WO2015011732A2 - Plant poly-phenol and copper (ii) mediated degradation of dna and rna - Google Patents

Abstract

A composition comprising a plant polyphenol, especially Resveratrol, and copper (II) for the degradation of or preventing or reducing a rise in the level of one or more of circulating fragments of chromatin, DNA and RNA. The molar ratio of plant polyphenol to copper (II) is in the range 1 :1 to 10^-10 Higher levels of degradation of chromatin fragments, DNA and RNA occur as the molar ratio of the copper to plant polyphenol to copper (II) is reduced to an optimum level depending on the material to be degraded. The composition provides therapeutic treatment for conditions associated with raised levels of chromatin fragments including cancer and HIV, may enhance cell destruction therapies and, reduce the incidence of death of live cells in the presence of dead cells or fragments of chromatin from dead cells.

Description

PLANT POLY-PHENOL AND COPPER (II) MEDIATED DEGRADATION OF DNA AND RNA

Field of invention:

The present invention provides a method for degradation of DNA and RNA by Resveratrol- Copper (II) (R-Cu) in vitro and in vivo. Further, the invention provides a method for prevention and /or treatment of various pathological conditions associated with elevated blood levels of extra-cellular DNA and chromatin as well as viral RNA.

Background:

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a phytoalexin present in red wine, grapes, berries, peanuts etc. Resveratrol exists in both cis and trans isomeric forms and, in plants, is mostly present in glycosylated forms (3-O-B-D-glucosides) along with minor conjugated forms containing 1-2 methyl groups (pterostiibene), a sulfate group (trans-resveratrol-3-sulfate) or a fatty acid.

Copper is an essential micronutrient; and because of its role as a metal co-factor it is believed to play a central role in the formation of reactive oxygen species (ROS), such as 0₂ ^~ and -OH radicals [Li et al., 1995]. Resveratrol, in solution, has been shown to exhibit pro-oxidant activity in presence of copper so as to cause oxidative DNA damage. Fukuhara and Miyata were the first to report a pro-oxidant activity of resveratrol (3,5,4-trihydroxy-trans-stilbene) in a plasmid- based DNA cleavage assay in the presence of copper. Resveratrol forms a complex with Cu(ll), leading to its reduction to Cu(l) with concomitant formation of reactive oxygen species. Furthermore, the reaction presumably leads to the formation of Oxidized product(s)' of resveratrol, which also appear to catalyze the reduction of Cu(ll).

Studies by Fukuhara showed that resveratrol is capable of binding to DNA possibly through electrostatic and hydrogen bonding forces, and that the Cu-dependent DNA damage is more likely caused by a copper-peroxide complex rather than by a freely diffusible oxygen species [Fukuhara K., Miyata N.; Bioorganic and Medicinal Chemistry Letters; 1998; 8 (22), 3187]. Binding of copper with resveratrol in solution has been speculated in a quantum-chemical computational study and has been demonstrated in solution by UV-visible spectra, positive ion mode ESI-MS spectrum, and HPLC performed on 'Ultrasphere-ODS' columns [Mikulski D., Molski M.; Journal of Molecular Structure: THEOCHEM; 2010; 956 (1-3); 66; Zheng L. F., Wei Q. Y., Cai Y. J., Fang J. G., Zhou B., Yang L., Liu Z. L.; Free Radical Biology and Medicine; 2006; 41 (12); 1807; Ahmad A, Asad F. S., Singh S„ Hadi S.M.; Cancer Letters; 2000; 154 (1); 29; Tamboli V., Defant A, Mancini I., Tosi P.; Rapid Communications in Mass Spectrometry; 2011 ; 25 (4); 526; Belguendouz L, Fremont L, Linard A; Biochemical Pharmacology; 1997; 53; 1347].

Strand scission by the R-Cu system was also found to be active as assayed by bacteriophage inactivation [Ahmad A, Asad F. S., Singh S., Hadi S.M.; Cancer Letters; 2000; 154 (1); 29]. Using the Comet assay for DNA damage, R-Cu system was demonstrated to be capable of causing DNA degradation in cells such as lymphocytes, using a cellular system of lymphocytes isolated from human peripheral blood [Azmi A.S., Bhat S.H., Hadi S.M.; FEBS Letters; 2005; 579 (14); 3131 ]. The DNA degrading effects could be abolished in the presence of ROS scavengers. Resveratrol and its synthetic analogues have been shown to have pro-oxidant effects on pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions [Zheng L. F., Wei Q. Y., Cai Y. J., Fang J. G., Zhou B., Yang L, Liu Z. L; Free Radical Biology and Medicine; 2006; 41 (12); 180].

Oral administration of copper to rats resulted in elevated copper levels in lymphocytes. When such lymphocytes were isolated and treated with Resveratrol in vitro, increased level of DNA fragmentation was detected using the comet assay. Pre-incubation of copper-loaded lymphocytes with neocuproine, a copper chelator, resulted in inhibition of Resveratrol induced DNA fragmentation [Khan HY, Zubair H, Ullah MF, Ahmad A, and Hadi SM (201 1 ). Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism. Biometals. 24: 1 169- 1 178]. However, copper overload (30mg / kg b.w.) over a period can cause severe toxicity, especially neuronal and hepatic damage, and copper overloading cannot be used for therapeutic purposes [Brewer GJ (2010). Copper toxicity in the general population. Clin Neurophysiol. 121 : 459-460].

Copper binds to both native and synthetic DNA molecules with high affinity [Coates JH, Jordan DO, Srivastava VK (1965). The binding of copper (II) ions to DNA. Biochem Biophys Res Commun. 20: 61 1-615; Eichhorn GL, Clark P (1965). Interactions of metal ions with polynucleotides and related compounds, v. the unwinding and rewinding of DNA strands under the influence of copper (ii) ions. Proc Natl Acad Sci U S A, 53: 586-593]. This binding is dependent upon the size, charge electron affinity and geometry of the DNA [Coates JH, Jordan DO, Srivastava VK (1965). The binding of copper(ll) ions to DNA. Biochem Biophys Res Commun. 20: 61 1 -615; Eichhorn GL, Clark P (1965). Interactions of metal ions with polynucleotides and related compounds, v. the unwinding and rewinding of dna strands under the influence of copper (ii) ions. Proc Natl Acad Sci U S A. 53: 586-593]. It has been demonstrated that copper ions primarily bind to the N7 and O at C6 atom of guanine and the N3 and O of C-2 atom in cytosine, with no direct interaction with the A-T pairs of DNA [Zimmer C, Luck G, Fritzsche H, Triebel H (1971). DNA-copper (II) complex and the DNA conformation. Biopolymers. 10: 441-463]. Complex formation between copper with G-C bases is accompanied by a conformational change of the DNA double-helical structure as demonstrated by EPR, UV- VIS, CD and fluorescence spectroscopy studies [4, 5]. The binding efficiency increases dramatically when Cu(ll) is reduced to Cu(l) in the presence of reducing agents, such as Resveratrol and other plant polyphenols as well as Dopamine and Ascorbate. The conformational change from square planar coordination with guanine to tetrahedral geometry is attributed to the increase in the binding capacity of copper to DNA [4,5].

Mittra et al., showed that when circulating nucleosomes in the form of chromatin fragments (Cfs) isolated from plasma/serum of normal subjects and patients suffering from different malignancies are added to a variety of cells in culture, they freely enter the recipient cells without assistance and induce a DNA damage response (DDR) which results in incorporation of exogenous chromatin into the host cell genomes [I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases as described in WO2007/049286]. A DDR induced by Cfs is observed in all cell types tested, including those of mesenchymal, epithelial, neuronal, endothelial, myocardial, hepatic and adipose tissue origin, as well as in isolated lymphocytes, suggesting that Cfs may be universal DNA damaging agents. Cells exposed to Cfs showed evidence of chromosomal instability, senescence, apoptosis and oncogenic transformation. When chromatin treated cells were injected into immuno-deficient mice, tumor development was observed in a proportion of the injected animals [I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases as described in WO2007/049286].

Conventional therapies for treatment of cancer typically involve radiotherapy and/or chemotherapy, for example with adriamycin, with the intention of destroying cancer cells which leads to cleavage and fragmentation of chromatin and raised levels of circulating Cfs. Ready uptake of Cfs in recipient cells and concomitant DNA damage response may be implicated in metastasis. Whilst chemotherapies and radiotherapies may be necessary to destroy primary tumours, such therapies themselves may increase the risk of metastatic disease or other disorders related to elevated Cfs.

Lipopolysaccharide (LPS), a component of bacterial cell wall, has been used to induce sepsis in experimental animal models, especially in mice, to induce a condition that mimics sepsis in humans. LPS is known to induce cell death and, therefore, leads to cleavage and fragmentation of chromatin and raised levels of circulating Cfs.

The above findings of Mittra et. al. suggests that reduction in circulating Cfs by an appropriate methodology would be a desirable therapeutic approach in treating cancer and in treating other conditions associated with elevated levels of Cfs such as cancer, systemic autoimmune disorders, diabetes, Parkinson's disease, Alzheimer's disease, cerebral stroke, myocardial infarction, inflammation, sepsis, critical illness, trauma, renal failure, HIV/ AIDS, as well as ageing and age-related disorders [Rykova EY, Laktionov PP and Vlassov W 2010 Circulating nucleic acids in health and disease; in Extracellular nucleic acids: Nucleic acids and molecular biology (eds) Y Kikuchi and E Rykova (Berlin Heidelberg: Springer-Verlag) 25 93-128; Mittra I, Nair NK, Mishra PK.2012. Nucleic acids in circulation: are they harmful to the host? J Biosci. 37:301-312].

In summary, the prior-art shows the following features:

• Resveratrol reduces Cu (II) to Cu (I) to generate free radicals.

· Resveratrol in the presence of Cu (II) is capable of degrading DNA in vitro via the medium of free radicals.

• Lymphocytes isolated from rats fed with copper show evidence of DNA fragmentation when treated with Resveratrol in vitro.

• Copper binds to DNA with high affinity changing the conformation of DNA.

· The DNA binding efficiency increases when Cu (II) is reduced to Cu (I) in the presence of Resveratrol.

• Circulating chromatin fragments (Cfs) can induce DNA damage in vitro leading to genomic instability, senescence, apoptosis and oncogenic transformation in the recipient cells.

The term "degradation" as employed herein includes cleavage and/or fragmentation of DNA RNA. For example plasmid DNA has a round configuration and upon cleavage opens into a linear conformation. Further cleavage results in shorter lengths of DNA fragments or oligomers being formed. When subjected to electrophoresis, a cleaved plasmid DNA molecule will generally tend to have lower mobility than the uncleaved plasmid whereas fragments, being smaller tend to have greater mobility than the plasmid, thereby providing a means of determining whether cleavage, fragmentation, both or neither has occurred.

Summary of the invention:

An object of the invention is to provide a material which is able to degrade chromatin and to provide a means for degrading Cfs selectively with potential therapeutic benefits. Another object of the invention is to provide a method for degradation of DNA and RNA by R- Cu, especially wherein the amount of Cu is relatively low compared to that of R, and its use in providing a method for the prevention and / or treatment of various pathological conditions associated with elevated blood levels of DNA, Cfs and viral RNA.

Another object is to provide a means of increasing Cfs, DNA or RNA degradation with a low level of Cu (II) with respect to Resveratrol.

Yet another object of the invention is to degrade plasmid DNA by R-Cu in vitro, under varying pH and in different solvents.

Yet another object of the invention is to investigate the plasmid DNA-degrading activity of plant poly-phenols including Resveratrol in combination with Cu in vitro.

Yet another object of the invention is to investigate the plasmid DNA-degrading activity of R in combination with heavy metals other than Cu (II) in vitro. ^

Yet another object of the invention is to investigate whether R-Cu, especially in varying ratios of R to Cu is capable of degrading eukaryotic genomic DNA in vitro.

Yet another object of the invention whether R-Cu, especially in varying ratios of R to Cu is capable of degrading eukaryotic apoptotic DNA in vitro.

Yet another object of the invention is to investigate the DNA-degrading effects of R-Cu in vivo. Yet another object of the invention is to investigate whether R-Cu, especially at varying ratios of R to Cu, can be used to provide a method for reducing the level of, or rate of increase of, or preventing the increase of Cfs levels, neutropenia and increased inflammatory cytokines following a tissue-damaging therapy such as chemotherapy, for example after treatment with Adriamycin or radiotherapy as a therapeutic strategy for cancer.

Yet another object of the invention is to investigate whether R-Cu, especially at varying ratios of R to Cu, can be used to provide a method for reducing the level of, or rate of increase of, or preventing the increase of Cfs levels, and for the prevention of lipopolysaccharide (LPS)- induced inflammatory cytokines and lethality as a therapeutic strategy.

Yet another object of the invention is to investigate whether, R-Cu, especially at varying ratios of R to Cu, can be used to provide a method for the prevention of lung metastasis in a mouse melanoma model.

Yet another object of the invention is to investigate whether, R-Cu especially at varying ratios of R to Cu, can be used to provide a method for enhancing anti-tumour activity of Adriamycin in a mouse xenograft model.

Yet another object of the invention is to explore RNA degrading activity of R-Cu in vitro. Yet another object of the invention is to employ the RNA degrading activity of R-Cu to reduce levels of HIV RNA-associated reverse transcriptase and virus caspid protein p24 in vitro.

We have now found that a composition containing a plant polyphenol, for example Resveratrol, and copper (II) is surprisingly beneficial in degrading the DNA component of chromatin, for example extra-cellular Cfs, and provides potential therapeutic benefits in treating conditions associated with elevated Cfs levels. Further the composition provides beneficial improvements in certain known tissue-damaging therapies. We have also found that these effects whilst present at certain ratios of the plant polyphenol to copper, the effects are maintained at reduced ratios of copper to the plant polyphenol, for example of Resveratrol. Degradation of DNA component of Cfs is sufficient to preclude uptake in a healthy cell and provides a basis for therapeutic treatment of conditions associated with uptake of Cfs.

We have now found that a composition containing a plant polyphenol, for example Resveratrol, and copper (II) is surprisingly beneficial in degrading RNA, and provides potential therapeutic benefits in treating conditions associated with RNA viruses such as HIV. We have also found that these effects whilst present at certain ratios of the plant polyphenol to copper, the effects are maintained at reduced ratios of copper to the plant polyphenol, for example of Resveratrol. Degradation of RNA component of HIV is sufficient to preclude further infection of cell by the virus and provides a basis for therapeutic treatment of HIV/AIDS.

In a first aspect, the invention provides a composition comprising a plant polyphenol and copper (II) for use in a method of treatment to degrade one or more of fragments of chromatin / DNA and RNA.

Examples of plant polyphenols include flavanols, anthocyanins, chalcones, flavandiols, flavanonols, flavanones, flavonols, flavones, isoflavones, hydroxytyrosol. Suitably, the plant polyphenol is selected from beta-carotene, catechin, curcumin epicatechin, proanthocyanosides, pelargonidin, cyanidin, delphinidin, resveratrol, dihydroquercetin, dihydrokaempferol, myricetin, armadendrin, morin, hesperetin, naringenin, quercetin, kaempferol, apigenin, luteolin, genistein, daidzein, glycitein, hydroxytyrosol or mixtures thereof. Resveratrol, beta-carotene and curcumin are preferred, especially resveratrol. Resveratrol is trans-3,5,4'-trihydroxystibene.

Suitably, the DNA to be degraded is selected from plasmid DNA, eukaryotic genomic DNA and eukaryotic apoptotic DNA. Suitable RNA to be degraded includes eukaryotic RNA or HIV RNA- associated reverse transcriptase.

Composition according to the invention is suitable for use in treating conditions associated with an elevated level of circulating chromatin fragments and viral RNA. Examples of these conditions include cancer, systemic autoimmune disorders, diabetes, Parkinson's disease, Alzheimer's disease, cerebral stroke, myocardial infarction, inflammation, sepsis, critical illness, trauma, renal failure, HIV/AIDS, ageing and age-related disorders.

The invention is especially beneficial in treating or preventing lung metastasis and HIV/AIDS. In a second aspect, the invention provides a composition comprising a. plant polyphenol and copper (II) for use in preventing a rise in or reducing the circulating level of chromatin fragments and inflammatory cytokines or in preventing or reducing the level of neutropenia in a subject after a therapy for cell-destruction selected from chemotherapy and radiotherapy.

Suitable cell destruction therapies may include any known chemotherapy for example administration of Adriamycin or bacterial cell wall component such as lipopolysaccharide.

In a further aspect, the invention provide a composition comprising a plant polyphenol and copper (II) wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^{" 9}. This composition is suitable for use in a method of treatment of the human or animal body by therapy. The extremely low concentration of copper obviates the toxic side-effects of high levels of copper.

Preferably, the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"4. Suitably, the concentration of the plant polyphenol is from 10 microMolar to lOOmilliMolar.

Compositions of the invention may be employed in a method of treatment to degrade plasmid DNA and the concentration of the plant polyphenol is suitably from 50 to 200 microMolar and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"2 preferably in the range 1 : 10^"1 to 10^"2.

Where the concentration of the plant polyphenol is from greater than 200 microMolar to 2mM, the molar ratio of the plant polyphenol to copper (II) is preferably in the range 1 :1 to 10^"3.

The concentration of the plant polyphenol may be from greater than 2mM to 10 mM and the molar ratio of the plant polyphenol to copper (II) is preferably in the range 1 :1 to 10^"4 and more preferably in the range 1 : 10^"2 to 10^"4.

Although Resveratrol is especially preferred, curcumin and other plant phenols may be employed. Suitably curcumin is employed at a molar ratio of curcumin to copper (II) in the range 1 :1 to 10^"3.

In a preferred embodiment, a composition according to the invention may be used to degrade eukaryotic genomic DNA or eukaryotic apoptotic DNA wherein the concentration of the plant polyphenol is from greater than 2mMolar to 10mM and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"3, preferably from 1 : 10^"2 to 10^"3. It has been surprisingly foung that the benefits of the composition of the invention are greater as the ratio of copper to plant polyphenol decreases. This is beneficial as copper is toxic. At higher concentration of the plant polyphenol/copper composition, beneficial effects may be observed at lower ratios of copper to the plant polyphenol. In a preferred embodiment the molar ratio of the plant polyphenol to copper (II) is in the range 1 : 10^~3 to 10^'10, preferably from 1 : 10^"3 to 10^~8 and especially from 1 : 10 ³ to 10^"7. These ranges may be employed generally but are preferred when the concentration is at least 5 micromolar and preferably at least 1 millmolar. The composition of the invention may be used to reduce or prevent apoptosis of living cells upon or after contact with dead cells. Living cells undergo apoptosis because dead cells coming in contact with them release fragmented apoptotic chromatin which enter into the living cells and induce DNA damage leading to cell death (apoptosis) of the living cells.

In another embodiment, the composition is administered to a mixture of living and dead cells and the concentration of the plant polyphenol is from greater than I microMolar to 1 mM, the molar ratio of the plant polyphenol to copper (II) is in the range 1 :10^~1 to 10^"8 preferably 1 : 10^"1 to 10^"8 and the number of viable living cells is at least 50% greater than the number of viable living cells without administration of the composition.

The composition may be administered by any suitable method. The concentration of the composition as referred to herein is the concentration of the composition as administered which provides the desired cellular concentration level. Preferably, the composition is administered interperitoneally to a subject, preferably at a dose level of 10 to 100mg polyphenol per kg weight of the subject, or can be administered orally. Suitably, an oral dose is around 100 times greater than an ip dose due to the lower uptake.

Preferably, the composition comprises a solvent. Examples of preferred solvents include aqueous acetonitrile, aqueous ethanol, weak alkaline solution and water.

The composition suitably has a pH of 3 to 1 1.

Compositions of the invention are useful in preventing lung metastasis from intravenously injected cancer cells. The composition is suitably administered concurrently with the injected cancer cells to a subject and the molar ratio of the plant polyphenol to copper (II) is suitably in the range 1 :1 to 10^"6. Compositions according to the invention are suitable for use in a method of enhancing the anti-tumour effect of a cell destruction treatment. The cell destruction treatment may be selected from known cell destruction treatments, preferably chemotherapy treatment for cancer and/or a radiotherapy treatment for cancer. The composition is suitably administered to a subject concurrently with or sequentially, before or after, with a cell destruction treatment and/or a radiotherapy treatment. The composition may be employed with any known chemotherapy agents invluding such agents selected from alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors and mitotic inhibitors or any combination thereof.

Examples of suitable alkylating agents include Cyclophosphamide, Ifosfamide, Melphalan, Thiotepa, Cisplatin, Carboplatin, and Oxalaplatin and combinations thereof; Examples of suitable antimetabolites include 5-fluorouracil (5-FU), Gemcitabine (Gemzar®) and Methotrexate and combinations thereof. Examples of suitable anti-tumor antibiotic include Anthracyclines: Doxorubicin (Adriamycin®), Epirubicin, Actinomycin-D and Mitomycin-C and combinations thereof. Examples of suitable topoisomerase inhibitors include Etoposide (VP-16) and Mitoxantrone and combinations thereof. Examples of mitotic inhibitors include Taxanes: Paclitaxel (Taxol®) and Docetaxel (Taxotere®) Vinca alkaloids: Vinblastine (Velban®) and Vincristine (Oncovin®) and combinations thereof.

Compositions of the invention are useful in enhancing the anti-tumour effect of a chemotherapy agent. The composition is suitably administered with the chemotherapy agent concurrently or sequentially to a subject and the molar ratio of the plant polyphenol to copper (II) is suitably in the range 1 :1.

In another aspect, the invention provides a method of treatment of the human or animal body by therapy comprising administering to the subject a composition comprising a plant polyphenol and copper (II) wherein the molar ratio of the plant polyphenol to copper (II) is in the. range 1 :1 to 10^'9.

The invention provides a method for plasmid DNA degradation by R-Cu in vitro under varying pH and varying ratios of R: Cu in different solvents.

In vitro experiments were also conducted to investigate the effect of varying ratios of R to Cu in degrading eukaryotic genomic and eukaryotic apoptotic DNA.

In vivo experiments were conducted to investigate the effect of DNA-degradation by R-Cu, in varying ratios of R to Cu, on Adriamycin-induced rise in levels of Cfs and to explore a potential therapeutic strategy for the prevention of Adriamycin-induced neutropenia and inflammatory cytokines in mice. Investigations were also done to explore whether R-Cu, in varying ratios of R to Cu, will reduce inflammatory cytokines and prevent lethality in a mouse model of sepsis. Further experiments were conducted to investigate whether administration of R-Cu, at varying ratios of R to Cu, could be used for the prevention of lung metastasis in a mouse melanoma model. Further experiments were conducted to investigate whether administration of R-Cu, at varying ratios of R to Cu, could be used to enhance chemo-sensitivity of Adriamycin in a mouse xenograft model. The invention provides a method for eukaryotic RNA degradation by R-Cu, in varying ratios of R to Cu, in vitro in order to explore a potential therapeutic strategy in reducing levels of HIV RNA- associated reverse transcriptase and virus caspid protein p24.

Estimation of degradation of plasmid DNA.

Plasmid pTZ57R and/or pTRIPZ (as specified) were treated with R:Cu²⁺ at varying ratios of R to Cu²⁺ and under different conditions (Figures 1-13). The band-intensity of the intact plasmid was quantified using ImageJ software. The background (blank) values were subtracted, and the corrected intensity in each case, relative to the band-intensity of the untreated plasmid DNA, was represented as a percentage of the intact plasmid with an error of ± 10%.

Estimation of degradation of eukaryotic genomic DNA.

Eukaryotic genomic DNA was treated with R:Cu²⁺ at varying ratios of R to Cu²⁺ (Figure 14). The band-intensity of the intact genomic DNA was quantified using ImageJ software. The background (blank) values were subtracted, and the corrected intensity in each case, relative to the band-intensity of the untreated genomic DNA, was represented as a percentage of the intact genomic DNA with an error of ± 10%.

Estimation of degradation of eukaryotic apoptotic DNA.

Eukaryotic apoptotic DNA was treated with R:Cu²⁺ at varying ratios of R to Cu²⁺ (Figure 15). The band-intensity of the high molecular weight (HMW) fragment was quantified using ImageJ software. The background (blank) values were subtracted, and the corrected intensity in each case, relative to the band-intensity of the HMW fragment of untreated apoptotic DNA, was represented as a percentage of the HMW fragment with an error of ± 10%.

Estimation of degradation of Eukaryotic RNA.

Eukaryotic RNA was treated with R:Cu²⁺ at varying ratios of R to Cu²⁺ (Figure26). The band- intensity of the two RNA bands were quantified using ImageJ software. The background (blank) values were subtracted, and the corrected intensity in each case, relative to the band-intensity of the two RNA bands, was represented as a percentage of the two RNA bands with an error of ± 10%.

In the accompanying figures, the band of the untreated DNA or RNA appeared at an intermediate distance as indicated on the figures. Where the DNA was a plasmid DNA, the band travelled a shorter distance due to cleavage of the round plasmid to form a slower-moving open conformation. Where further degradation occurred and the DNA was fragmented into smaller components, these moved further and appear to the other side of the untreated material or as a streak on the plots.

The invention is illustrated with the following non-limiting examples. Example 1 : Degradation of plasmid DNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

(Figure 1A and 1 B): illustrate degradation of plasmid DNA pTZ57R (figure 1A) and pTRIPZ (figure 1 B) by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol wherein the starting molar ratio of R : Cu was R(1 ) : Cu(1 ) [R (100μΜ) -Cu (100μΜ )].

Plasmid DNA isolation was done as per manufacturer's instructions (Hi-Media). The harvested transformed bacterial culture (E. Coli DH₅a containing either plasmid pTZ57R or plasmid pTRIPZ) was lysed and centrifuged. The obtained pellet was applied onto a silica column and plasmid DNA were allowed to bind in presence of high salt concentration. The adsorbed plasmid DNA was washed to remove contaminants, and eluted in DNA binding buffer. 500ng of either plasmid pTZ57R or plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios of R to Cu, dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with O^g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact plasmid in each lane is presented in Table 1A and 1 B.

Table ,1A (plasmid pTZ57R):

)

Table 1 B (plasmid pTRIPZ): R:Cu molar Intact plasmid ratio (%)

Lane 1 : 1 kb marker

Lane 2: Plasmid DNA (500ng) 100

Lane 3: Plasmid DNA (500ng) + R (100μΜ) 100

Lane 4: Plasmid DNA (500ng) + Cu (100μΜ) 100

Lane 5: Plasmid DNA (500ng) + R (100μΜ) -Cu (100μΜ ) 1 1 0

Lane 6: Plasmid DNA (500ng) + R (100μΜ) -Cu (20μΜ) 1 0.2 0

Lane 7: Plasmid DNA (500ng) + R (100μΜ) -Cu (10μΜ ) 1 0.1 80

Lane 8: Plasmid DNA (500ng) + R (100μΜ) -Cu (2μΜ ) 1 0.02 100

Lane 9: Plasmid DNA (500ng) + R (100μΜ) -Cu (1 μΜ ) 1 0.01 100

Lane 1( . Plasmid DNA (500ng) + R (100μΜ) -Cu (0.2μΜ ) 1 0.002 100

Lane 1 ' Plasmid DNA (500ng) + R (100μΜ) -Cu (0.1 μΜ ) 1 0.001 100

Lane 1. ; Plasmid DNA (500ng) + R (100μΜ) -Cu (0.02μΜ ) 1 0.0002 100

(Figure 2A and 2B): illustrate degradation of plasmid DNA pTZ57R (figure 2A) and pTRIPZ (figure 2B) by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R : Cu was R(1) : Cud): R (500uM) : Cu (500uM)

500ng of either plasmid pTZ57R or plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1% agarose gel, stained with O^g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact plasmid in each lane is presented in Table 2A and 2B.

Table 2A (plasmid pTZ57R):

Lane 5: Plasmid DNA (500ng) + R (500μΜ) -Cu (500μΜ ) 1 :1 80

Lane 6: Plasmid DNA (500ng) + R (500μΜ) -Cu (100μΜ) 1 :0.2 0

Lane 7: Plasmid DNA (500ng) + R (500μΜ) -Cu (50μΜ ) 1 :0.1 0

Lane 8: Plasmid DNA (500ng) + R (500μΜ) -Cu (10μΜ ) 1 :0.02 0

Lane 9: Plasmid DNA (500ng) + R (500μΜ) -Cu (5μΜ ) 1 :0.01 0

Lane 10: Plasmid DNA (500ng) + R (500μΜ) -Cu (1μΜ ) 1 :0.002 60

Table 2B (plasmid pTRIPZ):

R:Cu Intact plasmid molar ratio (%)

Lane 1 1 kb marker

Lane 2 Plasmid DNA (500ng) 100

Lane 3 Plasmid DNA (500ng) + R (500μΜ) 100

Lane 4 Plasmid DNA (500ng) + Cu (500μΜ) 100

Lane 5 Plasmid DNA (500ng) + R (500μΜ) -Cu (500μΜ ) 1 1 0

Lane 6 Plasmid DNA (500ng) + R (500μΜ) -Cu (100μΜ) 1 0.2 0

Lane 7 Plasmid DNA (500ng) + R (500μΜ) -Cu (50μΜ ) 1. 0.1 0

Lane 8 Plasmid DNA (500ng) + R (500μΜ) -Cu (10μΜ ) 1 : 0.02 40

Lane 9 Plasmid DNA (500ng) + R (500μΜ) -Cu (5μΜ ) 1 : 0.01 100

Lane 10: Plasmid DNA (500ng) + R (500μΜ) -Cu (1 μΜ ) 1 : 0.002 100

Lane 1 1 : Plasmid DNA (500ng) + R (500μΜ) -Cu (0.5μΜ ) 1 : 0.001 100

Lane 12: Plasmid DNA (500ng) + R (500μΜ) -Cu (0.1 μΜ ) 1 : 0.0002 100

(Figure 3A and 3B): illustrate degradation of plasmid DNA pTRIPZ by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R : Cu was R(1) : CudV. R ( mM) : Cu ( mM)

500ng of plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1% agarose gel, stained with O^g/ml ethidium bromide and visualized using gel- documentation system.

Since the untreated plasmid (lane 2, Figure 3A) showed evidence of degradation, the results of this experiment were ignored and the experiment was repeated twice thereafter. Since these experiments gave similar results, only one of them is represented in Figure 3B. The percentage of intact plasmid in each lane is presented in Table 3B.

Table 3B (plasmid pTRIPZ):

(Figure 4): illustrates degradation of plasmid DNA pTRIPZ by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R : Cu was R(1) : Cud ): R (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1hr, run on a 1 % agarose gel, stained with O^g/ml ethidium bromide and visualized using gel- documentation system.

The percentage of intact plasmid in each lane is presented in Table 4.

Table 4 (plasmid pTRIPZ):

Lane 4: Plasmid DNA (500ng) + Cu (5 mM) 100

Lane 5: Plasmid DNA (500ng) + R (5m ) -Cu (5 mM) 1 :1 0

Lane 6: Plasmid DNA (500ng) + R (5mM) -Cu (1 mM) 1 :0.2 0

Lane 7: Plasmid DNA (500ng) + R (5mM) -Cu (0.5 mM) 1 :0.1 0

Lane 8: Plasmid DNA (500ng) + R (5mM) -Cu (0.1 mM) 1 :0.02 0

Lane 9: Plasmid DNA (500ng) + R (5mM) -Cu (0.05 mM) 1 :0.01 0

Lane 10: Plasmid DNA (500ng) + R (5mM) -Cu (0.01 mM) 1 :0.002 30

Lane 11 : Plasmid DNA (500ng) + R (5mM) -Cu (0.005 mM) 1 :0.001 60

Lane 12: Plasmid DNA (500ng) + R (5mM) -Cu (0.001 mM) 1 :0.0002 80

Conclusion from Example 1 (Figures 1-4): Increasing the ratio of R to Cu (i.e., by reducing Cu with respect to R) enhances degradation of plasmid DNA in 50% ethanol and this effect is dependent on the starting concentrations of R and Cu used.

Example 2: Degradation of plasmid DNA by R-Cu in the presence of varying molar ratios of R to Cu in different solvents.

(Figure 5): illustrate degradation of plasmid DNA pTZ57R by R-Cu in different solvents

Molar ratio of R : Cu was R(1) : Cu(1)R (5mM) : Cu (5mM)

500ng of plasmid pTZ57R was suspended in TE buffer, and R-Cu dissolved in 20 μΙ of following solvents: 50% ethanol; 50% acetonitrile; 3mM NaOH or distilled water was added. The mixtures were incubated at 37°C for 1 hr, run on a 1% agarose gel, stained with O^g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact plasmid in each lane is presented in Table 5. Table 5 (plasmid pTZ57R):

(Figure 6): illustrates degradation of plasmid DNA pTRIPZ by R-Cu in the presence of varying molar ratios of R to Cu in 50% acetonitrile.

Starting molar ratio of R : Cu was R(1) : Cud): R (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of 50% acetonitrile was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with 0.5 g/ml ethidium bromide and visualized using gel- documentation system.

The percentage of intact plasmid in each lane is presented in Table 6.

Table 6 (plasmid pTRIPZ):

(Figure 7A and 7B): illustrates degradation of plasmid DNA pTRIPZ by R-Cu in the presence of varying molar ratios of R to Cu in 3mM NaOH.

Starting molar ratio of R : Cu was R(1) : Cud): R (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of 3mM NaOH (aqueous) was added. The mixtures were incubated at 37°C for 1hr, run on a 1 % agarose gel, stained with 0.5Mg/ml ethidium bromide and visualized using gel-documentation system.

Since the untreated plasmid (lane 2, Figure 7A) showed evidence of degradation, the results of this experiment were ignored and the experiment was repeated twice thereafter. Since these experiments gave similar results, only one of them is represented in Figure 7B.

The percentage of intact plasmid in each lane is presented in Table 7B.

Table 7B:

(Figure 8A, and 8B): illustrates degradation of plasmid DNA pTRIPZ by R-Cu in the presence of varying molar ratios of R to Cu in water.

Starting molar ratio of R : Cu was R(1) : Cud ): R (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of water was added. The mixtures were incubated at 37°C for 1hr, run on a 1 % agarose gel, stained with 0.5 g/ml ethidium bromide and visualized using gel- documentation system.

Since there were some technical problems during the running of the gel (Figure 8A), this experiment was ignored and repeated twice thereafter. Since these two experiments gave similar results, only one of them is represented in Figure 8B. The percentage of intact plasmid in each lane is presented in Table 8B. Table 8 B:

We next examined the ability of R:Cu in degrading plasmid DNA pTRIPZ in 50% ethanol. This experiment has already been reported earlier. For gel picture, kindly refer to Figure 4 (page 4 of 31 ) and for tabulated results refer to Table 4 (page 13).

Conclusion from Example 2 (Figures 5-8): Increasing the ratio of R to Cu (i.e., by reducing Cu with respect to R) enhances degradation of plasmid DNA irrespective of the solvent used.

Example 3 (Figure 9A and 9B): illustrate degradation of plasmid DNA by R-Cu under different pH conditions.

Molar ratio of R : Cu was R(1) : Cud): R (5mM) : Cu (5mM)

500ng of either plasmid pTZ57R or plasmid pTRIPZ was suspended in TE buffer, and R-Cu dissolved in 20 μΙ of distilled water adjusted at the following pH with HCI or NaOH was added: 3.0; 5.0; 7.5; 9.0 and 11.0. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with O^g/ml ethidium bromide and visualized using gel-documentation system. The percentage of intact plasmid in each lane is presented in Table 9A and 9B.

Table 9A (plasmid pTZ57R): Intact plasmid

(%)

Lane 1 1 kb marker

Lane 2 Plasmid DNA (500ng) 00

Lane 3 Plasmid DNA (500ng) + R (5mM) -Cu (5mM) pH - 3 20

Lane 4 Plasmid DNA (500ng) + R (5mM) -Cu (5mM) pH - 5 10

pH -

Lane 5: Plasmid DNA (500ng) + R (5mM) -Cu (5mM) 0

7.5

Lane 6: Plasmid DNA (500ng) + R (5mM) -Cu (5mM) pH - 9 0

Lane 7: Plasmid DNA (500ng) + R (5mM) -Cu (5mM) pH - 11 0

Table 9B (plasmid pTRIPZ):

Conclusion from Example 3 (Figure 9): Degradation of plasmid DNA by R-Cu (molar ratio 1 :1) occurred under all pH conditions tested.

Example 4: Degradation of plasmid DNA by other plant poly-phenols (PPP) and Cu in the presence of varying molar ratios of PPP to Cu in 50% ethanol.

(Figure 10): illustrates degradation of plasmid DNA pTRIPZ by Curcumin-Cu in the presence of varying molar ratios of Curcumin to Cu in 50% ethanol.

Starting molar ratio of Curcumin:Cu was Curcumind) : Cud): Curcumin (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer and Curcumin-Cu, in varying ratios of Curcumin to Cu, was dissolved in 20 μΙ of 50% ethanol Was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with 0.5Mg/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact plasmid in each lane is presented in Table 10.

Table 10 (plasmid pTRIPZ):

(Figure 1 1): illustrates degradation of plasmid DNA pTRIPZ by β-Carotene -Cu in the presence of varying molar ratios of β-Carotene Carotene to Cu in 50% ethanol.

Starting molar ratio of B-Carotene:Cu was β-Carotened ) : Cud):

β-Carotene (5mM) : Cu (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer and β-Carotene-Cu, in varying ratios of β-Carotene to Cu, was dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with 0.5 g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact plasmid in each lane is presented in Table 1. Table 11 (plasmid pTRIPZ):

Conclusion from Example 4 (Figures 10. 11 ): Plant polyphenols Curcumin and β-Carotene in presence of Cu were also effective in degrading plasmid DNA.

Example 5: Absence of degradation of plasmid DNA by R and other heavy metals (HM) in the presence of varying molar ratios of R to HM in 50% ethanol.

(Figure 12): illustrates absence of degradation of plasmid DNA pTRIPZ by R-Zn in the presence of varying molar ratios of R to Zn in 50% ethanol.

Starting molar ratio of R:Zn was R(1) : Zn(1 ); R (5mM) : Zn (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer and R-Zn, in varying ratios of R to Zn, was dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1% agarose gel, stained with 0.5pg/ml ethidium bromide and visualized using gel- documentation system.

The percentage of intact plasmid in each lane is presented in Table 12.

Table 12 (plasmid pTRIPZ): Intact

R:Cu

plasmid molar ratio

(%)

Lane 1 : 1 kb marker

Lane 2: Plasmid DNA (500ng) 100

Lane 3: Plasmid DNA (500ng) + R (5mM) 100

Lane 4: Plasmid DNA (500ng) + Zn (5 mM) 100

Lane 5: Plasmid DNA (500ng) + R (5mM) - Zn (5 mM) 1 1 100

Lane 6: Plasmid DNA (500ng) + R (5mM) - Zn (1 mM) 1 0.2 100

Lane 7: Plasmid DNA (500ng) + R (5mM) - Zn (0.5 mM) 1 0.1 100

Lane 8: Plasmid DNA (500ng) + R (5mM) - Zn (0.1 mM) 1 0.02 100

Lane 9: Plasmid DNA (500rig) + R (5mM) - Zn (0.05 mM) 1 0.01 100

Lane 10 Plasmid DNA (500ng) + R (5mM) - Zn (0.01 mM) 1 0.002 100

Lane 1 1 Plasmid DNA (500ng) + R (5mM) - Zn (0.005 mM) 1 0.001 100

Lane 12 Plasmid DNA (500ng) + R (5mM) - Zn (0.001 mM) 1 0.0002 100

(Figure 13): illustrates absence of degradation of plasmid DNA pTRIPZ by R-Li in the presence of varying molar ratios of R to Li in 50% ethanol.

Starting molar ratio of R:Li was R(1) : Lid): R (5mM) : Li (5mM)

500ng of plasmid pTRIPZ was suspended in TE buffer and R-Li, in varying ratios of R to Li, was dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with 0.5 g/ml ethidium bromide and visualized using gel- documentation system.

The percentage of intact plasmid in each lane is presented in Table 13.

Table 13 (plasmid pTRIPZ):

Lane 4: Plasmid DNA (500ng) + Li (5 mM) 100

Lane 5: Plasmid DNA (500ng) + R (5mM) - Li (5 mM) 1 1 100

Lane 6: Plasmid DNA (500ng) + R (5mM) - Li (1 mM) 1 0.2 100

Lane 7: Plasmid DNA (500ng) + R (5mM) - Li (0.5 mM) 1 0.1 100

Lane 8: Plasmid DNA (500ng) + R (5mM) - Li (0.1 mM) 1 0.02 100

Lane 9: Plasmid DNA (500ng) + R (5mM) - Li (0.05 mM) 1 0.01 100

Lane 10 Plasmid DNA (500ng) + R (5mM) - Li (0.01 mM) 1 0.002 100

Lane 11 Plasmid DNA (500ng) + R (5mM) - Li (0.005 mM) 1 0.001 100

Lane 12 Plasmid DNA (500ng) + R (5mM) - Li (0.001 mM) 1 0.0002 100

Conclusion from Example 5 (Figures 12, 13): Heavy metals such as Zn and Li in presence of Resveratrol were ineffective in degrading plasmid DNA.

Example 6: Degradation of other forms of DNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

(Figure 14): illustrates degradation of eukaryotic genomic DNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R:Cu was R(1) : Cud ): R (5mM) : Cu (5mM)

Genomic DNA was isolated from Jurkat (human lymphocytic leukemia) cells using the Wizard® Genomic DNA purification kit (Promega). 2 x 10⁶ Jurkat cells were harvested, washed twice with PBS and treated with nuclei lysis solution. The cellular proteins were separated from high molecular weight genomic DNA by salt-precipitation. Finally, genomic DNA was concentrated and desalted by isopropanol treatment. 500 ng of genomic DNA was suspended in TE buffer, and R-Cu, in varying ratios of R to Cu, was dissolved in 20 μΙ of 50% ethanol was added. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with 0.5 g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of intact genomic DNA in each lane is presented in Table 14.

Table 14 (eukaryotic genomic DNA):

Lane 2: Genomic DNA (500ng) 100

Lane 3: Genomic DNA (500ng) + R (5mM) 100

Lane 4: Genomic DNA (500ng) + Cu (5 mM) 100

Lane 5: Genomic DNA (500ng) + R (5mM) -Cu (5 mM) 1 :1 100

Lane 6: Genomic DNA (500ng) + R (5mM) -Cu (1 mM) 1 :0.2 0

Lane 7: Genomic DNA (500ng) + R (5mM) -Cu (0.5 mM) 1 :0.1 0

Lane 8: - Genomic DNA (500ng) + R (5mM) -Cu (0.1 mM) 1 :0.02 0

Lane 9: Genomic DNA (500ng) + R (5mM) -Cu (0.05 mM) 1 :0.01 0

Lane 1 ( ): Genomic DNA (500ng) + R (5mM) -Cu (0.01 mM) 1 :0.002 50

Lane 1 ' : Genomic DNA (500ng) + R (5mM) -Cu (0.005 mM) 1 :0.001 100

Lane 1. >; Genomic DNA (500ng) + R (5mM) -Cu (0.001 mM) 1 :0.0002 100

(Figure 15): illustrates degradation of eukarvotic apoptotic DNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R:Cu was R(1) : Cud ): R (5mM) : Cu (5mM)

Apoptotic DNA was isolated using the Apoptotic DNA Ladder Kit (Roche). 2 x 10⁶ Jurkat cells were treated with adriamycin (5pg/ml) for 48 hr. Apoptotic cells were harvested, washed x 5 with PBS and treated with binding/lysis reagent. The lysate was applied to a filter tube with glass fiber fleece and centrifuged. DNA was allowed to bind specifically to the surface glass fibers in the presence of chatropic salts. Residual impurities were removed by wash buffer and subsequently eluted in elution buffer. 500ng of apoptotic DNA was suspended in TE buffer; R- Cu, in varying ratios of R to Cu, dissolved in 20 μΙ of 50% ethanol was added to the DNA. The mixtures were incubated at 37°C for 1 hr, run on a 1 % agarose gel, stained with O^g/ml ethidium bromide and visualized using gel-documentation system.

The percentage of HMW band of apoptotic DNA in each lane is presented in Table 15.

Table 15 (eukaryotic apoptotic DNA):

Lane 3: Apoptotic DNA (500ng) + R (5mM) 100

Lane 4: Apoptotic DNA (500ng) + Cu (5 mM) 100

Lane 5: Apoptotic DNA (500ng) + R (5mM) -Cu (5 mM) 1 1 100

Lane 6: Apoptotic DNA (500ng) + R (5mM) -Cu (1 mM) 1 0.2 0

Lane 7: Apoptotic DNA (500ng) + R (5mM) -Cu (0.5 mM) 1 0.1 0

Lane 8: Apoptotic DNA (500ng) + R (5mM) -Cu (0.1 mM) 1 0.02 0

Lane 9: Apoptotic DNA (500ng) + R (5mM) -Cu (0.05 mM) 1 0.01 0

Lane 1( ): Apoptotic DNA (500ng) + R (5mM) -Cu (0.01 mM) 1 0.002 100

Lane 1 ' : Apoptotic DNA (500ng) + R (5mM) -Cu (0.005 mM) 1 0.001 100

Lane 1. )· Apoptotic DNA (500ng) + R (5mM) -Cu (0.001 mM) 1 0.0002 100

Conclusion from Example 6 (Figures 14, 15): Increasing the ratio of R to Cu (i.e., by reducing Cu with respect to R) enhances degradation of eukaryotic genomic and apoptotic DNA in 50% ethanol.

Example 7 (Figure 16): illustrates prevention of apoptosis of living cells when dead cells are added to them in culture in the presence of R-Cu at varying ratios of R to Cu. Starting molar ratio of R:Cu was R(1 ) : Cud ): R (5uM) : Cu (5uM)

10 X 10⁶ MDA-MB-231 cells were induced to undergo apoptosis by treatment with Adriamycin (5 g/ml) for 48 hr. The dead cells were washed x 5 with PBS, and the apoptotic MDA-MB-231 cells (10 X 10⁵) were added in a ratio of 1 :1 to living MDA-MB-231 cells (10 X 10⁵) grown in DMEM medium in 3 cm petri dishes in duplicates. Immediately prior to the addition of apoptotic MDA-MB-231 cells, R-Cu in varying ratios of R to Cu was added to the culture dish. The various ratios of R to Cu were: 1 :1 ; 1 :10^"1; 1 :10^"2; 1 :10^"3; 1 :10^"4; 1 :10^"5; 1 :10^"6; 1 :10^'7; 1 :10^"8 and 1 :10^"9. After 96 hr, viable MDA-MB-231 cells were counted following Trypan-blue exclusion.

Conclusion from Example 7 (Figure 16): Mittra et. al. have shown that when dead cells are added to living cells, fragmented chromatin that emanate from dead cells enter freely into the recipient living cells and induce DNA damage, genomic instability, senescence and apoptosis of the living cells. The number of living cells is thus, depleted [I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases. US Patent Application No. FPAA819PCT dated 27.10.2006].

The current experiment shows that R-Cu can effectively degrade the chromatin fragments that emanate from dead cells thereby preventing apoptosis of living cells and preventing depletion in the number of living cells. This chromatin degrading activity of R-Cu increases as the molar ratio of R to Cu increases (i.e., when Cu is reduced with respect to R).

Example 8: Reduction of rise in Cfs and inflammatory cytokines following Adriamycin treatment and prevention of neutropenia by R-Cu in varying ratios of R to Cu.

Reduction of rise in Cfs following Adriamycin treatment by R-Cu in varying ratios of R to Cu:

(Figure 17A): illustrates Cfs profile following Adriamycin treatment over time

Six-week old C57BL/6 mice weighing ~ 20g were injected with Adriamycin (5mg/kg) intraperitoneal (i.p.). Three animals were sacrificed at different time-points and blood was collected through orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for Cfs estimation. The latter was performed using Cell Death Detection ELISA^plus kit (Roche Applied Sciences, Mannheim, Germany). The results were expressed in arbitrary units (Mean±S.E.). Number of animals = 42, 3 at each time-point. The results indicate that the peak of increased chromatin fragments occurred at around 36 hours. Studies were carried out administering Resveratol-Cu at 36 hours after Adriamycin treatment.

(Figure 17B): illustrates reduction of elevated levels of Cfs in mice following Adriamycin treatment by R-Cu at varying molar ratios of R to Cu at 36 hr.

Starting molar ratio of R:Cu was R(1) : Cud ): R (1mM) : Cu (1mM)

Six-week old C57BL/6 mice weighing ~ 20g in the experimental groups were given a single intra-peritoneal (i.p.) injection of Adriamycin (25mg/kg); while the control group was injected with saline i.p. One experimental group was given Resveratrol (0.67mg /kg = 1mM R per mouse) i.p., in addition 4-hours prior to Adriamycin treatment, and every 12-hour thereafter; another group was given Cu (0.717 mg/kg = 1mM Cu per mouse) in addition 4-hours prior to Adriamycin treatment, and every 12-hour thereafter. The other experimental groups were given R-Cu at varying ratios of R to Cu starting with a ratio of 1mM:1mM and in successively increasing ratios of R to Cu (i.e., successively decreasing the concentration of Cu with respect to R) up to a ratio of 1mM : 10^'9 mM. The R-Cu injections were given 4-hours prior to Adriamycin injection and every 12-hour thereafter. The animals were sacrificed at 36 hours after Adriamycin injection and blood was collected by orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for Cfs estimation. The latter was performed using Cell Death Detection ELISA^P|US kit (Roche Applied Sciences, Mannheim, Germany). The results were expressed in arbitrary units (Mean±S.E.). Number of animals = 73, 5 in each group except Adriamycin group which had 8 animals. Reduction of rise in inflammatory cytokine IL6 following Adriamycin treatment by R-Cu in varying ratios of R to Cu:

(Figure 18AV. illustrates profile of inflammatory cytokine IL6 following Adriamycin treatment over time.

Six-week old C57BL/6 mice weighing ~ 20g were injected with Adriamycin (5mg/kg, i.p.). Three animals were sacrificed at different time-points and blood was collected through orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for estimation of inflammatory cytokines by flow cytometry. The results were expressed as pg/ml (Mean±S.E.). Number of animals = 28, 2 at each time-point. The results indicate that the peak of increased IL6 occurred at around 36 hours. Studies were carried out administering Resveratol-Cu at 36 hours after Adriamycin treatment.

(Figure 18B): illustrates reduction of rise in inflammatory cytokine IL6 following Adriamycin treatment by R-Cu, at varying ratios of R to Cu, at 36 hr.

Starting molar ratio of R:Cu was R(1) : Cud): R (1 mM) : Cu (1mM)

Six-week old C57BL/6 mice weighing ~ 20g in the experimental groups were given a single intra-peritoneal (i.p.) injection of Adriamycin (25mg/kg); while the control group was injected with saline i.p. One experimental group was given Resveratrol (0.67mg /kg = 1mM R per mouse) i.p., in addition 4-hours prior to Adriamycin treatment, and every 12-hour thereafter; another group was given Cu (0.717 mg/kg = 1 mM Cu per mouse) in addition 4-hours prior to Adriamycin treatment, and every 12-hour thereafter. The other experimental groups were given R-Cu at varying ratios of R to Cu starting with a ratio of 1mM:1mM and in successively increasing ratios of R to Cu (i.e., successively decreasing the concentration of Cu with respect to R) up to a ratio of 1 mM : 10^"9 mM. The R-Cu injections were given 4-hours prior to Adriamycin injection and every 12-hour thereafter. The animals were sacrificed at 36 hours after Adriamycin injection and blood was collected by orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for IL6 estimation by flow cytometry and results were expressed as pg/ml. Number of animals = 42, 3 in each group.

Prevention of neutropenia by R-Cu in varying ratios of R to Cu following Adriamycin treatment:

(Figure 19A): illustrates profile of total leukocyte count (TLC) following Adriamycin treatment over time.

Six-week old C57BL/6 mice weighing ~ 20g were injected with single injection Adriamycin (5mg/kg) intra-peritoneal (i.p.). Three animals were sacrificed at different time-points and blood was collected through orbital puncture in 6% EDTA. Total leukocyte count was estimated by the standard method using the Neubauer chamber. The results were expressed as leukocyte count per cubic mm (Mean±S.E.). Number of animals in each group = 42, 3 at each time-point.

(Figure 19B): illustrates prevention of neutropenia following Adriamycin treatment and by R-Cu in varying ratios of R to Cu at 36 hr. .

Starting molar ratio of R:Cu was R(1) : Cud ); R (1mM) : Cu (1 mM)

The mice were treated as described in Figure 18B. Blood by orbital puncture was collected in 6% EDTA and total leukocyte counted by the standard method using the Neubauer chamber. The results were expressed as leukocyte count per cubic mm (Mean±S.E.). Number of animals = 75, 5 in each group except Adriamycin group which had 10 animals.

Conclusions from Example 8:

(Figure 17A, 17B): Adriamycin induced peak of circulating Cfs at 36 hr is reduced by R-Cu, and this activity is maintained even when R to Cu ratios are as low as 1 : 1 x 10^"7 mM. The 36-hr time-point was chosen since Cfs level peaks at 36 hr following Adriamycin treatment (Figure 18A).

(Figure 18A, 18B): Adriamycin induced peak of inflammatory cytokine IL6 at 36 hr is reduced by R-Cu, and this activity is maintained even when R to Cu ratios are as low as 1 : 1 x 10^"9 mM. The 36-hr time-point was chosen since IL6 level peaks at 36 hr following Adriamycin treatment (Figure 19A).

(Figure 19A, 19B): The dynamics of TLC following Adriamycin administration is bi-phasic, in that there is dramatic decline in the first 24 hr followed by a trough lasting for several days with recovery in TLC thereafter (Figure 20A). The 36-hr time-point was chosen for further study since TLC showed a marked decline at this time-point. Adriamycin-induced neutropenia at 36 hr is prevented by R-Cu, and this activity is maintained even when R to Cu ratios are as low as 1 : 1 x 10^"9 mM. - Example 9: Reduction of rise in Cfs and inflammatory cytokines following LPS treatment and prevention of lethality by R-Cu in varying ratios of R to Cu.

Reduction of rise in Cfs following LPS treatment by R-Cu in varying ratios of R to Cu:

(Figure 20A): illustrates Cfs profile following LPS treatment over time

Six-week old C57BL/6 mice weighing ~ 20g were injected with LPS (20mg/kg) intra-peritoneal (i.p.). Three animals were sacrificed at different time-points and blood was collected through orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for Cfs estimation. The latter was performed using Cell Death Detection ELISA^P|US kit (Roche Applied Sciences, Mannheim, Germany). The results were expressed in arbitrary units (Mean±S.E.). Number of animals in each group = 30, 5 at each time-point. (Figure 20B): illustrates reduction in elevated levels of Cfs following LPS treatment by R-Cu in varying molar ratios of R to Cu at 18 hr.

Starting molar ratio of R:Cu was R(1) : Cud): R (1 mM) : Cu (1mM)

Six-week old C57BL/6 mice weighing ~ 20g in the experimental groups were given a single intra-peritoneal (i.p.) injection of LPS (20mg/kg); while the control group was injected with saline i.p. One experimental group was given Resveratrol (0.67mg /kg = 1mM R per mouse) i.p., in addition 4-hours prior to LPS treatment, and every 12-hour thereafter; another group was given Cu (0.717 mg/kg = 1 mM Cu per mouse) in addition 4-hours prior to LPS treatment, and every 12-hour thereafter. The other experimental groups were given R-Cu at varying ratios of R to Cu starting with a ratio of 1 mM:1 mM and in successively increasing ratios of R to Cu (i.e., successively decreasing the concentration of Cu with respect to R) up to a ratio of 1mM : 10^"9 mM. The R-Cu injections were given 4-hours prior to LPS injection and every 12-hour thereafter. The animals were sacrificed at 18 hours after Adriamycin injection and blood was collected by orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for Cfs estimation. Cfs levels were measured using Cell Death Detection ELISA^plus kit (Roche Applied Sciences, Mannheim, Germany). The results were expressed in arbitrary units (Mean±S.E.). Number of animals = 54, 6 in each group. ^J

Reduction of rise in inflammatory cytokine IL6 following LPS treatment by R-Cu in varying ratios of R to Cu

(Figure 21A): illustrates profile of inflammatory cytokine IL6 following LPS treatment over time. Six-week old C57BL/6 mice weighing ~ 20g were injecte d with LPS (20mg/kg, i.p.). Three animals were sacrificed at different time-points and blood was collected through orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for estimation of inflammatory cytokines by flow cytometry. The results were expressed as pg/ml (Mean±S.E.). Number of animals in each group = 27, 3 at each time-point.

(Figure 21 B): illustrates reduction of rise in inflammatory cytokine IL6 following LPS treatment by R-Cu in varying ratios of R to Cu at 18 hr.

Starting molar ratio of R:Cu was R(1) : Cud): R (1 mM) : Cu (1mM)

The mice were treated as described in Figure 21 B. The animals were sacrificed at 18 hours after LPS injection and blood was collected through orbital puncture. Blood was allowed to clot at 4°C overnight and serum was separated for estimation of inflammatory cytokine IL6 by flow cytometry. The results were expressed in pg/ml. Number of animals = 45, 3 in each group except LPS group which had 6 animals.

Prevention of lethality following LPS treatment by R-Cu in varying ratios of R to Cu: (Figure 22): illustrates prevention of lethality by R-Cu, in varying molar ratios of R to Cu, in LPS treated mice.

Starting molar ratio of R:Cu was R(1 ) : Cud ): R (1 mM) : Cu (1 mM)

Six-week old C57BL/6 mice weighing ~ 20g were given a single lethal injection of LPS (20mg/kg) i.p. They were divided into 4 groups. Group 1 received injection saline i.p. twice daily starting 4 hr before LPS treatment; Group 2 received injection Resveratrol (0.67mg /kg = 1 mM R per mouse) i.p. twice daily starting 4 hr before LPS treatment; Group 3 received injection R- Cu (molar ratio 1 mM:1 mM) i.p. twice daily starting 4 hr before LPS treatment; Group 4 received injection R-Cu (molar ratio 1 mM:1x10^"4 mM) i.p. twice daily starting 4 hr before LPS treatment. The animals were monitored for lethality. Number of animals = 64, 16 in each group.

Conclusions from Example 9:

(Figure 20A, 20B): LPS-induced peak of circulating Cfs at 18 hr is reduced by R-Cu, and this activity is maintained even when R to Cu ratios are as low as 1 : 1 x 10^"4 mM. The 18-hr time- point was chosen since Cfs level peaks at 18 hr following LPS treatment (Figure 21 A).

(Figure 21 A, 21 B): The dynamics of IL6 levels following LPS administration is bi-phasic displaying two peaks one at 6 hr and the other at 18 hr (Figure 22A). LPS induced peak of inflammatory cytokine IL6 at 18 hr is reduced by R-Cu, and this activity is maintained even when R to Cu ratios are as low as 1 : 1 x 10^"2 mM.

(Figure 22): R-Cu reduced lethality in LPS treated mice. However, R-Cu at molar ratio 1 mM: mM reduced lethality even further, and this effect was retained even when R to Cu molar ratio was increased to 1 mM: 1 x 10^"4mM (i.e., concentration of Cu was decreased with respect to R).

Example 10 (Figure 23): illustrates anti-tumour effect of Adriamycin is enhanced by R-Cu

(molar ratio of R:Cu was R(1 mM) : Cu( 1 mM)).

A: Immune deficient mice (20 in number) were injected sub-cutaneously with MDA-MB-231 cells (5 x 10⁵). Once the tumours reached the size of ~ 500 mm³, they were measured in two dimensions every three days and animals were sacrificed on day 14.

B: Immune deficient mice (20 in number) were injected sub-cutaneously with MDA-MB-231 cells (5 x 10⁵). When tumours were ~ 500 mm³ in size, they were divided into 2 groups of 10 mice each. One group was given Adriamycin (0.65mg/kg) once daily i.p., while the other group was given Adriamycin (0.65mg/kg) once daily i.p. + R-Cu in a molar ratio of 1 mM: 1 mM twice daily i.p. Tumour size were measured in two dimensions every two days and animals were sacrificed on day 16.

Conclusion from Example 10 (Figure 23A and 23B): Adriamycin treatment reduced the rate of tumour growth via its anti-tumour effect (Figure 23A vs 23B). The anti-tumour effect of Adriamycin is significantly enhanced by concurrent administration of R-Cu (molar ratio 1 mM:1 mM) such that the growth rate of tumours is reduced.

Example 11 (Figure 24): illustrates prevention of oncogenic transformation of mouse fibroblast cells by R-Cu, at varying ratios of R to Cu, when dead cancerous cells are added to them in culture.

Starting molar ratio of R:Cu was R(1) : Cud ): R (5uM) : Cu (5uM)

10 X 10⁶ Jurkat (human lymphoblastic lymphoma) cells were induced to undergo apoptosis by treatment with Adriamycin (5^g/m\) for 48 hr. The dead cells were washed x 5 with PBS, and the apoptotic Jurkat cells (10 X 10⁵) were added to living NIH3T3 (mouse fibroblast) cells (10 X 10⁵) (ratio of 1 :1 ) that were grown in DMEM medium in 3 cm petri dishes. Immediately prior to the addition of apoptotic Jurkat cells, R-Cu in varying ratios of R to Cu was added to the culture dish. The various ratios of R to Cu were: 1 :1 ; 1 : 10^"1; 1 :10^"2; 1 :10^"3; 1 :10^"4; 1 :10^"5; 1 :10^"6; 1 :10^"7; 1 : 10 ^s, 1 : 10^"9 and 1 : 10^"10. After 96 hr, the recipient NIH3T3 cells were examined under a phase contrast microscope for oncogenic transformation of NIH3T3 cells and as to whether the latter is prevented by R-Cu in varying ratios of R to Cu.

Conclusion from Example 11 (Figure 24): Mittra et al. have shown that when dead cells are added to living cells, fragmented chromatin that emerge from dead cells enter freely into the recipient living cells and induce DNA damage, genomic instability and oncogenic transformation of the living cells [I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases as described in WO2007/049286 and US Patent Application No. FPAA819PCT dated 27.10.2006].

The current experiment shows that when dead Jurkat cells are added to NIH3T3 cells, the latter undergo oncogenic transformation as described by Mittra et al., above. Treatment with Resveratrol (5μΜ) alone prevented oncogenic transformation to some extent. Treatment with Cu (II) (5μΜ) alone, cause the recipient cells to show evidence of cell-death and the latter increased further in the presence of a combination of Resveratrol and Cu (II) (molar ratio 1 :1 ; 5μΜ: 5μΜ), and the phenomenon of cell death continued up to a R-Cu molar ratio of 1 :10^"3. Beyond this ratio, prevention of oncogenic transformation became increasingly evident, and this was entirely prevented at a molar ratio of R-Cu 1 :10^"7. At this latter R-Cu ratio the dead Jurkat treated NIH3T3 cells became indistinguishable from untreated NIH3T3 cells. The copper did not show any toxic effect and the level of breakdown of DNA reached a peak at this level. Beyond the R- Cu ratio of 1 :10^"7 evidence of oncogenesis began to appear again, and at a R-Cu ratio of 1 :10^"10 there was no oncogenic prevention and the treated cells appeared identical to NIH3T3 cells treated with dead Jurkat cells. A R-Cu ratio of 1 :1 to 1 : greater than 10^"10, preferably to 1 :10^"8, especially to 1 :10^"7 reduces or prevents oncogenic transformation and at a ratio of 1 :10 ³ or lower, whilst toxic effects of copper are reduced or minimized.

Example 12 (Figure 25): illustrates prevention of lung metastasis induced by administration of B16F10 malignant melanoma cells by R-Cu in varying ratios of R to Cu.

Fifty C57BL/6 mice were given i.v. injection of B16F10 melanoma cells (1x10⁵) as a single bolus. Animals were divided into five groups of 10 animals each. Group 1 were allowed drinking water ad libitum; in addition they were given injection saline i.p. starting 4 hr prior to B16F10 cell injection and twice daily thereafter for 3 days. Group 2 were given Resveratrol in drinking water at a molar concentration of 100mM (67mg /kg R per mouse, please see explanation below) starting 3 days prior to B16F10 injection and continued throughout the course of the experiment. In addition, the animals received i.p injection of Resveratrol at a molar concentration of 1 mM (0.67mg /kg R per mouse, please see explanation below) twice daily starting 4 hr prior to B16F10 injection and continued for three days after B16F 0 injection. Groups 3, 4 and 5 were given R-Cu in drinking water (in a molar ratios of 100mM: 1mM, 100mM: 1x10^"4mM and 100mM: 1x10^"6mM respectively) starting 3 days prior to B16F10 injection and continued throughout the course of the experiment. In addition the animals received i.p. injection R-Cu (molar ratio 1mM: 1 mM, 1mM: 1x10^"4mM and 1mM: 1x10^"6mM respectively) twice daily starting 4 hr prior to B16F10 injection and continued for three days after B16F10 injection.

Explanation of dosing: Since R given orally is poorly adsorbed, the molarity of R in drinking water was increased 100-fold (100mM) compared to the i.p. dose (1 mM). Since Cu is freely absorbed, the molarity of Cu were similar (1mM, 10^"4 mM and 10^"6 mM) when administered in drinking water and i.p.

Animals were sacrificed on day 21 and their lungs were excised. Number of black colonies in both lungs of all animals was counted. Results were depicted as box-plots showing median, 25 and 75 percentile and range of values. A)= B16F10 cells only; B)= B16F10 cells + R (100mM oral and 1 mM i.p.); C)= B16F10 cells + R-Cu (molar ratio 100mM:1 mM oral and 1 mM:1mM i.p.); D)= B16F10 cells + R-Cu (molar ratio 100mM:10^"4mM oral and 1mM:10^"4mM i.p.); E)= B16F10 cells + R-Cu (molar ratio 100mM:10^"6mM oral and 1mM:10^"6mM i.p.).

Conclusion from Example 12 (Figure 25): R alone caused a small reduction in the number of lung metastasis. However, R-Cu at molar ratios of lOOmM: 1 m orally + 1 mM: 1mM i.p. caused a further reduction, and this reduction was maintained, if not enhanced, when the concentration of Cu with respect to R was further reduced (100mM: 10^"4mM orally + 1 mM: 10^"4mM i.p. and 100mM: 10^"6mM orally + mM: 10⁶mM i.p.). Example 13: Degradation of RNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol and reduction in levels of RNA-associated reverse transcriptase in vitro.

Degradation of RNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol:

(Figure 26): illustrates degradation of RNA by R-Cu in the presence of varying molar ratios of R to Cu in 50% ethanol.

Starting molar ratio of R:Cu was R(1 ) : Cud ): R (5mM) : Cu (5mM)

Human lymphocytic leukemia cell line Jurkat was procured from ATCC, and was grown in RPMI 1640 with 10% FBS. Cells at exponential phase of growth (approximately 5 x 10⁶) were washed thrice in PBS and RNA was isolated using Trizol® reagent (Life Technologies, Carlsbad, CA, ^'" USA). Stock solutions of 20 mM of Resveratrol and Res-Cu were prepared in 50% ethanol. 2μg of RNA was suspended in TE buffer, and R-Cu, in varying molar ratios, dissolved in 20 μΙ of

50% ethanol was added. The reactions were incubated at 37°C for 1 h. Following incubation, RNA were subjected to 0.8% agarose gel electrophoresis at 75 volts for 90 mins. Image acquisition and analysis were performed using gel-documentation system. .

The percentage of intact RNA bands in each lane is presented in Table 16.

Table 16 (eukaryotic RNA):

Lane 10: RNA

+ R (5mM) -Cu (0.01 mM) 1 :0.002 0 0

Lane 1 1 : RNA (2μς) + R (5mM) -Cu (0.001 mM) 1 :0.0002 60 60

Lane 12: RNA (2 ig) + R (5mM) -Cu (0.0001 mM) 1 :0.00002 100 100

Reduction in levels of RNA-associated reverse transcriptase by R-Cu in vitro:

Table 18 and Table 19 illustrate reduction in HIV RNA-associated reverse transcriptase by R- Cu.

Molar ratio of R:Cu was R(1 ) : Cud )

Cell free virus supernatants from HIV-1 producing Indian isolate CRI GT50 T cell line was used in presence and absence of R and R-Cu to test its efficacy in inhibiting RT activity in vitro. The following established method was adapted for estimating the RT activity where the enzyme activity is expressed as net CPM of isotope incorporated [Johnson V.A., Byington R.E. , Kaplan J.C. Reverse transcriptase (RT) activity assay In Techniques in HIV research (ed. A. Aldovini and B.D. Walker); pp. 97-102. Stockton Press, New York; 1990].

METHOD IN DETAIL:

PEG precipitation of viral proteins.

1 ] 1 ml of cell free culture supernatant was taken out in a 1 .5 ml microvial and to this 0.5 ml of cold 30% PEG solution added. The vial is vortex and kept overnight on ice at 4°C.

2] Next day it is centrifuged at ~8000 x g for 45 min. in a refrigerated centrifuge (Plastocraft, Mumbai)

3] Supernatant was aspirated and pellet was lysed in 25 μΙ of solution B and then 100 μΙ of solution A was added, mixed by vortexing. The sample was stored at -20°C; 10 μΙ of this was used for RT assay after being thawed on ice.

R. T. Assay.

(Figure 27): illustrates flow-chart of Reverse Transcriptase assay.

1 ] A working cocktail for RT assay is made for viral RT activity determination in the following way.

Table 17 (working cocktail):

UB 22.5 22.5

³H-TTP(1.0 mCi/ml) 2.5 2.5 rA.dT 5 0 dA.dT 0 5

Total volume 90 μΙ 90 μΙ

2] 90 μΙ of rA or dA cocktail was taken in the tube and 10 μΙ of cold sample was added, the tube vortex and incubated at 37°C for 1 hr.

3] Reaction was terminated by transferring the sample tubes in ice-water bath.

4] 10 ml of cold tRNA was added to the tube followed by 1 ml of cold 10% TCA. Tube was vortex and left in ice-water bath for at least 30 min.

5] The reaction mixture was harvested on 0.45 μίτι filter using a vacuum manifold (Millipore, USA) followed by harvesting of each reaction tube washings carefully with equal volume of cold 5% TCA for three times.

6] Dried filters were suspended in 5 ml of scintillation fluid and counting recorded in a beta counter (Packard, USA). rA.dT and dA.dT primer based counts were calculated by multiplying the observed counts with dilution factor which in the assay mentioned above, is 7.5. The net RT activity estimation was arrived at by subtracting the net cpm of rA count - net cpm of dA count. Reagents

30% Polyethylene Glycol (PEG; Sigma, USA): 30% PEG-8000/0.4 M NaCI solution.

150 gm of PEG and 1 1.7 gm of sodium chloride was dissolved separately, mixed and the final volume made to a total of 500 ml of DW. Solution was filter sterilized and stored at 4°C.

0.2 M Dithiothreitol (DTT; Sigma, USA): 0.309 gm of DTT was dissolved in 10 ml of universal buffer and aliquots stored at -20°C.

0.2 M Magnesium cloride solution (MgCI₂.6H₂0; Sigma, USA): 4.066 gm of MgCI₂.6H₂0 was dissolved in 100 ml of DW, aliquots stored at -20°C.

0.5 M EDTA (Ethylene diamine tetra acetic acid-disodium salt; Life Technologies, USA): 93.06 gm of EDTA was dissolved in DW by raising pH of the solution to 8.0, using 10 N sodium hydroxide and final volume was made to 500 ml.

Solution A: The buffer containing 25 mM Tris.CI (pH 7.8), 0.25 mM EDTA, 0.025% Triton-X 100 (Sigma, USA), 50% glycerol (Life Technologies, USA), 10 mM DTT and 100 mM KCI was made by adding the following stock solutions in the given amount and making the final volume to 500 ml using DW. 1 M Tris.CI (pH 7.8)-12.5 ml, 0.1 M EDTA-1.25 ml, 10% Triton X-100-1.25 ml, glycerol-250 ml, DTT-0.77 gm, KCI-3.72 gm. Aliquots of 100 ml were stored at -20°C.

Solution B: The reagent containing 0.9% Triton and 440 mM KCI was made by adding the following stock solutions/reagents in the given amount and making the final volume to 100 ml using DW. 10% Triton X-100-0.9 ml and KCI-0.326 gm. The solution was stored at 4°C.

Universal Buffer (UB): The reagent containing 10 mM Tris.CI, pH 8.0, 15 mM NaCI was made by adding the following stock solutions in the given amount and making the final volume to 100 ml using DW. 1 M Tris.CI (pH 8.0)-1.0 ml, 5M NaCI-0.3 ml. The buffer was stored at 4°C.

10 mg/ml t RNA (Baker's yeast tRNA; Sigma, USA): 250 mg vial of Ribonucleic acid was reconstituted in 25 ml of Universal buffer, aliquots of 0.5 ml stored at -20°C.

Synthetic primers: (Pharmacia, Sweden)

10 units/ml poly rA oligo (dT) ₂-is template

10 units/ml poly dA oligo (dT)₁₂-is template

25 U of each was dissolved in 2.5 ml of universal buffer, aliquots were stored at -20°C.

10% TCA: 50 gm of Trichloroacetic acid and 4.46 gm of Sodium pyrophosphate (Sigma, USA) was dissolved in total of 500 ml of DW. Solution was stored at 4°C

5% TCA: 100 gm of Trichloroacetic acid and 17.8 gm of Sodium pyrophosphate was dissolved in total of 2 L of DW.

³ Ή-ΤΤΡ [methyl, V2'-³H : Thymidine 5'-triphosphate, ammonium salt in ethanol/water (1 : 1 ), sp. activity- 90-130 Ci/mmol (Amersham, UK).

Scintillation Fluid: POPOP & PPO (Sigma, USA) 0.5 gm of POPOP and 7.0 gm of PPO was dissolved in 1 L of Toluene.

Table 18 illustrates reduction in HIV RNA-associated reverse transcriptase by R-Cu Molar ratio of R:Cu was R(1 ) : Cud).

Table 19 illustrates dose response effect of R-Cu in reducing HIV RNA-associated reverse transcriptase.

Molar ratio of R.Cu was R(1 ) : Cud). RT activity/ml % Reduction

Only viral supernatant 18281

Viral supernatant + R(40 μΜ) - Cu(40 μΜ) 1 1966 34.5

Viral supernatant + R(4 μΜ) - Cu(4 μ ) 13310 27.2

Viral supernatant + R(0.4 μΜ) - Cu(0.4 μΜ) 14059 23.0

Conclusions from Example 13:

(Figure 26): Increasing the ratio of R to Cu (i.e., by reducing Cu with respect to R) enhances degradation of cellular RNA in vitro.

(Table 18 and Table 19): Treatment of HIV supernatent with R-Cu (molar ratio 40μΜ:40μΜ) significantly reduced reverse transcriptase associated with HIV-RNA indicating a reduction in viral load. R-Cu exhibited a dose response effect with respect to reduction in viral load.

Conclusion:

(1 ) It was surprisingly found that plasmid DNA degrading activity of R-Cu in vitro increases as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where Cu is so low that the DNA degrading activity of R-Cu is lost. This phenomenon was observed in the 4 different solvents that were tested (Figure 1 - 8). Complete fragmentation of plasmid DNA was observed as a smear in the gel images (Figures 3B, 4, 6, 7A, 7B and 8A).

(2) Even more surprisingly it was found that R-Cu was capable of degrading plasmid DNA under all pH conditions tested (Figure 9A and 9B).

(3) Even more surprisingly it was found that other plant polyphenols, such as Curcumin and β- Carotene, in the presence of Cu, were also effective in degrading plasmid DNA compared to R- Cu (Figure 10 and 1 1 ).

(4) Even more surprisingly it was found that R in the presence of other heavy metals, such as Zn and Li, were entirely ineffective in degrading plasmid DNA (Figure 12 and 13).

(5) Even more surprisingly it was found that eukaryotic genomic DNA degrading activity of R-Cu in vitro increases as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that the DNA degrading activity of R-Cu is lost (Figure 14). Complete fragmentation of eukaryotic genomic DNA was observed as a smear in the gel image.

(6) Even more surprisingly it was found that eukaryotic apoptotic DNA degrading activity of R- Cu in vitro increases as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that the DNA degrading activity of R-Cu is lost (Figure 15). Complete fragmentation of eukaryotic apoptotic DNA was observed as a smear in the gel image.

(7) Even more surprisingly it was found that R-Cu was capable of degrading chromatin fragments that emanate from dead cells in culture, thereby preventing apoptosis in living cells and preventing depletion in their number. This activity increases as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that the DNA degrading activity of R-Cu is lost (Figure 16).

(8) Even more surprisingly it was found that R-Cu prevented the rise in Cfs that follows Adriamycin treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that Cfs degrading activity of R-Cu is lost (Figure 17A and 17B).

(9) Even more surprisingly it was found that R-Cu prevented the rise in inflammatory cytokine IL6 that follows Adriamycin treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that Cfs degrading activity of R-Cu is lost (Figure 18A and 18B).

(10) Even more surprisingly it was found that R-Cu prevented neutropenia that follows Adriamycin treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), and this activity is retained even when the ratio of R to Cu is as low as 1 : 10^"9 (Figure 19A and 19B).

(11 ) Even more surprisingly it was found that R-Cu prevented the rise in Cfs that follows LPS treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that Cfs degrading activity of R- Cu is lost (Figure 20A and 20B).

(12) Even more surprisingly it was found that R-Cu prevented the rise in inflammatory cytokine IL6 that follows LPS treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that Cfs degrading activity of R-Cu is lost (Figure 21 A and 21 B).

(13) Even more surprisingly it was found that R-Cu prevented lethality following LPS treatment to mice, and that this activity is retained as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R) (Figure 22).

(14) Even more surprisingly it was found that the anti-tumour effect of Adriamycin is significantly enhanced by concurrent administration of R-Cu (molar ratio 1 :1 ) such that tumour growth in Adriamycin treated animals is further retarded by R-Cu over that achieved by Adriamycin alone (Figure 23). (15) Even more surprisingly it was found that R-Cu at a molar ratio of 1 : 10^"7 completely prevented oncogenic transformation of NIH3T3 mouse fibroblast cells treated with dead Jurkat (cancerous) cells (Figure 24). The successive reduction in the concentration of Cu (II) with respect to a constant molar concentration of Resveratrol (5μΜ) beyond this point showed increasing re-appearance of oncogenic transformation.

(16) Even more surprisingly it was found that Cu (II) at higher concentrations with respect to a constant molar concentration of Resveratrol (5μΜ) were highly toxic to cells leading to their death; the latter was particularly observed between a R-Cu molar ratio of 1 :1 - 1 :10 ³ (Figure 24).

(16) Even more surprisingly it was found that R-Cu prevented lung metastasis in a mouse melanoma model and that this activity was retained even when the molar ratio of R to Cu was of the order of 1 : 1x1 f ⁶ (Figure 25).

(17) Even more surprisingly it was found that eukaryotic RNA degrading activity of R-Cu in vitro increases as the ratio of R:Cu increases (i.e., Cu is decreased with respect to R), to a point where copper is so low that the RNA degrading activity of R-Cu is lost (Figure 26). Complete fragmentation of eukaryotic RNA was observed as a smear in the gel image.

(18) Even more surprisingly it was found that R-Cu (molar ratio 1 :1 ) was capable of reducing HIV RNA-associated reverse transcriptase in vitro indicating a reduction in viral load (Table 18).

Claims

1. A composition comprising a plant polyphenol and copper (II) for the degradation of or preventing or reducing a rise in the level of one or more of circulating fragments of chromatin, DNA and RNA.

2. A composition according to claim 1 wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^~10.

3. A composition according to claim 1 or claim 2 wherein the DNA is selected from

plasmid DNA, eukaryotic genomic DNA andeukaryotic apoptotic DNA; and the RNA is selected from eukaryotic RNA and HIV RNA.

4. A composition according to any one of the preceding claims for use in treating a condition associated with an elevated level of circulating chromatin fragments.

5. A composition according to claim 4 wherein the condition is selected from cancer, systemic autoimmune disorders, diabetes, Parkinson's disease, Alzheimer's disease, cerebral stroke, myocardial infarction, inflammation, sepsis, critical illness, trauma, renal failure, HIV/AIDS, ageing and age-related disorders.

6. A composition according to claim 5 for use in treating or preventing cancer.

7. A composition according to claim 6 for use in treating or preventing metastasis.

8. A composition according to claim 5 for use in treating or preventing HIV/AIDS

9. A composition according to any one of the preceding claims for use in preventing a rise in or reducing the circulating level of one or more of chromatin fragments and inflammatory cytokines or in preventing or reducing the level of neutropenia in a subject after a therapy for cell-destruction selected from chemotherapy and radiotherapy.

10. A composition according to any one of the preceding claims wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^~4

11. A composition according to any one of the preceding claims wherein the

concentration of the plant polyphenol is from 5microMolar to 10Omil!iMolar.

12. A composition according to any one of the preceding claims for use in degrade

plasmiding DNA wherein the concentration of the plant polyphenol is from 50 to 200 microMolar and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"2.

13. A composition according to claim 12 wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :10^~1 to 10^~2

14. A composition according to any one of the preceding claims for use in degrading plasmid DNA wherein the concentration of the plant polyphenol is from greater than 200 microMolar to 2mM and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"3.

15. A composition according to any one of the preceding claims for use in degrading plasmid DNA wherein the concentration of the plant polyphenol is from greater than ' 2mM to 10 mM and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^'4

16. A composition according to claim 15 wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 : 10^"2 to 10^"4

17. A composition according to claim any one of the preceding claims wherein the plant polyphenol comprises curcumin and the molar ratio of curcumin to copper (II) is in the range 1 :1 to 10^"3

18. A composition according to any one of the preceding claims wherein the plant

polyphenol comprises beta carotene and the molar ratio of beta carotene to copper (II) is in the range 1 :10 to 10^"4

19. A composition according to any one of claims 1 to 1 1 for use in degrading eukaryotic genomic DNA or eukaryotic apoptotic DNA wherein the concentration of the plant polyphenol is from greater than 2mMolar to 10mM and the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1 to 10^"3.

20. A composition according to any one of claims 1 to 9 wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :10^"3 to 10^"10.wherein the level of copper is such that the compositionis non-toxic to a human subject.

21. A composition according to claim 20 wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 : 10^"3to 10^"8.

22. A composition according to any one of the preceding claims comprising a plant polyphenol and copper (II) for use in reducing or preventing apoptosis of living cells upon or after contact with dead cells

23. A composition according to claim 22 wherein the concentration of the plant

polyphenol is from greater than 2mMolar to 10mM, the molar ratio of the plant polyphenol to copper (II) is in the range 1 :10^~1 to 10^"9 and the number of viable living cells is at least 50% greater than the number of viable living cells without administration of the composition.

24. A composition according to any one of the preceding claims wherein the composition is administered interperitoneally to a subject at a dose level of 10 to 100mg polyphenol per kg weight of the subject

25. A composition for use in enhancing the anti-tumour effect of a cell destruction

treatment, the composition comprising a plant polyphenol and copper (II).

26. A composition for use according to claim 25 wherein the cell destruction treatment is selected from chemotherapy treatment for cancer and/or a radiotherapy treatment for cancer, the method comprising administering to a subject a composition as defined in any one of the preceding claims concurrently with or sequentially with the chemotherapy treatment and/or a radiotherapy treatment.

27. A composition for use according to claim 26 wherein the chemotherapy agent is selected from alkylating agents, antimetabolites, anti-tumor antibiotics,

topoisomerase inhibitors and mitotic inhibitors or any combination thereof.

28. A composition for use according to claim 27 wherein: i) the alkylating agent is selected from one or more of

Cyclophosphamide, Ifosfamide, Melphalan, Thiotepa, Cisplatin, Carboplatin, and Oxalaplatin; ii) the antimetabolite is selected from one or more of 5-fluorouracil (5- FU), Gemcitabine (Gemzar®) and Methotrexate; iii) the anti-tumor antibiotic is selected from one or more of

Anthracvclines: Doxorubicin (Adriamycin®), Epirubicin, Actinomycin-D and Mitomycin-C; iv) the topoisomerase inhibitor is selected from one or more of Etoposide (VP-16) and Mitoxantrone ; and v) the mitotic inhibitor is selected from one or more of Taxanes:

Paclitaxel (Taxol®) and Docetaxel (Taxotere®) Vinca alkaloids: Vinblastine (Velban®) and Vincristine (Oncovin®).

29. A composition for use according to claim 26 or claim 27 wherein administration of the composition degrades or prevents or reduces a rise in the level of chromatin fragments, inflammatory cytokine IL6 and/or neutropenia wherein the molar ratio of the plant polyphenol to copper (II) in the composition is in the range 1 :1 to 10^"9.

30. A composition for use according to claim 29 wherein the chemotherapy agent is an anti-tumour antibiotic.the molar ratio of the plant polyphenol to copper (II) in the composition is in the range 1 :1 to 10^"6 in preventing or reducing a rise in IL6.

31. A composition for use according to claim 29 wherein the chemotherapy agent is an anti-tumour antibiotic, the molar ratio of the plant polyphenol to copper (II) in the composition is in the range 1 :1 to 10^"7 in degrading or preventing or reducing a rise in chromatin fragments.

32. A composition for use according to claim 25 wherein the cell destruction treatment comprises administering lipopolysaccharide, the molar ratio of the plant polyphenol to copper (II) in the composition is in the range 1 :1 to 10^~4 degrading or preventing or reducing the level of chromatin fragments, preventing or reducing a rise in IL6 or in the prevention of lethality.

33. A composition for use according to any one of claims 26 to 32 wherein the subject is treated for cancer and administration of the said composition prevents or reduces the likelihood of metastasis.

34. A composition for use in the prevention or reduction of oncogenic transformation of normal cells in the presence chromatin fragments from dead cancer cells , the composition comprising a plant polyphenol and copper (II).

35. A composition for use according to claim 33 wherein the molar ratio of the plant polyphenol to copper (II) in the composition is in the range 1 : 10^"6 to 10^~10

36. A composition for use according to claim 1 for degradation of eukaryotic RNAwherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :10^"1 to 10^"4

37. composition for use according to claim 1 for degradation of or reducing HIV RNA- associated reverse transcriptase wherein the molar ratio of the plant polyphenol to copper (II) is in the range 1 :1to 10^"1.

38. A composition according to any one of the preceding claims wherein the plant

polyphenol is selected from trans-3,5,4'-trihydroxystibene, curcumin and beta- carotene.

39. A composition according to any one of the preceding claims further comprising a solvent.

40. A composition according to any one of the preceding claims having a pH of 3 to 11.