGMP PDE inhibitors

As explained above, exisulind is a compound that exhibits desirable antitumor properties. Its efficacy and use as an antitumor agent was discovered before it was understood that the compound works to inhibit cGMPspecific PDE activity in tumor cells.

During clinical trials in cancer patients, it has been verified, inter alia, that the selection procedure of the present invention can be used to select compounds for the treatment of humans. Having determined that exisulind had antitumor properties (in vitro) and because it had the profile of the desired compound meeting the selection criteria of the present invention, it was determined based on the success of the compound in two human clinical trials that other compounds could be selected using the criteria of the invention.

As indicated above, many tumors carry an APC mutation. Among other things, the selection procedures of the present invention have been verified in clinical trials in patients with a tumor bearing an APC mutation.

The APC mutation was first discovered in patients with a hereditary tumor, adenomatous polyposis of the intestine (“APC”). APC disease is characterized by the presence of hundreds to thousands of polyps in the colon during the second decade of life, and routine treatment means surgical removal of the colon before 20 years of life.

The first clinical trial included patients with sAPC. In this study, each patient had a large intestine except a small part of the intestine adjacent to the rectum (where the small intestine is attached) to protect rectal function. However, in such a patient, polyps are commonly formed in the remaining small portion of the colon that need periodic removal (e.g., electrocautery).

The exisulind test was a preventive test designed to evaluate the antitumor characteristics of the drug by comparing the degree of accumulation of new polyps formed over 12 months in the drug and non-treatment groups. Patients who formed between 9 and 44 polyps per year were eligible. At the beginning of the study, at the end of 6 months and at the end of 12 months, all polyps were removed. The study was conducted with 34 patients. Based on the established number of polyps produced over 1 year in APC patients who produced 9 to 44 polyps per year, exisulind was clinically and statistically significantly superior to placebo in terms of decreasing polyp formation rate.

Based on the average number of polyps produced during the first six months of the study, approximately one third of the number of polyps compared to placebo-treated patients were found in exisulind-treated patients (mean 9 polyps / year and 26 polyps / year; p = 0.013) . Based on the average number of polyps generated over the 12 months of the study, exisulind-treated patients produced approximately half the number of polyps compared to placebo-treated patients (mean values of 18 polyps / year and 38 polyps / year; p = 0.020).

A special clinical trial was also conducted with male patients who had prostate cancer, resulting in prostate removal. The study was conducted in patients with detectable levels of PSA (prostate specific antigen) that were elevated after radical prostatectomy, suggesting recurrence of prostate cancer.

96 patients were evaluated for prostate cancer: double-blind, placebo-controlled, and multicentre trials were performed with exisulind administered at 500 mg / day. The data below shows a statistically significant difference in PSA levels between the exisulind-treated group and the

9 placebo was administered. PSA levels in the exisulind-treated group were significantly reduced compared to PSA levels in the untreated group. Although elevated PSA is not a disease in itself, it is closely monitored in the medical field as a surrogate indicator of the recurrence of prostate cancer in these men.

In addition to evaluation based on differences in PSA levels between exisulind treated and untreated groups that were evaluated as a whole, ongoing analyzes also included subgroup analyzes. In this study, patients were divided into high, medium and low risk groups in terms of the risk of developing their metastatic disease. This distinction was made using a methodology published in the Journal of the American Medical Association (JAMA, May 5, 1999, pp. 1591-1597). In order to determine which study patients belonged to which group, the medical histories, if they did not know whether the patients were given the drug or placebo, had medical history; the patients studied were assigned to the corresponding risk group according to the published methodology described above. In the medium and high risk groups, statistical analyzes showed statistically significant differences in PSA levels between exisulind-treated patients and placebo-treated patients.

The data from the prostate study are as follows:

Table 10

Effect of exisulind on PSA levels in men after prostatectomy with increasing PSA

Group Placebo Exisulind "P" value Overall 4.49 2.85 0.0004 With great risk 4.98 2.91 0.0002 With medium risk 6.24 2.95 0.0053

In these exisulindic trials and several others, including the drug in other indications, she was monitoring adverse events (AE), clinical laboratory tests (hematology, serum chemistry and urine analysis), using vital signs (blood pressure, pulse, breathing, temperature and weight) , physical tests and upper endoscopy evaluated the safety of the substance.

In safety terms, no significant deficiencies were found in clinical trials conducted with exisulind for more than 400 patients. Exisulind did not cause any blood discrasions, vomiting due to dosing, or neurological or renal toxicity associated with conventional chemotherapeutic agents. It also did not cause any clinically significant changes in life traits. In paired polyp biopsies and normal colon tissues in APC patients, exisulind was found to increase apoptosis rate in polyp,

9 9 but not in normal colon tissues, suggesting minimal effects of exisulind on normal tissues.

At doses higher than the maximum tolerated dose (MTD = 600 mg in patients with subtotal colonectomy, 400 mg in patients with intact colon; 350 mg in pediatric patients), only adverse drug-related adverse events were seen as increased liver function tests (LFTs) that were observed early during treatment. Experience has shown that the LFT increase was reversible and did not occur when the dose was reduced. The remaining events (eg occasional abdominal pain) were mostly of short duration, were mild to moderate in intensity and did not require discontinuation of treatment or reduction of exisulind dose.

Briefly, these tests have shown that exisulind administration is an effective, well-tolerated long-term therapy for the clinical management of tumor processes. These results indicate that selecting another compound that inhibits cGMP-specific PDE activity (as well as meeting other selection criteria of the present invention) can result in a therapeutically effective drug, in vivo.

The second drug is a substance that has been shown to inhibit cGMP and which was also discovered before the mechanism of action was known and before it was known to meet the selection criteria of the invention. It is (Z) -5-fluoro-2-methyl- (4-pyridylidene) -3- (N-benzyl) indenylacetamide hydrochloride (Compound I). In in vitro and in vivo evaluations, Compound I has been shown to be an anticancer drug with activities against a broad spectrum of tumors. It is also safe for animal and human studies using a single dose escalation.

One of ordinary skill in the art will recognize from the data below that Compound I can be safely administered to animals at doses far beyond teralable (and in many cases toxic) doses of conventional chemotherapeutic or anti-tumor NSAIDs. E.g. no signs of toxicity were observed in the acute toxicity study in rats when Compound I was administered at a single oral dose (0.5% carboxymethylcellulose vehicle) of 2000 mg / kg or higher. At 4000 mg / kg, body weight gains were slightly reduced. A single dose of 1000 mg / kg administered intraperitoneally resulted in a decrease in body weight gain and mesenteric adhesions were observed at necropsy of some animals in this group.

In the group consisting of two bitches and two dogs, administration of a dose of 1000 mg / kg of Compound I in the form of capsules did not show any signs of toxicity. Due to the nature of the Compound I capsules, this dose required administration of at least 13 capsules to each animal, which was considered the maximum number without stressing the animals. Therefore, these dogs were given 7 consecutive doses of 1000 mg / kg / day. No apparent drug-related symptoms were observed during dosing.

9 9 9 99 99 99 99 99 99 99 99 99 99 99 99 99 99

9

9 9 9 9 • 9 9 9

9

999 99 99

At one dose, Compound I is not acutely toxic. Based on the results of these studies, Compound I oral LD50 was considered to be greater than 1000 mg / kg in dogs and 4000 mg / kg in rats, and intraperitoneal LD50 in rats was considered to be greater than 1000 mg / kg.

In a seven-day study in rats and the compound was evaluated at 0, 50, 500 or 2000 mg / kg / day, no observed signs of toxicity occurred at 50 mg / kg / day. In female rats, at 500 mg / kg / day, the compound-related effects were limited to an increase in absolute and relative liver weights. At a dose of 2000 mg / kg / day, the effects included respiratory and / or abnormal respiratory murmurs, decreased weight gain and reduced food consumption in male rats and increased liver weight in female rats. No hematological or chemical changes in the blood, nor any microscopic pathological changes were observed at any dose.

A 28-day study was also conducted in rats at doses of 0, 50, 500 and 2000 mg / kg / day. There were no abnormal clinical observations attributed to CP-461, and there were no particular changes in body weight, ophthalmoscopic tests, haematological and chemical values of blood and urine tests. No macroscopic tissue changes were observed in necropsy. Organ weight data showed a statistically significant increase in liver weight in the 2000 mg / kg / day group and a statistically significant increase in thyroid weight in the 2000 mg / kg / day group. A slight increase at lower doses was not statistically significant. Histopathological evaluation of the tissues indicated the presence of traces of follicular cell hypertrophy, an increased number of mitotic images (indicating possible cell proliferation) in the thyroid gland, and mild centrilobular hypertrophy in the liver. These changes were generally limited to a small number of animals at 2000 mg / kg / day, although one female had elevated mitotic images in the thyroid at 500 mg / kg / day. Liver findings may indicate a very mild stimulation of microsomal enzymes, leading to increased thyroid hormone metabolism, which in turn leads to thyroid stimulation. Thus, one skilled in the art will recognize that these effects are extremely small compared to those expected at similar doses of conventional chemotherapeutics or NSAIDs.

To further determine the safety profile of Compound I, a study was performed to assess whether Compound I-induced apoptosis of prostate tumor cells was comparable to their effects on normal tissue-derived prostate epithelial cells. Androgen-sensitive prostate tumor cells, LNCaP (from ATCC (Rockville, MD)) were propagated under standard conditions using RPMI 160 medium containing 5% FCS and 2 mM glutamine. Primary prostate epithelial cell (PrEC) cultures, derived from normal prostate (from

Clonetics lne. (San Diego, CA)) were grown under the same conditions as the tumor cell lines except that serum-free medium optimized for the growth of these cultures (Clonetics Inc) was used. For experiments, LNCaP or PrEC cells were placed in a 96-well microtiter plate such that the cell density was 10,000 cells per well. After 24 h, cells were exposed to either vehicle (0.1% DMSO) or 50 μΜ Compound I (free base) dissolved in DMSO. After various drug treatment periods (4, 24, 48, 72 or 99 h), cells were lysed and measured for histone-linked DNA as an indicator of apoptotic cell death (see Piazza et al., Cancer Research 57: 2452-2459, 1977).

Giant. 27 shows a time-dependent increase in the amount of fragmented, histone-linked DNA in LNCaP cell cultures due to treatment with 50 μΜ of Compound I (free base). A significant increase in fragmented DNA was detected after 24 h treatment and induction was maintained for up to 4 days of continuous treatment. In contrast, up to 4 days of treatment with Compound I (50 μ působení) on PrEC cells ("normal" prostate) did not affect DNA fragmentation. These results show selective induction of apoptosis in tumor cells as opposed to normal cells. This is in contrast to conventional chemotherapeutic agents that induce apoptosis or necrosis in rapidly growing normal and tumor cells as well.

In conclusion, patients are required to demonstrate anti-tumor effects in a single escalating clinical trial in humans, a human safety study during which the drug is administered orally, Compound I, which produces insignificant side effects at any dose including doses in excess of predicted levels.

As indicated above, Compound I also exhibits potent antitumor properties. The growth inhibition IC 50 obtained for Compound I was 0.7 µΜ for the SW-480 cell line. This result was confirmed by evaluating Compound I in rodents using aberrant crypt foci ("ACF") as an indicator of carcinogenesis (see Bird, Cancer Leti. 37: 147-151, 1987). This rodent model with azoxymethane-induced carcinogenesis ("AOM") was used to determine the effects of Compound I (free base and salt) on the development of malignant colon cancer in vivo. ACFs are precursors of colon tumors and ACF inhibition is a factor predicting chemopreventive efficacy.

In the rat experiment, ACF initiation was achieved by two consecutive weeks of carcinogen administration. Compound I was administered one week before the start of ACF and for the duration of the experiment. ACF was recorded after 5 weeks of treatment. Compound I was administered orally to male Fisher 344 rats as part of the diet. The daily food consumption (mg / kg body weight) varied throughout the study and, in order to provide a basis for comparing individual doses, the dose of Compound I was expressed in grams per kg of food. To determine if she •

Compound I adverse effect on growth and / or diet, body weight was determined during the experiment. Experimental groups had less weight gain than controls, indicating bioavailability. However, the weight differences were less than 10% and therefore were not expected to affect ACF production.

Compound I free base inhibited ACF production as measured by colonic crypt reduction. The data are summarized in Tab. 11. In addition to the low-dose group (only 0.5 g / kg food), the differences between the drug group and the control group were substantial, and in the 1.0 and 2.0 g / kg groups, respectively. even statistically significant.

Table 11

Inhibition of aberrant crypt foci by Compound I

Dose of compound n diameter of ACF / colon % control p (t-test) (g / kg food) (+ SO) vs.Control Control 10 149 ± 9 - 0.5 7 149 ± 14 100 ALIGN! 0.992 1 10 111 ± 9 75 0.008 1.5 10 132 ± 4 89 0.101 2,0 10 107 ± 15 72 0,029

Compound I also retroactively met the selection criteria of the present invention and was one of the compounds used to determine the accuracy of the selection criteria. For example, using the protocols described above, Compound I has an IC 50 cGMP-specific PDE of 0.68 µΜ using cGMP-specific PDE from HT29 cell extracts. Compound I inhibited COX I (at 100 μΜ) by less than 25%.

Because Compound I is pro-apoptotic, the EC50 value of DNA fragmentation was 15 μΜ. The percent apoptosis of SW-480 cells for Compound I at various drug concentrations is shown in Tab. 12.

Table 12

Morphologically induced induction of HT-29 cell apoptosis from SW-480 colon adenocarcinoma cells by Compound I

Action Dose % apoptosis Vehicle (0.1% DMSO) - 1 Compound I 0,35 μΜ 16 Compound I 0,7 μΜ 27 Mar: Compound I 1.5 μΜ 88

Compound 1 activity is not limited to activity against colon cancer cell lines or colon cancer models in animals. Compound I has a wide range of anti-tumor effects on various tumor cell lines. Various types of human carcinoma cell lines were propagated under sterile conditions in RPMI 1640 medium with 10% fetal bovine serum, 2 mM L-glutamine and sodium carbonate. To determine the inhibitory effects of Compound I on cell growth, cells were dispensed into a 96 well microtiter plate such that the cell density was 1000 cells per well. 24 hours after cell placement, different concentrations of Compound I free base dissolved in DMSO (final concentration 0.1%) were dosed to the cells. The effect of the drug on tumor cell growth was determined by a neutral red cytotoxicity assay following 5 days of continuous treatment. Neutral red is a dye that selectively stains living cells by being transmitted by an ATP-dependent transport mechanism.

As summarized in Table 13, Compound I (free base) exhibits potent growth inhibitory activity when evaluated for a variety of human cell lines derived from various tissues. Compound I exhibits comparable growth inhibitory effects regardless of histogenesis of the tumor from which the cell lines were derived. The GI50 (drug concentration that causes 50% growth inhibition compared to control vehicle), calculated for all cell lines, was 1-2 μΜ.

In addition to the data presented in the table below, we obtained comparable sensitivity to Compound I (HCl salts) in the human leukemia cell line (CCRF-CEM, K562 and Molt-4), myeloma cell line (RPMI8226), pancreatic tumor cell line (PAN- 1) and ovarian tumor cell line (OVCAR3).

Table 13

Inhibition of growth of various human tumor cell lines by Compound I

Cell line Origin of the tumor GI ₅ o μΜ GI90 μΜ Colo 205 Colon 1.6 2.4 HCT-15 Colon 1.7 3.0 HT-29 Colon 2.1 8.0 SW-620 Colon 1.7 2.5 DU145 Prostate 1.6 2.8 PC-3 Prostate 1.7 82.5 NCI-H23 Lung 1.7 2.5 NCI-H321M Lung 2.1 13.2 NCI-H460 Lung 1.9 30.0 NCI-H82 Lung 1.7 5.8 MDA-MB-231 Breast 1,8 77.6 MDA-MB-435 Breast 1.6 2.3 UISO-BCA-1 Breast 1.5 4.7 Molt-4 Leukemia 1.6 not specified CCRF-CEM Leukemia 1.4 not specified K-562 * Leukemia 1,8 not specified RPMI-8226 Myeloma 1,2 not specified OVCAR * Ovary 1,2 not specified PANC-1 Pancreas 2.2 not specified

* Except in the cases marked with an asterisk, where testing was performed with HCl salt, testing was performed with the free base of the compound.

Given the human and animal safety characteristics and the very broad effect of Compound I on animals and cell cultures, it is clear that compounds meeting the selection criteria of the present invention (including inhibition of cGMP-specific PDE) may be useful anti-tumor agents. therapeutics.

To identify structurally complementary compounds inhibiting cGMP-specific PDEs that may be therapeutically effective as antitumor agents, one of ordinary skill in the art has a number of useful model compounds disclosed herein (as well as analogues thereof incorporated by reference) that can be used. as a basis for computer modeling f f f f f f f f f f f f f f f f f f f f f f f f f Complementary compounds with the same conformation but which are different chemically. For example, software that is sold by Molecular Simulations Inc. as WebLab® ViewerPro ^1M includes molecular visualization and chemical communication capabilities. This software includes functionality, including 3D visualization of known active compounds, to validate the accuracy of the sketched or imported chemical structure. The software further allows the structures to be sorted according to user-defined characters. Distances, angles or dihedrals can also be measured.

Since the structures of the other active compounds have been disclosed above, this software can be used to identify a new additive compound which can then be tested and selected according to the selection criteria of the present invention, group analysis and selection techniques based on two-dimensional and three-dimensional similarity. These software methods assume that compounds that look similar or have similar properties are more likely to have similar activity, which can be confirmed by the selection criteria of the present invention.

In addition, when these additive compounds are computer modeled, many of these compounds and variants thereof can be synthesized using known combinatorial chemical techniques commonly used by those skilled in the pharmaceutical industry. As examples of several companies offering combinatorial chemical services, for example, New Chemical Entities, Inc. of Bothell Washington, Protogene Laboratories, Inc. of Palo Alto, California, Axys, Inc. of South San Francisco, Calif., Nanosyn, Inc. of Tuscon, Arizona; of San Diego, California and RBI, Inc. of Natick, Mass. However, there are many other companies. Many large pharmaceutical companies have similar, if not better, inherent options. Briefly, one skilled in the art can readily prepare many compounds for testing, from which he will select compounds promising for the treatment of tumor processes having the features of the compounds disclosed herein. It is of interest to identify the binding of selected antitumor compounds to the PDE5 protein in identifying compounds that can be tested and then selected using the criteria of the invention. As described below, it has been found that the more preferred desirable compounds that meet the selection criteria of the present invention bind to the cGMP catalytic region of PDE5.

A PDE5 sequence that does not include the catalytic domain was used for this determination.

One way to produce this sequence is to express this sequence as a fusion protein, more preferably with glutathione S-transferase ("GST"), to be noticeable.

The RT-PCR method is used to obtain the cGB domain of PDE5 with forward and reverse primers derived from the cDNA sequence of bovine PDE5A (McAllister-Lucas L.M. et al., J. Biol.

Chem. 268, 22863-22873 (1993)) and in the selection of PDEs 1 to 10. Total RNA assay kits, followed by purification of the oligo (dT) mRNA. A direct primer (GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG) is used to synthesize a 1484 bp fragment encoding a phosphorylation site and cGMP binding sites with both low and high affinity human PDE5A (203-1686 bp, cGBPDE5). -AGAAAT-G, 203-227) and antisense primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGCTG, 1664-1686). The synthesized cGB-PDE5 nucleotide fragment encodes 494 amino acids with 97% similarity to bovine PDE5A. The fragment is then cloned in the pGEX-5X-3glutathione-transferase (GST) fusion vector (Pharmacia Biotech) with the promoter and restriction sites EcoRI and XhoI. The fusion vector is then transfected into E. coli BL21 (DE3) (Invitrogen). The transfected BL21 is cultured to the log phase and then IPTG is added as an inducer. The induction takes place at 20 ° C for 24 hours. Then the bacteria are harvested and lysed. The soluble cell lysate is incubated with Sepharose 4B and GSH conjugate (GSH-Sepharose 4B). The GST-cGB-PDE5 fusion protein can bind to GSH-Sepharose beads and the other proteins are washed out of the beads with excess cold PBS.

The expressed GST-cGB-PDE5 fusion protein is shown as a 85 kD protein by electrophoresis in a 7.5% polyacrylamide SDS gel. It is characterized by binding cGMP and may be phosphorylated by protein kinase G and A. It has two cGMP binding sites and a Kd of 1.6 ± 0.2 μΜ, which is close to a Kd = 1.3 μΜ for native bovine PDE5. GST-cGBPDE5 on GSH-conjugated Sepharose beads can be phosphorylated in vitro with cGMP-dependent protein kinase and cAMP-dependent protein kinase A. K _m phosphorylation of GST-cGBPDE5 by PKG is 2.7 μΜ and V _max is 2.8 μΜ, while K _m BPDEtid phosphorylation is 68 μΜ. In PKG phosphorylation, the ratio of molecular phosphate incorporated into the GST-cGB-PDE5 protein and one to one protein.

The binding of cGMP to the compounds of interest (Francis SH et al., J. Biol. Chem. 255, 620-622, 1980) was determined in a total volume of 100 μΐ containing 5 mM sodium phosphate buffer (pH = 6.8). mM EDTA, 0.25 mg / ml BSA, ³ H-cGMP (2 μΜ, NEN) and GST-cGBPDE5 fusion protein (30 pg / assay). Each test compound is added at the same time as the ³ H-cGMP substrate and the mixture is incubated for 1 h at 22 ° C. The mixture is then transferred to a Brandel MB24 with GF / B filter, followed by a 2-fold wash with 10 mL of cold 5 mM potassium buffer (pH 6.8). The membranes are then excised, transferred to scintillation vials ("vials"), and 1 ml of water and 6 ml of Ready Safe ™ scintillation fluid are added to each vial. The vials are placed in a Beckman LS6500 scintillation device.

• * ·

For calculations, they were by binding the binding protein for 5 min. blank samples were prepared, the results being less than 1% compared to the uncooked protein.

Quenching by the filter membrane or other residues was also calibrated.

PDE5 inhibitors, sulfide, exisulind, Compound B, Compound E, E4021 and zaprinast and cyclic nucleotide analogues, cAMP, cyclic IMP, 8-bromo-cGMP were selected to test whether they could compete for cGMP binding sites of GSTcGB-PDE5. , cyclic UMP, cyclic CMP, 8-bromo-cAMP, 2'-O-butyl-cGMP and 2'-O-butyl-cAMP. The results were shown in Figure 24. CGMP specifically binds the GST-cGB-PDE5 protein. Cyclic AMP, cUMP, cCMP, 8-bromo-cAMP, 2'-O-butyl-cAMP and 2'-O-butyl-cGMP do not compete for cGMP binding. Cyclic IMP and 8-bromo-cGMP may partially compete with cGMP (2 μΜ) at high concentrations (100 μΜ). None of the PDE5 inhibitors showed any competition with cGMP in binding GST-cGBPDE5. Therefore, they do not bind to cGMP binding sites of PDE5.

Compound E, however, competitively (with cGMP) bound to PDE5 (i.e., peak A). (Compound E also competitively (with cGMP) binds to PDE peak B). Since Compound E does not bind to the cGMP-binding site of PDE5, there is a competitive binding between Compound E and cGMP where desirable compounds such as Compound E bind to the cGMP catalytic site of PDE5. However, this information, which can be readily obtained by one skilled in the art (using conventional competitive binding experiments), may facilitate modeling of other compounds. Using the chemical structures of the desired compounds disclosed herein and cGMP binding site information, one of ordinary skill in the art can model, identify, and select (using the selection criteria of the present invention) other compounds for use as therapeutics.

The compounds selected in accordance with the methodology of the present invention can be formulated into pharmaceutical compositions. Commonly, the term "pharmaceutical composition" is understood to mean a compound (eg, as a solid listed above) and a pharmaceutically acceptable carrier which is administered to a patient by oral administration in solid or liquid form, IV or IP administration in liquid form, by topical administration in the form of an ointment or in the form of a rectal or topical application in the form of a suppository. Most preferred are carriers for oral administration. As is well known in the art, pharmaceutically acceptable carriers for pharmaceutical compositions for oral administration are capsules, tablets, pills, powders, lozenges, and granules. In such solid dosage forms, the carrier may comprise at least one inert diluent such as sucrose, lactose or starch. These carriers may also contain, as in normal practice, other additives such as lubricating agents such as magnesium stearate. In the case of capsules, tablets, lozenges, and pills, the carriers may also contain buffering agents. Carriers such as tablets, pills, and granules can be prepared by film coating the surfaces of tablets, pills, or granules. Occasionally • • •

Being film coated with the compound which is then compressed into a tablet, pill or granule and the tablet, pill or granule used for administration to the patient. Preferred coatings include those that dissolve or degrade under the influence of colon pH such as shellak or Eudraget S.

Pharmaceutically acceptable carriers for pharmaceutical compositions include liquid dosage forms for oral administration such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents, such as water, which are commonly used in the art. In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, coloring, and perfuming agents.

Pharmaceutically acceptable carriers for pharmaceutical compositions for IV or IP administration include conventional pharmaceutical saline solutions.

Pharmaceutically acceptable carriers for pharmaceutical formulations for topical administration include DMSO, alcohol or propylene glycol and the like, which may be used in combination with a patch or other liquid-retaining material and which are required to keep the drug in place on the skin such that to keep it dry.

Pharmaceutically acceptable carriers for pharmaceutical compositions for rectal administration are more preferably suppositories which may contain, in addition to the compounds of the present invention, excipients such as coconut butter, suppository wax or gel.

The pharmaceutically acceptable carrier and compounds of the present invention are formulated into pharmaceutical compositions in individual dosage forms for administration to a patient. The dosage level of the active ingredient (i.e., the compound selected in accordance with the invention) in the dosage may vary, i. the dosage must contain an amount of active ingredient that is consistent with the desired method of administration (e.g., oral or rectal) effective to achieve tumor-eliminating activity. The dose level selected depends on the origin of the active compound administered (eg, its IC50, which can be readily ascertained), the route of administration, the desired duration of treatment, and other factors. If desired, the unit dose may be such that the daily requirement of the active compound is in a single dose or is divided between multiple doses, eg, two to four per day. For IV administration, the starting dose can be determined based on the dose required to achieve an IC 50 in terms of plasma content of an average adult male (i.e., about 4 liters). The initial doses of active compound selected in accordance with the present invention may range from 0.5 to 600 mg.

More preferably, the pharmaceutical compositions of the present invention are packaged in containers (eg, box, bottle or both) with suitable printed material (eg, inside the package) containing indications, instructions for use, and the like.

9 9 9 9 9 9 9 9 9

Numerous modifications and variations of the present invention are possible based on the above teachings. Thus, the present invention may be practiced other than as specifically described herein within the scope of the appended claims.

Claims (14)

PATENT CLAIMS A pharmaceutical composition for the treatment of a tumor process comprising a pharmaceutically acceptable carrier and a compound selected from: determining the activity of a cyclooxygenase (COX) inhibiting compound; determining the activity of a PDE inhibiting compound, wherein the PDE is characterized by: (a) higher specificity for cGMP than cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; and selecting a compound for treating tumors that exhibits less COX inhibitory activity than said PDE activity. The pharmaceutical composition of claim 1, further characterized in that said compound is selected for treating tumors based on determining whether it inhibits tumor cell growth in a tumor cell culture; and by selecting a compound that inhibits tumor cell growth. 3. A pharmaceutical composition according to claim 2, wherein the inhibition of growth is determined by evaluating whether the compound induces apoptosis. 4. A pharmaceutical composition for the treatment of a tumor comprising a pharmaceutically acceptable carrier and a compound selected based on determining the activity of the compound that inhibits tumor cell growth; determining the activity of a PDE inhibiting compound, wherein the PDE is charaterized by: (a) higher specificity for cGMP than cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; and (e) selecting a compound having tumor cell growth inhibitory activity and activity inhibiting said PDE The pharmaceutical composition of claim 4, further comprising determining whether the compound induces apoptosis in tumor cells; and selecting a compound that induces apoptosis. The pharmaceutical composition of claim 5, further comprising determining if the compound exhibits COX-inhibiting activity; and selecting a compound having a lower COX-inhibiting activity as compared to the activity of a compound inhibiting said PDE. 9 9 9 9 9 9 9 9 9 9 9 9 · » 9 9 9 9 • 9 9 9 7. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected from the PDE inhibiting activity of a compound, wherein the PDE is characterized by: (a) higher specificity for cGMP than for cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; and (e) selecting a compound having this inhibitory activity. The pharmaceutical composition of claim 7, wherein the compound is further selected based on determining whether the compound induces apoptosis in the tumor cell and by selecting a compound having activity that induces apoptosis. A pharmaceutical composition for the treatment of a tumor comprising a pharmaceutically acceptable carrier and a compound selected from: determining the activity of a cyclooxygenase (COX) inhibiting compound; determining the activity of a PDE2 inhibiting compound; and selecting a tumor treatment compound having less COX-inhibiting activity than said PDE-inhibiting activity. The pharmaceutical composition of claim 9, further characterized in that said compound is selected based on determining whether the compound inhibits tumor cell growth in a tumor cell culture; and selecting a compound for treating a tumor that exhibits tumor cell growth inhibition. The pharmaceutical composition of claim 10, wherein the inhibition of growth is determined by evaluating whether the compound induces apoptosis. 12. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected from: determining the activity of a tumor cell growth inhibiting compound; determining the activity of a PDE2 inhibiting compound and selecting a compound that exhibits tumor cell growth inhibiting activity and an activity inhibiting said PDE The pharmaceutical composition of claim 12, further comprising determining whether the compound induces apoptosis in tumor cells; and selecting a compound that induces apoptosis. 14. The pharmaceutical composition of claim 13, further comprising: • * * *. 22nd 23. 24. determining whether the compound exhibits COX-inhibiting activity; and selecting a compound having a COX inhibiting activity that is lower than that of said PDE inhibiting activity. A pharmaceutical composition for the treatment of a tumor comprising a pharmaceutically acceptable carrier and a compound selected by determining PDE2 inhibitory activity and selecting a compound having such inhibitory activity. The pharmaceutical composition of claim 15, further characterized in that the compound is selected based on determining whether it induces apoptosis in the tumor cell and by selecting a compound having activity that induces apoptosis. A method of selecting a compound for treating a tumor comprising: determining the activity of a cyclooxygenase (COX) inhibiting compound; determining the activity of a PDE2 inhibiting compound; and selecting a tumor treatment compound having a COX inhibitory activity less than that of said PDE. The method of claim 17, wherein said compound is selected based on determining whether the compound inhibits tumor cell growth in a tumor cell culture; and by selecting a compound for treating a tumor that inhibits tumor cell growth. The method of claim 18, wherein growth inhibition is determined by evaluating whether the compound induces apoptosis. A method of selecting a compound for treating a tumor, comprising determining the activity of the compound to inhibit tumor cell growth; determining the activity of a PDE2 inhibiting compound; and selecting a compound that exhibits tumor cell growth inhibiting activity and an activity inhibiting said PDE. The method of claim 20, further comprising determining whether the compound induces apoptosis in tumor cells; and selecting a compound that induces apoptosis. The method of claim 21, further comprising: determining whether the compound exhibits COX-inhibiting activity; and selecting a compound having a COX inhibitory activity that is lower than that of a PDE inhibitory compound. A method of selecting a compound for treating a tumor, comprising determining the activity of the compound inhibiting PDE2 and selecting a compound having such inhibitory activity. The pharmaceutical composition of claim 23, wherein the compound is further selected based on determining whether the compound induces apoptosis in the tumor cell and by selecting the compound having activity that induces apoptosis. • « 9 9 9 9 9 9 25. A method of identifying a compound for the treatment of a tumor, comprising determining a cyclooxygenase (COX) inhibitory activity of the compound; and determining the activity of the PDE inhibiting compound, wherein the PDE is characterized by: (a) higher specificity for cGMP than for cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; selecting a compound having a COX-inhibiting activity lower than that of said PDE 26. The method of claim 25, further comprising determining whether the compound inhibits tumor cell growth in culture; and selecting a tumor treatment compound that inhibits tumor cell growth. The method of claim 25, further comprising: (a) determining whether the compound induces apoptosis of the tumor cell; and (b) selecting a compound for treating a tumor that induces apoptosis. 28. A method of selecting a compound for the treatment of a tumor, comprising determining the activity of inhibiting the growth of the compound; determining the activity of a PDE inhibiting compound, wherein the PDE is characterized by: (a) higher specificity for cGMP than for cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; and selecting a compound that exhibits this growth inhibitory activity and the activity inhibiting said PDE. 29. A method of testing a compound effective for treating a tumor comprising determining the PDE inhibitory activity of a test compound, wherein the PDE is characterized by: (a) higher specificity for cGMP than for cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase; and selecting a compound having this inhibitory activity. 30. The method of claim 29, comprising determining whether the compound induces apoptosis in the cell and further selecting the compound with apoptosis inducing activity. 99 99 9 «• · 9 9 « 9 9 9 99 ··· 31. The method of claim 29, further comprising determining whether the selected compound inhibits prostaglandin synthesis and the selection of a compound having low prostaglandin synthesis inhibiting activity. 32. The method of claim 29, wherein the activity of said PDE is detected by isolating cGMP-specific PDE activities from a tumor cell line comprising PDE5 and said new PDE by inhibiting PDE5 activity by exposing said PDE isolate to a PDE5 inhibitor at a concentration. which does not inhibit new PDE and by determining the inhibitory activity of the test compound to the remaining cGMP activity, whereby the inhibitory activity of the test compound to the new PDE can be determined. 33. An isolated phosphodiesterase characterized by: (a) higher specificity for cGMP than cAMP; (b) positive cooperative kinetic behavior in the presence of a cGMP substrate; (c) submicromolar affinity for cGMP; and (d) insensitivity to incubation with purified cGMP-dependent protein kinase. 34. The isolated phosphodiesterase of claim 33, further characterized by reduced susceptibility to inhibition by zaprinast and E4021. 35. The isolated phosphodiesterase of claim 33, further characterized in that it can be separated from classical PDE5 activity by anion exchange chromatography. 36. The isolated phosphodiesterase of claim 35, further characterized in that it is hardly activated at all by the calcium / calmodulin system. 37. The isolated phosphodiesterase of claim 36, further characterized by being insensitive to rolipram, vinpocetin and indolidane. 38. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected from: assessing the antitumor activity of the compound against the tumor to be treated; assessing if the compound increases PKG activity in the tumor to be treated; and selecting a compound that exhibits anti-tumor activity and has the ability to cause an increase in PKG in the tumor being treated. 39. The pharmaceutical composition of claim 38, wherein said compound is further selected based on the evaluation of whether the compound inhibits PDE5 and the selection of a compound that inhibits PDE5. • • • • * v «in 9 ·> • 9 9 9 »• * 40. The pharmaceutical composition of claim 38, wherein said compound is further selected based on evaluating whether the compound reduces β-catenin in the tumor to be treated and by selecting a compound that reduces β-catenin. 41. The pharmaceutical composition of claim 38, wherein said compound is further selected based on evaluating whether the compound inhibits cGMP-specific phosphodiesterase (&quot; PDE &quot;) and selecting a compound that inhibits said PDE. 42. The pharmaceutical composition of claim 38, wherein said compound is further selected based on evaluating whether the compound increases PKG expression and based on the selection of the compound if it increases PKG expression. 43. The pharmaceutical composition of claim 38, wherein said compound is further selected based on evaluating whether the compound increases PKG activation and based on the selection of the compound if it increases PKG activation. 44. The pharmaceutical composition of claim 38, wherein said compound is further selected based on the evaluation of whether the compound inhibits PDE2 and the selection of a compound that inhibits PDE2. 45. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected from: assessing whether the compound increases PKG activity in the intact tumor cell of the tumor to be treated; assessing whether the compound decreases β-catenin in tumor cells; selecting a compound which causes an increase in PKG activity in the intact tumor cell and causes a decrease in β-catenin in the tumor to be treated. 46. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected based on the selection of a compound that increases PKG activity in the tumor; and evaluating the tumor growth inhibitory activity of the compound, wherein the compound that increases PKG activity and has tumor growth inhibitory activity has the ability to inhibit the tumor process without inhibiting the growth of normal cells. 47. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected from determining the activity of a cyclooxygenase (COX) inhibiting compound and determining whether the compound increases PKG activity in tumor cells; and selecting a compound for treating a tumor with less COX-inhibiting activity than its ability to increase PKG activity. 48. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected based on determining the activity of the compound inhibiting the tumor. 4 · 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 44 cell growth; determining whether the compound increases PKG activity in tumor cells; and selecting a compound that exhibits an activity that inhibits tumor cell growth and increases PKG activity in the tumor cells. 49. A pharmaceutical composition for treating a tumor comprising a pharmaceutically acceptable carrier and a compound selected based on the assessment of whether PKG causes phosphorylation of β-catenin in the tumor being treated and the selection of a compound that causes phosphorylation of β-catenin as the compound. for treating the tumor to be treated. 50. A method of selecting a compound for treating a tumor to be treated, comprising: (a) assessing the antitumor activity of the compound on the tumor to be treated; (b) assessing whether the compound increases PKG activity in the tumor to be treated; and (c) selecting a compound that exhibits anti-tumor activity and is capable of causing an increase in PKG activity in the tumor to be treated. 51. The method of claim 50, further comprising evaluating whether the compound inhibits PDE5 and selecting a compound that inhibits PDE5. 52. The method of claim 50, further comprising assessing whether the compound reduces β-catenin in the tumor to be treated, and selecting a compound that thereby reduces β-catenin. 53. The method of claim 50, further comprising assessing whether the compound inhibits cGMP-specific phosphodiesterase ("PDE") and selecting a compound that inhibits said PDE. 54. The method of claim 50, further comprising evaluating whether the compound increases PKG expression and selecting the compound if it increases PKG expression. 55. The method of claim 50, further comprising assessing whether the compound increases PKG activation and selecting the compound if it increases PKG activation. 56. The method of claim 50, further comprising assessing whether the compound inhibits PDE2 and selecting a compound that inhibits PDE2. 57. A method of selecting a compound for treating a tumor to be treated, the method comprising: (a) assessing whether the compound increases PKG activity in the intact tumor cell of the tumor to be treated; (b) assessing whether the compound reduces β-catenin in the tumor cells; and (c) selecting a compound which causes an increase in PKG activity in the intact tumor cell and causes a decrease in β-catenin in the tumor to be treated. • · · · · · · · · · · · · · · · · · · · · · · · · 58. A method of identifying a compound effective for treating a tumor comprising: selecting a compound that increases PKG activity in the tumor; and evaluating the activity of the tumor growth inhibiting compound, wherein the compound that increases PKG activity and has tumor growth inhibiting activity has the ability to inhibit the tumor process without inhibiting the growth of normal cells. 59. A method of identifying a compound effective for treating a tumor comprising: determining a cyclooxygenase (COX) inhibitory activity of the compound; and determining whether the compound increases PKG activity in tumor cells; low COX-inhibiting activity and an increase in PKG activity suggest that the compound is effective for the treatment of tumor. 60. A method of selecting a compound for treating a tumor, comprising determining the activity of the compound to inhibit tumor cell growth; determining whether the compound increases PKG activity in tumor cells; and selecting a compound that exhibits activity that inhibits tumor cell growth and increases activity PKG in tumor cells. 61. A method of selecting a compound for treating a tumor to be treated, comprising evaluating whether PKG causes phosphorylation of β-catenin in the tumor to be treated, and selecting a compound that causes PKG to phosphorylate β-catenin, as a compound for treating a tumor to be treated.