Three points for HAT chemotherapy
- stage
- bloodstream trypomastigotes dividing
- CNS phase “domrant” parasites
- the parasite when it causes the early stages of the disease is primarily in the bloodstream or lymphatic system, or do you go directly for the CNS phase?
- blood-brain barrier
- defines physico-chemical properties of a drug
- very difficult for drugs/compounds to get across
- a lot of drugs can’t cross this barrier so usefulness restricted to blood and lymph form
- if not lipophilic enought to get across the epithelial barrier of the blood-brain barrier can you come up with a drug that can get across
- defines physico-chemical properties of a drug
- sub-species
- difference in drug sensitivity of T. gambiense and T. b. rhodesiense
- the 2 forms have slighlty different chemistries
- want to develop a drug to target both sub-species, but most drugs target only one
- so must decide if want to develop a drug against themore common form (gambiense is 95%) or the more dangerous form (rhodesiense) that kills patients very rapidly
- difference in drug sensitivity of T. gambiense and T. b. rhodesiense
Current drugs against early stage HAT
- suramin
- pentamidine (PMD)
- melarsoprol (MelB)
Suramin
structure
- symmetrical
- derivative of urea
- links up the 2 arms
- naphthylamine
- 2 benzyl groups stuck together with an amine group on the end
- polysulfonated
- negatively charged at physiological pH
- restricts the use of this compound to being used against the bloodstream stage
- may be responsible for why it’s a good drug in the first place - positivley charged molecules attracted to negative charge
Suramin
history
- introduced in 1922
- Bayer (brand name Germanin)
- drug of choice against early stage East African trypanosomiasis (T. b. rhodesiense)
- example of drug used only for early stage when the parasite is in the blood and lymphatic system
- does work against gambiense form but use is restricted in West africa because suramin can also be used to treat other parasitic infections
- eg river blindness - Onchocerca volvulus
*
- eg river blindness - Onchocerca volvulus
Suramin
(absorption)
- weekly 1g intravenous injections over 5-6 weeks
- $10/g
- need medical supervision which adds to cost
Suramin
distribution
- 99% protein bound - due to charge
- isn’t free within the blood plasma or the lymph
- tends to stick to positively charged particles that are on cell surface or other molecules within the blood
- only 1% is free within the plasma
- passes poorly through blood-brain barrier
- can’t be used to treat once the parasite is in the cerebrospinal fluid
Suramin
elimination
- not metabolized
- ourlivers won’t touch thisb lood
- can be excreted in sweat
- long half-life (approx. 35-60 days)
- half-life is the time for half of a given amount of administered drug to disappear
Suramin
side effects
albuminuria (protein in urine) - clears up after treatment stops
Suramin
uptake
- invovles molecules hidden beneath the VSG coat
- ont he cell surface have the invariant surface glycoprotein (ISG75)
- ISG75 acts as a magnet for suramin
- suramin is recruited/sticks to ISG75
- as a result of the fluid mosaic model (membrane molecules constantly in motion) the charged suramin molecules end up in the flagella pocked
- when the receptors and ligands are within the flagella pocket the ISG75 undergoes a post-translational modification resulting in ubiquitination
- chemical tag added on to the receptor when it’s within the flagella pocket and bound to suramin
- this ubiquitin acts as an attractant for adaptins which tehna ct as a scaffold for laying down clathrins
- clatharin is a key marker of endocytosis
- clatharin-coated vesicles form pits that bud off
- clatharin-coated pits become clatharin-coated vesicles
- uptake via receptor-mediated vesicle uncoats
- uncoated vesicle fuses with endosome before suramin/ISG75 passes on to lysosome\then have neaked vesicle still containing suramin-charged ISG75 that passes through the endosomal system to a lysosome (packed with enzymes, proteases, nucleases that chop up things that have been taken up from outside the cell)
- in lysosome proteases act on suramin?ISG75
- serine CSB) peptidase
- cysteine (CatL) protease
- degrade ISG75 that causes suramin to be temporarily released from that molecule
- suramin and ISG75 disassociated
- suramin transported out of lysosome by MFST (major facilitator superfamily transporter)
- transported out of lysosomal lumen into the cytoplasm
- ISG75 recycled back to cell surface/degraded
Suramin
mode of action
- suramin inhibits enzyme encountered in the endocytic pathway
- 3’-nucleotidase
- protein kinase
- acid phosphatase
- acid pyrophosphate
- phospholipase A1
- suramin inhibits glycolytic enzymes (keeps from making ATP chemical energy)
- effectively an aerobe - its mitochondrial function shut down in terms of oxidative phosphorylation pathways so relies upon glycolysis to make energy
- suramin inhibits polyamine synthesis
- stabilizes DNA
- suramin inhibits N-acetylglucosamine synthesis
- one fo the sugars in the GPI anchor
- so suramin could be reducing GPI anchor biosynthesis so the parasite mightnot be able to pack VSGs as close together as it wants
- all due to suramin’s negative charge (polysulfonated groups)
- acting and interacting with positively charged molecules
- resistance in the field has not been detected
- could be that these downstream inhibitor effects may be doing many things, not just one specific thing (pleiotrophic)
Suramin
picture
Pentamidine (PMD)
structure
- aromatic
- has benzyl groups
- diamidine
- 2 amidine groups on the ends
- positively charged at physiological pH
Pentamidine
history
- introduced in 1937
- drug of choice against early stage West African trypanosomiasis (T. b. gambiense)
- also antimony-resistant Leismania and Pneumocystis carnii (yeast pneumonia) (PCP)
Pentamidine
absorption
- 7-10 daily intramuscular injections ($20/course)
Pentamidine
distribution
- ~70% protein bound (due to charge bind to proteins associated with plasma - 30% free in plasma)
- passes poorly through the blood-brain barrier
Pentamidine
elimination
- not metabolized but can be excreted in its native state
- ~15% clearance via urine in 24 hours
- half-life 9-13 hours
Pentamidine
side effects
- allergic reactions
- stomach upset
- loss of appetite
- nausea
- vomiting
- diarrhea
- dizziness
- cough
Pentamidine
uptake
3 transporters
- P2 = purine transporter (pentamidine, adenine/adenosine)
- AQP = aquaglyceroporin 2
- non-selective waterphil channel that allows small molecules through, localized to flagella pocket
- LAPT = low affinity pentamidine transporter
- once the pentamidine is in the cell it appears to have a tropism/preference for the mitochondria
- possible mechanisms:
- binds to mitochondrial DNA, prevents synthesis
- mitochondrial membrane potential collapses
- because of its charge pentamidine sticks to negative moleculse
- the biggets negative molecule we have is DNA
- with 2 arms it intercalates the kDNA of the mitochondria
- the drug prevents the synthesis of new DNA molecules - prevents transcription o fDNA elements within the mitochondrial genome
- mitochondrial membrane potential collapses
- it affects the redox wellbeing of the cell
Melarsoprol (MelB)
structure
- trivalent melaminophenyl arsenical
- melanin ring, part for uptake, part that kills the parasite - has arsenic in trivalent form
- highly toxic - 5-10% of patients die from drug alone (more like 5-7%)
- convulsions, fever, loss of consciousness, rashes, bloody stools, nausea, vomiting
- have alternative for West africa, but no alternative for East Africa
Melarsoprol
history
- introduced in 1949
- only drug available against late stage of both HATs
Melarsoprol
absorption
- very painful intraveous injections
- resuspend in adjuvant - PEG (essentially antifreeze)
- scars veins and blood vessels collapse
Melarsoprol
treatment schemes
scheme 1
- 1 per day for 4 days
- rest period for 7-10 days
- repeat 3 or 4 times
scheme 2
- daily injections for 10 days
- both cost $50/course
Melarsoprol
distribution
- in plasma, can cross blood/barrier (hydrophobic nature of the drug)
- ~50 fold lower in cerebrospinal fluid relative to blood plasma
- only a fraction gets across, but in therapeutic levels
Melarsoprol
elimination
- converted to melarsen oxide (changing valence states of melarsoprol)
- rapidly excreted in urine
- half-life ~35 hours
Melarsoprol
uptake
uses 2 transporters that pentamidine uses
- P2 purine transporter
- AQP2 - aquaglyceroporin
Melarsoprol
mode of action
- gets into the parasite, is converted into active form melarsen oxide
- MelB is pro-drug
- don’t know what mediates conversion of pro-drug to drug
- on the parasiate surface or within the mammalian cell?
- 2 transporters can take up the drug itself, so don’t know where this activaton event occurs
- here assume it occurs within the cytoplasm of the parasite, probably the result of some oxido-reductive enzyme
- melarsen oxide complexes with thiols
- thiol = molecule containig reactive cysteine (SH)
- protein thiol
- free thiol trypanothione
- main thiol found within parasites is trypanothione
- so melarsen oxide readily sticks with trypanothione in its oxidative state to form a metal-thiol conjugate
- the metal-thiol conjugate can stick to trypanothione enzymes
- complex inhibits trypanothione-dependent enzymes
- trypanothione reductase (TR) activity falls (key enzyme that takes oxidized trypanothione and convertes it to its reduced active form) → trypanothione levels fall, downstream processes reduced considerably including:
- DNA synthesis (trypanothione in reduced form is a reductant inDNA’s biosynthetic pathways)
- reduced trypanothione is the driver of all of the peroxide-metbaolizing pathways
- trypanothione in its reduced form plays a key role in protecting the parasite from oxidative stress, so if you reduce the amount of this the cell suffers
- if you can prevent the formation of trypanothione it has a pleiotrophic effect of whether the cell survives or not
- dihydrotrypanothione (T[SH]2) levels fall
- causes oxidative stress
- reduced ribonucleotide reductase activity (DNA synthesis)
- trypanothione reductase (TR) activity falls (key enzyme that takes oxidized trypanothione and convertes it to its reduced active form) → trypanothione levels fall, downstream processes reduced considerably including:
Suramin and Pentamidine
vs
Melarsoprol
- suramin and pentamidine are against early stage HAT
- melarsoprol against CNS stage HAT
- (also eflornithine)
Resistance to melarsoprol and pentamidine
- know about the P2 transporter because of resistance studies
- if you grow parasites in the lab on an increasing concentration of melarsoprol you can select out cell lines - and those cell lines when analyzed generally have a reduced uptake capacity for melarsoprol (and pentamidine)
- cells selected on melarsoprol (and pentamidine) have reduced uptake of adenine, melarsen oxide, and melarsoprol via P2 transporter
- Carter and Fairlamb - Nature - 1993
- when you look at those the gene that appears to bemutated is the P2 transporter gene
- wild type P2 gene cloned
- single copy gene
- 10 transmembrane domains
- Maser et al. - Science - 1999
- the protein is a transmembrane-bound protein
- cand o functional work in yeast that lack this particular protein by re-expressing the trypanosome enzyme in a yeast P2 non-mutant that can now take up melarsoprol and melarsen oxide and become sensitive to these compounds
- parasite P2 exprssed in yeast strains unable to synthesize purines
- recombinant yeast took up adenine, melarsen oxide, and melarsoprol
- recombinant yeast made sensitive to melarsen oxide
- P2 gene from melarsoprol resistant line closed
- P2 is mutated, 6 point mutations alter amino acids
- expression of mutant P2 in yeast did not allow uptake of adenine or arsenical drugs
- these 6 point mutations are sufficient to stop P2 binding and interacting with melarsoprol
Gene knockout experiments
Enock Matovu et al, 2003
- both copies of the P2 gene have been deleted from the T. brucei geome
- P2 is a single copy gene per haploid genome
- growth experiments of mutant
- in culture, null mutant parasites were more resistant to arsenicals
- gives further weight to teh argument that P2 is involved in transport
- in animal models, null mutant parasites showed resistance
- to melarsoprol in an in vivo model
- in culture, null mutant parasites were more resistant to arsenicals
Gene knockout experiments
- Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populaitons and their cross-resistance
- Bernhard et al., 2007
- Detection of mutant P2 adenosine transporter (TbATI) gene in Trypanosoma brucei gambiense isolates from northwest Uganda using allele-specific polymerase chain reaction
- Nerima et al.
- further if you look at the P2 gene in a clinical context using clinically resistant strains
- if you look at the P2 transporter, many of those clinically resistant strains havem utations in the P2 gene
- also gives cross-resistance to pentamidine
How does P2 transport arsenicals/pentamidine?
- all comes down to structure
- Koning and Jarvis, 1999
- Maser et al., 2003
- arsenicals/adenosine/pentamidine - structurally conserved amidine motif
- H-bodning occurs between P2 and arsenical/pentamidine/adenosine at this motif is sufficient to at least initiate the interaction between the transporter and the drug or negative substrate
- P1 (other purine transporter) recognizes motifs in the ribose ring (absent from arsenical drugs)
Resistance… not so simple
- Half of MelB resistance cases NOT due to P2 transporter
- Matovu et al., 2001
- Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in Africa trypanosomes
- Baker et al., 2012
- loss of AQP2 activity in culture leads to melarsoprol/pentamidine cross-resistance
- half of clinically resistant cases attributed to P2 - leaves a lot of other cases that we don’t actually know what causes the resistance - but….
- aquaglyceroporin - in the lab you can knock out aquaglyceroporin2 gene from the T. brucei parasite and renders the cells resistant to melarsoprol and pentamidine
- P2 has been categorically proven to produce resistance due to reduced uptake
- demonstrated that AQP2 plays a role in clinical resistance as well
- resistance in the lab using genetically defined lines or drug selected lines IN THE LAB has infuromes us what to look out for in the field
- has given us 2 mechanisms - P2 and AQP2 - if you lose a component of those activities you generate a degree of resistance (particularly worrying if you have the East African form of the disease because there’s no other form of treatment)
- potential eflux mechanism proposed
- Shahi et al., 2002
- a putative thiol-conjugate transporter protein (ThMRPA) was identified found on the surface of parasite
- transporter can pump out the drug-thiol conjugate of the cell
- over exressoin leads to MelB resistance
- reduced levels associted with hypersensitivity
- not linked to resistance in field isolates
Eflornithine
- alternative drug against CNS stage HAT
Eflornithine
(general/structure)
- difluoromethylornithine (DFMO) - analogue of ornithine
- originally developed as cancer drug
- very few side effects
- “miracle” or “resurrection” drug
- very effective for late stage West African trypanosomiasis
Eflornithine
history
- introduced in 1981 (Aventis)
- used against late stage disease T. b. gambiense
Eflornithine
absorption
- 4 daily intravenous infusions for 7-14 days at high dose
- 400mg/kg
- $500/course
Eflornithine
distribution
- in plasma
- can cross blood/brain barrier
- up to 50% in cerebrospinal fluid
Eflornithine
elimination
- not metabolized
- rapidly (80% in 24 hours) excreted in urine
- half-life ~3.5 hours
Eflornithine
uptake
- AAT6 = amino acid transporter
- precise location not known
Eflornithine
mode of uptake
- taken up into the parasite by AAT6
- reduction/loss of TbAAT6 leads to resistance (in clinical samples???)
- once within the cytoplasm of the parasite eflornithine binds to an enzyme ornithine decarboxylase (ODC)
- this enzyme is involved in polyamine biosynthesis
- ourbodies have ODC and the parasites have ODC
- what’s the basis of selectivity, why isn’t it affecting humans?
Eflornithine
mode of action
- binds to ornithine decarboxylase (ODC) irreversible inhibitor
- eflornithine acts as an irreversible inhibitor of the enzyme
- affinity of eflornithine to mammalian ODC is similar to trypanosomal ODC
- the mammalian enzyme is turned over and replaced at a rapid rate
- mammalian ODC turned over at faster (20 minutes) rate than T. brucei ODC
- have the ODC within a human cell and every 20 minutes you’re effectively replenishing half the amount of ODC
- if you have that ODC bound to eflornithine every 20 minutes you’re getting rid of the inhibited enzyme and repalcing it with fresh enzyme - so repalcing with fresh uninhibited enzyme
- why the drug works against the parasite is this replenishment takes a lot longer in the parasite
-
T. b. rhodesiense 4-5 hourse, T. b. gambiense 18-19 hours
- the inhibited enzyme is staying around in the parasite longer compared to the mammalian form
- why it’s not used against rhodesiense is that it’s replenished faster
- the basis of selectivity - how quicly you can replace the inhibited enzyme
- mammalian ODC turned over at faster (20 minutes) rate than T. brucei ODC
- mammalian ODC/eflornithine complex removed rapidly and replaced rapidly in mammalian cells
- occurs slowly in T. brucei
- trypanosomes treated with eflornithine have reduced ODC activity
*
Eflornithine doesn’t actually kill the parasite..
- it puts the parasites in a state of suspended animation
- acts as static agent so the parasite stops dividing
- in that static state our immune system comes in and wipes the parasite out
- does rely upon the patient having an active healthy immune system
- not for people with immune systems that don’t funcion properly
- eg with HIV
- not for people with immune systems that don’t funcion properly
Eflornithine
actions
- reduces production rate of polyamines
- drug does not kill parasite - rapid growth rate is reduced (cytostatic)
- patient’s immune system kills parasite
Eflornithine
stopped…
- Aventis stopped making the drug due to cost (1995)
- BUT Vaniqa Cream (eflornithine hydrochloride) - a topical prescription treatment for facial hair
- “Drug firm wakes up to sleeping sickness” - campaign persuades aventis to give five years’ worth of medicine and help WHO research
- a cost of $24million for 60,000 does (~$420 per course)
- how now released the patent in the hopes of streamlining the 15-step process
Eflornithine and chagas
- what’s kicked off the use of eflornithine to treat West HAT is partnerine it up with a drug used for chagas disesae
- this drug and eflornithine - although they work antagonstically - actually help each other to cross the blood/brain barrier
- now can use a combination therapy to treat West HAT
- the cost of treatment for 1 person is around $30
Current drug treatments are problematic
- toxic
- melarsoprol - based on arsenic!
- 5-10% of patients die
- pentamidine, suramin
- melarsoprol - based on arsenic!
- limited efficacy
- suramin only works agains East HAT
- pentamidine only works against West HAT
- pentamidine and suramin are only effctive on non-cerebral stages of sleeping sickness
- eflornithine is not effective against T. b. rhodesiense
- resistance
- melarsoprol
- medical supervision
- ALL
- expensive
- eflornithine
New/modified treatments
- may rely on combinational therapies
- eg NECT (nifurtimox-eflornithine) combinational therapy
- added to WHOs List of Essential medicines
- alternative to melarsoprol for late stage West HAT
- chaper than eflornithine monotherapy
- encouraging pharmaceutical companies to manufacture “uneconomical” drugs