Progress 06/01/24 to 05/31/25
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A veterinary student was involved in the analysis of the T. gondii samples and presented her final work of the Veterinary degree in July 2024. A Master´s student is now involved in these studies and will present her final work of the Master in 2025. The BKI sarcocystosis/EPM project is being conducted in an academic setting, and all of the investigators have demonstrated commitments to the education of future scientific researchers. The project activities and resulting data have been integrated into teaching and training activities. Currently, there is one postdoctoral scientist and three graduate students participating in the project at the University of Kentucky. These trainees have gained guidance and experience with experimental preparations, sample collections, data management, and animal handling, with oversight from the principal investigators and renowned equine veterinary clinician scientists.Dr. Izabela de Assis Rocha attended the 2025 Conference of Research Workers in Animal Diseases in Chicago, IL to network and present the findings from this study. How have the results been disseminated to communities of interest?The results for the recent HORSE BKI-1708 PK experiments were presented at the 2025 Conference of Research Workers in Animal Diseases. PHARMACOKINETICS OF BUMPED-KINASE INHIBITOR 1708 IN HORSES FOR EQUINE PROTOZOAL MYELOENCEPHALITIS (EPM) THERAPY I De Assis Rocha1, J McPeek1, S Reed1,2, M Hulverson3, R Choi3, L Barrett3, S Arnold3, W Van Voorhis3, D Howe1, A Page1 We presented a poster with results from theT. gondiiproject at the"The International Congress on Toxoplasmosis" which took place in Berlin in May 2024. What do you plan to do during the next reporting period to accomplish the goals?For Objective1: We are completing the studies of the late BKI-1748 treatment of pregnant ewes and writing a paper to describe the chronic infection. For Objective 2:A study to investigate the use of BKI-1708 to treat horses with S. neurona-related EPM is not yet warranted given that therapeutic levels of drug were not achieved in CSF and CNS tissues from horses dosed with up to 15 mg/kg. However, BKI-1708 is a promising candidate for EPM prophylaxis given the high levels of the drug observed in plasma. Experiments to assess BKI-1708 as a preventative will be conducted similar to published studies examining potential protection provided by intermittent administration of Protazil® (diclazuril) and Marquis® (ponazuril), two drugs that are FDA-approved for EPM treatment. BKI-1708 at 9mg/kg in a 36% paste will be administered orally twice weekly for four months to 10 horses; an additional 10 horses will serve as untreated controls. Blood will be collected daily during the first month to assess plasma levels of BKI-1708 between doses. Serum endpoint titers against S. neurona will be monitored every two weeks as a proxy for infection. It is expected that a portion of the non-treated horses (~40-50%) will exhibit increases in serum endpoint titers due to natural exposure to the parasite, while diminished or no increase in endpoint titers are expected in the treated population. This animal work will be conducted at the University of Kentucky and has been approved by the UK IACUC (#2018-3079), and reviewed and approved by the University of Washington IACUC under approval # 2154-01.
Impacts
What was accomplished under these goals?
Objective 1:Test lead BKI(currently BKI-1748)for PK, safety and efficacy in a pregnant sheep model of congenital transmission ofT. gondii Last period, we reported we had completed the BKI-1748 treatment of pregnant EWES at 7d after infection with T. gondii and there was evidence of high efficacy with protection of the unborn fetuses and Ewes. This has now been published in Sánchez-Sánchez R, Huertas-López A, Largo-de la Torre A, Ferre I, Dini FM, Re M, Moreno-Gonzalo J, Choi R, Hulverson MA, Ojo KK, Arnold SLM, Hemphill A, Van Voorhis WC, Ortega-Mora LM. Treatment with BKI-1748 after Toxoplasma gondii systemic dissemination in experimentally infected pregnant sheep improves fetal and lamb mortality and morbidity and prevents congenital infection. Antimicrob Agents Chemother. 2025 Feb 13;69(2):e0144824. doi: 10.1128/aac.01448-24. Epub 2024 Dec 31. PMID: 39745365; PMCID: PMC11823607. Efficacy against congenital infection in sheep. During this reporting period we also conducted a new study on the efficacy of BKI-1748 against congenital transmission ofT. gondiiinfection in sheep. In this study, the drug was administered from day 14 post-infection, when IgM levels peak in sera and parasite DNA can be detected in placenta. The results are being processed and preliminary results indicate that the drug significantly reduced the number of dead lambs in the infected sheep. Results of this will be reported in the next reporting period. Efficacy against chronic infection in sheep. In the efficacy studies of BKI-1748 against congenital toxoplasmosis in sheep carried out in previous years, we also collected tissue samples at day 60 post-infection to evaluate the efficacy of the compound against the establishment of chronicT. gondiiinfection in ewes. Parasite DNA was widely detected in the tissues analysed (biceps femoris muscle, heart, tongue and brain) from infected/untreated animals, while it was not detected in the infected/treated sheep. These results are now being written up for publication in 2025 Objective 2:Test lead BKI(currently BKI-1748)for its effect in Equine Protozoal Myeloencephalitis (EPM) at the University of Kentucky with support by Univ of WA The project included treating horses with our Bumped-Kinase Inhibitor lead for Sarcocystosis/Equine Protozoal Myeloencephalitis (EPM), but it took us longer than expected to establish the best lead and test its safety and pharmacokinetics in horses. This was due to: 1) the low bioavailability of the original candidate (BKI-1748) in horses; 2) safety off-target issues that were discovered in other leads considered, that included bone toxicity, CNS depression, and cardiac toxicity; and 3) having to find an optimal oral route for reliable oral bioavailability in horses. Experiments with BKI-1708 has solved some of the issues associated with: 1) poor bioavailability in blood where > 50% oral bioavailability could be obtained; 2) BKI-1708 lacked any signal in small animals or horses for bone toxicity, CNS depression, or cardiac toxicity; and, 3) a lipid based paste at 20% to 36% 1708, administered orally to horses was able to give reliable and reproducible blood levels that were safe in horses treated with a dose of 9mg/kg BKI-1708 of daily dosing for 10d, giving 6 to 3 μM blood plasma peak and trough levels. We enrolled and treated another group of horses with 15mg/kg daily therapy for 5 days, and found the administration was safe and was associated with 7 to 4 μM peak and 3 to 4 μM trough values in plasma. Surprisingly, BKI-1708 was undetectable or near the lower limit of detection (~7nM) in cerebrospinal fluid (CSF) and CNS tissues collected at necropsy, despite therapeutic levels in blood and previous findings in mice.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
de Sousa MCF, Imhof D, H�nggeli KPA, Choi R, Hulverson MA, Arnold SLM, Van Voorhis WC, Fan E, Roberto SS, Ortega-Mora LM, Hemphill A. Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice. Int J Parasitol Drugs Drug Resist. 2024 Aug;25:100553. doi: 10.1016/j.ijpddr.2024.100553. Epub 2024 Jun 19. PMID: 38917582; PMCID: PMC11254172.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
S�nchez-S�nchez R, Imhof D, Hecker YP, Ferre I, Re M, Moreno-Gonzalo J, Blanco-Murcia J, Mej�as-L�pez E, Hulverson MA, Choi R, Arnold SLM, Ojo KK, Barrett LK, Hemphill A, Van Voorhis WC, Ortega-Mora LM. An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii. J Infect Dis. 2024 Feb 14;229(2):558-566. doi: 10.1093/infdis/jiad470. PMID: 37889572; PMCID: PMC10873186.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
S�nchez-S�nchez R, Huertas-L�pez A, Largo-de la Torre A, Ferre I, Dini FM, Re M, Moreno-Gonzalo J, Choi R, Hulverson MA, Ojo KK, Arnold SLM, Hemphill A, Van Voorhis WC, Ortega-Mora LM. Treatment with BKI-1748 after Toxoplasma gondii systemic dissemination in experimentally infected pregnant sheep improves fetal and lamb mortality and morbidity and prevents congenital infection. Antimicrob Agents Chemother. 2025 Feb 13;69(2):e0144824. doi: 10.1128/aac.01448-24. Epub 2024 Dec 31. PMID: 39745365; PMCID: PMC11823607.
|
Progress 06/01/23 to 05/31/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student, Izabela de Assis Rocha, participated in the project and has gained experience conducting pharmacokinetic experiments. How have the results been disseminated to communities of interest?The results presented in the XIX Congress of Veterinary and Biomedical Sciences 2023 (https://eventos.ucm.es/91241/section/40493/xv-congreso-de-investigacion-de-estudiantes-de-grado-en-ciencias-de-la-salud-xix-congreso-de-cienci.html). Results of pharmacokinetics experiments were also presented at the EPM Special Interest Group session of the 2023 meeting of the American College of Veterinary Internal Medicine. Izabela de Assis Rocha, Matthew A. Hulverson, Ryan Choi, Lynn K. Barrett, Samuel L. Arnold, Tom Kennedy, Wesley C. Van Voorhis, Stephen M. Reed, Daniel K. Howe, and Allen E. Page. Pharmacokinetics of Bumped Kinase Inhibitor-1708 In Horses: A Candidate for Treatment of Equine Protozoal Myeloencephalitis Meeting of the American College of Veterinary Internal Medicine, Philadelphia, PA, June 2023. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will submit a manuscript to a journal indexed in the JCR, gathering the protection of BKI-1748 against the establishment of the chronic phase of Toxoplasma gondii infection. To address the issue of low drug levels in the CNS, a PK study will be conducted using an increased oral dose of 15 mg/kg. Intrathecal catheters will be placed in the atlantoaxial space, to allow for a greater number of CSF samples collected to better determine peak and trough levels of BKI-1708 in the CNS. Once we are confident that effective levels of BKI-1708 can be achieved in the CNS, a therapeutic dosing regimen will be developed. Horses with an antemortem diagnosis supportive of EPM will be recruited for a non-inferiority trial, with the BKI being compared to the FDA-approved treatment, ponazuril.
Impacts
What was accomplished under these goals?
Objective 1b. Efficacy of the lead BKI (BKI-1748) in a pregnant sheep model of toxoplasmosis. To support this study of BKI-1748 in sheep, we had synthesized 100 grams BKI-1708 at 99% purity and confirmed its purity by NMR and HPLC and biológical activity by enzyme and anti-parasitic assay before use. Experimental design Nineteen Rasa Aragonesa breed sheep on day 90 of pregnancy and seronegative for the most common abortifacient agents were distributed into three experimental groups: Sheep from group 1 (G1, n=8) were infected with 1,000 TgShSp1 sporulated oocysts and 48 hours later treated with 10 doses of BKI-1748 at 15 mg/kg every 48 hours using 60% Phosal 53MCT, 30%PEG400 and 10% Ethanol 96 as vehicle. Sheep from group 2 (G2, n=8) were infected with 1,000 TgShSp1 sporulated oocysts, but not treated. Sheep from group 3 (G3, n=3) were neither infected nor treated. At necropsy, 60 days post infection, the following samples were collected from the dams: heart, biceps, femoris muscle, tongue, and brain and then storage at -20ºC. 50 grams of each tissue were subjected to acid-pepsin digestion, followed by DNA extraction, and quantitative PCR. Results All tissues from G2 (infected/untreated) were PCR positive and parasite loads were significantly higher in the brain compared to biceps femoris muscle. By contrast, all the samples from infected/treated group (G1) were PCR negative. Therefore, there is full protection from BKI-1748 against the establishment of the chronic phase of the Toxoplasma gondii infection in sheep. Objective 2: Test lead BKI for its effect in Equine Protozoal Myeloencephalitis (EPM). Sub-Objective 2a. Test late lead BKIs (currently BKI-1708) in horse PK. BKI-1708 multi-dose oral PK experiment in horses: Four, 4-year-old horses were treated daily for ten days by oral administration of a 36% suspension of BKI-1708 made in a blank commercial paste at a dose of 9 mg/kg. Blood and CSF were collected daily and 24, 48, and 72 hours after the final dose. Blood collection was also performed pre-dose and 8 hour post-dose to assess trough and peak drug levels. CSF collection was performed by lumbar puncture on days 1, 3, 7, 10, and 48 hours after the final dose. CSF was collected from only one horse per time-point on each day to allow a 48-hour interval between spinal taps. Horses were monitored daily by physical exams and serial CBC/blood chemistry. No significant adverse effects were noted. Peak and trough levels of BKI-1708 in plasma were 6 mM and 2 mM, respectively, while CSF levels were low and near the lower limit of detection (0. 07 mM) for all time points. To support the planned study of BKI-1708 vs. Ponazuril, for EPM therapy, we have synthesized 3 kg of BKI-1708 at 99% purity and we confirmed its purity by NMR and HPLC and biológical activity by enzyme and anti-parasitic assay.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
S�nchez-S�nchez R, Imhof D, Hecker YP, Ferre I, Re M, Moreno-Gonzalo J, Blanco-Murcia J, Mej�as-L�pez E, Hulverson MA, Choi R, Arnold SLM, Ojo KK, Barrett LK, Hemphill A, Van Voorhis WC, Ortega-Mora LM. An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii. J Infect Dis. 2024 Feb 14;229(2):558-566. doi: 10.1093/infdis/jiad470. PMID: 37889572; PMCID: PMC10873186.
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Imhof D, Pownall WR, Schlange C, Monney C, Ortega-Mora LM, Ojo KK, Van Voorhis WC, Oevermann A, Hemphill A. Vaccine-Linked Chemotherapy Approach: Additive Effects of Combining the Listeria monocytogenes-Based Vaccine Lm3Dx_NcSAG1 With the Bumped Kinase Inhibitor BKI-1748 Against Neospora caninum Infection in Mice. Front Vet Sci. 2022 Jun 27;9:901056. doi: 10.3389/fvets.2022.901056. PMID: 35832325; PMCID: PMC9272043.
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
M�ller J, Anghel N, Imhof D, H�nggeli K, Uldry AC, Braga-Lagache S, Heller M, Ojo KK, Ortega-Mora LM, Van Voorhis WC, Hemphill A. Common Molecular Targets of a Quinolone Based Bumped Kinase Inhibitor in Neospora caninum and Danio rerio. Int J Mol Sci. 2022 Feb 21;23(4):2381. doi: 10.3390/ijms23042381. PMID: 35216497; PMCID: PMC8879773.
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
de Sousa MCF, Imhof D, H�nggeli KPA, Choi R, Hulverson MA, Arnold SLM, Van Voorhis WC, Fan E, Roberto SS, Ortega-Mora LM, Hemphill A. Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice. Int J Parasitol Drugs Drug Resist. 2024 Aug;25:100553. doi: 10.1016/j.ijpddr.2024.100553. Epub 2024 Jun 19. PMID: 38917582; PMCID: PMC11254172.
|
Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Our Target audience was Scientists following progress in drug development and they were reached by presentation of abstracts at meetings and a publication in the Journal of Infectious Diseases this year (see report). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Completense University of Madrid: As stated in the second budget period of the project, Yanina P. Hecker (https://orcid.org/0000-0001-9316-0588), a postdoctoral scientist, was hired by and is carrying out the pregnant sheep models of toxoplasmosis (including animal monitoring and collection of samples) and sample processing (cell culture, molecular and serological techniques). University of Kentucky: One PhD student, Izabela de Assis Rocha, has participated in the project and gained experience conducting pharmacokinetic experiments. How have the results been disseminated to communities of interest?Our results have been published as an original article in the Journal of Infectious Diseases: Sánchez-Sánchez et al., 2023. An early treatment with BKI-1748 exhibits full protection against abortion and congenital infection in sheep experimentally infected with Toxoplasma gondii. The Journal of Infectious Diseases, jiad470. doi: 10.1093/infdis/jiad470. Our results were also presented at two international meetings, the Apicowplexa 2022 (http://www.apicowplexa.net/) held in Bern (Switzerland) October 2022. This meeting focused on Apicomplexa in farm animals. Our pharmacokinetic results were also presented at the 2023 Conference of Research Workers in Animal Diseases. I. De Assis Rocha, M.A. Hulverson, R. Choi, L.K. Barrett, S.L. Arnold, W.C. Van Voorhis, S. Reed, D.K. Howe, and A.E. Page. Pharmacokinetics of Bumped Kinase Inhibitor-1708 in Horses: A Candidate for EPM Treatment. Conference of Research Workers in Animal Diseases, Chicago, IL, January 2023. What do you plan to do during the next reporting period to accomplish the goals?We plan to analyze samples collected from the dams to evaluate the efficacy of BKI-1748 against establishment of the chronic phase of the Toxoplasma gondii infection. Based on the encouraging results of the initial PK investigations of BKI-1708 in horses, a follow-up study will be performed to assess the cumulative effects of repeated dosing, as well as assess both safety and distribution in the central nervous system.
Impacts
What was accomplished under these goals?
Objective 1b: Efficacy of the lead BKI (BKI-1748) in a pregnant sheep model of toxoplasmosis. Experimental Design Nineteen Rasa Aragonesa breed sheep on day 90 of pregnancy and seronegative for the most common abortifacient agents were distributed into three experimental groups: Sheep from group 1 (G1, n=8) were infected with 1,000 TgShSp1 sporulated oocysts and 48 hours later treated with 10 doses of BKI-1748 at 15 mg/kg every 48 hours using 60% Phosal 53MCT, 30%PEG400 and 10% Ethanol 96 as vehicle. Sheep from group 2 (G2, n=8) were infected with 1,000 TgShSp1 sporulated oocysts but not treated. Sheep from group 3 (G3, n=3) were neither infected nor treated controls. The following parameters were evaluated throughout the experiment: Rectal temperatures: Rectal temperatures were recorded daily from 2 days before infection to 14 days after infection and then weekly after the end of the experiment. Fetal viability: Ultrasound scanning was carried out twice weekly from infection until delivery to evaluate fetal viability. Cellular and humoral immune responses: Blood samples were collected on days 0, 3, 5, 7, 10, 14 and then weekly until the end of the experiment to evaluate IgG responses in sera and IFN-? responses in supernatants from in vitro blood stimulation. Parasite detection by PCR: Parasite was detected by PCR in cotyledons from the placenta and in fetal target tissues (brain and lungs). RESULTS Rectal temperatures: Sheep from G1 (infected/treated) showed lower rectal temperatures from day 5 (p < 0.0001) through day 10 post-infection (p < 0.05) than sheep from G2 (infected/untreated) (Figure 1). ? Figure 1. Rectal temperatures. Sheep from G1 and G2 were infected with T. gondii oocysts. Sheep from G1 received the BKI-1748 treatment and sheep from G2 did not. Fetal viability: All sheep from G2 (infected/untreated) aborted (7/8 on day 8 post- infection and 1/8 on day 45 post-infection). By contrast, all sheep from G1 (infected/treated) and G3 (uninfected/untreated) gave birth to healthy lambs. Therefore, the treatment showed full protection against abortion. Cellular and humoral immune responses: Sheep from G1 (infected/treated) showed higher IFN-? levels on day 7 post-infection (p < 0.05) than sheep from G2 (infected/untreated). Regarding IgG responses, sheep from G2 (infected/untreated) seroconverted on days 14-21 post-infection. By contrast, sheep from G1 (infected/treated) and G3 (uninfected/untreated) remained seronegative throughout the experiment. Parasite detection by PCR: In abortions on day 8 post-infection in G2 (infected/untreated) parasite DNA was detected only in 1 out of 78 samples (parasite did not have enough time infect the placenta). By contrast, in the abortion on day 45 post-infection in G2 (infected/untreated) parasite DNA was detected in all samples. In G1 (infected/treated), parasite DNA was not found in any of the lambs born. Therefore, the treatment showed full protection against congenital infection. Objective 2: Test lead BKI for its effect in Equine Protozoal Myeloencephalitis (EPM). The new lead BKI for treatment of horses, BKI-1708, was tested in in vitro growth assays with Sarcocystis neurona. These experiments revealed an approximate IC50 of 42 nM for BKI-1708, indicating it would be a good candidate for pharmacokinetic analyses in horses. Sub-Objective 2a. Test late lead BKIs in horse PK. BKI-1708 single-dose oral PK experiment in horses: BKI-1708 was suspended at 25% in a commercial paste, and 3 mg/kg was administered orally to two, 3-year-old horses once. Blood samples were collected over a 72-hour period, and the drug levels in plasma were measured. These results estimated the bioavailability of BKI-1708 at 50%, with an approximate half-life of 15 hours. BKI-1708 multi-dose oral PK experiment in horses: A 20% suspension of BKI-1708 was made in a commercial paste, and 3 mg/kg was administered orally to two, 3-year-old horses daily for a 5-day period. Blood was collected multiple times on day 1, pre-dose and 8h post-dose on days 2-4, multiple times on day 5, and then daily until day 10 of the study period (5 days after final dose). Analysis of BKI-1708 in plasma revealed that steady state levels were achieved rapidly (48-72 hours), with peak and trough levels of 1 mM and 0.3 mM, respectively.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
S�nchez-S�nchez et al., 2023. An early treatment with BKI-1748 exhibits full protection against abortion and congenital infection in sheep experimentally infected with Toxoplasma gondii. The Journal of Infectious Diseases, jiad470. doi: 10.1093/infdis/jiad470.
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Completense University:As stated in the second budget period of the project, Yanina P. Hecker (https://orcid.org/0000-0001-9316-0588), a postdoctoral scientist, has been hired by the Complutense University of Madrid. She is carrying out the pregnant sheep models of toxoplasmosis (including animal monitoring and collection of samples) and sample processing (cell culture, molecular and serological techniques). University of Kentucky:One PhD student, Izabela de Assis Rocha, is participating in the project and has gained experience with conducting pharmacokinetic experiments. How have the results been disseminated to communities of interest?Results of the pharmacokinetics studies were presented at the EPM special interest group session of the 2021 American College of Veterinary Internal Medicine conference and at the 2021 Conference of Research Workers in Animal Diseases. Rocha, I., A. Page, S.M. Reed, D.K. Howe, W.C. Van Voorhis, S. Arnold, R. Choi, M. Hulverson, T. Kennedy. Updates on the Bumped-Kinase Inhibitors for Equine Protozoal Myeloencephalitis Therapy: Preliminary from the Pharmacokinetics Study. EPM Special Interest Group, 2021 Meeting of the American College of Veterinary Internal Medicine. Rocha, I., M.A. Hulverson, R. Choi, L.K. Barrett, S.L. Arnold, W.C. Van Voorhis, S. Reed, D.K. Howe, A.E. Page. Pharmacokinetics of bumped kinase inhibitor 1748 in horses: investigational therapeutic for EPM. Conference of Research Workers in Animal Diseases, Chicago, IL 2021. The results from the studies carried out will be presented in the international meeting Apicowplexa 2022 (http://www.apicowplexa.net/) about Apicomplexa in farm animals that will take place in Bern (Switzerland) next October, 2022. In addition, by the end 2022 a manuscript about these experiments will be submitted to a journal indexed in the JCR. What do you plan to do during the next reporting period to accomplish the goals?University of Completense and Univ of WA: Along the next year of the project, we will finish the analysis of samples to evaluate the humoral and cellular immune responses, the histological lesions in placental and fetal target tissues and the protection of the drug against the congenital infection (detection of the parasite in placental and fetal target tissues by PCR and quantification by qPCR). In addition, we will disseminate the results from the experiments through an international meeting (Apicowplexa 2022, Apicomplexa in farm animals) and the submission of a manuscript to a journal indexed in the JCR. University of Kentucky and Univ of WA:An experiment planned for mid-2022 will examine the PK for two alternative BKIs after intravenous administration in horses. Additional follow-up PK experiments will address other vehicles for oral administration of BKIs in horses.
Impacts
What was accomplished under these goals?
Objective 1:Test lead BKI(currently BKI-1748)for PK, safety and efficacy in a pregnant sheep model of congenital transmission ofT. gondiiat CUM Madrid with support by Univ of WA. Tasks to be accomplished during the project by SALUVET-Complutense University of Madrid with support from University of Washington: Year 1: Determine pharmacokinetics and toxicity of lead the BKI (BKI-1748) in pregnant sheep. Year 2 and year 3: efficacy of the lead BKI (BKI-1748) in a pregnant sheep model of toxoplasmosis. Tasks developed by SALUVET-Complutense University of Madrid Pharmacokinetics and toxicity of BKI-1748) in sheep As an initial attempt to know the pharmacokinetic and toxicity of BKI-1748 in sheep we tested an oral unidose of BKI-1748 at 5 mg/kg (solved in a vehicle based on 60% Phosal 53 MCT, 30% PEG400 and 10% Ethanol) in three non-pregnant sheep. Plasma levels of BKI-1748 peaked at 12 hours after administration with levels of 2.04 ± 0.53 µM, and no detectable plasma levels at 72 hours after administration. Concerning safety, rectal temperatures were in the physiological range (38.5-39°C) and there were not clinical signs of toxicity or abnormalities in the fecal consistency. After that, we carried out the study of the pharmacokinetic and toxicity of BKI-1748 in sheep at 90 days of pregnancy, testing two different oral regimens (using the same drug vehicle as above). The experimental design comprised three experimental groups; group I: 10 doses of 5 mg/kg every 24 hours (3 pregnant sheep), group II: 10 doses of 15 mg/kg every 48 hours (5 pregnant sheep) and group III: 3 pregnant sheep receiving the vehicle alone. Pharmacokinetics was characterized by maximum plasma levels of 1.5-2 µM in group I and 3-3.5 µM in group II and levels of 0.5 µM prior to the administration of the next dose in both treated groups. Concerning safety, no changes in rectal temperatures were found between treated and untreated animals and there were not abortions, clinical signs of toxicity, abnormalities in the fecal consistency or changes in the hematological (erythrocytes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin, packed cell volumen, platelets, leukocytes, segment neutrophils, lymphocytes, monocytes, eosinophils and basophils) and biochemical parameters such as proteins, albumin, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, creatine kinase, urea, creatinine, calcium, phosphorus, sodium, potassium. Efficacy of BKI-1748 in a pregnant sheep model of toxoplasmosis The regime of 10 doses of BKI-1748 at 15 mg/kg by the oral route every 48 hours was chosen to test the efficacy in a pregnant sheep model of Toxoplasma gondii infection since concerning safety it shows maximum plasma levels substantially above the inhibitory concentration 50 (IC50) and substantially lower than the levels showing side effects in a zebrafish (Danio rerio) embryo development model and a regime of 5 doses at 20 mg/kg every 24 hours was safe in pregnant mice. Concerning efficacy, according to previous experiments in a pregnant mouse model of T. gondii infection, BKI-1748 administration (5 doses at 20 mg/kg every 24 hours) showed high efficacy against pup mortality and vertical transmission of T. gondii. In pregnant sheep, we chose the regime of 10 doses every 48 hours trying to cover 22 out of 60 days with efficacious plasma levels trying to avoid the negative effects of the reactivation of the replication of the parasite after drug removal. The experimental design to test the efficacy of BKI-1748 in a sheep model of T. gondii infection at 90 days of pregnancy comprised three experimental groups; group I (n=8): infected with 1,000 T. gondii oocysts (TgShSp1 isolate) and treated (starting the treatment 48 hours after infection), group II (n=8): infected with 1,000 T. gondii oocysts (TgShSp1 isolate) and untreated and group III (n=3): uninfected and untreated animals. Objective 2:Test lead BKI(currently BKI-1748)for its effect in Equine Protozoal Myeloencephalitis (EPM) at the University of Kentucky with support by Univ of WA. The initial pharmacokinetic (PK) experiment was conducted on four horses fitted with intravenous and intrathecal catheters. The lead compound, BKI-1748, was administered orally at approximately 4 mg/kg, and blood and CSF samples were collected over a 72-hour period. Samples were analyzed by LC-MS/MS to assess the amount of BKI-1748 present. At two hours post administration, only one horse had detectable BKI in the blood and none had detectable values in the CSF. These findings suggested that the majority of BKI-1748 either was poorly absorbed and/or was cleared by "first-passage" by portal circulation through the horse liver. A second PK experiment was conducted in two horses that were administered BKI-1748 intravenously at 1 mg/kg. Analysis of blood samples revealed drug at peak values of >20 mM at five minutes post administration and still detectable at 12 hours, with a terminal half-life approximating two hours. Collectively, the results suggest that BKI-1748 clearance is moderate in horses and is likely poorly absorbed when administered orally. Future PK experiments will examine other BKIs along with different vehicles to improve absorption after oral dosing.
Publications
|
Progress 06/01/20 to 05/31/21
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? Dr Hemphill's and Dr. Van Voorhis' groups will collaborate to repeat the dosing and PK measurements with BKI-1748 in mice to achieve the exposure of BKI-1748 during therapeutic treatment of neosporosis in pregnancy Dr. Arnold has predicted the brain permeability of various BKI-backup compounds and Dr. Van Voorhis' group is testing the predictions in mice dosed with the BKIs. Dr. Howe is measuring the activity of these backups against Sarcocystis neuronae in vitro. Drs. Howe, Allen, and Reed are exploring vehicles for improved BKI-1748 oral exposure in horses to conduct a multidose PK experiment to direct the Sarcocystis therapy experiments. The IV PK obtained suggests that with better absorption of solubilized compound, efficacy with twice daily dosing can be achieved.
Impacts
What was accomplished under these goals?
Additional safety tests were run with BKI-1748 by the consortium including: Dog cardiovascular safety testing with no effect up to 17.5 uM; safety in pregnancy sheep demonstrated by Dr. Ortega Mora's group at 5 mg/kg CD x 10 days with no issues; no zebrafish embryotoxicity noted until trace effects at 20 uM, no mouse pregnancy interference at 20 mg/kg/day x 5 days, and no mouse toxicity observed at 100 mg/kg/day x 7 days with negative biochemical and histopathology toxicity. The conclusions of these tests were that the safety profile of BKI-1748 remains outstanding in the range needed to treat toxoplasmosis and probably sarcocystosis and that BKI-1748 remains our best lead BKI compound for the treatment of these infections. Dr. Hemphill's group treated pregnant mice for 5 days in mid-gestation, beginning 48 hours after infection with 100 T. gondii Spain7 oocysts per pregnant mouse. The efficacy outcome was outstanding, in that there was no fetal loss, compared with 100% fetal loss in the 100 oocyst T. gondii infected and untreated control groups. A PK profile for mice treated with BKI-1748 needs to be obtained to further define the therapeutic exposure required. A follow up PK study is planned to help define the exposure needed (see Plan #1 below). Drs. Howe, Allen, and Reed performed an initial PK study of horses orally dosed with BKI-1748. The horses were dosed 1X with 2.5 mg/kg of BKI-1748 suspended in 3% ethanol /7% Tween-20/ 90% saline. Unfortunately, the compound was in a slurry and no compound was found in the peripheral blood, perhaps suggesting poor absorption or first pass complete clearance. Horses were then dosed once by IV with BKI-1748 compound dissolved in PEG-400(90%)/DMSO(10%) at 1 mg/kg. The compound was rapidly cleared, but there was still detectable compound at 12 hrs after dosing. This suggest the previous vehicle did not lead to sufficient absorption and exposure. We will work on finding a better oral vehicle in the next period.
Publications
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