Figure 7 - available via license: Creative Commons Attribution 4.0 International
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Illustration of the P-glycoprotein (P-gp) interaction with bumped kinase inhibitors (BKIs) in the gastrointestinal tract. A, Low absorption of BKI1318 into the enterocyte as a result of P-gp-mediated efflux. B, In the presence of the P-gp inhibitor elacridar, BKI1318 absorption into the intestinal epithelium allows for targeting of Cryptosporidium.
Source publication
Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cry...
Contexts in source publication
Context 1
... association with the increased BKI1318 GI exposure, P-gp inhibition improved BKI1318 efficacy in a mouse model of Cryptosporidium infection. The improved outcome with elacridar coadministration is likely due to the increased BKI1318 enterocyte exposure, because intestinal P-gp efflux will reduce the exposure of Cryptosporidium to BKI1318 (Figure 7). As expected, since BKI1369 was not a P-gp substrate, the efficacy of BKI1369 was not affected by elacridar coadministration. ...
Context 2
... inhibition of P-gp-mediated drug efflux in the GI tract, blood-brain barrier, and kidney is targeted to increase tissue drug concentrations, drug efflux transporters expressed by bacteria, fungi, and protozoa can also modulate drug disposition, leading to decreased efficacy [35][36][37]. Cryptosporidium organisms reside in a vacuole in the cytoplasm at the tip of enterocytes (Figure 7). A feeder organelle separates Cryptosporidium from the host cell, and this folded membrane structure regulates access to both nutrients and drugs. ...
Citations
... 19 Pavlova et al. 20 revealed that p-glycoprotein can reduce the accumulation of antimicrobial drugs, leading to the emergence of drug-resistant strains of pulmonary tuberculosis. Arnold et al. 21 demonstrated that p-glycoprotein can decrease bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Pglycoprotein can downmodulate dendritic cells and result in an impaired immune response. ...
In this study, we investigated whether matched and mismatched multidrug resistance gene (MDR1) genotypes (G2677TA, C1236T and C3435T) were associated with prognosis in patients after allogeneic haematopoietic stem cell transplantation (allo‐HSCT). One hundred patients after transplantation and their donors were enrolled. Matched MDR1 G2677TA donor–recipient was associated with an increased risk of non‐relapse mortality (NRM) (29.5% vs. 6.2%, p = 0.002), poor overall survival (OS) (51.7% vs. 63.8%, p = 0.024) and disease‐free survival (DFS) (38.6% vs. 67%, p = 0.005). There were no differences in OS, DFS or NRM between MDR1 C1236T‐ and C3435T‐matched and ‐mismatched groups. Subgroup analysis suggested that within the matched MDR1 G2677TA group, male gender, haematopoietic cell transplantation‐specific comorbidity index ≥1, serum creatinine >137.2 μmol/L and post‐transplantation thrombocytopenia were associated with poor survival. Our results demonstrated that patients receiving matched MDR1 G2677TA allo‐HSCT experienced a poorer prognosis compared with the mismatched group. The potential mechanism may involve increased expression of P‐glycoprotein, leading to decreased accumulation of antimicrobial agents and ultimately contributing to the progression of inflammation. This identification of MDR1 G2677TA genotype compatibility holds promise as a valuable molecular tool for selecting donors for allo‐HSCT.
... Parasites generated using this method have several advantages over animal generated parasites notably they provide a novel method for the generation of parasite stages needed for vaccine development, which has shown to have great promise for a malaria vaccine [44]. In vitro cultured parasites permit extended (beyond 48 h) drug testing protocols to be employed and provide a method for preliminary pharmacokinetic/pharmacodynamic data to be obtained for candidate chemotherapeutic agents [47,48]. Currently no other in vitro culture method provides access to sufficient oocyst numbers (10 7 to 10 8 oocysts/mL) that can be generated in a GLP facility for use in human clinical trials. ...
Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.
... Therefore, clofazimine observed in plasma would reflect compound that went into solution in the intestinal lumen and likely transited through the gastrointestinal epithelium by a transcellular pathway. Transcellular transit is important given the localization of Cryptosporidium spp. to intracellular but extracytosolic parasitophorous vacuoles located beneath the apical plasma membrane of infected intestinal epithelial cells, as this localization suggests that therapeutic exposure within intestinal epithelial cells is essential for therapeutic efficacy (30)(31)(32)(33). ...
Infection with Cryptosporidium spp. can cause severe diarrhea leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a Phase 2a clinical trial in HIV infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine’s lack of efficacy in the Phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using E max and logistic models which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the Phase 2a study participants were well below concentrations associated with anti- Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the Phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed.
... To understand the ideal pharmacokinetic profile for an anti-Cryptosporidium drug, researchers have explored various approaches including empirical observations of systemic and intestinal exposure, physiologically based pharmacokinetic modeling [35], and consideration of efflux pumps [36]. Although a broadly applicable profile remains elusive, in some cases, such as the BKIs and PI(4)K inhibitors, compound concentrations in intestinal epithelial cells seem to drive efficacy. ...
Purpose of review:
Substantial progress has been made recently on the development of new therapeutics for cryptosporidiosis, an infection by the protozoan parasite Cryptosporidium that is associated with diarrhea, malnutrition, growth stunting, cognitive deficits, and oral vaccine failure in children living in low-resource settings.
Recent findings:
Various drug discovery approaches have generated promising lead candidates. The repurposed antimycobacterial drug clofazimine was tested in Malawian HIV patients with cryptosporidiosis but was ineffective. Target-based screens identified inhibitors of lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, methionyl-tRNA synthetase, and calcium-dependent protein kinase 1. Phenotypic screens led to discovery of a phosphatidylinositol 4-kinase inhibitor, the piperazine MMV665917, and the benzoxaborole AN7973. The relationship between pharmacokinetic properties and in-vivo efficacy is gradually emerging. A pathway to clinical trials, regulatory approval, and introduction has been proposed but additional work is needed to strengthen the route.
Summary:
Several lead compounds with potent activity in animal models and a favorable safety profile have been identified. A sustained effort will be required to advance at least one to clinical proof-of-concept studies. The demonstrated risk of resistance indicates multiple candidates should be advanced as potential components of a combination therapy.
... 14 Interestingly, efflux by Pgps (P-glycoprotein efflux pumps) appears to reduce efficacy. 15 BKIs that are effective in mice have been shown to dramatically reduce diarrhea and parasite excretion in both calf 16,17 and piglet models 18 of established Cryptosporidium infection. ...
In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
Cryptosporidium is an opportunistic protozoan, with many species of cross‐human infectivity. It causes life‐threatening diarrhoea in children and CD4‐defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti‐cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular–extracytoplasmic) and down‐regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin‐D (GSDM‐D) immunohistochemical expression, IFN‐γ, IL‐1β and IL‐18 blood levels by ELISA, and via parasite scanning by modified Ziehl–Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected‐control and drug‐control (nitazoxanide‐treated) mice. H&E staining of intestinal sections from romidepsin‐treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM‐D expression in vivo, and higher serum/culture IFN‐γ, IL‐1β and IL‐18 levels in romidepsin‐treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.
Introduction
Cryptosporidium species are protozoan parasites that are important causes of diarrheal disease including waterborne outbreaks, childhood diarrhea in resource-poor countries, and diarrhea in compromised hosts worldwide. Recent studies highlight importance of cryptosporidiosis in childhood diarrhea, malnutrition, and death in resource-poor countries. Despite this, only a single drug, nitazoxanide, has demonstrated efficacy in human cryptosporidiosis and its efficacy is limited in malnourished children and patients with HIV.
Areas covered
In this review, we highlight work on potential targets for chemotherapy and review progress on drug development. A number of new targets have been identified for chemotherapy and progress has been made at developing drugs for these targets. Targets include parasite kinases, nucleic acid synthesis and processing, proteases, and lipid metabolism. Other groups have performed high-throughput screening to identify potential drugs. Several compounds have advanced to large animal studies.
Expert Opinion
Development of drugs for cryptosporidiosis has been plagued by lack of success. Barriers have included poor correlations between in vitro activity and clinical success as well as frequent unanticipated adverse effects. Without a clear pathway forward, it is wise to maintain a diverse development pipeline. Drug developers should also realize that success will likely require a sustained, methodical effort.
Bumped Kinase Inhibitors (BKIs), targeting Calcium-dependent Protein Kinase 1 (CDPK1) in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology, that have shaped our selection of pre-clinical candidates.
