Case study - Natural Molecules - candidates to drugs

why should we care about traditional medicine and drug discovery?

• majority of global population lives in developing countries

• majority of people in developing countries use plants to meet their primary healthcare needs

what are the 2 different approaches to finding medicinal plants?

• random search

• targeted search

what is a random search? what is the successful for this method?

• a broad net is cast and plants from a specific region are collected and screened for potential medicinal properties

• no regard for taxonomic status, ethnobotanical use or any other quality

• low success rates

give an example of a drug that was discovered using the random search method

taxol = anti cancer drug

what are the different types of targeted searches?

• phylogenetic surveys: close relatives of plants known to produce useful compounds are collected → likely to get positive results

• ecological surveys: plans that live in a particular habitat or have particular characteristics (e.g. immunity to predation) are selected

• ethnobotanical surveys: plants used by indigenous people in traditional medicine for specific diseases are selected → often has positive results

what is the most common plasmodium species that infects human? how dangerous is this species and why?

plasmodium falciparum

• most dangerous → cause of majority of malaria deaths

• can cause cerebral malaria = fatal

describe the life cycle of the plasmodium falciparum

• infected female anopheles mosquito bites human

• sporozoites injected which is taken up by the liver

• develops in the liver

• parasites are released to invade and develop in RBC

• parasites multiplies in RBC :. clinical illness develops

• some parasites do not multiply but become gametocytes to be taken up by mosquitos :. when next mosquito bites infected human, the mosquito becomes infected → parasite develop to sporozoite stage in mosquito

what are some of the complications of falciparum?

• fever

• low blood sugar

• cerebral malaria

• anaemia

what is a distinct adhesive phenotype of P. falciparum? what does this cause?

platelet-dependent clumping

• when parasite is released into the blood to infect new RBCs, they can cause platelet clumping :. blocks capillaries and smaller blood vessels

• there are lots of smaller blood vessels in the brain :. damage to endothelial lining occurs :. cell contents are released

• immune system is activated :. cytokine release :. inflammatory response

• causes cerebral malaria

what is the blood brain barrier maintained by?

• pericytes which attach to the underside of the lumen

• tight junctions between endothelial cells

what was historically first line treatment for malaria?

• quinine

• chloroquine

what caused the global spread of chloroquine resistance in P. falciparum?

• chlorquine acted as a selection pressure :. via natural selection, P. falciparum mutated and produced resistance strands

• resistance strands thrived because there was less intraspecific competition for resources

how were new antimalarials for P. falciparum discovered?

ethnobotanical survey

• started by collecting information on traditional chinese medicine to reduce fever → looked at ancient chinese medical literature, folk recipes, interviewing experienced chinese medical practitioners

• over 2000 potential hits were identified

• screened these potential hits for antimalarial activity

which successful antimalarial drug resulted from this ethnobotanical survey? what is its plant of origin?

artemisinin → comes from qinghaosu plant remedy for fever → qinghaosu is orginally extracted from artemisia annua

what are the 2 parts of the artemisinin structure that are of interest? how do these groups affect the structure-acivity relationship?

• peroxide bridge → break this = no activity :. must be conserved

• carbonyl group → optimising this can improve activity

artemether is derived from artemisinin. how does the structure of artemether differ from artemisinin?

• carbonyl group is converted to ether group → methyl group attached to the O makes the compound more lipophilic :. can directly act on membranes

• different to mode of action of quinones and chloroquines (even though they also have peroxide bridge)

how do quinines work?

• targets parasites when they’re in the RBC stage

• parasite needs a source of iron whilst attacking RBC :. it takes up haemoglobin in vacuoles and breaks this down to access iron

• iron liberated is toxic :. parasite detoxifies it by polymerising it

• quinines prevent polymerisation by breaking their own peroxide bridge :. kills parasite → :. bridge needed for activity

how does artemether work?

• drug is taken up inside parasite

• peroxide bridge is broken :. makes it into a very reactive species → carbon centred free radical

• methyl group attached to O makes drug very lipophilic :. reactive species goes towards mitochondiral membrane of parasite → disrupts mitochondria membrane potential

• :. mitochondria loses function :. no ATP

what are the sources of artemisinin?

rely purely on extraction of backbone frm plant material

• chemical synthesis of artemisinin is not yet competitive in price with the natural source because of its high abundance in the plant

• :. field production of A. annua is the main source

how are plant based substances extracted using liquid-liquid extraction?

• liquid-liquid extraction through various polarities of solvent

• dry off solvent and do solvent exchange

• add more refined polarities of solvent through purification process

• end up with a pure compound which can be crystallised

how is artemisinin extracted frm artermisia annua?

solid-liquid extraction

• take plant material and crush it up (increases SA) and add 1st ethanol solvent

• dry off solvent :. left with raffinate

• re-extract raffinate in hexane = different polarity to ethanol → polar to non polar

• sterilise and filter

• do ethyl acetate chromatography

• crystallisation

how are green chemistry principles applied in the extraction of artemisinin from artemisia annua?

• traditional artemisinin extraction often uses harsh organic solvents that can be harmful to the environment → alternative solvents = carbon dioxide under supercritical conditions, which is a non-toxic

• use of more efficient extraction techniques that reduce energy consumption and waste generation e.g. microwave assisted extraction

what are the main chemical contaminants in the artemisinin extraction process and what unit operation removes these?

• plant lipids and waxes are co-extracted with artemisinin from the Artemisia annua plant → can interfere with purification and reduce the purity of the final product

• chlorophyll →can impart unwanted colour and impurities.

• chromatography is used to remove these

why is artemisinin no longer used as monotherapy for P. falciparum malaria?

• have to wait a long time before we get signficant undetectable level

• artemisinin is active against resistance strains but it takes ~2 weeks to do this and takes 1 week to clear sensitive organisms → 7 day treatment is too long → if patient stops taking treatment prematurely, recrudescence can occur

what is recrudescence of P. falciparum?

return of malaria symptoms after a period of apparent clearance of the infection due to the survival and multiplication of the original population of parasites in the host's bloodstream

• occurs when concentration of antimalarial drug in the blood below the threshold required to eliminate all parasites → can be due to: inadequate dosing, poor adherence, resistance

• often believe the infection has cleared however there is small number present → below limit of detection :. cannot decect them → test is not sensitive enough

what is the difference between recrudescence and relapse?

• relapse: occurs when dormant liver-stage parasites (hypnozoites) of Plasmodium vivax or Plasmodium ovale become reactivated and re-enter the bloodstream

• recrudescence: parasites that caused the initial infection were not fully killed and begin to multiply again

how is the peroxide bridge decomposed in artemisinin?

haem mediated

• decomposition catalysed under influence of iron going from ferrous to ferric state (Fe²⁺ → Fe³⁺)

• breaking of bridge gives the carbon centred free radical

what artemisinin resistance in P. falciparum associated with?

polymorphisms encoding the K13 propeller domain

• parasite sequesters haemoglobin from inside RBC using active endocytosis → vesicle pinches off from RBC membrane using K13 protein

describe the PK of artemisinin. what does this mean for the treatment regimen?

• fast clearance → C_max is in less than 1 day :. combination therapy required

• need secondary drug that isn’t as potent as artemisinin (:. prevent resistance) but is longer acting

• :. artemisinin will kill off most of the parasites (rapid initial parasite clearance) and secondary drug will kill off remaining undetectable parasites :. prevents recrudescence

• Artemisinin-based Combination Therapy (ACT) = current standard treatment

what is a co-formulated drug?

2 different drugs are combined in 1 tablet

give an example of a companion drug in artemisnin-based combination therapy (ACT). how does it work?

lumefantrine

• interfere with this haem detoxification process by inhibiting the formation of hemozoin crystals → buildup of toxic haem within the parasite :. death

what are the APIs in coartem?

artmether and lumefantrine

why should coartem be given for at least 3 days?

timeframe in which artemether works and lemefantrine will stay in system long enough to have appropriate kill effect (7 days)

which has a better cure rate: 6 dose or 4 dose regimens or coartem? 3 day course or 5 day course?

6 dose

• highly effective and very well tolerated

• 5 day regimen is better than 3 days but may not be economically feasible for some patients :. want to ensure 3 day regimen is suitable

which region and population group is most vulnerable to infection by P. falciparum?

• sub-saharan africa bears highest burden

• children are most vulnerable

why do children often have their own formulation of coartem ACT therapy? which formulation is child friendly?

• tablets are too big and have bitter taste → crushing can lead to under dosing

• instead use dispersible tablets are suitable for all ages with sweet tasting additives to mask bitterness