Cryptosporidium parvum, an apicomplexan parasite and the causative agent of cryptosporidiosis, contributes to a high burden in mortality and morbidity for humans and livestock. Currently, there are no reliably successful parasite-specific treatments for the debilitating diarrhea associated with the infection. Bumped kinase inhibitors (BKIs), which selectively target parasite calcium-dependent protein kinases (CDPKs), have been shown to decrease infections caused by several medical and veterinary-relevant parasites, including Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. In the present study, various dosing regimens of BKI-1708 were evaluated for safety and clinical efficacy in the calf model for cryptosporidiosis, specifically with the aim of finding a minimum effective dose. The majority of the different BKI dosages produced notable improvements across nearly all clinical and parasitological measures, including diarrhea severity, oocyst shedding, and overall health status. These results provide strong evidence for advancing BKI-1708 as a preclinical candidate for treatment of cryptosporidiosis.

BACKGROUND:Cryptosporidiosis is a leading cause of severe diarrhea and mortality in young children with no vaccine or effective treatment. Cryptosporidium controlled human infection model (CHIM) in healthy adults provides an opportunity to evaluate the safety and efficacy of novel chemical entities prior to testing in young pediatric patients. However, CHIM development has been hindered by the lack of current good manufacturing practice (cGMP)-compliant Cryptosporidium parvum oocysts, which are required for filing an Investigational New Drug Application (IND) with the U.S. Food and Drug Administration. METHODS:To overcome this barrier, we developed a cGMP-compliant process for producing C. parvum oocysts. The approach involved sourcing well-characterized oocysts purified from neonatal calves under controlled non-GMP conditions, followed by cGMP processing that included peracetic acid-based surface sanitization, comprehensive microbial testing and release as a non-sterile oral product for human use. RESULTS:Peracetic acid (0.5% for 20 minutes) treated C. parvum oocysts demonstrated acceptable microbial safety and preserved viability. Viability was assessed through oocyst excystation, HCT-8 cell infection and mouse infectivity studies. The cGMP produced C. parvum oocysts with stringent batch-release test criteria for quantity, purity, identity, potency and bioburden was released as ABO809 for clinical use under an IND. ABO809 was successfully used in human challenge studies, producing reliable infection and characteristic clinical symptoms in healthy adult volunteers. CONCLUSIONS:We describe a robust cGMP-compliant and qualification process for C. parvum oocysts that enables CHIM studies in healthy adults. Thereby opening avenues to evaluate efficacy of novel therapeutics and vaccines to treat cryptosporidiosis.

BACKGROUND:The protozoan parasite Cryptosporidium parvum is a leading cause of diarrhea and mortality in young children and neonatal calves. There are no effective drugs to treat cryptosporidiosis, and no vaccines are available to prevent infection or reduce transmission. The substantial global public health burden of cryptosporidiosis calls for urgent efforts to develop effective treatments and prevention strategies. This includes identifying and functionally characterizing new parasite targets and evaluating vaccine candidates in relevant animal models. Recently, we identified the important role of C. parvum calcium-dependent protein kinase 5 (CDPK5) in the parasite's sexual cycle and found that genetic deletion of this kinase reduced virulence and oocyst shedding in immunocompromised mice. METHODS:In this study, we evaluated the genetically attenuated C. parvum CDPK5-knockout (CDPK5-KO) strain in a natural and clinically relevant calf model of cryptosporidiosis. We compared clinical health, diarrhea, dehydration, and oocyst shedding in calves infected with the CDPK5-KO strain to those infected with the control strain expressing a functional kinase (CDPK5-HA). RESULTS:We observed a significant improvement in overall clinical outcome, with less diarrhea and dehydration in calves infected with the CDPK5-KO strain compared with those infected with CDPK5-HA strain. Although total fecal oocyst shedding was lower in CDPK5-KO-infected calves than in the control group, diarrhea and oocyst shedding did not fully resolve during the trial. CONCLUSION:These findings show the effectiveness of a neonatal calf model for evaluating genetically attenuated C. parvum strains and for guiding future challenge studies to better understand their role in reducing diarrheal disease.

BackgroundDiarrheal pathogens, such as Cryptosporidium, impose a heavy burden of disease in resource-limited regions. Cryptosporidiosis often causes chronic infection in immunocompromised people and gastrointestinal injury in malnourished children, leading to wasting, stunting, and cognitive impairment. Current treatment for cryptosporidiosis fails in these vulnerable populations, highlighting the need for new medicines. Here we describe the anti-Cryptosporidium efficacy, pharmacokinetics, and safety of a bumped kinase inhibitor BKI-1708. BKI-1708 inhibits the essential molecular target, calcium-dependent protein kinase 1 (CDPK1), which is highly expressed in the major proliferative stages of the parasite life cycle.Methods and findingsEfficacy was demonstrated in the Cryptosporidium parvum IFNγ-KO mouse infection and calf diarrhea models. Dose response in the mouse model demonstrated oral doses as low as 15 mg/kg administered daily for 3 days completely suppressed oocyst shedding. Metabolite profiling in pre-clinical species and human hepatocytes identified an active metabolite, M2, which retains sub-micromolar activity against C. parvum. Pharmacokinetic analysis of BKI-1708 and M2 in mice demonstrates good systemic exposure, important for treating biliary and upper respiratory infections in some cases of cryptosporidiosis. In mice, M2 reaches 7-fold and >3-fold higher levels over BKI-1708 in plasma and the gastrointestinal tract, respectively. Oral administration of M2 completely suppressed oocyst shedding in the mouse model at doses as low as 8 mg/kg for 3 days. Wide safety margins are demonstrated in mice, rats, and dogs.ConclusionsBKI-1708 has characteristics of a safe and effective drug for treating Cryptosporidium infections in animal models and shows promise for use in humans. Moreover, BKI-1708 and M2 formed in vivo, offer an attractive prospect of a dually active preclinical candidate for the treatment of cryptosporidiosis.

Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum, a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.

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.

The gastrointestinal disease cryptosporidiosis, caused by the genus Cryptosporidium, is a common cause of diarrheal diseases in children, particularly in developing countries and frequently fatal in immunocompromised individuals. Cryptosporidium hominis (Ch)-specific bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) has been a molecular target for inhibitor design. (Note that this bifunctional enzyme has also been referred to as TS-DHFR in previous literature since the functional biochemical reaction first involves the conversion of methylene tetrahydrofolate to dihydrofolate at the TS site.) While nanomolar inhibitors of Ch DHFR-TS have been identified at the biochemical level, effective delivery of these compounds to achieve anticryptosporidial activity in cell culture and in vivo models of parasite infection remains a major challenge in developing new therapies. Previous studies, using a nanotherapy approach, have shown a promising Ch DHFR-TS inhibitor, 906, that can successfully target Cryptosporidium parasites in cell culture with nanomolar anticryptosporidial activity. This formulation utilized poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with 906 (NP-906) and conjugated with a Cryptosporidium monoclonal antibody (MAb) on the nanoparticle surface to specifically target the glycoprotein GP25-200 in excysting oocysts. However, a limitation for in vivo use is antibody susceptibility to gastric acidity. To address this gap, a prodrug diethyl ester form of 906 (MAb-NP-Prodrug) was synthesized that allowed higher compound loading in the MAb-coated PLGA nanoparticles. An oral formulation was prepared by loading lyophilized MAb-NP-Prodrug into gelatin capsules with an enteric coating for gastric stability. Proof-of-concept studies with this oral formulation demonstrated antiparasitic activity in a chronic mouse model of Cryptosporidium infection. Efficacy was observed after a low daily dose of 2 x 8 mg kg-1 for 5 days, when examined 6 and 20 days postinfection, offering a new avenue of drug delivery to be further explored.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

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.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a moderate-to-severe diarrheal disease now recognized as one of the leading causes of morbidity and mortality in livestock globally, and in humans living in resource-limited parts of the world, particularly those with AIDS or malnourished individuals. This recognition has fueled efforts for the discovery of effective therapeutics. While recent progress in drug discovery has been encouraging, there are presently no acceptably effective parasite-specific drugs for the disease. The urgent need for new drug discovery or drug repurposing has also increased the need for refined animal models of clinical disease for therapeutic efficacy evaluation. Here, we describe an acute model of cryptosporidiosis using newborn calves to evaluate well-defined clinical and parasitological parameter outcomes, including the effect on diarrhea severity and duration, oocyst numbers produced, and multiple measures of clinical health. The model is highly reproducible and provides unequivocal direct measures of treatment efficacy on diarrhea severity and parasite replication.

The protozoan parasite Cryptosporidium is a leading cause of diarrheal disease in those with compromised or under-developed immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection, and then tested the isolated effects of these metabolites on C. parvum invasion and growth. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, while long-chain unsaturated fatty acids enhance C. parvum invasion. The influence of these two classes of metabolites on C. parvum infection likely reflects the streamlined metabolism in C. parvum , which is unable to synthesize fatty acids. Hence, gut metabolites, either from diet or produced by the microbiota, play an important role in the early susceptibility to cryptosporidiosis seen in young animals. Importance Cryptosporidium occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development, and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced growth of C. parvum in vitro and may increase susceptibility to infection in young mice. Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium is unable to synthesize de novo. The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids provide further insight into reliance on fatty acid salvage and metabolism of this enteric parasite.

In the field of global health, physiologically based pharmacokinetic (PBPK) modeling has improved the confidence in malaria and tuberculosis (TB) drug development programs by assisting with first in human dose predictions, assessing potential drug‐drug interactions, and designing efficacious dosing regimens. PBPK models are composed of a compound file and a virtual physiology/population, and the impact of PBPK modeling in malaria and TB research is largely based on the substantial effort to improve both components. For example, compound files for commonly used therapeutics in malaria and TB have been developed through initiatives led by Medicines for Malaria Venture (MMV) and the Critical Path to TB Drug Regimens (CPTR). In addition, there has been considerable effort to develop virtual populations for these disease areas. Specifically, the focus of physiology and population model development for malaria has been on pediatric and pregnancy models. For TB, a virtual South African population has been generated which captures relevant genetic variants and TB‐related physiological changes that affect drug distribution and metabolism in this population.While the successful application of PBPK modeling in malaria and TB drug development has demonstrated the usefulness of this approach in global health, PBPK modeling has not been extensively applied to the area of drug development for enteric pathogens. In 2015, diarrhea caused > 688 million illnesses in children under 5 and was a leading cause of death among all ages (>1.3 million deaths). Despite advances in both the development of vaccines and small molecule therapeutics, new treatments are urgently needed for the treatment of multi drug resistant (MDR) bacterial pathogens and disease areas without an efficacious vaccine treatment. In symptomatic patients, we expect diarrhea to be associated with a reduction in gastrointestinal (GI) transit times, altered luminal pH, increased luminal volume, and disrupted epithelial integrity. However, the extent of these changes is often not clear. In addition, there has been insufficient clinical data to support how the pathophysiology may impact therapeutic disposition. To initially address this lack of clinical data, an aim of a recently completed clinical study in Malawi (NCT03341767) was to characterize the PK of clofazimine in HIV+ adult populations divided into two groups (diarrhea vs. non‐diarrheal). These clinical data were used to inform PBPK modeling efforts to predict the impact of diarrhea on clofazimine disposition and to relate the clinical study PK and efficacy outcomes to those obtained with pre‐clinical animal models of disease. While this work primarily focused on participants with cryptosporidiosis, our results provide a foundation for future efforts to use PBPK models in the development of therapeutics intended for various study populations with diarrhea.Support or Funding InformationBill and Melinda Gates Foundation Award OPP1172544

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 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.

The protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals.IMPORTANCECryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvumin vitro Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite's interactions with exogenous fatty acids during the initial stages of infection.

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.

ABSTRACT Cryptosporidium parvum calcium-dependent protein kinase 1 ( Cp CDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar Cp CDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro . Correlation between anti- Cp CDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.