Download Nitro drugs and the trypanosomatids

Parasitology 2017
Nitro drugs and the trypanosomatids
Susan Wyllie
([email protected]
Neglected Tropical Diseases:
What are they?
Disease
(millions)
(million DALYs)a
(per annum)
35
89
1.5 million
Tuberculosis
2,000
36
1.5 million
Malariac
198
82.7
0.6 million
Sleeping sickness
Chagas' disease
Leishmaniasis
0.4
8.0
12
1.6
0.6
2.0
10,000
13,000
30,000
Schistosomiasis
Onchocerciasis
Filariasis
200
18
120
1.8
1.0
5.6
15,000
0
0
BACTERIAL
PROTOZOAL
a Disability-adjusted
Health burden Deaths
HIV / AIDSb
VIRAL
HELMINTHIC
Infected
life years lost to the community, c.f. War = 20 million DALYs
b
80% of all deaths in Africa
c
90% of all deaths in Africa
*
World Health Reports, 2014
African Sleeping Sickness
(Trypanosoma brucei)
Long slender
trypomastigote
(blood and lymph)
Short stumpy
trypomastigote
(bloodstream)
CNS Stage
HUMAN
Blood stage
TSE TSE FLY
Metacyclic
trypomastigote
(salivary glands)
Epimastigote
(salivary glands)
Procylic
trypomastigote
(mid-gut)
Human African Trypanosomiasis
Early stage
Late stage
CNS invasion
Few weeksa to few
monthsb
aTrypanosoma
brucei rhodesiense (10% of cases)
bTrypanosoma brucei gambiense (90% of cases)
Current Sleeping Sickness Drugs
Suramin
(1916)
Pentamidine
(1937)
Melarsoprol
(1946)
Eflornithine
(1977)
(plus Nifurtimox
2009)
Inactive in all late stage disease
Parenteral administration (i.v.)
Prolonged treatment (3 weeks)
Toxicity (fatal anaphylaxis ~ 1 in 20,000; skin reactions,
reversible renal damage)
Future availability (Bayer) and cost
Inactive in all late stage disease
Parenteral administration (i.m. for 10 days)
Inactive in some T.b.rhodesiense cases
Toxicity (myalgia, diabetes, sterile abscess)
Cost ($ 60-150)
Severe toxicity (death 5% of patients) Parenteral
administration (slow i.v. infusion)
High relapse rate (drug resistance?)
Prolonged hospitalisation (> 1 month)
Future availability (Aventis) and cost
Inactive against T.b.rhodesiense late stage
Parenteral administration (i.v. infusions)
Prolonged treatment (4 x / day for 2 weeks)
Reversible toxicity (convulsions; bone marrow suppression;
GI symptoms; nerve deafness)
Future availability (Aventis) and cost ($700)
Chagas’ Disease (T. cruzi)
Amastigotes
(tissue stage)
Trypomastigotes
(bloodstream)
release and reinvasion
Infected
macrophage
Trypomastigote
Amastigote
HUMANS
TRIATOMINE
BUGS
Metacyclic
trypomastigotes
(rectal lumen)
Epimastigotes
(rectal lumen)
Pathology of Chagas’ Disease
Acute stage
Indeterminant stage
Chronic stage
>10 years
Cardiomyopathy
Normal
>10 years
Romana’s sign
Mega-organ disease
Treatment of Chagas’ Disease
Acute stage
Chronic stage
O
O2N
O
N N
S
O
H3C
Nifurtimox (1964)
NO2
N
H
N
N
O
Benznidazole (1972)
None
Life cycle of Leishmania spp.
Macrophage
Amastigotes
(skin, bone marrow
liver & spleen)
Promastigote
Infected macrophages
HUMAN
SAND FLY
Phlebotomus spp
Metacyclic
promastigotes
(proboscis)
Promastigotes
(midgut)
Pathology of the Leishmaniases
CUTANEOUS
MUCOCUTANEOUS
VISCERAL
L.major
L.mexicana
L.tropica
L.braziliensis
L.donovani
L.infantum
Drugs for Leishmaniasis
Pentostam
(SAG)
Pentamidine
Liposomal
Amphotericin B
(AmBisome)
Drug resistance
Parenteral administration
Prolonged treatment (up to 4 weeks)
Toxicity (liver and pancreas, HIV patients)
Compliance / Cost ($ 120-150)
HCOH
HCO
OH
O-
OCH
HCO
Sb O
Sb
OCH
HCO
+
3Na
OCH
COO-
Parenteral administration (i.m.)
Poor response rates (around 75%)
Prolonged treatment (up to 4 weeks)
Toxicity (e.g. myalgia, diabetes, sterile abscess)
Cost ($ 60-150)
Parenteral administration (slow iv infusion)
Severe toxicity (e.g. renal failure, cardiotoxicity)
Cost (AmBisome > $1000)
Prolonged hospitalisation
CH2OH
CH2OH
HCOH
COO-
NH
NH
H2N
NH2
O
O
OH
H3C
HO
OH
O
O
CH3
OH
OH
OH
OH
Miltefosine
Parenteral administration (i.m.)
Prolonged treatment (up to 3 weeks)
Toxicity (nephrotoxicity; nerve deafness)
Availability / Cost ($50)
Toxicity (teratogenic, pregnancy)
Ease of resistance (long half life of drug)
Compliance / Cost ($50)
O
H
H3C
O
OH
O
HO
HO
NH2
O
O
H2 N
H 2N
O
HO
HO
HO
O
H2N
OH
Paromomycin
(Aminosidine)
OH
NH2
OH
O
NH2 O
OH
O
H3C
(CH2)14 O
P
O
O-
N+(CH)3
CH3
OH
The Need for New Drugs - Summary
Limitations of current anti-parasitic drugs
• Toxicity (melarsoprol 5% deaths in HAT)
• Teratogenicity (miltefosine for leishmaniasis)
• Drug resistance (pentostam for leishmaniasis)
• Efficacy (nifurtimox for Chagas’ disease)
• Route of administration (all HAT drugs are by injection)
• High cost (Ambisome >$1,000, Eflornithine ~$700)
• Availability (eflornithine, melarsoprol)
• Compliance (antimalarials)
• Policy (artemesinin combination therapy)
Pharmaceutical industry & unmet medical need
• 17 new drugs developed for all tropical diseases 1975-2004
• Most inappropriate for resource poor settings
• Diseases of the poor
• Cost to develop new drug ~$800m (no market)
The Ideal Target Product Profile for NTDs
Safe for use: men, women, children and foetus
Minimal toxicity: tolerable side effects; better than current drugs
Few contraindications: drug-drug interactions; HIV or TB co-infections
Efficacy: better than current drugs
Compliance: short treatment; once per day
Resistance: low potential to generate drug resistance
Orally active: avoid needles and hospitalization
Broad spectrum: all disease-causing species, including resistant lines
Stable: 2 years shelf life at 40C and 75% relative humidity
Affordable: diseases of poverty (cheaper than existing drugs)
Nitro-heterocyclic Drugs
 Compounds which contain a nitro group attached to an aromatic ring system
Therapeutics for a number of indications, particularly infectious diseases
Metronidazole
Antibiotic
Antiprotozoal
Nitrofurantoin
Antibiotic
(UTIs)
Azathioprine
Immunosuppressive
Tolcapone
Parkinson’s disease
Chloramphenicol
Antibiotic
Flutamide
Prostate cancer
Nifedipine
Angina
Hypertension
Nitro-heterocyclics in the treatment of
infectious disease – the future
Renewed interest in the use of nitroheterocyclics for the treatment of infectious disease
PA-824 – phase II clinical trials against tuberculosis
Nitazoxanide – used in the treatment of giardiasis, cryptosporidiosis and is in
clinical trials for viral hepatitis
Nitro-drugs and Chagas’ disease
Nifurtimox and benznidazole have been used in the treatment of Chagas’
disease since the 1970’s
Estimated global population infected with T. cruzi, 2009
*
Nfx
nitrofuran
Bnz
nitroimidazole
Efficacy of Nfx and Bnz
~70% against acute stage
<20% against chronic stage
Low efficacy attributed to naturally occurring nitro-drug resistant strains
(Filardi and Brener (1987) Trans. R. Soc. Trop. Med. Hyg. 81, 755-759.)
Nitrodrugs against trypanosomiasis
•
Clinical trials of nifurtimox against HAT (1970s):
• first clinical trial (Janssens and Demunynck (1977)
– inconclusive, toxicity concerns
– recommended only for melarsoprol-refractory patients
• subsequent trials in the 1980s
– variable efficacy (30 – 80%)
– toxicity increasing with dose and treatment duration (30 – 60 days)
– last resort for melarsoprol-refractory cases
• clinical trials in 2001 – 2009:
– three drug combinations - nifurtimox:eflornithine:melarsoprol
(Priotto et al. (2006) PLoS Clin. Trials 1, e39)
- (nifurtimox plus eflornithine, NECT) was found to be safer than
(melarsoprol plus nifurtimox or plus eflornithine)
– NECT is as effective as eflornithine monotherapy
Nifurtimox-eflornithine combination therapy
(NECT) for the treatment of HAT
Eflornithine (1981)
NECT (2009)
Regimen
4 x daily for 14 days
(56 slow IV infusions)
IV 2 x daily for 7 days +
oral Nfx 3 x 10 days
Cost
£407 / patient
£203 / patient
Side effects
neuropenia
anemia
reduced
Eflornithine (DFMO)
Nifurtimox (Nfx)
NECT
•
Nifurtimox-eflornithine combination therapy (NECT) added to the WHO list of essential
medicines in 2009
Advantages of NECT
•
•
•
•
•
•
•
improved efficacy (>96%)
reduced hospitalisation time
reduced side effects
reduced risk of infections
reduced cost
simplified logistics *
reduced risk of drug resistance *
NECT has become the treatment of choice for stage II T. b. gambiense HAT and is used to treat
>90% of infections
First new treatment for TriTryp diseases in >20 years
Why do combination therapies protect against the
emergence of drug resistance?
Far easier for resistance to develop against a single drug (single mutation) than against a
combination (chances of two advantageous mutations happening in one parasite
exponentially higher)
Drugs are often given in combination to protect against the development of resistance
Drug combinations used to treat parasitic diseases include:
Malaria: artesunate (oral artemisinin) should always be given in combination (mefloquine
and amodiaquine often used)
Malaria: Malarone contains a combination of atovaquone and proguanil
Toxoplasmosis (in immuno-compromised patients): Pyrimethamine/sulfadiazine and folinic
acid
Proposed mechanism of action of NECT
Targeting trypanothione metabolism
Intracellular thiols
COO-
Monothiol
O
+
H3N
-
COO
N
H
SH
H
N
+
N
H
O
O
Dithiol
OH
O
+
H3N
COO
Mammals
O
H3N
O
Glutathione
GSH
H
N
-
N
H
H
N
O
SH
SH
H
N
O
NH2+
O
N
H
Trypanothione
T[SH]2
Trypanosomatids
Functions of Trypanothione
Maintenance of thiol redox status
• Reduction of protein and other disulphides (TryR)
• Regulation of thiol redox potential (TryR and TryX)
Defence against oxidant stress
• Removal of peroxides via:
1.Type I tryparedoxin peroxidases (TryP)
2.Type II tryparedoxin peroxidases (TDPX)
3. Ascorbate peroxidase
4.Trypanothione S-transferase (eEF1B complex)
Defence against chemical stress
• Detoxification of methylglyoxal (glyoxalase I & II)
• Xenobiotic metabolism (trypanothione S-transferase)
Deoxyribonucleotide synthesis
• Electron donor to ribonucleotide reductase via TryX
TryR
TryP
TryX
TDPX
Glyoxalase 1
Proposed mechanism of action of NECT
Eflornithine
H2N
F
H2N
F
O
OH
Ornithine ODC Putrescine
SpdS
Spermidine
N
CH3
N
N
O
OH
NH2
O
NH
S
COOH
NH2
S
O
H3C
Nifurtimox
NTR
NADP+
OH
Glutamate
+ Cysteine GCS
H3C
N N
H2O
TryP/TDPX
TryX
TryS
Trypanothione
Trypanothione
disulphide
TryR
NH2
N
O
H2O2
SAM
AdoMet
AdoMetDC
N
O
O2N
Glutathione
NADPH
NECT mechanism of action summary
Eflornithine inhibits trypanothione biosynthesis – key in the parasite’s defence
against oxidant stress
Nifurtimox results in the generation of high levels of oxidant stress
With impaired anti-oxidant defences (eflornithine), the parasite is far more
susceptible to nifurtimox
This is a great example of synergy between two drugs – work better in
combination than individually
Fexinidazole – the future
Phase I clinical trials completed
Phase II and III trials on-going in the Democratic Republic of Congo
Proposed for use as the first orally available treatment for stage 1 and 2 HAT
Nitro-drugs - key role in the future treatment of HAT
Can fexinidazole also be an effective treatment for
visceral leishmaniasis?
In vivo metabolism of fexinidazole
[O]
[O]
fexinidazole
sulfoxide
sulfone
Fexinidazole is rapidly oxidised in vivo to sulfoxide and sulfone metabolites
These metabolites accumulate to high levels in the blood following oral dosing
of mice and likely to the therapeutically relevant species in vivo
Sokolova et al., Antimicrobial Agents and Chemotherapy 54, 2893-2900 (2010)
In vitro sensitivity of L. donovani to fexinidazole
and its metabolites
L. donovani EC50, µM
Compound
Structure
Promastigote
Amastigote
(in macrophage)
Fexinidazole
sulfoxide
3.1 ± 0.3
5.3 ± 0.1
Fexinidazole
sulfone
4.8 ± 0.1
5.3 ± 0.3
Miltefosine
6.1 ± 0.2
3.3 ± 0.2
5.6 ± 0.2
>50
Fexinidazole
In vitro potency of fexinidazole sulfoxide and sulfone compares well with miltefosine
100
300,000
Suppression, %
Parasite burden, LDU
In vivo sensitivity of L. donovani to fexinidazole
and its metabolites
200,000
100,000
0
75
50
ED50 – 12 mg kg-1
ED90 – 57 mg kg-1
25
0
50
100
on
t
C
150
Fexinidazole, mg kg -1
ro
lv
eh
Pe
ic
le
nt
(0
os
)
ta
m
M
(1
ilt
ef
5)
os
i
ne
Fe
xi
(1
ni
2)
da
z
Fe
ol
e
xi
(2
ni
5)
da
zo
Fe
le
xi
ni
(5
da
0)
zo
Fe
le
xi
(1
ni
00
da
)
zo
le
(2
00
)
0
Drug treatment (mg kg -1)
Fexinidazole proved to be an extremely effective, dose-dependent inhibitor
Five single doses of 200 mg kg-1 suppressed infection by 99%
ED50 estimated at 12 mg kg-1
At the lowest doses tested – fexinidazole equivalent to miltefosine
200
Pharmacokinetic properties of fexinidazole and its
metabolites
Blood concentration, ng ml-1
105
EC99 Sulfone
104
EC99 Sulfoxide
sulfone
sulfoxide
103
Oral dosing – 200 mg kg-1
102
fexinidazole
101
0
10
20
30
40
50
Time, h
Blood levels of fexinidazole and major metabolites were determined by UPLC/MS/MS
Fexinidazole has potential as a once daily, oral treatment for visceral leishmaniasis
Fexinidazole: Determination of tissue distribution
-
Male albino rats dosed with [14C]-FEXINIDAZOLE
-
Tissue distribution determined by whole body autoradiography
-
Radioactivity distributed to all the body and was found in all organs and
tissues analysed
“Following oral dosing, highest radioactivity levels were
measured in the intestinal wall, stomach, liver, kidney,
prostate, pancreas, bladder, heart, muscle, spleen,
thyroid and lung”
Fexinidazole may be particularly suited for use in treatment of visceral leishmaniasis
Torreele et al., PLoS Negl Trop Dis. 2010,4, e923 (2010)
Cytocidal effect of fexinidazole sulfone on L.
donovani
108
control
Cell number, ml-1
7
10
106

+ drug
105
104
103
0
10
20
30
Time, h
Amastigotes incubated with 10x [EC50] fexinidazole sulfone
At defined intervals, parasites were removed, washed and sub-cultured without drug
Cells lost viability after 24h in the presence of drug
Incredibly important that anti-trypanosomal drugs are cytotoxic
The Ideal Target Product Profile for NTDs
Safe for use: men, women, children and foetus
Minimal toxicity: tolerable side effects; better than current drugs
Few contraindications: drug-drug interactions; HIV or TB co-infections
Efficacy: better than current drugs
Compliance: short treatment; once per day
√
√
Resistance: low potential to generate drug resistance
Orally active: avoid needles and hospitalization
√
Broad spectrum: all disease-causing species, including resistant lines
Stable: 2 years shelf life at 40C and 75% relative humidity
Affordable: diseases of poverty (cheaper than existing drugs)
√
34
√
Fexinidazole for VL - conclusions
Fexinidazole has potential as a much needed additional oral therapy for visceral
leishmaniasis
Biological and pharmacokinetic properties of fexinidazole appear to be ideally
suited for use against the severest form of leishmaniasis
Comprehensive preclinical studies already completed on fexinidazole (phase I
clinical trials for HAT)
Every reason to hope that fexinidazole can progress rapidly into clinical
development for use against visceral leishmaniasis
Wyllie et al., Science Translational Medicine, 4, 119re1 (2012)
Fexinidazole for visceral leishmaniasis
DNDi undertook a Phase II proof of concept study to determine the efficacy and
safety of fexinidazole in VL patients in 2013
Study was carried out in Sudan where current therapies (Amphoptericin B and
Miltefosine are less effective)
Advantages of trialling fexinidazole in Africa – effective treatments for VL are
urgently needed for this region
Outcome:
12 patients enrolled in the trial
All demonstrated reduced parasite load during treatment although 9/12 relapsed
following cessation of treatment
Length of dose and general pharmacokinetics are to be further investigated
Also, fexinidazole will be trialled as a partner drug for miltefosine (combination)
Drugs for Neglected Diseases Initiative
http://www.dndi.org/
WHO
http://www.who.int/tdr/en/
[email protected]
Medicines for Malaria Venture
http://www.mmv.org/
Bill and Meilinda Gates Foundation
http://www.gatesfoundation.org/
Reading list
Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects.
Patterson, S and Wyllie, S. Trends Parasitol. (2014) 30, 289-298.
Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense
trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Priotto et al., Lancet. (2009)
374, 56-64.