Methods for Detection of Pathogenic Bacteria PDF

METHODS FOR THE DETECTION OF Evidence-based prescribing and PATHOGENIC BACTERIA dispensing could be informed by Multidrug resistant organisms (MROs) and effective, low-cost diagnostic tests, drug resistance...

METHODS FOR THE DETECTION OF Evidence-based prescribing and PATHOGENIC BACTERIA dispensing could be informed by Multidrug resistant organisms (MROs) and effective, low-cost diagnostic tests, drug resistance capable of integration into clinical, US approx 4% of acute care hospital pharmacy and veterinary practices patients have at least 1 Healthcare The 2016 O’Neill report on ‘Tackling Associated Infection (HAI) Drug-Resistant Infections Globally’ Europe: prevalence of HAI is 7.1 per 100 suggests that by 2020 all clinicians patients should perform a rapid diagnostic test ○ European Centre for Disease before prescribing antimicrobials Prevention and Control estimates that more than 4M patients are WHO Global action plan on AMR affected by HAI every year in Europe Concern → transmission of resistance between multi-resistant organisms, which could ultimately lead to strains which have limited or no susceptibility to antibacterial agents (MDR/XDR/PDR) Global initiatives for AMR UK Gov: reinstituted the Longitude Prize in 2014 → UK public voted on which global problem should be the subject of Rapid surveillance techniques the ￡10M prize Need to inform use of antimicrobial Successful challenge: “to create a agents cost-effective, accurate, rapid and Results used to identify easy-to-use test for bacterial infections colonised/infected patients and inform that will allow health professionals their directed (rather than empirical worldwide to administer the right [broad range agents]) treatment antibiotics at the right time” Surveillance should form basis for an Assessments of submissions made organism-specific approach to every 4 months; the first team to meet all transmission-based precautions of the criteria will win the prize Effective infection prevention relies on The $20M Antimicrobial Resistance the introduction of contact precautions Diagnostic Challenge seeks “new (such as patient isolation in a innovative and novel laboratory single-patient room or cohorting patients diagnostic tests that identify and with the same strain of MRO in characterise antibiotic resistant bacteria designated patient-care areas) and/or distinguish between viral and bacterial infections Antimicrobial stewardship Systematic approach to optimising the Evidence-based prescribing should be use of antimicrobials in hospitals → use informed by diagnostic testing old generation first line to prevent WHO global action plan on antimicrobial resistance to new generation antibiotics resistance suggests that antibiotic Includes prescriptions are rarely based upon ○ Implementing clinical guidelines effective diagnoses and that the standard that are consistent with the latest of care should be evidence-based version of TGs prescribing and dispensing ○ Establishing formulary restriction and approval systems that  include restricting From 2010 The Department of Health broad-spectrum and later (DoH) introduced mandatory screening generation antimicrobials for MRSA for all admissions to English ○ Educating prescribers, hospitals pharmacists and nurses about A national audit in 2011 found good antimicrobial prescribing ○ Poor compliance (61%) practice and antimicrobial ○ That only approx 50% of new resistance positive patients were isolated, with approx 25% not receiving MRSA decolonization therapy MRSA is susceptible to very few agents, ○ 37% of newly identified MRSA including glycopeptides (vancomycin and patients were discharged prior to teicoplanin), quinupristin-dalfopristin and their test result being available linezolid ARHAI now recommends that in England Cases of methicillin and only ‘checklist-activated screening’ (i.e. quinupristin-dalfopristin resistant staph of patients with high MRSA risk factors or aureus already reported in Europe admission to high risk units) is employed Does universal surveillance work? YES Requirements of a Bacterial Surveillance US study looked at rates of MRSA Method clinical disease in 3 consecutive periods Sensitive (correctly identifies - baseline (no surveillance), MRSA microorganism) surveillance for all ICU admissions, Specific (identifies ONLY microorganism universal MRSA surveillance (all of interest) admissions) - in 3-hospital organisation Rapid with 40,000 annual admissions Reliable PCR-based nasal surveillance for MRSA Cost-effective followed by topical decolonisation and Simple to perform contact isolation for patients who tested Does not require specialist interpretation positive for MRSA Baseline prevalence density of MRSA Current detection methods disease was 8.9 cases per 10,000 Phenotypic methods patient days ○ Biochemical testing During ICU surveillance prevalence ○ Chromogenic media density fell to 7.4 and during universal ○ MALDI-TOF MS surveillance to 4.7 per 10,000 days Genotypic (molecular diagnostic) Universal surveillance also led to a methods reduction in disease occurring up to 30 ○ Real time polymerase chain days after discharge reaction (PCR) If no surveillance estimated that further ○ Whole genome sequencing 1319 transmission of MRSA would have (WGS) occurred ○ Multi-omics approaches Prevented MRSA transmission and prolonged disease Microscopy/staining 0.14 patient-per-day rate of MRSA transmission if patient not subject to contact isolation Universal MRSA surveillance is no longer recommended by the Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infection (ARHAI)  Electronic transitions correspond to electrons in molecule being excited from one energy level to another As conjugation increased, the difference in energy between the highest occupied (HOMO) and lowest unoccupied (LUMO) decreases so λmax increases Gram stain (pink or purple) ○ Gram positive or gram negative UV-VIS Spectrum of beta-carotene in Ethanol Chromophore/fluorophore detection Enzyme substrate; colour/fluorescence muted or absent due to linkage to targeting molecule; must be stable in medium Chromophore/fluorophore released only if specific bacterium present; should be retained by bacterial colonies Phenolphthalein The Visible Region Requirements for chromogen Free chromogenic molecule must have strong colour Chromophores Colour must be muted or absent when Part of the molecule which contains the attached to targeting molecule electrons involved in an electronic Targeting molecule recognised by transition is called the chromophore specific bacterial enzyme  Link between targeting molecule and chromogen broken by a specific bacterial enzyme Colour chromogen released thus confirming presence of specific bacteria Specific bacterial enzyme targets Peptidases (aminopeptidases cleave N-terminal amino acid from a peptide) ○ L-alanyl aminopeptidase ○ L-pyroglutamyl aminopeptidase ○ L-glutamyl aminopeptidase Glycosidases ○ 𝜷-glucuronidase ○ 𝛂- or 𝜷-glucosidase Target molecules: Other enzymes e.g., deaminase, 1,2-Substitued-7-aminophenoxazinones nitroreductase 9-(4-N’-L-Ala-L-Ala-L-Ala-aminophenyl)-10-m ethylacridinium bis(trifluoroacetate) Differentiates between gram negative (hydrolysed to red) and gram positive (grow, but no hydrolysis, so colourless) R1 or R2 = H, alkyl C1-C12, aralkyl C6-C14, aryl, CO2H, CO2R2, or NR3R4 Colour muted when N7-acylated Colour released when amide hydrolysed All incorporate unnatural amino acids, 𝜷-alanine Pseudomonas aeruginosa Pseudomonas aeruginosa is a pathogenic bacterium that secretes thick mucus (biofilm) and is a complicating infection in lungs of cystic fibrosis patients, burns victims and AIDS patients Difficult to treat once established and leads to high mortality Greater clinical success in treating P. aeruginosa infection if detect and treat early Why 𝜷-Ala-Containing substrates? L-alanyl aminopeptidase: bacterial enzyme ○ Gram negative bacteria have L-alanyl aminopeptidase and Natural phenoxazinone hydrolyse substrate to release colour  ○ Differentiates gram positive (no colour) from gram negative bacteria 𝜷-Alanyl aminopeptidase ○ Much less common in bacteria but expressed in Pseudomonas aeruginosa Fluorogenic detection using self-immolative ○ Greater selectivity for detection linkers (Agar media) 1-N-(𝜷-Alanyl) amino-1-pentylresorufamine (𝜷-Ala-1-PRF) Liquid media Origin of colour Effective detection of pseudomonas aeruginosa Slight detection of Burkholderia cenocepacia Good chromogen with bright purple colour Molecular diagnostic methods Often based on polymerase chain reaction (PCR) technology Mass spectrometry-based methods also offer promise These techniques offer specificity and speed but are complex, costly and require specialist interpretation 4-Methylumbelliferyl 𝜷-D-galactopyranose - use in detection of coliform bacteria PCR technology Produces millions of copies of DNA sequence in 2 hours Series of cycles involving: ○ Denaturing of double stranded DNA at >90°C ○ Annealing of upstream and downstream primers for DNA Hydrolysis of 𝜷-alanylaminonaphthyridine by sequence of interest pseudomonas aeruginosa ○ Thermus aquaticus (Taq) DNA polymerase then catalyses DNA replication Real-time PCR Detection of MRSA in a Real-time PCR Detection of Other Mixture of Staphylococci Organisms) Employs DNA primers and a series of BD GeneOhm VanR assay: Detection of molecular beacon probes vancomycin resistant enterococci (VRE) Primers are specific for the mecA and S. based on the VanA gene aureus specific orfX genes to allow for Oxazolidinone-resistant E. faecalis and variations in the staphylococcal cassette E. faecium (based on G to U mutation at chromosome (SCCmec) residue 2576 of 23S ribosomal DNA) SCCmec is a mobile genetic element P. aeruginosa (based on outer which carries the mecA gene (which membrane lipoprotein genes, oprI and encodes the 𝜷-lactam-resistant penicillin oprL) binding protein PBP2’) Vancomycin Drawbacks with quantitative PCR (qPCR) Hydrophilic and forms hydrogen bonds to Involves high levels of amplification so the terminal D-Ala-D-Ala sequence - contamination or the detection of nucleic preventing crosslink formation and acids which remain from a previously blocking the release of the disaccharide cleared infection is a concern from the carrier lipid The discrimination between an Resistance: occurs through alteration in asymptomatic colonisation and a the ligase activity clinically relevant infection relies upon The Vancomycin Resistant Enterococci the development of standardised (VRE) Van A phenotype produces a quantitative cut off cycle-threshold (Ct) D-Ala-D-Lactate ligase which synthesis values but no clear relationship between an ester (D-Ala-D-Lac) rather than an number of microorganisms present in amide (D-Ala-D-Ala) specimen and the presence of healthy D-Ala-D-lactate sequence has 1000-fold carriage or disease within the individual reduction in affinity for vancomycin but can still be added to L-lys and act as PCR technology precursor for crosslink formation Requires some means of detection of amplified DNA sequence (traditional MALDI-TOF Mass Spectrometry PCR assays utilised immunoassays or Matrix Assisted Laser Desorption agarose gels but these added 1-3 hours Ionisation - Time of Flight (MALDI-TOF) to assay) Mass Spectrometry Real-time (qPCR) assays use molecular ○ Energy is transferred to matrix beacons - single - stranded probes which from a laser beam have a DNA recognition sequence with a ○ Matrix employed has a fluorescent dye at one end and quencher chromophore which absorbs at at other the wavelength of the laser ○ If recognition sequence matches ○ Matrix absorbs a pulse of energy DNA, probe opens up and and undergoes rapid heating fluorescence is no longer which leads to the vaporisation quenched and ionisation of the analyte molecules Real-time PCR detection of MRSA in a ○ Molecular weights of ions mixture of staphylococci analysed by time taken to reach 98.7% of 1,657 MRSA isolates were detector detected in under 1 hour using this technique Only 26 of 569 methicillin-susceptible S. aureus (MSSA) strains were mistakenly identified as MRSA  Discovery of the mecC gene (a homologue of the mecA gene, that is responsible for methicillin resistance in MRSA) - redesign of PCR assays to improve sensitivity and avoid false negatives Sanger Methods All ions given same kinetic energy (KE) so lighter ions travel faster Spectral fingerprints vary between microorganisms Among the compounds detected in the spectrum, some peaks (molecular masses) are specific to genus, species, and sometimes to subspecies Spectra can be obtained from intact cells MS Detection of MRSA Number of studies have attempted to differentiate MRSA and MSSA MS profiles are different, with MRSA containing more peaks Point-of-Care (POC) Tests/Devices No specific profile for MRSA but POC test: is performed near the patient individual strains have very similar or treatment facility, has a fast profiles turnaround time, and may lead to change in patient management MS Detection of Carbapenemase activity Do not require access to centralised laboratory facilities and should be sufficiently rapid to allow clinically meaningful (e.g., the initiation of directed, as opposed to empirical, antibacterial treatment) to be implemented at the place at which the patient is being treated WHO Criteria Whole genome sequencing (WGS) Affordable WGS provides comprehensive Sensitive information allowing for the: Specific ○ Identification of pathogens User-friendly ○ Exact profiling of resistance Rapid and robust genes Equipment-free ○ Recognition of outbreaks and Deliverable ○ The immediate design of PCR probes based on the generated Biorecognition elements used as biosensors in genetic data in the event of POC devices outbreaks Requires culturing of clinical samples  Magnetic Nanoparticles for Detection of S. aureus protease 1. Black coloured MNPs incorporating a terminal carboxyl group are conjugated to the N-terminus of a peptide substrate for S. aureus protease 2. Immobilisation on a gold sensor platform (Au-S interaction) 3. Biosensor exposed to S. aureus, enzymatic cleavage of the amide bond releases the nanoparticle Electrochemiluminescence (ECL) detection 4. Nanoparticles are attracted to external of antibody sandwich complex magnets located at the back of the sensor platform and so expose the gold coloured surface The colour change from black to gold occurs in 1 minute and can be detected with the naked eye Aptamer-based Latent TB Infection Test Smartphone app using colorimetric detection of a 3’-biotin-labelled aptamer which recognises mannose-capped lipoarabinomannan (ManLAM), a glycolipid from the M. tuberculosis cell wall ○ M. tuberculosis (Mtb) immobilised on a nitrocellulose membrane. ○ Incubated with the biotin-labelled aptamer, followed by streptavidin-labelled horseradish Requirements of a bacterial surveillance peroxidase (HRP). method ○ Quantitation uses the oxidation of Sensitive (correctly identifies the colourless microorganism) 3,3′,5,5′-tetramethylbenzidine Specific (identifies ONLY microorganism (TMB) reduced (red) to its blue of interest) oxidised (ox) form by HRP in the Rapid, reliable, and cost-effective presence of hydrogen peroxide. Simple to perform (does not require Aptamer is specific for binding to specialist interpretation) ManLAM so does not detect other bacteria, including E. coli, S. aureus and E. faecalis; quantitation limit of 10^4 CFU/mL and can be performed in 5 hours. NANOSCALE DRUG DELIVERY SYSTEMS The scale of things What is special about nanoscale? Atoms and molecules are generally less Historical timeline of clinical-stage than one nanometer → chemistry nanoparticle technologies Condensed matter physics deals with solids with infinite array of bound atoms Nanoscience deals with the in-between meso-world Surface to volume ratio Size-dependent properties Percentage of surface atoms Lipid nanoparticle-mRNA formulations as COVID-19 vaccines Nanoscale = high ratio of surface area to volume Recombinant hepatitis B vaccine Licensed in US in 1986, and was the first Gold NPs licensed vaccine in the US produced by Melting point recombinant DNA technology Start from an energy balance; assume Recombinant hep B vaccine is produced the change in internal energy (∆U) and by inserting a plasmid containing the change in entropy per unit mass during gene for hep B surface antigen (HBsAg) melting are independent of temperature into a recombinant strain of common Melting point depends on particle size baker’s yeast (Saccharomyces ○ E.g., the melting point of gold cerevisiae) particles decreases dramatically The antigen is harvested and purified as the particle size gets below 5 from fermentation cultures of the nm recombinant yeast containing the gene  Localised surface plasmon resonance When the resonance is coherent with that of the external optical field, nanoparticles exhibit strong absorption at this wavelength of the external field Absorption is transferred into heat Why are gold nanoparticles dispersion red in colour? Surface plasmon resonance (SPR) and localised surface plasmon resonance (LSPR) Localised surface plasmon resonance (LSPR) is SPS on nanosized structures Gold nanoparticles as a quencher Surface plasmon resonance (SPR) is the collective oscillation of conduction band electrons that are in resonance with the oscillating electric fields of incident light, which will produce energetic plasmonic electrons through non-radiative excitation Size dependence Surface plasmon resonance of metal nanoparticles red-shifts with increasing nanoparticle sizes Uneven excitation of free electrons for bigger nanoparticles leads to the retardation effect and correspondingly the red-shift (move the longer wavelength) of the SPR Shape effect Quantum size effect Au nanorods exhibit both transverse and When the size of the QD is smaller than longitudinal surface plasmon resonances the excitation bohr radius (electron and (along their two axes) hole), energy band gap of the QD Their longitudinal peaks are very exhibits strong quantum size sensitive to their aspect ratio (L/W ratio - confinement and becomes larger ratio leads to longer wavelength) size-dependent and can cover a wide spectral range Energy level of QDs Aggregation state The band gap of QDs is size-dependent Surface plasmon band red-shifts upon and decrease with increasing sizes aggregation of nanoparticles due to Smaller band gap corresponds to longer interparticle coupling wavelength For 14 nm Au nanoparticles, the dispersion colour experiences red-to-blue transition during aggregation, which is widely used in Au nanoparticle-based colorimetric biosensors Quantum dots (QD)s Semiconductor quantum dots A QD is a semiconductor nanostructure that confines the motion of conduction band electrons, valence band holes in all three spatial directions Quantum dots emits light when electrons that were excited into the conduction return to valence band and combine with holes Light that can excite smaller QDs also can excite larger QDs QD emission colour evolves from blue, green to red when its size increases Optical properties  Phospholipids bilayers spontaneously close in on themselves to form sealed compartments, which are called liposomes Methods for the preparation of liposomes Liposomes and lipid NPs What is a liposome? Spherical vesicles with a phospholipid bilayer Liposomes are concentric bi-layered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids Doxil (FDA 1995) for AIDS-related kaposi Phospholipids and cholesterol sarcoma, multiple myeloma, ovarian cancer (IV) High concentration up to 2:1 molar ratios Doxorubicin hydrochloride encapsulated of cholesterol to PC in Stealth liposomes (100 nm) composed Hydroxyl group of cholesterol orientated of N(carbonyl-methoxypolyethylene towards the aqueous surface and glycol aliphatic chain aligned parallel to the acyl 2000)-1,2-distearoyl-sn-glycero3-phosph chains in the center of the bilayer oethanolamine sodium, fully hydrogenated soy phosphatidylcholine, and cholesterol Current liposomal drug preparations The bilayer phospholipids theory Liposomes are formed when thin phospholipid films are hydrated Liposomes vs lipid nanoparticles Liposomes are spherical vesicles formed mainly by phospholipids and other physiologic lipids Lipid nanoparticles are solid particles are room and body temp, consisting of solid lipids (SLN) or a mixture of solid lipid and Bottom up: nanoprecipitation a liquid lipid (NLC) Liposomes and lipid nanoparticles are similar by design, but slight different in composition and function LNPs are liposome-like structures especially geared towards encapsulating nucleic acids (RNA and DNA) Traditional liposomes include one or more rings of lipid bilayer surrounding an aqueous pocket, but not all LNPs have a Bottom up: spray-drying contiguous bilayer that would qualify them as lipid vesicles or liposomes Top-down: PRINT (Particle Replication in Non-wetting Templates) Development of LNPs using microfluidics Fabrications Bottom up: self-assembling delivery systems Amphiphilic molecules The next advance was to construct ○ Surfactant materials that would self assemble with ○ Lipid drugs to create controlled drug delivery ○ Block-copolymer vehicles Self assembly is typically approach using Liposome formulation a molecule that has a hydrophilic head and hydrophobic tail to form a shell Self assembly E.g, doxorubicin Bottom up: ruby red colloidal gold Graphene - electrochemical exfoliation Surface engineering Water-solubility Colloidal stability Surface-functionality Repulsion of surface charges on Nanoparticle-cell interaction nanoparticles surface prevents the Bio-distribution and PK nanoparticles to aggregate Increase of salt concentration leads to Stabilising mechanisms for nanoparticles in thinner electrical double layer and thus aqueous medium reduced electrostatic repulsion (poorer Key features of successful surface colloidal stability) coating ○ Highly stable at high-salt medium ○ Resistant to non-specific protein bindings ○ Easy bioconjugation schemes Polymer ligands: steric stabilisation As the distance of separation between the particles decreases in the coagulation step, the polymer coatings Charged-stabilised nanoparticles begin to overlap, which is entropically Electrical double layer refers to two unfavourable, and thus push the parallel layers of charge surrounding the nanoparticles away from each other object Ultimately, it is the crowding of the The first layer, the surface charge (either polymer chains within this overlap positive or negative), comprises ions volume that produces the steric absorbed directly onto the object due to stabilisation effect a host of chemical interactions Polymer ligand should have good The second layer is composed of ions solubility in aqueous medium attracted to the surface charge via the Higher graft density of polymer ligands coulomb force, electrically screening the leads to better stabilisation (stronger first layer steric stabilising effect) The Stern Layer is the first (internal) layer of the electric double layer, which forms at a charged surface in an ionic solution Protein corona on nanoparticles: non-specific binding Nanoparticles, when exposed to biological fluid, became coated with proteins and other bio-molecules to form a ‘protein corona’ The non-specific binding from a monolayer on nanoparticles surfaces and has micromolar affinity, and this non-specific binding can be detrimental to the built-in molecular recognition in the nanoparticles PEGylation: one way to reduce non-specific binding PEGylation: the process by which poly(ethylene glycol) chains are attached to carriers The ability of PEG to reduce non-specific protein binding is believed to result from the preferred (polar) gauche Nanocarriers as an emerging platform for conformation of PEG in water, which cancer therapy offers two hydrogen bond acceptors in The peptide VATANST (STP) can ideal distance for hydrogen bonding with specifically bind with vimentin, which is water highly expressed on the osteosarcoma This conformation leads to extensive cell surface hydration in aqueous environments The nanoparticle is core-shell structured which, along with good conformational to protect the loaded DOX during blood flexibility and high chain mobility, causes flow a steric exclusion effect that prevents the THe disulfide bonds within the absorption of proteins nanoparticles are sensitive to the osteosarcoma microenvironment, which Nanomedicine has high glutathione (GSH) levels Under the enhanced permeability and retention and active tumour targeting effects, the STP-decorated DOX-loaded NPs accumulated in tumour tissues High GSH levels can rupture disulfide bonds, resulting in the controlled release of DOX, which will cause necrosis of tumour cells  The nanoparticles could increase the tumour inhibition efficiency against osteosarcoma and reduce the side effects of DOX to major organs Advantages of nanoparticles for drug delivery Improved bioavailability by enhancing ADME - Summary aqueous solubility Nanoparticles: absorbed through Suitable for intravenous delivery endosomal process; in circulation longer Increase circulation time in the body time, less toxicity (targeting effect) Extravasate through the endothelium in inflammatory sites, epithelium (e.g., Nanoparticles enhanced targeting intestinal tract and liver), tumour, or Particle sizes ranging from 10 nm and penetrate microcapillaries 200 nm Allows for efficient uptake by a variety of Particle size alone can affect cell types and selective drug biodistribution accumulation at target sites ○ Particles less than 10 nm are 400 time more likely to cross the Naked drugs vs formulated drugs with intestinal wall than 1-micron nanoparticles particles ○ Particles between 1-10 micron deposit in the deep lung via impaction while other escape during breath or deposit in the mouth Nanoparticle too small - can escape through the breath - too big, can not reach the deep lung ○ Particle below 500 nm can escape the filtering of the liver and kidney for several cycles Nanoparticle are highly charged coupled with surface to volume ratio - this Sequential biological barriers to property can affected cellular interaction nanoparticles Nanoparticle design for overcoming delivery Gene delivery vectors barriers Viral: harmless and not cause disease ○ DNA is inserted into a virus which is then sent to a target cell ○ Retrovirus ○ Adenovirus ○ Adeno-Associated virus ○ Herpes simplex virus Non-viral ○ DNA in a circular plasmid is sent into a target cell PLGA PRINT particles ○ Liposome Print method: make different ○ Polymers nanoparticles ○ Naked DNA ○ Physical methods Example: insert transgene into adenovirus Cell association, biodistribution, tumour accumulation First generation of Ad vectors were engineered by replacing the E1A/E1B region with transgene 2nd generation: with further deleting the other early gene regions (E2a, E2b, or E4), permitting additional space for larger transgene cassettes Gene delivery 3rd generation vectors, referred to as Central dogma of molecular biology ‘gutless’ or ‘helper-dependent’ vectors, Genes are template to make proteins have all viral sequences deleted, except Introduce genes to make proteins such for the inverted terminal repeat as antibodies to target viruses sequences (ITR) and the packaging signal Gene therapy: cystic fibrosis A transgene is a gene that has been transferred naturally, or by genetic  engineering techniques, from one 2. The transgene of interest which organism to another when expressed in cells, serves to confer a desired effect Viral gene therapy modalities 3. The “regulatory cassette”, the In vivo gene therapy entails the direct combined administration of vector carrying a enhancer/promoter/auxiliary therapeutic transgene into the patient elements that controls stable or Ex vivo gene therapy involves extraction transient somatic expression of of a patient’s cells or from an allogenic the transgene as an episome or source, genetic modification by a vector as a chromosomal integrant carrying a therapeutic transgene, selection and expansion in culture, and infusion to re-introduce the engineered cells back into the patient Four gene therapy approaches as follows ○ Gene replacement: the delivery of a functional gene to replace a non-working gene ○ Gene silencing: inactivation of a mutated gene that has become toxic to cells ○ Gene addition: overexpression of a “foreign” or exogenous gene to Viral based vectors impact cellular function Adeno-Associated Viral (AAV) Vectors ○ Gene editing: a permanent AAVs are usually non-integrating = DNA manipulation of a gene in a they carry doesn’t typically insert itself patient’s genome into the cell’s genome If the vector is taken up by a cell that divides, the therapeutic gene won’t be copied with each cell division and may be lost over time, thereby diluting the treatment effect AAVs are commonly used to target non-dividing cells, such as cells in the liver, nervous system, eyes and skeletal muscles AAVs can persist in patients for a prolonged period - possibly even a lifetime Adenoviral vectors are like AAV vectors Lentiviral and retroviral vectors Viral vector for gene delivery Can carry larger genetic packages of Typically a viral vector is defined by three RNA, which is converted into DNA components as follows: During this process, the vectors integrate 1. The protein capsid and/or into the genome of the target cell envelope that encapsulates the The ability to integrate into the cell genetic payload, and defines the genome makes lentiviral and retroviral vector’s tissue or cell tropism and vectors best suited for dividing cells, antigen recognition which are targets for an ex vivo treatment approach  For example, these vectors are used to Use restriction endonucleases to digest Target T cells, which are a type of PCR product and Vector DNA immune cell, and stem cells, which are Purify expected fragments from digestion special cells that can develop into many mixes cell types Use ligase to ligate the fragments from source DNA and the fragment from vector DNA Non-viral vectors: physical methods First FDA-approved gene therapy (2017) Inside eye, the RPE65 gene provides instructions to make a protein for normal Gene gun vision People with RPE65 gene mutation cannot make the protein Luxturna loads normal genes (a DNA strand) into a vector, an inactivated AAV2 virus The vector, injected into the eye, delivers the normal RPE65 gene to eye to make the protein to restore eyesight FDA-approved gene and cellular therapy Non-viral gene delivery: chemical methods products Cationic lipid-based delivery system Lipofectamine consists of a 3:1 mixture Molecular cloning of DOSPA Use PCR reaction to amplify a DNA (2,3-dioleoyloxy-N-[2(sperminecarboxami fragment  do)ethyl]-N,N-dimethyl-1-propanimunium trifluoroacetate) and DOPE They complex with negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane Structure and function of small interfering RNAs RNA interferences (RNAi) is a biological Lipid nanoparticles for siRNA delivery process in which RNA molecules inhibit Small interfering RNA (siRNA) is an RNA gene expression, typically by causing the sequence destruction of specific mRNA molecules ○ In vivo degradation by nucleases Two types of small ribonucleic acid (half-life ~15 min in serum) (RNA) molecules - microRNA (miRNA) ○ Renal clearance and small interfering RNA (siRNA) - are ○ Too negatively charged to cross central to RNA interferences cell membranes to enter cells Long double-stranded (ds)RNA or hairpin RNA substrates are cut by Dicer into smaller (~21-nucleotide (nt)) small interfering (si)RNAs Alternatively, siRNA duplexes (19-23 nt) can be introduced into cells, where they are phosphorylated at the 5’ ends by cellular kinases These small dsRNAs assemble into the First siRNA drugs approved by US FDA RNA-induced silencing complex (RISC), (2018) which contains AGO2, Dicer and other Patisiran is a medication for peripheral cellular factors nerve disease (polyneuropathy) Then siRNA forms activated RISC Polyneuropathy is a rare, debilitating, (siRISC) that contains an antisense and often fatal genetic disease affecting (guide) strand approx 50,000 people worldwide, Activated RISC finds its target mRNA affecting peripheral nerves (peripheral and uses the antisense strand to guide neuropathy) in roughly the same areas cleavage of the target mRNA on both sides of the body, featuring RISC is recycled and could carry out weakness, numbness and burning pain several cleavage events It is characterised by the buildup of abnormal amyloid protein in peripheral nerves, the heart and other organs The FDA has approved infusions of Patisiran to treat peripheral nerve disease caused by hereditary  transthyretin-mediated amyloidosis Both Comirnaty and the Moderna (hATTR) vaccine deliver synthetic mRNA Is is the first-ever approval of a new molecules into cells, instructing them to class of drugs, small interfering make antigens, or foreign invaders that ribonucleic acid (siRNA), used to treat the immune system recognises as not rare and genetic diseases being part of itself Cells are instructed to make only the Deliver siRNA using lipid nanoparticles - SARS CoV-2 spike protein, which is just patisiran enough to activate the immune system 1. After IV administration of patisiran, PEG Then, if a person is exposed to dissociates from the LNP COVID-19, the immune system will 2. Removal of the PEF coating allows detect the familiar antigens and produce endogenous ApoE to associated with the antibodies to attack them LNP, facilitating uptake into hepatocytes Because the vaccine introduces new via an ApoE-dependent process genetic material into cells for a short 3. On internalisation via endocytosis, the period of time to induce antibodies, it is a ionisable lipid is protonated (positively gene therapy as defined by ASGCT charged), as the pH decreases in the The mRNA in Comirnaty does not alter endosome the recipient’s genetic material and is 4. The positively charged lipid reacts with only present in the body transiently the negatively charged endosomal lipid, resulting in disintegration of the LNP, First mRNA vaccine delivered using lipid destabilisation of the endosome nanoparticles membrane, and release of the siRNA The siRNA is very different from mRNA into cytoplasm in structure and size 5. The siRNA binds RNA-induced silencing mRNA is at least 100-fold larger than complex (RISC), leading to the targeted siRNA, and this affects the structure of degradation of TTR mRNA and the LNPs subsequent reduction in the target The mRNA is in th core of the LNPs protein levels while siRNA is more towards the surface 6. A proportion of internalised LNPs Currently, mRNA-LNPs are best undergo exocytosis egress from late described by the core-shell model (b & c) endosome/lysosomes back into the and the nanoparticles have a surface circulation layer and an amorphous, isotropic core The lipids in th core could be homogeneously dispersed with small water pockets in between (c) First mRNA vaccine approved by US FDA (2021) First ever mRNA vaccine or drug to receive approval  chains or replacement of the sugar residues e.g., PEGylation The first generation of therapeutic proteins The second generation of therapeutic proteins Protein/peptide therapeutics Global Protein Therapeutics Market Size was Valued at USD 341.4 Billion in 2023 Worldwide Protein Therapeutics Market Size is expected to reach USD 605.38 Billion by 2023 Nanotechnology for protein delivery Protein degrade Protein must be injected - they are not taken orally (digest in the GI tract) Proteins rapidly degrade in the body by natural mechanisms This means that to have a sustained affect patients must endure many injections Protein/peptide therapeutics Oral delivery of insulin The 1st generation has the native sequences, identical with the proteins/peptides naturally occurring in the body tissues e.g., insulin The 2nd generation has improved properties, especially PK, biodistribution, higher specificity, efficacy and minimised side effects To achieve these desired features, the structures are deliberately modified by covalent attachment of the chemical compounds, introduction of certain changes within the sequences of the protein, fusions of two or more peptide  (changes in pH, enzyme concentration or redox gradients) ThermoDox (Celsion) Breast cancer recurrence at chest wall (microwave hypothermia) Hepatocellular carcinoma (radiofrequency ablation) Traditional insulin replacement therapy Liver tumours (mild hypothermia) Refractory solid tumours (magnetic resonance high intensity focused ultrasound) Glucose-responsive nanoparticles for insulin delivery Liposomal encapsulation of doxorubicin Third generation: nanotechnology-based protein and peptide delivery Stimuli-responsive nanocarriers for drug delivery To respond to specific stimuli, either exogenous (variations in temperature, magnetic field, ultrasound intensity, light or electric pulses) or endogenous Glucagon-like peptide-1 (GLP-1) Mechanism of oral absorption of semaglutide Glucagon-like peptide-1 (GLP-1) is a As the tablet is eroded, SNAC causes a 30-amino acid peptide hormone, which is local increase in pH via a buffering action secreted by enteroendocrine L cells in This increases in gastric pH may protect the GIT and by pre-proglucagon neurons semaglutide from enzymatic degradation located in the nucleus of the solitary tract by reducing the conversion of in the hindbrain pepsinogen to pepsin GLP-1 promotes satiety and reduces In addition, SNAC promotes energy intake by virtue of its monomerisation of semaglutide by neurotransmitter role in weakening the hydrophobic interactions brainstem-hypothalamus pathways that would otherwise promote signalling satiety semaglutide oligomerization Pharmacologically, GLP-1 receptor The enhanced absorption of semaglutide agonists exhibit glucoregulatory is due to the indirect action of SNAC, functions by: which is incorporated into the lipid ○ Stimulation of insulin release in a membrane of local gastric cells and glucose-dependent manner fluidized the plasma membrane of the ○ Suppression of glucagon activity gastric epithelium (a solid-to-fluid during hyperglycemia (glucagon structural transition), allowing is a peptide hormone to stimulate transcellular passage of semaglutide glucose production in the liver The action is transient and reversible and thereby to maintain adequate plasma glucose concentration) Semaglutide oral administration experiment ○ A minor delay of gastric emptying data resulting in slower glucose absorption Stomach absorption of semaglutide An oral formulation of semaglutide, a GLP-1 receptor agonist, was approved by the FDA in 2019 as a peptide therapy for the treatment of type 2 diabetes It works by activating GLP-1 receptors in the pancreas, which leads to enhanced Semaglutide absorption via stomach not insulin release and reduced glucagon intestine release-responses that are both glucose-dependent-with a consequent low risk for hypoglycemia A new era for oral peptides The development of an oral semaglutide formulation with a similar exposure-response relationship to the well-established injectable formulation of semaglutide is a significant milestone in  the treatment of T2D and in overcoming the barriers to oral peptide therapy The availability of the first oral GLP-1RA not only expands the repertoire of highly effective treatments for patients with T2D but may also mark the beginning of a new era for oral peptides REGULATION Current directions and influence on pharma industry Science ○ Oncology ○ Rare disease ○ Cell and gene therapy ○ mRNA-based therapies ○ AI in drug development ○ Precision medicine and data Policies ○ WHO: universal health coverage ○ WHO: future pandemic Pharmaceuticals - traditional development preparedness (pandemic treaty) pathway ○ Sovereign manufacturing capability ○ Industrial and innovation ○ Health funding and regulatory ○ Science and research ○ Export and trade Corporate ○ Value ○ ESG and climate change impacts ○ EDI and increasing the patient voice Product development plan ○ New ways of working “If it’s not documented, it didn’t happen” ○ Digital integration ○ Codes of conduct and ethics ○ Transparency ○ Business and corporate policies Top therapeutic classes by sales - 2023 Top Medtech market segments - 2021 Medical devices development pathway Initiation Formulation Design and development Verification and validation Manufacturing and testing Clinical development Expenses and revenues curve for a new Regulatory submissions medicine Launch Post-market compliance  Reimbursement Development, maintenance and monitoring systems for listing of Legal basis of medicines regulation in medicines Australia Assessment of medicines for export Definition of a therapeutic good A good which is represented in any way to be, or is likely to be taken to be, for therapeutic use (unless specifically excluded or included under Section 7 of the Therapeutic Goods Act 1989) “Therapeutic use” means use in or in connection with: ○ Preventing, diagnosing, curing or alleviating a disease, ailment, defect or injury Australian Legislation ○ Influencing, inhibiting or Therapeutic Goods Act, 1989 modifying a physiological process ○ To provide a national framework ○ Testing the susceptibility of for the regulation of therapeutic persons to a disease or ailment goods so as to ensure their ○ Influencing, controlling or quality, safety and efficacy and preventing conception timely availability ○ Testing for pregnancy or ○ Replacement or modification of parts of the anatomy Classes of therapeutic goods Prescription medicines OTC or non-prescription medicines Complementary medicines Sunscreens Legislative instruments Medical devices and in vitro diagnostics ○ Therapeutic Goods Regulations, (IVDs) 1990 Biologics ○ Therapeutic Goods Orders Blood and blood components (various) Australian Regulatory Guidelines for Risk-based approach to regulation Prescription Medicines Australian Regulatory Guidelines for Medical Devices Australian Regulatory Guidelines for Biologicals Adopted International guidelines Therapeutic Goods Administration (TGA) Pre-market evaluation and approval of therapeutic goods Licensing of manufacturers (GMP) Post-market surveillance through sampling Adverse event reporting Medicine Scheduling  Submission basics Which registration pathway? Inclusion on ARTG All goods must be entered in the ARTG before they can be supplied in, imported to, or exported from Australia Standard/PR/PA Pathway Elements Prescription Medicines Registration Process Designations and determinations - orphan, priority review, provisional approval Medical Devices A medical device is any material instrument, apparatus, appliance, implant etc, including any component  part of accessory including software, and ○ Declaration of Conformity in-vitro diagnostics, which is used in ○ Manufacturer's evidence health care (compliance with Essential Principles) Medical device classification Submit application for inclusion Print out your ARTG certificate Supply your device In vitro diagnostic (IVD) medical device definition A medical device that is: ○ A reagent, calibrator, control material, kit, specimen receptacle, software, instrument, apparatus, equipment or system, whether used alone or in combination with another Essential Principles diagnostic product for in vitro use Fundamental regulatory requirements ○ Intended by the manufacturer to List in Schedule 1, Therapeutic Goods be used in vitro for the (Medical Devices) Regulations 2002 examination of a specimen Composed of: derived from the human body, ○ General principles to show solely or principally for: benefits outweigh risks Giving information about a ○ Principles regarding design and physiological or construction pathological state or a ○ Principles regarding labelling congenital abnormality or ○ Principles regarding standards Determining safety and and testing of the device compatibility with a potential recipient Conformity Assessment Procedures Monitoring therapeutic Schedule 3, Therapeutic Goods (Medical measure Devices) Regulations 2002 ○ Not a product that is: Help demonstrate compliance with the Intended for general Essential Principles laboratory use Quality Management System Not manufactured, sold or requirements e.g., ISO 13485 presented for use as an Design examination IVD medical device Third party assessment Manufacturer evidence: IVD regulation by TGA ○ Declaration of conformity ○ Conformity assessment certificate Supplying a medical device in Australia Confirm your product is a medical device that needs to be included in the ARTG Determine the kind of medical device - check GMDN code Determine the classification of the medical device - which Class? Prepare all documents required for Case study medical device inclusion: Biologicals Cell therapies A biological comprises, contains or is Alliance for Regenerative Medicine H1 derived from human cells or human 2022 report: tissues (or specified by the Secretary to https://alliancerm.org/sector-snapshot-ap be a biological) ril-2024/ And is used to: Project A-CELL: ○ Treat or prevent disease, ailment, https://alliancerm.org/manufacturing/a-ce defect or injury ll-2022 ○ Diagnose the condition of a person Genetically modified organisms (GMOs) ○ Influence, inhibit or modify a Regulatory integration of GMOs physiological process in persons ○ Test the susceptibility of persons to a disease or ailment ○ Replace or modify parts of the anatomy in persons Biologics are grouped into classes based on level of risk Gene technology (GT) Act 2000 Application process (Class 1) Passed in December 2000 Must be listed in Schedule 16 of Came into effect on 21 June 2001 Therapeutic Goods Regulation, 1990 A national scheme for the regulation of Currently only faecal microbiota GMOs transplant (FMT) material listed Legislation mirrored in State/Territory Submit application form Acts Pay appropriate fees GT Regulations 2001 - day to day GMP not required information on operation of the Act User’s Guide - plain English aid to Application process (Class 2, 3 or 4) interpretations of Act, Regulations What is a GMO? An organism that has been modified by GT An organism that has inherited modified traits from a modified organisms Anything declared by the regulations to be a GMO Does not include humans who have received somatic cell gene therapy or an organism declared by the regulations not to be a GMO What are dealings with GMOs? Propagate the GMO Make, develop, produce or manufacture the GMO Grow, raise or culture Breed the GMO Use the GMO in the course of manufacturer of a thing that is not a GMO Importation Future trends in therapeutic product development GMO Authorisations Cell and Gene Therapy Products Delivery methods for gene therapy Add slides from iPad Regulatory pathways Genetically modified human therapeutics Class 4 biological cell therapies - examples MEDICATION ACCESS PATHWAYS S19A Access to drugs in Australia Section 19A of the Therapeutic Goods TGA registered Act 1989 SAS ○ Temporary approvals to import or Authorised Prescribe supply a medicine that is not Clinical Trials registered on the ARTG Personal Importation Unapproved therapeutic goods SSSI Serious scarcity substitution instrument Registered Medications Allow for schedule 4 medications to be Therapeutic Goods Administration (TGA) substituted ○ Australian Register of ○ Lower or higher strength Therapeutic Goods (ARTG) ○ IR vs SR Safe and effective for a specified ○ Different salts indication Legal to market, prescribe, dispense National Medicines Policy Availability according to Schedule S2, Quality use of medicines S3, S4, S8 Framework based on partnerships Off label use - non approved indication between Governments, health Unapproved drugs cannot be advertised educators, pharmaceutical industry and health practitioners Structure of drugs in clinical practice TGA - quality and safety of products PBS PBS - access (subsidy) Origins Pharmaceutical industry - Purpose innovator/manufacturer of drug product ○ Section 85 (General Schedule) Health practitioners - prescribing habits and Section 100 (National Health Act 1953) Personal Importation Process Importing unapproved therapeutic goods Criteria for subsidy for personal use or use by someone in your immediate family Pharmaceutical Benefits Scheme The following must be met: TGA registration required evidence of ○ The goods are an individual or safety and efficacy the treatment of immediate family ○ Only approved for a TGA that you are travelling with (such approved indication as a parent travelling with their PBS listing also required cost child’s medicine) effectiveness ○ They are not sold or given to An acceptably cost-effective medicine another person can be recommended for listing if: ○ Where possible, the medicines ○ It treats or prevents significant are kept in their original medical conditions that are not packaging covered, or only partially covered, ○ The goods do not contain a by currently listed drug(s) controlled substance ○ It is more effective and/or less ○ The total quantity of the goods harmful than a currently listed imported within a 12-month drug period does not exceed ○ It is as effective and safe as an 15-month’s supply existing listed drug ○ If the goods are medicines in S4 or S7 of the Poisons standard, a Shortage prescription from an Australian-registered MP is held  ○ You cannot import more than a 3-month supply per order ○ If you wish to bring more than 3-month supply into Australian, an Australian-registered doctor will need to apply for SAS approval Special Access Scheme Provides import and supply of an unapproved therapeutic good to a single patient on a case by case basis Clinical trials Approvals required: ○ Animal or Human Ethics Research Approval (HREC) ○ TGA Clinical Trial notification Categories (CTN) scheme (allows Category A: notification for a patient importation) defined as seriously ill Or CTA (uncommon) Category B: application pathway ○ Governance approval Category C: notification of use of ○ Drug committee approvals specified therapeutic goods ○ MOH equivalent health department approval Authorised prescriber ○ OTGR approvals To apply for an individual patient → ○ IBC approvals (SAS) For multiple patients with same condition Important points to note → authorised prescriber Conduction a trial still has to comply with: Required endorsement from the local ○ Australian laws human research committee (HREC) for Therapeutic Goods Act those goods not on the established 1989 history of use list Therapeutic Goods Involves reporting on: Regulation 1990 ○ Any suspected AE or product ○ State based defects Medicines, Poisons and ○ Number of patients treated every Therapeutic Goods Act 6 months 2022 Only available to medical practitioners Poisons and Therapeutic Can only prescribe once formal approval Goods Regulation letter is received ○ International guidelines PIC/s annex 13 Who can prescribe ICH GCP ○ Local policies Advanced therapeutics policy Medication handling policy ○ SUSMP ○ PBS Evaluation of New Drugs Clinical trials  Pre-evaluation Genotoxicity and ○ Early pharmacological studies carcinogenicity studies Clinical evaluation - HUMAN studies Reproductive toxicity ○ Pre-registration studies ○ Post-registration Phase 1 studies Phases of clinical trials First in man Human pharmacology Small number (10-100) of participants (cohorts) Highly monitored, specialist units These studies are investigating ○ Safety ○ Toxicity ○ Maximum tolerated dose ○ PK/PD Highly pretreated patients OR young healthy volunteers Preclinical evaluation Single ascending dose (Phase 1a) or Involves Multiple ascending dose (Phase 1b) ○ Evaluation of new chemical entity ○ Identification code or generic Phase II studies name given Early phase ○ Application for a patent by the Therapeutic exploratory sponsor 100-300 Preclinical research studies can include; Early pharmacology/efficacy ○ Toxicology screening Dose finding in an indication ○ In vitro cell lines Toxicity monitoring ○ Pharmacogenomic probes Patient in whom no alternative treatment ○ In vivo animal studies is available Preclinical studies Phase III studies Animal studies require animal ethics Comparative studies approval and safety processes Therapeutic confirmatory Aim: to predict clinical (human) 300-3000 patients effectiveness and toxicity Randomised clinical trials (RCT) ○ Design human clinical trials Test hypothesis ○ Pre clinical safety evaluation is ○ Difference between two treatment essential for a safe starting dose arm for phase I studies and ○ Investigational agent vs gold identification of potential adverse standard comparator effects that may occur ○ Design strategies: blinded, ○ Studies include: parallel or crossover design Single dose toxicity studies Trial design Repeat dose toxicity Randomisation studies In clinical trials, participants are generally Safety pharmacology allocated to different arms of the trial (for studies example, to receive the study medicine PK and toxicology studies or the placebo) randomly Local tolerance studies Each participant has an equal chance of being in any of the arms of the trials  It is an important method to reduce the pharmacy and goods are risk of bias in the outcomes of the trial provided to the public Pharmacist employed by a public hospital who manufacture for supply in that organisations for use within the same state or territory A person who applies supplementary labelling to a manufactured product Labelling has to be PIC/s annex 13 compliant Blinding ○ TGO 69, TGO 91 and TGO 92 do Blinding is a procedure in which one or not apply to investigational more parties in a trial are kept unaware agents of which treatment arms participants have been assigned to Pharmacists cannot produce goods for Blinding is an important aspect of any supply via wholesale trial done in order to avoid and prevent ○ I.e., pharmacists can supply PER conscious or unconscious bias in the patient off a script design of a clinical trial Pharmacists cannot make bulk products to ship across state borders Comparator ○ This would be wholesaling A comparator may be on a placebo or on Supplementary labels should be added an active treatment at point of dispensing Placebo control ○ Labelling in bulk required a GMP ○ A placebo is a fake or sham licence treatment specifically designed All other pharmacy laws and regulations without any active element around supply are applicable Active control An exemption DOES NOT mean a site ○ An active control is an has capacity or is willing to take on the established treatment that is risk of phase 1 studies outside of GMP known to have efficacy and is an ○ We require the product to be alternative to a placebo control GMP compliant regardless of phase Placebo effect The placebo effect is a psychosomatic Phase IV effect brought about by relief of fear, Often after registration (i.e., post anxiety or stress because of study marketing approval) participation ○ Postmarketing surveillance Unapproved drugs ○ Therapeutic use ○ Controlled cohort trial vs ‘real Manufacturing and labelling world’ experience Therapeutic Goods Act 1989 ○ 1000’s ○ Investigational agents must be ○ New toxicities/adverse reactions produced under GMP (rare) identified ○ Exemption to part 3-3 Pharmacists who Human subjects in clinical trials: ethical manufactures in a considerations and concerns Hippocratic oath: First do not harm The four pillars of bioethics  ○ Autonomy ○ Investigators ○ Beneficence ○ Pharmacy ○ Non-maleficence ○ Ethics committee review ○ Justice ○ Governance Advertising of any agent not on ARTG is Ethics approval an offence All research, including trails, require ○ Ads reviewed by HREC ethics committee approval National Health and Medical Research Ethics Committees Council (NHMRC) Committees (hospital, university or both) National statement on the Ethical Statutory constitution conduct of human research ○ Researchers The National Statement requires many ○ Clinical practitioners types of human research to undergo ○ Science training ethics review ○ Lay persons It also sets out the requirements for an ○ Minister of religion HREC’s establishment operation and membership Human Research Ethics Committees (HREC) Purpose of ethics review is to advocate National statement for Values and principles in ethical conduct ○ Scientific merit and integrity Themes in research ethics: risk and ○ Ethical framework benefit, consent ○ Risks and benefits Section 3: ethical considerations in the ○ Adherence to international and design, development, review and national guidelines conduct of research Ensure participants of the clinical trial: ○ Give informed consent (written) Ethics under no duress Informed consent ○ Provided appropriate information Placebo about risks, benefits, procedures Randomisation and blinding Clinical trials Clinical equipoise Guideline for good clinical practice Provides the ethical basis for clinical International conference on research that involves assigning patients harmonisation (ICH) of technical to different treatment arms of a clinical requirements for registration of trial pharmaceuticals for human use There is no “better” intervention present ○ International ethical and scientific during the design of the RCT quality standard for designing, A true state of equipoise exists when one conducting, recording and