Treatment of trypanosomiasis - 2b (5)

Question 1

Three points for HAT chemotherapy

Answer 1

• 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

Question 2

Current drugs against early stage HAT

Answer 2

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

Question 4

Suramin

structure

Answer 4

• 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

Question 5

Suramin

history

Answer 5

• 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
      *

Question 6

Suramin

(absorption)

Answer 6

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

Question 7

Suramin

distribution

Answer 7

• 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

Question 8

Suramin

elimination

Answer 8

• 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

Question 9

Suramin

side effects

Answer 9

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

Question 10

Suramin

uptake

Answer 10

• 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

Question 11

Suramin

mode of action

Answer 11

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)

Question 12

Suramin

picture

Answer 12

Question 13

Pentamidine (PMD)

structure

Answer 13

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

Question 14

Pentamidine

history

Answer 14

• 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)

Question 15

Pentamidine

absorption

Answer 15

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

Question 16

Pentamidine

distribution

Answer 16

• ~70% protein bound (due to charge bind to proteins associated with plasma - 30% free in plasma)
• passes poorly through the blood-brain barrier

Question 17

Pentamidine

elimination

Answer 17

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

Question 18

Pentamidine

side effects

Answer 18

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

Question 19

Pentamidine

uptake

Answer 19

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

Question 20

Melarsoprol (MelB)

structure

Answer 20

• 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

Question 21

Melarsoprol

history

Answer 21

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

Question 22

Melarsoprol

absorption

Answer 22

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

Question 23

Melarsoprol

treatment schemes

Answer 23

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

Question 24

Melarsoprol

distribution

Answer 24

• 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

Question 25

Melarsoprol

elimination

Answer 25

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

Question 26

Melarsoprol

uptake

Answer 26

uses 2 transporters that pentamidine uses

• P2 purine transporter
• AQP2 - aquaglyceroporin

Question 27

Melarsoprol

mode of action

Answer 27

• 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)

Question 28

Suramin and Pentamidine

vs

Melarsoprol

Answer 28

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

Question 29

Resistance to melarsoprol and pentamidine

Answer 29

• 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

Question 30

Answer 30

• 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

Question 31

Answer 31

• 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

Question 32

Gene knockout experiments

Enock Matovu et al, 2003

Answer 32

• 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

Question 33

Gene knockout experiments

Answer 33

• 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

Question 34

How does P2 transport arsenicals/pentamidine?

Answer 34

• 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)

Question 35

Resistance… not so simple

Answer 35

• 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

Question 36

Eflornithine

Answer 36

• alternative drug against CNS stage HAT

Question 37

Eflornithine

(general/structure)

Answer 37

• 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

Question 38

Eflornithine

history

Answer 38

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

Question 39

Eflornithine

absorption

Answer 39

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

Question 40

Eflornithine

distribution

Answer 40

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

Question 41

Eflornithine

elimination

Answer 41

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

Question 42

Eflornithine

uptake

Answer 42

• AAT6 = amino acid transporter
• precise location not known

Question 43

Eflornithine

mode of uptake

Answer 43

• 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?

Question 44

Eflornithine

mode of action

Answer 44

• 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

*

Question 45

Eflornithine doesn’t actually kill the parasite..

Answer 45

• 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

Question 46

Eflornithine

actions

Answer 46

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

Question 47

Eflornithine

stopped…

Answer 47

• 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

Question 48

Eflornithine and chagas

Answer 48

• 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

Question 49

Current drug treatments are problematic

Answer 49

• 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

Question 50

New/modified treatments

Answer 50

• 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