Beyond immune escape:a variant surface glycoprotein causes suramin resistance in Trypanosoma brucei

Suramin is one of the first drugs developed in a medicinal chemistry program (Bayer, 1916), and it is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense. Cellular uptake of suramin occurs by endocytosis, and reverse genetic studies with T. b. brucei have linked downregulation of the endocytic pathway to suramin resistance. Here we show that forward selection for suramin resistance in T. brucei spp. cultures is fast, highly reproducible and linked to antigenic variation. Bloodstream-form trypanosomes are covered by a dense coat of variant surface glycoprotein (VSG), which protects them from their mammalian hosts' immune defenses. Each T. brucei genome contains over 2000 different VSG genes, but only one is expressed at a time. An expression switch to one particular VSG, termed VSGSur , correlated with suramin resistance. Reintroduction of the originally expressed VSG gene in resistant T. brucei restored suramin susceptibility. This is the first report of a link between antigenic variation and drug resistance in African trypanosomes

Pharmacokinetic Comparison To Determine the Mechanisms Underlying the Differential Efficacies of Cationic Diamidines against First- and Second-Stage Human African Trypanosomiasis

This is the published version.Human African trypanosomiasis (HAT), a neglected tropical disease, is fatal without treatment. Pentamidine, a cationic diamidine, has been used to treat first-stage (hemolymphatic) HAT since the 1940s, but it is ineffective against second-stage (meningoencephalitic, or central nervous system [CNS]) infection. Novel diamidines (DB75, DB820, and DB829) have shown promising efficacy in both mouse and monkey models of first-stage HAT. However, only DB829 cured animals with second-stage infection. In this study, we aimed to determine the mechanisms underlying the differential efficacies of these diamidines against HAT by conducting a comprehensive pharmacokinetic characterization. This included the determination of metabolic stability in liver microsomes, permeability across MDCK and MDR1-MDCK cell monolayers, interaction with the efflux transporter MDR1 (P-glycoprotein 1 or P-gp), drug binding in plasma and brain, and plasma and brain concentration-time profiles after a single dose in mice. The results showed that DB829, an azadiamidine, had the highest systemic exposure and brain-to-plasma ratio, whereas pentamidine and DB75 had the lowest. None of these diamidines was a P-gp substrate, and the binding of each to plasma proteins and brain differed greatly. The brain-to-plasma ratio best predicted the relative efficacies of these diamidines in mice with second-stage infection. In conclusion, pharmacokinetics and CNS penetration influenced the in vivo efficacies of cationic diamidines against first- and second-stage HAT and should be considered when developing CNS-active antitrypanosomal diamidines

In Vitro and In Vivo Evaluation of 28DAP010, a Novel Diamidine for Treatment of Second-Stage African Sleeping Sickness

This is the published version.African sleeping sickness is a neglected tropical disease transmitted by tsetse flies. New and better drugs are still needed especially for its second stage, which is fatal if untreated. 28DAP010, a dipyridylbenzene analogue of DB829, is the second simple diamidine found to cure mice with central nervous system infections by a parenteral route of administration. 28DAP010 showed efficacy similar to that of DB829 in dose-response studies in mouse models of first- and second-stage African sleeping sickness. The in vitro time to kill, determined by microcalorimetry, and the parasite clearance time in mice were shorter for 28DAP010 than for DB829. No cross-resistance was observed between 28DAP010 and pentamidine on the tested Trypanosoma brucei gambiense isolates from melarsoprol-refractory patients. 28DAP010 is the second promising preclinical candidate among the diamidines for the treatment of second-stage African sleeping sickness

Synthesis and Antiprotozoal Activity of Dicationic 2, 6-Diphenylpyrazines and Aza-Analogues

Dicationic 2,6-diphenylpyrazines, aza-analogues and prodrugs were synthesized; evaluated for DNA affinity, activity against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) in vitro, efficacy in T. b. r. STIB900 acute and T. b. brucei GVR35 CNS mouse models. Most diamidines gave poly(dA-dT)2 ΔTm values greater than pentamidine, IC50 values: T. b. r. (4.8 to 37 nM) and P. f. (10 to 52 nM). Most diamidines and prodrugs gave cures for STIB900 model (11, 19a and 24b 4/4 cures); 12 3/4 cures for GVR35 model. Metabolic stability half-life values for O-methylamidoxime prodrugs did not correlate with STIB900 results

Indole and benzimidazole bichalcophenes: synthesis, DNA binding and antiparasitic activity

A novel series of indole and benzimidazole bichalcophene diamidine derivatives were prepared to study their antimicrobial activity against the tropical parasites causing African sleeping sickness and malaria. The dicyanoindoles needed to synthesize the target diamidines were obtained through Stille coupling reactions while the bis-cyanobenzimidazoles intermediates were made via condensation/cyclization reactions of different aldehydes with 4-cyano-1,2-diaminobenzene. Different amidine synthesis methodologies namely, lithium bis-trimethylsilylamide (LiN[Si(CH3)3]2) and Pinner methods were used to prepare the diamidines. Both types (indole and benzimidazole) derivatives of the new diamidines bind strongly with the DNA minor groove and generally show excellent in vitro antitrypanosomal activity. The diamidino-indole derivatives also showed excellent in vitro antimalarial activity while their benzimidazole counterparts were generally less active. Compound 7c was highly active in vivo and cured all mice infected with Trypanosoma brucei rhodesiense, a model that mimics the acute stage of African sleeping sickness, at a low dose of 4 × 5 mg/kg i.p. and hence 7c is more potent in vivo than pentamidine

Isothermal Microcalorimetry, a New Tool to Monitor Drug Action against Trypanosoma brucei and Plasmodium falciparum

Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly

In vitro studies and in vivo evaluation of novel diamidines for 2nd stage sleeping sickness

Summary: African sleeping sickness is one of the most neglected tropical diseases. Transmitted by the tsetse fly it exclusively occurs in sub-Saharan Africa. It is caused by two different parasite subspecies causing two different forms of African sleeping sickness. Trypanosoma brucei gambiense is prevalent in West and Central Africa while Trypanosoma brucei rhodesiense is prevalent in East and South Africa. Sleeping sickness is classified in two main stages. In the first stage, the parasites reside in the lymph and blood system. In the second stage, the parasites additionally infect the brain. Untreated sleeping sickness is fatal. Drugs are available for this fearsome disease, however, most of them are old and have many drawbacks, such as severe adverse effects, treatment failures and complicated treatment schedules, which is a problem in remote rural areas where the disease primarily occurs. African sleeping sickness is a communicable disease that can be controlled. In 1998, there were an estimated 300,000 cases. By 2012 the prevalence has decreased to about 30,000, by different control measures such as vector control, improved surveillance and free drug distribution. Elimination seems possible, but safe and effective drugs are needed to reach this goal. One of the current drugs is the diamidine pentamidine which is in use since the early 1940s. However, it works only in patients with first stage disease and it has to be injected. The Consortium for Parasitic Drug Development (CPDD) was founded in the year 2000 to find novel diamidines with better characteristics than the existing drugs. We improved oral absorption, which makes it possible to use pills instead of injections, and central nervous system (CNS) penetration. One compound (pafuramidine) has been tested in patients with first stage infections. It was the first compound that cured sleeping sickness orally, which is of great help for rural areas. Unfortunately, pafuramidine caused kidney and liver problems, and it did not cure second stage infections. In the meantime, we have identified superior compounds especially for the second stage. As described in Chapter 3, two compounds, the prodrugs DB868 and DB844, given orally, cured all mice with CNS infections. However, both prodrugs were too toxic at the high doses required to cure both stages in monkeys. Nevertheless, DB868 is a good candidate drug to cure first stage sleeping sickness by an oral treatment, as demonstrated in mice and monkeys with first stage infections. Chapter 4 shows data of another CNS potent prodrug, DB1227 which was, however, less effective than DB868 in CNS infected mice. Chapters 3, 7, 8 and 9 deal with two unmasked diamidines, DB829 and 28DAP010, which were highly effective in mice with second stage infections after i.p. injection. This was unexpected since diamidines are rather unlikely to cross the blood brain barrier in sufficient concentrations by diffusion. These two diamidines may penetrate into the brain by specific transporter proteins. The advantage of the two diamidines is that both cure with a short treatment course which could shorten the time of hospitalization of the patients. We have already tested DB829 in monkeys with promising results. It was safe and effective at low doses and a short treatment schedule in monkeys with second stage disease. 28DAP010 seems to be similarly effective as DB829 on both T. brucei subspecies in vitro and in mouse models. In Chapter 6 we established a new in vitro method to measure the kinetics of drug action on pathogenic protozoa on a real time basis. We exploited the capacity of viable cells to produce heat and measured the heat flow using microcalorimetry. 28DAP010 inhibited the heat production of trypanosome cultures faster than DB829. The parasite clearance time of 28DAP010 was also faster than of DB829 in mice. The required effective treatment duration was still similar in mice with single dose for first stage and 5 days for second stage infections. Upcoming efficacy studies will reveal if 28DAP010 is as curative in monkeys as DB829 and toxicity studies of 28DAP010 and DB829 side by side will shed light on their toxicity profile. These studies will help to select the better of these two compounds as a clinical drug candidate for the treatment of second stage sleeping sickness. ---------- Zusammenfassung: Die Afrikanische Schlafkrankheit ist eine Tropenkrankheit, welche durch die Tsetsefliege übertragen wird und daher ausschliesslich im tropischen Afrika vorkommt. Sie gehört zu den vernachlässigsten Krankheiten überhaupt und wird deshalb auch “vergessene Seuche“ genannt. Der Erreger ist ein einzelliger Parasit. Es gibt zwei verschiedene Unterarten, die zu etwas unterschiedlichen Schlafkrankheitsformen führen. Ohne wirksame Medikamentenbehandlung sind beide Formen tödlich. Trypanosoma brucei gambiense kommt nur in West- und Zentralafrika vor, während Trypanosoma brucei rhodesiense in Ost- und Südafrika zu finden ist. Der Krankheitsverlauf kann in zwei Stadien unterteilt werden. Im ersten Stadium findet man die Parasiten im Blut- und Lymphsystem und im zweiten Stadium zusätzlich im Gehirn. Zwar gibt es für diese Krankheit Medikamente, jedoch sind die meisten davon veraltet, haben ausgeprägte Nebenwirkungen und sind wegen Rückfällen oder der komplizierten und aufwendigen Behandlung problematisch. Die Bekämpfung der Afrikanischen Schlafkrankheit ist möglich. 1998 gab es geschätzt etwa 300.000 Krankheitsfälle. Durch verbesserte Überwachung mit anschliessender medizinischer Behandlung der Infizierten, kostenlose Medikamentenverteilung und Vektorkontrolle, liess sich die Krankheit auf etwa 30.000 Krankheitsfälle im Jahr 2012, eindämmen. Für eine Eliminierung sind wirksame und verträgliche Medikamente notwendig. Ein Diamidin, das schon seit den frühen 40-er Jahren eingesetzt wird ist Pentamidin. Es wirkt noch heute, aber nur in Patienten die sich im ersten Stadium befinden, zudem muss es injiziert werden. Im Jahr 2000 wurde das Konsortium CPDD, für die Entwicklung neuer Wirkstoffe zur Behandlung parasitärer Erkrankungen, vor allem für die Schlafkrankheit, gegründet. Neuartige Diamidine mit verbesserten Eigenschaften wurden gesucht und es war uns möglich, die orale Bioverfügbarkeit und die Bluthirnschrankengängigkeit, chemisch zu verbessern. Pafuramidin, war einer der neuen Wirkstoffe, das erste oral einzunehmende Medikament gegen Schlafkrankheit, das im Menschen getestet wurde. Ein orales Medikament hat grosse Vorteile für diese Krankheit, die hauptsächlich in abgelegenen Gebieten Afrikas vorkommt, wo ein ausgebautes Gesundheitssystem oft fehlt. Pafuramidin heilte nur das erste Schlafkrankheitsstadium und dabei wurden Leber- und Nieren-Unverträglichkeiten festgestellt. Während der klinischen Studie testeten wir weitere Diamidine und fanden verbesserte Substanzen, vor allem bezüglich der Wirksamkeit des zweiten Krankheitsstadiums. Kapitel 3 und 4 beschreibt die wirksamsten Moleküle, die das Zweitstadium bei oraler Verabreichung heilten. Diese Moleküle, DB844, DB868, DB1227, aber auch das Pafuramidin sind Medikamenten-vorstufen (Prodrugs). Diese wurden entwickelt, um die orale Aufnahme und Gehirn-gängigkeit zu verbessern. Die aktivsten waren DB868 und DB844 in Mäusen, jedoch zeigten beide Moleküle toxische Wirkungen im Affen ohne dabei ausreichend die Gehirninfektion zu heilen. Dennoch war DB868 im Affenmodell deutlich besser verträglich als Pafuramidin und ist somit ein guter Ersatzkandidat für eine orale Wirkstoffentwicklung fürs erste Stadium. Unerwartet konnten wir jedoch zwei Diamidine (ohne Vorstufenergänzung) identifizieren, die ebenfalls Mäuse mit Gehirninfektionen heilten. Da Diamidine unter physiologischen Bedingungen protoniert sind, ist es unwahrscheinlich, dass sie durch die Bluthirnschranke diffundieren. Möglicherweise werden sie über spezifische Mechanismen ins Gehirn transportiert. Kapitel 3, 7, 8 und 9 befassen sich mit den beiden aktivsten Diamidinen, DB829 und 28DAP010. Ihre hohe Wirkung und die kurze Behandlungszeit nach parenteraler Verabreichung (i.p. oder i.m) sind vielversprechend. DB829 war gut verträglich und wirksam bei niedrigen Dosen und heilte die infizierten Affen mit dem zweiten Krankheitsstadium bereits bei einer 5-tägigen Behandlung. In vitro und im Mausmodel war 28DAP010 auf beide Trypanosomen Unterarten ähnlich wirksam wie DB829. Um die Wirkungszeit neuer Substanzen auf Trypanosomen zu testen, entwickelten wir eine neue Methode, die in Kapitel 6 beschrieben wird. Dabei nutzten wir die Eigenschaft der Zellen, Wärme zu produzieren und massen diese mit einem Kalorimeter auf Echtzeit. 28DAP010 reduzierte die Wärmeentwicklung einer Trypanosomenkultur deutlich schneller als DB829. Auch in infizierten Mäusen wirkte 28DAP010 schneller. Die Behandlungsdauer und Dosierung war bei beiden Diamidinen trotzdem vergleichbar. Eine Einzeldosis heilte das erste und eine 5-tägige Behandlung das zweite Stadium in Mäusen. Weitere Studien sind nötig, um die Wirksamkeit von 28DAP010 im Affenmodel zu überprüfen und die Verträglichkeit beider Diamidine zu analysieren. Diese Ergebnisse werden zeigen, welches der bessere klinische Kandidat für die Behandlung des zweiten Schlafkrankheitsstadiums sein wird

A well-plate format isothermal multi-channel microcalorimeter for monitoring the activity of living cells and tissues

Design and properties are reported for a novel type of multi-channel isothermal microcalorimeter. It is equipped with 48 calorimetric units (channels) and is primarily intended for use as a monitor of the activity of living cells, tissues and small animals. Calorimetric vessels are positioned in a holder with the format of a 48-well microtiter plate. At most, 47 samples can be measured simultaneously; one vessel is then used as reference. The standard configuration is 32 sample positions using 16 channels as references in a twin calorimeter setup. The detection limit is then 0.1 μW. Sample volumes are usually 100 μl–300 μl. The 24 h baseline stability is typically 0.2 μW (room temperature variation ≤1 °C). The instrument was designed considering feasible uses in applied biology, especially in pharmaceutical and clinical laboratories and in environmental work. However, it can be employed as a general monitor of slow processes in different fields of biology and for non-biological systems, including accurate determination of their thermal powers (heat production rates). In the present report, properties of the instrument are characterized by chemical calibration experiments and in measurements of growth of bacteria and mammalian cells

Synthesis and antiparasitic activity of new bis-arylimidamides : DB766 analogs modified in the terminal groups

Fifteen novel bis-arylimidamide derivatives with various 6-membered (7a-c) and 5-membered (7d-o) heterocyclic rings replacing the terminal pyridyl rings of the lead compound DB766{(2,5-bis[2-i-propoxy-4-(2-pyridylimino)aminophenylfuran]}, were prepared and evaluated versus Trypanosoma cruzi, Leishmania amazonensis, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Compound 7a with pyrimidine replacing the pyridine rings showed good activity versus T. cruzi, T. brucei rhodesiense and P. falciparum (IC50 = 200 nM, 32 nM and 8.5 nM, respectively). Three compounds (7g, 7i, 7j) with thiazole replacing the pyridine rings gave low micromolar (0.17-0.3 μM) IC50 values versus L. amazonensis, however only 7g exhibited an acceptable selectivity index (SI = 27). Compounds 7a, 7j and 7m exhibited potent activity against T. brucei rhodesiense (IC50 = 12-60 nM). Ten of the 15 compounds with pyrimidine, pyrrole, thiazole and imidazole terminal units were highly active against P. falciparum (IC50 = 9-87 nM). Both pyrimidine and pyridine terminal groups are advantageous for anti-T. cruzi activity and several different heterocyclic terminal units are effective versus P. falciparum, both findings merit further investigation

Antiprotozoal activity of dicationic 3,5-diphenylisoxazoles, their prodrugs and aza-analogues

Fifty novel prodrugs and aza-analogues of 3,5-bis(4-amidinophenyl)isoxazole and its derivatives were prepared. Eighteen of the 24 aza-analogues exhibited IC₅₀ values below 25 nM against Trypanosoma brucei rhodesiense or Plasmodium falciparum. Six compounds had antitrypanosomal IC₅₀ values below 10 nM. Twelve analogues showed similar antiplasmodial activities, including three with sub-nanomolar potencies. Forty-four diamidines (including 16 aza-analogues) and the 26 prodrugs were evaluated for efficacy in mice infected with T. b. rhodesiense STIB900. Six diamidines cured 4/4 mice at daily 5 mg/kg intraperitoneal doses for 4 days, giving results far superior to pentamidine and furamidine. One prodrug attained 3/4 cures at daily 25 mg/kg oral doses for 4 days