Treatment of trypanosomiasis - 2b (5) Flashcards

Question

Melarsoprol elimination

Answer 1

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

Answer 2

uses 2 transporters that pentamidine uses * P2 purine transporter * AQP2 - aquaglyceroporin

Answer 3

* 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)

Answer 4

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

Answer 5

* 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

Answer 6

* 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

Answer 7

* 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

Answer 8

* 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

Answer 9

* 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

Answer 10

* 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)

Answer 11

* 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

Answer 12

* alternative drug against CNS stage HAT

Answer 13

* 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

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

* AAT6 = amino acid transporter * precise location not known

Answer 19

* 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?

Answer 20

* 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 *

Answer 21

* 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

Answer 22

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

Answer 23

* 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

Answer 24

* 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

Answer 25

* 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

Answer 26

* 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