Anthelmintics Flashcards
LO
- Briefly state the importance of anthelmintic therapy for human health. Describe common animal parasites of humans and the pathophysiology associated with infection
- Describe the mechanism of action of levamisole, piperazine and ivermectin and how these drugs achieve selective toxicity
- Have an appreciation of the different experimental approaches used to assess selective toxicity for anthelmintics and the use of C. elegans for mode of action studies for new resistance breaking anthelmintics
Papers to read
ivermectin papers:
- ivermectin review.pdf
- avr-15 paper
- ivermectin 20 years on
What are helminths?
Helminth is a general term meaning worm.
What classes do helminths fall into?
What are some general characteristics of helminths?
Where do they live in the body?
Fall into three different classes
- Nematodes (round worms)- free living and in parasitic form
- Trematodes (flukes)- blood flukes, schistosomes which live in blood
- Cestodes (tape worms)- live in the intestine
- They are simple, not segmented, simple nervous systems
The scale of the problem of helminth infections
dont need to know figures as are out of data, but pattern is still roughly the same
In what ways is disease transmitted?
Provide examples for each
- Ingestion of contaminated material
- Contaminated water e.g., water flea, ingestion
- Via an intermediate host e.g., snails (water snail is the intermediate for schistosomes) and insects (biting flies and mosquitoes carrying malaria)
- Direct invasion through the skin e.g., hookworm (hookworm larvae is buried in soil and can detect soil of your feet and burrow through to infect)
Using Ascariasis, go through the infection and diagnostic stages of disease…
- Adult worms live in the lumen of the small intestine. A female may produce approximately 200,000 eggs per day, which are passed with the feces
- Unfertilized eggs may be ingested but are not infective. Larvae develop to infectivity within fertile eggs after 18 days to several weeks
- , depending on the environmental conditions (optimum: moist, warm, shaded soil). After infective eggs are swallowed ,
- the larvae hatch
- , invade the intestinal mucosa, and are carried via the portal, then systemic circulation to the lungs
- . The larvae mature further in the lungs (10 to 14 days), penetrate the alveolar walls, ascend the bronchial tree to the throat, and are swallowed
- . Upon reaching the small intestine, they develop into adult worms. Between 2 and 3 months are required from ingestion of the infective eggs to oviposition by the adult female. Adult worms can live 1 to 2 years.
What is the pathophysiology of helminth infection due to?
- due to presence of adult
- due to migrating larva
- due to allergic reaction
Ascariasis
postoperative period of paediatric patients after ascaris extraction by enterotomy
What are the major diseases caused by Helminth infections?
Lymphatic Filariasis (elephantitis)
Schistosomiasis (Bilharzia)
Tell me about Lymphatic filariasis (elephantitis)
- Lymphatic filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring disease. It is caused by infection with parasites classified as nematodes (roundworms) of the family Filariodidea that are transmitted through the bites of infected mosquitos
- Mosquito-transmitted larvae are deposited on the skin from where they can enter the body. The larvae then migrate to the lymphatic vessels where they develop into adult worms, thus continuing a cycle of transmission
- It causes the lymph node to become blocked and then swollen
Tell me what Schistosomiasis (Bilharzia) can cause?
bathing in contaminated water, bladder cancer, liver cancer, blockage of blood supply, inflammation in gut
What is Onchocerciasis (river blindness) caused by?
What symptoms does it cause?
How is it transmitted?
- Caused by the worm Onchocerca volvulus
- Dermatitis
- Subcutaneous nodules
- Eye lesions- caused by migrating larvae known as microfilarae
- Transmitting by biting black fly which breeds in fast flowing rivers
What can be done in order to try and control Helminthiasis?
- controlling the disease vectors
- improved sanitation
- health education
- vaccination programmes
- chemotherapy
What are some potential considerations for selective toxicity?
- Infections of children, adults, pregnant women… (drugs need to be well tolerated by all ages)
- Poor access to medical facilities
- Parasites invade different tissues
What are the mechanisms for selective toxicity, explain a bit about each
- Distributional- i.e., of drug poorly absorbed in the gut, if used to treat nematode there. If it got to host it shouldn’t have a massive effect as it is poor absorbed. Therefore, limiting toxicity to host but selectively toxic to nematode
- Biochemical- differences between parasite and host can be exploited for selective toxicity
A table to show the key drugs registered for the treatment of parasitic worms in humans
Most drugs act on the nervous system or muscle
These lectures will focus on the following compounds:
Levamisole
Piperazine
Ivermectin
New generation anthelmintics
What are some approaches used in anthelmintic drug discovery?
What does each mean?
- Serendipity- by chance
- Screening natural products- if you look at soil samples around world for e.g., you can grow crops to see if any natural products from microorganisms have an antihelminthic activity
- Target selection and rational drug design- no example yet as to when its’ been successful, but is being used more now
What are some approaches used to define pharmacology of anthelmintics?
When was Levamisole discovered?
1966 by Janssen Cilag
In 1970, what was Levamisole shown to cause?
1970 Levamisole causes spastic paralysis (hypercontraction) of nematodes
Mutations in what receptors lead to resistance to Levamisole?
*Harbour mutations in nicotinic receptors (those which are resistant to levamisole)
Tell me about the experiment, in 1974, that Benner did on C.elegans that were resistant to Levamisole
- 1974 Brenner isolates levamisole resistant C. elegans
- Mutants carry mutations in nicotinic acetylcholine receptor subunits
- Self-fertilising hermaphrodites: the mutant worms so can carry on mutant line
- Go to F2 line as the mutations are recessive
- Then introduce levamisole to agar plates
- Wt worms are paralysed by levamisole but if some worms carry mutations against the paralytic action of levamisole on C. elegans then they breed true to map and identify the gene which had resistance to levamisole
- Mutants in C.elegans are names after phenotype they have
- ‘Unc-63’ stands for uncoordinated and these are resistant to levamisole
- Nicotinic acetylcholine receptor in nematode has a pentameric structure (like in mammals), a gated cation channel, a mutation in this subunit is what is resistant against levamisole (‘unc-63’)
What is important for nematode survival?
Use Ascaris suum as an example
Ascaris suum (must move in intestine to hold position in host). So would be expelled from host if unable to move)
Tell me about the motoneuron, nerve cord connections in nematodes and how the NMJ forms
- Motor neurones which regulate activity of muscle is within the nerve cord
- Motor neurones are organised in simple way, no extensive processes, bipolar structure, NMJ to be forms (muscle cells send arm to motor neuron which wrap around motor neuron process and form NMJ)
Explain the circuitry for control of locomotion
- dorsal node cord regulates dorsal muscle and ventral node cord regulate ventral muscle
- circuit explains how worm can make coordinated, sinusoidal bend to keep position
VE= ventral excitor
VI= ventral inhibitor
DE= dorsal excitor
DI= dorsal inhibitor
- VE (NT is Ach), NT acts on nicotinic Ach receptors bringing about muscle contraction
- VE –> excited DI
- DI inhibits dorsal muscle via the NT GABA (in mammals acts on GABA ligand-gated chloride channel, causes hyperpolarisation and muscle relaxation, this receptor is similar to GABA A receptors in the mammalian brain)
- Excitatory NT, Ach and inhibitor NT, GABA
- Mammals don’t have inhibitory transmission directly on muscle, instead is acts on motor neurons on spinal cord
Measuring the effects of levamisole on muscle
- Pushes pipette, containing levamisole, against patch of membrane on vesicle and able to record currents flowing through channel when it opens. Can look at opening and closing of channel as current flows through
- Current recordings show typical behaviour of non-selective nicotinic acetylcholine receptor channels
Explain selective toxicity of levamisole
Absorbed across gastro-intestinal tract
Well tolerated by host, doesn’t kill host
side-effects include dizziness (levamisole is a weak agonist at autonomic ganglia, NMJ receptors) and nausea (probably due to activation of nicotinic receptors at autonomic ganglia)
selective toxicity, therefore, relies on differences between host and parasite nicotinic acetylcholine receptors
Potent activator of nicotinic Ach receptor
What does the drug piperazine cause?
What is it marketed as?
What does it cause?
Piperazine causes flaccid paralysis (muscle relaxation) of nematodes
Marketed as Pripsen
Used for thread worm (prevalent in school kids)
Simple structure
Causes a signalling change in motor NS
What receptors in Piperazine through to act via?
Piperazine thought to act via Ach receptor antagonist (NOT)
however it acts via GABAergic system and acts directly to body wall muscle on GABA gates chloride channels to bring about flaccid paralysis
Ivermectins structure
What is Ivermectin very effective against?
Microfilariae
Is Ivermectin or Diethylcarbamazine better for treating microfilariae?
Ivermectin
Tell me about chloride channel activation with ivermectin in invertebrate muscle?
- Dihydroavermectin is the full name for ivermectin
- Hyperpolarisation when ivermectin added, ivermectin acts on membrane and opens ion channels which is what leads to hyperpolarisation
- Suggests that ivermectin acts on Cl- channels which is what leads to hyperpolarisation
- Effect looks irreversible, doesn’t wash out
- Ivermectin has a prolonged effect on increasing membrane conductance
- Ivermectin is an irreversible activator of Cl- channels
What techniques were used in order to identify the gene(s) which encoded ivermectin sensitive receptors?
Prepared mRNA from C.elegans –> reverse transcription to cDNA –> pool of 5000 cDNAs (hypothesis was that one of these proteins would encode a binding site for ivermectin)
Expression cloning: 5000 cDNAs –> injected into xenopus oocytes (single cell model) –> challenged oocytes with glutamate (used as this is a NT in the insect NMJ and acted on chloride channels) and also used ivermectin –> split pool –> saw response –> split pool –> tested again until got down to a few cDNAs in order to identify the genes that encodes a glutamate/ ivermectin sensitive receptor
Iterative screening to identify genes encoding ivermectin sensitive receptors
What did the results of expression cloning of C.elegans show?
What type of receptor is the ivermectin receptor?
Tell me its structure
A glutamate-gated chloride channel
It has 5 subunits so is a pentameric structure
What are the 6 C.elegan GluCl genes and their products?
Tell me the steps to making C.elegan transgenics
Transgenic means that one or more DNA sequences from another species have been introduced by artificial means.
State one location where the glutamate-gated chloride channel is expressed that causes inhibition of feeding?
Pharynx
There are glutamate-gated chloride channels in motoneurons, when treated with ivermectin what effect does it cause here?
When inhibited with ivermectin it causes an inhibition of locomotion
Tell me the EC50 of C.elegans which are the wild type and also a GluCl mutant for 3 GluCl channels
Tell me about the new generation anthelmintic Emodepside
What are they derived from?
Whats their structure?
Emodepside- natural product, derived from plant, structure is a cyclo-octadepripeptide, hard to synthesise so has to be extracted naturally
Tell me the effects that emodepside has on C.elegans as well
Paralysed C.elegans as well
slows development
inhibits locomotion
inhibits feeding
inhibits egg-laying
Tell me about the genes/ chromosomes involved in emodepside resistance
Resistance to emodepside mapped to chromosome V
Locus contains several candidate genes, including slo-1
slo-1 null is highly resistant to emodepside
Another new generation anthelmintic is
Amino-aceto-nitrile derivates AADs
AADs have drug-resistance breaking properties
The putative AAD receptor
