Progress 09/01/14 to 08/31/20
Outputs
Target Audience:Our target audience was those scientists engagedin drug development for animal and humanhealth, as well as parasitologists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?These results have been presented at numerous meetings including theNIFA Annual Meetings, and international animal health meetings such as Apicowplexa 2015, 2017, & 2019, andWAAVP 2019. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major Accomplishments In this final budget year, we continued our characterization of BKIs to find the right combination of activity, potency, and PK/ADME/toxicity without inhibiting hERG. We focused on fine tuning the pharmacological properties of compounds with either the pyrazolo [2, 3-d] pyrimidine (PP) scaffold like BKI-1294 and -1369 or the 5-aminopyrazole-4-carboxamide scaffold as BKI-1770, -1748, or -1708 with a goal to provide alternatives as preclinical candidates. For cryptosporidiosis therapy, in the PP scaffold, we characterized BKI-1812 and -1814. Both were promising at 60 mg/kg orally x 5 d in clearing infection in the IFN-γ-KO mouse model with n-Luc-tagged-C. parvum. Unfortunately lower doses of BKI-1812 (e.g. 30 mg/kg x 5d) were ineffective at clearance of C. parvum. Exposures of BKI-1812 in plasma during 60 mg/kg therapy was as high as 43 µM and cardiotoxicity was associated with 4-fold higher levels in the rat cardiovascular test. Thus, we abandoned this lead. For BKI-1814, we found efficacy at 30 mg/kg x 5 days and, with a delay of 1-2 days, at 15 mg/kg x 5 days. Unfortunately BKI-1814 is converted very efficiently to BKI-1649, e.g. levels reached greater than 100 µM after dosing at 30 mg/kg. This BKI-1649 had been previously associated with gut and neurotoxicity, and thus BKI-1814 was abandoned as a lead. In the AC series, we studied BKI-1770 and BKI-1708. BKI-1770 and BKI-1708 were both very promising, with BKI-1770 working well at 30mg/kg PO QD x 5 d and BKI-1708 working well at 8mg/kg PO QD x 5 d in the IFN-γ-KO mouse model with n-Luc-tagged-C. parvum. Both of these were tested in the newborn calf model of C. parvum infection and were effective at stopping diarrhea and reducing oocyst excretion at as low as 3 mg/kg PO BID x 5 days. However, when calf safety studies were performed, both compounds when dosed at 7.5 mg/kg BID, calves demonstrated plantigrade stance. When these BKI-1770 and -1708 treated calves were sacrificed, epiphyseal plates showed enlargement and chondrocytes were disordered. We are concerned about this effect because of the target human population with cryptosporidiosis is 6-18 month old children, who would be susceptible to epiphyseal growth plate disorders, like the calves. When we compared the exposure of the treated calves at 3 mg/kg bid to the exposure of the calves given 7.5 mg/kg, the exposures of these were overlapping, suggesting there was no margin of safety for this bone toxicity issue. We have begun a mouse model for bone toxicity to help us further explore this issue with BKIs. For toxoplasmosis and neosporosis therapy, we found that much broader systemic exposure to treat these infections than was necessary than for cryptosporidiosis. We demonstrated that our PP BKI-1294 gave proof-of-concept of efficacious therapy for BKIs in both toxoplasmosis and neosporosis in the mouse peritoneal infection, the mouse brain infection, the pregnant mouse model, and the sheep pregnancy model (see publications). However BKI-1294 turned out to have cardiotoxicity issues in rodents and dogs so we abandoned it and looked for other molecules beyond BKI-1294 that lacked cardiotoxicity issues. Our work for a toxoplasmosis and neosporosis lead has coalesced around BKI-1748. This lead was found to work extremely well in both a mouse peritoneal and a mouse brain infection model of both toxoplasmosis and neosporosis. We found this lead was safe when given to pregnant mice and sheep. Future studies will study the BKI-1748 lead in the mouse and sheep pregnancy models of Toxoplasma gondii and Neospora caninum. Thus, in terms of accomplishments, we delivered a number of advanced preclinical leads, demonstrated efficacy in several animal models, and defined toxicity issues, some of which were solved during the project period and some of which we are currently investigating. We believe the results of these studies will be advanced to a late pre-clinical lead in the next year or two and can proceed into clinical trials.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
256. Shrestha A, Ojo KK, Koston F, Ruttkowski B, Vidadala RSR, Dorr CS, Navaluna ED, Whitman GR, Barrett KF, Barrett LK, Hulverson MA, Choi R, Michaels SA, Maly DJ, Hemphill A, Van Voorhis WC, Joachim A. Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro. Int J Parasitol Drugs Drug Resist. 2019 Apr 2;10:9-19. doi: 10.1016/j.ijpddr.2019.03.004. [Epub ahead of print] PubMed PMID: 30959327; PubMed Central PMCID: PMC6453670.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
257. S�nchez-S�nchez R, Ferre I, Re M, Ramos JJ, Regidor-Cerrillo J, D�az MP, Gonz�lez-Huecas M, Tabanera E, Benavides J, Hemphill A, Hulverson MA, Barrett LK, Choi R, Whitman GR, Ojo KK, Van Voorhis WC, Ortega-Mora LM. Treatment with Bumped kinase inhibitor BKI-1294 is safe and leads to significant protection against abortion and vertical transmission in sheep experimentally infected with Toxoplasma gondii during pregnancy. Antimicrob Agents Chemother. 2019;63(7):e02527-18 doi: 10.1128/AAC.02527-18. PubMed PMID:31061151.
|
Progress 09/01/18 to 08/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?See publications and also at CRWAD conference talk, Dec 2018. What do you plan to do during the next reporting period to accomplish the goals?In the coming months, we will continue work on our BKIs scaffolds to determine the best efficacy and PK/ADME/Toxicity profiles in both the sheep and calf models of clinical cryptosporidiosis and toxoplasmosis in the aim of finalizing a candidate to move forward with to GLP trials.
Impacts
What was accomplished under these goals?
In the past budget year, we have continued characterization of BKIs to find the best combination of activity and potency, and the most favorable PK/ADME/toxicity without inhibiting hERG. We focused on fine tuning the pharmacological properties of compounds with the pyrazolo [2, 3-d] pyrimidine (PP) scaffold such as 1294 and 1369. We also worked on the 5-aminopyrazole-4-carboxamide scaffold with compounds such a 1770 and 1708 with the larger goal of providing alternatives as preclinical candidates. We provided synthetic support for large scale preparation of compounds (WuXi) selected for animal PK and efficacy studies. Additional compounds were tested in both the Toxoplasma and Cryptosporidium models. New BKIs with the best parameters, PK/ADME and in vivo efficacy were tested in the newborn mouse C. parvum acute infection model, the immunosuppressed mouse chronic C. parvum infection model, and the mouse T. gondii infection model. Leads were tested in the calf-C. parvum clinical model at Arizona.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hulverson MA, Bruzual I, McConnell EV, Huang W, Vidadala RSR, Choi R, Arnold SLM, Whitman GR, McCloskey MC, Barrett LK, Rivas KL, Scheele S, DeRocher AE, Parsons M, Ojo KK, Maly DJ, Fan E, Van Voorhis WC, Doggett JS. Pharmacokinetics and In Vivo Efficacy of Pyrazolopyrimidine, Pyrrolopyrimidine and 5-Aminopyrazole-4-Carboximide Bumped Kinase Inhibitors against Toxoplasmosis. J Infect Dis. 2019 219(9):1464-1473. doi: 10.1093/infdis/jiy664. PubMed PMID: 30423128. {Original Research}
Huang W, Hulverson MA, Choi R, Arnold SLM, Zhang Z, McCloskey M, Whitman G, Hackman R, Rivas K, Barrett LK, Ojo K, Van Voorhis WC, Fan E. Development of 5-aminopyrazole-4-carboxamide-based bumped-kinase inhibitors for Cryptosporidiosis therapy. J Med Chem. 62(6):3135-3146.. doi: 10.1021/acs.jmedchem.9b00069. PubMed PMID: 30830766. {Original Research}
Arnold SLM, Choi R, Hulverson MA, Whitman GR, McCloskey MC, Dorr CS, Vidadala RSR, Khatod M, Morada M, Barrett LK, Maly DJ, Yarlett N, Van Voorhis WC. P-glycoprotein mediated efflux reduces the in vivo efficacy of a therapeutic targeting the gastrointestinal parasite Cryptosporidium. J Infect Dis. 2019 Jun 8. pii: jiz269. doi: 10.1093/infdis/jiz269. [Epub ahead of print] PubMed PMID: 31180118.
|
Progress 09/01/17 to 08/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We trained a graduate student, Kelly Hennessey, who graduated in April 2018 and an undergrad, Grant Whitman, who graduated and is now working with us as a full time research scientist before he goes to Grad School. How have the results been disseminated to communities of interest?We have published six papers in peer-reviewed journals (see publications on Page 3-4). What do you plan to do during the next reporting period to accomplish the goals?In the next budget year, we will continue our careful characterization of BKIs to find the right combination of activity, potency, and PK/ADME/toxicity without inhibiting hERG. We will focus on fine tuning the pharmacological properties of compounds with either the pyrazolo [2, 3-d] pyrimidine (PP) scaffold like 1294 and 1369 or the 5-aminopyrazole-4-carboxamide scaffold as 1770 and 1708 with a goal to provide alternatives as preclinical candidates. We will also continue to provide synthetic support for large scale preparation of compounds selected for animal PK and efficacy studies. Additional compounds will be tested in the Toxo and Crypto models over the next year. New BKIs with the best parameters, PK/ADME and in vivo efficacy will be selected for testing in the newborn mouse C. parvum acute infection model, the immunosuppressed mouse chronic C. parvum infection model, the mouse T. gondii infection model.Leads will continue to be tested in the calf-C. parvum clinical model in the next 12 months.
Impacts
What was accomplished under these goals?
Scale-up synthesis for the AC and PP scaffold series inhibitors. We designed and synthesized 25 new AC scaffold and 25 new pyrazolo-pyrimidine (PP) compounds as well as 15 new pyrrolo-pyrimidine (PrP) compounds in the past year to address issues identified in efficacy and toxicology studies. Additionally, to support various in vitro and in vivo evaluations, resynthesis or scale-up was performed 51 times: 23 compounds at 10 mg, 5 compounds at 10-100 mg, 5 compounds at 200 mg, and 1 compound at >1 gram scale. The group purchased large quantities of two PP scaffold BKIs 1369 and 1294 as well as a large scale synthesis of one AC scaffold BKI, 1770, from a CRO to perform both large animal efficacy and toxicity studies with a larger amount of animals per group (n=3). We have a new lead compound 1708 which is showing excellent parameters and will be further tested in large animalsTesting lead BKIs for appropriate PK/ADME, efficacy and toxicology properties. In the past year the UW performed EC50 determinations and characterization on approx. 65 unique compounds, 27 PK mouse studies on inhibitors and Crypto efficacy studies on approx. 20 individual compounds with some follow-up dose determination for efficacy studies. We added Dr. Bob Hackman, a board certified pathologist to our team to read blinded BKI safety organ slides and report on any potential toxicity in the mouse multi-dose safety experiments of BKI 1369 and BKI 1770.Determine the efficacy, toxicity, and PK/ADME of BKIs in calf clinical cryptosporidiosis. The UA conducted efficacy and toxicity testing of lead BKI 1369 in the newborn calf clinical model of cryptosporidiosis at varying concentrations and dosages. Evaluation for efficacy against Crypto was determined as follows. Each calf (n = 6) was infected by oral inoculation with 5 x107 purified C. parvum (Iowa) oocysts on day 0, when 36-48 h of age. At day 2 post-infection (PI), and every 12-24 h thereafter until day 4 PI, calves (n =3 or 4) were administered BKI 1369 in vehicle orally at a 2 or 5 mg/kg dosage either QD or BID for 3 or 5 days (3, 6, or 10 doses total). For comparison, control calves (n = 3 or 4) were identically infected and treated with vehicle alone. Calves were examined twice daily to assess and assign numerical scores for the following variables: clinical symptoms; general health observations; presence or absence of diarrhea; and fecal consistency. Calves were weighed at birth and at termination to compute average bodyweight gain/loss. The total volume of feces excreted for successive 24 h collections was determined to provide an objective, quantitative index of diarrhea volume and severity for each calf. Total daily oocyst counts for each calf were determined by qPCR. Calves were euthanized on day 10 PI. Serum and whole blood samples were collected from all calves for CBC and serum biochemistry. RESULTS: For BKI 1369 at 5 mg/kg BID 5 days dosing: Daily oocyst counts were lower in treated calves for days 3-8 PI when compared to control calves. Total oocyst counts were also lower in treated calves. Clinical health scores (days 5-9 PI and all days combined) were better in treated calves. The total fecal volume was lower for days 4-8 PI and all days combined in treated calves. There was a significant difference in average % weight gain between control and treated groups with the treated group gaining weight. Duration of diarrhea was shortened for the treated group compared (days 4-8 PI). Daily fecal consistency for treated calves was better (less severe diarrhea) for days 5-8 PI when compared to controls. For BKI 1369 Dosing trial (2 mg/kg and 5 mg/kg QD, and 5 mg/kg BID for 3 days of dosing): Significant differences in clinical health scores were only seen for days 6 and 7 and overall for 5 mg/kg BID treated calves when compared to controls. The other groups did not show significance when compared. The total fecal volumes was lower for day 3 (5 mg/kg BID and 6 (5 mg/kg QD) PI in treated group. The average total daily fecal volume was significant for the 5 mg/kg BID group. There was significant difference in average % weight gain between control and the 5 mg/kg BID treated group. Duration of diarrhea was shortened for all the treated groups compared to control group, based on a dose response. Daily fecal consistency in treated calves was better (less severe diarrhea) for days 3, 6, and 7 PI for 5 mg/kg BID and day 6 PI for 5 mg/kg QD when compared to controls. All parameters showed a trend toward improvement with an increase in dosing concentration and dosage number. The number of significant events would have increased with a larger amount of animals per group (n=3). Complete the preclinical package and select a clinical candidate enabling an IND. Working towards this goal.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Hennessey KM, Rogiers IC, Shih H-W, Hulverson MA, Choi R, McCloskey MC, et al. (2018) Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia
and Cryptosporidium parvum. PLoS Negl Trop Dis 12(8): e0006673. https://doi.org/10.1371/journal. pntd.0006673
Scheele S, Geiger JA, DeRocher AE, Choi R, Smith TR, Hulverson MA, Vidadala RSR, Barrett LK, Maly DJ, Merritt EA, Ojo KK, Van Voorhis WC, Parsons M. 2018. Toxoplasma
calcium-dependent protein kinase 1 inhibitors: probing activity and resistance using cellular thermal shift assays. Antimicrob Agents Chemother 62:e00051-18. https://doi.org/10
.1128/AAC.00051-18.
Lee S, Ginese M, Beamer G, Danz HR, Girouard DJ, Chapman-Bonofiglio SP, Lee M, Hulverson MA, Choi R, Whitman GR, Ojo KK, Arnold SLM, Van Voorhis WC, Tzipori S. 2018.
Therapeutic efficacy of bumped kinase inhibitor 1369 in a pig model of acute diarrhea caused by Cryptosporidium hominis. Antimicrob Agents Chemother 62:e00147-18. https://doi
.org/10.1128/AAC.00147-18.
Hulverson MA, Choi R, Arnold SLM, Schaefer DA, Hemphill A, McCloskey MC, Betzer DP, Muller J, Vidadala RSR, Whitman GR, Rivas KL, Barrett LK, Hackman RC, Love MS, McNamara CW, Shaughnessy TK, Kondratiuk A, Kurnick M, Banfor PN, Lynch JJ, Freiberg GM, Kempf DJ, Maly DJ, Riggs MW, Ojo KK, Van Voorhis WC. 2017. Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis. Int J Parasitol 47:753763. https://doi.org/10.1016/j.ijpara.2017.08.006.
M�ller J, Aguado-Mart�nez A, Ortega-Mora L-M, Moreno-Gonzalo J, Ferre I, Hulverson MA, Choi R, McCloskey MC, Barrett LK, Maly DJ, Ojo KK, Van Voorhis W, Hemphill A. 2017. Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis. J Antimicrob Chemother 72:23342341. https://doi.org/10.1093/jac/dkx134
.
Vidadala RS, Rivas KL, Ojo KK, Hulverson MA, Zambriski JA, Bruzual I, Schultz TL, Huang W, Zhang Z, Scheele S, DeRocher AE, Choi R, Barrett LK, Siddaramaiah LK, Hol WG, Fan E, Merritt EA, Parsons M, Freiberg G, Marsh K, Kempf DJ, Carruthers VB, Isoherranen N, Doggett JS, Van Voorhis WC, Maly DJ. 2016. Development of an orally available and central nervous system (CNS) penetrant Toxoplasma gondii calciumdependent protein kinase 1 (TgCDPK1) inhibitor with minimal human ether-a-go-go-related gene (hERG) activity for the treatment of toxoplasmosis. J Med Chem 59:6531 6546. https://doi.org/10.1021/acs.jmedchem.6b00760.
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Journal Publications as listed. What do you plan to do during the next reporting period to accomplish the goals?In the next budget year, we will continue to explore and characterize the series of BKIs to find the right combination of activity, potency, and PK/ADME/Toxicity. We will also continue to provide synthetic support for large scale preparation of compounds selected for animal PK and efficacy studies. Additional compounds will be tested in the Toxo and Crypto models over the next year. New BKIs with the best parameters, PK/ADME and in vivo efficacy will be selected for testing in the newborn calf C. parvum infection model, pregnant sheep model on toxoplasmosis and the mouse infection models of T. gondii and N. caninum.
Impacts
What was accomplished under these goals?
Accomplishments Aim 1: BKIs. UW performed 28 batches of scale-up synthesis of lead AC compounds for various in vivo toxicity and efficacy studies, and re-synthesized 22 compounds for hERG test and 27 compounds for PK studies. Also 30 new AC compounds were designed/synthesized to fill backup pipeline and to address PK/toxicity issues found during lead optimization. For PP series, we continue to reduce hERG activity of compound 1294. Two promising leads were scaled to .25 kg level and tested. Backup compounds for Crypto and Toxo will be tested in DMPK, toxicology, and rodent models of PK/ADME/Toxicity, and efficacy. Aim 2: 1517 and 1553 for efficacy in non-pregnant and pregnant mouse models for N. caninum infection. 1517 and 1553 inhibited the tachyzoite form of the parasite as show by using a transgenic beta-gal reporter strain with 50% inhibitory conc. (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 μM, respectively. Control fibroblast IC50s were above 20 μM; however, morphological changes occurred in cultures treated with >5 μM 1517 after prolonged exposure (>6 days), indicating some interference with host cell metabolism. Treatment of intracellular tachyzoites with 5 μM 1553 for 6 days inhibited endodyogeny by inhibiting the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 μM 1517 did not form large complexes and showed more evidence of immediate cell death. However, after 10 d treatment in vitro, both compounds failed to completely prevent regrowth of parasites from culture. Balb/c mice experimentally infected with N. caninum then treated6 days with 1517 or 1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by 1517 when applied at 50 mg/kg per day. 1517 20 mg/kg/day significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. 1553 was less detrimental to fertility, and also provided significant, but clearly less pronounced protection of dams and offspring. These results demonstrate this compound class protects offspring from vertical transmission and disease, but to a lower degree and with side effects compared to the previously studied compound 1294. Regrowth of N. caninum following long-term 1294 treatment. 1294, 1553 and 1517 do not act parasiticidal in cells, even after 5 µM treatment over 10 d. Upon treatment, parasites form long-lived, multinucleated complexes (MNCs), which have a large parasite mass, novel preformed apical parts, but separation of newly formed tachyzoites is inhibited. It is not known whether re-emerging parasites originate from single parasites or from MNCs. A follow-up study using immunofluorescence staining and N. caninum antibodies directed against SAG1, IMC1, CDPK1, etc. showed regrowth after drug treatment originates from the capacity of the MNC to overcome their cytokinesis block and continue in the cell cycle after few days of drug release. Immunolocalization and CDPK1 expression in N. caninum treated with 1294. Using a polyclonal anti-TgCDPK1 antibody, the expression of CDPK1 enzyme was monitored during 1294 treatment for 5 days and after drug was stopped and parasite regrowth occurred. This was done on the mRNA level by reverse transcription RT-PCR, and on protein level by immunofluorescence. In both cases, expression of CDPK1 is downregulated during treatment, while for other antigens (e.g. SAG1), upregulation is observed. Aim 3: Efficacy of BKIs in a pregnant sheep model of N. caninum. 49 pregnant sheep were randomly allocated in 8 groups for testing 1294 (2 doses) and 1553 (1 dose). On gestation day 90: Groups 1, 3, 5 and 7 were inoculated IV with 106 N. caninum tachyzoites in PBS and Groups 2, 4, 6 and 8 were inoculated IV with PBS only. Treatment started 48 h post infectionI. 70% Tween 80/30% EtOH was used as vehicle. Ewes were monitored by ultrasound twice a week until lambing or the occurrence of fetal death. Lambs and ewes were culled 48 h after birth or immediately after detection of fetal mortality. Most inoculation points showed subcutaneous reactions at 24 h, disappearing by day 8. No changes in hematology and biochemistry found during time of drug administration. Concentration of 1294 found in the blood stream was monitored. Humoral immune response in dams showed a significant decrease of IgG levels compared to controls. Cellular immune response was also measured and showed lower IFN-g levels. No fetal mortality was observed in control Groups 2, 4, 6 and 8 with PBS inoculattion only. Fetal mortality was observed in 5 of 8 ewes in G3, 4 of 8 ewes in G5 and in all infected animals in control groups 1 and 7.The results suggest that BKI 1294 and 1553, at the doses given in group G3 and G5, provide partial protection from fetal mortality due to N. caninum. Drug delivery needs to be optimized to see if further protection can be achieved. Efficacy and toxicity testing of 1534 and 1369 in newborn calf clinical model. Three cohorts of calves were completed this reporting year (one for 1534 and two for 1369) Evaluation for efficacy against C. parvum determined as follows. Each calf (n = 6) was infected by oral inoculation with 5 x107 disinfected C. parvum (Iowa) oocysts on day 0, when 36-48 hours of age. At day 2 post-infection) (PI), and every 12 to 24 h thereafter until day 6, calves were administered 1553 orally at a 5 mg/kg dosage (10 doses total).For comparison, control calves (n = 6) were identically infected and treated with vehicle alone. Calves were examined twice daily to assess and assign numerical scores for the following variables: clinical health scores; general health observations; presence or absence of diarrhea; and fecal consistency. Calves were weighed at birth and at termination to compute average bodyweight gain/loss. The total volume of feces excreted for successive 24 h collections was determined to provide an objective, quantitative index of diarrhea volume and severity for each calf. Total daily oocyst counts for each calf are being determined by real-time PCR. Calves were euthanized on day 10 PI. Serum and whole blood samples were collected from calves for CBC and serum biochemistry.1534 Clinical health scores were worse in treated calves compared to controls for all individual days PI and mean score for all days combined. This may be due to evidence of gastrointestinal toxicity (constipation followed by explosive diarrhea in some calves, while other calves displayed severe bloating). Daily and mean score for all days combined fecal volumes were not significantly different in treated calves (P >0.05). There was no significant difference in avg % weight gain between groups. No difference in duration of diarrhea was seen for the treated group. Daily fecal consistency scores in treated calves were not significantly better on any day PI (P >0.05).1369 Oocyst counts showed a reduction in the number shed daily (days 3-8 PI; P <0.001) and total number (P <0.0001).Clinical health scores on days 5-9 PI were significantly better in treated calves (P <0.05). Daily (days 4-8 PI) and total (all days combined) fecal volumes were significantly lower in treated calves (P <0.05). There was a difference in average % weight gain between control and treated groups with 0.3 lb. control vs. 4.7 lb. gain treated, however the result was not statistically significant. Duration of diarrhea was shortened for the treated group (2 days vs 7-8 days in controls). Daily fecal consistency scores in treated were significantly better (less severe diarrhea) for days 5-10 PI (P <0.025). Overall (all days) fecal consistency was significantly better for treated (P <0.035). Experiments using the calf clinical model of cryptosporidiosis are ongoing for determining the lowest possible dose and number of days for treatment.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Schaefer DA, Betzer DP, Smith KD, Millman ZG, Michalski HC, Menchaca SE, Zambriski JA, Ojo KK, Hulverson MA, Arnold SL, Rivas KL, Vidadala RS, Huang W, Barrett LK, Maly DJ, Fan E, Van Voorhis WC, Riggs MW. Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis.(2016)J Infect Dis. 15;214(12):1856-1864. Epub 2016 Oct 17.
PMID:27923949
M�ller J, Aguado-Mart�nez A, Balmer V, Maly DJ, Fan E, Ortega-Mora LM, Ojo KK, Van Voorhis WC, Hemphill A.Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites.(2017)Antimicrob Agents Chemother. 61(4). pii: e02324-16. doi: 10.1128/AAC.02324-16. Print 2017 Apr.
PMID:28137808
Hulverson MA, Vinayak S, Choi R, Schaefer DA, Castellanos-Gonzalez A, Vidadala RSR, Brooks CF, Herbert GT, Betzer DP, Whitman GR, Sparks HN, Arnold SLM, Rivas KL, Barrett LK, White AC Jr, Maly DJ, Riggs MW, Striepen B, Van Voorhis WC, Ojo KK.Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.(2017)J Infect Dis. 2017 215(8):1275-1284. doi: 10.1093/infdis/jix120.
Huang W, Choi R, Hulverson MA, Zhang Z, McCloskey MC, Schaefer DA, Whitman GR, Barrett LK, Vidadala RSR, Riggs MW, Maly DJ, Van Voorhis WC, Ojo KK, Fan E. 5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum.(2017)Antimicrob Agents Chemother. 61(8). pii: e00020-17. doi: 10.1128/AAC.00020-17. Print 2017 Aug.
PMID:28533246
Arnold SLM, Choi R, Hulverson MA, Schaefer DA, Vinayak S, Vidadala RSR, McCloskey MC, Whitman GR, Huang W, Barrett LK, Ojo KK, Fan E, Maly DJ, Riggs MW, Striepen B, Van Voorhis WC. Bumped kinase inhibitor gastrointestinal exposure is necessary to treat Cryptosporidium infection.(2017)J Infect Dis. doi: 10.1093/infdis/jix247. [Epub ahead of print]PMID:28541457
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Many papers were written and published in this period (see bibliography). What do you plan to do during the next reporting period to accomplish the goals?In the next budget year, we will continue to explore and characterize the series of BKIs to find the right combination of activity, potency, and PK/ADME/Toxicity. We will also continue to provide synthetic support for large scale preparation of compounds selected for animal PK and efficacy studies. Additional compounds will be tested in the Toxo and Crypto models over the next year. New BKIs with the best parameters, PK/ADME and in vivo efficacy will be selected for testing in the newborn mouse C. parvum acute infection model, the immunosuppressed mouse chronic C. parvum infection model, the mouse T. gondii infection model, and Mongolian gerbil C. hominis rodent models. We plan a trial with BKI-1294 treatment (50mg/kg/day for 6 days) of T. gondii oocyst infected pregnant mice. Infection dose will be 25 oocysts per mouse. In addition to clinical signs in dams and offspring, and parasite loads in different organs, we will also assess the immune responses (humoral response and cytokine responses) in the different treatment groups. To test efficacy of BKIs in pregnant sheep model of toxoplasmosis, 80 Rasa Aragonesa sheep one year old from a high health status closed flock have been selected. All animals were seronegative for most common transmissible sheep abortifacients and are kept in isolation until the beginning of the experiment. This experiment will be carried out in early 2017.
Impacts
What was accomplished under these goals?
Scale-up synthesis and testing of lead BKIs for appropriate efficacy, PK/ADME, and toxicology properties. For the 5-aminopyrazole-4-carboxamide (AC) series compounds, we performed 16 batches of scale-up synthesis of various leads for animal PK, toxicity, and efficacy studies, and re-synthesized 31 compounds for hERG test. In addition, 49 new AC compounds were designed and synthesized to fill the backup pipeline of the project and to address PK and toxicity issues identified during lead optimization. For the pyrazolo [2, 3-d] pyrimidine PP series, we worked to reduce hERG activity of compound 1294. To maintain properties of 1294-including the basic piperidine ring at the N-1 position and the hydrophobic substituent at the C-3 position-we pursued two strategies. (1) making the naphthyl ethoxy group of 1294 more polar for increased hydrophobicity leads to hERG activity. So, naphthyl ring was replaced by a quinolone, reducing activity against hERG by 10-20-fold without loss of TgCDPK1 activity. The ethyl group was replaced by a difluoromethoxy group. The difluoromethoxy analog should be more favorable as lower hydrophobicity and more metabolically stable than ethyl or cyclopropyl. Also ifluoromethoxy analogs are potent inhibitors of TgCDPK1 and CpCDPK1 and effective in cellular assays. (2) explored introducing substituents into the 1294 piperidine ring. We explored the 4-position because substituents could be introduced without making compounds chiral. Initial efforts looked at introducing either a cyano or hydroxy group at the 4-position. A 4-cyano group does not appear to reduce hERG activity, but a 4-hydroxy group leads to a pronounced decrease. We generated numerous analogs containing a 4-hydroxy group (with various C-3 substituents) and the potency and selectivity for TgCDPK1 and CpCDPK1 and activity against parasites is being determined. Finally, a similar strategy was used with 3-azetidine analogs of 1294 and again, a 3-hydroxy group leads to reduced hERG activity. We scaled up two promising leads to the 0.25 kg level and began testing. Simultaneously, backup compounds for both Crypto and Toxo implications will be tested in drug metabolism and pharmacokinetics (DMPK), toxicology, and rodent models of PK/ADME/Toxicity, and efficacy.Pharmacokinetics and toxicity of BKIs in non-pregnant sheep To test PK and toxicity of higher doses of BKIs we carried out the following experiment not included in the original proposal. Animals and their health status: six, 9-12 month-old, non-pregnant Rasa Aragonesa sheep seronegative for Toxoplasma gondii (ELISA test in house), Neospora caninum (ELISA test in house), Coxiella burnetii (LSIVet® Ruminant Q Fever Serum/Milk ELISA Kit), Chlamydophila abortus (ID Screen® Chlamydophila abortus Indirect Multi-Species), Schmallemberg virus (ID Screen® Schmallemberg virus Indirect Multi-Species) and pestivirus (ID Screen® BVD P80 Antibody Competition). Experimental design: BKIs 1294 and 1553 were dosed at 40 mg/kg and 35 mg/kg respectively using SQ administration. 70% Tween 80 and 30% Ethanol was used as drug vehicle. Samples and procedures: Sheep were observed daily to evaluate overall health, rectal temperatures and local reactions in the inoculation points. Blood samples with heparin were collected to obtain plasma before drug administration and 1, 2, 4, 8, 12, 24, 30, 48, 72, 96, 120 and 144 hours after administration. Plasma samples were sent to University of Washington (UW) to determine BKIs bioavailability.Cryptosporidiosis: We continued conducting efficacy and toxicity testing of 1553 in the newborn calf clinical model. Two calf cohorts were completed in this reporting year. Each calf (n = 6) was infected orally with 5 x107 disinfected C. parvum (Iowa) oocysts on day 0, when 36-48 hours of age. At day 2 post-infection (PI), and every 12-24 h thereafter until day 6 PI, calves were administered 1553 orally at a 5 mg/kg dosage (10 total). Control calves (n = 6) were identically infected and treated with vehicle alone. Calves were euthanized on day 10 PI. Serum and whole blood samples were collected from all calves for CBC and serum biochemistry. Results: Oocyst count analyses showed a reduction in the number shed daily (days 3, 8, 9, and 10 PI; P< 0.02) and total number shed for the duration (P< 0.04). Clinical health scores on days 6, 7, and 8 PI were better in treated calves (P< 0.01). Daily (days 4 and 6 PI) and total (all days combined) fecal volumes were lower in the treated (P< 0.03). No significant difference in average % weight gain between groups. Duration of diarrhea was shortened (4 days in treated vs 7-8 days in controls). Daily fecal consistency scores in treated calves were significant (less severe diarrhea) for days 3, 6, 7, and 8 PI (P< 0.01). Overall fecal consistency was better for treated (P< 0.0001). Toxoplasmosis: 1) Mouse infection with T. gondii. Female Balb/c mice were randomly placed in 5 groups and inoculated IP with T. gondii ME49 tachyzoites: group 1 (n=25) 1000/mouse, group 2 (n=40) 500/ mouse, group 3 (n=40) 100/mouse, group 4 (n=40) 10/mouse and group 5 (n=5) 0-1/mouse. In addition, 30 female Swiss mice were randomly placed in 3 groups and inoculated: group 1 (n=10) 500 tachyzoites per mice, group 2 (n=10) 100/mice, group 3 (n=10) 10/per mice. Mice were examined daily for clinical signs of toxoplasmosis. To avoid severe clinical signs, mice were treated with sulfadimidine in drinking water at 0.3 mg/ml from day 7-18 PI. Necropsy blood was collected to determine T. gondii infection. Cardiac and semitendinous muscle and brain tissues were collected for PCR and histopathological analyses. 2) Prepare T. gondii oocysts for infection. Domestic cats were purchased and fed either body carcasses or T. gondii brain cysts (100 and 200) from mice above. Feces was collected daily and oocyst shedding tested until 17d PI. Oocysts are purified by conventional floatation methods. Oocysts were sporulated in 2% K2Cr2O7 for 5 days at RT and stored at 4ºC. T. gondii oocysts were sent to U of Bern for experiments. 3) Infection of pregnant mice. Female CD-1 mice (10/grp) were rendered pregnant and infected PO with 5, 25, 100, 500 or 2000 T. gondii ME49 oocysts one week post-mating. Samples (lung and brain) were taken at the following points: A.) Death before 3 wks post infection B.) At 3 wks post infection C.) At 4 wks post-partum (40 days PI) Results and Conclusions: i). It is possible to orally infect mice with T. gondii oocysts at a titer as low as 5 oocysts/mouse. ii). All infected dams were lung and/or brain positive for T. gondii. iii). Seroconversion occurred around d 21 PI. iv). Animals infected with high titers (500 to 2000 oocysts) have an increased early mortality. v).Vertical transmission occurs at the lowest titers. iv.) The survival of the pups is strongly impaired by the infection. A small number of pups persist until the end of the experiment. v.) So far as analyzed, all surviving pups were brain positive for T. gondii, and in some cases, T. gondii could also been detected in DNA extracted from eyes. This could be a useful pregnancy model for T. gondii oocyst infection of dams. In order to avoid increased dam mortality, the oocyst titer should not be higher than 25 oocysts per dam, ideally between 5 and 10.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hines S.A., Ramsay J.D., Kappmeyer L.S., Lau A., Ojo K.K., Van Voorhis W.C., Knowles D.P. and Mealey R.H. (2015). Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor. Parasites & Vectors. 8(1):33.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Winzer P., M�ller J., Aguado-Mart�nez A., Rahman M., Balmer V., Manser V., Ortega-Mora L.M., Ojo K.K., Fan E., Maly D.J., Van Voorhis W.C. and Hemphill A. (2015). In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum. Antimicrobial Agents and Chemotherapy. 59(10):6361-6374.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Yacoob C., Ojo K.K., Vidadala R.S., Zhang Z., Fan E., Maly D.J., Van Voorhis W.C., and Shen H. (2015). Partition-optimized single emulsion particles improve sustained release of amphiphilic bumped kinase inhibitors to control malaria transmission. International Journal of Applied Biology and Pharmaceutical Technology. Volume-6, Issue-4.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Huang W., Ojo K.K., Zhang Z., Rivas K., Vidadala R.S., Scheele S., DeRocher A.E., Choi R., Hulverson M.A., Barrett L.K., Bruzual I., Siddaramaiah L.K., Kerchner K.M., Kurnick M.D., Freiberg G.M., Kempf D., Hol W.G., Merritt E.A., Neckermann G., de Hostos E.L., Isoherranen N., Maly D.J., Parsons M., Doggett J.S., Van Voorhis W.C. and Fan E. (2015) Studies of 5-Aminopyrazole-4-carboxamide Analogues as Potent and Selective Inhibitors of Toxoplasma gondii CDPK1. ACS Med Chem Lett. 2015 Oct 22;6(12):1184-1189. eCollection 2015 Dec 10. PubMed PMID: 26693272; PubMed Central PMCID: PMC4677665.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Pedroni M.J., Vidadala R.S., Choi R., Keyloun K.R., Reid M.C., Murphy R.C., Barrett L.K., Van Voorhis W.C., Maly D.J., Ojo K.K., and Lau A.O.T. (2016) Bumped kinase inhibitor prohibits egression in Babesia bovis. Veterinary Parasitology 215: 2228.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Vidadala R.S., Rivas K.L., Ojo K.K., Hulverson M.A., Zambriski J.A., Bruzual I., Huang W., Zhang Z., Scheele S., DeRocher A.E., Choi R., Barrett L.K., Siddaramaiah L.K., Hol W.G., Fan E., Merritt E.A., Parsons M., Freiberg G.M., Marsh K., Kempf D., Isoherranen N., Doggett J.S., Van Voorhis W.C. and Maly D.J. (2016) Development of an Orally Available and Central Nervous System (CNS) Penetrant Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitor with Minimal Human Ether-a-go-go Related Gene (hERG) Activity for the Treatment of Toxoplasmosis. Journal of Medicinal Chemistry (59): 6531-6546.
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
"Pharmacokinetics, safety and efficacy of Bumped Kinase Inhibitor 1553 in a pregnant sheep model of neosporosis . Oral communication. COST Action 1307. Chemotherapy towards diseases caused by endoparasites. Targeting metabolism and transport in parasites: drugs and delivery systems . Porto, Portugal. May 5-6, 2016.
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
"Fetal pharmacokinetics of Bumped Kinase Inhibitor 1553 in pregnant sheep . Poster presentation. COST Action 1307. Chemotherapy towards diseases caused by endoparasites. Targeting metabolism and transport in parasites: drugs and delivery systems . Porto, Portugal. May 5-6, 2016.
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Year two of this project will see us: 1) performing less chemistry to obtain 2 backup compounds to push forward if needed. The important in vitro testing will be collected on all backups to determine toxicity. The necessary small animal toxicity testing will also be performed on backups. 2) Determining compound exposure required for the treatment N. caninum infection in a murine model for 3 BKI compounds. We need to do efficacy studies of our preclinical leads in neosporosis, before going on to test farm animals, as required by IACUC principals of using lower, less sentient animals (rodents) before going to larger farm animals. For small animal in vivo efficacy testing for neosporosis, our 2-4 preclinical lead compounds will be selected from those already shown to be effective in murine models of toxoplasmosis and cryptosporidiosis. The compounds with the best efficacy and PK and toxicity will advance to large animal studies. 3) Leading BKI compounds will be studied for therapy of infected animals: calves for C. parvum and pregnant sheep for T. gondii and N. caninum. If our first two BKI clinical candidates are successful in the large animal models, subsequent effort will focus on completing preclinical testing. Ultimate goal of this entire project is to in the have a preclinical drug candidate and one or two backups to advance to final preclinical testing and IND/NADA registration for veterinary and human use.
Impacts
What was accomplished under these goals?
Work accomplished according to project AIMS: 1) Scale-up synthesis and testing of lead BKIs for appropriate efficacy, PK/ADME, and toxicology properties; We were able to purchase up to 250 gms of several of our leads to support the many necessary animal studies and our UW Chemistry labs were able to scale up 10-15 additional compounds for animal efficacies in all models. 2) Determine efficacy of BKIs in a rodent model of neosporosis. We assessed the effects of 1294 on pregnancy outcome in non-infected mice. Treatment with BKI 1294 (50 mg/kg/day) orally for 8 days once a day did not affect pregnancy, the number of offspring in the different groups were virtually identical (56 offspring in 1294-treated group with 3 stillborn and 56 in the control group with 3 stillborn). No negative impact could be detected 1 month after birth. Second we assessed treatment effects of 1294 in non-pregnant and pregnant mice infected with the N. caninum Nc-Liv strain. We detected no clinical signs of N. caninum infection in any of the 1294 treatment groups (50 mg/kg/day orally for 8 days once a day). Adults and pups were tested for N. caninum presence in their brains with real time PCR. Of the treated non-pregnant mice 1/6 was Nc positive compared to 6/6 in control group, 1/6 dams were positive but 0/27 pups. Number of stillborn was decreased by 1294 treatment (6/27 compared to 8/39). Respective numbers of animals in control and 1294-treated groups by chi-square tests (stillborn, p>0.1; dams, p<0.005; pups, p<0.001). Finally we looked at the effects of treatment of 1294, 1553 and Buparvaquone in non-pregnant and pregnant Balb/c mice infected with another N. caninum strain Nc-Spain7. All females were infected with 105 tachyzoites of the Nc-Spain7 isolate at mid gestation. Two days after infection, mice were either given Buparvaquone (50mg/kg/ day), 1294 (50 mg/kg/day), or 1553 (20mg/kg/day) applied 5 times once daily. Pregnancy was confirmed between days 15-18, mice give birth on days 20-22 and reared pups for 30 days. Blood from dams and non-pregnant mice was recovered to assess humoral immune responses. Brains from dams, non-pregnant mice and surviving pups were sampled for parasite detection and quantification by real time PCR. 1294 treatment had a profound impact on the CNS infection. 7/7 dams control dams and 3/10 1294 treated dams were positive, 8/8 non-pregnant controls and 1/6 1294 treated non-pregnant mice were positive.43/43 control pups and 11/58 1294 treated pups were positive. These mouse studies have paved the way for the sheep model and testing these compounds for Nc infection and Toxoplasma gondi infection (Aim 3). 3) Determine the efficacy and PK/ADME/Toxicity of BKIs in sheep and calf models of clinical cryptosporidiosis, toxoplasmosis, and neosporosis; UA conducted efficacy and toxicity testing of BKI 1517 in the newborn calf clinical model of cryptosporidiosis. Due to the late start for the grant, only 2 cohorts of calves were completed. Evaluation for efficacy against C. parvum was determined as follows. Each calf (n = 4) was infected by oral inoculation with 5 x107 disinfected C. parvum (Iowa) oocysts on day 0, when 36-48 hours of age. At day 2 post-infection) (PI), and every 12 to 24 h thereafter until day 6 PI, calves were administered BKI 1517 orally at a 10 mg/kg dosage (10 doses total). For comparison, control calves (n = 4) were identically infected and treated with vehicle alone. Calves were examined twice daily to assess and assign numerical scores for these variables: clinical symptoms; general health observations; presence or absence of diarrhea; and fecal consistency. Calves were weighed at birth and termination to compute average bodyweight gain/loss. Total volume of feces excreted for successive 24 h was determined to provide an objective, quantitative index of diarrhea volume and severity for each calf. Total daily oocyst counts for each calf are being determined by real-time PCR. Calves were euthanized on day 10 PI. Serum and whole blood samples were collected from all for CBC and biochemistry. Clinical health scores (overall and days 7 and 8 PI) were significantly better in treated when compared to controls (P < 0.04). Daily (days 4, 5, and 6 PI) and total (all days combined) fecal volumes were significantly lower in treated calves when compared to controls (P < 0.025). There was no significant difference in average % weight gain between control and treated groups. Duration of diarrhea was shortened for treated group compared to control group (2-3 days vs 7 days). Daily fecal consistency in treated calves was significantly better (less severe diarrhea) for days 4, 5, and 7 PI when compared to controls (P < 0.025). Overall (all days) fecal consistency was significantly better for treated compared to controls (P < 0.0425). Overall urine output was significantly greater in treated calves when compared to controls (P < 0.035), indicating less dehydration in the treated group. In the sheep model, the focus of the first year was to get accurate PK of the BKIs. This has been accomplished in uninfected sheep and non-pregnant sheep.At least oneof the drugs gave excellent PK in sheep, whether doses were given SQ, IV or PO.
Publications
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