KR100644365B1 - Methods for identifying compounds for inhibition of neoplastic lesions, and pharmacetical compositions containing such compounds - Google Patents

Abstract

The present invention is directed to phosphodiesterase-2 (PDE2), phosphodiesterase-5 (PDE5) and / or protein kinases for the identification of compounds useful in the prevention and treatment of precancerous and cancerous lesions in mammals. The present invention relates to the use of at least one of G (PKG), to a method for treating neoplasia of the neoplasm using the compound, and to a pharmaceutical composition containing the compound.

Inhibitory activity of neoplasms.

Description

Methods for identifying compounds for inhibition of neoplastic lesions, and pharmacetical compositions containing such compounds}

1 is a graph showing the cGMP activity of cGMP phosphodiesterase obtained from SW480 neoplastic cells, as eluted using a DEAE-triacrylic M column.

FIG. 2 is a graph of cGMP activity of reloaded cGMP phosphodiesterase obtained from SW480 tumor cells, as eluted using a DEAE-trisacryl M column.

3 is a graph showing the kinetic aspects of the novel PDE of the present invention.

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

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

FIG. 6 illustrates the inhibitory effect of sulfindac sulfide and excisulin on PDE4 and PDE5 purified from cultured cancer cells.

Figure 7 illustrates the effect of sullindac sulfide on HT-29 intracellular cyclic nucleotide levels.

8 illustrates the phosphodiesterase inhibitory activity of Compound B.

9 illustrates the phosphodiesterase inhibitory activity of Compound E.

FIG. 10 illustrates the effect of sulfinad sulfide and excisulin on apoptosis and necrosis of HT-29 cells.

FIG. 11, measured by DNA fragments, illustrates the effect of sulfinact sulfide and excisulin on HT-29 cell growth inhibition and apoptosis induction.

12 illustrates apoptosis induction properties of Compound E. FIG.

Figure 13 illustrates apoptosis induction properties of Compound B.

Figure 14 illustrates the effect of sulfindac sulfide and excisulin on cancer cell growth.

FIG. 15 illustrates growth inhibitory activity and apoptosis inducing activity for sulfinact sulfide and control (DMSO).

Figure 16 illustrates the growth inhibitory activity of Compound E.

FIG. 17 is an illustration of the inhibition of pre-melignant, hyperproliferative lesions in mouse mammary gland organ culture by sulfinac metabolites.

FIG. 18A shows SDS protein gel of SW480 lysate treated with drug without cGMP added, where cells were DMSO (0.03%; lanes 1 and 2), excisulin (200, 400 and 600 μM; lanes). 3, 4, 5), and E4021 (0.1, 1 and 10 μM; lanes 6, 7, 8) were incubated for 48 hours.

18B is an SDS (X-ray film exposure) gel PKG assay of SWGMP lysate treated with cGMP and drug, where cells were DMSO (0.03%; lanes 1 and 2), exisulind (200, 400 and 600 μM; lanes 3, 4, 5), and E4021 (0.1, 1 and 10 μM; lanes 6, 7, 8) were incubated for 48 hours.

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

FIG. 20 is a graph showing the cGMP activity of cGMP phosphodiesterase obtained from HTB-26 tumor cells, which was eluted using DEAE-triacrylic M column.

FIG. 21 is a graph showing the cGMP activity of cGMP phosphodiesterase obtained from HTB-26 tumor cells as eluted using DEAE-trisacryl M column according to the concentration of substrate.

FIG. 22 is a graph showing cGMP activity of cGMP phosphodiesterase obtained from LnCAP tumor cells as eluted using DEAE-triacrylic M column.

FIG. 23 is a graph showing the cGMP activity of cGMP phosphodiesterase obtained from LnCAP tumor cells as eluted using DEAE-trisacryl M column according to the concentration of substrate.

FIG. 24 is a bar graph illustrating the specific binding of noncatalytic cGMP binding sites of PDE5 to cyclic nucleotide derivatives and selected PDE5 inhibitors.

FIG. 25 is a graph showing cGMP activity of cGMP phosphodiesterase obtained from SW480 tumor cells as eluted analysis using DEAE-trisacryl M column with ethylene glycol buffer.

FIG. 26 is a graph showing cGMP activity of cGMP phosphodiesterase obtained from SW480 tumor cells grown in roller bottles, as eluted using a DEAE-trisacryl M column.

27A shows an increase in the amount of histone related fragment DNA over time in LNCaP cell medium treated with 50 μM of Compound I.

27B shows the treatment of PrEC prostate cells treated with Compound I (50 μM) for 4 days without affecting the DNA fragment.

The present invention is directed to phosphodiesterase-2 (PDE2), phosphodiesterase-5 (PDE5) and / or protein kinases for the identification of compounds useful in the prevention and treatment of precancerous and cancerous lesions in mammals. It relates to the use of at least one of G, to a method for treating neoplasia of a neoplasm using said compound, and to a pharmaceutical composition containing said compound.

Currently, in the case of non-surgical cancer treatment, one or more chemotherapeutic agents or hormones with highly toxic effects if the cancerous condition of the patient reaches the therapeutic benefit of the chemotherapeutic or hormonal agent beyond the severe side effects of these agents. Agents are administered to patients to treat cancer patients. The side effects are already well known to oncologists and the types of side effects vary depending on the drug. Typically, however, chemotherapeutic agents are used over a short period of time and allow the patient to pause the drug to withstand the side effects of the drug. Therefore, in consideration of both the risks and benefits of the drug, if the patient has a precancerous lesion, he or she does not use the chemotherapeutic agent due to its side effects, and in order to prevent recurrence after the cancer has been removed. Do not continue to use chemotherapy or hormones.

In the early 1990s, the light of hope for cancer patients began to appear as nonsteroidal anti-inflammatory drugs ("NSAIDs"). Many studies have been published on cancer and prostate cancer that introduce various biochemical molecules overexpressed from tumor tissue. Accordingly, the results of studies on whether overexpressed specific molecules are associated with cancer and whether neoplasms can treat aberrant proliferation when the excessive expression is suppressed have appeared. For example, with respect to familial adenomatous polyposis (FAP), Waddell et al., 1993, showed that prostaglandins are excessively expressed in the polyps. It has been assumed that cancer-related symptoms can be alleviated by inhibiting enzyme (PGE ₂ ) activity (Waddell, WR et al., "Sulindac for Polyposis of the Colon," Journal of Surgical Oncology, 24: 83-87, 1983). Thus, Waddell et al. Administered a nonsteroidal anti-inflammatory drug, sulindac (PGE ₂ inhibitor), to several FAP patients. Accordingly, Waddell et al. Found that the production of polyps was eliminated and showed no relapse during the therapy. The phenomenon of inhibiting PGE ₂ is a result of suppressing cyclooxygenase (COX) by the nonsteroidal anti-inflammatory agent. Wadel et al.'S success with the administration of sulfinac and its relationship with PGE ₂ / COX provided an opportunity to confirm the role of two biochemical objects, PGE ₂ and COX, in carcinogenesis. I have proven my opinion.

The minimal side effects of sul-dang compared to conventional chemotherapeutic and hormonal drugs have been encouraging for patients suffering from neoplasia, thus opening the door for the possibility of treating them early in the tumor. Compared with conventional chemotherapeutic agents, the advantage of long-term administration is added. However, the hopes have eased as public questions about whether the compound may have therapeutic effects on cancers that have already occurred in light of the fact that Waddell et al. Administered sulfinac only to precancerous patients (FAP patients).

In addition, the hope has been eliminated by the side effects of nonsteroidal anti-inflammatory drugs. Prolonged administration of sulfinac or other nonsteroidal anti-inflammatory drugs may exacerbate the digestive tract in which PGE ₂ functions in gastrointestinal protection. In addition, the long-term administration of the nonsteroidal anti-inflammatory drugs inhibits renal failure and normal blood coagulation. As Waddell et al. Experienced, some patients receiving sulfinac stopped taking medication due to side effects, which forced them to rely on surgical procedures to remove polyps. Waddell, WR et al., " Sulindac for Polyposis of the Colon, "The American Journal of Surgery, 157: 175-79, 1989]. Thus, patients with neoplastic dysplasia, such as patients with FAP, patients with hepatic polyp formation, or men who have undergone prostatectomy, who have increased PSA levels (an increase in PSA levels indicates a recurrence of the disease, but the transition to cancer It is not known whether the nonsteroidal anti-inflammatory agent could actually be a long-term treatment. In addition, the side effects of the non-steroidal anti-inflammatory drugs are limited to their use in patients with abnormal growth of other neoplasms that require long-term drug treatment. Recently, some scholars have recommended the administration of nonsteroidal anti-inflammatory drugs specific to COX-2, such as celecoxib. However, since the drug causes kidney disorders and other side effects, limitations such as taking the dose and duration of the drug into consideration when administering to a neoplastic dysplasia patient over a long period of time are being applied. In addition, recently published literature suggests that a high dose of a drug, such as celecoxib, should be administered in order to obtain a therapeutically effective effect on the colon polyps at the site of the colon's rectum.

More important in the treatment of colon cancer is that aberrant proliferation of certain colonic neoplasms (eg HCT-116) does not express COX-2 and the inhibitor is ineffective against this type of neoplasm. It has been reported [Sheng, et al. "Inhibition of Human colon Cancer Cell Growth by Selective Inhibition of Cyclooxygenase-2," J. Clin. Invest., 99 (9): 2254-9, 1997].

Scholars have recently discovered that COX / PGE ₂ , which has been the subject of previous research, is not a fundamental target for treating neoplastic dysplasias that require long-term treatment. (Probably a secondary target). Pamuku et al. Unexpectedly found that sulfonyl compounds, which have been reported to be substantially incapable of inhibiting PGE ₂ and COX, inhibited the growth of various tumor cells, including polyps in colons [US Pat. No. 5,401,774]. . The sulfonyl derivatives have been shown to be effective in the rat model of carcinogenesis in the colon, and other analogues (hereinafter referred to as "exisulind") have been shown to be effective in clinical trials in FAP patients. More surprisingly, it has been shown to be effective against cancers already occurring in clinical trials, prostate cancer.

In addition, recently published studies strongly demonstrate that COX I and / or COX II are not substantially expressed in all neoplasms, suggesting that inhibitors specific for COX I or COX II may be useful in treating neoplasia. The hope of a therapeutically useful application has been removed. Lim et al., "Sulindac Derivatives Inhibit Growth and Induce Apoptosis in Human Prostate Cancer Cell Lines," Biochem. Pharmacology, Vol. 58, pp. 1097-1107 (1999) in press].

Thus, as with many other proteins overexpressed in neoplasms, overexpression of PGE2 / COX is the result of a combination of these rather than the causes of some neoplasia. However, synthesizing the findings raises the question of how compounds such as excisulind (having activity against both COX- and tumor-free tumors) work. What does the compound do in tumor cells?

In Piazza et al., Compounds such as excisulind inhibit cyclic-specific GMP phosphodiesterases (eg PDE5), so other compounds such as these compounds can be detected using this enzyme, which is an anti-tumor It has led to the discovery of other compounds that can be developed and formulated into pharmaceutical compositions that are effective (US Application Nos. 08 / 866,027 and 09 / 46,739).

The pharmaceutical composition is extremely excellent in anti-tumor effect, and if the patient wants to avoid the occurrence of side effects, there can be no side effects associated with conventional chemotherapeutic agents and side effects associated with COX or PGE ₂ inhibition. In addition, compounds that have anti-tumor effects and inhibit PGMP specific to PGMP induce apoptosis of tumor cells (form of apoptosis or suicide of cells) but are harmless to normal cells. Thus, the novel compounds are newly defined as selective apoptotic anti-neoplastic drugs (SAANDs) that induce selective apoptosis. Therefore, the SAAND preparations argue against a number of existing problems such as: (1) normal cells also die to see the effects of anti-tumor compounds. (2) COX is responsible for neoplasia; And (3) to prevent colon cancer caused by NSAIDs by inhibiting COX of one or both types.

However, according to the new findings presented below, not all compounds that inhibit PDE5 induce apoptosis in tumor cells. For example, as well known PDE5 inhibitors, japrinast and sildenafil alone do not induce apoptosis and do not inhibit the growth of tumor cells. However, PDE5 inhibitors that cause pre-apoptosis selectively induce apoptosis (ie, tumor cells rather than normal cells), and are not substantially involved in COX inhibition. There is no question that it can be.

However, the screening of PDE5 for the development of safe compounds that share anti-tumor and pre-apoptotic properties for the formulation of new pharmaceutical compositions for use in the treatment of neoplasia, proliferative and already-occurring cancers. It is desirable to improve the technique.

The present inventors have conducted research with the question that some PDE5 inhibitors alone induce apoptosis, but other compounds do not, and therefore, phosphodides specific to cyclic GMP which have not been reported before. Therapeutic active forms have been discovered. Without being bound by a particular theory, the inventors believe that the novel PDE activity may be a novel tertiary structure of PDE2 that is substantially devoid of c-AMP hydrolytic activity, ie cGMP specific. Conventional PDE2 (classic PDE2) is not specific for cGMP (which also hydrolyzes cAMP) and is found in tumor cells. The novel PDEs and PDE2s are useful for screening pharmaceutical compositions with desirable anti-tumor effects. Basically, PDE5 and PDE2 activity (new or existing tertiary structures) in tumor cells are inhibited by compounds that inhibit PDE5 with antitumor effect, resulting in apoptosis. If only PDE5 suppresses (not several forms of PDE2), apoptosis does not occur.

In a broad aspect, the novel PDE tertiary structure has the following characteristics.

A) shows specificity for cGMP rather than cAMP,

B) show cooperative kinetic aspects in the presence of a cGMP substrate;

C) exhibits a submicromolar affinity for cGMP;

D) insensitivity during incubation with purified cGMP dependent protein kinase

Other features of the novel PDE are as follows.

-Reduce the susceptibility to inhibition of japrinas and E4021.

Can be separated from conventional PDE5 activity by anion exchange chromatography.

Not activated by calcium and calmodulin.

-It is insensitive to rolipram, vinpocetin and indolidan.

A preferred embodiment of the present invention is to assess whether one compound increases cGMP-dependent protein kinase G (PKG) activity and / or reduces beta-catenin of tumor cells. As an unexpected feature of selective apoptotic anti-neoplastic drugs (SAAND), it has been found that when tumor cells are exposed to SAAND, PKG activity increases and beta-catenin decreases. The inventors believe that an increase in PKG activity is at least partially elevated in PKG activity, as described above, by an increase in cGMP caused by SAAND inhibiting the appropriate PDEs.

Other characteristics of SAAND are:

(1) inhibition of PDE5 reported in the patent;

(2) inhibition of PDE structures specific to novel cGMP;

(3) inhibition of PDE2;

(4) elevation of intracellular cGMP in tumor cells;

(5) reduction in cAMP concentrations in some tumor cells.

Thus, one embodiment of the novel method according to the present invention is to assess the fact of which compounds increase PKG activity in tumor cells and whether they inhibit PDE5.

Another embodiment of the novel screening method according to the present invention is to assess whether a compound increases PKG activity in tumor cells and whether it inhibits PDE and / or PDE2 specific to the novel cGMP mentioned above. . A third embodiment also evaluates whether a compound increases PKG activity in tumor cells and whether cGMP increases or decreases cAMP. This successfully evaluated compound is then applied as SAAND.

First of all, the present invention relates to a novel method of selecting compounds for in vivo and in vitro test methods that can prevent and treat cancer, particularly pro-cancer lesion sites safely. In particular, the present invention relates to methods of selecting compounds that can be used to treat and prevent neoplasia, including prostate cancer. Identified compounds can minimize side effects due to COX inhibition and other nonspecific interactions by using existing chemotherapeutic agents. Compounds of interest can be tested by exposing new PDEs to the target compounds, and if specific compounds inhibit the new PDEs, specific experiments will continue to investigate the antitumor effects (eg, Designing or conducting in vivo or in vitro animal experiments and clinical trials).

Accordingly, the present invention includes a search and selection method for identifying compounds effective for treating aberrant proliferation of neoplasia that confirms the inhibition of the novel PDE and / or compounds for PDE2 and COX. Preferably, the search and selection method according to the invention comprises a method of determining whether the compound inhibits the growth of tumor cells in vivo or in vitro.

By selecting a compound as described above, it is possible to develop a compound for treating aberrant proliferation of neoplasia, and to identify a potentially beneficial and improved compound more quickly and accurately than ever before. Specific advantages will be apparent in the detailed description of the invention that follows.

Still another object of the present invention is to provide a pharmaceutical composition containing the compound as well as a therapeutic method associated with the compound.

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

A. Separation of new PDE tertiary structure

Phosphodiesterase specific to cGMP (which would be a new PDE2 form) was first isolated from human carcinoma cells commonly referred to as SW480 [American Tissue Type Collection in Rockville, Maryland, U.S.A.]. SW480 is a human colon cancer cell derived from a properly differentiated epithelial adenocarcinoma. As described below, structures similar to the tertiary structure were isolated from neoplasms of the breast (ie, HTB-26 cells) and prostate (LNCAP cells).

As used herein, the term "isolated" (which can be understood from existing invention) includes not only isolated from tumor cells but also made by recombinant methods (eg, expressed in cell lines of bacteria or other non-human cell host vectors). that). However, separation from human tumor cells is more desirable because the protein of interest thus separated has a similar structure (ie, tertiary structure or anatomy), if not identical to one of its natural forms in the tumor cells. This structure facilitates the selection of antitumor compounds that will inhibit the enzyme in vivo.

Novel PDE activity was first discovered in SW480 colon cancer cell line. To isolate novel phosphodiesterases from SW480, approximately 4 million SW480 cells were cultured, washed twice with 10 mL of cooled phosphate buffer (PBS), pelletized by centrifugation and plated with 150 cm 2 tissue culture dishes. Scraped off.

The cells were homogenized 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 μM benzamidine, 10 μM TLCK, 2000 Resuspend in U / mL aprotinin, 2 μM pepstatin A) and homogenize in an ice bath using polytron tissueizer (3 times, 20 seconds / pulse). Homogenized material was centrifuged at 105,000 g for 60 minutes at a temperature of 4 ° C. using a Beckman L8 ultracentrifuge, and then the supernatant was diluted with TMPI-EDTA (60 mL) and pre-equilibrated with TMPI-EDTA buffer. -Applied to Trisacryl M column. The column used was washed with 60 mL TM-EDTA, and the PDE activity was eluted with 120 mL linear gradient of NaOAC (0-0.5 M) in TM-EDTA at a flow rate of 0.95 mL / minute, 1.4 mL / fraction. Eight fractions are collected and immediately quantified for cGMP hydrolysis (ie, in minutes). Figure 1 shows the elution of the column showing the peaks of the first two cGMP PDE activities, peak A and peak B eluted with 40-50 mM and 70-80 mM NaOAC, respectively.

As described below, the cyclic nucleotide PDE activity of the fractions was measured using a modified two-step radioisotope method of Thompson et al. (Thompson WJ, et al., Adv. Cyclic Nucleotide Res. 10: 69-92, 1979). It was. The reaction was always carried out with a suitable substrate (200,000 cpm) with Tris-HCl (40 mM; pH 8.0), MgCl ₂ (5 mM), 2-mercaptoethanol (4 mM), bovine serum albumin (30 μg), cGMP (0.25 μM to 400 μl containing 5 μM). The incubation time was adjusted to less than 15% hydrolysis. The mixture was incubated at 30 ° C. and boiled for 45 seconds before the reaction was stopped. Subsequently, the mixed solution was cooled, incubated at 30 ° C. for 10 minutes after the addition of snake venom (50 μg). After stopping the reaction by adding MeOH (1 mL), the mixture was transferred to an anion exchange column (Dowex 1-X8, 0.25 mL resin). The eluate was mixed with MeOH and applied to the resin. After addition of 6 mL scintillation solution, tritium activity was measured using Beckman LS 6500 for 1 minute.

In addition, 15-30 fractions of the original 80 were refilled with a DEAE-Trisacry M column to fractionate the cGMP hydrolysis activity of peak A and peak B, and the linearity of NaOAC (0-0.5 M) in TM-EDTA. It is eluted with a concentration gradient. The fractions were again immediately analyzed for cGMP hydrolysis (by the process using 0.2, 2, 5 μM substrate described above) and the results are graphically shown in FIG. 2. In the vicinity of peak B shown in FIG. 2, the activity was dramatically improved by increasing the substrate concentration of cGMP when in contrast with peak A. The results are consistent with those of PDE2, but in view of the fact that the enzymes characterized in FIG. 2 are cGMP-specific (see below), a new form can be compared to the classical PDE2 reported in the literature. It suggests the fact. Peak A activity shows a clear substrate saturation at the high affinity catalyst site.

B. Separation of classic PDE2 from SW480

By developing two ways to isolate peak B from SW480, it was found that the enzyme had conventional PDE2 activity (ie only cGMP-nonspecific stimulation of cGMP). The first method is to incubate SW480 in a 850 cm 2 Corning roller bottle instead of a 150 cm 2 tissue culture flask. SW480 was incubated at a rate of 0.5 rpm in bottles containing 200 mL RPMI 1640, 2 mM glutamine and 25 mM HEPES, respectively. Cells were harvested according to the following procedure. PBS medium was warmed to 37 ° C. for at least 15 minutes. Prepare 5% FBS / RPMI 1640 (200 mL) medium and add 5 mL of glutamine. In addition, 5 mL of antibiotic and antifungal were added together.

70 mL of PBS solution was added to 10 mL of 4 × pancreatin. The temperature of the mixed solution was kept at room temperature. The medium was removed and washed with 4 mL of phosphate buffer to protect the bottom of the flask. All solutions were removed by pipette. Add 4 mL of diluted pancretin to the flask and take care to protect the bottom. The flask was incubated at 37 ° C. for 8-10 minutes. After incubation, the flask was checked with an inverted microscope to confirm that all cells were grown quickly. The face of the flask was carefully impacted several times to allow cells to separate. Pancreatin proteolysis was stopped by adding 10 mL of cooled medium to the flask. The solution was shaken at the bottom to recover the cells. The medium is removed using a 25 mL pipette and the cells are placed in a 50 mL centrifuge tube containing ice. The tube was spun at a speed of 1000 rpm in a clinical centrifuge for 5 minutes at 4 ° C. to pellet the cells. The supernatant was removed and each pellet frozen in liquid nitrogen for 15 seconds. Recovered cells are stored at -70 ° C.

PDEs were isolated from recovered SW480 cells using the EPLC method. Sample filling and elution rates were controlled on an 18 mL DEAE TrisAcryl M column using AKTA EPLC (Pharmacia). About 6 million cells contained in SW480 were used for the experiment. Cell 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 μM benzamidine, 10 μM TLCK, 2000 U / The sample was manually homogenized after resuspension with mL aprotinin, 2 μM leupetin, 2 μM pepstatin A). EPLC buffer A consists of 8 mM TRIS-acetate, 5 mM magnesium acetate, 0.1 mM EDTA, pH 7.5 and buffer B consists of 8 mM TRIS-acetate, 5 mM magnesium acetate, 0.1 mM EDTA, pH 7.5. It became. The supernatant was packed into a column at a rate of 1 mL per minute and washed with 60 mL buffer A at 1 mL per minute. The content was set to 0-15% Buffer B in 60 mL, 15-60% Buffer B in 60 mL, and 50-100% Buffer B in 16 mL. 1.5 mL fractions were recovered according to the slope.

The experimental results obtained are similar to the experimental results for the novel PDE activity (e.g., Figure 1) already presented above (Figure 26). However, the cAMP hydrolysis activity at 0.25 μM substrate concentration, which can be activated 2-3 times in this separated peak B 5 μM cGMP, was excluded.

The second method used to classify classic PDE2 in SW480 was performed using the non-PFLC DEAE column method supplied above, but in this case 30% ethylene glycol, 10 mM TLCK and 3.6 mM β -Performed with a buffer solution containing mercaptoethanol (see section IA). By adding the reagent to the buffer, the elution rate (Fig. 25) results in peak A from 40 to 150 mM, peak B from 75 to 280 mM, and peak C from 200 to 500 mM sodium acetate (see Figure 25). ) From low concentrations to high concentrations of sodium acetate. Peak B, shown in FIG. 25, can be analyzed with 2 μM cAMP substrate and showed twice the activity by 5 μM cGMP (see FIG. Y). EHNA, a selective PDE2 inhibitor with 1.6 μM IC ₅₀ at peak B, inhibited 2 μM cGMP PDE activity and also peaked at 3.8 μM IC ₅₀ (and 2.5 μM IC _{50 with} 10 μM rolipram). Inhibits 2.0 μM cAMP PDE activity in B.

C. cGMP-specific and Novel Peak B Activity of PDE Peak A

Each fraction from the DEAE column in section IA also measured cGMP hydrolytic activity (0.25 μM cGMP) with or without Ca ⁺⁺ or Ca ^++- CaM and / or EGTA, and with 5 μM cGMP Measurements were made for cAMP (0.25 μM cAMP) with or without conditions. PDE peak A and peak B (fractions 5 to 22; see FIG. 1) do not significantly hydrolyze cAMP, demonstrating that both do not have the activity of conventional PDE, a family of typical cAMP hydrolysates (ie PDE 1, 2, 3).

Ca ⁺⁺ (with or without calmodulin) did not stimulate the cAMP or cGMP hydrolysis activity of peak A and peak B, and cGMP did not activate or inhibit cAMP hydrolysis. Peaks A and B indicate that they correspond to cGMP-specific PDE activity but are different from classical or known PDE1, PDE2, PDE3 or PDE 4 activity.

As described below, for the novel PDE peak B, the annular GMP does not stimulate the cGMP hydrolysis activity of the enzyme or any cAMP hydrolysis activity (as opposed to the case of peak B from section IB above). Since the known isoforms of PDE2 hydrolyze both cGMP and cAMP, these results indicate that novel PDE peak B-the novel phosphodiesterase of the present invention-cAMP hydrolysis ("cGS") that cGMP activates. Or no known or known PDE2 family activity.

D. Peak A is classic PDE5 but not new peak B (new cGMP-specific PDE).

To characterize certain PDE isotopes, the kinetics and substrate affinity should be measured. Peak A showed the characteristics of the typical "PDE5". For example, the Km of the enzyme of cGMP is 1.07 μM and Vmax is 0.16 nmol / min / mg. Further, Jaffe Lena host (IC ₅₀ = 1.37 μM) and _{E4021 (IC 50 = 3 nM)} , and the side napil as described below inhibit the activity of the peak A. Moreover, Japrinat showed an inhibition on cGMP hydrolytic activity of peak A, consistent with the results reported in the literature.

PDE peak B in section IA shows significantly different kinetic properties when compared to PDE peak A. For example, cyclic GMP hydrolysis, which means the Michaelis-Menten dynamic pattern in the Eddy-Hopsti plot of peak A, decreases linearly with slope as the concentration of the substrate increases. However, peak B shows novel properties for cGMP hydrolysis under the absence of cAMP. That is, as the cGMP substrate increases (ie, see Figure 3), the slope of the Eddy-Hopsti plot decreases (apparently Km = 8.4) and then increases (Km <1). Therefore, such a phenomenon exhibits a submicromolar affinity of peak B to cGMP (ie, Km <1).

Consistent with kinetic studies in the presence of cGMP substrates (FIG. 3) and positive-cooperative kinetic behavior, cGMP hydrolytic activity increases with increasing concentrations of cGMP substrates. This was found by comparing 0.25 μM, 2 μM and 5 μM concentrations of cGMP in the presence of PDE peak B after the second DEAE separation to exclude cAMP hydrolysis and exclude that the new enzyme is already identified PDE5. As shown in Figure 2, the higher the concentration of cGMP, the greater the cGMP hydrolysis of PDE peak B.

This finding suggests that cGMP bound to the enzyme of peak B causes a structural change in this enzyme. This confirms the advantage of using native enzymes obtained from tumor cells, but the present invention is not limited to native forms of enzymes having the above characteristics.

E. Effect of PDE peak B on insensitive to other pride and sildenafil and other PDE inhibitors compared to peak A

Other PDE inhibitors were studied using 12 drug concentrations from 0.01 to 100 μM and substrate concentrations of 0.25 μM ³ H-cGMP. IC ₅₀ values are calculated from the various slopes and S-shaped curves used for Prism 2.01 (graphpad). The results are shown in Table 1. Compounds E4021 and japrinast inhibit peak A (high affinity), while the IC ₅₀ value calculated for new PDE activity at peak B (section IA) is significantly increased (> 50-fold). This confirms that peak A is PDE5. This data demonstrates that the novel PDE activity of the present invention, despite all practical purposes, exhibits insensitivity to japrinast and E4021.

TABLE 1

Comparison of PDE Inhibitors of Peak A and Peak B (cGMP Hydrolysis) of Section IA

In contrast, Sulindac sulfide and Compound E competitively inhibit the phosphodiesterases of peaks A and B to the same titer (IC ₅₀ = 0.38 for PDE peak A and 0.37 μM for PDE peak B). While both peaks A and B are susceptible to sulindac sulfide and compound E, the fact that peak B (or a form thereof) is insensitive to japrinast is important for tumor treatment and selection of compounds useful for such treatment. Do. To examine whether Japrinat, E4021 and sildenafil induce apoptosis and inhibit the growth of tumor cells, they were tested and the same experiment was conducted for compound E. As described below, japrinast alone does not induce significant apoptosis or have growth-inhibitory properties, whereas sulfindax sulfide and Compound E are in stark contrast. That is, if a compound (i.e., japrinast) has specificity only for PDE peak A, the compound alone will not induce apoptosis, but the compound's ability to inhibit both PDE peaks A and B is not a tumor cell. Is related to the ability to induce apoptosis in humans.

F. Insensitivity of novel PDE peak B in culture with cGMP-dependent protein kinase G

A greater difference between PDE peak A and new peak B (section IA) is observed at each cGMP-hydrolysis activity under varying concentrations of cGMP-dependent protein kinase G (which phosphorylates existing PDE5). In particular, peaks A and B from section IA were incubated at different concentrations of protein kinase G at 30 ° C. for 30 minutes. Hydrolysis of cyclic GMP of two peaks is measured after phosphorylation is attempted. Consistent with the already published information on PDE5, peak A shows increased cGMP hydrolytic activity in response to incubation with protein kinase G, indicating that peak A is phosphorylated. However, peak B was unchanged (ie unphosphorylated and insensitive to incubation with cGMP dependent protein kinase G). These data are consistent with that peak A is isotype like the PDE5 family already known, and peak B of section IA has novel cGMP-specific PDE activity.

G. New Peak B in Prostate and Breast Cancer Cells

A similar process used to separate from the new peak B SW480 was isolated from two different tumor cells, breast cancer cells, HTB-26 and prostate cancer cells, LnCAP. This protocol has changed in many ways. To provide comparably greater fertility of other cell lines, AKTA FPLC (Pharmacia) was used for sample filling and 18 mL DEAE TrisAcryl M column to control elution. The SW480 performed the same procedure several times to provide a reference for Peak B. Between 200 and 400 million cells of SW480 are used for this experiment. Seventy million LnCAP cells are used in one profile (see Figures 22 and 23), and in another experiment 32 million HTB-26 cells are used in one profile (see Figures 20 and 21). After resuspending the cells in homogenization buffer, the samples are manually homogenized. FPLC buffer A is 8 mM TRIS-acetate, 5 mM magnesium acetate, 0.1 mM EDTA (pH7.5), and buffer B is 8 mM TRIS-acetate, 5 mM magnesium acetate, 0.1 mM EDTA, 1 M sodium acetate ( pH 7.5).

The supernatant is charged to the column at a rate of 1 mL per minute and then washed with 60 mL buffer at a rate of 1 mL per minute. The content is 60 mL of 0-15% buffer B, 60 mL of 15-50% buffer B, and 16 mL of 50-100% buffer B elute. Recover in a 1.5 mL gradient. The peak of cGMP PDE activity is eluted near fraction 65, 400 mM sodium acetate (see also 20-23). This activity is measured at 0.25 μM cGMP (indicative of ultra-affinity with cGMP).

Rolipram, a PDE4-specific drug, inhibits most cAMP PDE activity (ie, cAMP activity is due to PDE4), indicating that cGMP activity of peak B is more specific for cGMP than cAMP. All three peaks B (SW480, HTB-26, and LcCAP) are not stimulated by calcium / calmodulin and are directed to 100 nM E4021, a specific PDE5-specific inhibitor such as japrinat (see Figures 20 and 22). Resistant Peak B also shows a dramatic increase in activity as the substrate increases from 0.25 μM to 5 μM cGMP (shows positive cooperative kinetics, see 21 and 23). In addition, these three peaks show a similar inhibition pattern by excisulind and compound I below.

II. Relationship between protein kinase G and β-catenin-general

This series of experiments demonstrated that in tumor cells where an anti-tumor cGMP-specific PDE inhibitor, such as exisulind, lacks a polytumorous coli gene (“APC gene”) deficiency of the adenocarcinoma or a gene coding for β-catenin, cGMP Execute to determine the effect on dependent protein kinase G ("PKG"). As described below, the inhibitor increases PKG activity in the tumor cells. The cause of the increase in activity is not only because of increased activation of PKG in cells including the two defects, but also due to increased expression of PKG in cells containing APC defects. In addition, PKG obtained from these defective tumor cells is precipitated with β-catenin when immunoprecipitated.

β-catenin has been associated with a variety of other cancers since researchers have been found in high concentrations in patients with tumors containing mutations in the APC tumor-suppressor genes. People with mutations in these genes at birth often develop thousands of small tumors in the colon. When the APC gene works properly, it encodes a common APC protein known to bind and regulate β-catenin. Therefore, the discovery that PKG in tumor cells with APC gene deletion or β-catenin deficiency is associated with β-catenin strongly suggests that PKG is involved in one of the major cellular pathways leading to cancer. In addition, cGMP is associated with PKG / β-catenin / APC deficiency of these cells due to the relationship of increased PKG following cGMP-specific inhibition upon treatment with selective apoptotic anti-neoplastic drugs (SAANDs).

This association is further supported by the observation that β-catenin itself decreases when tumor cells, including APC defects or β-catenin defects, are exposed to SAAND. This decrease in β-catenin is initiated by PKG. Another novel discovery associated with the present invention is that PKG phosphorylates β-catenin. β-catenin phosphorylation causes β-catenin to be degraded by the system of ubiquitin-proteasomes.

Β-catenin phosphorylation by PKG is important in tumor cells because it bypasses the effects of APC and β-catenin mutations. Mutations in the APC protein are believed to affect binding to β-catenin bound to the mutated APC protein, resulting in a change that interferes with the phosphorylation of β-catenin by GSK-3b kinase. In the case of mutated β-catenin, an increase in PKG activity causes the mutant β-catenin to be phosphorylated. Increased PKG activity in tumors due to cGMP-PDE inhibition results in β-catenin phosphorylation (causing degradation) in tumor cells containing both types of mutations.

In summary, the findings not only lead to new pharmaceutical screening methods for identifying SAAND candidate compounds, but also solidify the role of cGMP-specific PDE inhibition in therapeutic approaches to tumors. The findings, as described above, may explain the wide range of therapeutic effects of unexpected tumors that SAANDs can suppress because tumors in both cases can be treated with or without APC disease.

III. Screening of Pharmaceutical Compositions Using PDE

A. General

The novel PDEs and PDE2s of the present invention can identify compounds useful for the prevention and treatment of neoplasms with or without PDE5 and do not cause severe side effects.

Cancer and prostate cancer are known to be diseases that cannot regulate cell growth. Cell growth depends on many factors. One factor is the rate of cell proliferation and the other is the rate of cell death. Cells die by necrosis or apoptosis in their own cells, depending on the stimulus of the external environment. Cell differentiation is another factor influencing tumor growth kinetics. Determining which compound affects one of several aspects of cell growth is an important factor in finding relevant targets for pharmaceutical treatment. The screening method according to the above technique can be used in conjunction with other assays for selecting other compounds that inhibit cell growth or contribute to cell necrosis.

The present invention is the product of several important discoveries. First, the inventors found that preferred growth inhibitors of tumor cells induce early death of cancer cells by apoptosis [Piazza, GA, et al., Cancer Research , 55 (14), 3110-16, 1995. ]. Secondly, we accidentally found a compound that selectively induces apoptosis without actually inhibiting COX and also inhibits PDE5. In particular, unlike the main results of the previous experiments, it is a fact that the preferred compounds for treating neoplastic lesions inhibit PDE5 (EC 3.1.4.17). PDE5 is one of at least 10 gene families for phosphodiesterases. The PDE5 and novel PDEs of the present invention are unique in that they selectively degrade cyclic GMP but not cAMP, while other PDE groups selectively degrade or hydrolyze cAMP and not cGMP, or both cGMP and cAMP. Decompose non-selectively. Preferred compounds for use in the treatment of neoplasia are substantially nonselective or do not inhibit the phosphodiesterase forms that degrade cAMP.

B. COX Search

One preferred embodiment of the present invention is to determine the cyclooxygenase inhibitory ability of a specific compound, and to determine the cGMP specific PDE inhibitory ability of the compound. By comparing this test compound with a compound already known to be useful for treating neoplastic lesions, it is possible, directly or indirectly, to evaluate the neoplastic inhibition of the test compound. 5-fluoro-2-methyl-1- (p-methylsulfonylbenzylidene) -3-indenyl acetic acid (Exescis) is an example of a standard drug known to be effective in treating neoplastic areas without causing gastrointestinal disorders. Lind). In addition, other useful drugs for comparison include indomethacin or sulfide metabolites (5-fluoro-2-methyl-1- (p-methylsulfonylbenzylidene) -3-indenylacetic acid (sullindax sulfide) Other useful drugs are known to inhibit 1-3- (3-chloroanilino) -4-phenylphthalazine (MY5445) as cGMP specific PDEs.

As used herein, the term "cancer constitutive lesion" refers to a syndrome caused by abnormal neoplasms including dysplastic or tissue changes. Examples of the syndrome include dysplasias occurring in the colon, breast, prostate or lung tissue, dysplastic nevus syndrome, or precursors of malignant melanoma of the skin. In addition to polymorphism syndrome, colon polyps and cervical cancer metastatic lesions (e.g., cervical dysplasia), dysplasia of the esophagus, lung and prostate, prostate Internal tumors, breasts and / or skin or related symptoms (eg photokeratosis keratosis).

In the present invention, the term "carcinoma" or "cancer" means a cancer-induced lesion. Specific examples of the cancer include melanoma, breast cancer, prostate cancer and colon cancer. As used herein, "neoplasia" and "neoplasm" refer to cancer precancerous and cancerous lesions.

Abbreviations used in the present invention are as follows.

PG; Prostaglandins

PS; Prostaglandin synthase

PGE ₂ ; Prostaglandin E ₂

PDF; Phosphodiesterase

COX; Cyclogenase, cyclic nucleotides

RIA; Radioimmunoassay

The fact that a compound inhibits COX can be determined by measuring one of two methods. That is, one method is to measure the PGE ₂ released by the cells by exposing the searched compound to the original HL-60 cells, and another method is the activity of purified cyclogenase (COX) in the presence of the compound. To measure. The two methods include the protocol already described in the literature, but the preferred planning process is as follows.

By measuring PGE ₂ , the compound can assess whether it inhibits the production of prostaglandin E ₂ (“PGE ₂ ”). An enzyme immunoassay (EIA) kit for measuring PGE ₂ can be purchased from Amersham, Inc., based in Arlington, Illinois, USA. Examples of preferred cells include cells that produce a large amount of PG, such as HL-60 cells. HL-60 cells are human promyelocytic cells that are differentiated by DMSO into mature granulocytes. Collins, SlJ., 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. The differentiated cells are stimulated by the calcium ion carrier A23187 to produce PGE ₂ [Kargman, S., Prasit, P. and Evans, JF, "Translocation of HL-60 Cell 5-Lipoxygenase", J Biol. Chem ., 266 : 2374-23752, 1991]. HL-60 cells can be produced by ATCC (ATCC: CCL240). The HL-60 cells were added 37 ° C. and carbon dioxide (5%) in RPMI medium containing 20% fetal placental serum (heat inactivated), 50 U / ml penicillin and 50 μg / ml streptomycin. And incubated.

To induce myeloid differentiation, the cells were exposed to 1.3% DMSO for 9 days and washed and resuspended in Dulbecco's phosphate buffer at a concentration of 3 × 10 ⁶ cells / ml.

The differentiated HL-60 cells (3-10 ⁶ cell number / ml) were incubated for 15 minutes at 37 ° C. in a medium containing the tested compound at an appropriate concentration. Followed by the stimulation of the cells for 15 minutes ^{A23187 (5 ~ 10 -6 M)} . PCE ₂ secreted into the external medium was measured according to the method described above.

As described above, a second method for assessing COX inhibition of one compound is to measure COX activity in the presence of the test compound. Cyclooxygenases, already published in the literature, exist in two forms (COX-1 and COX-2), which are known to regulate the synthesis of prostaglandins. COX-2 is a changeable form of COX, and COX-1 is a fixed form of COX. The activity of COX-1 was determined in the seminal vesicles of rams according to the method for purifying and characterizing the cyclooxygenase in positive platelets described by Bupati and Balasbramanion ( Biochem . J., 239 : 371-377). Mitchell et al. [Selectivity of Nonsteroidal Anti-inflammatory Drugs as Inhibitors of Constitutive and Inducible Cyclooxygenase, ” Proc. Natl. Acad. Sci. USA ., 90 : 11693-11697 using purified COX-1 . The activity of COX-2 can be measured using COX-2 purified from sheep placenta according to the method of Mitchell et al. [1993, supra ].

Cyclooxygenase of certain drugs can be measured according to conventional methods. For example, in the above-mentioned method of bupati and balasbramanion [ supra ], prostaglandin H synthase 1 (Cayman, Ann Arbor, Michigan) was used to obtain arachidonic acid (Sigma) of 100 μm. , Co-incubated with 1.0 mM glutathione, 1.0 mM hydrokinone, 0.625 μm hemoglobin and 1.25 mM CaCl 2 in 100 mM Tris-HCl under conditions of pH 7.4 and the drug to be tested. After incubation, the reaction was terminated with trichloroacetic acid. After the reaction is stopped by adding thiobarbituric acid and malaldehyde, the enzyme activity can be measured by a spectrophotometer at 530 nm.

Clearly, compounds exhibiting low activity on COX-1 and COX-2 are preferred, as compared to high activity on PDE5 / new PDE / PDE2.

The amount of COX inhibition is measured by comparison with the activity of cyclooxygenase in the presence and absence of the test compound. The presence of some activity (ie less than about 25%) or no inhibitory activity of COX at a concentration of about 100 μM suggests that the compound is useful for treating neoplastic sites.

C. Determination of phosphodiesterase inhibitory activity

The inhibitory effect of the compounds on the novel phosphodiesterase activity according to the present invention can be searched using PDE and / or PDE2 together with enzymes, recombinant enzymes or PDE5 isolated by the above-mentioned methods. In addition, the amount of cyclic nucleotides for all cells is measured by RIA comparing cells treated or untreated by japrinast.

Phosphodiesterase activity can be measured according to known methods, for example using radiation ³ H cyclic GMP (cGMP; cyclic 3'5'-guanosine monophophate) as a substrate for PDE enzymes. See Thompson, WJ, Teraski, WL, Epstein, PM Strada, SJ Advances in Cyclic Nucleotide Research, 10: 69-92, 1979].

In sum, the substrate ³ H-cGMP specific active solution as defined above (0.2 μM; 100,000 cpm; 40 mM Tris-HCl, pH 8.0), 5 mM MgCl 2 and 1 mg / mL BSA, was tested at a total volume of 400 μl. Mix with medicament. The mixture was incubated at 30 ° C. for 10 minutes using PDE isolated in the present invention. The reaction was stopped by boiling the reaction mixture for 75 seconds. After cooling on ice, 100 μl in 0.5 mg / mL snake venom ( O. Hannah venom , purchased from Sigma) was added and incubated at 30 ° C. for 10 minutes. The reaction was stopped by adding alcohol, ie 100% methanol. Analytical samples were applied with a 1 mL Dowex 1-X8 column and washed with 1 mL 100% methanol.

Radioactivity of freshly discovered and washed from the column was measured with a scintillation counter. Phosphodiesterase inhibition was determined by measuring radioactivity of drug-treated reactions and comparing them with control samples (compounds except for the use of drug solvents tested due to lack of reaction mixture).

Optionally, the function of the target compound for inhibiting the phosphodiesterase of the present invention was reflected by an increase in cGMP in neoplastic cells exposed to the searched compound. The amount of PDE activity can be determined by analyzing the content of cyclic GMP in extracts of treated cells using radioimmunoassay (RIA).

In the process, HT-29 or SW-480 cells were placed on a plate and sufficiently incubated.

As indicated above, SW-480 includes both PDE5 and novel PDEs of the present invention, and when PDE activity was assessed by this method, bound cGMP hydrolysis activity was simultaneously analyzed. Test compounds were incubated using cell culture at concentrations between about 200 μM and 200 pM. After about 24 to 48 hours, the culture medium was removed from the cells and the cells were lysed. The reaction was stopped using 0.2 N HCl / 50% MeOH. Samples were removed for protein analysis. Cyclic GMP was purified from acid / alcohol extracts of cells using anion exchange chromatography such as Dowex columns. The cGMP was dried and acetylated by known methods such as using acetic anhydride in triethylamine [Steiner, AL, Parker, CW, Kipnis, DM, J. Biol. Chem. , 247 (4) : 1106-13, 1971].

Iodide ligand (tyrosine methyl ester) of induced cyclic GMP was incubated by standard or unknown methods in the presence of antiserum and appropriate buffer. Antisera can be produced using direct techniques using cyclic nucleotide-hapten. Antiserum was obtained from sheep injected with the succinyl-cGMP-albumin complex and diluted to 1 / 20,000. Dose-interpolation and error analysis from standard curves were applied by known methods [Seibert, AF, Thompson, WJ, Taylor, A., Willbourn, WH, Barnard, presented below as evidence] , J. and Haynes, J., Applied Physiol. , 72 : 389-395, 1992].

In addition, the culture medium can be acidified, frozen (-70 ° C.) and analyzed for cGMP and cAMP.

In addition, an increase in the cGMP content in tumor cells caused by the target compound was observed, and also a decrease in the cGMP content was observed. Specific target compounds (ie, ones that are not essentially normal but which selectively induce apoptosis in tumor cells) are one of the initial actions resulting from an increase in cGMP content in a few minutes, resulting in cGMP-specific PDE inhibition over time. Is observed to proceed consistently. Second, tumor cell treatment with the desired anti-tumor compound leads to a reduced cAMP content within 24 hours. Intracellular targets for drugs are being further studied, but data to date indicate that initially increased cGMP content in tumor cells exposed to the target compound and subsequent subsequent decreases in cAMP content preceded apoptosis in tumor cells. ) Is shown.

The ratio change of the two cyclic nucleotides is not only to determine the absolute value of cGMP, cGMP-specific phosphatidase inhibition, or cGMP hydrolysis level, but rather inhibition of the desired cGMP-specific phosphatasease of the test compound. Measuring activity may be a more accurate means. Anti-tumor compounds are not treated together in tumor cells, and the ratio of cGMP content / cAMP content is in the range of 0.03 to 0.05 (ie cGMP content of 300-500 fmol / mg protein is cAMP content of 6000-8000 fmol / mg protein). That's it.

After exposure to the preferred anti-tumor compound, the ratio is increased several times (preferably at least about three times) as a result of an increase in cyclic GMP initially and then a decrease in cyclic AMP.

In particular, it can be observed that the target compound has an initial increase in the cGMP content of tumor cells treated with a level of cGMP greater than about 500 fmol / mg protein. Moreover, each target compound later decreases in the cAMP content of tumor cells treated with a level of cAMP less than about 4000 fmol / mg protein.

The determination of the content of the cyclic AMP used a radioimmunoassay technique similar to the method described for cGMP above. Basically, cyclic nucleotides were purified and dried from acid / alcohol extracts of cells using anion exchange chromatography, acetylated by known methods and quantified using radioimmunoassay methods. Induced cyclic AMPs and iodinated ligands of cyclic GMP were incubated by standard or unknown methods in the presence of specific antisera and appropriate buffer solutions.

Identification of cyclic nucleotide content can be obtained by measuring the turnover or accumulation of cyclic nucleotides in intact cells. To measure complete cell cAMP, pre-labeled ³ H-adenine was used in a known manner [Whalin, ME, Garrett Jr., RL, Thompson, WJ, and Strada, SJ "Correlation of cell-free brain cyclic nucleotide phosphodiesterase activities to cyclic AMP decay in intact brain slices ", Sec. Mess. and Phos. Protein Reserch, 12 : 311-325, 1989]. The procedure can measure adenyl cyclase or cyclic nucleotide phosphodiesterase activity in intact cells of a specific protocol by measuring the excess outflow of labeled ATP to cyclic AMP. The cyclic GMP accumulation is too low to be studied using complete cells pre-labeled by known methods [Reynolds, PE, SL Strada and WJ Thompson, “Cyclic GMP Accumulation In Pulmonary Microvascular Endothelial Cells Measured By Intact Cell Prelabeling ", Life Sci. , 60 : 909-918, 1997].

In addition, the effect of the compound's PDE inhibitory activity can be determined from tissue samples. Tissues from Humans Tissues from animals that have been biopsied or anesthetized are collected from test substances exposed to the test compound. In short, tissue samples were homogenized in 500 μl of 6% TCA. Known amounts of homogenate were removed for protein analysis. The remaining homogenate was placed on ice for 20 minutes to precipitate the protein. The homogenates were then centrifuged at 15,000 g at 4 ° C. for 30 minutes. The supernatant was recovered and the pellet was recovered. The supernatant was washed four times with 5 times the volume of saturated diethyl ether of water. The upper ether layer was discarded for each wash step.

The aqueous ether extract was dried in vacuo. The dried sample may be frozen for later use or may be used immediately. The dried extract was dissolved in 500 μl of assay buffer. The cGMP-specific inhibitory amount can be determined by assaying for the amount of cyclic nucleotides using the RIA method, as defined above.

The amount of inhibition was determined by comparing the activity of the new PDE (or PDE2) in the presence or absence of the compound. Inhibitory activity of the novel PDE (or PDE2) appears to be a useful compound for the treatment of neoplasms. The inhibitory activity of a compound, which is higher than the level of excisulind, preferably higher than 50% at a concentration of 10 μM or less, indicates that the antitumority of the compound is stronger.

Preferably, the IC ₅₀ value for novel PDE inhibition should be less than 50 μM for compounds in which potential potential further consideration should be taken into account.

D. Determining Whether Compounds Reduce Cancer Cell Proliferation

In an alternative embodiment, the methods of the present invention include a method of determining whether the compound reduces the proliferation of cancer cells. Several cell lines can be used as samples of the tissue to be tested. For example, such cell lines include: adenocarcinoma of SW-480- colon; Adenocarcinoma of HT-29-colon, A-427-lung adenocarcinoma carcinoma; MCF-7-breast adenocarcinoma; And the UACC-375-melanoma family; And DU145-weak carcinoma. Cytotoxic data obtained using the cell line suggests an inhibitory effect on tumor lesions.

This cell line is so characteristic that it was used by the US National Cancer Institute as a screening program for new anticancer drugs.

The inhibitory capacity of cancer cell proliferation of the compounds can be measured using the HT-29 human colon carcinoma cell line obtained from ATCC. HT-29 cells are characterized as related colon cancer cell culture models [Fogh, J., and Trempe, G. In: Human Tumor Cells in Vitro, J. Fogh (eds.), Plenum Press, New York, pp. 115-159, 1975. HT-29 cells were treated with 5% fetal bovine calf serum (Gemini Bioproducts, Inc., Carlsbad, Calif.) And 2 mm glutamine, and 1% antibiotics at 37 ° C., 95% air wet atmosphere and 5% CO ₂ . It was maintained in RPMI medium supplemented with antifungal agent. Briefly, HT-29 cells were placed on plates at a density of 500 cell counts / well in 96 wells and incubated at 37 ° C. for 24 hours prior to addition of compounds. Determination of each cell number involves six replicates. After 6 days of incubation, the cells were fixed at 10% concentration by adding cold tricloacetic acid, and protein levels were determined using a sulforhodamine B (SRB) color protein dye assay by methods known in the literature. [Skehan, P., Stroreng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, JT, Bokesch, H., Kenney, S., and Boyd, MR, "New Colorimetic Assay For Anticancer-Drug Screening", J. Natl. Cancer Inst . 82 : 1107-1112, 1990.

In addition to the SRB assay, several other methods can be used to measure proliferation inhibition and can replace the SRB assay. These methods include the following calculation of live cells following trypan blue staining, labeling cells capable of DNA synthesis using BrdU or radiolabeled thymidine, intermediate red staining of live cells, or MTT staining of live cells.

Significant cancer cell proliferation inhibition of greater than about 50% at a dose of 100 μM or less indicates that the compound is useful for treating tumor lesions.

Preferably, the IC ₅₀ value is determined and used for comparison purposes. This value is the relative drug concentration required to inhibit cancer cell proliferation by 50% compared to the control.

Preferably, the IC ₅₀ value should be less than 100 μM for the compound for possible uses to treat tumor damage.

E. Determination of Compound Induced Apoptosis

As a second alternative embodiment, another screening method of the present invention comprises determining whether the compound induces apoptosis in cancer cell culture.

Two distinct cell death forms can be explained by morphological and biochemical criteria, namely necrosis and apoptosis. Cell necrosis can be carried out by increasing the permeability of the plasma membrane, and the cells swell and the plasma membrane breaks down in minutes. Apoptosis is characterized by bleeding membranes, cytoplasmic condensation and endogenous endonucleases activation.

Apoptosis occurs spontaneously during the conversion of normal tissues and during the embryogenesis of organs and limbs. Apoptosis is also induced by cytotoxic T-lymph cells and natural killer cells, ionizing radiation and certain chemotherapeutic agents. Misregulation of apoptosis plays an important role in many pathological conditions, including cancer, AIDS, or Alzheimer's disease. Compounds can be searched for induction of apoptosis using cultures of cancer cells maintained under conditions as described above. Cell treatments with test compounds include pre- or post-fusion cultures and treatment for 2-7 days at various concentrations. Apoptotic cells can be measured in both the fixation and "floating" compartments of the culture.

The compartment can remove the supernatant, trypsinize the adhered cells, and collect the cells via the following centrifugal washing step (10 minutes, 2000 rpm). Protocols for treating cancer cells with Sullidac and related compounds to obtain significant amounts of apoptosis are described in the literature (Piazza, GA, et al., Cancer Research, 55: 3110-31116, 1995). Features of the novel method include harvesting suspended and fixed cells to identify appropriate treatment times and dose ranges and appropriate cell culture conditions to observe apoptosis.

In the next treatment of the compound, the apoptosis and necrosis can be analyzed using a fluorescence microscope by labeling with acridine orange and ethidium bromide. Methods for measuring apoptotic cell numbers have been described in the literature (Duke & Cohen "Morphological and Biochemical Assays Of Apoptosis", Current Protocols In Immunology, Coligan et al., Eds., 3.17.1-1.17.16 (1992).

For example, it can be collected by treating trypsin to suspended and fixed cells and washing three times with phosphate buffer (PBS). Fractions of cells were centrifuged, and then the precipitate was redissolved in a staining mixture comprising acridine orange and ethidium bromide in medium and PBS and mixed lightly. The mixture was then placed on a microscope slide and the shape of apoptosis was observed.

Apoptosis can also be quantified by measuring the increase in DNA fragments of cells treated with experimental compounds. Commercial photometric EIA was used for in vitro determination of cytoplasmic histone related DNA fragments (mono- and oligonucleosomes) (apoptosis detection ELISA ^okys , Cat. No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay is based on the principle of sandwich-enzyme-immunoassay using mouse monoclonal humers for DNA and histones. This allows the mono- and oligonucleosomes to specifically bind in the cytosolic fraction of the cell lysate.

According to the invention, apoptosis was measured as follows. Samples (cytolysates) were placed in streptavidin-coated mito titer plates (“MTP”). Then, a mixture of anti-histone-biotin and anti-DNA peroxidase complex was added and incubated for 2 hours. During the incubation period, the anti-histone antibody binds to the histone component of the nucleosome and simultaneously immobilizes the immune complex to streptavidin-coated MTP via biotinylation.

In addition, anti-DNA peroxidase antibodies react with the DNA component of the nucleosomes. After removal of unbound antibody by washing, the amount of nucleosomes can be measured by peroxidase remaining in the immunocomplex. Peroxidase is determined by photoreaction with the substrate ABTS7 (2,2'-azido- [3-ethylbenzthiazoline-sulfonate]).

For example, SW-480 colon adenocarcinoma cells were placed in 96 well MTPs at a density of 10.000 cells per well. Cells were then treated with experimental compounds and incubated at 37 ° C. for 48 hours. After incubation, MTP was centrifuged and the supernatant was removed. Thereafter, cell precipitates from each well were re-dissolved in lysis buffer for 30 minutes. Lysates were centrifuged and fractions of the supernatant (ie, cytoplasmic fraction) were transferred to streptavidin-coated MTP. Care must be taken not to shake dissolved precipitates in MTP (ie, cell nuclei containing high molecular weight, unfragmented DNA). After transfer, the samples were analyzed.

Fold stimulation (FS = OD _max / OD _veh ), which is an indicator of the apoptosis response, is determined for each compound tested at a given concentration. In addition, EC ₅₀ values are determined by measuring concentration series of experimental compounds.

Statistically significant increase in apoptosis (ie, doubling stimulation at 100 μM concentration) is an additional indicator that the compound is useful for the treatment of tumor lesions. Preferably, the EC ₅₀ value for apoptosis activity should be less than or equal to 100 μM of a compound that is considered suitable for the treatment of tumor injury. Here, EC ₅₀ is defined as the concentration that causes 50% induction of apoptosis for vehicle treatment.

F. Mammary gland organ culture model experiment

Experimental compounds identified by this method can be tested for antitumor activity that inhibits pre-tumor damage in mammary organ culture systems. Mouse gland organ culture technology has been successfully used by other researchers to study the effects of known anti-tumor agents such as NSAIDs, retinoids, tamoxifen, selenium and natural products and is effective for the screening method of the present invention. .

For example, female BALB / c mice can be treated with compounds of estradiol and progestrone daily to increase gland activity in response to hormones in vitro.

The animals were killed and the mammary glands of the thorax were sterile cut and incubated for 10 days in growth medium supplemented with insulin, prolactin, hydrocortisone and aldosterone.

DMBA (7, 12-dimethylbenzanthracin) is added to the medium to induce prostate malignancy. Removal of prolactin, hydrocortisone and aldosterone from fully developed glands results in degeneration of the glands but of pro-malignant tumors.

Experimental compounds were dissolved in DMSO and added to the culture medium during the incubation period. At the end of the incubation period, the glands were fixed with 10% formalin on a glass slide, stained and fixed with alum carmine. The incidence of lesions in the mammary gland is the ratio of lines with lesions to those without lesions. The incidence of lesions on the lines treated with the test compound is compared to the incidence of untreated lines.

The extent of the site where the mammary gland lesion has occurred can be measured by projecting an image of a line on the ground tissue pad. The area covered by the line was placed on the pad and the area was 100%. The space covered by each of the degenerate structures can also be contoured in the ground digits and measured by a computer.

Experiment result

Numerous compounds have been investigated by various protocols and searched for potential use in the treatment of tumors. The experimental results are as follows. This test compound is labeled with the corresponding character code as follows:

A: lac-tereo- (E) -1- (N, N'-diethylaminoedethio) -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-trimethosylmenzylidene) -3-acetic acid;

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

D: lac- (E) -1- (butane-1 ', 4'-olido)-[3', 4 ': 1,2] -6-fluoro-2-methyl-3- (p-methyl Sulfonylbenzylidene) 1S-indanyl-N-acetylcysteine;

E: (Z) -5-fluoro-2-methyl-1- (3,4,5-trimethooxybenzylidene) -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': 1 '', 3 '']-1- (butane-1 ', 4'-olido)-[3', 4 ' : 1,2] -6-fluoro-2-methyl-3- (p-methylsulfonylbenzylidene) -indane; And

H: lac- (E) -1- (butane-1 ', 4'-olido)-[3', 4 ': 1,2] -6-fluoro-2-methyl-3- (p-methyl Sulfonylbenzylidene) -1S-indanyl-glutathione).

Example 1 COX Inhibition Measurement

Comparative compounds and experimental compounds were measured for COX inhibitory activity according to the planning procedure [ supra ] for COX determination. 4 shows the effect on various concentrations of sulindac sulfide or exisulind on purified cyclooxygenase (type 1) activity. Cyclooxygenase activity was measured by using cyclooxygenase purified from ram semen cysts as described previously [Mitchelle et al, supra ]. The IC ₅₀ value for the excisulind is greater than 10,000 μM, whereas the IC ₅₀ value for the sulfingac sulfide is calculated to be about 1.76 μM. These data show that sulindac sulfide but not excisulin is a COX-I inhibitor. Similar data are obtained for COX-2 isozymes (Thompson, et al., Journal of the National Cancer Institute, 87: 1259-1260, 1995).

5 shows the effect of experimental compounds B and E on COX inhibition. COX activity is determined as the compound shown in FIG. The data show that experimental compounds B and E do not significantly inhibit COX-1.

TABLE 2

Cyclooxygenase Inhibitory Activity on the Kinds of Compounds

According to the planned process [ supra ] A to E compounds were measured for COX inhibitory activity as shown in Table 2 above. Compound C was found to inhibit COX greater than 25% at a 100 μM dose and was no longer selected for screening.

Example 2 cGMP PDE Inhibition Measurement

Comparative compound and test compound was assayed for [supra] cGMP PDE inhibitory activity in accordance with the planning process for the measurement described above. FIG. 6 illustrates the effects of varying concentrations of sulindac sulfide and exisulind on purified PDE4 or cGMP PDE activity from tumor cells cultured with human colon HT-29, as previously described [WJ Thompson et al., Supra ]. Shows. The Sulfondak IC ₅₀ value for PDE 4 inhibition is 41 μΜ and the value for cGMP PDE inhibition is 17 μΜ. The IC ₅₀ value of exisulind for PDE 4 inhibition is 181 μM and the value for cGMP PDE inhibition is 56 μM. These data show that both sulindac sulfide and excisulind inhibit phosphodiesterase activity. These two compounds show selectivity for cGMP PDE isoenzymes over PDE4 isoforms.

7 shows the sulindac sulfide effect on cGMP or cAMP production as determined in HT-29 cells cultured according to the above described assay [ supra ]. HT-29 cells are treated with sulfindac sulfide for 30 minutes and cGMP or cAMP is measured by conventional radioimmunoassay. As suggested, sulfindac sulfide increases the concentration of cGMP by more than 50% with an EC ₅₀ value of 7.3 μM (FIG. 7A). Rolipram, known as a PDE4 inhibitor, increases cAMP, but the concentration of cAMP (Figure 7B) is not affected by this treatment. This data demonstrates the pharmacological significance of inhibiting cGMP PDEs, compared to PDE4.

8 shows the effect of phosphodiesterase on the indicated dose of experimental compound B on cGMP PDE or PDE4. The calculated IC ₅₀ value is 18 μΜ for cGMP PDE and 58 μΜ for PDE4.

Figure 9 shows the effect on the indicated doses of experimental compound E on PDE4 or cGMP PDE. The calculated IC ₅₀ is 0.08 μM for cGMP PDE and 25 μM or more for PDE4.

TABLE 3

CGMP PDE Inhibitory Activity Among Compounds

Compounds of Table 3 were measured for supra PDE inhibitory activity according to the planning process described above. Among the compounds that do not inhibit COX, only Compound E showed greater than 50% inhibition at 10 μM. As noted in FIG. 8, Compound B exhibits greater than 50% inhibition at a dose of 20 μM. Thus, depending on the dose concentration used in a single dose experiment, some compounds may be detected, otherwise they may be activated at slightly higher doses. The dose used is subjective and may be lowered if the activated compound is found at a level that can identify a large number of effective compounds.

Example 3 Apoptosis Analysis

Comparative and experimental compounds were analyzed for novel PDE inhibitory activity according to the planned process [ supra ]. According to the above process, FIG. 10 shows the effect of sulindac sulfide and excisulin on apoptosis due to apoptosis and necrotic. HT-29 cells were treated for 6 days with the indicated doses of sulindac sulfide or excisulind. Apoptosis due to apoptosis and cell necrosis has been previously determined (Duke and Cohen, In: Current Protocols in Immunology, 3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The data show that both sulindac sulfide and excisulin can induce apoptosis without causing necrosis. All data was collected from the same experiment.

FIG. 11 shows the effect of sulfindac sulfide and excisulin on tumor growth inhibition and apoptosis induction measured by DNA fragments. The chart 11A by excisulind; Growth inhibition (open, left) and DNA fragments (close, right). The following chart (11B) by sulfindax sulfide; Growth inhibition (open symbol) and DAN fragment (close symbol). Growth inhibition is determined by SRB assessment 6 days after treatment. DNA fragments are measured 48 hours after treatment. All data were collected in the same experiment.

Figure 12 shows the property of inducing apoptosis of Compound E. HT-29 colon adenocarcinoma cells are treated with the indicated concentrations of Compound E for 48 hours and apoptosis is measured by DNA fragment analysis. The calculated EC ₅₀ value is 0.05 μM.

FIG. 13 gives the property of inducing apoptosis for Compound B. HT-29 colon adenocarcinoma cells are treated with indicated concentrations of Compound B for 48 hours and apoptosis is measured by DNA fragment analysis. The calculated EC ₅₀ value is 175 μΜ.

TABLE 4

Inhibitory Activity of Apoptosis Between Kinds of Compounds

According to the drainage induction planning process [ supra ], compounds from A to E were measured for apoptosis inducing activity as shown in Table 4 above. Compounds B, C and E exhibit significant apoptosis inducing activity at 2.0 fold or more at 100 μM dose. Of these three compounds, only B and E at this dose did not inhibit COX but inhibited cGMP-specific PDE.

Apoptotic inducing activity for the phosphodiesterase inhibitor family was determined, and the data is shown in Table 5 below. HT-29 cells were treated for 6 days with several inhibitors of phosphodiesterase. Postapoptosis and necrosis were labeled morphologically with acridine orange and ethidium bromide according to the supra described above. The data show that new cGMP-specific PDEs are useful for screening for compounds that induce apoptosis in HT-29 cells.

TABLE 5

Apoptosis-Inducing Data for PDE Inhibitors

Example 4 Growth Inhibition Assay

Comparative compounds and experimental compounds were analyzed for PDE5 inhibitory activity of the [supra] compound according to the planning process for the assay. 14 shows the inhibitory effect of the growth of HT-29 cells with varying concentrations of sulfindac sulfide and excisulin. HT-29 cells were treated for 6 days at various excisulind (denoted) or sulindac sulfide (denoted) doses as indicated. Cell number is measured by sulforhodamine analysis as previously described (Piazza et al., Cancer Research, 55: 3110-3116, 1995). The IC ₅₀ value for sullindac sulfide is about 45 μΜ and 200 μΜ for exisulind. The data show that both sulindac sulfide and excisulin can inhibit tumor cell growth.

FIG. 15 shows growth inhibition and apoptosis-inducing activity of sulfindac sulfide. Time course experiments showed that the medium contained HT-29 treated with 0.1% DMSO (marked with open symbol) or Sullivan sulfide, 120 μM (marked with closed symbol). Growth inhibition (above 15A) was determined by counting live cells after trypan blue staining. Apoptosis (below 15B) was measured by morphological crystals after staining with acridine orange and etideum bromide, as previously described [Duke and Cohen, in: Current Protocols in Immunology, 3.17.1 ?? 3.17.16, New York, John Wiley and Sons, 1992]. These data show that sulfinact sulfide can inhibit tumor cell growth and have the effect of an increase in apoptosis. All data were collected in the same experiment.

Figure 16 shows the growth inhibitory activity of experimental compound E. HT-29 colon carcinoma cells were treated with the indicated concentrations of Compound E for 6 days and the cell number was measured by SRB analysis. The calculated IC ₅₀ value is 0.04 μΜ.

TABLE 6

Growth-inhibitory activity of compounds

According to the screening process [ supra ], the compounds from A to E were tested for growth inhibitory activity as shown in Table 6 above. All experimental compounds show activity exceeding 100 μM alone dose.

Growth inhibitory activity for the phosphodiesterase inhibitor family was determined and the data is shown in Table 7 below. HT-29 cells were treated for 6 days with various phosphodiesterase inhibitors. Cell growth was determined by the described SRB assay. The data below for the above results indicate that the novel PDE inhibitors are effective at inhibiting tumor cell growth.

TABLE 7

Growth Inhibition Data of PDE Inhibitors

Compounds were tested in different cell lines to demonstrate the effectiveness of this screening method for various types of neoplasms. The effects of sulfinad sulfide and excisulind in various cell lines were determined and the data is shown in Table 8 below. IC ₅₀ values were determined by SRB analysis. The data show the broad effectiveness of these compounds against various tumors effectively at comparable dose ranges. Thus, the compounds identified and selected by the present invention are useful for treating various forms of neoplasms.

TABLE 8

Inhibit growth of multiple cell lines

Example 5 Activity in Mammary Gland Culture Model

Figure 17 shows the inhibition of cancer-like lesions in the mammary gland culture model of sulfinac metabolites. Mammary gland culture model experiments were conducted by the methods described previously (Mehta and Moon, Cancer Research, 46: 5832-5835, 1986). These results demonstrate that sulfides are inactive, whereas suldinak and excisulin effectively inhibit the formation of cancerous lesions. The data support the hypothesis that cyclooxygenase inhibition is not required for antitumority of the preferred compounds.

analysis
To select compounds for the treatment of tumors, the present invention provides a theoretical explanation of comparing experimental compound data from various planning processes. In the overall construction of the invention, experimental compounds can be classified according to the potential for treating human tumors. Compounds with the desired effect will be selected for further experimentation and use in humans.

Qualitative data of various experimental compounds and various planning processes are shown in Table 9 below. These data show that exisulind, Compound B and Compound E have adequate activity to pass four searches, namely COX non-inhibition, effective cGMP-specific PDE inhibition, growth inhibition and induction of apoptosis. The activity of these compounds in mammary gland cultures validates the effects of the present invention. Qualitative evaluation of the screening process ranked Compound E first, Compound B second, and then exisulind.

TABLE 9

Activity Profiles of Different Compounds

In addition, new assays for PKG activity have emerged, which are used in the screening methods of the present invention, but are more commonly used for PKG activity assays for other purposes (e.g., to study the role of PKG in normal cellular function). useful. For purposes of explanation, it is necessary to first describe the PKG assay before explaining how useful PKG activity is in drug assays to determine whether a compound is useful in treating tumors.

New PKG Analysis

The new PKG assay of the present invention involves binding a plurality of amino acid sequences to the solid phase, each having at least a cGMP binding site and a phosphorylation site of phosphodiesterase type 5 ("PDE5"). Such sequences are known and described in the references below. Preferably, the bound PDE5 sequence does not comprise a catalytic site of PDE5, which will be described below. One way of binding a PDE5 sequence to a solid phase is to express the sequence as a fusion protein using one member of the PDE5 sequence and an amino acid binding pair, and chemically link the other member of the amino acid binding pair to the solid phase (eg beads). It is.

One binding pair that can be used is glutathione S-transferase ("GST") and glutathione ("GSH"), where GST is present as a fusion protein expressed together with the PDE5 sequence described above, and GSH is shared on a solid phase. Combined. In this way, the PDE5 sequence / GST fusion protein can simply bind to the solid phase by passing through a solution containing the solid fusion protein as described below.

The RT-PCR method was performed by Bovine PDE5A cDNA sequence selected from the PDE 1-10 family [ McAllister-Lucas LM et al, J. Biol. Chem. 268, 22863-22873, 1993, to prepare cGB sites of PDE5 using forward and reverse primers. Oligo (dT) column purification for mRNA at 5 'to 3' for total RNA was used for HT-29 cells. Forward primer (GAA-TTC-TGT-TAG- AAA-AGC-CAC-CAG-AGA-AAT-G, 203-227) and reverse primer (CTC-GAG -CTC-TCT-TGT-TTC-TTC-CTC- TGC-TG, 1664-1686) is used to synthesize 1484 bp fragments encoding the phosphorylation site of human PDE5A (203-1686 bp, cGB-PDE5) and the low and high affinity cGMP binding sites. The synthesized cGB-PDE5 base fragment encodes 494 amino acids with 97% homology to bovine PDE5A. The synthetic base was then introduced to the EcoRI and XhoI cleavage sites of the glutathione-S-transferase (GST) fusion vector [Pharmacia Biotech] with a tac promoter. The fusion vector was then transfected into E. coli BL21 (DE3) bacteria (Invitrogen). The transfected BL21 bacteria grew in log phase and induction was performed at 20 ° C. for 24 hours after IPTG was added as an inducer. The bacteria were harvested and lysed. Lysable cells were incubated with GSH complex Sepharose 4B (GSH-Sepharose 4B). The GST-cGB-PDE5 fusion protein binds to GSH-Sepharose beads and other proteins are separated from the beads by adding cold PBS.

The expressed GST-cGB-PDE5 fusion protein appeared as 85 Kd protein on 7.5% SDS-PAGE. The protein is characterized by cGMP binding and phosphorylation by protein kinases G and A. The prepared protein shows two cGMP binding sites and K _d = 1.6 ± 0.2 μM which is close to K _d = 1.3 μM of bovine original PDE5. It can be phosphorylated by cGMP-dependent protein kinase and cAMP-dependent protein kinase A in GST-cGB-PDE5 in vitro bound to GSH conjugated sepharose beads. The K _m of GST-cGB-PDE5 phosphorylation by PKG is 2.7 μM and Vmax is 2.8 μM, while the K _m of BPDEtide phosphorylation is 68 μM.

The binding ratio of one molecule of phosphate to one GST-cGB-PDE5 protein is phosphorylated by PKG.

To analyze liquid samples that are believed to include PKG using the PDE5-binding solid phase described above, the sample and solid phase were mixed with phosphorylation buffer comprising ³² P-γ-ATP. This solution is incubated at 30 ° C. for 30 minutes to phosphorylate the PDE5 sequence by PKG, which would occur if PKG was present. The solid phase was then separated from the solution (eg, by centrifugation or filtration) and washed with phosphate-buffered saline (“PBS”) to remove the remaining solution and unreacted ³² P-γ-ATP. .

The solid phase can then be tested to see if ³² P is bound directly (by a liquid scintillation counter). If the above binding is confirmed, it suggests that the sample contains PKG because PKG phosphorylates PDE5. As described above, if PDE5 is bound through a fusion protein, the fusion protein comprising PDE5 can be eluted from the solid phase using SDS buffer and the eluate can be analyzed with ³² P binding. This method has the advantage that, if there is a possibility that other proteins are present, the eluate can be separated into several proteins (eg by gel separation) so that the fusion protein fraction can be analyzed with ³² P bonds. have.

Phosphorylated fusion proteins can be eluted from the solid phase using SDS buffer and reisolated by electrophoresis. If gel separation is performed, the protein can be stained to locate the protein and ³² P phosphorylation of the PDE5 portion of the fusion protein by PKG can be measured by X-ray film exposure to the gel. If ³² P is seen as an X-ray film, this indicates that PKG is present in the raw sample containing PKG that phosphorylates the PDE5 portion of the fusion protein eluted from the solid phase.

Preferably, in the assay, an excess of protein kinase inhibitor ("PKI") (eg, 100-fold) that specifically inhibits protein kinase A ("PKA") without inhibiting PKG is added to the assay buffer. Must be added. PKA inhibition is preferred because it phosphorylates PKG substrates (eg PDE5). By adding PKI, phosphorylation by PKA will be eliminated and the phosphorylation detected will only be high by PKG alone.

Instruments that may be prepared for analysis of the present invention include the following reagents prepared in each container:

1. Cell Lysis Buffer: 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na ₃ VO ₄ , 1 mM NaF, 500 μM IBMX, proteolytic enzyme inhibitor.

2. Protein Kinase G Solid Phase Substrate: Sepharose 4B (50% Suspension) with Recombinant GST-cGB-PDE5

3. 2 × phosphorylation buffer: ³² P-γ-ATP (3000 mCi / mmol, 5-10 μCi / assay), 10 mM KH ₂ PO ₄ , 10 mM K ₂ HPO ₄ , 200 μM ATP, 5 mM MgCl ₂

4. PKA Protein Kinase I Inhibitors

Portable containers and the like for carrying out the reaction should also be provided.

By virtue of the above, it is possible to immediately plan various methods for applying the analytical forms described in the other forms by the analytical prior art.

In summary, the presence and relative amount of PKG (control) by measuring the phosphorylation of a protein phosphorylated with a labeled phosphorylation agent using at least a portion of PDE5 (or other protein that can be selectively phosphorylated by PKG). In comparison to) can be identified.

SAANDs increase PKG activity in tumor cells.

Using the PKG assay described above, the following experiments revealed that SAANDs exhibited PKG activity due to increased PKG expression or increased cGMP levels (or both) in tumor cells treated with selective apoptotic anti-neoplastic drugs (SAAND). It was performed to reveal the increase.

Experiment process

Two different types of PDE inhibitors were measured for their effect on PKG in tumor cells. Excisulin, a SAAND, was measured because it has anti-tumority.

In addition, E4021, a classic PDE5 inhibitor, not SAAND, was measured to determine whether PKA increase was simply due to PDE5 inhibition, or whether PKG increase caused pro-apoptosis in the inhibition of SAANDs of PDE5. US patent application Ser. No. 09 / 173,375, filed Oct. 15, 1998, to Liu et al.

To test the effect of cGMP-specific PDE inhibition on tumors containing APC mutations, SW 480 colon cancer cells were used. SW 480 is known to contain APC mutations. About 5 million SW480 cells in RPMI 5% serum were added to each of the eight test dishes.

2-10 cm test dish-30 μl DMSO vehicle control (untreated drug)

3-10 cm test dish-200 μM, 400 μM, 600 μM excisulin in DMSO, and

3-10 cm test plates-E4021; 0.1 μM, 1 μM and 10 μM in DMSO.

The test dish was incubated for 48 hours at 37 ° C. in a 5% CO ₂ incubator.
The test dish was incubated for 48 hours at 37 ° C. in a 5% CO ₂ incubator.

The liquid medium was aspirated from the test dish (the cells themselves would stick to the dish). The frozen cells were washed with cold PBS and 200 μl cell lysis buffer (ie 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM with proteolytic inhibitor added). NaF, 500 μΜ IBMX) was added to each dish. Immediately after adding the lysis buffer, the cells adhered to each dish were scraped, and the collected cell lysate was placed in a microcentrifuge tube, incubated at 4 ° C. for 15 minutes, and gently stirred to allow the cells to completely dissolve. After the cells were completely lysed in the microcentrifuge tube, the tubes were centrifuged at 14,000 rpm to transfer each supernatant to a new tube.

Thereafter, the contents of each microcentrifuge tube are analyzed for protein. If the drug inhibits cell growth, the total protein content of the control group will be higher than that of the drug-treated sample. Obviously, if the drug works, then the total protein amount of the drug treated sample should also be the same as the control. In this case, the microcentrifuge tubes of the control and E-4021 were diluted to normalize with the group of samples treated with excess excisulin (group of samples treated with a small amount of exisulind and excess of exisulind). Standardized samples should be standardized). Therefore, after protein analysis, the total protein concentration of each sample must be normalized (eg, diluted).

For each drug concentration and control two kinds of PKG assays are performed, one with cGMP added and the other without cGMP as follows. The reason for performing both types of assays is because cGMP activates PKG. When the activity of PKG is measured through the novel PKG assay of the present invention, it is generally due to the increase in PKG activity due to an increase in intracellular cGMP (which may be due to cGMP-specific PDE inhibition) or an increase in PKG protein expression It's hard to find out.

By investigating the PKG activity of the cGMP-added and non-sampled samples, it is possible to determine if the increase in PKG activity is due to an increase in PKG protein expression. Thus, if an anti-tumor drug improves PKG activity compared to a control, it can be seen that the increase in PKG activity in the drug treated sample is due to (instead of due to) increased PKG protein expression in the following cases;

(1) The drug treated sample containing cGMP has greater PKG activity than the control sample containing cGMP.

(2) Untreated drug containing cGMP has greater PKG activity than control sample

Samples containing cGMP and samples without cGMP are prepared and 50 μL of each cell lysate is added onto the 20 μL PDE5 / GST solid slurry substrate. To determine the dissolution samples of the drug treatment and control groups, each mixture was added with a phosphorylation buffer containing 10 μCi ³² P-γ-ATP solution (200 μM ATP; 4.5 mM MgCl ₂ ; 5 mM KH ₂ PO 4; 5 mM K ₂ HPO 4). The reaction begins.

The resulting mixture is incubated at 30 ° C. for 30 minutes, centrifuged to separate the solid layer, and then discarded in a supernatant. The solid layer in each tube is washed with 700 μL of cold PBS and then Ramley sample buffer (Bio-Rad, 30 μL) is added. The mixture is boiled for 5 minutes and then placed in a 7.5% SDS-PAGE gel and transferred to 150 V for 1 hour. The gel stains 85 Kd GST-PDE5 fusion protein bands with Kozami Blue.

The gel is dried and placed on an X-ray film. If PDE5 is phosphorylated, a corresponding black band appears, indicating the degree of phosphorylation as the intensity of each band.

As can be seen in FIGS. 18A and 18B, compared to the control group with and without the excess cGMP, SAAND excisulins showed PKG activity in proportion to the drug dose in both the cGMP and non-added samples. It can be seen that this is more pronounced as the 85 Kd band appears darker in each sample treated with the drug. In addition, SW480 samples treated with exisulind showed greater phosphorylation activity than those treated with only exisulind without adding cGMP by adding cGMP. Thus, the increase in PKG activity in drug-treated samples is due not only to the increase of intracellular cGMP by SAAND inhibiting cGMP specific PDE, but also to the increase of PKG protein expression in tumor cells with APC mutations.

SW480 samples treated with E-4021 did not show PKG activity compared to the control group (see FIGS. 18A and 18B), indicating that the increased activity of PKG by SAAND in tumor cells with APC mutations was simply due to the inhibition of existing PDE5. It shows no.

In addition to the method of the X- ray film, SDS-PAGE through a 85 Kd band from the gel, and can measure the PKG activity, this time after cutting the band by using the radiation detector an amount of ³² P in the binding band from the gel Can be measured.

To test the inhibitory effects of cGMP-specific PDEs in tumor cells containing β-catenin mutations, HCT116 tumor cells were used, HCT116 containing β-catenin mutations but not APC mutations.

HCT116 was cultured in the same manner as the SW480 procedure, and only excisulin and control were used in this experiment. Excisulin-treated cells produced much more phosphorylated PKG compared to the control group, indicating that PKG activity in drug-treated cells was independent of APC mutations. Thus, for the purposes of the present invention, the "reducing β-catenin" of the claims is intended to represent the wild type and mutant type of the protein.

Confirmation of increased PKG expression and decreased β-catenin through western blot

As described above, selective apoptotic anti-neoplastic drugs (SAAND) result in increased expression of PKG and increased cGMP, both of which increase the PKG activity of SAAND-treated tumor cells, via Western blots. More confirmed.

SW480 cells treated with excisulin were harvested from microcentrifuge tubes by rinsing with ice-cold PBS as before, and the cells were stirred and lysed under modified RIPA buffer for 15 minutes. After lysing the lysate, the supernatant was transferred to a new microcentrifuge tube again, and BioRad DC protein analysis (Temecula, Calif.) Was performed to measure the protein concentration of the sample and normalized as before.

50 μg of each sample was taken and separated on 10% SDS-PAGE gel, and the protein was transferred to nitro cellulose membrane. The nitro cellulose membrane was fixed with freshly prepared TBST solution containing 5% skim milk powder with stirring for 1 hour at room temperature.

Chlorine anti-PKG primary antibody was diluted with fresh 5% skim milk powder, and then the nitro cellulose membrane was added and incubated with stirring for 1 hour at room temperature. The nitro cellulose membrane was washed three times with TBST solution for 10 minutes and then incubated with stirring in a solution containing secondary POD bound to rabbit anti-chlorine antibody for 1 hour at room temperature. The nitro cellulose membrane was washed three times for 10 minutes with TBST solution and then detected with a Boehringer Mannheim BM blue POD substrate.

As shown in FIG. 19, excisulind reduced β-catenin and increased PKG, which was found through Western blot. SW480 cells were treated with excisulin or mediator (0.1% DMSO) for 48 hours, 50 μg of each supernatant of each cell lysate was removed from the 10% SDS-PAGE gel, and the protein was transferred to a nitro cellulose membrane. It was read using anti-β-catenin and anti-PKG antibody.

SAANDs reduce β-catenin in tumor cells.

These results were observed by culturing SW480 cells with 200, 400 or 600 μM excisulin or mediator (0.1% DMSO).

Cells were harvested 48 hours after treatment as above and immunoblotting was performed. Immunoblotting was detected by Western blot, and as a result, it was found that the expression of β-catenin was reduced by 50% in the group treated with excisulin compared to the control group, and thus, β-catenin was decreased by SAAND treatment. have. Along with the above drug treatment and confirmation of increased PKG activity and the fact that it is phosphorylated by the following PKG, the above results indicate that the reduction of β-catenin in tumor cells is initiated by activating PKG. Therefore, PKG activity in tumor cells can be usefully used as a tool for the inspection of anti-tumor components.

Phosphorylation of β-catenin by PKG

In the laboratory, PKG phosphorylates β-catenin. The experiment that established this is to immunoprecipitate the complex containing β-catenin from SW480 cells which are not drug treated by a method such as "β-catenin immunoprecipitation" described below. The immunoprecipitated complex is still immobilized on a solid layer (ie beads) but mixed with ³² P-γ-ATP and pure PKG (100 units).

The corresponding controls without PKG were prepared.

After the protein is separated from the solid layer using SDS buffer, the mixture containing the protein is transferred to a 7.5% SDS-PAGE gel. Moving the mixture out of the gel removes the excess amount of ³² P-γ-ATP. Therefore, any ³² P-γ-ATP detected in the 93 Kd β-catenin band is due to the phosphorylation of β-catenin. Any increase in any of the ³² P-γ-ATPs found in the 93 Kd β-catenin bands treated with extra PKG compared to the control without extra PKG treatment phosphorylation of β-catenin in the extra PKG treated bands It is due to.

The results indicate that β-catenin can be phosphorylated by PKG because the increase in phosphorylation is significant in the bands treated with PKG compared to the control group that exhibits minimal virtually undetectable phosphorylation.

Phosphorylation of Mutant β-Catenin by PKG

HCT116 cells without APC mutations but with β-catenin mutations were performed according to the method described above, and the results showed that β-catenin mutations were phosphorylated by PKG.

Thus, for the purposes of the present invention, phosphorylation of β-catenin in the claims is intended to represent wild and mutant forms of the protein.

β-catenin precipitates with PKG.

The supernatant of the cell lysates of SW480 and HCT116 is prepared by the same method as the Western blot. The cell lysate is cleaned in advance by adding 150 μL of protein Sepharose bead slurry (50%) for each 500 μg of cell lysate, and then incubated for 10 minutes in a tube mixer at 4 ° C. Protein beads are removed by centrifugation for 10 minutes at 14,000 g at 4 ° C, and the supernatant is placed in a new centrifuge tube. 10 μg of rabbit anti-β-cacanine antibody (Upstate Biotechnology, Lake Placid, New York) is added to 500 μg of cell lysate. The lysate / antibody mixture is slowly mixed in a tube mixer at 4 ° C. for 2 hours. The immune complex is captured by slow mixing overnight in a 150 μL protein Sepharose beads slurry (75 μL of dense beads) and a tube mixer at 4 ° C. The Sepharose is harvested by impact centrifugation (5 seconds with fine centrifugation at 14,000 rpm), the supernatant is discarded and the beads washed three times with 800 μL of ice cooled PBS buffer. Sepharose washes the beads twice in 50 μL of sample buffer, mixes lightly and boils for 5 minutes to separate the immune complexes from the beads.

Beads are harvested by centrifugation and SDS-PAGE is performed using the supernatant.

Western blot is performed with supernatant. The membrane is read with rabbit anti-β-catenin antibody and then washed three times with TBST for 10 minutes to remove excess rabbit anti-β-catenin antibody. After adding goat anti-rabbit antibody coupled with horseradish peroxidase, incubate for 1 hour at room temperature. At the end of this process, it is possible to visually identify the presence of β-catenin as an HRPO substrate, and the experiment clearly shows the presence of β-catenin.

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To detect PKG in the membrane, anti-β-catenin antibody conjugates were separated in 62 mM Tris-HCl (pH7.6) buffer of 2% SDS and 100 μM 2β-mercaptoethanol for 0.5 hour in water at 55 ° C. I was. The membrane was then fixed with stirring with TBST of 5% skim milk powder. This fixed and isolated membrane is probed with rabbit polyclonal anti-PKG antibody [Calbiochem, LaJolla, CA], which is probed with goat, anti-rabbit secondary antibody bound to HRPO. The presence of PKG on the membrane can be visually identified as a substrate. In this experiment, PKG was visually identified. Considering that the proteins on the membrane are immunoprecipitated with β-catenin obtained only from the supernatant of cells, the results clearly show that PKG is physically bound to a protein containing β-catenin obtained from the supernatant of cells. gave.

The western blot membrane was also separated as described above and then anti-GSK3-β antibody was used to co-precipitate with β-catenin. In this experiment, GSK3-β was detected on the membrane, indicating that GSK3-β precipitates together with β-catenin and PKG, thus it can be assumed that the three proteins are part of one complex. Since GSK3-β and β-catenin form part of the APC complex in normal cells, PKG is thought to be involved in the phosphorylation of β-catenin as part of the complex.

Antitumor Pharmaceutical Compositions Containing cGMP PDE Inhibitors

As described above, excisulind is a compound that exhibits desirable anti-tumor properties. It is already known that the effects and uses of these drugs as antitumor agents are exerted by inhibiting cGMP specific PDE activity in tumor cells.

Above all, the fact that a compound effective for human treatment can be selected according to the selection criteria of the candidate compound according to the present invention has been verified in clinical trials for patients with abnormal growth of neoplasms. After finding out that the excisulin has an anti-tumor effect (in the laboratory), the compound was found in other clinical trials other than the excisulin in two clinical trials by searching for a compound meeting the selection criteria of the candidate compound according to the present invention. It was found that it can be selected according to the selection criteria of the invention.

As mentioned above, the neoplasia is likely to manifest mutations in the majority of adenomatous polyposis coli (APC). Above all, the selection criteria of candidate compounds according to the present invention have been established in human experiments in patients with neoplasia of proliferative cells capable of mutation of APC. The APC disease is characterized by the appearance of hundreds of thousands of polyps in the colon in the teenager, the conventional treatment is removed by surgical resection of the colon before turning 20 years old.

The first clinical trial was carried out using exisulind in APC patients. In the trial, patients had already undergone colon resection, except for a small portion of the colon around the rectum (attached to the small intestine), and the rectal function was maintained. However, these patients had polyps formed in small areas of the colon, which typically remained, requiring periodic removal (eg, electrocautery).

The use of exisulind in the first clinical trial was a preventive clinical trial to evaluate the anti-tumor effect of exisulind by comparing the cumulative number of new polyps formed over 12 months in the drug and placebo groups. Patients who met the clinical criteria developed 9 to 44 polyps in the colon each year. All polyps were removed at 6 months and 12 months after the start of the trial. 34 eligible patients participated in the clinical trial. According to the predicted average number of polyps formed over 12 months in APC patients with 9 ?? 44 polyps in the colon each year, the rate of reduction of exisulind polyp formation is clinically or statistically significant compared to the placebo group. Was high. According to the average number of ribs produced during the first six months of the study, the incidence of polyps was one-third higher than that of the placebo group in patients receiving exisulind (9 annual averages) And 26; p = 0.13). In addition, according to the average number of polyps formed over the 12 months in the clinical trial, the incidence of polyps was 1/2 of the placebo group compared to the placebo group (the annual average was 18 and 38; p = 0.020).

A separate clinical trial was conducted on male patients with prostate cancer and all prostate glands were removed. This study was conducted in patients whose prostate specific antigen (PSA) levels could be detected after prostatectomy with radiation, all suggesting recurrence of prostate cancer.

96 patients participated in the evaluation of prostate cancer. In the above clinical trials, patients with exisulind were subjected to a multicenter clinical trial with 500 mg / day for double blind and placebo control. As shown in the data shown below, there is a statistically significant difference in the case of PSA concentrations between the excisulind and placebo groups. PSA concentrations in the excisulind group were significantly reduced compared to that of the placebo group. The fact that elevated PSA levels do not in itself represent a disease, but it is well known in the medical community as an indicator suggestive of recurrence of prostate cancer in the subject.

Overall, the mean PSA concentrations were assessed to differ between the excisulind and placebo groups, followed by an interim evaluation of the subclasses. The patients who participated in this study were classified into severe, moderate and mild risk groups in light of the risk of cancer metastasis. This classification is based on JAMA methodology, a magazine published by the American Medical Association [JAMA May 5, 1999, pp. 1591-97. To identify which risk group the subjects belonged to in this trial, the clinical practitioners were given a history of the patients and did not know what drugs were being administered to the patients. Clinical practitioners assigned subjects to appropriate risk groups according to the JAMA methodology mentioned above. According to the statistical analysis, there was a statistically significant difference in the average PSA concentration between the exisulind group and the placebo group. The results are shown in Table 10 below.

TABLE 10

Effect of excisulin on mean PSA level after prostatectomy with increased PSA

Adverse reactions, clinical tests (liver function tests, blood tests and urine tests), basic clinical tests (blood pressure, pulse rate, respiratory rate, temperature and weight), physical examination in the above clinical trials and several other clinical trials administered exisulind And upper endoscopy, the safety of excisulin was established.

Several clinical trials in which 400 patients were treated with exisulind did not develop safety-related problems. When administering exisulind, there were no side effects such as blood disorder, dose-limiting vomiting or neurological or renal toxicity caused by administration of conventional chemotherapeutic agents. In addition, excisulind did not produce a clinically significant change even in the basal clinical. In paired biopsies of colonized polyps and normal tissues in patients with APC, it has been found that exisulind has minimal effect on normal tissues because it increases the rate of apoptosis in polyps.

Initial doses with adverse effects on dose limits at doses above the maximum allowed dose of exisulind (600 mg for patients with partial colon resection, 400 mg for patients without colon surgery, and 350 mg for pediatric patients) Only elevations in liver function tests (LFTs) are seen. Other mild to moderate adverse events (eg, sometimes stomach pain) were typically short, and therefore there was no need to stop or reduce the dose of excisulin.

In short, the clinical results show that exisulind is effective in clinical treatment of neoplasia of the neoplasia, and has excellent tolerability and long-term treatment. In addition, the clinical results showed therapeutically effective results in in vivo experiments, particularly when selecting additional compounds that inhibit cGMP specific PDE activity (suitable for other selection criteria of the present invention).

In addition, additional compounds invented before the mechanism of action is known or whether the selection criteria of the present invention are found to be suitable are known as (Z) -5-fluoro-2-methyl- (4-pyridylidene) which inhibits cGMP activity. ) -3- (N-benzyl) indenylacetamide hydrochloride (Compound I). The compound has been shown to have a wide range of tumor effects on neoplasia in human and laboratory experiments. In animal or clinical trials, the safety of this compound has been established with single and extended therapy.

As can be seen by those skilled in the art according to the data presented below, the compound I is in a much higher dose than the conventional chemotherapeutic or anti-tumorally effective nonsteroidal anti-inflammatory drugs (mostly toxic). It can be safely administered to the animal. For example, in acute toxicity experiments with rats, no toxicity symptoms were observed when Compound I was administered 2,000 mg / kg in a single dose (0.5% carboxy-methylcelulose as a medium). Weight gain was slightly reduced at the dose of 4,000 mg / kg. Body weight gain was reduced when administered at a single dose of 1,000 mg / kg, and mesenteric adhesion was observed in some animals at necropsy.

In animal experiments with dogs, no toxicity was observed in a single group consisting of two males and two females when Compound I was administered at a dose of 1,000 mg / kg. Given the properties of the compound I in capsule form, the dose was administered to each animal with at least 13 capsules, which is considered to be the maximum dose that does not stress the animal. Thus, the animals were administered seven consecutive times at a dose of 1,000 mg / kg per day. No drug-related adverse events were observed at the individual dose levels.

Therefore, no acute toxicity was observed when Compound I was administered to animals in a single dose. According to the animal test results, oral LD ₅₀ of Compound I is considered to be 1,000 mg / kg in dogs and 4,000 mg / kg in rats, and LD ₅₀ is considered to be 1,000 mg / kg or more in rats intraperitoneally. do.

No toxicity was observed at a dose of 50 mg / kg per day when Compound I was administered at a dose of 0, 50, 500 or 2,000 mg / kg per day in a seven-day dosing experiment using rats. Absolute and relative liver weights were elevated in females due to drug-related effects when doses of 500 mg / kg daily in animals. Adverse events observed when administering a dose of 2,000 mg / kg per day were dyspnea and / or abnormal breath sounds, weight gain, and decreased feed intake in males and increased liver weight in females. No liver function, hematologic changes, or pathological changes observed under a microscope were observed at any dose level.

In addition, rats were administered Compound I at a dose of 0, 50, 500 or 2,000 mg / kg per day in a 28-day clinical trial. No abnormal clinical symptoms related to CP-461 were observed, and no unusual symptoms were observed in body weight changes, optometry, liver function and blood tests, and urinalysis. When the animals were visually observed after necropsy, no tissue change was observed. When examining the weight of each organ, liver weight was statistically significantly increased at the dose of 2,000 mg / kg, and thyroid weight was statistically significantly increased at the dose. No statistically significant changes were observed at doses lower than this dose. Histopathological examination of each tissue showed hypertrophy of follicle cells and an increase in the number of mitosis (possibly cell proliferation) in the thyroid gland and mild lobular hypertrophy in the liver. In general, the number of mitosis of the thyroid gland increased in one female at a dose of 500 mg / kg per day, but this change was limited to a small number of animals at a dose of 2,000 mg / kg per day. In the liver investigation, the enzymes present in the microsomes were slightly stimulated to increase the thyroid metabolism, resulting in thyroid stimulation. Therefore, those skilled in the art will understand that such a drug effect is extremely small than the drug effect seen when a similar dose of a conventional chemotherapeutic agent or nonsteroidal anti-inflammatory agent is administered.

To specifically establish the safety of Compound I, a clinical trial was conducted in which apoptosis of the prostate cancer cell line induced by Compound I could be compared with the drug effect on prostate epithelial cells derived from normal tissues. Androgen-sensitive prostate cancer cell line LNCaP [ATTC, Rockville, MD] was placed in RPMI 160 medium containing 5% fetal placental serum and 2 mM glutamine and cultured under standard conditions. Primary prostate epithelial cell cultures (PrEC) derived from normal prostates [Clonetica, San Diego, CA] were grown under the same reaction conditions using tumor cell lines excluding serum-free media optimized for growth of the cultures. It was made. During the experiment, LNCaP or PrEC cell lines were inoculated in 96 well plates at a density of 10,000 cells per well. 24 hours after inoculation, the cells were treated with 50 μM of compound I (freebase) dissolved in vehicle (0.1% DMSO) or DMSO. After each hourly drug treatment (4, 24, 48, 72 or 99 hours), the cells were lysed to determine histone-associated DNA as an indicator for apoptosis. [Piazza et al., Cancer Research 57: 2452- 2459, 1997].

FIG. 27 shows that the amount of histone-related DNA fragments in LNCaP cell line cultures increases over time after treatment with 50 μM of compound I (freebase). Significant increases in DNA fragments were observed after drug treatment over 24 hours. In contrast, when the PrEC cells (normal prostate) were treated with Compound I (50 μM), the amount of DNA fragments did not increase even after 4 days of drug treatment. The results show selective induction of apoptosis in tumor cells as opposed to normal cells. This is in stark contrast to existing chemotherapeutic agents that induce apoptosis or necrosis in both rapidly proliferating normal and tumor cells.

Finally, in clinical trials following oral administration of the drug, Compound I did not show any significant adverse reactions at any dose level beyond the doses considered necessary for exerting antitumor effects.

As described above, Compound I has a strong antitumor effect. IC ₅₀ for tumor growth inhibition obtained by administration of Compound I was 0.7 μΜ in SW-480 cell line. The results were also confirmed when the antitumor effect of Compound I was evaluated on amphibians using modified aberrant crypt foci (ACF) as an indicator of cancer formation. The animal experiments using amphibians for cancer generation induced by azoxymethane (AOM) were to evaluate the antitumor effect of Compound I (free base and salt) on colon cancer expression in vivo. The ACF is a precursor of colon tumors, suggesting that inhibiting it may have a chemically protective effect.

The initiation of ACF in animal experiments with rats occurs by injecting cancer-producing factors for two consecutive weeks. Compound I was administered one week before the onset of ACF and during the duration of the experiment. ACF was found 5 weeks after administration. Compound I was administered orally to 344 male rat Fisher species in the nursery. Daily feed intake (mg / kg body weight) was supplied at different intervals during the study, so the dose of Compound I was expressed in grams per kilogram of feed to compare the dose. Body weights were measured during the experiment to determine whether Compound I had an effect on animal growth or feed. The animals lost weight compared to the control group, indicating the bioavailability of the drug. However, the weight difference between the two groups was less than 10%, which did not affect ACF formation.

The free base of compound 1 inhibited ACF formation because it was determined that the potential factor per colon was measured and is summarized in Table 11. Except for the low-dose group (feed intake: 0.5 g / kg), the difference between the treatment and control groups was significant and statistically significant when feeding amounts of 1.0 and 2.0 g / kg were provided.

Table 11

Inhibition of aberrant crypt foci by Compound I

Compound I is also one of the compounds retroactively meeting the selection criteria of the present invention and used to validate the selection criteria. For example, using the aforementioned protocol, Compound I has an IC ₅₀ of 0.68 μM for cGMP-specific PDE using cGMP-specific PDE from H29 cell extract. The COX I inhibitory capacity (for 100 μM) of the compound is less than 25%.

The EC ₅₀ for the DNA fragment of Compound I is 15 μM in relation to causing pre-apoptosis. In addition, the percentage of apoptosis for Compound I in SW-480 cells is shown in Table 12 at each concentration.

Table 12

Induction of Apoptosis of HT-29 in SW-480 Colon Adenocarcinoma Cells by Morphologically Determined Compound I

The activity of Compound I is not limited to colon cancer cell lines or animal models with colon cancer. The compounds show a wide range of antitumor effects against various tumorous cell lines. Various types of human cancer cell lines were placed in RPMI 1640 medium containing 5% fetal placental serum, 2 mM L-glutamine and sodium bicarbonate and cultured under standard conditions.

In order to measure the inhibitory effect of the compound I on tumor cells, the cell line was inoculated in 96 well plates with a density of 1000 cells per well. 24 hours after inoculation, compound I (final concentration: 0.1%) as free base dissolved in DMSO was administered to the cell lines at various concentrations. Drug effect on tumor cell growth was measured by neutral red cytotoxicity test through 5 days continuous administration. Neutral red dye is selectively absorbed by living cells by ATP-dependent transport mechanisms.

As summarized in Table 13 below, Compound I (freebase) exhibits potent tumor growth inhibitory effects when tested against a series of cultured human cell lines derived from various tissues. Compound I exhibits a tumor suppressor effect that can be compared regardless of the tissue production of tumors derived from cell lines. GI ₅₀ concentrations for all cell lines (up to 50% tumor growth inhibition of the median control) were calculated to be 1-2 μM.

Along with Table 13 shown below we describe human leukemia cell lines (CCRF-CEM, K562 and Molt4), myeloma cell line (RPMI8226), pancreatic cancer cell line (PAN-1) and ovarian cancer cell line (OVCAR-3) for compound I (hydrochloride). ), We compared sensitivity.

Table 13

Inhibition of proliferation of several human tumor cells by compound I

Given the safety of animals and humans and the very broad cell culture effects of Compound 1, compounds that meet the selection criteria of the present invention (inhibiting cGMP specific PDEs) can be used as useful antitumor agents.

Those skilled in the art to structurally identify additional PDE inhibitors specific for cGMP that may exhibit therapeutically effective activity as anti-tumor agents are those of the numerous model compounds listed in the present invention (the analogues thereof listed in the references). Additional compounds having the same shape but chemically different may be used as the base compound for computer modeling. For example, Molecure Simulation's software (WebLab ViewerPro ™) is equipped with molecular transparency and chemical exchange capability. The software has a variety of functions, including 3D perspective views of existing active compounds to verify the accuracy of the capture state or the chemical structure introduced. In addition, the software can arbitrarily adjust the structure according to user-defined shapes, and can measure distances and angles or two plane images.

As such, the structure of the other active compounds is shown so that the user can identify new additional compounds that can be searched and selected according to the selection criteria of the present invention according to the group analysis and the 2D / 3D similarity searching technique. According to the software method, it is necessary to adhere to the principle that compounds having similar structure or similar or identical properties exhibit similar activities and can be identified by the selection criteria of the present invention.

When additional compounds are identified according to computer modeling as described above, many of the compounds or derivatives thereof use conventional combination chemistry techniques commonly used by those skilled in the pharmaceutical industry. Synthesis is possible. The combination chemical technique can be rented from a number of companies [New Chemical Entities of Bocchel Washington, Protogen Laboratory of Palo Alto, California, Axys of San Francisco, Southern California, Nanosyn of Tucsan, Arizona, Trega of San Diego, California and RBI of Massachusetts Natick]. Many pharmaceutical companies have facilities to carry out related projects. In short, one of ordinary skill in the art will readily find numerous compounds to be searched and select promising compounds to treat neoplasia that possess the properties of the compounds listed herein. It would be interesting to note the identification of compounds that can be searched and the binding of selected antitumor compounds to the PDE5 protein to meet the selection criteria of the present invention. According to the procedure described below, a compound meeting the selection criteria of the present invention is bound to the cGMP catalyst site of PDE5.

To do this, a PDE5 sequence containing no catalytic region was used. One way of forming the sequence is to express the sequence as a fusion protein, preferably by using glutathione S-converting enzyme (GST) together, which will become apparent soon.

The RT-PCR method was performed by bovine PDA5A cDNA sequence [McAllister-Lucas L. M. et al., J. Biol. Chem. 268, 22863-228873, 1993, together with the forward and reverse primers designed from, to obtain the cGB region of PDE5, and also select from 5 'to 3' of the PDE 1-10 family. The kit for total RNA is used with HT-29 cells according to oligo (dT) column purification of mRNA. Forward primer (GAA-TTC-TGT-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G, 203-227) and reverse primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC- CTC-TGC-TG, 166401686) was used to synthesize a 1484 bp fragment encoding a phosphorylation site and a low and high affinity cGMP binding site of human PDE5A (204-1686 bp, cGB-PDE5). The PDE5 nucleotide fragment thus synthesized encodes 494 amino acids with 97% similarity to bovine PDE5A. The encoded PDE5 nucleotide fragment was cloned into a pGEX-5X-3 glutathione-S-transferase (GST) fusion vector (Pisia Biotech Co., Ltd.) using a tac promoter with EcoRI and XhoI cleavage sites. The fusion vector is transduced with E. coli BL21 (DE3) bacteria [Invitrogen]. The transduced BL-21 bacteria grow in log phase and then IPTG is added as an inducer. The induction is made at 20 ° C. for 24 hours. The bacteria are extracted and dissolved. Lysed cell lysate is incubated with GSH bound to Sepharose 4B, and other proteins are washed from the beads by excessively cooled phosphate buffer.

The expressed GST-cGB-PDE5 fusion protein appears on a 7.5% SDS-PAGE gel as an 85 Kd protein. The protein is characterized by cGMP binding and phosphorylation according to protein kinases G and A. In addition, the protein shows two cGMP binding sites and the K _d is 1.6 ± 0.2 μM, which is close to Kd = 1.3 μM of bovine native PDE5. GST-cGB-PDE5 for GSH bound sepharose beads can be phosphorylated in the laboratory by cGMP-dependent protein kinase and cAMP-dependent protein kinase A. The K _M of GST-cGB-PDE5 phosphorylation by PKG is 2.7 μM and Vmax is 2.8 μM. On the other hand, the K _M of BPDEtide phosphorylation is 68 μM. Phosphorylation by PKG shows that phosphate molecules are bound to GST-cGB-PDE5 fusion proteins in a ratio of 1: 1.

CGMP binding assays for compounds of interest [Francis SH et al., J. Biol. Chem. 255, 620-626, 1980], 5 mM sodium phosphate buffer (pH = 6.8), 1 mM EDTA, 0.25 mg / ml BSA, H ³ -cGMP (2 μM, NEN) and GST-cGB-PDE5 fusions The total amount containing protein (30 μM per dose) is performed at 100 μM. Individual test compounds are added simultaneously as ³ H-cGMP substrates and the mixture is incubated at 22 ° C. The cultured compound is transferred to a Brandel MB-24 cell harvester equipped with GF / B as a filtration membrane and washed twice with 10 mL of cooled 5 mM potassium buffer (pH 6.8). The membrane of the washed mixture is cut and transferred to a scintillation vial and a scintillation mixture containing 1 ml of water and 6 ml of Ready Safe (trademark) solution is added to each vial. The vials are counted in a Beckman LS 6500 scintillation counter.

To calculate this, a blank sample is prepared by boiling the bound protein for 5 minutes and the bound count is less than 1% compared to the protein that does not boil. Assays are quenched by filtration membranes or other devices.

Sulfides, excisinsins, compounds B, compounds E, E4021 as PDE inhibitors and cAMP, cyclic IMP, 8-bromo-cGMP, cyclic UMP, cyclic CMP, 8-bromo- as japrinat and cyclic nucleotide analogs In the case of cAMP, 2'-0-butyl-cGMP and 2'-0-butyl-cAMP, they are selected to test whether they can bind competitively with the cGMP binding site of the GST-cGB-PDE5 fusion protein. The results are shown in FIG. cGMP specifically binds to the GST-cGB-PDE5 fusion protein. Cyclic AMP, cUMP, 8-bromo-cAMP, 2'-0-butyl-cAMP and 2'-0-butyl-cGMP do not compete with cGMP at the binding site. Cyclic IMP and 8-bromo-cGMP can partially compete with cGMP binding sites at high concentrations (100 μM). PDE5 inhibitors do not compete with cGMP for binding sites of the GST-cGB-PDE5 fusion protein. Thus, these inhibitors do not bind to the cGMP binding site of PDE5.

However, compound E does not competitively bind with cGMP to PDE5 (ie peak A).

Compound E also competitively binds PDE with cGMP (peak B). Given that Compound E does not bind to the cGMP binding site of PDE5, there is a competitive binding site between Compound E and cGMP, so that a preferred compound like Compound E binds to the cGMP catalytic site on PDE5. This may help to search for information already known to those of ordinary skill in the art (existing competition experiments) or other compounds. Therefore, the person having ordinary knowledge in the art through the information on the chemical structure and cGMP binding site of the preferred compound group presented in the present invention can search for, identify, and select other chemicals for therapeutic use (selection criteria of the present invention). Can be used).

Compounds selected according to the invention can be formulated as pharmaceutical compositions, which typically refer to pharmaceutical compositions. For example, the compound (solid agent mentioned above) and a pharmaceutically acceptable substance may be used in solid or liquid form, intravenous or intraperitoneal injection, topical administration in ointment form, or rectal or topical administration in suppository form. It is called. Of these, oral formulations are most preferred.

Conventionally well known pharmaceutically acceptable formulations for oral pharmaceutical compositions include capsules, tablets, pills, powders, troches and granules. In the case of the solid preparation, the carrier contains at least one diluent such as sucrose, lactose and starch. The carrier may typically additionally contain a lubricant, such as magnesium stearate. Capsules, tablets, troches and pills can contain buffers as carriers. Formulations such as tablets, pills and granules can be prepared by applying an enteric coating on their surfaces. In addition, enteric coated compounds may be prepared by compression into tablets, pills or granules for administration to a patient. Enteric coatings may be chosen that dissolve or dissolve at pH in the colon, such as shelllac or Eudragit S.

Pharmaceutically acceptable formulations for pharmaceutical compositions are oral liquid formulations, for example emulsifiers, solutions, suspensions, syrups, and elixirs containing inert diluents that are pharmaceutically acceptable and commonly used in the art. Priests. In addition to the inert diluent, the composition may contain wetting agents, emulsifiers and suspending agents, sweeteners, flavors and fragrances.

In pharmaceutical compositions for intravenous or intraperitoneal injection, the pharmaceutically acceptable carrier contains conventional pharmaceutical saline.

Pharmaceutically acceptable carriers of pharmaceutical compositions for topical administration include DMSO, alcohols, or propylene glycol, and are used in combination with a patch or liquid-holding substance to preserve the drug on the skin so that the drug does not dry. do.

Pharmaceutically acceptable formulations of the pharmaceutical compositions for rectal administration may contain cocoa butter, suppository waxes, or gels in addition to the compounds of the invention as suppositories.

Pharmaceutically acceptable carriers and compounds of the invention are formulated as pharmaceutical compositions in unit dosage form for administration to a patient. Amount of active ingredient effective to obtain activity to treat abnormal growth of tumor according to the preferred method of administration (ie oral or rectal administration) by varying the content of active ingredient (ie, the compound selected according to the present invention) as a unit dosage form. Will be determined.

Thus, the dosage chosen is determined in view of the nature of the active compound administered (eg, IC ₅₀ which can be quickly identified), route of administration, preferred duration of treatment and other factors. If desired, the dosage may be determined by a single daily dose for the active compound or by splitting multiple doses (eg 2-4 times daily). For intravenous administration, the initial dose is based on the dose at which IC ₅₀ is obtained from the mean adult male blood concentration (ie 4 L). The initial dose of the active compound selected according to the present invention can be selected from 0.5 to 600 mg.

The pharmaceutical composition of the present invention is preferably packaged in a container (e.g., a box or a bottle or both) in which an appropriate packaging material (e.g., instructions) carrying indications and instructions for use is inserted.

In the case of the present invention, it is clear that modifications and variations are possible in light of the above description. Accordingly, other matters other than those mentioned by the present invention may be enclosed in the claims to be described below.

As described above, the method of identifying a compound according to the present invention and a pharmaceutical composition comprising the compound obtained thereby have a useful effect in the prevention and treatment of precancerous and cancerous lesions in mammals.

Claims (61)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete And measuring the tumor inhibitory activity of the compound, and measuring the PDE2 inhibition activity of the compound, the anti-tumor effect which comprises an IC ₅₀ with respect to the cancer cells, select less than 100 μM, and the PDE2 IC ₅₀ is 10 μM or less compound Method for selecting compounds with The method of claim 20, further comprising determining that the EC ₅₀ for apoptotic activity in the cancer cells of the compound is 100 μM or less and selecting the compound. 22. The method of claim 21, further comprising measuring cyclooxygenase (COX) inhibitory activity of the compound and selecting the compound having an inhibitory activity of less than 25% at 100 μM. delete delete Selecting a compound having a cyclooxygenase (COX) inhibitory activity of less than 25% at 100 μM and having a PDE IC ₅₀ of 10 μM or less, The PDE is A) exhibits more specificity for cGMP than cAMP; B) shows a positive cooperative kinematic aspect in the presence of a cGMP substrate; C) shows the affinity of the ultrafine mass for cGMP; And, D) A method of identifying a compound having an anti-tumor effect, characterized by insensitivity during incubation with a protein kinase dependent on purified cGMP. 27. The method of claim 25, further comprising determining whether the compound has an IC ₅₀ of less than 100 μM for cancer cells and selecting the compound. The method of claim 25, further comprising determining that the EC ₅₀ for apoptotic activity in the cancer cells of the compound is 100 μM or less and selecting the compound. Measuring the inhibitory activity of cancer cells on the compound, To measure PDE inhibitory activity against a compound, The PDE is A) shows specificity for cGMP rather than cAMP; B) shows a positive cooperative kinematic aspect in the presence of a cGMP substrate; C) shows the affinity of the ultrafine mass for cGMP; And, D) exhibit insensitivity upon incubation with protein kinases dependent on purified cGMP; And, Method of selecting with the anti-tumor effect which comprises an IC ₅₀ with respect to the cancer cells, select less than 100 μM wherein said PDE IC ₅₀ is less than or equal to 10 μM compound compound. Measuring the PDE inhibitory activity of the test compound, The PDE is A) shows specificity for cGMP rather than cAMP; B) shows a positive cooperative kinematic aspect in the presence of a cGMP substrate; C) shows the affinity of the ultrafine mass for cGMP; And, D) exhibit insensitivity upon incubation with protein kinases dependent on purified cGMP; And a compound having an antitumor effect comprising selecting a compound having the PDE IC ₅₀ of 10 μM or less. The method of claim 29, further comprising determining whether the compound induces apoptosis in cancer cells and selecting a compound having an EC ₅₀ of 100 μM or less for the apoptosis activity. delete delete Isolated phosphodiesterases are: A) shows specificity for cGMP rather than cAMP; B) shows a positive cooperative kinematic aspect in the presence of a cGMP substrate; C) shows the affinity of the ultrafine mass for cGMP; And, D) isolated phosphodiesterases characterized by insensitivity during incubation with protein kinases dependent on purified cGMP. 34. The isolated phosphodiesterase of claim 33, wherein said isolated phosphodiesterase additionally exhibits reduced susceptibility to inhibition by japriast and E4021. 34. The isolated phosphodiesterase of claim 33, wherein said isolated phosphodiesterase can be further separated from exhibiting classic PDE5 activity in anion exchange chromatography. 36. The isolated phosphodiesterase of claim 35, wherein said isolated phosphodiesterase is additionally not substantially activated by calcium / calmodulin. 37. The isolated phosphodiesterase of claim 36, wherein said isolated phosphodiesterase is additionally insensitive to rolipram, vinpocetin, and indolidan. delete delete delete delete delete delete delete delete delete delete delete delete A) determining whether the compound has an IC ₅₀ for cancer cells of less than 100 μM; B) determining whether the compound increases PKG activity in cancer cells; And, C) A method of selecting a compound having an anti-tumor effect comprising selecting a compound having an IC ₅₀ of less than 100 μM against the cancer cells and increasing PKG activity in the cancer cells. 51. The method of claim 50, wherein the compound further comprises determining whether to inhibit PDE5 and selecting a compound that inhibits PDE5. 51. The method of claim 50, wherein the compound further comprises determining whether to reduce the β-catenin of the cancer cells upon treatment, and selecting the compound that reduces the β-catenin. 51. The method of claim 50, wherein the compound additionally comprises determining the inhibition of phosphodiesterase (PDE) specific for cGMP and selecting the compound having a PDE IC ₅₀ of 10 μM or less. 51. The method of claim 50, wherein said compound further comprises determining whether to increase PKG expression and selecting a compound that increases said PKG expression. 51. The method of claim 50, wherein said compound further comprises determining whether to increase PKG activation and selecting a compound that increases said PKG activity. 51. The method of claim 50, wherein the compound additionally measures the inhibition of PDE2 and selects a compound having a PDE2 IC ₅₀ of 10 μM or less. delete Selecting compounds that increase PKG activity in cancer cells and measuring the inhibitory activity of the compounds against cancer cells to select compounds that increase PKG activity and have an IC ₅₀ of less than 100 μM for cancer cells without substantial disruption to normal cells Method for identifying a candidate compound having an anti-tumor effect, including that. Cyclooxygenase (COX) inhibitory activity against the compound was measured, and the cyclooxygenase (COX) inhibitory activity was measured to less than 25% at 100 μM by measuring whether to increase PKG activity in cancer cells for the compound. And identifying a compound having an antitumor effect comprising selecting a compound that increases PKG activity. To determine the inhibitory activity of tumor cells against the compound and to determine whether to increase the PKG activity in the tumor cells for the compound, the IC ₅₀ for the tumor cells is less than 100 μM and PKG activity in the tumor cells A method of selecting a compound having an anti-tumor effect comprising selecting a compound to increase. delete

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