WO2014049604A2 - A novel biomedical device for cancer therapy - Google Patents

Description

BIOMEDICAL DEVICE FOR CANCER THERAPY.

FIELD OF THE INVENTION:

The present invention is in the field of medical devices. Particularly, the invention relates to in-dwelling therapeutic device. The therapeutic device is basically a drug eluting device and may be a stent or a catheter. The invention further relates to a therapeutic device having microorganisms harboured therein, useful in cancer therapy. The microorganisms may be attenuated in their virulence factors and with cloned genes encoding specific proteins with anticancer activity. The microorganisms in suspension or biofilm form in the devices may be encased in membranes that allow only diffusion of proteins with molecular masses of 25, 50, 75 or 100 kDa but not live biofilm organisms per se. The device of the present invention is intended to deliver drugs of microbial origin directly to the tumor site without reaching the blood stream.

BACKGROUND OF THE INVENTION:

Live bacteria that have been used for treating cancers, primarily in animal models, are strictly anaerobic such as Clostridia and Bifidobacteria or facultative anaerobic such as Listeria monocytogenes or Salmonella typhimurium (1, 2). Al- R strain of S. typhimurium had significant tumor regressing effect, such as that of orthotopic human pancreatic tumors, in nude mice. In addition, Al-R strain was also shown to significantly reduce the metastasis of lung and high grade osteosarcoma in nude mice as well as spinal cord gliomas (3, 4). Genetically improved avirulent mutants had also been shown to retain the tumor targeting properties (4) and will be excellent candidates for further testing in humans. However, so far, earlier phase I human clinical trials have not shown significant effects in colonization or cancer regression in metastatic melanoma, due to certain toxicity associated with injections of live bacteria in humans (5). Such injections trigger both innate and adaptive immune responses for clearance of the foreign bacteria, thereby enhancing side effects in already debilitated patients. The reasons bacteria such as Salmonella, Listeria, Clostridia and others allow cancer regression are believed to be due to the activation of the immune system elicited by the bacteria as well as active growth of the bacteria in the hypoxic regions of the core of the tumors. Usually, virulence deletion mutants are rapidly cleared from the host compared to the wild type. So lack of toxicity in the blood stream has a negative effect on the retention and proliferation of live bacteria in the host tumors. For example, mutants attenuated in actA and plcB genes in Listeria monocytogenes have been shown to have a reasonable safety profile in adult volunteers (5), although no tumor regression effects were studied.

Phase I human clinical trials of a live, attenuated L. monocytogenes strain in 15 patients with recurrent metastatic squamous cell carcinoma of the cervix at doses of 1X10⁹, 3.3X10⁹ or 1X10¹⁰ cfu showed toxicity with flu-like syndrome such as fever, chills, nausea and/or vomiting/headache (6). Such symptoms are typical of administration of IL-2, indicating the involvement of innate immune response to the i.v. infusions of live bacteria.

The use of Clostridia in cancer therapy has shown efficacy in tumor regression, but with considerable toxicity. Consequently, Clostridial strains have been used more as diagnostic agents for detection of tumors because of their, high specificity for targeting tumor cells (7).

A very promising Clostridial strain, C. novyi-NT (8) was used as spores in phase I human clinical trials in 2006 (NCT 00358397, www.clinicaltrials. gov), but the study was suspended for safety issues, again demonstrating the problem of using live bacteria through the i.v. or i.m. route.

From the above discussion, it is interesting to note that most of the efforts, lasting for more than 60 years, for cancer therapy basically involve anaerobic bacteria. Yet, the most successful practical use of bacteria in cancer therapy involves the use of an aerobe, Mycobacterium bovis BCG, the vaccine strain for tuberculosis, to treat superficial urothelial carcinoma of the bladder. Live BCG cells are administered through a urethral catheter directly into an emptied bladder. The catheter is then removed, leaving the live bacteria in the bladder, which then induce an immune reaction in the bladder, leading to tumoricidal activity (9, 10). However, BCG therapy for bladder cancer is associated with a variety of complications, ranging from minor cystitis to life-threatening BCG sepsis, occurring in up to 90% of patients (11, 12).

As of today, there are no live bacteria, other than M. bovis BCG which has a very limited application only in superficial bladder cancer therapy, that have gone past phase ΙΔΙ human clinical trials successfully and none is on the horizon, given the toxicity associated with intravenous injections of live bacteria in the blood stream evoking strong immune reaction. Attenuated bacteria, with or without cloned genes, get cleared by the immune system quickly, thereby reducing their effectiveness and staying power in the tumor vicinity.

A major problem in the application of live bacteria in cancer therapy (1, 2) is a lack of understanding of how bacteria actually cause tumor regression. Spectacular antineoplastic effect, when C. «ovy -NT was used along with mitomycin C and dolastatin-10, instead of just mitomycin C and dolastatin-10, certainly points out to the active involvement of bacteria in tumor regression and lysis (13). Similarly, the exquisite targeting of tumor and growth in the core of the tumors is known to be a hallmark of Salmonella' ability to cause tumor shrinkage (2, 4, 14). However, such therapies are often known as immunotherapy because the tumor regression is believed to be due to bacteria's preferential growth in the hypoxic core of the tumor, thereby depriving the tumor cells "of nutrients, and eliciting immune action that causes tumor regression.

Do certain bacteria actually fight tumors by elaborating anticancer agents, rather than simply targeting tumors or invoking a strong immune response? indeed, an enzyme arginine deiminase (ADI) produced by Mycoplasma arginini has been shown as early as in 1990 to have anti-tumor activity (15). Since then, many studies including phase I/II human clinical studies, have been performed with ADI and a polyethylene glycol (PEG)-conjugated ADI, termed ADI-PEG20, in patients with hepatocellular carcinoma and malignant melanoma (16). In general, such results have shown modest anticancer effect of ADI-PEG20 in such patients with tolerable side effects and the clinical trials are continuing with additional patient recruitment (16). ADI action is believed to be due to depletion of arginine in such cancer cells as hepatocellular carcinoma, melanoma or renal cell carcinoma which do not express arginino-succinate synthetase in vivo,

The successful use of a bacterial protein, ADI, rather than live bacteria in human clinical trials raises an interesting question: do other bacteria produce similar proteins or small molecule compounds to fight cancer? Arginine deiminase is not unique to Mycoplasma, including M arginini. The ADI from Pseudomonas aeruginosa, an aerobic opportunistic pathogen, has also been shown to have anticancer activity against a range of cancers such as fibrosarcoma, breast and ovarian cancers (17). Most interestingly, a 17 kDa truncated N-terminal part of ADI, called Pa-CARD because it harbors a caspase recruitment domain (CARD), has even higher anticancer activity than ADI in such cancers and in liquid-borne cancers such as chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) cell lines (17; 18).

Pseudomonas aeruginosa, not previously known or tried in any live cell anticancer drug development, not only produces ADI, but another protein with significant anticancer activity called azurin (19). Azurin has not only anticancer activity against a range of cancers, but also strong growth suppressing activity against viruses such as the HIV/AIDS virus HTV-1 or parasites such as the malarial parasite Plasmodium falciparum or the toxoplasmosis parasite Toxoplasma gondii (19). Most interestingly, azurin, which is an intracellular periplasmic protein, is secreted by P. aeruginosa when P. aeruginosa cells are exposed to cancer cells, suggesting that azurin is a weapon that P. aeruginosa uses to keep cancer cells in check. Pseudomonas aeruginosa is a biofilm-forming extracellular pathogen that prefers to establish long term residence in human tissues without causing much harm to the host normal cells but becomes very protective of the host as its habitat and has developed promiscuous protein weapons to target other invaders of the human body such as cancers, viruses and parasites (19). A similar azurin-like protein, termed Laz, is produced by gonococci/meningococci which also retains such activity against cancers, HlV-1, P. falciparum and T. gondii (19). A 28 amino acid peptide derived from azurin, azurin 50-77 termed p28, has no toxicity or immunogenicity in animals, including non-human primates (20). In phase I human clinical trials in Chicago, p28 (NSC 745104, www.clinicaltrials.gov) has shown no side effects in 10 advanced cancer patients where no drug is working and who have an average life expectancy of 8 weeks. p28 has shown significant beneficial effect in such patients in a dose dependent manner without demonstrating any side effects.

The therapeutic efficacy of azurin in mouse xenograft models, and the lack of side effects of the p28 peptide with anticancer activity in both animal and phase I human clinical trials raises an important question: can live cells of P. aeruginosa fight cancer similar to the anaerobic or facultative anaerobic bacteria such as Clostridia, Salmonella, etc, as mentioned earlier? As a known pathogen, P. aeruginosa, even attenuated strains lacking its major virulence factors such as Exotoxin A, elastase, and other toxins such as ExoS, ExoT and ExoU, that are injected in host cells by a type III secretion mechanism, will evoke strong immune response resulting in considerable toxicity when given intravenously. A unique feature of many pathogenic bacteria such as P. aeruginosa, Streptococcus and many others is their ability to form biofilms which allows surface adhesion of such bacteria on biotic or abiotic surfaces and acquisition of enough nutrients to allow a slow mode of growth. Indeed, biofilm formation of P. aeruginosa in both the lungs of cystic fibrosis patients and in-dwelling medical devices is well known for its pathogenesis, allowing the biofilm bacteria to resist both immune attack and antibiotic treatment. It is also conceivable that P. aeruginosa biofilms in an in-dwelling medical device such as a catheter or stent inserted near a tumor can keep the tumor growth in check through elaboration of weapons such as azurin. This approach can contribute to effective cancer therapy, alone or in combination with common anticancer drugs.

Since azurin is a small 14 kDa protein secreted in presence of cancer cells, other pathogenic bacteria with long term residence in human bodies that can secrete small (less than 25 to 30 kDa) protein with anticancer activity may proe to be a potential component for cancer treatment. Having access to such bacterial protein weapons, with inhibitory activity against a range of cancers, viruses, parasites and pathogenic bacteria, perhaps even multiply drug resistant ones, will not only provide us with promiscuous, multi-disease-targeting drugs, but will provide a general principle of method development for isolation of such potential drugs. Indeed, we have recently described the isolation of a 17 kDa protein, MPT63, produced and secreted by Mycobacterium tuberculosis and M. bovis BCG, and a 30 amino acid peptide derived from MPT63, termed MB30, that, similar to azurin- p28, have strong anticancer activity against a range of cancers such as bladder, colon, etc.. Similar to azurin, MPT63 demonstrates promiscuity by strongly inhibiting the growth of the HIV/AIDS virus HIV-1. The production of such a protein weapon by M. bovis BCG, widely used in the treatment of superficial bladder cancer as mentioned earlier (9, 10), clearly suggests that bacterial regression of cancer is not just due to growth inside tumors or induction of an immune response, as is widely believed (24), but is due to active participation by the microorganisms in secreting or exposing on the surface various protein or other weapons to suppress invasion and growth of a variety of invaders of the human body including cancers, viruses, parasites and pathogenic bacteria/fungi. It is thus quite possible that anaerobic/facultative anaerobic bacteria such as Clostridia, Salmonella, etc, mentioned earlier, may produce and secrete similar weapons to fight cancers and other invaders. This kind of an approach will allow them to be used as biofihns on catheters/stents or other in-dwelling devices and inserted in the vicinity of the tumor, singly or in combination with other in- dwelling medical devices harboring other types of bacteria as biofilms. The protein weapons can also be isolated and used as diagnostic agents and/or therapeutic compounds. The in-dwelling devices containing the biofilms of P. aeruginosa, M. tuber culosis/M. bovis, Clostridia, Salmonella, etc, either wild type or attenuated by mutations/deletions in various genes contributing to their virulence, can be used as is, or preferably encased by membrane filters with pore sizes that allow diffusion of only molecules with a mass of 25 kDa or 40 kDa. For example, a catheter with attenuated P. aeruginosa harboring deletions in exotoxin A, elastase (lasA/lasB) and type III secretion systems (but with or without hyperexpression of the azurin gene), and encased by a membrane filter with a cutoff molecular mass of 25 kDa will allow secretion and diffusion of azurin to reach the tumor but not any residual toxins with molecular mass higher than 25 kDa. In none of the cases, the biofilm bacteria can escape from the catheter to reach the blood stream but remains very close to the tumor to sense its presence. Such an approach greatly reduces any toxicity associated with an immune response, as is normally observed with live bacteria given orally, intravenously or by other means. Multiple catheters with multiple different bacterial biofilms will produce a synergistic effect without concomitant side effects, in absence or in presence of treatments with other drugs.

OBJECTIVES OF THE INVENTION:

The main objective of the present invention is to provide a biomedical device for cancer therapy.

Specifically the objective of the invention is to provide an in-dwelling or inserted / catheterized therapeutic device with regulated delivery of drug of microbial origin. Further, neither the microbes nor the drug enter the blood stream. Thus, the invention provides a device with novel drug delivery system. The other objective is to provide a therapeutic device that may be a stent or a catheter.

Yet another objective is to provide a therapeutic device with microorganisms harboured therein in the form of suspension or bio films useful in cancer therapy.

The device may be made of any physiologically acceptable material comprising plastic, ceramic, wood, metal, polymer (natural or synthetic) or hydrogel. Still another objective is to provide a therapeutic device containing live Pseudomonas aeruginosa biofilms. Pseudomonas aeruginosa may be wild type, or virulence compromised or mutated to reduce toxin and/or cloned with the azurin gene under a strong promoter to hyper express the desired protein or derivative thereof..

The mutations may be in genes encoding type III secretion and Exotoxin A and the elastase genes las A/las B that are important virulence factors for P. aeruginosa. For other biofilms comprising of other bacteria such as Salmonella, Clostridia, M. bovis BCG, etc, in the in-dwelling device, appropriate attenuated strains may be used.

Yet another objective is to insert a therapeutic device at the tumor site wherein the biofilm microorganisms be encased in a membrane surrounding the device. The membrane may be provided with pore sizes capable of preventing diffusion of macromolecules with masses greater than 25 KDa to 40 kDa. Such membranes with defined pore sizes will prevent the release of any bacteria from the biofilm to the blood stream. It is to be noted that while biofilm bacteria are resistant to antibiotics or immune attack, in the absence of membrane encasing, any released bacteria from the device will ₎be susceptible to antibiotics or immune attack, facilitating their clearance.

SUMMARY OF THE INVENTION: Accordingly the present invention provides a biomedical device for cancer therapy comprises indwelling medical device encased in a filter with cut off molecular mass of 25 kDa, the said device harbor live microorganisms selected from Pseudomonas aeruginosa, Streptococcus, Staphylococcus, Salmonella, Clostridia Mycobacterium bovis BCG.

In one of the embodiment, the device may be a stent or catheter or any other similar means that can be implanted at tumor site. In other embodiment, the device could be made of any physiologically acceptable biocompatible material that will not hamper the growth of the microorganism and selected from plastic, wood, polymer (natural or synthetic) or hydrogel.

In yet other embodiment, the therapeutic device may harbor suspension or biofilms of microorganism either wild type or attenuated or modified, wherein the organisms preferably be Pseudomonas aeruginosa. Pseudomonas aeruginosa may be virulence compromised or mutated to eliminate toxin production, and cloned with azuringene under a strong promoter to hyperproduce azurin protein. The mutations may be in genes encoding type III secretion and Exotoxin A and the elastase genes las A/las B.

The biofilms may be composed of different sets of bacteria such as Salmonella, Clostridia, M. bovis BCG, etc. Such medical devices can be used in conjunction with other traditional anticancer drug treatment.

DETAILED DESCRIPTION:

There are emerging reports on the use of live and virulence attenuated bacteria for the treatment of cancers. Few bacterial species such as Salmonella, Clostridia, Mycobacterium bovis, when injected intravenously, intramuscularly or other means, have the ability to enter into the human tumors and allow tumor regression. Mycobacterium bovis BCG is used in the treatment of superficial bladder cancer and is thought to induce cancer regression through activation of the immune system.

However, there is no report on the use of live wild type or attenuated Pseudomonas aeruginosa for the treatment of cancers.

In prior art, the organisms being administered parenterally via injections or orally pose problem in reaching and targeting the poorly vascularized tumors, which have no or low level oxygen. Additionally, eradication of the disseminated/migrated organisms to other parts of the body is another problem. Further, these pathogenic organisms, even when attenuated, evoke strong immune response, leading to toxicity problems.

The biomedical device of the present invention having confinement of microorganisms encased in the membrane of the device eliminates the problem of dissemination in other parts of the body or blood stream and toxicity development. Further the right choice of microorganism, attenuated Pseudomonas aeruginosa with mutations/deletions in various toxin genes helps in solving certain problems of accumulation of toxins.

The novel device of the present invention is a novel drug delivery system (NDDS) that provides the benefits of protein / peptide therapy via a combination medical device. The device is a drug eluting oncology stent a first-ever combination medical device designed to deliver in situ microbial therapy directly to the tumor site without reaching the blood stream. This revolutionary approach to cancer therapy will provide an important alternative or adjunct treatment to surgery, radiation or chemotherapy, particularly for inoperable or hard to reach cancer tumors.

The device preferably encases a biofilm in a membrane filter, enabling secretion and diffusion of bacterial proteins/peptides to reach the tumor and blocking release of any residual toxins with molecular mass higher than 25 kDa. The concentration of anticancer molecule released from bacteria to the tumor site allow tumor regression effectively, while the device will prevent the bacterium as well as the toxic molecules having size more than 25 kDa to be released thereby minimizing risk of potential side effects of using live bacterium in cancer therapy. A range of cancers may be treatable by this method, either as an alternative or adjunct to surgery, radiation or chemotherapy.

The device was tried in preclinical trials wherein the experiments were conducted on nude mice. The two experimental nude mice (Figures 1 A, B) were subcutaneously injected with one million cells of human breast tumor (cancer cell line MCF-7) to instate infection. After 5 days, when both the mice showed small visible tumors, a device of the present invention (drug eluting stent) harboured with Pseudomonas aeruginosa was inserted in the experimental mouse, while the other (control) mouse was devoid of such stent. The sizes of tumors in both mice were measured twice a week by an electronic caliper over a period of five weeks. Such measurements demonstrated that the size of the tumor in the experimental mouse (with catheter) was about half the size of the tumor in the control mouse up to about three and a half weeks. Most surprisingly, the tumor in the experimental mouse went necrotic and burst after about three and a half weeks (Figure IB, experimental mouse) while the tumor in the control (without any catheter) mouse kept growing (Figure 1A, control mouse). At about 5 weeks, while the tumor in the control mouse kept growing and the mouse was extremely lethargic and sick, the necrotic tumor and the wound surrounding it of the experimental mouse gradually healed (Figure 1C, experimental mouse). This mouse exhibited good mobility and activity like any mouse without tumor.

The device was also tested on human subjects with high grade prostate cancer. Insertion of a catheter containing pseudomonas proved to be promising i regression of tumor The applicant had recently described the production of anti-cancer proteins, or peptides derived from microorganisms,, such as azurin, arginine deiminase (ADI), Pa-CA D and azurin-p28 that allow tumor regression both in vitro and in vivo in mice (1, 17, 18, 19). Although, as described earlier, live bacteria such as Clostridia, Salmonella, etc., have been shown to allow tumor regression, there is no report on the use of live or attenuated Pseudomonas aeruginosa for the treatment of cancers. Pseudomonas aeruginosa is known to form biofilms on the abiotic or biotic surfaces and can grow under microaerophilic conditions. The use of a stent, catheter or other similar therapeutic device that contains biofilm bacteria of the wild type or virulence compromised P. aeruginosa strains may provide potential and viable treatment of cancers in humans. The stent, catheter, hydrogel or other similar therapeutic device containing P. aeruginosa is administered in the human body at the site of the tumors or near to the tumors where the anticancer proteins or small molecules secreted by the bacterium allow regression of solid tumors. The use of wild type or virulence compromised P. aeruginosa is an excellent way to treat a variety of human cancers. For example, a patient with prostate cancer may have a catheter containing P. aeruginosa biofilms in the uro vesicular region near the tumor. While the P. aeruginosa persists in the catheter, the patient's condition will be relatively stable with the tumor either stabilized or undergoing shrinkage. Additional therapy with anticancer drugs will help in the complete elimination of the tumor.

Further, it is clear that such a stent, catheter, or similar therapeutic device with P. ' aeruginosa also may be encased in membranes with pore sizes that allow diffusion of only 25 Kda to 40 kDa macromolecules. Because the P. aeruginosa toxins such as Exotoxin A or ExoS/T are larger than 40 Kda, the live cells of P. aeruginosa or their released toxins cannot come out of the catheter to enter the tumors or surrounding tissues, leaving the release of low molecular weight anticancer proteins such as azurin to fight cancer. Here, we focus on the use of plastic, ceramic, wooden or metal stent, catheter, or similar therapeutic device containing a biofilm of the live bacterium Pseudomonas aeruginosa that is either wild type or genetically modified to eliminate its pathogenicity and to be inserted at the site of the tumors to allow tumor regression in cancer patients. The strain of the P. aeruginosa in the biofilm could be laboratory or clinical isolates of wild-type bacteria or laboratory-derived mutants of such bacteria defective in the production of toxins such as Exotoxin A, Elastase or toxins elaborated through the type III secretion system (ExoS, ExoT, ExoU, etc). P. aeruginosa may have cloned azurin gene under a strong promoter to hyperexpress azurin gene with its signal sequence to allow secretion of the azurin.

Other biofilm-forming bacteria such as Streptococcus, Staphylococcus, Mycobacterium bovis BCG, etc, also may be used in place of P. aeruginosa. The cancers to be treated by the live cells of P. aeruginosa or other biofilm- forming bacteria as part of catheters or stent surfaces are prostate, melanoma, sarcoma, breast, lung, ovarian, kidney, cervical, liver, bladder, colon, pancreas or other tumor types. The use of catheter/stent or similar therapeutic device containing P. aeruginosa or other bacterial biofilms inserted at the site of the tumor(s) is a treatment modality that can be given singly or in conjunction with other treatments such as radiation, chemotherapy or the emerging treatments with bacterial proteins or peptides given via intravenous/intra muscular routes.

References

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Claims

WE CLAIM:

1. A biomedical device for cancer therapy comprises indwelling medical device encased in a filter with cut off molecular mass of 25 kDa, the said device harbor live microorganisms selected from Pseudomonas aeruginosa, Streptococcus, Staphylococcus, Salmonella, Clostridia, Mycobacterium bovis

BCG.

2. The device as claimed in claim 1 wherein the microorganisms are either in the form of biofilm or suspension.

3. The device as claimed in claim 1 wherein the indwelling medical device is a stent or a catheter or any other similar means that can be implanted at tumor site.

4. The device as claimed in claim 3 wherein the indwelling medical device is made of any physiologically acceptable biocompatible material selected from plastic, ceramic, wood, metal, polymer (natural or synthetic) or hydrogel.

5. The device as claimed in claim 1 wherein the live microorganisms harbored are either wild type or attenuated or modified

6. The device as claimed in preceding claims wherein the organism is Pseudomonas aeruginosa.

7. The device as claimed in claim 6 wherein Pseudomonas aeruginosa is virulence compromised, mutated to eliminate toxin production, or cloned with azurin gene under a strong promoter to hyperproduce azurin protein.

8 The device as claimed in claim 7 wherein the mutations are in genes encoding type III secretion and Exotoxin A and the elastase genes las

A/las B. The device as claimed in claim 1 for use to treating cancers of prostate, melanoma, sarcoma, breast, lung, ovarian, kidney, cervical, liver, bladder, colon, pancreas or other tumor types and HIV/AIDS without entering in blood stream. The device as claimed in claim 9 for use in conjunction with other traditional anticancer drug to treating cancers of prostate, melanoma, sarcoma, breast, lung, ovarian, kidney, cervical; liver, bladder, colon, pancreas or other tumor types and HTV/AIDS.