Treatment of human african trypanosomiasis (HAT)

Three considerations for HAT therapy
1. 1) what stage to target?
  1. Stage; bloodstream (dividing trypomastigotes) or CNS 'dormant' parasites
    1. Major clical manifestations often present during the CNS phase - stage to target?
2. 2) blood brain barrier
  1. Defines physico-chemical properties of the drug (e.g. Polar etc.)
3. 3) sub species
  1. Difference in drug sensitivity of T. b. gambiense (W african) and T. b. rhodesiense (E african)
Current drugs against early stage HAT
1. SURAMIN (trade name = germanin)
  1. Introduced in 1922 (by Bayer - phamaceutical company)
  2. Drug of chce against early stage E African trypanosomiasis (T.b. rhodesiense)
    1. Not used for W African (T.b. gambiense) - instead it is reserved for river blindness (caused by Onchocerca volvulus)
  3. Absorption/dose
    1. Weekly 1g (IV), for 5-6 weeks [$10/g]
  4. Structure
    1. Symmetrical
    2. Based on a core molecule of urea (CO(NH2)2)
    3. Features two naphthylamine (x2 six carbon rings with an NH2 sidechain)
    4. Polysulfonated (features six sulfonate groups)
      1. Make drug negatively charged at physlogical pH
        Important for function! - negative charge allows for interaction with parasite proteins
  5. Distribution
    1. 99% is protein bound due to negative charge
    2. Passes poorly through BBB due to negative charge
  6. Elimination
    1. Not metabolised (long half life) - approx. 35-60 days
  7. Side effects
    1. Albminuria (protein in urine) - stops after end of treatment course
  8. Mode of action (MOA)
    1. Uptake
      1. Suramin binds to the parasite receptor ISG7S (invariant surface glycoprotein 7S)
        Receptor moves theough he fluid mosaic membrane and ends up in the flagellal pit - here the suramin bound ISG7S is ubiquitinated
        This receptor modification (Ub) recruits adaptins to the inner surface of the membrane
        Adaptins recruit a clathrin coat to form beneath the surface of the membrane around the receptor-suramin complex
        Clathrin coated pit forms and the receptor-surmain complex enters the cell through receptor-mediated endocytosis (forms clathrin-coated vesicle)
        In cytoplasm: clathrin disassociates from the vesicle, the uncoated vesicle then fuses with an endosome then a lysosome
        In lysosome: proteases (serine peptidase [CSB] and cysteine protease [CatL]) act on the receptor, causing suramin to disassociate from ISG7S
        The acidic lysosome causes suramin to loose its charge - it then passes through the major facilitator superfamily transporter (MFST) to the cytoplasm
        In cytoplasm: the MOA od suramin still unclear - preseumed effects;
        Inhibition of endocytotic enzymes (3'-nucleotidase, protein kinase, acid phosphatase, acid pyrophosphatase, phospholipase A1)
        Ihibition of glycolytic enzymes (affecting glycolysis - demonstrated in test tube)
        Inhibits polyamine synthesis
        Inhibits N-acetly glucasamine (important part of the VSG glycosylphosphatidylinsositol (GPI) anchor - immune evasion
        ISG7S is receylced
  9. So far there have been no reports of resistence to suramin
    1. Probably due to its numerous effects
2. Pentamidine (PMD)
  1. Structure
    1. Symmetrical
    2. Two aromatic terminals which feature an amidine group (R(=NH)NH2) - making the molecule a diamidine
    3. Positively charged at physlogical pH
  2. Introduced in 1937
  3. Drug of choice against early stage W. African trypanosomiasis (T.b. gambiense)
    1. Also used for resistent leishmaniasis and Pneumocystis carinii pneumonia (PCP)
  4. Absorption/dose
    1. 7-10 daily IM injections ($20/course)
  5. Distribution
    1. 70% bound to proteins - due to charge
    2. Passes poorly through the BBB (due to charge) - cannot reach therapeutic levels and therefore not used for CNS stage
  6. Elimination
    1. Not metablised - ~15% clearance via urine in 24hrs (half life is abot 9-13hrs)
  7. Side effects
    1. Most people (>90%) have allergic reaction
    2. Stomach upset, loss of apetite, nausea, vomiting, diarrhoea, dizziness and cough
  8. MOA
    1. Uptake is via three membrane transporters
      1. P2 (purine transporter)
      2. Low affinity pentamidine transporter (LAPT)
      3. High affinity pentamidine transporter (HAPT)
        Recently proposed as being an aqua-glycero porin (meaning its constanly open)
      4. Possible MAOs in parasite
        Binds to mitochondrial DNA - preventing synthesis on new mitochondria (affects splicing)
        mtDNA is found within the mitochondira of all eukaryotic cells
        Reduces the mitochondrial membranepotential - causing release of ROS and induce apoptosis
        Mitochondrial transporter not known
3. Melarsoprol (MelB)
  1. Structure
    1. Trivaelent, melaminophenyl arsenical (contains arsenic)
      1. Highly toxic - 5-10% of patients die from drug alone
  2. Absorption/dose
    1. Given with adjuvant polyethylene glycol (PEG) and given as IV injection
      1. Very painful! (Arsenic mixed with antifreeze)
        Scars blood vessels and causes collapse
    2. Scheme 1: one injection per day for 4days, then a rest period of 7-10days - repeat 3-4 times
    3. Scheme 2: daily injections for 10 days
      1. Both cost $50/course
  3. Introduced in 1949
  4. Only drug available against late stages of both HATs
  5. Distribution
    1. In plasma - can cross BBB (concentration is 50 times lower in the cerebrospinal fluid)
  6. Elimination
    1. Converted to melarsen oxide - rapidly excreted in urine (half life is about 35hrs)
  7. Side effects
    1. Arsenic poisoning (convulsions, fever, loss of conciousness, rashes, bloody stools, nausea and vomiting)
  8. MOA
    1. Uptake into the parasite is via the membrane receptors; P2 and HAPT
      1. MelB is a prodrug -> converted to malarsen oxide (not known whether by the host or parasite)
        Melarsn oxide complexes with thiols (any molecule that features reactive cysteine residues)
        Protein thiols
        Free thiol trypanothione - involved in protection from oxidative stress (effectively two glutathioones)
        = two glutathiones (protect against ROS) and a spermadine linker
        Complex inhibits trypanothione-dependent enzymes
        Trypanothione reductase (TR) which converts trypanothine to dihydrotrypanothne (T(SH)2)
        Ribonucleotide reductase (DNA nucleotide synthesis)
        Requires trypanothione for redox reaction (electrons taken from thiol groups)
  9. Resistance to melarsoprol
    1. Cells resistant to melarsoprol have a reduced adenine, melarsoprol and melarsen oxide uptake via P2 transporter
      1. Wild type P2 comes from a single copy gene that codes a channel protein with 10 transmembrane domains
        In recombinant yeast unable to synthesise purines: expression of parasite P2 allowed yeast to take up adenine, melarsoprol and melarsen oxide
        Yeast made sensitive to melarsen oxide
        6 point mutations that alter the amino acid sequence of P2 identified - this causes drug resistance
        Yeast expressing this mutated P2 did not become sensitive melarsen oxide
        In P2 gene knockout experiments
        In culture null mutant parasites were more resistant to arsenicals
        In animal models, null mutant parasites showed resistance
        How does the P2 transport arsenicals?
        Due to the structure of the arsenical (e.g. Melarsoprol) - arsenicals share a structural motif similar to adenosine
        Feature an aromatic group with a N=C(NH2)
        This motif H-bonds with the P2 transporter
        The other membrane purine transporter P1 recognises motifs in the ribose sugar ring of the nucleotide which is absent in arsenicals
        However, half of drug resitance cases are not due to P2 mutations - therefore other mechanisms of resistance
        Potential efflux mechanism proposed - thiol conjugate transporter protein (TbMRPA) was identifiedon the surface of parasites
        Melarsoprol binds to thiols
        Eliminates conjugated thiols from the cell
        Overexpression is associated with resitance, whereas low expression of MRPA is associated with hypersensitivity
Other drugs against CNS (late) stage HAT
1. Eflornithine (difluoremethylornithine, DFMO)
  1. Structure
    1. Structural analgoue of ornithine (amino acid involved in urea cycle and important for the metabolism of toxic nitrogen)
      1. Fluorinated
  2. Introduced in 1981 (tradename - aventis)
  3. Designed to be an anti-cancer drug
  4. Very few side effects and termed the "miracle" or "resuscitation" drug due to its ability to seemingly revive patients with CNS stage HAT
  5. Absorption/dose
    1. Four daily IV injections (for 1-2weeks, at high dose - 400mg/kg)
      1. $500/course - due to improved production techniques its not about $300
  6. Distribution
    1. In plasma - can cross the BBB (up to 50% of dose in CSF)
  7. Elimination
    1. Not metabolised, rapidly excreted in urine (about 80% in first 24hrs) - half life is 3.5hrs
  8. MOA
    1. Parasite uptake by the amino acid transporter 6 (AAT6)
      1. Down regulation by RNAi of TbAAT6 leads to resistance
      2. Irreversibly binds to onithine decarboxylase (ODC)
        Reduces the production rate of polyamines (e.g. Trypanothione) - many knock-on effects
        Doesnt directly kill parasite - decreses growth (cytostatic) allowing the immune system to control the infection
        Binds to mammalian ODC with same affinity - so how do humans tolerate the drug?
        Human ODC turnover is much faster (about 20mins) than trypanosomal ODC turnover (about 4-5hrs in T.b. rhodesiense and 18-19hrs in T.b. gambiense)
        In mammals ODC/eflornithine complex is removed and replaced with new ODC faster (barely effecting activity)
        In trypanosomes ODC turn around is much slower and there is subsequent loss of ODC activity
  9. Aventis stopped producing eflornithing due to the cost
    1. However vaniqa cream (eflornithine hydrochloride) a treatment for facial hair on women is in production
      1. Market in the Western world
      2. Aventis agreed to donate 5yrs worth of eflornithine to WHO
        At a cost of $25M
    2. No market in the Western world
Current drug treatments are problematic
1. Toxic
  1. Majorly: melarsoprol (melaresn oxide) (based on arsenic - 5-10% of patients die outright)
  2. Minorly: suramin and pentmaidine
2. Limited efficacy
  1. Suramin only effective against E African trypanosomiasis (T.b. rhodesiense)
  2. Pentamidine only effective against W African trypanosomiasis (T.b. gambiense)
  3. Suramin and pentamidine only effective against non-cerebral forms of HAT
  4. Eflornithine only effective against T.b. gambiense (CNS stage) - T.b. rhodesiense (CNS stage) has a less stable ODC
3. Resistance
  1. Melarsoprol - mutation of P2 and TbMRPA overexpression
4. Medical supervision
  1. All!
5. Expensive
  1. Eflornithine (~$300/course)
6. Future of HAT treatment
  1. May rely on combinational therapies (i.e. NECT = nifurtimox eflornithin combinatnal therapy)
    1. NECT is used against late stage W African trypanosomiasis (T.b. gambiense)
      1. Alternative for melarsoprol
      2. Cheaper than elfornithine monotherapy (~$150/course)

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Treatment of human african trypanosomiasis (HAT)

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	Created by maisie_oj over 11 years ago