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Two molecules (no longer in active development within GlaxoSmithKline) were chosen to study mechanisms of DIMT, based on their potent and selective effect on mitochondrial function. Furthermore, the mechanisms by which they affect mitochondrial function (respiratory chain inhibition and uncoupling) are commonly observed in preclinical assessment of drugs for mitochondrial activity. These compounds are introduced in section 1.9.1 and 1.9.2 below.

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1.9.1. GSK932121A(C

IIII

)

GSK932121A (shown in Fig.1.6 below) is a 4(1H)-pyridone molecule that was in development as an anti- malarial medicine. In FTIH trials, GSK932121A was well tolerated however adverse toxicity was observed in pre-clinical studies with a pro-drug of GSK932121A, designed to increase oral bioavailability. This toxicity profile was later observed using GSK932121A itself when dosed IP (to provide similar systemic exposure levels to those achieved with pro-drug). These data resulted in GSK932121A’s termination from development due to a potentially narrow therapeutic index [127].

FIGURE 1.6: Chemical structure of GSK932121A (mw: 425.786). This pyridone molecule was designed for the treatment of the Plasmodium falciparum malaria infection.

Mitochondria are seen as an attractive target for anti-infective medicines. Due to the integral function mitochondria play in cellular biochemistry a sound therapeutic approach is to induce mitochondrial dysfunction in the infectious species (e.g. parasite), whilst avoiding crossover to the mammalian target. This approach has shown proof of principle by the atovaquone/proguanil (GlaxoSmithKline’s Malarone) combination therapy, used today as a treatment of multi-drug-resistant malaria. The drug works via inhibition of the Qo site of the cytochrome bc1 complex thus inhibiting mitochondrial electron transport.

As the ETC is located in the plasma membrane in prokaryotes as opposed to within the mitochondria in eukaryotes it is hypothesised that drugs will effect mitochondrial processes with much higher potency in prokaryotic systems and thus provide a wide therapeutic window.

Similar to atovaquone, GSK932121A is a selective inhibitor of the mitochondrial ETC. As described by Bueno and colleagues in 2012, GSK932121A inhibits the plasmodial cytochrome bc1 complex in the low nanomolar range (IC50= 0.007µM). GSK932121A is significantly more potent against the P.falciparum

N H O C H₃ OH Cl O O F F F

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target compared to mammalian cytochrome bc1 isolated from either human HEK293 cells (IC50= 0.4µM,

57x less potent) or rat H9C2 cells (IC50= 0.6µM, 86x less potent). In whole cells the trend continued,

wherein, cytotoxicity was observed at significantly higher doses in mammalian derived HepG2 cells (IC50=10µM) and H9C2 cells (IC50=14µM) compared to plasmodium derived cultures (3D7A and

FCR3A) which both showed an IC50 for cytotoxicity of 0.006µM. This represents between a 1600-2400x

higher potency against plasmodium cells compared to mammalian cells, suggesting a potentially wide therapeutic margin, based on in vitro data.

FTIH study with GSK932121A: There was no evidence of dose-limiting toxicity in oral pre-clinical

studies and therefore GSK932121A was progressed to a FTIH study. The purpose of the FTIH study was to evaluate the safety and tolerability of the parent drug GSK932121A in comparison with placebo and to investigate the pharmacokinetics of the compound in man. Eight healthy adult male subjects received single doses of GSK932121A parent (3mg, n=4 or 30mg, n=4) with four separate subjects receiving placebo alone. GSK932121A was well tolerated.

The pro-drug approach: Improved bioavailability causes an adverse toxicological profile: Despite

excellent solubility, GSK932121A demonstrated non-linear pharmacokinetics in preclinical species with decreased oral bioavailability at high doses of the solid dosage form. In attempts to improve the oral bioavailability of GSK932121A a pro-drug approach was investigated. A water soluble phosphate ester pro-moiety was attached to the C6-CH2-OH hydroxyl as shown in Fig.1.7 below. Water soluble phosphate

esters increase solubility and stability and undergo rapid bio-conversion (by endogenous membrane bound alkaline phosphatases) causing the release of high concentrations of the parent drug. Early indications in rats showed an approximately 10-fold greater oral bioavailability of the pro-drug at high dose compared with the parent alone. Due to this outstanding physiochemical and pharmacokinetic profile, the pro-drug of GSK932121A was progressed into full pre-clinical testing as a backup for the parent GSK932121A candidate.

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FIGURE 1.7: Schematic of the approach taken to manufacture a GSK932121A pro-drug. (A) A water soluble phosphate ester was added to the C6-CH2OH group of GSK932121A in order to improve both stability and solubility for the purposes of increasing oral bioavailability. (B) The resulting pro-drug showed increased bioavailability and improved PK profile but the increased systemic exposure levels resulted in acute toxicity consistent with mitochondrial impairment, which led to GSK932121A’s termination due to a potentially narrow therapeutic index. Diagram adapted from [127].

During these pre-clinical studies unexpected, acute toxicity was observed in rats after oral administration of the pro-drug. The toxicity was characterized by hypothermia, cardiovascular abnormalities and at high- doses, sudden death. Upon further investigation, the severe, acute toxicity exhibited by the pro-drug could be replicated by the parent compound alone following IP administration, which induced similar exposure levels to those achieved with the pro-drug given orally. One hypothesis was that the toxicity observed in rats was due to inhibition of the mammalian bc1 mitochondrial complex by GSK932121A. FTIH studies with GSK932121A were put on hold and the compound later terminated despite its good tolerability profile at the two dose levels tested. A package of investigative work was initiated to explore the acute toxicity caused by GSK932121A within which the present work (chapters 3-5) forms an integral first stage.

1.9.2. GSK2617971A(OXPHOSU

)

Unlike GSK932121A (described above), GSK2617971A was withdrawn from active development early, during the preclinical stage. GSK2617971A is a pyrazole compound which was designed as a selective androgen receptor modulator (SARM) for the treatment of cachexia (muscle wastage). GSK2617971A

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(Fig.1.8) has shown potent uncoupling activity in vitro and is acutely toxic in vivo in rats, when dosed at 60mg/kg–100mg/kg. GSK2617971A was terminated early in development following observations of toxicity in oral dose range finding (DRF) tolerability studies.

FIGURE 1.8: Chemical structure of GSK2617971A (mw: 371.118). This pyrazole molecule was designed as a selective androgen receptor modulator (SARM) intended for the treatment of cachexia.

Since GSK2617971A’s termination from development, it has been used as a tool molecule for the study of DIMT. As compounds with some degree of OXPHOS uncoupling activity are regularly identified in preclinical screening, it was imperative to gain further understanding of its mechanism of toxicity in vivo, in order to better inform discovery groups of the potential safety liability of this class of mitochondrial toxicant (chapter 6). Furthermore, adverse effects caused by uncoupling of OXPHOS have been observed in the clinical situation following administration of medicines such as doxorubicin and therefore improved mechanistic understanding of this class of mitochondrial toxicant is translatable to effects observed in patients.