WO2024210859A1

WO2024210859A1 - Use of embryonated toxocara canis eggs inactivated by different methods as a drug candidate

Info

Publication number: WO2024210859A1
Application number: PCT/TR2023/051684
Authority: WO
Inventors: Kübra ASLAN; Ahmet Eken; Alparslan YILDIRIM; Halit CANATAN
Original assignee: Erciyes Universitesi
Current assignee: Erciyes Universitesi
Priority date: 2023-04-05
Filing date: 2023-12-25
Publication date: 2024-10-10
Anticipated expiration: 2025-10-05
Also published as: TR2023003766A2

Abstract

This invention is for use of the embryonated Toxocara canis eggs inactivated by different methods as a drug candidate.

Description

USE OF EMBRYONATED TOXOCARA CANIS EGGS INACTIVATED BY DIFFERENT METHODS AS A DRUG CANDIDATE

TECHNICAL FIELD

Multiple Sclerosis (MS) is an inflammatory chronic disease characterized by demyelination of the central nervous system. Immunologically, a patient's own helper T cells, known as CD4+, play a major role in MS pathology. In MS, which is an autoimmune disease, Th1 and Th17 cells, known as pro-inflammatory cells, and the cytokines secreted by them such as IFN-y, IL-ip, IL-17, IL-22 and GMCSF increase, while the numbers or functions of Tregs, which are regulatory cells, and the levels of IL-10 and TGF-p, regulatory cytokines, are decreased. Th cells, which play an important role in the pathology of Experimental Autoimmune Encephalomyelitis (EAE) and Multiple Sclerosis (MS), are basically divided into four groups: Th1 , Th2, Th 17, Th22. However, there are also hybrid cells such as Th1/Th17, Th17/Th22. In MS, which is an autoimmune disease, Th1 , Th17 and Th22 cells and the cytokines produced by them, such as IFN-y, IL-17A and IL-22, come to the fore. This imbalance between the pro-inflammatory cells and the regulatory cells causes a repeated tissue damage and neurodegeneration. It manifests itself with numbness, tingling, low vision, urinary and fecal incontinence, and cognitive failure. Although MS is frequently seen in young women, it is 3 times more common in women than in men.

MS, which seriously negatively affects the lives of young individuals, also causes significant costs to the healthcare system in the developed countries.

The most common type of MS is known as Relapsing-Remitting MS, which alternates with periods of attacks and remissions. Many drugs used in the treatment of MS aim to reduce complications during these periods of attacks. However, there is no effective drug to stop the progression of the disease.

There are 20 drugs approved by the FDA for use in the treatment of MS. These drugs are generally characterized in that they are intended to reduce the attacks during the periods of relapse. However, they do not directly affect the progression of the disease. Therefore, it is necessary to prevent the development of the disease or to develop new agents that have protective properties.

Fingolimod is the first MS drug approved by the FDA. T and B cells intensely express the S1 P1 receptor on the cell membrane, which promotes their egress from the lymph nodes. Fingolimod-phosphate, the active form of fingolimod, acts as an antagonist of S1 P1 receptors, downregulates the receptor and prevents lymphocyte egress from the lymph nodes. These lymphocytes are not able to exhibit autoreactivity as they cannot enter the central nervous system. Due to the expression of the S1 P receptor in the vessels and myocardium, the use of Fingolimod slows the heart rate and causes a slowdown in the electrical conduction of the heart. Also, fingolimod has been reported to cause macular edema in the eye and thinning of the nerve fibers of the retina. The side effects of fingolimod include liver deformation and tumor development such as basal carcinoma. It is an oral tablet.

Apart from fingolimod, drugs frequently used in MS treatment include glatiramer acetate, lnterferon-i p blockers, teriflunomide, natalizumab, ocrelizumab, and alemtuzumab. These drugs are generally administered by injection. These are common drugs such as destroying effector T cells, inhibiting costimulators required for the activation of T cells, and suppressing pyrimidine synthesis that will prevent DNA synthesis in the cell.

Although the mechanism of action of the inactivated T. canis eggs used in our study is unknown, it is predicted that they have a more specific mechanism in the treatment of MS other than the mechanisms mentioned above. However, it has a completely different mechanism than said drugs. In the EAE model of MS disease applied to mice, the heat- inactivated T. canis eggs were administered to mice by oral gavage. As a result of our studies, the inactive T. canis eggs have been observed to reduce the disease scores, that is, the prognosis and the incidence of the disease. Also, it has been found to generally increase CD4 helper T cells and Treg cells, which play an important role in MS pathology, when examined in the spleen and lymph nodes. It also reduced the GMCSF cytokine, which has pathogenic properties in MS prognosis. Our project work to understand its mechanism of action and the effects on the brain continues. STATE OF THE ART

1. Borhani Zarandi, M, Hoseini, S.H, Jalousion, F, Etebar, F, Vojgani, M. Evaluate Toxocara canis excretory-secretory antigens in experimental allergic encephalomyelitis (EAE). Iran J.Vet Med.2018;1 1 (2):175-182.

It is not known whether the team in Iran obtains a patent regarding this study. Unlike our study, T. canis antigens were tested in the EAE model in this study. It is not the egg that is inactivated. In this study, the secreted antigens were shown to reduce clinical scores. In our study, the cell levels, protein amounts, etc. have been flow cytometrically demonstrated using more techniques than this study. The parasite products used in this study and in our study are completely different.

2. Jan Novak, Tomas Machacek, Martin Majer, Marie Kostelanska , Katerina Skulinova, Viktor Cerny , Libuse Kolafova, Jiri Hrdy and Petr Horak. Toxocara canis infection worsens the course of experimental autoimmune encephalomyelitis in mice. Parasitology. Accepted Manuscript- DOI: 10.1017/S0031 182022001238

This study is the closest study to ours. However, T. canis used in this study has provided a T. canis larva which has the ability to actively cause a disease. Additionally, as a result of this study, Toxocariosis, a T. canis infection, has been shown to worsen EAE scores and did not have a curative effect on MS.

3. CalogeroEdoardo Cicero, Francesco Patti, Salvatore LoFermo, Loretta Giuliano, Cristina Rascuna, Clara Grazia Chisari, Emanuele D’Amico, Vincenza Paradisi, Benoit Marin, Pierre Marie Preux, Antonia Mantella, Alessandro Bartoloni, Mario Zappia, and Alessandra Nicolett. Lack of association between Toxocara canis and multiple sclerosis: A population-based case-control study. Multiple Sclerosis Journal.2020; 26(2):258-259. https://doi.org/10. 1177/1352458518825394

In this study, the incidence of MS in people with T. canis infection was examined and no correlation was found between MS and T. canis infection. This study is a study in which T. canis antigens are measured serologically. Our study is a study conducted by creating an in vivo mouse model. Most of the detailed flow cytometric analyzes used in our study were not performed throughout these studies. These studies are generally in vitro studies. However, in our study, a EAE model was created and the eggs inactivated by different methods have been administered to mice, and the protective and curative effects of these eggs were tested and observed.

Drugs currently used in the treatment of MS are generally aimed at reducing the severity and frequency of attacks. Many anti-inflammatory, immunomodulatory and immunosuppressive drugs used in the treatment of MS reduce relapses but have limited effect in the progressive disease.

Although there are 20 drugs approved by the FDA for the treatment of MS, these drugs do not have a fully curative effect in the treatment of MS. The inactivated T. canis eggs included in our study are a part of helminthic therapy. Helminthic therapy has been used for decades in the treatment of autoimmune diseases such as autoimmune bowel disease and MS.

In the later stages of our study, it will be investigated which of the components in the inactive egg has a curative effect on the disease, and the necessary studies will be initiated to turn this product into a drug.

Many drugs used in the treatment of MS are administered by injection. These drugs have many problems such as inflammation at the injection site, as well as many side effects such as heart diseases and visual disorders.

The inactivated T. canis eggs used in our study did not cause any problems when tested in mice. It also reduced the clinical scores of the disease and delayed the onset of the disease. It also increased the number of regulatory T cells responsible for suppressing autoimmunity and decreased the GMCSF cytokine, a pathogenic cytokine, in the treated groups.

The inactivated T. canis eggs function as an immunomodulatory molecule.

Fingolimod, alemtuzumab or natalizumab are used in the treatment of MS. Fingolimod is an immunomodulating drug mostly used in the treatment of MS. Fingolimod is a sphingosine-1 -phosphate receptor modulator that blocks the egress of the lymphocytes from the lymph nodes and prevents them from contributing to an autoimmune reaction. The most common side effects of fingolimod are cold, headache and fatigue. Fingolimod has also been associated with potentially fatal infections, bradycardia, and, most recently, a case of focal encephalitis, an inflammation of the brain with bleeding.

Alemtuzumab reduces T and B cells carrying CD52 marker, monocytes, macrophages and other bone marrow cells. It is not specific. Alemtuzumab is used in the selected patients with high MS disease activity who does not recover despite the existing treatments. It is not suitable for newly diagnosed patients. It is applied to the patients who do not benefit from the first and second-line treatments and to the patients for whom it is not used due to the side effects or special reasons.

Apart from fingolimod and alemtuzumab, drugs frequently used in MS treatment include glatiramer acetate, lnterferon-ip blockers, teriflunomide, natalizumab, and ocrelizumab. These drugs are generally administered by injection. These are common drugs such as destroying effector T cells, inhibiting costimulators required for the activation of T cells, and suppressing pyrimidine synthesis that will prevent DNA synthesis in the cell. In our study, the inactivated T. canis eggs were tested in a mouse model, that is, on animals, and no side effects were observed.

DEFINITION OF THE INVENTION

The exact cause of MS disease is unknown. Only some T cell subsets are known to have an effect. The drugs used today are the ones that act these substances. Our active ingredient also affects these T cell subsets. The currently used MS drugs offer a definitive treatment method. Reducing the number and severity of attacks is not a definitive solution for the treatment of patients.

Thus, new drug candidates are needed. T. canis eggs inactivated by different methods have been a part of helminthic therapy that has been applied for years. In our study, we have shown that these eggs have some immunosuppressive and immunomodulatory effects, such as increasing the number of Treg cells and reducing clinical scores. In addition, the T. canis eggs used in our study provide immunomodulation, reduce pathogenic lymphocyte migration to the central nervous system, and do not non- selectively inactivate many immune cells like some other drugs used (Fingolimod, natalizumab, alemtuzumab). The immune depression/suppression characteristic of these drugs is not present in the inactivated T. canis eggs. Again, some S1 PR blockers used have heart-related side effects, but such a side effect of T. canis is not known.

The inactivated T. canis eggs have an immunomodulatory effect.

FIGURES

Fig. 1 : A) Clinical score chart obtained from EAE Scoring B) Calculations for area under the curve

According to the EAE Clinical score chart, the disease had a late onset in the EAE prophylactic group and the scores were lower than other groups. In the EAE therapeutic group, the scores decreased towards the end of the disease and recovery was observed. When the area under the curve is calculated, there is a significant decrease, especially in the EAE Prophylactic group.

Fig. 2: Incidence of disease among EAE groups. The incidence of EAE was lower in the therapeutic group than in the control group, especially in the prophylactic group.

Fig. 3: Effect of the inactivated T. canis eggs on Treg cells. FOXP3+ Treg cells increased especially in the spleen both in the prophylactic and therapeutic groups.

Fig. 4: Effect of the inactived T. canis eggs on GMCSF Cytokine production. The inactived T. canis eggs significantly reduced GMCSF, a pathogenic cytokine, in CD8 cells, especially in the therapeutic group (p<0.0001 ).

DESCRIPTION OF THE INVENTION

Method:

C57BL/6 male mice were maintained under pathogen-free conditions in accordance with welfare conditions in the animal facilities at the Experimental Research and Application Center of Erciyes University in Kayseri, Turkey. Animals were kept at room temperature in plastic propylene cages and in a sterile condition. 6-8 week-old male animals weighing 20-25 g were used in the research. All procedures were approved by the Institutional Ethics Committee for Animal Experiments (HADYEK, Erciyes University).

Feeding:

Our mice were divided into groups as in the table below and the specified administrations were carried out. Control group mice were administered 100 uL of tap water orally (as gavage) to prevent any changes due to treatment stress.

Experimental Groups

Inactivation Methods of T. canis eggs Heat Inactivation

In our study, heat-inactivated eggs have been tested on mice at this stage. As the eggs were administered to the mice as 1000 eggs, 1000 eggs were taken into the tubes of 1 .5 ml, washed with 1 ml of sterile 1 X Phosphate buffered saline (PBS), centrifuged at 400 G for 10 minutes, and then dissolved in 100 ul of sterile tap water and inactivated by being kept in a 60 °C heat block for 1 hour, and the inactivation control was performed by an inverted microscope.

Inactivation with paraformaldehyde

After 1000 eggs are washed twice again with 1X PBS, they will be incubated in 4% paraformaldehyde for 30 minutes in an incubator at 37 °C, and the inactivation process will be checked with an inverted microscope.

Sonication After Heat Inactivation

The eggs inactivated by heat as described above will then be lysed in a sonicator at 20- 30 kHz for 5 minutes and administered to mice.

Inactivation with Microwave

After 1000 eggs are washed twice again with 1 X PBS, the inactivation process will be checked by applying a microwave at 350-650 W for 10-75 seconds. This method was previously tried on another parasite by Kernou et al (2022) (Kernou et aL, 2022).

Reference: Kernou, O. N., Belbahi, A., Sahraoui, Y., Bedjaoui, K., Kerdouche, K., Amir, A., ... Rijo, P. 2022. "Effect of Sonication on Microwave Inactivation Kinetics of Enterococcus faecalis in Dairy Effluent". Molecules, 27(21 ).

Development of acute EAE with MOG35-55 peptide immunization and EAE scoring X volume of MOG35-55 peptide prepared in 1 mg/ml PBS and X volume of Complete Freund’s Adjuvant prepared and boosted by adding 40mg/ml inactivated Mycobacterium T uberluosis were mixed in a glass injector by means of a stopcock to obtain an emulsion, and 100 pl emulsion was injected subcutaneously into the right and left hip/abdomen junction area of the mice. Additionally, on the day of immunization (day 0) and day 1 , 200 ng of Pertussis Toxin (in 200pl PBS) was injected intraperitoneally and the mice were monitored every day for the development of EAE. EAE development was scored according to the following criteria:

• Tail weakness: 1 ;

• hind limb weakness and inability to right itself if turned upside down: 2;

• paralysis of one of the hind limbs: 2.5;

• paralysis of both hind limbs: 3;

• paralysis of one of forelimbs: 3.5;

• paralysis of both forelimbs: 4;

• moribund:5

Obtaining, Inactivating and Administrating T. canis Eggs

T. canis eggs were isolated from canines and the embryo development was achieved for 4-5 weeks at 25 °C.

The embryonated eggs were used in this study. The eggs were inactivated by incubation at 60 °C for 60 min. Temperature and time were modified from the study with Ascaris.

In the literature, in mouse models obtained with T. canis eggs, 500-2500 eggs were administered per mouse. In our study, we administered 2000 inactived T. caniseggs per mouse.

A total of 2000 inactived T. canis eggs in 200 pl of tap water per mouse were administered to mice by oral gavage in 2 pieces. To the mice, 1000 inactived T. canis eggs were administered via oral gavage in the EAE prophylactic group on days 0 and 5, and 1000 inactived T. canis eggs were administered in the same way in the EAE Therapeutic group on days 10 and 15. The control group was administered only 100 pl of tap water. In this study, the heat inactivation method has been tried so far and successful results have been obtained. In the study, egg inactivation will also be carried out using paraformaldehyde, microwave application and sonication.

Obtaining Lymphocytes from Brain and Spinal Cord Mice were sacrificed by cervical dislocation and quickly perfused through the left ventricle with 10 ml 1 X PBS, and this process was repeated 3 times. Then the brain and spinal cord were removed and placed in PBS.

1 ml of DMEM medium containing 2.5 mg/ml collagenase was taken and transferred to a 10 cm culture dish, and the organs were cut into 1 mm pieces with a scalpel. The single cell mixture resulting from enzymatic treatment for 30 minutes at 37 °C was passed through a 70 pm filter, centrifuged at 1200 rpm for 5 minutes, and the pellet was dissolved in 5ml of 70% Percoll and transferred to a 15 ml falcon tube. It was brought up to the volume with 5ml of 37% Percoll and rotated at 1800 rpm for 20 minutes at room temperature without brakes. Lymphocytes were collected from the middle fraction, dissolved in RPMI 1640 containing 10% FBS, counted and used for experiments.

Obtaining Lymphocytes from Spleen and Lymph Node

The spleen and lymph node were crushed with 1X PBS with the back of a syringe, and the supernatant was discarded by centrifuging at 400 RCF for 5 minutes. 1 ml of lysis buffer was added to the spleen tissues and incubated at room temperature for 5 min., then centrifuged at 400 RCF for 5 minutes and the supernatant was discarded. The spleen and lymph nodes were washed twice with sterile PBS and then dissolved in 1 ml complete medium and used in the experiments.

Determination of the Quantity of Th1 , Th 17, Th22 and Treg Cells in Central Nervous System and IL-22, IL-17A, IFN-y, GMCSF Cytokine Production

In order to determine the quantity of Th1 , Th17 and Th22 cells, the cells obtained from the central nervous system as mentioned above were placed in 96-well round-tipped plates and induced with PMA (50ng/ml), lonomycin (1 pg/ml) and Golgi Plug (1 pl/ml) in 10% FBS+RPM1 1640+ Anti-anti culture medium for 4 hours. Samples in the culture plate taken on ice were treated with 1 pg/ml anti-mouse CD16/CD32 for 5 minutes, followed by blocking Fc receptors, and then surface staining with anti-TCRa/b-FITC for 15 minutes on ice. After centrifugation at 1500 rpm for 3 minutes, the cells were washed twice with the staining buffer (2% FBS 1X PBS), dissolved in 10OpI Fix/Perm Buffer (BD), fixed and permeabilized for 20 minutes at 4 °C, then centrifuged at 1500 rpm for 3 minutes, and washed twice with Perm Buffer and centrifuged. Then, PE-IL-17A, APC-IFN-y or PE-IL- 22, APC GMCSF antibodies diluted in 1 OOpI Perm buffer (500x) were added and stained for 30 minutes at 4 °C, then washed 2 times with the staining buffer and analyzed in the flow cytometry device.

Method:

Feeding:

Experimental Groups

Experimental Group Number of Animals Administration Day of Administration

Naive Control 7 100 ul tap water gavage/animal were administered 2 times Days -10 And -5

Naive inactivated T. canis egg 8 1000 eggs gavage / animal were administered 2 times Days - 10 and -5

EAE Control 15 100 ul gavage/animal were administered 4 times Days 0 and 5 Days 10 and 15 EAE inactivated T. canis eggs prophilactic 15 1000 eggs gavage / animal were administered 2 times Days 0 And 5 EAE inactivated T. canis egg therapeutical 1000 eggs gavage / animal were administered 2 times Days 10 and 15 Development of acute EAE with MOG35-55 peptide immunization and EAE scoring X volume of MOG35-55 peptide prepared in 1 mg/ml PBS and X volume of Complete Freund’s Adjuvant prepared and boosted by adding 40mg/ml inactivated Mycobacterium T uberluosis were mixed in a glass injector by means of a stopcock to obtain an emulsion, and 100 pl emulsion was injected subcutaneously into the right and left hip/abdomen junction area of the mice. Additionally, on the day of immunization (day 0) and day 1 , 200 ng of Pertussis Toxin (in 200pl PBS) was injected intraperitoneally and the mice were monitored every day for the development of EAE. EAE development was scored according to the following criteria:

• Tail weakness: 1 ;

• hind limb weakness and inability to right itself if turned upside down: 2;

• paralysis of one of the hind limbs: 2.5;

• paralysis of both hind limbs: 3;

• paralysis of one of forelimbs: 3.5;

• paralysis of both forelimbs: 4;

• moribund:5

Obtaining, Inactivating and Administrating T. canis Eggs

A total of 2000 inactived T. canis eggs in 200 pl of tap water per mouse were administered to mice by oral gavage in 2 pieces. To the mice, 1000 inactived T. canis eggs were administered via oral gavage in the EAE prophylactic group on days 0 and 5, and 1000 T.canis eggs were administered in the same way in the EAE Therapeutic group on days 10 and 15. The control group was administered only 100 pl of tap water. In this study, the heat inactivation method has been tried so far and successful results have been obtained. In the study, egg inactivation will also be carried out using paraformaldehyde, microwave application and freezing with silicic acid.

Obtaining Lymphocytes from Brain and Spinal Cord

Mice were sacrificed by cervical dislocation and quickly perfused through the left ventricle with 10 ml 1 X PBS, and this process was repeated 3 times. Then the brain and spinal cord were removed and placed in PBS.

Obtaining Lymphocytes from Spleen and Lymph Node

Determination of the Quantity of Th1 , Th 17, Th22 and Treg Cells in Central Nervous System and IL-22, IL-17A,

IFN-y, GMCSF Cytokine Production

In order to determine the quantity of Th1 , Th17 and Th22 cells, the cells obtained from the central nervous system as mentioned above were placed in 96-well round-tipped plates and induced with PMA (50ng/ml), lonomycin (1 pg/ml) and Golgi Plug (1 pl/ml) in 10% FBS+RPM1 1640+ Anti-anti culture medium for 4 hours. Samples in the culture plate taken on ice were treated with 1 pg/ml anti-mouse CD16/CD32 for 5 minutes, followed by blocking Fc receptors, and then surface staining with anti-TCRa/b-FITC for 15 minutes on ice. After centrifugation at 1500 rpm for 3 minutes, the cells were washed twice with the staining buffer (2% FBS 1X PBS), dissolved in 10OpI Fix/Perm Buffer (BD), fixed and permeabilized for 20 minutes at 4 °C, then centrifuged at 1500 rpm for 3 minutes, and washed twice with Perm Buffer and centrifuged. Then, PE-IL-17A, APC-IFN-y or PE-IL- 22, APC GMCSF antibodies diluted in 100pl Perm buffer (500x) will be added and stained for 30 minutes at 4 °C, then washed 2 times with the staining buffer and analyzed in the flow cytometry device.

Antibodies Used:

1 ^st Mix 2^nd Mix 3^rd Mix 4^th Mix

APC-CD11 b APC/Cy7 CD3APC/Cy7 CD3 APC/Cy7 CD3

PE-Ly6G PE CD4 APC CD4

APC CD4

FITC CD19 APC F0XP3 FITC CD8 FITC CD8

PerCp/Cy5.5 CD138 PE/Cy7 IFN-g PE GMCSF

PE IL-22

PerCp/Cy5.5 TNF-a

PerCp/Cy5.5 IL-17A

Spinal Cord Histopathology

1 cm spinal cord removed after perfusion was placed in 10% formalin and transferred to 70% alcohol after 24 hours. Then, they were frozen in OCT medium, cut with a cryostat, mounted on a slide, stained with Hematoxylin-Eosin, and the pathology and infiltrated lymphocytes were scored.

Molecular Studies:

1. RNA isolation

2. cDNA synthesis 3. The amount of cytokines IL-4, IL-5, IL-13, IL-17A, IL-22, GMCSF, IFN-g, TGF-B, IL-6, IL-10, FOXP3 will be determined using RT qPCR.

Multiplex Elisa

By means of the Legendplex Th mouse cytokine panel, 13 cytokines belonging to Th cells will be determined simultaneously from the serum (Biolegend 741044).

As a result of these experiments, the inactivated T. canis eggs reduced the clinical scores of the disease and delayed the onset of the disease. It also increased the number of regulatory T cells responsible for suppressing autoimmunity and decreased the GMCSF cytokine, a pathogenic cytokine, in the treated groups.

1 It is not known to be effective in multiple sclerosis (MS). By performing clinical scoring in the MS mouse model, the MS clinical scores of the mice decrease significantly.

2 By performing an incidence analysis in the MS mouse model, the incidence of MS in mice significantly decreases.

3 It is unknown how it affects the CD4+ T cells and cells in the tissues it affects in the animal model of MS. Th17 cells decrease. Treg cells increase. The total number of CD4+ T cells decreases.

4 Its effect on the MS-associated cytokines is unknown. In our study, it reduced the amount of GMCSF, a pathogenic cytokine.

Claims

1 . Use of the embryonated Toxocara canis eggs inactivated by inactivation methods such as heat, paraformaldehyde, sonication after heat inactivation, and microwave as drug candidates, characterized in that the eggs are used for the treatment of multiple sclerosis.