FUTURE SCOPE OF ANTIOXIDANTS IN CLINICAL MEDICINE

Dr Indla Yogananda ReddyProfessor of Physiology

Objectives 1. Free Radicals Friends or Foes2. Pro-oxidant effect of antioxidant3. Why to look for the future scope of the

antioxidants in clinical practice4. What are the real future prospects of

antioxidants in clinical practice

Free Radicals Friends or Foes1. Generation of ATP – oxidative

phosphorylation2. Detoxification of xenobiotics-Cytochrome

P4503. Apoptosis of effete or defective cells4. Killing of micro-organisms and cancer cells5. Oxygenases (Cox, Lox)- for the synthesis of

prostaglandins & leukotrines6. ROS-second messengers7. Redox signaling pathway

Pro-oxidant effect of Antioxidant1. Ascorbate-high ferric ion-↑ lipid peroxidation2. Ascorbate-↑ DNA damage3. Vitamin E- pro-oxidant4. Beta-carotene-pro-oxidant-lungs-smokers5. Signal transduction for ROS-inhibited

Meta-anlyses of RCT of N-Acetylcystine in various diseases

Publication No of RCT/randomized patients

Disease Results conclusion

Adabag et al 10/1163 Post cardiac surgery

No change in mortality

No conclusive evidence on the ben Rx

Nigwekar et a 12/1324 post-cardiovascula

No significant change in ARF or mortality

No conclusive evidence on the ben Rx

Mckay et al 6/ Liver transplantati

No improvement in clinical outcome

No conclusive evidence on the ben Rx

Ho et al 10/1193 After major surgeries

No significant decrease in ARF or mortality

No conclusive evidence on the ben Rx

Sutherland et 8/2214 COPD Significant decrease in exacerbation

Treatment is beneficial

Why many antioxidants have failed to show efficacy in interventional human studies 1. Oxidative stress is not the primary cause of the disease2. Oxidative stress is not the only cause of the disease3. Patients do not equally benefit from antioxidant

therapy4. Administered antioxidant is not able to lower oxidative

stress5. Antioxidant molecule has harmful effects that Mask

its useful antioxidant actions6. Certain antioxidants are not effective in well-nourished

population 7. The target is not well selected

Oxidative stress is not the primary cause of the diseaseEarly or late stage of the tissue injury

Oxidative stress is not the only cause of the diseaseEg; 1. lack of success in lung disease 2. ischemic cell death in stroke

Patients do not equally benefit from antioxidant therapyOxidative stress status is different from patient to patientPatient selection should be based on their oxidative stress status↓ efficacy of vitamin EPharmacogenomics should be considered Eg; selection of diabetic patients-haptoglobin-vitamin E

Administered antioxidant is not able to lower oxidative stressDNA oxidative markers in urine ↓- Brussels sprouts but not with β-carotene, vitamin C, α-tocopherolCombination of antioxidants ↑ DNA oxidative damage1. Antioxidant molecule has low bioavailability2. Time and duration of therapy are not optimala. population-oxidative stress for decadesb. optimal duration of therapy3. Oxidative stress is hard to overcome in certain diseaseEg; balance between pro-oxidants and antioxidants

4. Antioxidants have poor target specificityEg; cancer, neurodegenerative diseases5. Reaction products of the antioxidants are toxicPro-oxidants+antioxidants=free radicalsEg; Edaravone 6. Single antioxidant is not enough to overcome oxidative stressEg; Vitamin E with vitamin C7. Physiological mechanisms prevent the achievement of high tissue level of antioxidantsEg; endogenous control mechanisms

Antioxidants have harmful effects that masks their useful antioxidant actionsi. β-carotene can have unwanted effects on lipid profileii. Vitamin E- may prevent the increase in HDL-2Certain antioxidants are not effective in well nourished populationEg; 1. Vitamins may prevent cancer in patients with poor

nutritional status 2. Should not generalise this notion to all antioxidantsEg; B12-dementia-↓ plasma B12-cognition 3. Antioxidants are probably more effective in developing

countries 4. Lutein and green tea in adequately nourished patients-plasma

oxidative stress parameters are not altered

The target is not well selectedRight target for therapyEg; SOD mimetic may not be successful-endogenous SOD functions very effectively

Near future directions of antioxidants should be in overcoming these drawbacks

Oxidative stress is not the primary cause of the disease-selection of diseases in which oxidative stress is the core pathology

Oxidative stress is not the only cause of the disease-combination of antioxidants with other drugs

Patients do not equally benefit from antioxidant therapy-with the use of biomarkers of oxidative stress select the patients with high oxidative stress

Antioxidants have low bioavailability-optimization of antioxidant levels (nano-carriers)

Time and duration of therapy are not optimal-optimization of the time and duration of antioxidant therapy, use of biomarkers to monitor the response to antioxidant therapy

Oxidative stress is hard to overcome in certain diseases-use of combination of antioxidants

Antioxidants have poor target specificity-use of antioxidants on disease specific pathways rather universal pathways (nano-carriers)

Reaction products of the antioxidants are toxic-optimization of antioxidants

A single antioxidant is not enough to overcome oxidative stress-use of combination of antioxidantsPhysiological mechanisms prevent the achievements of high tissue levels of antioxidants-selection of antioxidants that can overcome this effect Antioxidants have harmful effects that masks its useful antioxidant action-use of a better antioxidantCertain antioxidants are not effective in well nourished people-use of a proper antioxidantThe target is not well selected-to select the target (vectors in gene therapy, nano-carriers)

Antioxidant drugs approved for clinical use in various diseases

Edaravone Ischemic stroke

Idebenone Alzheimer disease

N-Acetylcystine Mucolytic

α-Lipoic acid Diabetic neuropathy

Daflon 500 Persistent ulcers

WHAT ARE THE REAL FUTURE PROSPECTS OF ANTIOXIDANTS IN CLINICAL PRACTICE1. Gene therapy2. Nano technology3. Genetically engineered plant products4. Synthetic antioxidant enzymes5. Functional food

Gene therapyi. Transductionii. Vectorsiii. Common antioxidant genes that are

transduced TransductionDNA is transferred from one cell to another cell with the help of a virusiv. Generalisedv. Specialised

Vectors used for gene transferVector Advantages Disadvantages

Naked plasmid DNA Easy to produce Very low transduction efficacy

Adenovirus High transduction efficacy Inflammation with high dose

Adeno-associated virus Long term gene expression, moderate immune response, transduces quicent cells,

Limited trans gene capacity

Lentivirus Long term gene expression, transduces quicent cells, relatively high trans gene capacity

No specific integration

Retrovirus Long term gene expression Non specific integration, limited tropism

HSV-1 High transduction efficacy, high trans gene capacity

Transduces only dividing cells, cytotoxic

Common antioxidant genes that are transducedi. Heme oxygenase-1ii. Superoxide dismutase'siii. Catalaseiv. Glutathione peroxidasev. Repair genes-a. Lipoprotein-Associated Phospholipase A2b. Guanosine 5-Triphospate Cyclohydrolase I

Limitations in antioxidant gene therapy1. Limited duration of Transgene expression2. Limited Transduction of cells3. Immune Responses4. Role of ROS in cell signalingLimited duration of Transgene expressioni. Time course of the disease progressionii. Selection of the individuals without any signs of the

disease-benefit from gene therapyiii. Gene therapy-long term expression of desired targetsiv. Current diagnostic tests and gene therapy vectorsv. I/R injury or thrombosis-so suddenvi. High risk patients-refinement of current diagnostic tests

Limited transduction of cells1. Limited efficacy of current vectors in

transduction of cells2. Wide spread delivery of trans genes is neededImmune ResponsesGene therapy vectors and transgenesIndividual variability in immune responseRole of ROS in Cell signalingROS-pathological events, normal cell signaling

Future directions of gene therapy1. Ex vivo gene transfer of stem and progenitor cells-unwanted transduction of cells & tissues2. Identification of patients with genetic weakness in antioxidant defense system3.Vector modifications-alternate serotypes and capsid4. Transient immune suppression

…..continue5. Use of transcription factors to elicit pleiotropic effects6. Development of regulatable vector systems-on and off as needed-a. promotors-orally administered drugsb. promotors-physiological stimuli (hypoxia)7. Development of novel gene delivery methods-biodegradable gels, gene-eluting stents8. Clinical potential of antioxidant gene therapy-EC-SOD-outside transduced cells-HO-1

Nano technologyNanocarrier Antioxidants-low bioavailability, low biocompatibilityNanoparticles + antioxidants 1. Nanoantioxidants prepared by encapsulation2. Nanoantioxidants prepared by surface-functionalization methodNanoantioxidants prepared by encapsulationa. Chemical methods-polymerizationb. Physical methods-spray & freeze dryingc. Physicochemical-liposomeTwo methods1. Top-down methods2. Bottom-up methodsMaterials used for the preparation of nano-encapsulesi. Synthetic polymersii. Natural polymers

Nano-antioxidants

Chemical synthetic polymers used for antioxidants encapsulation

Nanoparticles Antioxidants Superiority Experimental mode

Eduragi E & polyvinyl alcohol

Quercetin ↑ Antioxidant activity In vitro

Poly & polyethylene glycol-5000

Curcumin ↑ cellular uptake, bioactivity & bioavailability

Rat, cell lines

Poly-b-poly Resveratrol ↑ solubility, stability Cell cultures

Gum Arabic-maltodextrin

Epigallocatechin gallate

Integrity & biological activity maintained

Cell lines, in vitro

Stearyl ferulate Β-carotene, α-tocopherol

↑ Stability In vitro

Poly acid Vitamin E, C ↑ Antioxidant activ In vitro

Polysulfide SOD ↑ Antioxidant activ, stability

In vitro

Significance of Nanoantioxidants encapsulation1. Permeability2. Aqueous solubility 3. Stability4. More surface5. Improvement of control release6. Target specificity7. Enhances therapeutic antioxidant efficacy

Liposomes used in antioxidant encapsulation

Significance of using liposomesi. Synthesized from natural productsii. Non-toxiciii. Biodegradableiv. No-immune response v. Target specific

Nanoantioxidants prepared by surface-functionalization method1. Covalently linked to the surface of nanoparticle2. Enhances radical scavenging activity3. Kinetic enhancement effect of antioxidant activity

Future directions of Nano-antioxidants 1. Having a targeting group that can reach the diseased organ or tissue specifically2. Avoiding release of the antioxidant monomers located inside nanocarriers during long-term circulation before they reach the target point3. Having high antioxidative activity4. No in vitro and in vivo cytotoxicity5. Having an appropriate administration route6. Easily be excreted from the body7. The rational design of nanotechnology materials and tools based around a detailed and thorough understanding of biological processes.

Genetically engineered plant products1. Vegetables with high antioxidant compoundsEg; tomatoes-with up to 3 times lycopene concentration, longer shelf life2. Orange cauliflower-rich in carotene3. The Vegetable and Fruit Improvement Center at Texas-Super vegetables-purple carrot breed-40% more β-carotene4. Anti-carcinogenic properties of Citrus fruits5. Increasing the carotenoid content of watermelons6. Onions-↑ quercetin, anthocyanin7. Pepper-↑ quercetin8. Stone fruits-↑ anthocyanin

Functional foodsThey provide additional physiological benefits apart from the simple nutrition1. Whole foods- supplements or processed foods2. Indian foods constitute spices and medicinal plants-vitamins, nutrients and antioxidants3. Memory decline-low levels of dietary antioxidants4. Intake of fruits and vegetables with Oxygen Radical Absorbance Capacity (ORAC) 3000-5000/ day-significant impact

4.Bakery products-white breads with vitamins, fibers, microelements

5. Functional drinks-a. non alcoholic beverages fortified with-A,C, Eb. cholesterol lowering drinks with Omega-2 &

soyc. Eye health drinks-lutein6. Functional eggs-enriched with Antioxidants,

omega 3 FA

Synthetic antioxidant enzymes1. They reduce tissue damage by reperfusion2. Lower molecular weight3. More stable4. No immune response5. Tempol-SOD mimetic drugi. Used in acute and chronic painii. Enhances the antitumour effects of –

interleukinsiii. Efficient radioprotector

Summary1. Free radicals are having positive effects also2. Antioxidants are having negative effects also3. Appropriate use of antioxidants is required4. Gene therapy, nano technology, genetically

engineered plant products, synthetic enzymes, functional foods

Thank you