Treatment of trypanosomiasis - 2b (5) Flashcards Preview

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Flashcards in Treatment of trypanosomiasis - 2b (5) Deck (50):
1

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
  • 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

 

 

2

Current drugs against early stage HAT

  • suramin
  • pentamidine (PMD)
  • melarsoprol (MelB)

 

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4

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

 

5

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
      •  

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Suramin

(absorption)

  • weekly 1g intravenous injections over 5-6 weeks
  • $10/g
  • need medical supervision which adds to cost

 

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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

 

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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

 

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Suramin

side effects

albuminuria (protein in urine) - clears up after treatment stops

10

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

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Suramin

mode of action

  1. suramin inhibits enzyme encountered in the endocytic pathway
    • 3'-nucleotidase
    • protein kinase
    • acid phosphatase
    • acid pyrophosphate
    • phospholipase A1
  2. 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
  3. suramin inhibits polyamine synthesis
    • stabilizes DNA
  4. 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)

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Suramin

picture

A image thumb
13

Pentamidine (PMD)

structure

  • aromatic
    • has benzyl groups
  • diamidine 
    • 2 amidine groups on the ends
  • positively charged at physiological pH

 

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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)

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Pentamidine

absorption

  • 7-10 daily intramuscular injections ($20/course)

 

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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

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Pentamidine

elimination

  • not metabolized but can be excreted in its native state
  • ~15% clearance via urine in 24 hours
  • half-life 9-13 hours

 

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Pentamidine

side effects

  • allergic reactions
  • stomach upset
  • loss of appetite
  • nausea
  • vomiting
  • diarrhea
  • dizziness
  • cough

 

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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

 

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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

 

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Melarsoprol

history

  • introduced in 1949
  • only drug available against late stage of both HATs

 

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Melarsoprol

absorption

  • very painful intraveous injections
  • resuspend in adjuvant - PEG (essentially antifreeze)
  • scars veins and blood vessels collapse

 

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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

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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

 

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Melarsoprol

elimination

  • converted to melarsen oxide (changing valence states of melarsoprol)
  • rapidly excreted in urine
  • half-life ~35 hours

 

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Melarsoprol

uptake

uses 2 transporters that pentamidine uses

  • P2 purine transporter
  • AQP2 - aquaglyceroporin

 

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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)

28

Suramin and Pentamidine

vs

Melarsoprol

  • suramin and pentamidine are against early stage HAT
  • melarsoprol against CNS stage HAT
    • (also eflornithine)

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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 

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Q image thumb

  • 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

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Q image thumb

  • 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

32

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

 

33

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

34

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)

 

35

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

 

36

Eflornithine

  • alternative drug against CNS stage HAT

 

37

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

 

38

Eflornithine

history

  • introduced in 1981 (Aventis)
  • used against late stage disease T. b. gambiense

 

39

Eflornithine

absorption

  • 4 daily intravenous infusions for 7-14 days at high dose 
  • 400mg/kg
  • $500/course

 

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Eflornithine

distribution

  • in plasma
  • can cross blood/brain barrier
    • up to 50% in cerebrospinal fluid

 

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Eflornithine

elimination

  • not metabolized 
  • rapidly (80% in 24 hours) excreted in urine
  • half-life ~3.5 hours

 

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Eflornithine

uptake

  • AAT6 = amino acid transporter
  • precise location not known

 

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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?

 

44

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/eflornithine complex removed rapidly and replaced rapidly in mammalian cells 
    • occurs slowly in T. brucei
  • trypanosomes treated with eflornithine have reduced ODC activity

 

  •  

45

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

 

46

Eflornithine

actions

  • reduces production rate of polyamines
  • drug does not kill parasite - rapid growth rate is reduced (cytostatic) 
  • patient's immune system kills parasite

 

47

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

 

48

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

 

49

Current drug treatments are problematic

 

  • toxic
    • melarsoprol - based on arsenic!
      • 5-10% of patients die
    • pentamidine, suramin
  • 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

 

50

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