WO2006022460A1

WO2006022460A1 - Composition and method of use on the deuterium oxide and deuterium oxide related products for prevention, suppression, and treatment of recurrent urothelial cancer and urothelical cancers including bladder cancer

Info

Publication number: WO2006022460A1
Application number: PCT/KR2004/002153
Authority: WO
Inventors: Jong Yoon Bahk
Original assignee: Jong Yoon Bahk
Priority date: 2004-08-26
Filing date: 2004-08-26
Publication date: 2006-03-02

Abstract

This invention is about the components and usages of deuterium oxide (D2O) and compounds mixed with D2O and some materials which are directed to prevention or suppression of recurrence of urothelial cancer and treatment of early urothelial cancer, which developed at urotheium that is covering the inner superficial surface of urethra, bladder, ureter or renal pelvis. The components of this invention are pure D2O, mixture of D2O and solvents which are not harmful to human, or mixture of D2O and anticancer drug or immunostimulants. The components mixed with D2O to compose the D2O compound include inert salts, sterile distilled water, saline or many kinds of isotonic solution including dextrose solution, and other solution that does not damage the normal cells. Those other solutions include sesame oil, cottonseed oil, peanut oil, palm oil, coconut oil, animal oil and inorganic oils that are not harmful to human. The state of D2O or D2O products for usage will be D2O only, or mixtures of D2O and above mentioned components including anticancer agents. Those D2O products would be applied by retrograde fashion immediate or some times after the treatment. For better cytocidal effect, D2O products should be contact with urothelium more than one hour and can be applied repeatedly. These inventions, which are D2O or D2O products, can induce the treatments of urothelial cancer with fewer side effects and can reduce the former amount of anticancer drug dose.

Description

Composition and Method of Use on the Deuterium oxide and Deuterium oxide related products for Prevention,

Suppression, and Treatment of Recurrent Urothelial Cancer and Urothelical cancers including Bladder Cancer Technical Field

[1] The present invention involves compositions andmethods to use deuterium oxide

(D2O, heavy water) or compounds containingdeuterium oxide for the prevention or suppression of the recurrence ofurothelial cancer, or treatment of the early superficial urothelial cancers inurinary system Background Art

[2] After the first report on the discovery on deuteriumoxide (D2O, heavy water) was made around 1930, the purification and productionof D2O became possible from several decades ago. The studies with D2O inbiological field have long history. Majority of studies were performed at thefields of physio-chemistry but small portion of them are studied at thebiomedical areas. Although there are several reports, the studies related withhuman cells or tissues related with D2O are not many until recently.

[3] The exact mechanisms related with characteristicbehavior related with cy- toreduction have not exactly defined yet, there weremany theories. Some suggested the mechanism by the stabilization of tubulin(GChakrabarti et al. Biochemistry 1999, 38:3067-3072), mitotic arrest(PR Grossand W Spindel, Sciece 1960, 131:37-39; J Lamprecht et al. Eur J Cell Biol 1990,51:303-312; BL Sailer et al. Cytometry 1996, 25:164-172), impairment ofmemebrane function(D Schroeter et al. Eur J cell Biol 1992, 58:365-370) orinterference of cell division and the depression of the uptake ofDNA-precursors in mammalian cells(T Uemura et al. 2002 J Neurosurg96:900-908).

[4] From several decades ago, the animal study resultswhich showing shrinkage of the xenografted cancer tissues were observed fromthe nude mice which were intaken D2O orally(MW Biggs et al. Cancer Res 1963,23:1059-1062; AM Hughes et al. Bio-chem Biophys Acta 1958, 28:58-61; JJ Katz etal. JNCI 1957, 18:641-659; JA Laissue et al. Cancer Res 1982, 42:1125-1129).The survival of the experimental animal was prolonged more when D2O was giventogether with several cytostatic agents(HJ Altermatt et al. Laryngol RhinolOtol (Stuttg) 1987, 66:191-4). The biologically toxic concentrations of D2O onliving organisms are different according to the species. In rodents who drunkmore than around 20 - 25 % of the total water volume was D2O(J Hans et al.Cancer 1988, 62:462-466: LA Manson et al. Proc Soc Exp Biol Med 1960,105:481-483). Some fishes in deep sea could maintain good survival al- thoughthey have many chances to contact with D2O.

[5] Deuterium oxide works favorably on survival of themice whom hypertension was induced from salt or ethanol(S Vasdev et al. Can JCardiol. 1993 9:802-808), or gamma irradiated ones(JA Laissue et al. Radiat Resl983, 96:59-64). And in some drugs, D2O has the drug metabolic switchingeffects(DJ Kushner et al. Can J Physiol Pharmacol 1999 77:79-88) and reducesgenotoxicity of anticancer drugs(M Jarman et al. Car¬ cinogenesis 199516:683-688). It was reported that some man who drink small amount of 99.9 %pure D2O and some other person who directly infused D2O into blood stream didnot experienced any disorders(RJ Peppard et al. J Pediatr 1993, 123:457-462; ANorberg et al. Alcohol Clin Exp Res 2001, 25:1423-1430). Several reporters(MWBiggs et al. Cancer Res 1963, 23:1059-1062; AM Hughes et al. Bio- chem BiophysActa 1958, 28:58-61; JJ Katz et al. JNCI 1957, 18:641-659; JA Laissue et al.Cancer Res 1982, 42: 1125-1129) studied on hunan cancer related on D2O and mostof them are pancreatic cancer, gastric cancer and etc. and they were ad- vancedcancers and they had the characteristics of easy infiltration andmetastasis.

[6] Urinary system is composed with kidney, renal pel vis, ureter, urinary bladder

(bladder) and urethra. After the urine has producedfrom the kidney, it passes renal pelvis, ureter, bladder and urethra to bediscarded outside of the body. The roles of these organs are a conduit or areservoir for the passage or temporary storage of urine before urine has passedto outside of the body. Renal pelvis, ureter and urethra have the role ofconduit to pass the urine. So, they contact with urine with short period andthis means short contact with urinary carcinogens. Although it is not clearlydefined that renal pelvic, ureteral or urethral wall has the ability toreabsorb the urine which was secreted from the kidney, if these organs have there-absorption ability, the amount of re-absorption will be minimal.

[7] Compared to renal pelvis, ureter or urethra, bladderhas the function of storage and it has relatively long contact time (about 4 to 5 hours) and this means long contact with urinary carcninogen. And damage tothe bladder wall exposing muscle, either it was caused from surgery or others, usually causes re-absorption of some amount of urine into blood stream, generalcirculation.

[8] In urothelial cancers, the causes are diverse andclose related with various chemical substances such as 2-naphthylamine,4-diaminobiophenyl benzidine or alinine, tobacco, coffee, saccharin, cyclamate,phenacetin etc(AM Kamat and DL Lamm. Urol Clin North Am. 2002 29:157-68) andthe carcinogenetic processes are same regardless on the site of the urotheliumthey locate, pelvis, ureter, bladder or urethra. In case of smoking, it hasknown that they have very close relation between urothelial cancer(MP Zeegerset al. World J Urol. 2004 21:392-401) and the long history of smoking and theduration of smoking parallel with incidence of urothelial cancer. The latenttime for appearance of urothelial cancers are different according to thecarcinogen and duration of the contact time between carcinogen and urotheliumbut they have long duration of latent period (20 to 30 years) from carcinogencontact and this become a cause of different appearance of multiple onset overtcancers.

[9] The mucosa of the urinary systems, renal pelvis, ureter, bladder and part of urethra are transitional cell epithelia. Amongthese organs, bladder is the representative organ covered by urothelia and haswidest field of urothelia. The mucosa of the organs covered with urotheliacould be visually accessed by urologists using various endoscopic instnmentsand we can easily detect the changes of the urothelia in urinary mucosa. Thebladder mucosa has the same biological characteristic with other si- teurothelia. The bladder mucosa are coved by 6-7 layers of urothelium, most ofthem are transitional cell epitheliim, and the most superficial cells exfoliatecontinuously and the basal cell produces new cells being pushing up the upperolder cells. So, the localized damage of the mucosa of bladder caused bycertain stimuli or chemical agents could be regenerated very easily within notlong time and these conditions do not have the long term harmful effect onbladder function, usually. The damage, exfoliation and death of the mostsuperficial urothelia are not a big problem in medical aspect in long termalthough they have the local irritating symptom instantly.

[10] The bladder cancer is the representative cancer ofthe urotheliun. Bladder cancer covers more than 90 % of the urothelial cancersand most of bladder cancers are transitional cell cancers. The incidence of thebladder cancer is about 3.5 % among human cancer in developed country and theincidence increased as age advanced.

[11] Cystscopic observation on bladder detect the cancersin usual and confirm the diagnosis with the assistance of biopsy. Thecystoscopy is performed in patient showing painless hematuria or severe bladderirritating symptoms etc. Recently, non¬ invasive biomarkers, telomerase,hyaluronic acid, cell surface antigen, Lewis X antigen or DNA ploidy, havedeveloped but inferior than cystoscopic diagnosis. Transurethral resection(TUR) of bladder cancer is the standard treatment of bladder cancer and it hasthe basic and most important principle that all visible cancers should beremoved. During transurethral resection of bladder cancer (TURB), doctorsresect the bladder cancer with some underlying rruscle layer( superficial rrusclelayer). The obstacle in transurethral resection of the bladder cancer (TURB) isthat we could not remove the invisible cancer through magnified endoscope, suchas non-typical cancers, flat early superficial very small cancers and veryearly cancers. For those invisible overt cancers and some conditions justbefore to be an overt cancer, to treat them, intraviesical in¬ stillation of theanticancer agents, Adriamycin, mitomycin C, thiotepa, epirubicin or etc. orirrmmostimulants, interferon, bropirimine, KLH were used(O Alfthan et al. WorldJ Urol. 1997, 15:89-95).

[12] After the operative treatment of bladder cancer, there are many recurrences up to 70

% - 80 % of patients(RA Jr Thompson et al.J Urol. 1993, 149:1010-1011) and there are many theories for their recurrence.There are several theories on the recurrence of the post-TURB and they havesome rationale each other. Among those bladder cancer which has recurred, some(about 30 % of them) are progress to infiltrating (invasive) cancers. Invasivecancer means high possibility of life threatening. So, to prevent or reduce therecurrence after operative treatment of bladder cancer, above mentionedin- travesical instillation methods are used, too.

[13] With intravesical instillations, there are manydifferent outcomes are reported. But there is suspicion on the true long termeffect of instravesical instillation according to the recent statistic data. When we consider the complication of the chemotherapeutic agents(JB Thrasherand ED Crawford Urol Clin North Am. 1992, 19:529-539), there is possibility that intravesical instillation is meaningless in long term except a few agents. So, many urologists recommend that at high risk patients for recurrence,attempt for intravesical instillation is recommended but low risk patients, intravesical in¬ stillation does not. Moreover, most of the agents used inintravesical instillation used for other systemic cancer treatment and theyhave various complications although they are different each other. Thesecomplications cause many disorders and usually became an important reason tostop intravesical instillation of various agents.

[14] Due to these reason, development or discovery of theagents which is more effective in anticancer therapy and less or non-toxic topatient compared to conventional one is most desirable in future. And manyagents are studying for these goals Disclosure of Invention Technical Problem [15] Purification of the D2O is an open technique in general at present time but the medical use of the D2O is very rare, the research step for cancer treatment. The studied fields of the D2O in cancer are related with pancreatic and gastric cancers which have tumor biology easy to be metastasis.

[16] This invention on the deuterhm oxide and deuteriim oxide products which aimed to suppress or prevent the recurrence of the superficial urothelial timor and treat the flat or early urothelial cancers. As mentioned at background, bladder cancer is the rep¬ resentative urothelial cancer but urothelial cancer biology is same regardless of their origins or sites. Bladder cancer developmental incidence is increased with advance of age but aged people are not strong to accommodate complications of the anticancer agents. So, selection of the agents which has strong anticancer effect and low toxicity to patient is very important.

[17] There are many theories on the recurrence of the bladder cancer. It is known that the latent periods to the appearance of overt cancer in urothelial cancer from initiation in process of carcinogenesis needs 20 to 30 years. So, different initiation time means new appearance of the overt cancer in different time. Incomplete resection of the bladder cancer remaining remnant cancer and internal seeding during transurethral resection are other theories included in the hypothesis for recurrence of the bladder cancer. It is generally agreed that these are the causes of recurrence of the bladder cancer that is over 70 % up to 80 %.

[18] At present time, under the goal that discovery of the agents which has lower toxicity to patient and simultaneously showing high toxicity to cancer cell / tissue, many studies are performed to reduce, suppress or treat the recurrence of the bladder cancers. Among them, topical application (contact) of the anticancer agents or im- munostimulants for several hours to bladder cancer directly or area which is suspicious for harboring cancer / precancerous lesions is well known. But until now, the agents that have low toxicity to patient and high cytotoxicity to cancer has not discovered.

[19] Present invention is related with the deuteriim oxide (D2O, heavy water) and deuterium oxide products, and usage of them to prevent or suppress recurrent su¬ perficial tumor of the urothelial timor and treat the early small or flat indistinct su¬ perficial urotherlial cancer including urinary bladder cancer.

[20] The present invention involves composition and method of manufacturing or using deuterium oxide or its products to prevent or suppress the recurrence of urothelial cancer or treat the early superficial urothelial cancers including bladder cancer.

[21] Re-absorption of solution within the urinary bladder in certain condition is great, such as in case of exposed transected vein, but minimal in general in normal condition. Before retrograde instillation of any solutions into bladder, re-absorption should be considered due to the unexpected side effects. We should expect some unwanted side effects, such as hemolysis etc. when entry of the hypotonic solution into blood stream has occurred. When we use D2O or D2O products, we have to consider the tonicity of them because pure D2O has low tonicity. Before we use the D2O or D2O products, the isotonic condition should be maintained with nontoxic sodiim chloride or other inert materials including other salts. The re-absorption of isotonic D2O or D2O products would be much less toxic than non-isotonic ones. When some patients require maintenance therapy after some course of treatment, he can infuse several times more into bladder.

[22] From below, the inventor will explain figures that contained in this submitted ap¬ plication for patent which is a example among many experiments using various materials that inventor has performed.

[23] Figures are the results of bladder cancer cell T-24 exposed to the media, containing

D2O at different concentration, for two hours. After the bladder cancer cell T-24 was thawed, it was amplified at media made of normal water (H2O, light water) as usual. Among these amplified cells, 1 x 105 cells were seeded on the culture dishes, and cultured 24 hours and the media were sucked out and washed gently with phosphate buffered saline (PBS) for two times and replaced them with media containing D2O of different concentrations, 0 % (control), 25 %, 50 %, 75 % and 100 %, and cultured for two hours at incubator. After two hours, the D2O containing media was sucked out completely and washed gently with PBS for two times and replaced media made of normal water for further culture for 6 days more with three media changes. At post- subculture 7th day, photographs were taken and media were sucked out, then, cells were washed with PBS for two times and Trypsin-EDTA solution was applied and cells were detached cells from the culture dishes. The detached cells were counted under the microscope with hemacytometer. As seen at figure 1 (experiment 1), control group (0 %) showed very high growth rate and 25 % and 50 % showed high growth too compared to seeded nimber of the cells. But media containing 75 % and 100 % D2O showed growth less than 10 % of seeded nimber of the cells. This means high concentrated D2O itself has the highly effective cytocidal effects. As seen in figure 1, with increase in D2O concentration, arrest of cancer cell growth expanded until 50 %, but it would be worth to mention as "increased cell death rather than growth" in 75 % and 100 %. [24] The figure 2 is the results of the cell, T-24, growth in mitomycin C dissolved in

D2O solutions of different concentration (experiment 2). Dose of mitomycin C was 20mg in 40ml solvent. The solvents were composed with pure water and different proportion of D2O. In clinical setting, mitomycin C is commonly used for intravesical instillation to prevent and suppress the recurrence of the bladder cancer after TURB. The dose of mitomycin C is different according to the reporters, from 10 mg / 40 ml water to 40 mg / 40 ml. Experiment 2, middle of those doses was selected and experiment was performed as same as experiment 1, except one more control, control 2, for solvent composed with pure water and mitomycin C. When we observe the data from experiment 2 and compare them with data in experiment 1, the growth of experiment 2 was much low than experiment 1. The concentration of D2O in solvents of experiment 2 was just same as that of experiment 1. The data at 25 % group in experiment 2 showed only a slight difference with control 2 but between 25 % and 50 %, there was significant difference. In 75 % and 100 % of there were little difference between experiment 1 and experiment 2, very low growth in both experiments. In those 75 and 100 %, the mention on additive or synergistic effect could be meaningless because it was difficult to find living cancer cells remained on culture flask in microscope. When we compare figure 1 and figure 2 for cancer cell treatments, single use of D2O or mitomycin C is less effective than compound composed of D2O and anticancer agent mitomycin C. It means D2O and mitomycin C compound syner- gistically enhance the cytocidal effect of the D2O and mitomycin C.

[25] As mentioned before, intravesical instillation of some materials which have the cy- toreductive effects after primary treatment (transurethral resection) of urothelial timor including bladder cancer is common procedure to prevent, suppress or treat urothelial cancers recurrence. Although several decades have passed, except some agents, the true effect of others are suspicious in long term respect.

[26] As seen in figure 1 and 2, D2O and mitomycin C have their own cytoreductive effects but when they are used together as a compound, D2O and mitomycin C compound synergistically enhanced the cytoreductive effect. This result showing that D2O compound could be used as the agent for prevention, suppression and treatment of urothelial cancers or urothelial cancer recurrences.

[27] The components used for D2O compounds are D2O and some materials which have cytotoxic effects but medically permitted to use for man including anticancer agents.

[28] This invention is related with D2O or D2O related compound manufacturing and their use, which did not used until now for the prevention, suppression and treatment of urothelial cancer recurrence including bladder cancer.

Advantageous Effects

[29] The present invention which is involving compositions and methods using deuterium oxide (D2O, heavy water) or compounds containing deuteriim oxide for the prevention or suppression of the recurrence of urothelial cancer, or treatment of the early superficial urothelial cancers in urinary system would lessen the severity of the complications which resulting from the intravesical instillation therapy with con¬ ventional anti-cancer agents. And as we can see at the figure 2, when we combine deuterium oxide and other conventional anticancer or imαiinostimilatns, we can expect the synergistic effect of the anticancer agents. Brief Description of the Drawings

[30] Figure 1 : As mentioned before, T-24 bladder cancercells were cultured at culture dishes and divided into 5 groups. 5 group cellswere seeded at 1 x 105 cells in a plate and cultured at 37°C and 5% CO2incubator for 24 hours with media made of light water as conventional media. Atsubculture 24 hour, culture media in all culture dishes were removed and washedwith phosphate buffered saline (PBS) for two times and each culture dished werereplaced by media made of light water(cont.-l, control) and deuterium oxide andlight water mixture (25 % for D2O 25 % and 75 % light water, 50 % for D2O 50 %and 50 % light water, 75 % for D2O 75 % and 25 % light water and 100 % for 100D2O only). After replacement of media, they are incubated immediately at thesame incubator for tow hours. After two hour has passed, the all culture disheswere removed and all media were sucked out completely and washed with PBS fortwo times gently and media change was performed with media made of light wateronly. After then, every two day, media change was performed with media of lightwater only. At post- subculture 7 day, all cells were washed gently with PBS fortwo times and trysin-DETA solution was added and all survived cell were re- movedfrom the culture dish and counted with hemacytometer.

[31] Control group (cont-1) showed cell count 11.3 x 105cells, D2O 25 % shows 8.8 x

105 cells, 50 % shows 4.9 x 105 cells, 75 % showsO.l x 105 cells and 100 % shows 0.1 x 105 cells.

[32] This figure shows as concentration of D2O increase, cytotoxicity has increased.

[33] Figure 2 : As similar to experiment in figure l,T-24 bladder cancer cells were cultured at culture dishes and divided into όgroups. 6 group cells were seeded at 1 x 105 cells in a plate and cultured at37°C 5% CO2 incubator for 24 hours with media made of light water asconventional media. At subculture 24 hour, culture media in all culture disheswere removed and washed with phosphate buffered saline (PBS) for two times andeach culture dished were replaced by media made of light water(cont.-l forcontrol of light water only without add of mitomycin C, Cont.-2 for control oflight water only media mixed with mitomytcin C) and deuteriim oxide and lightwater mixture (25 % for D2O 25 % and 75 % light water media with mitomycin 20mg/ 40 ml of total media, 50 % for D2O 50 % and 50 % light water media withmitomycin 20mg / 40 ml of total media, 75 % for D2O 75 % and 25 % light watermedia with mitomycin 20mg / 40 ml of total media and 100 % for 100 D2O only media with mitomycin 20mg / 40 ml of total media). After replacement of media,they are incubated immediately at the same incubator for tow hours. After twohour has passed, all culture dishes were removed and all media were sucked outcompletely and washed with PBS for two times gently and media change wasperformed with media made of light water only. After then, every two day, mediachanges were performed with media of light water only. At post- subculture 7day, all cells were washed gently with PBS for two times and trypsin-DETAsolution was added and all survived cell were removed from the culture dish andcounted with hemacytometer.

[34] Control with light water only (Cont.-l) showsl0.8xl05 cells, control with light water only with mitomycin C shows 6.7xlO5cells, 25 % shows 6.1x105 cells and 50 % shows 4.3x105 cells and 75 % shows0.2xl05 cells and 100 % shows 0.1x105 cells. This figure 2 shows that, when D2Ohas combined with anticancer agent mitomycin C, there is appearance of synergistic cytocidal effects. Industrial Applicability

[35] This invention can be used for the bio-pharmaceutical company for the manu¬ facturing new kind of anticancer agentsfor intravesical instillation to prevent, suppress and treat recurrence ofurothelial cancers.

[36]

Claims

Claims[1] What is claimed is:

1. In the invention of the composition and method of usage on deuterium oxide (D20) and deuterium oxide (D20) related compound to prevent or suppress the recurrence of urothelial cancer or treat the early superficial urothelial cancers in urinary system including bladder cancer, allow the deuterium oxide only or deuterhm oxide compound to contact with the urothelial cancer, site that urothelial cancers were located and suspicious area for urothelial cancer for some hours.

2. The deuteriim oxide or deuteriim oxide compounds according to Claim 1, the materials used for the manufacturing deuterium oxide compound are salts (NaCl), distilled light water, saline solution, dextrose solution, some isotonic solution and the other solution that does not damage the normal cells and these deuterium oxide compound are used for prevention or suppression of the recurrence of urothelial cancer or treat the early superficial urothelial cancers in urinary system including bladder cancer.

3. The deuteriim oxide or deuteriim oxide compounds according to Claim 1, the materials used for the manufacturing deuterium oxide compound are oils which are isotonic or does not harmful to human and they derived from plant (or vegetable), animal or inorganic materials which including sesame oil, cottonseed oil, peanut oil, palm oil and coconut oil. When these oils mixed with deuteriim oxide, these compounds are used for prevention or suppression of the recurrence of urothelial cancer or treat the early superficial urothelial cancers in urinary system including bladder cancer.

4. The deuteriim oxide or deuteriim oxide compounds according to Claim 1, the materials used for the dissolving in or mixing with deuteriim oxide for compound manufacturing are anticancer agents or irrmmostimulants which are used in the fields of medicine for treatment of human cancers. When these anticancer agents are dissolved or mixed with deuteriim oxide, these compounds are used for prevention or suppression of the recurrence of urothelial cancer or treat the early superficial urothelial cancers in urinary system including bladder cancer.